ACTIONS OF RADIATIONS ON LIVING CELLS ACTIONS OF RADIATIONS ON LIVING CELLS Bx THE LATE DE LEA SECOND EDITION CAMBRIDGE AT THE UNIVERSITY PRESS 1962 PUBLISHED pry THE BYNDICS OF THE CAMBRIDGE UNIVERSITY PRESS Bentley House 200 Euston Road London NW 1 American Branch 32 Hast 57th Street New York 22 NY West African Office PO Box 33 Ibadan Nigeria Fust Edition 1916 Second Edihon 1955 Reprinted 1962 Printed in the United States of America CONTENTS Prefaces page it Last of Illustrations xm Chapter I Physical Properties and Dosimetry of Different Radiations 1 Jonization excitation and point heat, p 1 Ultraviolet hght, p 3 X rays p & yrays p 13 # rays and cathode rays p 15 2 rays p 16 Protons and neutrons, p 19 Dostnetry, p 21 Primary ronization ranges and energy dissipation p 23 Secondary 1omzation, clusters andé rays p 26 Number and total range of 1oaiz1ng particles per unit volume of tissue p 30 Chapter YE Chemical Effects of Tomzing Radiations, and Possible Mechunisms of Biological Action 33 The sone yreld p 32 Ges reactions p 34, Liquidsand sohds p 37 Solutions of water, p 40 Indirect action in aqueous solution p 42, p 39 Decomposition Tonic yieldain aqueous solution p 45 Chemical mechaniam of the indirect action m aqueous solution p 47 Spatial distribution and recombination of the active radicals,p 48 Kaneties of indirect actions in aqueous solution p 62 Competr tion between two solutes protective action p 65 Reduced yield in dilute solutions and with densely ionizing radiations p 57, Direct and indirect actions of radiation p 60 PossiBLz Mops or BroLoaicar AcTION OF Rapiation p 64 Cell poisons p 64 Activated water reactions p 64 Durect action on large lecul P 5 Localizat of p 86 The target theory p 66 Spread of the effect of an sonization p 67 Chapter II The Target Theory 69 Recognition of the single sonzation type of action p 71 Shape of survival curve, p 72 Time intensty factor p 78 Dependence on type of radiation p 78 Relation between target size and dose, p 80 8 d -y lonuzation p 88 The multi target thedry p 90 arays and y rays p 9I The assumptions of the single ronization target theory, p 92, Tho valtdity of the simple model, p 98 Chapter TV The Inactivation of Viruses by Radiation 100 The viruses p 100 The eizes of the viruses, p 102 The estumat: sctinity,p. 164, Technique of virus radiator te p 108 Disect and indirect actions of radiation on v: ruses p 108 Evivence ruar Virus Inactivation 1s Dug To A Srvore Ionization p 111 Exponential survival curves p_ 1 112 Independence of inactivation dose mntenait P 114 Dependence of inactivation dose on 10n density of radiation, P. ns lation between virus size and activation dose p 216 of The status the viruses p 123 Inactivation of viruses by ultra violet hight p 124 PUBLISHED BY THE SYNDICS OF THE CAMBRIDGE UNIVERSITY PRUSS Bentley House 200 Euston Road London NW 1 American Branch 32 East 51th Street New York 22 NY West African Office PO Box 23 Ibadan Nigeria First Edition 1946 Second Edition 1055 Reprinted 1962 Printed in the Umted States of Amervea CONTENTS vn Appendiz I Supplementary Calculations page 345 Appendix IT Textual Revisions and Additions 364 Muratiovs p 364 Tho of Pp p 364, % yrelds in diffe cells, p 364 Domrvant Litas, p 365 Crromosome Srrucrunat Damace, p 366 Location of breaks p 366, Frequency relations, p 366 Modifying factors, p 367, Sensitivity at different cies: Pp 309 Dose relations, p 570 Rolative typos frequency, p 372 Alternative derivation of G, Pp 373 Bacrerta p 373 Effect of intensity, p 373, Metabolic disturbances, p 374 Bibliography 375 Additions to Bibhography 396 Author Index 399 Subject Index 404 Note A marginal asterisk (*) refera to Appendix II vi CONTENTS Chapter VV Genetcal Effects of Racdhation page 126 The mechanism of horedity, p 126 Mutations1? 131 The nature of genes “aaa The one visible bychanges, and the p 140, position effect lethal mutations in Drosophila p 144 Relation between lothals and chromo some stroctural change p 154, Dominant lethals in Drosophila p 161 Deductions concerning the size of'the gene p 172 Cosmic rays and mutations Pp 180, Genetical effects of ultra violet hght, p 181 Chapter VI The Product of Ch Structural Changes by Radiation 189 P Is p 189 changes and phystological changes in chromosomes p 192 , ‘Tyrzs or Staucrunan Cuancr, p 197 a ee breaks p 199 Chromatid, breaks p 201 Teochromatid pee Faterchanges p 207 Tonteee i y of and 207 Pp 211, Location ofbreaks in p 2 1 p 217, ‘Modifying factors i 220 Dependence of the Ma old of structural changes on efficiencies of diferent wave feng and types of ionizing Tadiation a,237, 226 of yreld on dose p 229, Relative coer by ultra violet hght, Pp 242 o! p 240, shanges Chapter VII The Mechanism of Induction of Chromosome Structural Changes 245 Reasons for beheving that a break 1s caused by a single 1onzing particle, p 246 Distance epart at the moment of breakage of breaks which exchange p 249 Proportion of breaks which reatitute m Tradescantic, p 253 Dependence of the yield of aberrations im Tradescantia upon the duration of exposure p 262, The relatavo efficiencies of different radiations p 269 Isochromatid breake, p 277 Recapitulation p 279 Chapter VIII Delayed Division 282 Introduction, p_282, Delay of first cleavage in soa urchin eggs p 284 Stage ‘of divmion which 8 subject to delay p 293 Rapidly dividing tissues p 295, Inerease of cell mze p 300, The relative efficiencies of different radiations mn cautang delay of divinon p 303 Chapter 1X Lethal Effects 307 Death precipitated by division, p 307, The cause of desth at division p 311 Leraat. Muratrons p 313 of bactena mterpreted aa Tedbad Botton p 324 The wactivation of viruses ‘The kallmg of bacteria, p 316 The killing Leraal Caromosome StrucevraL Caances p 328 hale Le Pp 320 T p 332 The bean root p 335 Lethal 1 p 337 partial ateriity P,os Recapitulation and apphestion to rapidly dividing Rmmal tissues p 3: PREFACE TO THE FIRST EDITION My intention in writing this book has been to give an account of certain of the simplest and most fundamental actions of X-rays andother1omzing radiations onhving cells Thavenot attempted to survey the whole field of the biological effects of radiations Instead, I have thought that a useful purpose would be served by grving a rather detailed discussion of the mechamsm of those actions of radiation which are suffinently well understood for such a treatment to be profitable at the present time After introductory chapters desenbing the relevant physical properties and chemnecal effects of 1omzing rachations, the bulh of the book 1s occupied by the discussion of the effects of radia- tions on viruses, and on the genes and chromosomes of ngher cells In the concluding chapter the kulling of cells by radiation 18 discussed in so far as it can be understood in the hight of the preceding chapters One of the difficulties in writing the book has been to decide what background to assume the reader to possess In an en- deavour to make the book to some extent understandable by all classes of reader, J have wntten the physical chapter in an elementary fashton, and where it seemed practicable, have pre- faced my accounts of the various biological actions of radiation by a brief description of the properties and method of handhng of the organisms concerned While in general these mtroductory sections are brief, I have thought it advisable to provide a rather detailed troduction to the chapter on the genetical effects of radiation The rather techmcal vocabulary of the geneticist 1s not well understood by non-specialists, and im consequence many workers studymg the biological effects of radiation do not adequately appreciate the very considerable contributions which have been made to this subject by geneticists The study of the action of radiations on viruses, genes, and chro has reached a stage where the expemments are mamly quantitative, and the interpretations therefore neces- sarily to some extent mvolve elementary mathemati cs When developing such math tical interpretationg I have kept the algebraic detail in the background as far as possible, and have provided graphs and tables which enable the experrmental ist to PREFACE TO THE SECOND EDITION This book has been out of pnnt for some considerable time In attempting to meet the demand for a second edition, 1t was con- sidered desirable to make this challenging presentation of certain aspecta of radiolology available again in its orginal form, which bears on every page the mark of Lea’s clear mind and incisive logic The textual revisions, and some of the additions which Lea himself had written into his own copy of the book, have, however, been incorporated These additions appear as Appen- dix II, and the attention of the reader 1s directed to them by & marginal asterisk in the text Asa physicist Lea was acutely aware of the concentrated dis sipation of energy along the tracks of mdividual particles, which 1s a unique feature of the r1omzing radiations considered as cyto- toxic agents, and was especially mtercsted in exploring the implications of this fact for radiobiology In his writings, how- ever, at least as far back as 1941, he makes the distinction, which as exphotly discussed m the opening paragraph of Chapter II, between those forms of biological damage to which these con- cepts can and cannot usefully be apphed In his treatment of those forms of biological damage which he beheved to be mtiated by individual ionizing acts, he had the foresight to recogmze and ta allow for the spread of the energy absorbed within a molecule over distances of a few millimicrons, and for the effects of energy absorbed in the aqueous phase surrounding a biological structure Had he lived, the present edition would undoubtedly have been ennched in many ways, and particularly by a development of Ins ideas regarding the physical processes which underle the production of chemical change in aqueous solution As different forms of radiobiological damage yield one by one to quantitative expenmental mvestigation, 1t1s pertment to enquire what role, if any, the quantum nature of the primary physical proceases plays in determuung the reactions of the living cell to radiation This book records how such an enquiry was carned through by a bnlhant mind in relation to virus inactivation, gene mutation, and the production of vamious forms of chromo- some structural damage As such, I belreve it 18 of permanent value to the student of radiobiology, however far our knowledge x PREFACE interpret his results along the hnes suggested without any techmecal mathematical ability being required I have been at pains in the physical chapter to provide an adequate amount of numerical data concermng the amount and spatial distribution of 1onization in tissue exposed to the vanous radiations, such numerical data being constantly required in quantitative interpretations of the various biological actions of radiation The tables in Chapter 1 and the Appendix have been specially computed for this book While the physical prine:ples mvolved in the dissipation of energy by ronzing radiations in their passage throngh matter are understood, numerical data of the sort tabulated are not so accurately known as one would wish The tables are likely, therefore, eventually to need reviston in the light of more exact information It1s hoped that in the meantime they will be found of value by workers in this field of research T should not have been in a position to wnte this book had Inot, during the past ten years, been enjoying the collaboration of a number of frends and colleagues in studies of various biological effects of radiation I cannot adequately express my indebtedness to these collaborators, namely Dr D G Catcheside, the late Dr R B Hames, DrM H Salaman, Dr K M Smith, and my colleagues at the Strangeways Laboratory I am especially indebted to Dr L H Gray and Dr FG Spear, in frequent dis- cussions with whom, my ideas on the subject have taken shape For providing me with maternal for the plates, or for permitting the reproduction of published photographs, I am indebted to my collaborators already mentioned, and to Dr JG Carlson, Prof PI Dee, DrI Lasmtzki, DrR Markham, DrA Marshak, DrO F Robinow, DrJ E Smadel, Prof CT R Wilson, the British Inst:- tute of Radiology, the Radiological Society of North America, the Rockefeller for Medical Res +h, and the RoyalSociety Plate I zs # photograph taken at the National Physical Labora tory, and 1s reproduced by courtesy of the Director, Crown copy might reserved I should hke to thank Mrs D E Lea and Mr VC Norfield for preparing the figures and the plates respectively Finally, I acknowledge gratefully the support of the British Empire Cancer Campaign and the Prophit Trust DEL Sruanorways LasornaTory Cambridge July 1044 PREFACE TO THE SECOND EDITION This book has been out of print for some considerable time In attempting to meet the demand for a second edition, tt was con- sidered desirable to make this challenging presentation of certain aspects of radiobiology available again in its original form, which bears on every page the mark of Lea’s clear mind and inusive logic The textual revisions, and some of the additions which Lea himself had written mto his own copy of the book, have, however, been incorporated These additions appear as Appen- dix II, and the attention of the reader 1s directed to them by a margmal asterisk in the text As a physicist Lea was acutely aware of the concentrated dis sipation of energy along the tracks of mdividual particles, which 18 & umique feature of the 1onizing radiations considered as cyto- toxic agents, and was especially interested m exploring the implications of this fact for radiobiology In Ins writings, how- ever, at least as far back as 1941, he makes the distinction, which 18 explicitly discussed in the opening paragraph of Chapter III, between those forms of biological damage to which these con- cepts can and cannot usefully be applied In his treatment of those forms of biological damage which he believed to be mitiated by dividual somzing acts, he had the foresight to recognize and to allow for the spread of the energy absorbed within a molecule over distances of a few millmuicrons, and for the effects of energy absorbed in the aqueous phase surrounding a biological structure Had he lived, the present edition would undoubtedly have been enriched in many ways, and particularly by a development of his ideas regarding the physical processes which underlie the production of chemical change in aqueous solution As different forms of radiobiological damage yield one by one to quantitative expermmental mvestigation, 1 1s pertinent to en: quire what role, if any, the quantum nature of the primary physical processes plays in determining the reactions of the hving cell to radiation This book records how such an enquiry was carned through by a bnilhant mind in relation to virus inactivation Bene mutation, and the production of vanous forms of chromo. some structural damage As such, I behteve it 19 of perma 1 nent value to the student of radiobiology, howeve r far our knowledge x PREFACF interpret his results along tho lines suggested without any technical mathematical ability bemg required I have been at pains in the physical chapter to provide an adequate amount of numerical data concerming the amount and spatial distribution of 1onization in tissue exposed to the various radiations, such numerical data being constantly required in quantitative interpretations of the various biological actions of radiation The tables in Chapter 1 and the Append:x have been speoally computed for thia book While the physical principles involved in the dissrpation of energy by ionizing radiations m their passage through matter are understood, numerical data of the sort tabulated are not so accurately known as one would wish The tables are hkely, therefore, eventually to need revision in the light of more exact information Its hoped that.in the meantime they will be found of value by workers in this field of research I should not have been in a position to wnte this book had I not, during the past ten years, been enjoying the collaboration of a number of frends and colleagues in studies of various biological effects of radiation I cannot adequately express my indebtedness to these collaborators, namely Dr D G Catcheside, the late Dr R B Haines, Dr M H Salaman, Dr K M Smith, and my colleagues at the Strangeways Laboratory I am especially indebted to Dr LH Gray and Dr FG Spear, in frequent dis cussions with whom, my ideas on the subject have taken shape For providing me with matenal for the plates, or for permitting the reproduction of published photographs, I am indebted to my collaborators already mentioned, and to Dr JG Carlson, Prof PI Dee, DrI Lasmtzki, DrR Markham, DrA Marshak, DrC F Robmow, DrJ E Smadel, Prof CT R Wilson, the Bnitish Insti tute of Radiology, the Radzological Sonety of North America, the Rockefeller Institute for Medical Research, and the Roya) Society Plate I] £1s a photograph taken at the National Physical Labora tory, and 1s reproduced by courtesy of the Director, Crown copy nght reserved I should like to thank Mrs D E Lea and Mr VC Norfield for preparing the figures and the plates respectively Finally, I acknowledge gratefully the support of the British Empire Cancer Campaign and the Prophit Trust DE L Sraanoewarys LanonaToRY Cambridge July 1944 ILLUSTRATIONS Plate page I Distribution of tion produced by different radi II Viruses betueen III Chromosome structural changes 222 f 223 IV Bacteria viruses abnormal division figures Figure 1 Linear and exponential dose relations 38 2 Independence of absolute yield upon concentration of solute 43 3 Number of collisions per second made by an active radical 53 4 = Dimunution of ionic yield at low concentrations 58 5 Methods of plotting exper:mental data 73 6 Survival curves 76 7 Associated volume method of calculation 84 8 Relation between 37% dose and target diameter 9 Relation between 37% dose and molecular weight betueen 88 & 89 10 Relation of 37% dove to 37° dose with y rays 12 Relation of 37% dose to 37% dose with X rays 12 Target with indefinite boundary 97 13 Inactivation of plant ‘viruses by radiations 107 14 Dependence of inacti\ ation dose on protem concentration 109 15 Survival curves of irradiated plant viruses 112 16 Survival curves of irradiated animal viruses 13 17 Survival curves of irradiated bacteriophages 14 18 Relation between virus diameter and inactivation dose of X and y rays 7 19 Relation between virus diameter and mactivation dose of a rays 119 20 Visible mutations induced by X rays aa a function of dose 143 21 Sex linked recessive lethal mutations induced by % rays 145 220 ield of sex Inked jethals at d. 147 23 Induction of sex Inked recessive lethals by different radiations 148 24 Analysis of sex Inked recessive lethals 157 25 Survival of eggs fertized by irradiated sperm 161 26 Proportion of viable sperm hax ing chromosom e aberrations 168 x PREFACE of the phenomena treated by Lea may transcend that which was available to him LTamandebted toDrJ W Boagand DrJd G Neary for correct- ing a small error which ran through some of the data presented in Table 10, and for the amendment of Lea’s treatment of the overlapping factor F as it appears in Appendix I LH GRAY MOUNT VERNON HOSPITAL NORTHWOOD . MIDDLESEX May 1954 Chapter I PHYSIQAL PROPERTIES AND DOSIMETRY OF DIFFERENT RADIATIONS Ionization, excitation, and ‘point-heat’ The radiations with which we are concerned are the a-, £- and y radiations of radioactive substances, X-rays, protons and neutrons These may be grouped together as ionizing radiations Occasionally we deal with ultra-violet ght which 1s a non- 1omzing radiation Since the term sonzation will be used very frequently, we begin by explaiming its meaning An atom con- sists of a positively charged nucleus and a surrounding constella- tion of negative electrons, the whole being electneally neutral The principal means of energy dissipation by an ronizing radia- tion 1m ats passage through matter 1s the ejection of electrons from atoms through which it passes An atom 80 tonized 1s left positively charged, and is referredtoasanton It.s possible that, some actions of radiation of biological sigmficance are due to this separation of electrical charge, but im most cases it 18 more Plausible to attmbute it to chemical change resulting from the lomzation For, when an atom 1s tonized the molecule of which ut ws a part almost certamly undergoes chemical change Knowing that the chemical bonds which hold a molecule together are con- stituted by electrons shared between the two atoms joined by the bond, 11s to be expected that the removal of such a bonding electron from a molecule will Jead to its dissomation or other chemical change The removal of electrons other than bonding electrons may also be expected to lead to chemical change, since the energy mvolved an ionization, 10 electron-volts: or upwards depending on the atom 1omzed and the level init from which the electron 1s ejected, exceeds the energy required to remove an atom from the molecule 4 A second method by which radiations dissipate energy 1n tussne 1s by excitation This means the raising of an electron in an atom 1 The electron volt (eV )=1 602x 10-* erg, 19 a unit of energy of suitable magnitude for dealmg with energy changes in single atoms or molecules 1eMV =10'eV lekV =10%eV 1eV Per molecule = 23 05 kalogram calories per gram molecule Thus the statement that the ene: of dissotiation oftheC— bonds 94 lulocalories per gram molecule mens ‘that the energy required to dissociate a single bond 1s about 4 eV xiv ILLUSTRATIONS Figure poge 27 Dommant lethals as a function of dose 168 28 Depresaion of sex ratio Vt 29 Efficiencies of different wave lengths of ultra violet light 188 30 Structural changes un unsphit chromosomes 194 31 Structural changes in split chromosomes 195 32 Inf of on 1 changes 221 33° «wD upon di of of yield of ab 227 34 Breaks per cell as a function of dose 230 35 Yield of two break aberrations as a function of dose 231 36 ©Normal division figures as a function of dose 236 37° Yield of ultra violet induced deletions as # function of incident. energy 243 38 =Dimunution of yield of exch with di of exposure 266 39 Chromosome exchanges a4 a function of dose 208 40 Relations between 10n density and probability of breakage 271 41 Passage of an electron through a chromatid 2m4 42 Prumary chromatid breaks as a function of wave length 276 43 Cleavage delay in Arbacia 286 44 Cleavage delay as 4 function of time of irradiation 287 45 Cleavage delay as a function of doso 288 46 Decay of cumulative dose 290 47° Mitotic activity st various tumes after rrradiation 48 Dependence upon intenmty of dose required for a given delay 297 49 Duivinion delay as a function of dose 299 50} Inhubition of division in Bact cols 301 51 Inhibition of division in bean root tips 302 52 Reduction of mitotic count as a function of dose 304 53 Germination of pollen wradiated at different stages 64 Dividing and cells after 309 55 Survival curves of irradiated bactera 31g Survival curves (theoretical) 320 56 57 Bulling of Bact colt by soft X rays 323 6a Relation between chromosome structural changes and lethal effect 334 339 5! oS Hereditary partial atenhity 60 Illustrating target calculation 353 61 Relative efficiencies of sonuzing particles of different 1on dcnatios 354 Chapter I PHYSIOAL PROPERTIES AND DOSIMETRY OF DIFFERENT RADIATIONS Jomzation, excitation, and ‘point-heat’ The radiations with which we are concerned are the a-, #- and y-radiations of radioactive substances, X-rays, (protons and neutrons These may be grouped together as ionizing radiations Occasionally we deal with ultra violet hght which 1s @ non- lomzing radiation Smce the term ionization will be used very frequently, we begin by explaining its meaning An atom con- sists of a positively charged nucleus and a surrounding constella- tion of negative electrons, the whole bemg electrically neutral The principal means of energy dissipation by an ionizing radia- tion in its passage through matter 1s the ejection of electrons from atoms through which 1t passes An atom so zontzed 18 left positively charged, and is referred toasanton Its possible that some actaons of radiation of biological significance are due to this separation of electrical charge, but in most cases 1t 13 more plausible to attribute 1t to chemical change resulting from the 1onization For, when an atom rs tonized the molecule of which ut 18 a part almost certasnly undergoes chemical change Knowing that the chemical bonds which hold a molecule together are con- stituted by electrons shared between the two atoms jomed by the bond, at 1s to be expected that the removal of such a bonding electron from a molecule will lead to its dissociation or other chemical change The removal of electrons other than bonding electrons may also be expected to lead to chemical change, since the energy mvolved in ionization, 10 electron volts: or upwards depending on the atom 1omzed and the level init from which the electron 1s eyected, exceeds the energy required to remove an atom from the molecule 4 Asecond method by which radiations dissrpate energy 1n tisaue 1a by exestation This means the raising of an electron in an atom 1 The electron volt (eV )=1 602 x 10-18 suitable magnitude for oalng with energy changes maimgtente, of Tnolecules leMV =10teV , 1 ekV =10°eV LeV per molecule= 23 05 wlogram calories per gram molecule Thus the statement that the energy Sf dissociation of the C—H bondis 94 lulocalonespergram molecule means lat the energy required to dissociate a single bond 1s about 4 eV 2 PHYSICAL PROPERTIES OF RADIATIONS or molecule to a state of higher energy, and 1s & less drastic process than the complete ejection of an electron Ultra-violet hght, as well as 1onizing radiations, 18 capable of causing excita- tion In simple reactions (eg inorganic gas reactions) ultra- violet-nduced excitations are not much less effective than X ray-induced romizations in causing chemical change ‘There 18 some evidence, however, that in the decomposition of large organic molecules, an excitation 1s a good deal Jess efficient than an lomzation : In biological actions of radtation in which quantitative data are available both for 1omzing radiations and for ultra-violet hght, particularly the inact:vation of viruses and the killing of bactena, an excitation by ultra-violet hight 1s much less effective than an ionization Thus it eppears probable that when we are dealing with an ionizing radiation, excitation may usually be neglected as a cause of biological effect by companson with ionization The electron which 1s ejected from an atom in the process of 1c 1 ntually b attached to another atom and makes 1t a negative ion As far as the physical measurement of ionization 1s concerned, the positive and negative 10ns are equally significant, and one usually speaks of the production of an ron- pair But since the energy mvolved in the attachment of an electron to an atom to form a negative 10n 1s usually even less than the energy of excitation, 1t 1s probably safe to neglect negative 10n formation as a factor of biological importance Thus when we speak of an romzation we refer to the production of a positive 10n by the ejection of an electron This ejected electron may have sufficient energy to 1on1ze on its own account before 1t 1s brought to thermal energy and finally attached, and this secondary 1onszation 1s important and will be discussed later But again, it 1s the positive 1ons and not the negative tons which are of biological importance Practically all the energy dissipated by radiation in tissue ultimately becomes degraded to heat energy Thus a dose of 10° roentgens 18 sufficient to rayse the temperature about 0 25°C This small temperature mse for a large dose of radiation means that temperature change 1s quite madequate to explain the hio- logical effects of 1oniz1ng radiations in the way in which tempera- i Cp Jordan P (19389) ULTRA-VIOGLET LIGHT 3 ture mse accounts for most of the biological effects of, forexample, short-wave wireless waves On the other hand, the highly localized nature of the energy dissipation means that the energy which eventually causes a rise of temperature of 0 25°C im the tissue as a whole 1s intially confined to a small proportion of the atoms, and might be considered to produce a large rise m tem- perature of these atoms This 1s the basis of the “point-heat’ theory : The concept of an ionization as & hot spot 1s less satis factory than the concept of an atom iomized leading to chemical change m its molecule However, m the course of the degrada- tion of ionization and excitation energy to thermal energy, the posatbility must be borne in mind of molecules near to a high concentration of 1omzation suffering chemical change even although not themselves 1onized 2 Ultra-violet light In quantitative experiments 1t 1s necessary to use mono- chromatic ultra-violet light, since the biological effectiy eness per umt of energy vanes very much with wave-length and in par- ticular 1s very low above 3000A The physical measurement will usually give the energy incident upon the irradiated preparation in ergs per square centimetre If we are irradiating small objects such as bacteria or viruses, and can obtain a suspension in a, non- absorbing medium free from absorbing impurities, a satisfactory procedure is to use a stirred suspension sufficiently deep or sufficiently concentrated to absorb completely the incident ultra-violet light The total incident energy may then be divided either by the total number of suspended organisms or by the total volume of protoplasm in suspension to obtain the absorbed energy either in ergs per Organism or mn ergs per cube meron In other cases we may arrange to use a thickness of madiated material sufficiently small to absorb only a small proportion of the mudent radiation If we hnow the absorption coefficient of the irradiated material, or better, the absorption coefficient of that part of 1t absorption in which we beheve to account for the biological effect, we may caleulate the energy «bsorption in this part m ergs per cubic centimetre If J 1s the incident intensity m ergs/em 2, p the density of the absorbing substance in g /om 3, and # the absorption coefficient m em —! defined by the relation 1 Dessauer, F (1923} 2 Jordan P (1938¢) 4 PHYSICAL PROPERTIES OF RADIATIONS p= E log, fen28 logis%, where J 1a the mtenmty transmitted by alayer zcm thick, 7, beng the mtial mtenmty, then y/ 1s the energy absorption m ergs/cm 3 and (j:/p) I the energy absorption in ergs/g If (as happens, for example, in the irradiation of Droso- phila sperm m the male) it 1s not possible to avoid excessive absorption, either by too great thickness of the uradiated matenal or of intervening tissue, then quantitative experiments are not possible unless measurement can be made of the intensity of radiation reaching the ap under the condit of the experiment, To give some idea of the magmtude of the absorption ca- efficients, and the variation between different matenals, we het in Table 1 the absorption coefficients: of a number of substances for 2536A , this wave-length being chosen since 3t 18 in the biologically most effective region, and also 1s readily obtamable nearly monochromatic In the references cited the absorption coefficients for other wave lengtha may be found Ultra-violet light for biological experiments 1s usually ob- tamed from a mereury lamp If the efficioncy of different wave- lengths 1s being compared, then a quartz mercury aro in conyunc- tion with a large-aperture quartz monochromator 2s the usual equpment: An intensity of about 26x 104 ergs/om %/sec 18 possible with a lamp dissipatmg 1 kW If a monochromator 38 not available, or if one 1s not mntereated mn the relative efficiency of different wave lengths, the best type of lamp to use 1s the low- pressure discharge lamps neon and emitting 85-95%, 1 Ifthe ofa subst: has been din sol then the result will often be exp: d as the ti «in the formula logy, (I,/I) =2ex, ¢ bemg the concentration of solute in mg /cm * Orit may be expressed as the molar extinction coefficient ¢1n the formula, logo (Io/Z) =ecx, where ¢ 18 now the m gram per litre of a substance of molecular weight Mf In order to reduce the absorp tion toa basis table for energy ab sorption from knowledge of the ultra violet mtensity all have been reduced to # or z/p the absorption coefficient of the pure solute, by assuming that the absorption coefficient of a solution 13 proportional to the concentration of the absorbing solute Thus p/p=2300a= 2300¢/n1 2 Op for example, Gates, F L, (1929a), Benford, F (1936), Uber ¥ M & Jacobsohn, S (1938), Uber, F M (1940), Cannon, CV & Rice, OK (1936) Steane EWR & Philips, NWF (1938), ‘ 3 Tyetwille, HW Peel, 1942: Heidt LJ (1939), G.N (1939) ULTRA-VIOLET LIGHT 5 of its radiant energy in the line 2536A The monochromatism may be further improved, 1f desired, by the use of gaseous filters of chlonne and bromine,: or quid filters,2 or by using the radia- tion from the Jamp to excite resonance radiation 1 A convenient lamp. takes the form of a 30cm length of quartz tubmg about 8mm diameter wound in a close spiral of about 2 50m diameter Taste 1 Absorption coefficients for wave length 25364 Maternal em.aA og He % € %o oe Reference % trans Plant virus protein — 10 000 _ _ I Tyrosine _ 3,800 300 _ 2 Tryptophan _ 36 000 3,200 > z Bacterial protoplasm 3600 _ _ _ 3 Bacteral nucleoproten - 32 000 _ _ 4 Bacterial nbose nucleic _ 87,000 - _ 4 ecid Trypsin - 1,900 30000 - 5 Ribonuclease — 900 6,000 fd 6 Maize pollen contenta 1800 _ _ _ 7 Maize pollen wall _ _ _ 30 7 Vitelline membrane of _ _ ~ 8 8 hen’s egg Abdominal wall of Dro _ —_ - 67 9 aophila 1 Bawden FC & Pine, NW (1938) 2 Holtday EP (1936) 3 Gates FL (1930) 4 Lav, GL, Thompson RHS & Dubos, RI (1938) 5 Uber FM & McLaren AD (1941) 6 Uber FM & Ells VR (1941) 7 Uber FM (1939) 8 Uber FM, Hayashi, T & Ells VR (1941) 9 Durand E Hollaender A & Houlahan MB (1941) At 10 W this gives an intensity of about 10‘ ergs/em 2/sec at 10 cm distance, or about 5x 105 ergs/om */sec on a specimen inside the spiral It1s31mportant to understand the difference in spatial distribu- tion between the energy dissipation by ultra violet ght and by an lonizing radiation such as X-rays With ultra violet hght, the absorption coefficient depends on the molecular structure, and 1s, for example, different for nucleic acid and for protem The dose in ergs/em * absorbed energy may be very different, for 1 Peskoff N (1919) Oldenburg O (1924), Villars, DS (1926), Heidt LJ (1939) Svedberg,T & Pedersen, K O (1940), Mitchell, JS (1942) 2 A convement filter 1s a quartz cell lem thick contamng an aqueous solution of nickel sulphate (20%) and cobalt sulphate {6%) Houston, R A Q911) Backstrom H LJ (1940), Bowen EJ (1942), Lavin GI (1943) 3 Thomas LB (1941) 4 Procurable from the Thermal Syndicate Ltd » London 4 PHYSICAL PROPERTIES OF RADIATIONS pat log, fe23 logy 4, where J 1s the intensity transmitted by alayer x cm thick, J, being the imtial intensity, thon ji 18 the energy absorption in ergs/om 3 and (/p) Z the energy absorption inergs/g If (as happens, for example, 1n the irradiation of Droso- phila sperm in the male) it 1s not possible to avoid excessive absorption, either by too great thickness of the radiated material or of intervening tissue, then quantitative experiments are not possible unless measurement can be made of the intensity of radiation reaching the specimen under the conditions of the expermment To give some idea of the magnitude of the absorption co- efficients, and the variation between different matenals, we lst in Table 1 the absorption coefficients: of a number of substances for 2536A , this wave-length being chosen since it 18 in the biologically moat effective region, and also 1s readily obtaimable nearly monochromatic In the references cited the absorption coefficients for other wave lengths may be found Ultra-violet hght for biological experrments 18 usually ob- tained from a meroury lamp If the effinoncy of different wave lengths 1s beng compared, then a quartz mercury aro in conyunc- tion with a large aperture quartz monochromator 1s the usual equpment + An mtensity of about 25x 104 ergs/om ?/sec 13 posatble with a lamp dissipatmg 1 kW If a monochromator 3s not available, or if one 1s not interested in the relative efficiency of dofferent wave lengths, the best type of lamp to use 1s the low- Pp discharge lamps cont: ig neon and gg 85-95 % 1 H the absorption of a sub has been din sol then the result will often be exp das the @in the formula log), {Z,/I) = aex, ¢ bemg the concentration of solute im mg /em 5 Orit may be expressed aa the molar extinction coefficient e2n the formula logy, (Io/Z)=eex, where c is now the m gram. les per litre of a. substance of molecular rrewght M Inorder to reduce the absorp tion toa basis ble for energy ab sorption from knowledge of the ultra violet intensity, all have been reduced to # or #/p the absorption coefficient of the pure solute, by assuming that the absorption coefficient of a solution 18 proportional to the concentration of the absorbing solute Thus #/p= 23000 = 2300¢/af 2 Cp forexample, Gates, F L (1929a), Benford, F (1936) Uber,F M & Jacobsohn, S (1938), Uber FM (1940), Cannon CV & Rice, OK ¢ 3 Tetvalle, HW (1936) Steacre EWR & Phillips, NWF (1938) 1942) Heidt, LJ (1939), Peel, GN (1939) X-RAYS 7 electricity of erther sign’—1s the umit of dose employed « It corre- sponds to the hberation of 2 082 x 10° 10n-pairs per cm > of aur at 0° and 760 mm pressure, involving an energy dissipation of 0 1083 erg/cm 3 of air (taking 32 5eV per 1on-pair as the mean energy dissipation in air) Since the roentgen 1s already a umt of energy absorbed, there 1s no question of multiplymg dose in roentgens by absorption coefficrent What ts loosely spoken of as ‘intensity’ in roentgens per minute 1s stnetly dose rate, being a rate of energy absorption and not a rate of energy incidence For the convenience of the phy stcal measurement the roentgen 1s defined in terms of energy absorption per unit volume of air In the interpretation of expenments one 18 interested in the energy absorption per unit volume of tissue, which for the same incident intensity of radiation will be about 1000 times greater on account of the greater density of tissue The actual factor vanes with different wave-lengths, since the ratio of the absorp- tion coefficients of tissue and air vanes somewhat with wave- length It also depends on the elementary analysis of the tissue though not on the chemical nature of the compounds in which the elements are combined The way in which one calculates the amount of energy absorbed per gram of tissue per roentgen of radiation 18 explaimed in the Appendix, and m Table 2 the re sults of a calculation of this sort are given for water, for dry virus Protein, and for an undned soft tissue The percentage composi- tion by weight assumed for the virus protein is H 7, C 49, N 16, 0 25, P 1, § 0 5 and ash J 5, the ash being treated as having an average atomic number of 16 for the purpose of the calculation The wet tissue 1s taken as having a percentage composition by weight H 10, € 12, N 4, O 73, Na 01, Mg 004, P02, 8 02, ClO1, K 035 and Ca.001 With most expermental materials it will be found satisfactory to use the figures given in Table 2, either for water or for virus protein or for wet tissue, according to which approximates best to the composition of the tissue actually being irradiated Thus when irradiating a dned virus preparation the figures for virus protem will be used When radiating micro organisms 1n aqueous suspension the figures for water are appropriate, while the figures for wet ussue may be used when one 15 dealing with undried tissue in bulk ° 1 0001293 @ of ar occupies 1 em? at 0° and 760 mm pressure 6 PHX SICAL PROPERTIES OF RADIATIONS example, in different parts of an wradiated chromosome de- pending on the degree to which they are loaded with nucleic acid, and at different stages of the division cycle No such dif- ferences evust with X rays, apart from an increased energy absorption in and near to bone or other components containing atoms of elevated atomic number, since the absorption of X-rays by atoms is not affected by their chemical combinations A further difference 1s that with ultra violet light the excited atoms are distributed spatially at random in a homogeneous tissue irradiated by a uniform intenmty, with no tendency for excited atoms to occur m groups, produced si:multancously, or concentrated in a near path This 1s because each excited atom 1s produced by the complete absorption of a single quantum of ultra-violet hght and the quanta are emitted mndependently With ionizing radiations the ionizations are localized along the paths of 1onzing particles, and thus a number of ronizations may be concentrated in a cluster or a column of 1omzation The energy of a single quantum of ultra violet hght 1s con- nected with the wave length (A) im Angstroma by the relation Energy im electron volts=12,400/A Thus the wave length 2536A has quantum energy 4 89 eV X-rays X-radiation like ultra violet light 1s an electromagnetic radia- tion emitted in quanta, but the difference in wave length (0 05~ 10A for X-rays against 2000-3000A for ultra-violet hght) re- sults in there being little srmilanty m practice The absorption coefficient of X-rays depends not on the chemical combmation of the absorbing atoms but onlyontherratomicnumber Onaccount of the greater penetrating power of X rays 1t 13 not usually con- venient to measure the total energy tncrdent on a surface, but to measure the energy absorbed m a given volume In practice the energies involved are too small for a thermal method of measur- ing energies to be used and use 1s made of the fact that when the absorption takes place m air the latter becomes conducting and the saturation current through a given volume of airis a measure of the rate of energy absorption m that volume of ar The roentgen—defined as ‘the quantity of X- or y radiation such that the associated corpuscular emission per 0 001293 g of air pro- duces, 1n arr, ions carrying 1 electrostatic unit of quantity of X-RAYS 9 Jf the material being studied can be prepared in films of a few microns thickness, as with viruses, 1t 1g convement to ust long wave length X-rays (1-10A ), since these may be obtamed in high intensity from inexpensive apparatus, and have also the considerable advantage from the point of view of subsequent calculations that they are obtainable nearly monochromatic The wave-lengths 15, 41 and 83A hsted im Table 2, are ob- tainable in this way They are reduced to half their intensity by passage through layers of water of thicknesses respectively 695, 34 6 and 4 9 If more penetrating radiations are required an X-ray tube of the type manufactured for X-ray therapy will usually be used Such X-ray tubes ermt a range of wave-lengths The shortest wave length emitted 1s related to the peak hilovoltage on tho tube by the relation wave length m Angstroms=12 4/hilo- voltage, the quantum energy mm electron-hilovolts of this shortest wave length being equal to the kilovoltage on the tube The wave length emitted at greatest intensity 13 usually about twice the wave-length of the short-wave limit, and the longest wave- length emitted depends on the thickness of filter employed since long waves have a higher absorption coefficient than short Thus an X-ray tube operated at 160 kV wsth filters of 07mm Cu and 12mm Alemuits a continuous range of wave lengths from 0 078 to about 0 4A, the ‘effective’ wave-length, conventionally de- fined as that wave length which would, if monochromatic be reduced to half value by the same thichness of copper absorber as actually reduces the mixed radiation to half value, being 915A The corresponding quantum energies for the shortest longest and effective wave lengths are 160 ekV , about 30 and 83 ekV x The dissipation of energy in tissue iradzated by X rays of fairly long wave length (eg 15A ) takes place as tollows Some of the quanta which enter the tissue are absorbed by atoms The energy of a quantum, 8 ekV for an X-ray of 1 5 A , 18 com- pletely absorbed and results in the ejection of an electron The innermost electrons of the atom are most effective in this type 1 Fuller formation on the effective wave lengths of X rays emitted from tubes Operating at various hilovoltages and filtrations may an ow the article by Taylor be found Rete y L& m Duggar BE BY (19 (1936), Biological Effects PHYSICAL PROPERTIES OF RADIATIONS S€seLol$BgO9T0eFs86aEStTTO«BGISLTS«IOTsS1ae0UtT8r0OCI9STET«=slge1?9«og98HEegF165901L9FysOF1p9osBerEs1S5L8h092eS90kh1tae6tiO0lPU+)adABtRNYea@+98usyujorsndaeyra sTtoHeoEt2BsI9tSsTlsg«BosRrtTcFsoTehO1l8eSOWolTbz«g6tc0sE9Sg«GeT80LesbLo9gE0eS9«T0eO6sF7E9LRS0T8Ss9LEF16IZ9068TyvVioserie ocHzgF1InOroL0LoesET1Tlv—=|ou«S8SHsrLeUTSar3L99jlTord«OSSBGGraOILOoyOeOLIsRyLTTtE,§«==—oeO9€«n61z89zs9666e000)|u|S«68rLO6a90S08Rj8)T0Bo0rd«z2OFBHaF6qUo66eG8G00aOO0,}«=6aeo«G9TnDLSapleFIsTEs9tsS)Syur2O§S«e«6ShBj9F9UCaEoE8OusSSFdLS«=«EO§g«J6CPNcLo8FeIeSeE8yTSs)S,«==o6«GG)1«nL8G=Le0FOaU6atTSSR]«|aTeGEGSsejTo0A1E2na9L8R0e8rt«==1ibLBSIg3aogLZeksRG4GsAeege8L==GE§=VF8AE=BA6NRS—FI¥e‘E2OTuzyEBT0O5N0RBIPeY S‘GUPoLM3Etg:-=OTR1/2ZTe¥kLvAoTreztaO«o8ByULOTT>E»I=PES2$GaS9RTM6Do0)LTsByAuJMnSBOU6H{0TGJ2A)uTa{u0y7qO8VO6THP0ea+oTdutFOAsr)IsSnoi3EAmge0JOsoABoGFsaAN=G?—ET)aTtOUTeB.suorjdaic+EPoosGsAqSeeAB10aPFUjUsGeqdAasgg,atafTaa”zEeSveostoexersfudry AsXdye2a43w1duA0AAN9NRWnWnPRG‘eSaOpgSISsr9Taquosqe{LGZSG41FFOIO00O6OTTT1LLO0689z26TF510e1€39$$5s$1009o999s0.e029T9h9Sr2o19ITULeTtSl9LurOUuSi0aosETt3ettb5fTtTT0ro£1oLO9F0e1s88-eTetg83 69F6,OaeAgW9boehtTe6r1LDGoWsu89O1jWr9oL0g2LA¥1.NOIs89eSL6tT97£A6eR0O1S8l (suoaqnaujoestooq]UIBUNa{J0}) AWeOtSotoO} aTuopErULWorjnyos X-RAYS 11 mechamsm also contributes, this being the production of Compton recoil electrons In this process a quantum of radiation 1s scattered by an electron with reduced energy (1e increased wave-length), the balance of energy being transferred to the electron which 1s eyected from its atom Depending on the angle at which the quantum 1s scattered, the energy of the Compton recoil electron may vary from zero up to a maximum which 18 a fraction of the quantum energy depending on the wave length (0 327 for 01A, 0108 for 04A) Thus when X rays of O1A are irradiating tissue, in addition to & certain number of photo- electrons of energy 124 ehV , there will be Compton electrons of all energies between 0 and 40ehV Remembemng further that X rays of 01A cannot be obtarned monochromatic (except by means not usually practicable for biological experiments), 1¢ will be seen that calculations based on experments using these wave- lengths are more complicated than expermments with longer wave-lengths In Table 3 are listed the relative numbers of Compton and photoelectrons liberated in tissue (here taken as H,0) by different wave lengths of X-rays, together with the energy of the photoelectrons and the maximum and mean energies of the Compton electrons Plate IE shows recoil electrons (the shorter tracks) and photoelectrons (the longer tracks) The atom from which the photoelectron or Compton electron as ejected is of course ronized there 1s no reason, however, to attnbute any special virtues to an tomization caused by absorp- tion of an X-ray quantum, and there are very many more 10omzations caused by the ampact of recoil electrons or photo- electrons Thus if an effect, eg breakage of a chromosome, Occurs at a particular point in a cell there 1s no reason to attn- bute this to absorption of an X-ray quantum at that point, though 1t may very well be due to the passage of a photoelectron or recoil electron through the pomt In using Table 2 the composition of tissue relevant 18 that of the tissue in which the electrons arse Thus when dealing with micro organisms suspended in water, the hnear dimensions of the orgamsms being much smaller than the range of the electrons, and their total \olume being only a small fraction of the total volume of the water, most of the electrons which traverse the organisms wall have arisen in the water and not in the organisms, 10 PHYSICAL PROPERTIES OF RADIATIONS of absorption (photoelectric absorption), and an appreciable amount of energy 13 required to detach the electrons, depending on the atomic number of the absorbing atom It 1s 285eV for , a carbon atom, 528¢V for an oxygen atom, more for heavier elements, and may be taken as about 06 ekV on the average for tissue Thus the photoelectron on leaving the atom has energy 8-0 5=765ehV (or im general, 12 4/A—O@ 5ekV) This ejected electron has a range of | 5 1n tissue and enough energy to produce about 230 romizations Thus the ionization in tissue uradiated by X-rays of 1 5A 13 not distributed at random but 1s localized along tracks as in Plate Ir: This localization 13 very important m the theory of the biological action of radiations, and will enter into a good deal of the subsequent. calculations The distribution of 1onizations along the electron tracks 1s de- scribed later The energy of a few hundred electron volts needed to detach an electron from the atom which absorbs the quantum of rdia- tion eventually appears as 1omization The mechanism may either be the emission of @ second electron from the same atom (Auger effect), or, Jess frequently, the emission of a long wave- length X-ray quantum, which after travelling a distance of the order of 14 1n the tissue is itself absorbed by another atom with the production of a photoelectron having practically the whole energy of the long wave-length quantum Thus the absorption of the original quantum of 8 ekV im the tissue gives mise usually to two electrons, one of about 7 5ekV and the other of about 05ekV This division of the energy of the quantum between two electrons 2s rarely 1mportant, and we shall often, in Table 3 for example, calculate as if the energy of the original quantum were dissipated by a single photoelectron of the full energy 8 ekV Wath shorter wave lengths, eg 0 1A, absorption stall takes place to some extent by the photoelectric effect, but a second 1 The photographs in Plate I are Wilson chamber photographs (from Wilson, CTR 1923 Dee PI 1932 Cure P 1935) in which the ions an a gas have been made visible by the condensation of water drops upon. them The scale has been adjusted a0 that the photographs represent appr ly ly the rk of ronsin wrad: tissue Thus Plate Ir may be taken to rep the of mas section of tissue 15 x 12x 1 thick irradiated by a dose of lOr of X raya of wave length 15A No significance 1s to be attached to the twidth of the tracks Y-RAYS 13 and hence one should use the columns of Table 2 appropriate to water On the other hand, if having obtained the energy dissi- pation in ergs/g we wish to convert to ergs/em 9 by multiplying by the density, the density which 1s relevant 1s that of the tissue in which the 1onization 1s actually produced Thus if we have a suspension in water of micro organisms of density 12 g /em %, We convert a dose measured in roentgens to ergs/em > in the organisms by multiplying by 1 2 the figure given m the second column of Table 2 for the radiation concerned Tt will be noticed in Table 3 that a chango ir the wave-length of X-rays from 015 to 04A does not make a great deal of dif- ference in the mean energy with which the electrons are pro- jected in the tissue This 1s due to the replacement of electron recoil by photoelectron production as the principal means of absorption as we change from 015 to 04A In consequence, expermments designed to seo whether there 1s a vanation of bio- logical effect with w ave-length should not be confined to wave lengths in this range, which unfortunately 1s the range most readily available with standard X-ray equipment of the therapy type Ifthe nature of the expermental maternal permits, a much more valuable test 1s a comparison of soft X-rays (1A or greater) with medium X-rays (1e X-rays in the range 01-044), and y-raya y-tays The y-rays used in biological work are those of radium and its products, or sometimes radon and its products, sealed in a closed container with sufficient thickness of wall to absorb a- and # rays (at least 0 5mm_ platinum or equivalent) The radhations, which are natural X-rays of short wave-length, are emitted in a number of discrete wave lengths ranging, with thus filtration, in quantum energy from about 0 2 to about 2 2 eMV Absorption in tissue 1s practically entarely by Compton recoil, and electrons of all energies from zero up to about 20 eMV are thus produced By the use of a lead filter between the radium and the irradi- ated material it 1s possible to reduce the intensity of the longer more easily absorbed, wave lengths, and so reduce the average wave length and increase the proporti on of the 1onization in the uradiated tissue which 1s due to electrons of high energy In 12 2hz B83 58 38 pare’ SO00 e005 wYasuv=eaAvtns«B—yuaAUs0uoNITVu;IaePEtTpa:TSB]o,Y o£1908GLOS1I6gosL8r2O. £016IFkSE8B9t6oLesG0hO PHYSICAL PROPERTIES OF RADIATIONS DOM One Hae wODo voLe3i€g«9H0N2I1EiSOeT0SkLs0IToOeOp02e.L0«=—-1w8G21sLocn0ei1rg9Ft69sou6t£69ole0.F6se9ngn06OOoTeB60OoTtD0S81sG0io0«OénS0s0N-édGt8e16CT016o0O0Gin-0Oe1T-01“0$0s61o0-00-0N0=1GASIca6e2LOTdgs10iaBEF8eLe66HsisIto9u0OBetDzTF90s«=a9UGB8OSLs1I02lte9-L6T9Oicos$e690Srdt86L9efo1BLSe96SsaLerstFFeTo-0Oe$1668G£ead958y¢-ojoydpusjrosar 0FG9sOe0l0«=BgoosoteoaLtgF08608068900-0=ROeL66o-0010£00-096gfoe1lt8t03S6Le629da1YtaOoiEssFe seABaraueeuAaNPDaaUpho[oxohdnprue1uo£xq}gaJ3af3ye|uutSp1pr8a7y0ya}eui£ryaSosqruuyavoysaatudoowsdeoosrlpsgaeoqo—gds[u8p019xi041e9a29Jp)r;oeo9uOJoaTIysqJuIe=A0Oqgmldu7qoRmITnagE|ur3],aulayur $t6sES00lbrIt10OToeeOG1sale8otsrao0eP£9s3OIeFLn8-sisS0lte58O965L8008236-T06P¥£g1O090eLo-0a0ds$£6B1eS9a6L68be-90o0£98F0$90t1esT8Fo00OB0EBSL8OEoILS9FeIZOsS6S3LeCTS.H9gs8S9eor00Fler89ge8t0O3 OreTOL86br1-01630S610$08.0S16%F96080 oT6O0oS83st0iat01a1E08SL11T203S69Z6690218660081Fo021t00-00Bp6LI8E0--D0RT8H9I00O-.0LWEFsOLtst8Seo6eOi‘Eg_Ci$LBge1F8bSOTlSaee9lss Oe68c10sah8H8o90RosILT8o1Se08O1098SL9£¥t88e0--s00LoBe91tL19eO00$GT6S900T-)0c9OS0rr6eL0oo09e$s068oO1F9S2STeB0cSTo6O00 B-RAYS AND CATHODE-RAYS 15 a point source of 1 mg of radium filtered by 0 5mm of platinum the dose rate 1s 8 3 r /hr: The allowance to be made for absorp- tion in intervening tissue hke material 1s from 25 to 5% por cm, depending on the extent to whrch scattering reduces tho effective absorption coefficient For accurate estimation of doso an 1omzation measurement 1s to be preferred The electrons which dissrpate energy 1n a tissue irradiated by ‘y-rays may, on account of their high energy, have a range in tissue of several millimetres When irradiating small objects 1t 18 desirable that at least 3mm of tissue hhe maternal should sur- round the relevant part of the objects to serve as a source of electrons f-rays and cathode-rays X-rays and y-rays produce their effects in tigsue by the projec- tion at high speeds of electrons already 1n the tissue The same effects would be produced if electrons of similar speed could bo produced in other ways In the case of expenmental materials which are usable in thin layers, electrons may be introduced fiom anexternalsource Enther # rays, which are fast electrons emitted from radioactive materials, or cathode rays, which are artificially acrelerated electrons,s may be used The most convenient source of £ rays 1s a thin-walled vessel conta:ming radon gas, the # rays be:ng emitted by Ra B and RaC which deposit on the walls A wall thickness of 0 05 mm of glass or aluminium suffices to re- move the « rays, and the y rays which are also emitted produce neghgible 1onzation by comparison with the # rays The distn- bution of energy of the # rays emitted by such a source 1s given in Table 5, the figures in which are approximate only The second hne of the table gives the relative numbers of £-rays of different energies entering the tissue If one 1s interested in the proportion of the total energy dissipation which 18 produced by f-rays of various energies, the result 1s very different according to the ewe 1 Ma:T Bake Ww. ao” JE (1937) Gray, L H (1937) Kaye, 2 Cp Mayneord, WV & Honeyburne, J (1938) 3 Apparatus for the production of cathode rays suitable for biological experments has been described by Coolidge W D (1926) Cauchois Y (1932) Haskins, CP (1938), Cooper FS Buchwald CE Haskins, D (1939), Morningstar, O, Evans, RD & Haskins, CP & Evans, R CP (1941) 14 PHYSICAL PROPERTIES O¥ RADIATIONS Table 4: 1s given the enorgy distnbution of the recoil electrons produced in tissue by radium y rays with different degrees of filtrationIt 1s seen, for example, that with a filter of 05 mm of platinum only, 22% of the ionization 13 produced by recoil electrons of less than 0 26 eMV , but that when the filtration 13 imereased by 25 em Pb, only 5% of the 1onization 13 so pro- duced The efficiency of a lead filter in removing the longer wave lengths and so enabling the biological effectiveness of different Tanre 4 Electrons libersted in tissue by redium y rays The figures give the percentages of the total electron energy which are dissipated by electrons berated with the stated initiat energy Range of electron ener O- 005- O1- 025- ges (oMV ) 005 O1 025 05 OQ5-1 1-2 Total Filter 05mm Pt 15 38 249 263 264 1000 05mm Pt+lem Pb o4 12 71 «#203 330 330 000 O5mm Pt+25em Poh O2 06 131 341 480 1000 y ray wave lengths to be compared 1s reduced if scattered or secondary radiations from the filter contnbute much to the radiation falling on the irradiated object, smce such scattered radiation 1s of longer wave-length than the pnmary In experi- ments of this sort the diameter of the lead filter should be as small as possible by comparison with the distance between the source and the irradiated maternal y rays, like X rays, are measured in terms of roentgens, and the factors for converting doses in roentgens into energy dissipa- tion in tissue are given in Table 2 As analternative to measuring the dose-rate, 1t may often be calculated from the known content of the radium sources mahing use of the fact that at lem from 1 Table 428 calculated as follows By integration of the Klem Nishina formula one can calculate the energy distribution of recoil elections pro duced by y rays of any specified wave length The intensities of the principal ines in they ray apectra of Ra, Ra B and Ra C have been given by Elis CD & Aston GH (1930) and Stahel E & Johner W (1934) By use of the absorption coefficients of these wave lengths in platinum and lead which have been computed by Siz00 GJ & Willemsen, H {1938) and Kaye, GWC & Bmks W (1940), one can deduce the in tensities of the different Imes after any filtration Carrying through the energy distribution calculation for each wave length mn turn and com ping in the proportions appropriate one finally arrives at the figures given They do not allow for any scattered radiation from the filter lated } hing the @-RALS 17 For irradiation of thin films of material by a-rays emitted from an external source, polonium serves as a convenient source Its prepared from old radon seeds,: and emits @ rays of energy 5 298eMV with practically no other radiation a-rays of this energy have a range: of about 39, 1n tissue of unit density Alternatively, ‘active deposit’ sources may be prepared by exposing a metal disk to radon A source made in this way de- cays in a few hours and 1s only suitable therefore for short exposures After the lapse of half an hour, required for the com- plete decay of Ra A which emits less energetic a rays, the a rays emitted are those of Ra C’ which have an energy of 7 680 eMV and a range: in tissue of umt density of about 71 This source emits J-rays and y-rays as wellasa rays The romzation per unit volume due to the f rays 18, however, only about 1 % of that due to the a-rays and that due to the y rays 1s quite negligible Tastz 6 RaC’ a particles a@ ray energy at 3, Total absorber between source depth in tissue Relative 10n density and surface of tissue in oMV at 34 depth dem ar 677 100 lem air+8 84 Al 552 113 Lem awr+17 6p Al 403 142 lem air+26 4p Al 198 209 Experiments on the dependence of the efficiency of @-rays on their energy are most conveniently made by using an active de- posit source m conjunction with screens of aluminum foils By passage through the foil the energy of the a-ray 1s reduced and the 1on density (1¢ number of 1omzations per micron path) mereased Table 6 gives data for the 1on density and @-particle 1 For methods of preparation see for example, Rutherford, E Chadwick, J & Elus CD (1930), Whitaker, MD, Byorksteadt, W & Mitchell, AC G (1934), Kanne, WR (1937) 2 These ranges are calculated from the ly known mean ranges mm air and the relative stopping powers of air and water given in Table 12, P 25 The measured ranges in liquid water are lower and may be wrong, viz Po 324 (Michl, W 10914) and Ra C 604 (Pluhpp, K 1923) According to Philpp the range of Re C’ a rays in water vapour corresponds to 67 at denaty 1g jer ® 3 For apparatus for domg this see, for example, Rutherfor Chadwick, J. & Elle, CD (1930), Naidu, R (1934) nord, By 4 Cp Zirkle RE, (1936) 16 PH SICAL PROPERTIES OF RADIATIONS thichness of the layer of tissue used If the layer 1 only » few microns thick, then the total energy dissipation an the thin layer 1s divided among f rays of different energy in the manner shown in Line 3, in which the effect of the slower rays 1s enhanced by Tante 5 Energy distribution of A raya from Ra B+ C O1- 925- & ray energy (eVE¥ ) 0-01 025 05 05-1 1-3 Total Relative number of f rays of stated 8 29 30 19 14 100 energy as percentage of total number Energy diss:pation in thin layor of = 21 40 nDie) ~o “a 100 tissue by # rays of stated energy as percentage of total energy dia spation in the layer Energy dissipation in thick layer of 1 10 22 QT 40 100 tissue by f# raya of stated energy, as percentage of total energy dis sipated in the layer the greater number of 1omzations they produce per micron path Tf, however, a thicker layer 1s used, e g a layer of fluid several mm in thickness and kept stirred, the f-rays of ngher energy will become of more importance as shown in the fourth line While the definition of the roentgen does not contemplate its use for radiations other than X-rays and y-rays, an obvious ex- tension would be to describe as Ir of # raya that dose which in 0 001293 g of air hberates ions carrying 1 esu of charge of either sign Table 2 gives the energy dissipation in tissue corre- sponding to 1 r of # rays defined in this way a-Tays a-rays are the nucle: of helium atoms emitted spontaneously by radioactive materials and also obtamable artificially with the cyclotron Their range in tissue 1s very short and they are there- fore generally only usable with materials obtamable in thm films « rays produce many more iomzations per micron path in tissue than do electrons, and the comparison of the relative efficiencies, per 1onization, of electrons and a-rays 1s very valuable for testing theories of the biological action of radiations Hence m experi- mental werk :t 13 worth while gomg to considerable trouble to overcome the expernmental difficulties attendant upon the use of this feebly penetrating radiation Q-RAYS V7 For irradiation of thin films of matenal by a-rays emitted from an external source, polonium serves as a convenient source Itis prepared from old radon seeds,x and emits a-rays of energy 5298eMV with practically no other radiation a@-rays of this energy have a range: of about 394 mm tissue of unit density Alternatively, ‘active deposit’ sources may be prepared by exposing a metal disk to radon s A source made in this way de- cays im a few hours and 1s only suitable therefore for short exposures After the lapse of half an hour, required for the com- plete decay of Ra A which emits less energetic « rays, the a-rays emitted are those of Ra C’ which have an energy of 7 680 eMV and a range: in tissue of umt density of about 714 This source emits f-rays and y-rays as well as a-rays The ionization per unit volume due to the f rays 18, however, only about 1% of that due to the a-rays and that due to the y-rays 18 quite neglignble Taste 6 RaC aparticles a ray energy at 34 Total absorber between source depth mm tissue Relative ron density and surface of tissue wn eMV at 34 depth lem ar 677 100 lem air+8 84 Al 552 113 lem aw4+17 6p Al 403 342 Lem ew+26 4z Al 1938 209 Experiments on the dependence of the effi y of a-rays on their energy are most conveniently made by using an active de- Posit source in conjunction with screens of aluminium foil, By passage through the foil the energy of the a-ray 1s reduced and the 1on density (1e number of 1omzations per micron path) mereased Table 6 gives data for the 1on density and a particle 1 For methods of preparation see for example, Rutherford, E, Chadwick, J & Elks, CD (1930), Whitaker, MD, Byjorksteadt, W & Nhtchell, ACG (1934), Kanne, WB (1937) z These ranges are calculated from the accurately known mean, ranges in air and the relative stopping powers of ar and water given in Table 12, P 25 The measured ranges :n hquid water are Jower and may be wrong, wiz, Po 324 (Machi, W 1914) and Ra C 602 (Philipp, K 1923) According to Phlpp the range of Ra C’ a raya im water vapour corresponds to 674 at density 1g fem? 3 For apparatus for domg this see, for example, Rutherford, E, Chadwick, J & Elhs, CD (1980), Nadu, R (1934) 4 Cp Zirkle RE (1935) 18 PHYBICAL PROPERTIES OF RADIATIONS energy after passage through various thicknesses of aluminium foil By an obvious extension of the definition of the roentgen, we may define Ir of a-rays as that dose which lberates in 0 001293 g of air 1ons carrying 1 eeu of charge of either mga Table 2 gives the energy dissipation in tissue corresponding to this dose When the experimental matenal 1s not usable in thin films, two methods exst for the hberation of a rays in the body of the tissue, enabling « irradiation to be made without the necesaty of employing thin films The first method 13 to use radon gas dissolved 1n the tissue by immersing the tissue in a solution of radon in water enclosed in an air-tight vessel: The a rays are emitted by the radon itself and by ite products Ra A and Ra C’ in equal numbers In addition, to every three a-particles emtted two £ rays are emitted, and if the tissue 13 thick enough for their complete absorption, as will usually be the case, the total energy dissipation due to the f-rays 1s about 5% of that due to the a-particlea y-raya will usually add neglgrbly to the energy dissipation Table 7 gives the data of the energies Taste? @ and # particles trom dissolved radon Energy dueapation in tissue contaummg me of radon per g Toni Energy tions Energy of particle plua rf Ergs/g/ eV /10-* umite/ —10- Particle Emntter recoil nuclousin eMV sec fee. feo Bec & fee a@ray Ro 5 586 army ReA 6 no} 19622 1059-1157 7223 1243 20 6 aray RaC 7826 Max Mean Mean s ray RaB 065 023 089 062 587 366 0630 1k B ray RaC 315 076 The physical measurement will usually consist of the de- termination of the radioactive content of the tassue im milheunies per gram Reference to Table 7 enables this to be converted to energy dissipation im the tissue A second method: of obtamung a-particles berated in the bulk of the tissue 18 to impregnate the tissue with a salt of 1 See Grey LH & Read J (1942d) fora full account of this method 2 Cp Kroger PG (1940), Zahl P.A & Cooper, FS (1941) PROTONS AND NEUTRONS 19 lithium or boron and irradiate with slow neutrons «-rays are emitted as a product of the disintegration of the hthium or boron by the neutrons Methods of dosimetry have not yet been worked out Protons and neutrons Protons are hydrogen nucle: moving at high speed and are not emitted by radioactive materials but are obtainable as a beam from a cyclotron They have not as yet been used in biological work, but should be usable with thin layers of tissue They grve a number of 1onuzations per micron path mtermediate between electrons and a-rays, and should be valuable in the same type of experment for which a-rays are used Defining a roentgen of protons as for a-rays the energy dissipation in tissue corre- sponding to 1 r of protons 1s hsted in Table 2 In the same way that electrons already existing im the tissue are projected at high speed when it 18 radiated by X- or y-rays, hydrogen nucle already in the tissue are projected at high speed as protons when the tissue 1s 1rradiated by fast neutrons The neutrons also cause the projection of carbon, oxygen and other nuclei, and dissipate some energy in other ways, but most of the energy they dissipate in tissue 1s by the process of proton projection A roentgen of neutrons, if defined as the dose hberating in 0 001293 g of air ions carrying 1 e8u of charge, would not be a very surtable unit for neutron dosimetry, since the mechanism of romzation 1n aur is by projection of nitrogen and oxygen, and in tissue mamly by projection of hydrogen nuclei The factor for conversion of 1 r defined in this way to energy dissipation in tissue would be several times greater than the factors for other radiations, and would vary with neutron energy since the ratio of the collision cross sections of hydrogen and of the other gases vanes with neutron energy The disintegration of mtrogen by the neutrons would also complicate the determination of the factor These difficultzes can be largely avoided by defining the unit of neutrons in terms of a matenal contammng a proportion of hydrogen comparable to that in tissue To realize the unit ex- permentally it is y amply to the iomzation in the air in a small chamber the walls of which have the appro- pnate content of hydrogen, the dimension s of the chamber being 20 PHYSIOAL PROPERTIES OF RADIATIONS amall enough, or the gas pressure low enough, to ensure that the iomzation 1s almost entirely due to nucle: projected from the walls, and not to nucle: originating mn the aur Actutted by these considerations, Gray: has proposed the following unit for neutron dosimetry, provisionally designated by the symbol ly ‘The dose of neutrons at a point shall be lv when the associated corpuscular radiations produced in water liberate in air Le su of charge of either sign per 0 001293 g of ar’ The energy dissipation m various tissues corresponding to 1v of neutrona has been set out in Table 2 The unit used in America 1s an arbitrary umit based on the reading given by a ec cial X-ray di ter (the Victoreen) when exposed to neutrons It1s believed that 1 » unit equals 25 v units, but the conversion factor varies slightly with different instruments 2 Neutrons used in biological work up to the present have been obtamed from three types of generator (a) Acyclotron employing the Be + D reaction with deuterons of 8eMV or more (6) An ion tube employing the Li+D reaction with deuterons of about 1 eMfV {c) An 210n tube employing the D+ D reaction with deuterons of about 0 3 eMV Neutrons from any of these reactions liberate protons in tissue with a vanety of energies The energy distributions given in Table 8 are very approximate but may be used as a basia of calculation s A few expermments have been reported in which biological materials have been rradiated by slow neutrons Slow neutrons x Gray LH (private communieation) Gray LH (1944a} 2 Aebersald, PC & Lawrence JH (1942} 3 They are calculated aa follows Since the scattering of neutrons by protons 1s isotropic in centre of gravity coordinates, it follows that of the recoil protons produced by neutrons of energy Eos the proportion which are projected with energy between E, and EH, 13 (Z,—E,)/Ey, while the proportion of the total recoil proton energy carmed by protons of this range of energies 15 (Z,*—E,2)/E,* The data of Bonner T W (1941) and af Bonner, TW & Brubaker WV (1935, 1936) are used in calculating the vanous neutron energies Ey eraitted by the different radiations The D+D neutrons are supposed observed in the direction of the deuteron beam, the Li+D and the Be+D neutrons at night angles to the deuteron beam DOSIMETRY 21 have too httle energy to 1on1ze by setting protons into motion by simple collision, which 1s the principal process by which fast neutrons dissipate energy in tissue They are however captured by the atomic nucle: of various elements A nucleus which has captured a neutron emits an a-particle, a proton, an electron, or ay ray depending on the element concerned From the theo- retical pot of view there 1s not much interest in studying the Tants 8 Dustnbution of mitral proton in neutron PF Deuteron Proton energy mn eMV energy = -— ~ Reaction meMV Q-1 1-2 2-3 3-4 46 68 8-10 10-12 12-14 Total D+D 03 3131 ~«3r 7 -_- - — _ - 100 10 28 47 168 — —- — — _ 100 In¢D 09 61 19 «12 4 6 4 2 15 O56 100 1616 «4 WoW 12 9 7 4 100 Be+D 8 2 12 #12 #Y 2 19 9 _- _- 100 2 5 7 10 2 31 16 _ _ 100 Of the two lines of figures given for each reaction, the upper line gives the percentages of the total number of protons which have imtially the indicated energy and the lower line gives the percentages of the total proton energy dissipated by protons of the mdicated initial energy biological effect of slow neutrons, since the effects obtained will presumably be no different from those obtained when tissue 13 uradiated by & rays, protons, electrons or y-rays as the case may be There may, however, be some practical rmportance attaching to the use of slow neutrons, 1n that the tissue dose can be made greater ip one region than another by impregnating it with hthium or boron (see p 18) Dosimetry The roentgen 1s @ unit of dose internationally accepted for y Ttays and X-rays, and capable of obvious extension to cover most of the other ronizing radiations, in the manner + hich has been dicated separately for each radiation It 1s a unit chosen primanly for convenience in physical measurement, and while lr of any radiation represents the same amount of 1onization in air it does not always represent for different radiations the same ionization or energy dissipatio n in the tissue It 1s neces- sary therefore in comparing the efficiencies of different radia- - } 22° PHASICAL PROPLRTIFS OF RADIATIONS tions, to be able to cénvert roentgens into :onization in tissue or into energy dissipation in tissue There 1s no difficulty in principle in converting roentgens into energy dissipation in tissue, and if the elementary analysis of the tissue is known the conversion can probably bé made with an error of less than 10% The most obvious umt of energy to employ 1s the erg Ir of y rays or X rays involves the dissipation of about 90 ergs/g of tissue If we are concerned with the energy dissipated in a single cell or smaller entity, an erg becomes an inconveniently large umt, and it will be found more convement to employ the electron volt, which 1s 1 602 x 10-!% erg Thus lr of X- ory rays 1s about 60 eV /3 of tissue In view of the fact that 1t1s common physical practice to express energies of radiations in electron- volts, this unit 1s probably the most convement to employ Taste 9 Interconterston of units of energy dissipation in tissue leV 10-4 g = 1 602 ergs/g \ lenergy unit (Gray & Read 1 gram roentgen (May neord) == 83 78 ergs. 1 1onization per 10-21% ¢ @ ray proton or neutron=56 07 ergs‘ Xray y ray or electron=52 07 ergs/g As regards the expression of dosage m terms of ionization in tissue there 1s some uncertainty m the conversion, since the physical observations are in practice made on ionization of gases, and rt 1s necessiry to assume without experimental confirmation that the energy expended in the production of an ionization 1s the same m tissue as ma gas The values we take to apply to tissue are 32 6 eV per ionization by electrons or X- or y-rays, and 85 eV per ronizafion by a rays, protons or neutrons, these bemg the values commonly accepted for air Gray and Read: suggest that when the effects of different radiations are being compared, the doses be expressed in energy unis where by defimtion 1 energy-unit of any radiation dis- sipates the same energy in 1 g of the tissue concerned as 1r of y rays dissipates in 1 g of water ‘Lhe gram roentgen: 1s the energy dissipated by Ir of y-rays or hard X raysin 1 g of air In most of what follows we shall express tissue dosage either in tonizations or m eV /10- g of tissue (1072? g 1s the wanght 1 Gray LH Mottram JC Read J & Spear FG (1040) 2 Mayneord WV (1940) RANGE AND ENERGY DISSIPATION 23 of a cubic micron of water) Table 9 makes clear the relation between the various units, and in Table 2 we have given factors for convertmg doses measured in roentgens to any of the methods of expressmg tissue dosage hkely to be employed It willbe realized that although the factors are for convenience given to four figures, the precinon with which they are at present known 1s by no means so great Primary ionization, ranges, and energy dissipation The various 1omizing radiations we are considering all dissipate their energy by the passage through the tissue of either electrons, protons or @ particles These may be produced externally by a radioactive or an artificial source, or they may be liberated in the tissue as when the tissue 18 rrradiated by X-rays or neutrons, or 38 ympregnated with radon The pnneipal difference from the theoretical point of view 1s that in the former case, when a film of tissue thin compared with the range of the 1onizing particle 1s used, the efficiency of a particular part of the trach of the ionizing particle may be determined, while in the latter case the ionizing particle 1s completely absorbed in the tissue and one determines an average efficiency of the whole track Electrons, protons and « particles pass through tissue in paths which may be taken as straight for distances of a few microns, though the bending of electron tracks as a result of scatterng by atoms is considerable over Jonger distances As the particle traverses the tissue it loses energy as a result of collisions with atoms, excitation or 1omzation of the atom struck resulting As explained earher, excitation 13 probably of amall biological importance compared with ionization As the ionizing particle slows down, the energy dissipation per micron path, and so the number of :omzations per micron path (which we refer to as ton density, or specific tonrzation), increases In Plate I are shown photographs: of electron, proton, and a-ray tracks showing that the 1on-density1s greater for « raysand protonsthan for electrons, and 1s greater for a slow electron than for a fast electron Thus the 1on density increases towards the end of an electron track In Tables 10-13 are given numerical data: of the range, energy 1 Seep 10,n 1 . 2 The sources of these and subsequent tabulated data are deseribed am the Appendix 22° PHLSICAL PROPLRTIFS OF RADIATIONS tions, to be able to convert roentgens into ionization 1m tissue or into energy dissipation in tissue There isno difficulty in principle m converting roentgens into energy dissipation in tissue, and of the elementary analysis of the tissue 13 known the conversion can probably bé made with an error of less than 10% The most obvious unit of energy to employ 18 the erg Ir of y-rays or X-rays involves the dissipation of about 90 ergs/g of tissue If we are concerned with the energy dissipated in a single cell or smaller entity, an erg becomes an inconvemently large unit, and 1t will be found more convenient to employ the electron volt, which 1s 1 602 x 10-1 erg Thus I r of X- or y-rays 1s about 60 eV /3 of tissue In view of the fact that 1t 1s common physical practice to express energies of radiations in electron- volts, this unit 1s probably the most convenient to employ Tapre 8 Interconversion of units of energy dissipation in tissue leV /t0-* g =1 602 ergs/g 4 lenergy unit (Gray & Read) =93 12 ergyg 1 gram roentgen (Mayneord)= 83 78 ergs 1 ionization per 10-4 ¢ a Tay, proton or neutron =56 07 ergs/z ‘\ ray y ray or electron=52 07 ergs/g As regards the expression of dosage in terms of ionization m tissue, there 1s some uncertainty m the conversion, since the physica] observations are mn practice made on romzation of gases, and it 18 necessary to assume without expermmental confirmation that the energy expended in the production of an ionization 1s the same in tissue as in a gas The values we take to apply to tissue are 32 5 eV per 1omzation by electrons or X- or y rays, and 35eV per romizafion by « rays, protons or neutrons, these bemg the values commonly accepted for air Gray and Read: suggest that when the effects of different radiations are being compared, the doses be expressed mn énergy- unis where by definition I energy umtt of any radiation dis- sipates the same energy in 1g of the tissue concerned as 1 r of y rays dissipates in 1 g of water The gram roentgen: 1s the energy dissipated by lr of y rays or hard X-rays in 1 g of air In most of what follows we shall express tissue dosage either 1n 1omzations or in eV /10-** g_ of tissue (107? g 1s the weight 1 Cray LH Mottram JC, Read J & Spear FG (1940) 2 Mayneord W V (1946) RANGE AND ENERGY DISSIPATION 25 Taste 11 of prumary P d by per micron of path in tissue of d lensity 1 g /om * Electron Pnmary Electron Pnmary Electron Primary energy ions per ft energy ons per # energy tons per #t ekV ons/fe ehV 1ona/p ekV lons/p 1697 275 95 01 275 1270 1206 325 81 82 35 10 38 7801 375 71 96 45 8452 583 8 45 61 09 55 7 206 4095 55 60 99 65 6 332 304 2 65 43 86 15 5685 3406 76 38 64 85 5 186 300 5 85 34 41 95 4790 269 2 95 3190 105 4 603 2441 105 2921 115 4202 2145 115 26 96 135 3786 1851 125 25 06 165 3.349 163 0 135 23 43 195 3048 1459 45 22 01 225 2 846 1321 ve: 18 70 270 2 692 1134 225 15 06 330 2 382 390 2241 450 2166 xp xp xp For tissue density p g fem # multiply the figures given by p as indicated at the foot of each column Tanre 12 Range energy dissipation, and number of primary ronizations produced per micron path m tissue by « particles Tissue path Ene rticle dissipaton equivalent to Primary @ pa Imm ar at Range mn ionizations ener; per zof tissue 0° & 760mm tiague per jt tissue oe ekV jp B 10ns/jt 1 263 9 53 5207 2 1761 101 2883 3 46 168 2031 4 0) 261 1582 6 9377 35 2 1301 6 82 OL 470 1109 7 7310 603 968 0 8 66 09 75 6 860 5 2 60 41 916 715 $ 19 65 71 108 4 706 4 5 2984 (Po) 389 & 4860 (Rn) 411 59981 (Ra A) 470 7 6802 (Ra C) 708 xp —P Pp xp Above §igures apply to tissue of density 1 g fem * For denmty p g fem * they should bi e multiplied or divided by p as indicated at the foot of each colamn the curved path is some 40% greater than the extrapolated range de auced fro: 7m Fessurements on the penetration 0. foila by an electron beam 24 PHYSIOAL PROPFRTIES OF RADIATIONS dissipation, and number of :omzations produced per micron path by the three types of 1onizing particle, for various energies of the particle None of these data are known as accurately as one would wish, and the uncertainty attachmg to the number of Taste 10 Range and energy duspation per micron by electrons m tisaue of density 1gJom ’ Electron Energy Electron Ene en disapation Range energy dussipat on Range ek’ ekV jp ek’ ekV fp » OL 33 23 0.00301 9 2 436 2089 O15 3159 0 0045: 10 2 298 2517 o2 28 71 0.00622 i 2133 2969 03 23 86 0 01005 12 2993 3455 04 20 84 001455 13 1873 3972 06 18 62 0.01963 14 1768 4522 08 16 84 0 02528 15 1674 5 104 07 15 40 003150 20 1339 8 46£ 08 1417 0.03828 25 1127 12 55 09 13 20 0 04559 30 0 9803 1732 10 12:33 005344 40 O7811 28 82 12 10 94 0 07068 50 0 6698 42 69 14 9 859 0 08998 60 05879 58 66 16 8901 O1le 70 0 5280 76 63 18 8278 01344 80 04824 O8 47 20 7680 01595 90 0 4462 1180 25 6633 0 2304 100 04170 1412 30 5710 03124 110 03929 1660 35 5 086 0 4054 120 03727 1921 40 4596 0 5088 150 03278 2781 5 3872 0 7488 180 GO 2976 3743 8 3359 1025 210 0 2769 4789 7 2975 1342 240 Q 2602 5907 8 2676 3697 300 0 2381 8320 360 0 2239 1092 420 02142 1366 480 02073 1651 xp =p “Pp Pp For tissue of denaty p g jem * multiply or divide the figures given by p aa indicated at the foot of each column primary 1omzations per micron 1s considerable, since one has really very little information concermng the production of ionization 1m solids and hquids The range meant 1s the distance travelled by a particle of energy H in being brought to rest, and 1s measured along the curved path: The energy dissipation 19 1 Inthe case of electrons the paths of which are by no means atraight this range 18 not the same as the maximum penetration of a sheet of absorber According to Willams EJ (1930), the range measured along CLUSTERS AND 6-RAXS 27 The range of a slow electron of + hundred volts or go 18 only & few mullimicrons (Table 10), and the secondary 1omzations produced by this secondary electron are therefore produced close to the primary iomzation and with it form a cluster of 1omizations In Plate In the larger droplets are condensed on unresolved clusters of 1ons The relatr, e numbers of clusters of different sizes are given in Table 14: a-rays and slow protons produce their Taste 14 Frequency ofion clusters containing various numbers of ionizations No of iomzations in the cluster 1 2 4 >4 = Total Frequency of cluster of this size 0.43) «022 012 010 «015 106 pmmary ionizations at such close intervals that the successive clusters overlap and lose their mdividuabity, forming a column of ionizations rather than a senes of discrete clusters (Plate I4,R)} Occasionally the electron ejected at a pmmary Jonization has an energy of several hundred or even thousand electron volts Tt 1s then able to travel an appreciable distance and produce a large number of 1oruzations on its own account The secondary electron thus forms a separate trach branching off the main track, and 1s called a d ray Plate [a shows é revs branching from an a ray track Nearly half of the total number of 1on1za tions produced by the primary particle, whether it be electron, proton, or @ ray, are found in é rays of energy exceeding 100eV The other half of the total number of 1onizations are distributed among the isolated primary 1onizations and the clusters of two or three ionizations Since rays are slow electrons, they have an ion density greater than that of a fast electron and less than that of an a-particle It follows that the total range of all the d rays branching from an a ray track actually exceeds the range of the a-ray itself, but the total range of all the é-rays branching from an electron track 1s only a small percentage of the total range of the primary electron In Table 15 are given numerical data of the total range and total number of 1onizations produced by & TAS S2 of energy exceeding 100 eV érays turn out to play a rather important part in certain biological actions of radiation (see especially Chapters m1 and viz), 1 Experimental observations of Wilson, CTR (1923) 2 Including tertiary electrons,1e d rays produced by & rays 26 PHYSICAL PROPERTIES OF RADIATIONS computed in electron kilovolts per micron path and refers to a particle of the stated energy Z, and 13 not the avernge over all the energies between E and 0, thia average sf desired can be found by dividing the energy E by the range The number of Taste 13° Range energy dissipation, and number of prumary sonizations produced per micron path in tissue by protons Tissue path Energy equivalent to Primary Proton duwsipation Imm airat Range in somzations energy persgoftissue 0° & 766mm tissue per # tissue oly ekY fe B # ons/je 1 2769 1088 23 398 2 2 16 65 1100 73 2170 3 12 20 1106 M47 1518 4 9 742 1109 241 nqv7 5 8164 1112 355 06 58 8 7 058 13 486 213 7 6 236 1115 642 ‘71 63 8 5599 1116 813 63 62 2 5 089 1117 1004 5731 10 4672 1118 Tir 5219 xp 7p -p xp Above figures apply to tissue of density I g /em* For density p g fem ? they should be multiplied or divided by p as indicated at the foot of each column pmmary 1onizations per micron 1s similarly for the particle of energy E and not an average for all energies between H and 0 By promary ionization 1s meant an 10mzation caused by a col- lsion of the primary particle (electron, proton or a particle) not includmg secondary iomzation produced by 3 rays, which we shortly proceed to discuss The figures given in the tables are for a, tissue of density 1 g /om * and need to be multiplied or divided by p for a tissue of density p as indicated in the tables Secondary ionization, clusters, and 5-rays At each primary ionization an electron 1s ejected by collision of the prmary particle (electron, proton or a ray) with an atom of the tissue Often this electron 1s eyected with insufficient energy to make any ronizing collsions on its own account, and ultimately attaches itself to an atom to form a negative ion, the attachment being, however, probably without any biological significance (see p 2) If the electron 1s ejected with rather more energy 1t may produce a few onizations on its own account 6-RAYS Taste 16 (cont } 24 48 96 192 384 ekV 0 7579 04011 0 2326 01442 01012 0 4996 0 2657 0 1546 0 09600 0 06738 0 2935 01576 0 09215 005740 0 04036 0 2056 Ong 0 06543 0 04087 0 02880 0 1570 0 08570 0.05060 0 03169 0 02236 01261 0 06042 0 04117 0 02586 0 01826 01049 005817 003465 002182 0 01543 0 08938 0 04994 0 02987 001885 0 01336 007757 0 01366 0 02622 0 01659 001177 0 06828 003871 0 02334 001480 0.01052 eRWDWAe woPlere 0 05757 0 03299 002001 001274 0 009065 0 04722 002743 0 01677 0 01072 0 007651 0 03969 0 02338 001440 0 009245 0 006614 0 03399 0 02029 001259 0 008II8 © 005822 002952 0 01786 OO1ll7 0.007229 © 005197 0 02369 0 01467 0 009287 0 006055 0 004370 Aan 0 O1804 001155 0 007440 0 004898 0 003356 001421 0 009588 0006168 0 004099 0 602992 001146 0 007862 0 005240 0 003515 0 002579 0 009409 0 006689 0 004534 0 003069 0.002264 oOa 0 006024 0 004725 0 003340 0 002313 0 001728 0 004326 0 003724 0 001919 0.001448 0 003145 © 003006 0 001632 0 001243 0 002193 0 002468 0 001938 0001413 0 001086 0 001458 0 002052 0 001674 0 001242 105 0 0009031 0 0008423 0 001722 0 001462 0 001103 0 0008636 0 0002794 0001454 0 001289 0 0009900 0 00078168 0 001233 0 001145 0 0008945 tUPSaetQeTdbP tdSiItUTlP) tdSertetlr 0 0007129 0 001047 0 001024 0 0008140 0 0006544 0 0008887 0 0009205 0 0007449 0 0006042 0 0007517 0 0008314 0 0006850 0 0005595 © 0004227 0 0006858 0 0005865 0 0004883 0 0001077 0 0004429 0 0004195 © 0003648 0 0002944 0 0003158 00002871 0 0001937 0 0002457 0 0002338 0 0001541 0 0002186 0 0002130 0 00003168 0 0001422 0 0001537 0 00009512 00001167 0 0006294 0 00009152 0 00003890 0 00007334 0 00001929 © 00005962 8 00000172 0 00004495 0 00003678 ttS il 0 00003010 0 c0002450 0 00001971 © 00000837 xp 28 PHYSICAL PROPERTIES OF RADIATIONS Taste 16 dé raya exceeding 100eV. energy produced by i protons and 2 p Electrona @ particles Protons A ~ ¢ A” ~ c A + K & % pig s i abei Ae aks i, Be Be § Bq i aes B Be g ae i 8g §E % ges gb Boge e Spe g Bh gee Bh g $2 fa S tee aa ¢ ony °o 2? ia cy oO 3 Pes o% > pe oF 2 be 8% 2 fd of E 83 of Eg £3 os fo osER 2oh os «oh Zoe oh Boe ekV “ env # omy # 05 0064 0 225 1 252 O718 1 0302 0-905 15 0060 0504 2 201 0 832 2 0308 0 956 3 0 048 0597 3 173 0 879 3 0270 0-978 6 0038 0672 4 156 0907 4 0 236 0981 12 0031 0734 5 144 0925 6 0219 1006 24 0026 0780 6 135 0939 8 0 203 1013 48 0022 0802 q 129 0950 10 0193 1018 98 0022 0856 8 123 0059 Taste 16 ¢ ray production by electrons m tissue The table gives the number of 2 rays of energy exceeding W produced by & pmumary electron of energy E per micron path an tiaaue of density 1 g fem * For tissue density p g /em *, multiply the figures by p as indicated at the feet of the columns E 06 15 8 6 12 e&kV ekV 01 1650 8757 4964 2 662 1433 01S 7382 5191 3103 1712 09364 O25 — 2475 1650 0.9609 0 6412 035 — 1390 1050 0 6448 03734 045 _ 0 8203 0 7283 0 4723 0 2809 055 _ 0 4668 05308 0 3643 0 2226 065 _ 02131 0.3982 0 2910 0 1825 075 — — 04) 0 2381 01534 O85. _ Q 2343 0 1984 01314 095 _ 01789 0 1676 01141 11 _- _ 01167 0 1326 0 09432 13 —_— _ 0 05328 0 09956 007532 15 — — — 0 07602 0 06165 17 = _ _— 0 05858 005137 19 — — — 0 04497 0 04339 295 _ — 0 02605 0 03309 _ — _— 0 00817 0 02327 24 _ _ _ — 0 01664 346 — _ _ 001202 425 _ _ _— 0 008466 BB — _ — _ 0 002120 65 _ _ _— - > xp xe xp xp xp NUMBER OF IONIZING PARTICLES 31 It 13 also sometimes necessary to know the number of 1omizing particles (electrons, protons or « rays) which cross each square mucron of tissue irradiated by a given dose, or what amounts nearly to the same thing, the total path length in microns of all the ionizing particles which are berated in 1° of tissue, or which traverse 1,3 of tissue This information 1s also provided in Table 18, the calculation utilhzing the range energy and d 1ay data given in the present chapter Taste l7B é ray production by protons in tissue Number of é rays of energy exceeding 1} produced by a proton per micron path in tissue of density 1 g fem * For denmty pg /em* multiply by p Proton energy (in eM¥ ) Ww ekV 1 2 3 4 6 8 10 30 58 15 66 10 52 7921 5301 3984 3191 19 90 10 32 6 959 5 250 3 52 2 648 2122 1135 6 042 4110 3113 2 096 1580 1 267 7 b85 4211 2 889 2197 1485 1122 09011 5 650 3193 2211 1 689 1146 08673 0 6976 4355 2546 1778 1365 09304 07055 05681 3.458 2097 1480 1141 07810 05934 04784 2801 1789 1261 09764 06714 05212 04196 2298 1517 1093 08507 05876 04483 03624 1903 1319 09610 07514 05214 03987 09 322 1441 1089 08076 06364 04447 etRWoOAHeIRN 03412 02767 09923 08645 06582 05243 03700 02852 02318 wows 06636 O7001 05486 04421 03152 062441 91990 04122 05744 04648 03793 02733 0212 01738 02137 04752 03986 03297 02402 01879 01540 a — 03440 O3112 02641 O1965 O1651 01277 ~ 02145 wertA A 02248 01993 01533 01227 01018 = 01248 01651 01545 01234 01003 090839 — 00591 01212 01216 01615 00838 00707 — 00088 00877 00965 00848 00713 00607 CORATROE — _ —~ 00306 — 00536 00007 00562 00312 00198 004 _ —_ 00413 00386 9 oases —_ 00148 00303 00304 002801 — _— _ 00022 00219 00241 002298 VGeo ~ _ _ ~ 00153 00192 0O0T901 = _ _ _ 00100 00251 001580 - = _ _ _ 00055 00118 OO1314 e _ _ _ 00018 00090 001091 7 7 7 ~ ~ 00067 0 00901 = = = _ —~ 00048 0.00737 = = = > —~ 0.0028» -0 00595 175 7 - > — 000358 195 _ = a = _ _ 000171 04 oe _ = _ = 7 _ 0 00053 xp xp xp xp xp xp xp 30 PHYSICAL PROPERTIES OF RADIATIONS and it 18 necessary to have adequate data of their number and energy Tables 16 and 17 give for various energies of elec trons, protons and a raya the number of é rays produced per micron path in tissue of density 1 g femIf the actual tissue density 18 p g fem, then the number of é rays produced per micron path 1s p times greater than in Tables 16 and 17 Taste 174 4 ray production by a raya in tissue Number of é rays of energy exceeding W produced by an particle micron, path | tresue of denaity 1 g /em * For density pg jon bd multiply by p ps per v @ particle energy (in eMV } kv Ok 2 3 4 5 6 7 8 10 OL 4164 2314 1595 1215 9815 8231 7087 6222 5001 O15 2488 1466 1029 7908 6420 S401 4661 4099 3303 025 1108 7864 5759 4513 3703 3138 2721 2402 1944 035 5260 4963 3818 3057 2539 2167 1889 1874 1362 045 2026 3336 2740 2249 1892 I623 1427 1270 1039 O55 307 205 1734 1480 1285 1233 1012 8332 065 — 1595 1580 1378 1196 1048 9298 8343 6907 075 — 1072 1231 2117 9865 8737 7805 7037 5862 08 6720 9649 9173 8267 7406 6684 6038 5063 08 8 — 3575 «7548 «60-7596 «7006 «8354 «5763 «5250 4432 12 ~ — 5108 8768 45544 61396 4718 4336 3701 13 _ _ 2735 30987 4119 3949 3701 3446 2989 15 _ _ 0993 2681 3074 3078 2954 2793 2466 17 - 1682 2275 2412 2384 2293 2067 19 _ _ _ 0894 1644 1888 28993 1899 1761 225 — _ - —_ 0810 19! 1337 1378 43334 215 — —- _ — — 0505 0749 0863 0923 325 — ~ = = — 0031 0342 0507 0638 375 _ _ _ — _ i O14 0246 0429 425 — ~ ~ — _ — — 0046 0269 65 _ _ _ — — _ - _ _ xp xp xp xp xp xp xp xp xp Number and total range of ionizing particles per unit volume of tissue It 13 sometimes necessary to know the number of 1onzing particles generated per umt volume of 1rradnated tissue, 1e the number of electrons projected (by photoelectne or Compton effect} m unt volume of tissue by @ given dose of X or y-rays or the number of protons set mto motion per unit volume of tissue by a given dose of neutrons Table 18 provides the neces- sary data The calculations have been made for a ‘wet tussue’ of the composition assumed on p 7, and are based on the numerical data given in Tables 2, 3 and 8 Chapter IT CHEMICAL EFFECTS OF IONIZING RADIATIONS, AND POSSIBLE MECHANISMS OF BIOLOGICAL ACTION The ionic yield Experiments have been carned out on the chemical effects of romzing radiations: on pure substances in the gaseous, liquid and solid states, and also on solutions, particularly aqueous solutions Gaseous reactions have usually been studied using e-rays, radon, etther nuxed with the gas or else sealed in a thin-walled bulb at the centre of the gas, being the source of the rays Some expen- ments have been made with X-rays, cathode rays and /-rays, but there 1s much Jess information than one would hke on the relative efficiency of different radiations, and this is at present one of the most urgent requirements With the doses which 1t 1s practicable to give with X-rays, the percentage of the reactants which undergoes chemical change 1s usually small In the absence of complications, such as back reactions, the amount of chemical change then increases in direct proportion to the dose, and the yreld of the reaction 1s most conveniently expressed as the number of molecules of a specified substance formed or destroyed per ion-pair produced, a ratio conventionally repre- sented as M/N Other methods of representing the yield are sometimes found in the hterature If the energy of the radiation absorbed in the uradiated matenal has been measured calorimetnically, the yield may be given as gram-molecules reacting per kalogram- calome If1t has been measured by the complete absorption of a known number of ionizing particles of known energy 1t may be expressed as the number of molecules reacting per electron-volt of energy absorbed If radon 15 used as the source of radiation the yield may be expressed as gram-molecules reacting per milh- cunie of radon disintegrating, or as cm ° of gas reacting per millt- cure of radon disintegrating In X-ray experiments on dilute it Sometimes referred to as radtoch ui in dst, from toch 1; 4 r d by visible and ultra violet hght peneral references to this subject are Lind, SC (1928), The Chemical iffecte of a partycles and Electron#, Allsopp CB (1944) 32 PUYSICAL PROPERTIES OF RADIATIONS TantF 18 Numbera and ranges of ionizing particles per 4 per 1000r an wet tissuo of umt density Numbers of olectronst. Combined rangein g of all Wave length projected per tho electrons (including A # per 1000 r & rays) per uc? per 1000r y raya 00152 Om X rays 0.0809 190 360 0 1618 455 309 0 3033 431 352 04853 352 274 15% 670 10 83 41s 261 530 $32 373 286 1 Recoil plus photoelectrons Combined range in # of all tho romuzing particles per #? per 1000 v umts Deuteron Nhe of protons =————-—*~—_—___—. ener; projected per Protons eMY pz" per 1000u units Protons grays +2 rays Neutrons Li+D o9 00293 405 115 520 D+D 03 0 0364 227 085 312 Combined range m # of all the jonizing particies traversing the tissue, per 3 per 1000 r Energy & rays eMV @ rays é rays +8 rays I 027 067 094 2 039 O77 116 3 050 087 137 a rays iepediating thin layer ot 4 O61 095 156 which the a rays are not com pletely absorbed) 5 ont 10g itt 7 091 16 207 8 100 123 223 Range m # per pi} tissue re a rays of Rn+RaA+RaC completely a rays absorbed (1e radon im solution) @ rays é rays +8 rays per 1000 r 056 092 148 per sec for 1 cure radon present per 59 97 156 cm * tiasue for1 po of radon diaintegrated per em * 28 46 4 tissue GAS REACTIONS 35 experiments), and of the probable fate of excited molecules (from absorption spectra and photochemical studies) The following account desenbes the processes contributing to the total yield in typical gas reactions When an q-particle or a fast electron passes through a gas, energy 18 dissipated partly by 1omzation and partly by excitation or direct dissociation of the molecules with which st collides, the energy dissipated in ronization usually being of the order of one- half ofthe totalenergy In addition, a small fraction 1s converted directly into heat by elastic collisions Excitation often leads to the dissociation of the excited molecule, either spontaneously or when the excited molecule makes a colltsion with an ordinary molecule Thus, following excitation, H, may dissocrate into H+H, CO into C+0 These products, liberated in atomic form, are then likely to take part in chemical reaction The process of :onization of a gas molecule usually does not directly cause dissociation Thus m CQ, at low pressure the positive 1on CO# is more copiously formed than CO* or Ct, and m hydrogen the 10n Ht} 1s more copiously formed than H* When the positive 10n 3s neutralized by collision with either an electron or a negative ion, a large amount of energy 1s set free, and this almost always results in dissociation : The final result of positive ion formation 1s thus the dissociation of the ronized molecules, the free atoms hberated probably taking part in subsequent chemical reaction The electron which 1s projected from the molecule when jomization occurs may be fast enough to 1onize and excite on its own account, and the chemical changes so caused will not differ from those following romzation and excitation by the primary particle Some electrons will be eyected with insufficient energy for ionization or excitation, and all will eventually be slowed down In some gases, eg pure mitrogen, carbon monoxide carbon dioxide, neutral gas molecules have very little electron affimty, and the slow electrons therefore remain free until they collide with and neutralize a positive ion In other gases such as oxygen the electrons form negative 1ons,eg O, The energy of 1 If, however, the positive ions are neutralized on a surface, as when 4n electrically charged plate 1s mtroduced into the hamb then the molecule can get rid of the energy set free at neutralization, and dissociation may be avoided (Smith, C & Essex, H 1938) 34 OHEMICAL EFFECTS OF IONIZING RADIATIONS aqueous solutions the yield 1s sometimes expressed in terms of micromoles reacting per 1000 r per litre of solution To convert these units into molecules reacting per 10n-parr, it 1s necessary to know the mean energy dissipation per 1onization Taste 19 Ci factors for yields in radioch f reactions as M/N values (molecules per 10n pair) Molecules per ion pair X rays f rays a rays cathode rays 1 gram molecule per kilogram calone= 807 149 1 molecule per electron volt. = 5 325 1 gram molecule per milkcurie of radon diaintegrated = 5 80x 10ty _ 1 ml gas at wap per milicune of radon disintegrated = 259: — 1 micro mole per 1000 r per htre in dilute aqueous selutions 1 Soft X raye 03-05A = _ 038 Hard X rays and y rays = oad O34 « Assuming that the o rays and f rays and reco! nuclei from the dimntegra- hon of Rn, Ha A,Ra B Ra, RaC are all completely absorbed in the reacting system This will be sufficiently nearly true when the radon 1s dissolved 11 8 liquid p ding 1s made for th of the radon between the liquid and any gas space m communication with it For gas reactions the uo I since 6 prop of the @ particles are absorbed in the walls, either of the radon bulb or of the ges container, thus causing the factor to be greater than that given For the method of calculation 3m these cases see Lind SC (1928) In Table 19 this has been taken to have the value valid for air, viz 35eV per ion pair for a rays and 32 5eV per ton parr for electrons If the actual figure 1s known for the substance being used, say WW eV per jon par, then the conversion factor given in the table should be multiphed by W/35 for a-rays and W/32 5 for X-rays, cathode rays and f rays Gas reactions: The reactions which are best underatood are simple gas reac- tions in which either the decomposition of a single gas 1s studied, or the combination of two gases The interpretation of gaseous reactions is helped when wnformation 1s available of the types of positive and negative tons formed (from the mass spectrograph), of the 1omzation and excitation potentials (from electron impact 1 aqThe a by 1onzing of radthe induct of ch 1 change in gases d in this section was proposed by Eyrmg H Hrschfelder JO & Taylor HS (19364 6) LIQUIDS AND SOLIDS 37 1s hable to occur, and in which therefore the yreld obtamed 18 hkely to depend upon the experimental conditions Tapir 20 Tonic yields (1/4) in gas reactions Cathode Ultra violet Reaction @ rays X rays orf rays light NH, decomposition 1378 — 1204 02-0318 0 807 _- — — 1163 _— — _ NH, synthesis (including 02-035 _ ~9 26 — hydrazine} 0 283 — — ~~ N,0 decomposition 1 _- 397 pei) 44 _ — — HBr synthesis (great excess H,) 2 9t0 269 _— — HI decomposition ~6 809 — Bi C,H, poly merization 2033 — Qo 9 ort Q, from 0, 2-254 = 2215 gt H,O vapour decomposition 00517 136 — — CO, decomposition 007 _ 00495 123 1 Smith © & Essex, H* (1938) “r4 Lind SC & Bardwell DC (1929) 2 Wourtzel, E (1919) 1> Busse WF & Daniels F (1928) 3 Jungers JC (1932) 16 Gunther P & Holtzapfel, L 4 Gedye GR &Allibone T E (1930) (19394) (excess of xenon present) 5 Lind SC & Bardwell DC (1928) 17 Duane W & Scheuer O,(1913) 6 Gedye G R & Allibone T E (1932) 18 Wnug FO (1935) 7 Gedye GR (1931) 19 Noyes W A (1937) 8 Kolumban A D & Essex H (1940) 20 Lewis B (1928) 9 Gunther, P & Lewhter, H (1936) at Innd SC & Lavingston R (1932) vo Lind, SC & Livingston R (1936) 22 Vaughan WE & Noyes, WA 11 Brattam KG (1938) (1930) 12 Mund W & Jungers JC (1931) 23 Groth W (1937) 13 Lind, SC, Bardwell DC & Perry JH (1926) Liquids and solids Radiochemical actions in liquids and solids and solutions are less well understood than in gases, but are of more importance biologically As regards pure liquids and solids, data are rather meagre Kalan: has determined the ionic yield for a number of orgamec hquids exposed to # and y rays and finds values ranging from 0 1 to 8, that 18 to say, much of the same order as for gas reactions Lind and Ogg: state that the ronte yield for decom position by a-particles 1s approximately the same in liquid and in gaseous HBr Enzymes irradiated in the dry state by X-rays are mactiy ated Fig 1a shows the proportion of ribonuclease remaining active as & ant Panane Vain Lind 8 C (1928) The Chemical Effects of & particles 2 Lind SC & Ogg EF (1931) SOLUTIONS 39 A number of photochemical decompositions have been tudied in both the hquid and gaseous states: In some cases eg decomposition of H1) the yzeld per ultra-violet quantum s approximately the same, but cases are hnown (e g the decom- osition of ammonia) in which the yield 1s much less in the iquid state Solutions As regards reactions in solutions, analogy between photo- chemical and radiochemical reactions ts not very profitable In studying photochemical reactions, a solvent 1s usually chosen in which the absorption of ultra-violet light 1s negligible by com- parison with its absorption in the solute, so that the energy of the radiation 1s pnmarily liberated 1n the solute molecules only In such solutions at 13 often found that the quantum yield for decomposition of thé solute 1s rather less than for the decom- position of the same substance in the gaseous state This 1s plausbly explamed: either by recombination of the atoms or radicals resulting from the dissociation being facilitated by the caging effect of the solvent molecules, or by collisions between solvent molecules and excited solute molecules removing from the latter suffiaent vibrational energy to prevent decomposi- tion With ronzing radiations, however, tt 1s not possible to use a non-absorbing solvent, and to a first approximation the relative amounts of energy dissipated in solute and solvent are propor- tional to the masses of each present Consequently in a dilute solution the number of molecules of solute directly ronized or excited by the radiation 1s very small compared with the number of solvent molecules 1omzed or excited There 1s thus the Ppossi- bihty of an indirect effect on the solute, exther due to trans- ference of energy from excited or 1omzed solvent molecules, or due to chemical change occurring in the solvent and the products affecting the solute Photochemical reactions are known in which a non absorbing component suffers chemical change as a result of the absorption of the light m an absorbing substance, which may itself be unchanged and merely hands on the energy t Cp Dickinson RG (1935 1938) 2Faaa Gh J & Rabmnowitsch E (1934), Atwood, K< & Rollefson 38 CHEMICAL FFFICTS OF IONIZING RADIATIONS a function of the dose of radiation: The ionic yield 18 approxi- mately unity It appears, therefore, from the somewhat limited amount of deta available, that, as im gas reactions, the typical result of uPnecrhceantgaegde < wv] | a 3 o “I o 2 4 L i n i i 1 1 1 - 2 4 6 8 10 (2 4 16 18 a 10r Dose of * rays Fio 1 Linear and exponential dose relations A _ inactivation of mbonuclease (dry Lea Smith Holmes & Markham) B oxidation of ferrous sulphate (J0-* Mo Fricke & Morse) C of oO (12x10-! fo Fricke & Petersen) irradiating substances in the solid and liquid states 1s decomposi- tion at the rate of about I molecule per somzation It 1s notable that this simple result holds as « ell for a prote:n molecule as for a simple inorganic gas molecule 1 Lea DE Smith AM Holmes B & Markham, R (1944) SOLUTIONS 39 A number of photochemical decompositions have been studied in both the hquid and gaseous states: In some cases (eg decomposition of H1) the y:eld per ultra-violet quantum 18 approwmately the same, but cases are known (e g the decom- position of ammonia) m which the yield 1s much less in the liquid state Solutions As regards reactions in solutions, analogy between photo chemical and radiochemical reactions 1s not very profitable In studying photochemical reactions, a solvent 1s usually chosen in which the absorption of ultra violet light 1s negligible by com- parson with its absorption in the solute, so that the energy of the radiation 1s pnmarily hberated in the solute molecules only In such solutions it 1s often found that the quantum yield for decomposition of the solute is rather less than for the decom- position. of the same substance in the gaseous state This 1s plausibly explamed: either by recombination of the atoms or radicals resulting from the dissociation being facihtated by the caging effect of the solvent molecules, or by collisions between solvent molecules and excited solute molecules removing from the latter sufficient vibrational energy to prevent decomposi- tion With ronizing radiations, however, 1t 1s not posstble to use a non-absorbmg solvent, and to a first approximation the relative amounts of energy dissipated in solute and solvent are propor- tional to the masses of each present Consequently in a dilute solution the number of molecules of solute directly ionized or excited by the radiation 1s very small compared with the number of solvent molecules 1onized or excited There 1s thus the possi- bilty of an indirect effect on the solute, either due to trans- ference of energy from excited or ionized solvent molecules, or due to chemical change occurring in the solvent and the products affecting the solute Photochemical reactions are known in which a non absorbing component suffers chemical change as a result of the absorption of the hght in an absorbing substance, which may itself be unchanged and merely hands on the energy 1 Cp Dickinson RG (1935 1938) 2 Franck J & Rabmowitsch, E (1934), Atwood GK (ss) mess) K & Roll otefeony 40 OREMICAL EFFECTS OF IONIZING RADIATIONS it absorbs We shall not bo surprised to find that most radis- chomical reactions in dilute solution are bs t change in the solute occurring as the result of romzation and excitation in the solvent molecules Direct excitation or 1omza thon of the solute, when xt oceurs, will no doubt alao Jead to chermeal change, but since the number of direct. solute somza- tons and excitations in a dilute solution 1s very small compared with the number of ronizations and excitations of solvent mole- cules, direct action will be neghgble unless the probability of an tonzed or excited solvent molecule causing change in a solnte molecule 1s small The evidence 13 that this probability 1s quite high when water 1s the solvent Decomposition of water _ There are still some obscunties concernipg the production of 1 ge by radiations in water itself There 13 agreement that im ice at hquid air temperature the 1ome yield 13 low: It 1s not clear whether appreciable decomposition occurs in the vapour state (cp Table 20) As regards the hquid state, it 18 agreed that the irradiation of ordmary water not spenally fied and de-aerated leads to d Position with the produc fon of H, and G, gases and the formation of some H,O, With moderate doses of radiation not much oxygen 19 liberated as 19 to be expected in view of the H,O, formation The concentration of HO, does not, h , Increase indefinitely,s and with larger doses of radiation the reaction proceeds according to the equat 2H,0 =2H,+0, The ronic yield of the reaction, estimated as the her of molecules of H, produced per ron-pair, 13, for both a raya; and X-rays, about 10 The yreld of H,0, dumng the early stages of the Teaction with X-rays 1s about 1 molecule per ron-pars Ifa small y of a reducible substi 13 7 m solution, oxygen but no hydrogen will be evolved, while if an 1 005, the products of decomposition bemg 2H, +O, Becording to Duane, W & Scheuer O (1913) Di ip st to Gunther P & Holtzapfel, L (1939) 2 The of HyH,O, by tran is rapid, and increases with. mnereasing: HO;concentration (Fricke, H 119359) 3 Lanning, FC & Lind, 8 C (1938) give 0 87, Nurnberger,C E (1934) gives 678 Duane W & Scheuer, O (1913) give 106 4 Fricke, H (18340) gwes 0 8 molecule per ron par at acid pH, 02 ot ‘ikatine pH Clark, GL & Coe WS (1937) give 08 at acid pH DECOMPOSITION OF WATER 41 oxidizablo substance 1s present, hydrogen and no oxygen will be evolved : The behaviour of carefully punfied water freed from dissolved oxygen 1s different from that of water not free from oxygen, and the various experiments which have been reported are difficult to reconcile It has been stated: that no hydrogen and oxygen are produced when gas-free water 1s irradiated by X-rays A recent experiments with X-rays, however, reports a yreld of about 1 hydrogen molecule per 10n-parr Experiments with & rayas and f-rayas, using larger doses, also give an evolution of gas, but the yield appears to be lower than 1 molecule per ton- pair It seems to be established that the production of H,O, m uradiated water requires the presence of dissolved oxygen This was reported by Risse,s and Fricke found that the yield of H,O, camimshed on reducing the oxygen tension from 70 to 4com of mercury, and was undetectable in gas free water A trace of iodide or brome 10ns in gas free water catalyses the produc tion of H, and H,0, to the extent of 0 2 molecule of each per ion pair, which 18 less than the yield in aerated water, while traces of oxidizable or reducible substances lead to the evolution of gas, as already mentioned 1 Lanning, FC & Lind, SC (1938) 2 Risse O (1929), Friche H & Brownscombe, E R (19335) 3 Gunther, P & Holtzapfel, L (19396) 4 Nurnberger, CE (1934, 19360) The yield in the experiment usmg as free water can be calculated from the data gtven to be 006 assummg that the radon 1s distributed between liquid and gaseous phases in the pected ratio The it of radon 13 hable to disturbance by the evolution of gas which occurs leading to there bemg less radon m the solution than calculated It seems umprobable however, that this cor rection could rarse the yield to the order of 1 molecule per ion par Nurnberger, however, reports his experiments as consistent with a yield of umty 5 Kernbaum, M (1909) This early experiment appears to have been made with de aerated water If all the & rays reached the water the yield can be calculated from the data given to be 0 015 molecule per 10n pair A considerable proportion of the f ray energy would be absorbed an the wall of the vessel and some gas would remam dissolved in the solution It seems improbable that these could raise the yield to the order of 1 molecule per jon pair 6 pee © (1929) 7 FrekeFmcke H& H & wa Brownscombe ery fe ER { (1933 6) Fricke, H (1934), 40 ONEMICAL EFFECTS OF IONIZING RADIATIONS it absorbs We shall not be aurpnsed to find that most radio- chemical reactions in dilute solution are indirect, ck Ll change in the solute occurring aa the result of 1omzation and excitation in the solvent molecules Durect excitation or 10niza tion of the solute, when 1¢ occurs, will no doubt also lead to chemical change, but since the number of direct solute 10mza- tions and excitations in 2 dilute solution 1s very small compared with the number of :onizations and excitations of solvent mole- cules, direct action will be negligible unless the probability of an d or ted solvent molecul g change in a solute molecule 13 small The evidence 13 that this probability 1s quite high when water 18 the solyent Decomposition of water There are still some obscunties concerning the production of h hange by ig radiations in water itself There 13 agreement that in 1ce at hquid air temperature the ronic yield 13 low x It 1s not clear whether appreciable decomposition occurs in the vapour state (cp Table 20) As regards the hquid state, it 18 agreed that the irradiation of ordinary water not specially purtfied and de aerated leads to decomposition with the produc- tion of H, and O, gases and the formation of some H,0, With moderate doses of radiation not much oxygen 18 liberated as 18 to be expected in view of the H,O, formation The concentration of H,0, does not, h , Increase indefinitely,2 and with larger doses of radiation the reaction proceeds according to the equation 2H,0=2H,+0, The tonic yreld of the reaction, estimated as the number of molecules of H, produced per 10n pair, 1s, for both a rays; and X-rays, about 10 The yield of H,O, dumng the early stages of the reaction with X-rays 18 about 1 molecule per 1on pairs Ifa small qi y of a reducible subst: 1s present im solution, oxygen but no hydrogen will be evolved, while rf an 1 005 the products of decomposition bemg 2H,+0,, according to Duane W & Scheuer O (1913) Decomposition undetectable according to Gunther P & Holtzapfel L (19395) 2 The decomposition of H,O, by rachation 1s rapid and increases with increasing HO, concentration {Fricke H (19352)} 3 Lanning FC & Lind SC (1938) give 0 87 Nurnberger, CE (1934) gives 078 Duane W & Scheuer O (1913) give 106 4 Fricke H (1934a) gives 0 8 molecule per on pair at acid pH 04 at alkaline pH Clark GL & Coe WS (1937) give 06 at acid pH INDIRECT ACTION IN AQUEOUS SOLUTION 43 radiation, but their reaction follows excitation or romization of the solvent molecules: This conclusion 1s based principally ond theresults ofexpenments in which a given substance 1s wradiate sogublhutfpoHierMineartfo,rmdoles=8aLi|)}f] J e@ 3 1 1. L j l 5 Ww 5 1r 20 25 30 Dose of \ rays @ 10-4 M forme and 0 102M forrnre Lee re a a sent Fia 2 The 7 mdependence of absolute y1eld upo! yn concentr; m solutions of different concentration In such expersments it 15 found that the weight of solute reacting as a result of a given number of roentgens given to the solution 1s, over a wide range oO f concentrations, mdependent of the concentration of the solution Thus Fig 22 shows that 25 meromoles of hydrogen gas 1 Solute molecules which have been excited or 1omzed directly by the radiation fraction ofprobab} the tedly react eee also mebut these usually usu constitute a minute & 2¥Fneke H Hart EJ & Smith HP {1938) 42 CHEMICAL EFFEOTS OF IONIZING RADIATIONS The reported production of H,O, m gas free water irradiated by a rays: or # rays: 18 not necessanly inconsistent with the X-ray results, since in these experments higher doses were used and sufficient o.ygen was probably Iberated to serve in the production of H,0, We draw the following conclusions which, however, are to be considered surmises hable to correction when the expermmental contradictions aro resolved Pure gas free water 1s decom- posable by roming radtations, though the tonic yield 1s low and may be due to residual :mpurities In the absence of dissolved substances no appreciable amount of H,O, 1s produced By the use of considerable doses which are usual in experiments using a- and # raya, but not m experiments using X rays, sufficient oxygen accumulates in solution to lead to the production of H,0, If oxygen 1s present m the water at the start of the experiment, then production of H,O, with an rome yield of the order umty, and evolution of hydrogen and eventually of oxygen also, begin immediately Indirect action in aqueous solution A number of authors hive studied the chemical changes pro- duced by X rays and radioactive radiations in dilute aqueous solutions Among the principal researches may be mentioned those of Frickes using inorganic and armple organic compounds, and of Dales using enzymes Inorginic reactions which have been studied are mamly oxidations and reductions The change induced in a simple organie compound 1s usually oxidation to CO,, and 1s accompanied by an evolution of hydrogen The actual chemical change occurrmg when enzymes are uradiated has not been studied, the effect observed 1s the loss of enzyme activity All these actions of radiation on dilute aqueous solutions are inudtrect actions, that 18 to say, most of the molecules of solute which react have not been excited or ionized directly by the 1 Nurnberger C BE (19368) 3 era plone BW (1927), Fricke H & Morse & (1927,0 1929) Fricke H & Brownscombe E R (19334 5) Eriche H (19344 EJ (1934 1935¢ be d 1936) 193%a & 1938) Ericxo H & Hart (1938) 1942 1943@ 6) Dale WM Meredith, WI & Hart LJ & Smith HP Foes Je WALK (iodo (1943) Tweedie MC IONIC YIELDS IN AQUEOUS SOLUTION 45 original solute for the activated water, we should expect the amount of the first solute reacting per unt dose to be dimimshed This expectation has been confirmed which gives further sup- port to the view that the chemical changes in question involve an intermediary « Providing that there 1s no reverse reaction, and that the pro- ducts of the reaction do not compete for the actrv ated water, the quantity of the solute rema:ming unchanged diminishes linearly with the dose, as illustrated in Fig 1B: If, however, there is a protective agent present which secures most of the activated water, or if the products of the reaction themselves compete for activated water, then the gradient of the curve becomes less steep as the percentage of unchanged solute dimimuishes, owing to the solute securing a continually smaller proportion of the total amount ofactivated water avatlable s A curve of this ty pe 3s shown in Fig lcs Tonic yields in aqueous solution In Table 21 are collected values obtamed for the iome yield, 1e the number of molecules of solute reacting per 10n-panr in the solution, for a number of reactions investigated in dilute aqueous solution Many of the reactions are oxidation or reduction reve tions, and in these cases the number of equivalents reacting per ion pair has been listed as well as the number of molecules Some of the reactions could be caused by H,O, But the ionic yields are not consistent with this beng the whole explanation of oxidation and reduction in irradiated solutions, and some reac- tons (eg reduction of KIO,) take place which do not ocour with win H Hart, EJ & Smith HP (1938), Dale, WM (1942, a) 2 Fricke, H & Morse,S (1929) The reaction studied was the oxidation of FeSO, (10-3 Af in 0 8. N H,SO,) The departure of the points from the curve when the oxidation 1s 90% complete ts probably due to the back reaction also occurring under the irradiation 3 Cp Dale, WM, Meredith, WJ & Tweedie MOCK (1943) 4 Fnche H & Petersen, BW (1927) srrachating heemoglobin The curve 1s exp 1 Appr ly exp 1 curves have also been obtained by Dale, WM (1840, 1942), irradiating enzymes Probably this 1s the usual result when a large organic mole cule such as a protein 1s uradiated, since the products of the reaction will doubtless be usually capable of further reaction, and so will compete tor the activated wat er 44 CHEMICAL EFFECTS OF IONIZING RADIATIONS are berated per litre of a solution of formie acid given 10,000 r of X rays, irrespective of whether the solution contains 10~ or 10-1 gram molecules of formic acid per litre This emission of hydrogen corresponds tn either solution to the decomposition of 25 micromolcs of formic acid per litre In the more dilute solu ton this represents a large percentage change (25%), m the less dilute solution it represents a minute percentage change (0 026%) Similarly, 1t has been shown: that to produce comparable percentage nactivations in carboxypeptidase solutions of dif- ferent concentrations, much smaller doses of X-rays suffice for the more dilute solutions than for the less dilute solutions The dose required to inactisate a given weight of the enzyme 1s, however, approximately the same in the different solutions In experments of this sort the total energy dissipated by the jonizing radiation per gram of solution 1s practically the same in solutions of different concentration On the other hand, the energy dissipated by the radiation directly in the solute per gram of solution 1s proportional to the concentration, and with dilute solutions 1s only a minute fraction of the total energy dissipated per gram of solution Thus we see that the weight of solute reacting 18 proportional not to the energy dissipated directly in the solute alone, but to the energy dissipated in the solution altogether This result strongly suggests that energy dissipated In the solvent 1s eventually handed on to the solute, a conclusion which 1s strengthened by the fact that the 1ome yield calculated as molecules of solute reacting per-1oruzation in the solution 18 of the order unity, while an rome yield calculated as molecules of solute-reacting per molecule of-solute-directly 1omzed would he very much greater than unity (and would depend upon the concentration) Evidently the ionization or excitation of a water molecule by an ionizing particle causes the production of an mtermediary body of finite hfe, capable of causing reaction In many solutes The nature of this intermediary was for some years obscure, and it 1s usually referred to in the literature as actetated water The probable nature of activated water 18 discussed later By the addition to a solution of a substance capable of re- acting with activated water and so of competing with the 1 Dale WM (1940) MECHANISM OF INDIRECT ACTION 47 globin to methaemoglobin 1s stated to be independent of X-ray wave length from 0 25 to 076A The ionic yields obtained with a-particles and X rays mn the reduction of KMnO, agree The results for the irradiation of FeSO, suggest that the ionic yreld 1s lower with & particles than with X-rays, but this is a complicated reaction in which the yield depends upon the pH, the FeSO, concentration, the dose, and the degree of oxygenation of the solution, and no reliance can be placed upon a comparison made by different authors under different conditions The most stmking difference 1s that reported for the decomposition of & rays tyrosine by « particles and X rays also in the direction of being less efficient Inspection of the table shows that almost all the iomc yields he between 01 and 20 It appears probable that acti ated water 1s formed at the rate of about 1 molecule per 1on pair, and that reaction yields smaller than this are due to a proportion of collisions between reactant molecules and activated water lead- Ing to deactivation without reaction, or to other causes which are discussed later Chemical mechanism of the indirect action in aqueous solution Weiss: has suggested that activated water consists of free H and OH radicals To convert a water molecule into H and OH radicals requires 5eV (1¢ 115 hilocalones per gram-molecule), and two mechanisms may be suggested to account for this con Version by an ionizing radiation, following ionization or excita tion of water molecules respectively Tf an electron 1s ejected from an H,O molecule, the mole- cule may spkt into a hydrogen 1on and a hydroxyl radical H,0+ -> H+ + OH, thus producing a hydroxyl radical at the site of the ionization The electron which 1s ejected will travel a distance determined by the energy with which 1t was ejected, and eventually become attached either to a hydrogen ton or to a water molecule, in either event givimg rise to a hydrogen radical H+ +e-+H, or HO0+e+H+OH The H radical will thus be produced at an appreciable distance from the OH radical Its possible that some of the H,0 molecules which are excited ty the radiation decompose directly into H and OH radicals e radicals in this cace will be produce d close together 1 Wews J (1944) That H atoms and OH radi cals are involve suggested tentatively by Risse, d O (1929) and Fricke H (1938) was 46 CHEMICAL EFFECTS OF IONIZING RADIATIONS H,0, As poimted out by Allsopp, the assumption that H,0, 1s involved would not avoid the necessity for invoking the hypo thesis of activated water, since the formation of H,0, itself requires this hypothesis Taste 22 Jone yields in dilute aqueous aclution Vield per ion par Moles = Equi Reaction Radiation cules —_-vafents Reference HBr decompomtion ory 12 HI decomposition - :a 19 = Tanne, Fe & Tand, 8 C (1938) #Ma0, reduetion ae 05-09 2545 ” $ nO, CeO} reduction reduction rays se O74 3 aia” "6 Coe, 8 957)7) X10, reduction OL o¢e _ Nitrate reduction ' OG8t = 0-26T Clark, GL & Pickett LAY (1930) Nutatte oxdation ” 02 Ferrocyanide oxidation O4 — Prcke, H & Hart, E.J . O4 O4 Fricke, H & Hart, EJ {iea5e) (19958) Setemte oxidation ” O2 O4 » Arenute ondation : 02 O4 : " FeSO, oxidation6 , oxidations ; 1 32 10 3 1D Fricke H’ Gray, LE Lene Ff (unpublished) & Hart,EJ (19350) FeSO, oxidation 3 arays O8-17 O8-17 Numberger, CE (1934) Fe,(30,), reduction§ Mrays -05 8 ~10~ Gray, Lit & Weigert, F (unpublished) K,Cr,0, reduction \ rays o2 12 Fneke, H & Brownscombe ER {19330} Oxyhaemoglobin to , oe — =‘ Freke’ H & Petersen,BW (1927) methaemogiobin Various organic acids tu A 0412 — Freke H Hart, EJ & Smith HP H, and CO, H, yield (1838) H,O0+00 toH,,C0,,HCHO , 18 = : ' » d@ armno-acid oxdase inactivation Prosthetic group » 01 —~ Calculated from Dale, W.Mt (1942) ¢ protein > Ole —~ * » Tyrosine decomposition , O1 — — Stenstrom W & Lohmann, A (1928) Tyrosine decomposition rays 00035 — Nurnberger, CE (1937) Glutathione Xrays O417 — Kinsey, VE (1995) Ascorbic acid oT — Anderson RS & Harmison, B (1943) Ribonuclease rs 003 — Lea DE & Holmes B (unpublished) x Recaleulated ‘The authors report a yield of 0 2-0-3 molecule per ion pair but thrs appears to be baged on a mraconeeption ofthe felation between encrey absorption ‘and dose im roentgens zin oxygen free sclutlons The eld 1s about doubled in oxygenated solution 3 In 08M sulpbunc acid 4 In Dale’s preparation apprommately 20% of the protein was enzyme protem The calculation of ome yield bas'been made on the assumption that’ the 809% of non-enzyme protein has equal aflimty for activated water as the enzyme protein le (a, @ private commumucation) guggests that the afinity mught be lower In that event the rome yield for the specifie protein could be as low as 002 5 Thisas the figure given by Numberger Radon was used as the source ofa particles and about 5%, of the total imuzation would thus be due tof rays Accepting Stenstrom and Lohmann a for X rays as also applymg tof rays st follows that the whole of Nuraberger’s yreld can be accounted for by the B rays, with no decomposition at all produced by the rays reactions are complicated, the yrelds ferrous or fermic ammonium sulphate in 10~*.) H,5O, The given are the imittal yrelds There are very few data bearmg on the relative efficiennes of different radiations in promotimg reactions 1n solution, and work on this subject 1s urgently needed The conversion of oxyhaemo- 1 Allsopp, CB (1944) MECHANISM OF INDIRECT ACTION AT globin to methaemoglobin 1s stated to be independent of X-ray wave length from 0 25 to 076A The tonic yields obtained with a-particles and X rays in the reduction of KMnO, agree The results for the irradiation of FeSO, suggest that the ome yield 1s lower with particles than with X-rays, but this is a complicated reaction in which the yield depends upon the pH, the FeSO, concentration, the dose, and the degree of oxygenation of the solution, and no reliance can be placed upon a comparison made by different authors under different conditions The most stnlang difference 1s that reported for the decomposition of tyrosine by a particles and X rays also in the direction ofa rays being less efficient Inspection of the table shows that almost all the 1ontc j1elds he between 01 and 20 It appears probable that activated water 1s formed at the rate of about 1 molecule per ion pair, and that reaction yields smaller than this are due to a proportion of colhsions between reactant molecules and activated water lead- Ing to deactivation without reaction, or to other causes which are discussed later Chemica! mechanism of the indirect action in aqueous solution Weiss: has suggested that activated water consists of free H and OH radicals To convert a water molecule into H and OH radicals requires 5eV (1e 115 hilocalories per gram-molecule), and two mechanisms may be suggested to account. for this con version by an ionizing radiation, following ionization or excita tion of water molecules respectively If an electron 1s ejected from an H,O molecule, the mole cule may spht imto a hydrogen ion and a hydroxyl radical H,0* + H*+0H, thus producing a hydroxyl radical at the site of the ionization The electron which 13 ejected will travel a distance determined by the energy with which it was ejected, and eventually become attached either to a hydrogen ion or to a water molecule, in either event giving rise to a hydrogen tadical H+ +e +H, or H,O+e->H+OH The H radical will thus be produced at an appreciable distance from the OH radical Itas possible that some of the H,O molecules which are excited by the radiation decompose directly into H and OH radicals The radicals in this case will be produced close together 1 Weiss J (1944) That H atoms and OH radicals aro mvolved Suggested tentatively by Risse, O (1929) and Fricke H (1938) was 48 CHEMICAL FIEECTS OF IONIZING RADIATIONS . The peculinrities noted carher concerning the decomposition of water by ionizing radiations can largely be understood in terms of the production of H and OH radicals The low yield in pure gas free water 1s duo to the bach reaction H+OH—>H,0 For decomposition, it 18 necessary for collisions to occur between two hydrogen radicals or two hydroxyl radicals, H +HH, or OH+OH>H,0+0 followed by O+0+0, Proximity will favour the H + OH combination in the case of radicals produced by the decomposition of an excited molecule, though not so much in the case of radicals produced following an ionuzation Tf cussolved oxygen 1s present the reaction 1s H +0,->HO,, followed by 2HO,>H,0,+0, The removal of the hydrogen radicals by the oxygen reduces the rate of recombination of H and OH The OH radicals which accumulate combine to give oxygen as before The oxidation of morgame ions by OH radicals, or their reduc- tion by H radicals, explains most of the inorganic reactions which have been studied Thus Fet++OH>Fet+++OH , Cet+*+4.H—>Cett++H* Since oxidations remove OH radi- cals, the H radicals accumulate 1n the solution and combine with the evolution of gaseous hydrogen Simularly, reductions are accompamed by the evolution of gaseous oxygen, resulting from the combination of OH radicals, which accumulate in the solu tion when the H radicals are removed The oxidation of smple organic compounds, accompanied by an emission of hydrogen, 1s explained in a similar fashion The OH radical 1s Iighly reactive as an electron acceptor (OH +e>OHTM +3 7 eV ), and at 18 this which probably accounts for the fact that almost all organic fs aredi by wradiation in aqueous solutions Conversely almost any organic compound, 1f present in sufficient concentration, 1s able to act as a protective agent, by reducing the concentration of OH radicals m the solution by the above reaction The H radical reacts with solutes which are oxidizing agents, but 1s probably less reactive than the OH radical with most orgame mole- cules Spatial distribution and recombination of the active radicals In typical chemical reactions induced by 1omzing radiations in aqueous solution, the 10n1¢ yield 1s independent of the concen- SPATIAL DISTRIBUTION OF RADICALS 49 tration over a wide range of concentrations It has been shown, however, with some solutes, and 1s probably true in general, that at sufficiently low concentration the ionic yield 1s no longer constant but diminishes with diminution of concentration : The explanation 1s presumably that m suffinently concentrated solutions the H and OH radicals react with the solute before they have time to collide with each other, while in sufficiently dilute solutions the H and OH radicals collide with each other and combine before they have time to react with the solute mole- cules 2 A quantitative treatment requires a knowledge of the spatial distnbution of the radicals, which distnbution 1s far from uniform When a solution 1s irradiated, 1onizing particles pass through it (fast electrons in an X-ray experiment, a-rays in an a-ray expenment), and the H and OH radicals are produced along the paths of these ionizing particles The OH radicals (re- sulting from the posttive 10ns) are mmitially localized along the path of the 1onizing particle, the H radicals (resulting from the attachment to an H,O molecule or an H+ 10n of the electron ejected at ionization) are produced at a distance away de- pending on the distance travelled by the ejected electron before its attached The initial distribution of the OH and H radicals is thus the same as the inittal distribution of positive and nega- tiveions 3 This latter distribution can be studied in gases by the Wilson chamber method 4 It can also be studied in gases and insulating liquids by a less direct method depending on the com- parson of observed 1omzation currents with calculations of the 1 Stenstrom, W & Lohmann A (1928) usmg tyrosine, Kinsey, V E (1935) using glutathione Fricke, H, Hart, EJ & Smith, HP (1938) using formic acid oxalic acid, formaldehyde and methyl alcohol Lanmng, FC & Lind, SC (1938) using KMnOQ, 2 We are assuming that there is nothing in the diluting fluid with which the active radicals can react Precautions should be taken in experiments of this sort to use gas free water free from traces of organic impurity capable of acting as protective agents It 1s not certain that were adeq in all the exp we discuss 3 We are neglecting in this treatment any H and OH radicala pro. duced by rather than 4 perer, QO (1927) pl at 8 presaure of one tenth of an atmosphere and found thet the vadus hed a ray tracks m hydrogen of the column of iB ions was eq! t to 0002cm of ar at I atmospheric pressure mt t 50 OHEMIQAL EFFECTS OF IONIZING RADIATIONS proportion of ions which escape 1ome recombination, these cal- culations mvolving the imtial distribution of the ions: It 1s concluded that the number (n) of 1ons per cm of each sgn at a distance r from the aus of the romzing particle can be represented initially? by the formula n=Xe ene}, (If-1) N, 1s the number of tons of each sign produced by the ionizing particle per em path, and 6 1s a measure of the radius of the column of ions 6 has been found to have the values 179x 10-7 cm in ar (density 00012) and 2 34x 10-* cm in hexane (density 0 677) We shall take the value b=1 5x 10-* for water (donsity 1) We shall suppose formula (1) to represent also the inatial distribution of OH and H radicals The radicals rapidly diffuse away from the path of the 1omzing particle, and after time ¢ 1t can be shown; that the number of radicals of each sort per om 9 at a distance r from the path of the iomzing particle 8 _ "= NX ape napass | erable) . (1-2) where D 1s the diffusion constant, which for the purpose of our calculation we shall assume to be 2x 10-5 em ? sec ~! at room temperature for both H and OH radicals, though it may be Ingher for the former, The radius of the column 1s now A(4Dt+6%) instead of 6 N 1s the total number of ions per em length of path of the ron:zing particle, and progressively di- munishes from its intial value NV, as 4 result of recombination of the radicals or their reaction with any solute present x Jaffé, G (1913) Cp also Kara Michallova E & Lea DE (1940) 2 Strictly, not amtially, but after the very short tume interval needed for the positive rons which are to begin with closer to the axis of the ionizing particle than the negative tons to diffuse to a comparable distances 3 Here and elsewhere in this section the treatment of Jaffé, G (1913) 1s followed Jaffé worked out hus calculations for the diffusion and recombmation of 10ns, but the mathematics apply equally well to the diffe and it ofradicals pi dmg the Ip: values of the constants are used bvd: 4 im Water 4 The o 3, ry is 2x 10-* cm * sec ~1 at 15° (Orr, WI C & Butler, JA V 1935) RECOMBINATION OF RADICALS 51 After a short time (a fraction of a second) diffusion has broadened the columns sufiiently for adjacent columns to over- lap While the radicals are diffusing, their number 1s dimmishing owing to combination with each other and reaction with any solute which 1s present If, by the time adjacent columns over- lap, most of the radicals in a column have disappeared, then each column may be considered an wolated entity, and the concentra- tion of radicals we have to consider in calculating reaction rates 1s the concentration given by equations (1) or (2) Butif, by the time the columns overlap, only a small proportion of the radicals have disappeared, then the smtaal localization of the radicals in columns can be neglected and we can consider the reaction to occur in the hquid as a whole, and take as the concentration of radicals not the values m the column given by equations (1) or (2), but the much lower average values obtained by dividing the total number of radicals by the total volume of the solution It 18 necessary therefore to determine whether reaction occurs mainly before the columns mingle, or mainly after the columns mingle Jf the dose-rate 1s I roentgens per second, approwmately 2x 1012 Ty 1on-pairs (and therefore pairs of radicals) wall be pro- duced per em in ¢ seconds There beng N, 10n pairs per cm path, 2 x 10! Jt/N, 1omzing particles will cross each square em im ¢ seconds Now in t seconds the radius of the column is Vl4Dt+ 2), and its area 1s therefore 1(4Di+62) Thus for the columns to overlap in ¢ seconds we must have 1 (4Dt +6) x 2x 10% It/Ny=1 Tn practice 4Di> b?, and we deduce that t=lO-sx J(Nyf8rDI) and 4f/(4Di+-b%) = 10-8 x (2N,Djnl)t These expressions give respectively the tume required for the columns to overlap, and the radius of a column when adjacent columns overlap ‘To work out typical numencal values we insert im these for- mulae J=10r per second, D=2x 10-5 om ? sec “tl, Ny=3 x 107 lonzations per cm for a-rays or Ny=6x 108 iomzations per cm for Xrays We deduce that m X ray expen- ments adjacent columns overlap after 001 second when the column radius 1g 0 0012 cm » and that in a-ray experiments the 52 CHEMIOAL EFFECTS OF IONIZING RADIATIONS columns overlap after 0.08 second when the column radius 18 0 0025 em In the absence of a solute, the radicals disappear by the reac tion H+OH--H,0 The number of pairs of radicals disappeanng per cm? per second will, according to the mass action Jaw, be an®, where 71s the number of radicals of each kind per em * and a 13 a constant the value of which can be calculated on the Janetic theory assuming (provisionally) that every collision be tween H and OH 1s effective The value used: 18 a= 4x 1071? The number N of pairs of radicals which remain uncombined after a time ¢, during which the column diffuses to a radius {(4Dt-+ 6%), 1s related to the onginal number N, by the formula: NyN 214249 tog SEE 2 (13) Using the values of «, 6, Ny, and D already quoted, we find that by the time the columns overlap the proportion of radicals re- mainmg uncombined 3s 03% in an @-ray column and 13% in an X-ray column If a solute is present the radicals will disappear still more quickly We conclude therefore that the reaction takes place independently m the paths of the individual 1omzing particles, and that the radicals produced by different 1omizing particles do not mx appreciably We can calculate from formula (3) the time required for half of the radicals to combine, the times obtamed being 1 2 x 10-° second in an @ ray expermment and 2x 10-7 second in an X-ray experiment These times will be reduced if a solute 1s present of ind Kinetics in aq Fig 3 shows the calculated numbers of collisions per second undergone by a (specified) H or OH radical with solute mole- cules, the solute being of molecular weight Mf and present in 1 6x 10a as the im gram mol units of the reaction H+OH>H,O a 18 catculated from formulae given by Moelwyn Hughes, E A (1933), Kinetzes of Reactions n Solution 2 Jaffé, G (1913) 3 The calculation can be made only approximately and aesumes that the d of a molecule of x 10-* Aft em molecular weight Af 1s 1 33 Cp Moelwyn Hughes EA (1933), Kinetics of Reactions m Solution Hinshelwood CN (1940), Kunetecs of Chemical Change KINETICS OF INDIRECT ACTIONS 53 concentration either of 1 gram molecule per litre (curve A and left hand ordinate scale) or 1g per litre (curve B and nght-hand ordinate scale) For equal molar concentrations the collision Ts4 10 10. 10of “TTT Tt t J ty ry] e 4 6 = i B eg2 ‘Fh i s a 2 i 3 —& ah ~] % g = 3S s Cans) g 0 10° § 4 éo er)3 od = 5 2 6 4 e 8 S< ge: 7 2 et 6 24h 3: 2 8 3 8 2g 28 32 5 OP 4a x 5 5 =8 19H 8 10s a§ 2 & sh 4 2 ok 3 3 B + 2 88 4 4 s A gg 1 7 i = & = q A] N N10" Jatt Lai de Lnteldvenlattelaeelentet beeLietlth 17 10 B 10? 2 468 107 72 468 104 2 105 468 ,2 468 toe2 4$4 o J{=molecular weight of solute Fio 3 Number of collisions Sth solute molecules, the solute per second made by a (specrfied) being of molecular‘weight At ond pecent act: aa ration of A, 1 gram molecule per tre B 1g per htre rato mh different solutes mcreases with mereasing molecular rene t, but for equal concentrations in grams per litre the col- ison rate decreases with increasing molecular weight We shall eall Z the collision fre molecule per htre quency for a concentration of 1 gram- 54 CHEMICAL EFFEOTS OF IONIZING RADIATIONS A collision of an active radical and a molecule of a solute capable of reacting can result in ono of the following alternatives (a) Elimination of tho active radical and chemical change m the solute moleculo (6) Elmination of the active radical without chemical change in the solute molecule (c) Chango im nerther radical nor solute That not all collisions between a solute molecule and an active tadical which eliminate the latter lead to reaction in the solute 1s inferred from the fact that the :onic yield in many reactions 1s leas than unity under circumstances when radical recombina tion is not suspected Where large molecules such as enzymes aro concerned, 1t 18 possible that change in the solute molecule may occur which 1s not made evident by the method of estimation used In most of the reactions hsted in Table 21 1 appears that of those collisions in which the active radical 13 eliminated, a proportion between 01 and 1 0 result sn change in the solute molecule This proportion we designate by P That the probability of elrmination of an active radical at colhsion with a solute molecule 1s zero or very small in the case of many solutes, particularly snorganic substances, 1s evident from the fact that these substances could be present in Dale’s expenments in ngh concentration without exerting appreciable protective action on the enzyme We have no guarantee that this probabilty, which we shall call p, attams unity even with the most reactive solutes, but unity 1s at any rate its highest possible value When the concentration of solute 1s ¢ pram-molecules per htre, the probability that in time dé a (particular) active radical shall suffer a collision in which it 18 ehminated 1s eZpdt If there are » active radicals per em 5, neZpdt active radicals will be elum:- nated m time dt, and ncZpPdt solute molecules wall suffer chemical change in time dis If two solutes are competing, the concentrations being ¢, and ¢,, the number of molecules of solute 1 reacting will be e,Zyp,Py/(Zip1 + ¢e%P2), and of solute 2, NegLatyPo| (CyZyP, + %Z2P2) (II-4) x In these ae we are 1 the pr that an active radical shall be elurunated by combination with another active radical This 1s 3 ble p ding ¢ 13 suffi ly Ingh We discuss later the 8 case of low solute concentration COMPETITION BETWEEN SOLUTES 55 Competition between two solutes, pratective action The concentration ¢, of solute 2, at which the reaction of solute 113 reduced to 50% of 1ts value mn the absence of solute 2, 18, from equation (4), given 28 eae, 1, whence pilp=aFilee%, (1-8) Zr Freche, Hart and Smith: have irradtated mixtures of formic acid with caproic acid, methy] alcohol, formaldehyde, oxahe acid or acetone, plotting the yields of H, and CO, as a function of the relative concentrations of the formic acid and the other solute From their curves we can read off the ratio ¢,/¢, of the concentrations of the formic acid and the other solute at which the activated water appears to be equally shared between the two solutes The values of Z for the different solutes being taken from Fig 3, we are able to deduce that the values of p for the aix solutes caprote aad formic acid methyl alcohol formalde- hyde oxalic acid acetone are in the ratios 16 10 044 022 0067 0024 respectively The experiment 1s an illustra tion of one method by whrch relative (but not absolute) values of p may be determined A vanant of this method 1s to compare the efficencies of different substances in protecting the same solute Dale: lists the concentrations (c,) of a number of solutes which suffice to reduce to about 60% the number of molecules of alloxazin-adenme-dinucleotide reacting with a given dose, the dinucleotide itself bemg present in a concentration of ¢=6 1x 10-7 gram-molecule per litre With the aid of equation (5) and Fig 3 we can deduce the ratios p,/p, from these experi- mental values, p, referrmg to the dinucleotide The values of P./p, 80 obtained are given m Table 22, column 2 Dale also gives similar though less extensive date for the pro- tection of the specific protein component of d-amino-acid- omdase, and the values of p,/p,, where p, now refers to this Protein, are given m column 33 Companson of columns 2 and 3 1 Fncke, H, Hart, EJ & Smith, HP (1938) 2 Dale, W.M (1942) 3 Dale’s preparation contamed 204g of activo protein to 105xe total protein we have assumed that from the point of view of competing for activated water all the protein and not merely the enzyme protein 1s to 54 CHEMICAL EFFECTS OF IONIZING RADIATIONS A collision of an active radical and a molecule of a solute capable of reacting can result in one of the following alternatives (a) Ehmuination of the active radical and chemical change in the solute molecule (b) Eumination of the active radical without chemical change in the solute molecule (c) Change in neither radical nor solute That not all collisions between a solute molecule and an active radical which eliminate the latter Jead to reaction in the solute 1s inferred from the fact that the 1on1e yield in many reactions 1s leas than unity under circumstances when radical recombina tion 1s not suspected Where large molecules such as enzymes are concerned, it 1s possible that change in the solute molecule may occur which 1s not made evident by the method of estimation used In most of the reactions hsted in Table 21 :t appears that of those collisions in which the active radical 1s eliminated, a proportion between 02 and 10 result in change in the solute molecule This proportion we designate by P That the probabihty of elmiation of an active radical at colhsion with a solute molecule 1s zero or very small in the case of many solutes, particularly :morgante substances, 1s evident from the fact that these subst could be p t mn Dale’s experiments m high concentration without exerting appreciable protective action on the enzyme We have no guarantee that this probability, which we shall call p, attains umity even with the most reactive solutes, but unity 1s at any rate its highest posable value When the concentration of solute 1s ¢ gram molecules per hire, the probability that in time df a (particular) active radical shall suffer a collision in which it 1s elimmated 1s cZpdt If there are » active radicals per cm 3, neZpdt active radicals will be ehmi nated in time dé, and neZpPdt solute molecules will suffer chemical change m time df: If two solutes are competing, the concentrations being ¢, and ¢,, the number of molecules of solute 1 reacting will be nc,Z,p,P;/(,Zp, + ¢Z2P»), and of solute 2 ne gLop,Po| (CyZaP, + CZ2P2) (11-4) x In these lap we are the bility that an active radical shall be elumnated by combination with another active radical Thi: Bois ly ngh We discuss later the case of Jow solute concentration REDUCED YIELD IN DILUTE SOLUTIONS 57 should almost completely protect the dinucleotide, and not be noticeably protected by it This was in fact found by Dale If however, the concentration of the dinucleotide were to be in- creased ten times or more without changing that of the protein, some protection of the protein by the dinucleotide should be- come noticeable according to the present calculations As regards the absolute values of p, these experiments give no information Since the dinucleotide has the largest value of » for all the substances vestigated by Dale and since this cannot exceed unity, the figures in the second column may be taken to be maximum values of p for the corresponding solutes We shall give evidence later that the actual values are not very different from these maximum values Reduced yield in dilute solutions, and with densely ionizing radiations Providing that the solute concentration is sufficiently Ingh for nearly all the active radicals to be eliminated by collisions with solute molecules rather than by collisions with each other, the tonic yield will be independent of the solute concentration If, however, the solute concentration 1s so low that an appreciable Proportion of the total number of active radicals combine with One another rather than react with solute molecules, then the lonie yield will fall The determining factor 1s evidently the ratio of the number of solute molecules per cm ® to the number of active radicals per em 3 Since the latter number ts much higher man a-ray track than im the track of a fast election, 1t 1s evident that the dimmshed tonie¢ y1eld due to combination of radicals will become appreciable in a ray experiments at higher con- centrations than in X-ray experiments Thus effect probably accounts for the 1omie yields in certain of the reactions hsted mn Table 21 being less for « ray» than for X rays It 1s possible to make calculations of the rate at which active radicals disappear by the two processes of combmmation of radicals and reaction with the solute,: during the diffusion of the radicals aw ay from the axis of the column In Fig 4 the results of these calculations are given im the form of graphs showmg the Proportion of radicals which are el:minated by collision with the solute as a function of cZp cZp is a measure of the concentra 1 By an extension of the calculation given on pp 49-52 56 OMEMICAL EFFECTS OF IONIZING RADIATIONS of Table 22 enables us to determine the ratio of the values of p for the dinucleotide and the protem The ratio inferred from the glucose data appears to be anomalous The others agree faurly Taste 22 Relative deactsvation efficiencies of different solutes Relative affinities for activated water of solu Pap, where p, 18 Inferred ratio tiona containing value for of p values Ig mol Ig Specific dinucleotide per litre per litre Bolute 2 Dinucleotide _ protein ‘proton Zp/p, Zpip,M q) @) (3) (a) (5} @ ‘lycine 18x 10-4 85x10-4 48 46x10" 6 2x 105 ra oxalate 15x 10-¢ _ _ 46x 107 34x 10° Ta mtrate 18x 10-4 _ = 46x10? = 5 Bx 108 eucylglyeine 13x 10-3 2110-3 15 46x10" 23x10" Janine 17x 10-* 26x 10-3 15 46x10" 52x10" t ferncyanide 10x 19-8 _ - 46x109 = 14x 10+ < ferrocyamde 10x 10-* - - 46x 105 13x10" Ta hippurate 13x 10-* ~ _ 46x10" 23x10" ilucosa 43x10-! 21x10-+ 0005 15x10" 81x10" ucroae 32x 10-8 - - 15x 10% 43x10 £ thiocyanate 52x 10-2 - - 15x10% = 15x10 ta formate 59x 10-? _ _- 15x10" 22x10" ‘ructose 14x 1o-4 _- _ 46xtor 26x10! Ya nitnite 18x 10-8 - —_— 46x10 = 6 7x10 Ya nucleate 5Bx1o-*® _ _ 46x10" 42x10" ipecific protein of d 04 — - 38x10 54x10 amino acid oxidase Moxazin adenine di 10 - 76x 10% 83x lot nucleotide well and suggest that the value of p 1s about 2 5 times as great for the dmucleotide as for the protein, 1e the probability of an active radical being deactivated on colhsion 1s somewhat greater for the dinucleotide than for the protein The large size of the protera molecules more than Pp for the smaller value of p, and taking the values of Z from Fig 3, Zp is found to be five times greater for the protem than for the dinucleotide Thus when irradiatinga mixture containmng approximately equal molar concentrations, as in one of Dale’a experiments: the protein be allowed for If however the affinity for the active radicals of the non enzyme protem 13 smaller than that of the enzyme protem the figures in the third and fourth columns of Table 22 will need to be re duced by some factor between one and five and the value of p for the enzyme protemn might thus become equal to or shghtly m excess of that for the dinucleotide x The molar of the 2 de was 22x 10-7 gram molecule per tre of the active protem 11x 10-7 gram molecule per litre or of the total protem about five times this REDUCED YIELD IN DILUTE SOLUTIONS 59 for X-rays It 3s seen that the theory satisfacto rily fits the ex- penmental results In fitting the theory to the experiments, p 1s treated as an arbitrary constant Experiments of this sort thus serve to determine p, and values of p deduced in this manner are given in Table 23 The values of p for methyl alcohol and oxalic acid aro in the same ratio (7 1) as Was deduced on p 55 from a different experiment Tate 23 Values of p deduced from the dimmution of reaction yield at low solute concentrations Substance P Oxahe acid 0037 Methyl alcohol 025 Glutathione 0018 Tyrosine 0037 The absolute values of p given in Table 23 are subject to considerable numerical uncertainty, owing to the fact that any errors in the values assumed for Ny, 2, D and 6 will result in rather serious displacement of the scale of abscissae in the X-ray curve of Fig 44 Also, we have assumed throughout that a collision between an H and an OH radical will always result in combination If this 1s not so, the values of p m Table 23 will be too high It 1s unfortunate that no substance occurs in both Tables 22 and 23 Comparison of the values of p in Table 23 with the values of p,/p, for chemically related substances in Table 22 suggests that the former values are probably somewhat too high They also suggest that p, m Table 22 cannot be much less than unity, so that the figures in column 2 of this table can be tahen as values of p for the corresponding substances Having deduced the value of p for tyrosine from consideration of the manner m which the X-ray 1onie yield vanes with solute concentration (Fig 4x), 1t 18 posstble to predict the iomie yield with a rays by making use of the a-ray curve of Fig 44 In this way 1t 1s calculated that the 1onie yield in 2 2 2 x 10-* Af solution of tyrosine should be 18 times smaller to & rays than to X rays Experimentally it has been found to be about 30 trmes smaller 1 x Nurnberger CE (1937) found that the ronic yield in a 2 2x 10-"AL lone rare by a rays was 0.0029 Stenstrom W & 55x 10" A solut }) using Xrays found 1ome yields of 017 in a explanation ‘ution, and 008 ina 11x 10-4 M solution Nurnberger s was in principle the same as we have given 58 CHEMICAL FFFEOTS OF IONIZING RADIATIONS tion of the solute, and the graphs show, as anticipated, that the Proportion of the active radicals which react with the solute dimimishes with dimtrushing solute concentration, and (for con- centrations such that cZp<10) 18 less for o raya than for X-rays 10Fr Ryeilaetled 08F 0 6F ou o2F Ryelrateivled °o 0 oe a a2 a oe ne 10° 10% 104 1073 1072 10! 10-6 10-5 10-4 30-9 10-% 107F Molar concentration (0) Fic 4 Dunuuution of reaction yield at low solute concentrations A calew lated curves for X raysanda raya B E, companson of calculated curves and expenmental resulta (X rays) B oxalic acid (Fricke Hart & Smth) © methyl alcohol (Fricke Hart & Smuth) D glutathione (Kinsey), E, tyrosme (Stenstrém & Lohmann) In Figs 48, 0, D, R a comparison 1s made between the results of experiments: in which the iome yield has been studied as a function of the solute and the th 1 curve A (1928) using tyroame Kinsey VE r Steustrom 1 & Lohmann, (1835) using glutathione Fricke H Hart EJ & Srath, H.-P (1938) using oxalic acid and methyl aleohal DIRECT AND INDIRECT ACTIONS 61 fraction of solute 1 reacting 1s proportional to the dose increment. dn and 13 independent of the solute concentration ¢,,1€ =defey=dn Z,p,P,,¢:ZPe, {II-7) or coc enn Me, where Ng = C2L2Pol Map, P; (II-8) In other words, the concentration of solute ¢, diminishes from its imtial value as an exponential function of the dose of radin- tion, instead of as a linear function Its not always necessary for there to be an excess of a second solute for the curve to be of this type, for, as pomted out by Friche,: and in greater detail by Dale, Meredith and Tweedie, it may happcn that the products of the reaction are also capable of deactivating activated water, and if they are of approaimately the same efficiency as the onginal solute, c, in the brachet term of equation (6) will need to be replaced by its constant initial value, and so the bracket will remain constant without the necessity for the solute 2 to be in lirge excess, or even to be present at all Fig 1c is an example of an exponential curve explained in this way When, as in the present case, the amount of solute surviving & given dose dimmishes as an exponential function of the dose, it 1s convenient to indicate the rate of reaction by specifying the dose which reduces the amount surviving to a fraction e~! = 37 9%, of the initial amount, 1e by the dose referred to as vpn equation (8) As can readily be seen from equation (8) this 37 % dose s increases approximately in proportion to the concentration of the protecting solute 2, and at large concentrations of pro- tective solute would become very large However, in addition to reacting with activated water which may be termed the indirect action of the radiation, there 1s hittle doubt that a mole cule of solute will undergo chemical change if itself doreetly lonized by the radiation Normally in dilute solutions a dose which suffices to cause a considerable proportio n of a solute to react with the active radicals will only suffice to 10ni7e directly a negligible proportion of the solute molecules In the presence £ FrickeH (1934) 2 Dale WM, Meredith WJ & Tweedie, CK (1943) 3 Alternately referred to as the mactithon dose “here these terms are or mean lethal dose appropriate 60 CHEMICAL FFFECTS OF IONIZING RADIATIONS Tho low iome yield with a rays 18 thus fairly satisfactorily ac- counted for by the present calculation It1s highly desirable that the interpretation offered should be tested by uwradiating more concentrated solutions with « rays, when (according to Fig £4) the tonie yield should increase to values approaching thoge m X-ray experiments It 18 of interest to sce whether, with solute concentrations of the order found in hving cells, any appreciable difference be tween the omic yields of X ravs and a rays 1s to be anticipated on account of the mechanism we have been discussing By 1n spection of Table 22, column 6, it appears lhhely that there will be proteins, sugars and other cell constituents having values of Zp/M as high aa 108 If such constituents are present in @ con- centration of 10 g per litre, cZp=10° Fig 44 shows that for this value of cZp the ionic yield with @ rays 1s two thirds a8 great as with X rays It appears not impossible therefore that chemical changes induced in cells by the indirect action via the water may have smaller vields in a ray expenments than 10 X-ray experiments Direct and indirect actions of radiation We have mentioned that one of the charactenstics of the n direct action 1s that, in the absence of complications, the yield 18 directly proportional to the dose Fig 1, for example, shows how the percentage of ferrous sulphate decomposed. increases with the dose, the curve 1s linear from 0 to 90 % decomposition A back reaction probably accounts for the reaction not pro ceeding to completion Back reaction between the products of the primary action 1s one cause for a reaction curve being non- linear Another cause 1s the presence of protective agents Ifwe are estimating the proportion of a given solute 1 which survives wradiation with various doses in the presence of an excess of solute 2, then we see, from equation (4), that the number of gram-molecules of solute 1 ting tor a dose increment corre sponding to the production of dx gram-molecules of activated water 1s de, = One, Zp Pal Zipi + 2 Po) (II 6) With a sufficiently large excess of solute 2 the term im ¢, in the bracket can be neglected, and so we get the result that the DIREOT AND INDIRECT ACTIONS 63 By deactivating efficiency per umt mass we mean Zp/Jf, relative values of which may be read off from column 6 of Table 22 for the substances investigated by Dale Solid content imcludes the protective agent y/I refers to the solute being investigated We see from equation (9) that as the concentration of the solute being investigated 13 mcreased, the 37% dose in solutton increases, and tends as a muting value to the value for direct action only, which 1s presumably the value to be expected when the solute 1s radiated dry + In the event of the 1omic yseld for the indirect effect being considerably less than for the direct effect,1e y/I’<1, then the limit will be approached for concentrations of solute a good deal less than 100% Broda has recently published figures for the decomposition of ammonium persulphate in glycerine, the persulphate concentra- tion varying from 0 04 to 0 13 g /em $ The 37 % dose appears to be independent of concentration over this range, and it 1s in ferred that there 1s no indirect action in glycerine solutions such as occurs in aqueous solutions It 1s unwise, however, to mshe this deduction on the basis of observations which do not extend to low concentrations of the solute, and Broda’s expermments do not prove more than that the onic yield for the mdirect action 15 less than one-tenth of the tonic yield for the direct action As the concentration 1s diminished, the 37 % dose diminishes, but owing to recombination of radicals 1t does not do so in- defimtely If the solute 1s one for which the 1onz¢ yield for the indirect action (y) 1s considerably less than for the direct action (P), there may not be a great deal of difference between the 37% dose for the direct action (1e the 37% dose when the solute 15 utadiated dry, or m concentrated solution, or m the presence of sufficient protective agent), and the 37% dose in dilute solution This happens with virus protems, which appear to 1 Broda, E (1943), d gz p Ip} finds that the yield dry 1s much less than in concentrated solution in glycerme The doses of radiation he used ere not stated in roentgens but making an estimate from the data given, 1t appears that the ionic yield in solution 1s of the order of 25, suggesting @ chain reaction of some sort The negative result when the Ppersulphate 1s radiated dry probably means that the chain reaction cannot occur in the dry state The results do not rule out an iome yield of the order of umty in the dry state 62 CHEMICAL EFFEOTS OF IONIZING RADIATIONS of a protective agent however, reducing the efficiency of the indirect action via the water, it 1s clear that the relative m portance of the direct action mcreases From the foregoing theory the relation betweon the 37 % dose in eolution, when both direct and indirect effects occur, and the 37% dose which would be obtained when direct action only occurred, can readily be determined In a solution contammg only the one solute, the reaction producta of which are supposed to have an affinity for activated water equal to that of the un- changed solute, we obtain 37% dose for direct action only 14% ‘water content’ (it 9) 37% dose in solution L’\sohd content y 1s the 1ome yield for mdirect action,1¢ the number of solute molecules reacting, per ionization in the solvent, under condi- tions in which only the solvent contmbutes to the deactivation, while r as the 1ome yield for direct action, 1e the number of reacting per 10r directly produced in the solute, under conditions in which 1omzations not directly in the solute do not appreciably contmbute to the yreld The term (water content/solid content) amply means the ratio of the weight of water to the weight of solute in a given quantity of solution, eg 1s 90/10 for a solution contammg 10% by weight of solute If the sohd content is low enough for the 1ome yeld of the indirect action to be appreciably reduced by combination of the radicals, y must be reduced by mult:plying by the factor read from Fig 4a If the solution contains the solute being mvestigated at low concentration, and in addition contains @ protective agent m much higher concentration, then we obtain 37% dose for direct action only 37% dose in solution Y (Sra cone) =l tr solid content deactivating efficiency per umit nlass of solute * (seasnesung efhoiency per unit mass of protective agent, (I-10) MODES OF BIOLOGICAL ACTION 65 accomphshed by a moderate dose of radiation Thus dilute solutions of enzymes may be largely mactivated by doses of a few thousand roentgens which would have practically no effect upon a concentrated solution ora dry preparation Enzymes are present in cells m low concentration, and since their destruction would produce marked effects 1t 18 natural to suspect that enzyme destruction 1s of importance This argument has been developed by Dale : The low concentration of the en7y me in the cell does not in itself necessanly lead to a large percentage destruction by moderate doses, since other cell constituents present in larger amounts will protect it In fact, referring to equation (10) we see that the dose required to inactivate a given percentage of an enzyme present in concentration small com pared with that of the other cell solutes is dependent of the enzyme concentration, and 1s determined principally by the value of Zp/Af As may be seen from column 6 of Table 22, enzymes have the largest Zp/Jf values of any substances in vestigated by Dale It 1s quite possible, however, that other Pprotems are equally effective mn this respect It seems therefore that while the sensitivity of an enzyme in a cell will be Ingher than that of the enzyme in concentrated solution, 1t may not be as much higher as 1s sometimes supposed 2 Direct action on large molecules As will be explained in greater detail in Chapter m1, the dose Tequired to produce chemical change in a given proportion of the molecules of a substance by direct action 1s inversely proportional to the molecular weight, supposing that the 1ome yreld (number of molecules affected per ion pair) 1s constant As a rough working rule, a dose of 108 r will produce chemical change in half the molecules of a substance of molecular weight 10° (if the lome yield is about unity) Now the smallest viruses appear simply to be proteins of very high molecular weight, and in view of their high molecular weight the dose required to produce chemical change in a given proportion of the virus molecules 1s much smaller than 1s required to produce chemical change in & comparable proportion of a chemical of lower molecular weight We shall find in Chapter 1v that the inactivati on of these small viruses can be explained adequately on the view that x Dale, WM (1940, 1942) 2 Forssberg A (1945, 1946) 64 CHEMICAL EFFECTS OF IONIZING RADIATIONS have ¥ much Jess than unity, and will be discussed further 1 Chapter 1v From equation (10), we see that the 37 % dose in solution ma approach the 37% dose for direct effect even in a solution quite low solid content, mn the event of the protective agen having « deactivating efficiency per unit mass much higher tha that of the solute being investigated (or of y/J" being much les than umty) POSSIBLE MODES OF BIOLOGICAL ACTION OF RADIATIONS We shall take it for granted that the biological effects of 1onizin; radiations are due in some way to the chemical changes induce: by the radiations We are immediately faced with the problen of explaining why marked biological effects are produced by doses of radiation which produce only a smal] degree of chemica change Marked biologcal effects in different materials are pro duced by doses ranging from about 50 r to about 5x 108r The number of tomzations produced in @ cubic micron of tissue by & dose of 5x 10° r 13 about 10*, and judging from the results o! chemical experiments the ber of mol ting wall be of this order The number of atoms in 1 271s, however, about 10! ‘Thus even the very large dose of 8x 105 r produces 4 rather small percentage chemical change, and the dose of 50 r seems quite negligible from this pomt of view There are several ways, however, in which a small overall percentage chemical change may be smagined to be effective, and these we now proceed to discuss Celi poisons The products of decomposition of protems and other cell con- stituents by radiations have not been much investigated, but it 18 quite possible that they may be myurious m quite low concen- tration It is possible that there are some biological effects due to this cause There 13 not. much one can say, however, about their mechanism, and they will not be discussed 1n this book Activated water reactions As we have seen, by the use of a sufficiently dilute solution a large percentage of chemical change in the solute can be MODES OF BIOLOGICAL ACTION 65 accomplished by a moderate dose of radiation Thus dilute solutions of enzymes may be largely inactivated by doses of a few thousand roentgens which would have practically no effect upon a concentrated solution or a dry preparation Enzymes are present in cells in low concentration, and since their destruction would produce marked effects it 1s natural to suspect that enzyme destruction 18 of importance This argument has been developed by Dale : The low concentration of the en7jme in the cell does not in itself necessarily lead to a large percentage destruction by moderate doses, since other cell constituents present in larger amounts will protect it In fact, referring to equation (10) we see that the dose required to inactivate a given percentage of an enzyme present in concentration small com- pared with that of the other cell solutes 1s dependent of the enzyme concentration, and 1s determined prmerpally by the value of Zp/M As may be seen from column 6 of Table 22, enzymes have the largest Zp/Jf values of any substances in vestigated by Dale It 1s quite possible, however, that other proteins are equally effective in this respect It seems therefore that while the sensitivity of an enzyme in a cell will be higher than that of the enzyme i concentrated solution, it may not be as much higher as 1s sometimes supposed 2 Direct action on large molecules As will be explained in greater detail in Chapter m1, the dose required to produce chemical change in a given proportion of the molecules of a substance by direct action 1s inversely proportional to the molecular weight, supposing that the 1omic yreld (number of molecules affected per ion-pair) 1s constant As a rough working rule, a dose of 108 r will produce chemical change in half the molecules of a substance of molecular weight 10° Of the ionie yield 1s about unity} Now the smallest viruses appear simply to be proteins of very gh molecular weight, and in view of ther ngh molecular weight the dose required to produce chemical change in a grven proportion of the virus molecules 13 much smaller than is required to produce chemical change in & comparable proportion of a chemical of lower molecular weight We shall find in Chapter 1v that the mactivation of these small viruses can be explained adequately on the view that 1 Dale WM (1940 1942) 2 Forssberg A (1945 1946) 66 OHEMICAL EFFEOTS OF IONIZING RADIATIONS h I change in one fecule occurs whenever one or more romzations are produced in tt There are some effects of radiation on Ingher cells which are believed to be due to the direct 1omzation of large molecules in the cell These are reactions which may be included under the general heading of gene mutation, and will be discussed in Chaptera v and 1x Localization of ionization While the overall dose of radiation may be such that only minute proportion of chemical change occurs in the solution as a whole, in the immedsate nerghbourhood of the path of an 1onizing particle, particularly a densely 1on:zing particle such as an aray, practically every solute molecule may be affected This will be no less true for direct actions not dependmg on active radicals Thus, while the overall chemical change in the cell may be small, it may be high locally in the particular strac- tures through which the 1oning particle passes If effects in these structures are microscopically observable, or if the struc- tures are sufficiently vital for changes in them to affect the cell as a whole, then a biological effect will be recorded The beat example of this type of action so far studied 1s the breakage of chromosomes by radiation, discussed m Chapters vi and VII, in which & chromosome 1s broken by the passage through it of an ronizing particle, providing the latter 1s densely 1onwing and can produce (in the case of Pradescantia) something lke 20 1omza- tions 1n its passage through the chr thread of d t Oly These breaks are microscopically visible The target theory ‘When the biological effect observed 1s due to the production of ionization in some particular molecules, as m the mduction of gene mutations, or 1s due to the p of an ig particl through some particular structure, as 1n the mduction of chro- mosome breakage, 1t 18 possible to calculate the s1ze of the mole cule or structure involved from a knowledge of the proportion of the organisms irradiated which are affected by a given dose of radiation It 15 further possible to predict the variation of :ome efficiency of diff t radiat: m prod g effects of this sort The interpretation of biological effects of radiation along these SPREAD OF EFFECT OF IONIZATION 67 lines has become known as the target theory or Treffertheorte When making calculations in general terms one often speaks of the molecule or structure in which ronization has to be produced as the ‘target’, and the production of 1onization 1m it as a ‘hit’ This mechanusttc approach has been found unplausible by some workers in this field, but the successes of the theory in explaining in particular the different ionic efficiencies of different radiations make it evident that the model 1s not too crude to represent the facts adequately in the cases which are discussed in this book Spread of the effect of an ionization When a chromosome 1s broken by radiation, a phenomenon bnefiy referred to above, and which 1s discussed in detail in Chapter vit, the evidence 1s that the passage of a densely ronizing Particle, eg a proton or slow electron, anywhere through the chromosome thread causes a break Now the chromosome thread being (in the case of T'radescantia) of a diameter of about 0 1), it must be made up of a very large number of chain molecules, and only a small fraction of these chains will be broken by the direct 1onzation or excitation of bonding electrons by impact of the ionizing particle Some spread of the effects of 1onzation or excitation must therefore occur Transference of energy from one Part of a molecule to another 1s a process known to occur, and capable of interpretation on current quantum-mechanical theory A cruder representation 18 to regard the column of lonization produced by a densely 1onizing particle as a ne Source of heat, and to consider effects produced at a finite dis- tance from it as due to temperature rise calculable in terms of thermal conductivity and specific heat Jordan: has developed this point of view in a revival of the old ‘point heat’ theory of essauer 2 Stall a third mechanism, suggested by Gray,3 takes account of the fact that ionization produced in the water inside and 2m mediately outside the chromosome leads to the production of active radicals, and that the active radicals are capable of pro- dueing chemical changes, meluding presumabl y the disruption of bonds, at a finite distance from the place where 1onization Occurred As we saw on p 50, the H radicals are produced at 1 Jordan, P (1938¢) 2 Dessiuer, F (1923) 3 Gray, LH (unpubhshed) 68 CHEMICAL EFFEOTS OF IONIZING RADIATIONS distances of the order of 16mp fromthe path of thé particle Ifthe H radicals are effective in causing the change studied, then effects may be found at this distance from the path of the ionizing particle, especially in the case of a densely 1omzing diation such as a radiation If only the OH radicals are effective, then this will not be eo, since the OH radicals are produced much nearer to the path of the ronizing particle, and i a coll, where there is an appreciable concentration of protem in solution, the distance diffused by 8 radical before it 1s ehminated by collision with a solute molecule aa only of the order of 2 or Img: x Ifthe solute ts such that el: of active radicals by radical can be neglected by comp with el st by solute reaction, the number of radicals dimunishes according to the formula e“Z*! Talang eZp~10° in the cell (ep p 60) rt follows that the active radicals peraiat for a time of the order of 10-* sec , which euffices only for them to diffuse a distance of 2 or Imp Chapter IIT THE TARGET THEORY The target theory has been briefly introduced in Chapter Biological effects of radiation to which this theory 1s applicable are those in which the effect studied 1s due to the production of lonuzation by the radiation in, or in the immediate vicinity: of, some particular molecule or structure Thus the production of gene mutation by 1onization of the gene molecule, or of chromo- some breahage following the passage of an ionizing particle through the chromosome, are actions of radiation to which the target theory 1s apphcable There are many actions of radiation on hying organisms which are not to be interpreted on the target theory Thus, if the effect observed in a given cell 1s found to be due to changes im the surrounding tissue, or in the blood circula- ton, the target theory wall not be applicable to this effect Or if the effect observed im some cell structure 1 due to change in 1ts chemical environment as a result of 1omations produced in the cell fluids, then the theory will not be helpful It 1s clear that a large number of possible modes of action of radiation he outside the scope of the theory, and some wniters: have questioned whether there exist in fact any actions to which the theory 1s appheable It 1s the opmion of the present author, the basis of which will be made clear in the sequel, that the validity of the target theory 1s as certain as a scientific theory ever 1s in a tapidly developing subject, in the case of the imactrvation of small viruses by radiation, and the production of certarn chromo- ome aberrations m higher cells We shall apply it also to the killing of larger viruses and bacteria, and the production of gene mutations, where we regard its validity as highly probable Very Possibly there are other actrons of radiation which are correctly interpretable m terms of the target theory, but we do not con- sider that sufficient experimental evidence 1s yet available to give the theory more than the status of a working hypothesis in these cases Much needless controversy has ansen m the past 1 The quahfieation or in the immediate vicinity’ 1g made to take ore of the possibility of the spread, perhaps over distance s of the Pp na 8 few millmucrons of the effect of an ionization {as discusse d on 2 Eg Scott, cM (1937) The Biological Actions of X and Y rays 70 THE TARGET THEORY when biological effects of radiation have been interpreted on the target theory with madequate evidence, and we shall for this reason exclude from discussion instances where the application of the target theory has been based only on the determination of the shape of a survival curve Three investigations should form a part of any attempt to determine whether a given biological action of radiation 1s of the target-theory type One of these 13 the determmation of the mafner in which the number of organisms or cells affected sn- creases with the dose of radiation When a lethal action 1s being studied, this 1s essentially the same as determining the shape of the survival curve,1e the curve obtained by plotting the pro portion of organisms surviving against the dose The second 18 the determination of the manner in which the effect of a given dose depends upon the intensity at which it 1s administered The third 1s an investigation of the relative effectiveness of different types or wave lengths of radiation This Jast 1s of part:cular 1m portance, and it 1s from its success in explaining the manner 1n which the effect of a gtven dose vanes with wave length and type of radiation that the target theory derives its principal value We shall in this book be concerned to a considerable extent with this application of the theory, and if will be found that con siderable progress both on the experimental and theoretical sides has been made One class of action to which we apply the target: theory 13 the elass in which the biological effect 1s beheved due to a single iomzation We interpret im this manner the mactivation of viruses (Chapter 1y), the production of gene mutations (Chapter ¥), and the killing of bacteria (Chapter1x) From aradiochemical standpoint we may group these actions as those in which the biological effect 1s due to change produced 1n a single molecule by the ionization of that molecule From a biological standpornt we may consider all these actions to be of the nature of gene mutations A second class of action to which we apply the target theory 1s the production of certain chromosome aberrations im higher cells by radiation The aberrations follow the breakage of chromosomes caused by the p hrough the chr of 1omzing particles A single ionization in the chromosome has very small probabilty of causing breakage (in the case of ACTIONS DUE TO A SINGLE IONIZATION 7 Tradescantia, discussed in Chapter vii), but the passage of a single :onizing particle suffices, providing that it 1s densely 1onuzmg and so produces a sufficient number of 1onizations in the chromosome in its passage through it A third class of action to which the target theory has been apphed 1s one in which a large number of ionizatrons must be produced within the target, and therefore a number of ionizing particles must pass through it The more densely 1omzing 1s the tomzing particle,1e the more iomzations per meron it produces, the fewer 1omizing particles will be required, and this should be shown up by a change in the shape of the survival curve with different wave lengths or types of radiation Examples of actions of radiation which have been interpreted in terms of the existence of a target through which several ionizing particles TMmust pass, are the killing of bacteria,: of bean.seeds, of yeasts,3 of Protozoa,s and the inhibition of division m tissue cellss In the opmon of the present author the validity of the application of the target theory m the manner proposed 1s less well esta- bhshed in these cases, and we shall not discuss in detail the ‘multi-hit’ target theory in which the biological effect 1s sup posed due to the cumulative effect of several 1onizing particles separately passing through the target, but shall confine our attention to the actions which are attmbutable to a single lonization or a single :oniz1ng particle Recognition of the single-iomzation type of action Apart from the question whether the action studied can, on biological grounds, plausibly be beheved to be due to change Occurring in a single molecule discussion of which we defer until later, there are a number of Imes of evidence provided by the 1 Lacassagne, A & Holweck, F (1929a), using Pyocyanigue S Lea, DF, Hames, RB & Coulson, CA (unpublished) have repeated these experiments on a number of bacteria including the strain of Pyocyaneus used by Lacassagne & Holweck but have failed to confirm their exper) Mental results, and instead obtain data consistent with the interpretation. that a angle \onization 1s responsible for the lethal effect obsery ed 2 Glocker R (1832) 3 Glocker, R (1932), Lacassagne, A & Holweck, F (1930) 4 Crowther JA (1926) enpear, FG Gray LH & Read J (1938) The authors suggest that t & angie proton or a large number of electrons are required to pass 'rough the target, the total ionization required being almost the same 72 THF TARGFT THEORY radiation expenments themselves which should be mvestigated before concluding thats particular action studied 13 caused by a single iomation Tho following reaults aro to be expected for this type of action {a) The survival curve is expenentin] (6) The effect of a given dose 18 independent of the mtenaily at which it 18 given, or of the manner in which 128 fractionated {c) For the samo degree of effect, the dose required with dif ferent radyations increases in the order y-raya, hard X-rays, soft X-raya, neutrons, « rays Often the differenco 1s not detectable between -y-rays and the different wave lengths of X-rays, but becomes noticeable mth neutrons and a raya It1s probable also that the effect of a given dose will be independent of the temperature Shape of survival curve Its convement, while developing the theory im general terms, to speak of the region within which 1onzation has to be pro duced to obtain the mutation, killing, or other effect atudied #8 the target To romze the target xt 18 necessary for an ionizing particle (electron, proton, etc ) to pass through it, and the pas- sage of an somzing particle through the target producing romza- tion im 1t may be spoken of as 8 Att The type of action we are atudying 1a caused by a aingle hut Now it 1s evident that the number of hits 1s simply proportional to the dose of radiation given If the dose given is such that only a small proportion of the targets are Int, no distinction need be made between the total number of hits and the number of targets hit. The number of targets hit 1s then proportional to the dose, and a straight hne 38 obtained by plotting (asim Fig 6a) the yneld of the reaction against the dose If the dose used 1s larger so that the number of targeta hit 1s a considerable proportion of the whole number, cases will occur of several hits beng obtained i a emgle target The number of targets hit will thus be less than the total number of hits Although the total number of hits sncreases m strict proportionality to the dose, the number of targets hit increases more slowly, so that the yreld plotted agamst the dose gives a curve which is convex upwards {curve B, Fig 5) tending asymptotically to 200% at large doses If one 1s follownng, say, the Jallmg of bacteria or of other single celled organisms, the SHAPE OF SURVIVAL CURVE 13 numbers killed by successive increments of dose are not equal, but each merement of dose kills the same proportion of the number of orgamsms which have survived until then The 010 1 _, 08 A 2 0 8F = 2g O06} a @ 06E-- ~~ B g 3 a i \ g 004F- gor ' ke io H 0 02; 0 2b 1 1 1 1 oC i 1 ' 2x10'r 10 20 x10'r 10 0 8 g 08) c 3 it-~-- : D 3 ao. a a w 2 ' e $ ' g O4F.2 > IF ‘ g ' § H £0 7F \1 a wa 3s 2 if 1i J. i L 10 20 «106 0 2 3 é Ss. ae 8 x é & Top--~-------> « a 1 2 s c :\ & L . & 0 20 x10'r Dose Fro 5 Methods ofplotting exp 1 data for the single ionization type of action number of viable organisms falls off in a geometrical progression, or what 1s merely another way of saying the same thing, the survival curve 13 exponen tial (curve C, Fig 5) bor, 1f ny 1s the mutial number of organism s, and n the number which survive a dose D, then the proport ion of organisms not so far bit which wall be hit by an increm ent dD in the dose, will be 72 THF TARGFT THEORY radiation expermments themsclves which should be investigated bofore concluding that a particular action studied 19 caused by a single ronization The follow ing results are to be expected for this typo of action (z) The survival curve is exponential (8) The effect of a given dose 18 independent of the mtensity at which it 38 given, or of the manner in which it 3a fractionated (c) For the samo degree of effect, the dose required with dif ferent radiations increases in the order y rays, hard X rays, soft X-rays, noutrons, « rays Often the difference 1a not detectable between y rays and the different wave lengths of X-rays, but becomes noticeable with neutrons and & rays It1s probable also that the effect of a given dose will be mdependent of the temperature Shape of survival curve Its convenient, whilo developing the theory un general terms, to speak of the region within which ionization has to be pro duced to obtain the mutation, kullng, or other effect studied a3 the target To tone the target 1t 1s necessary for an ionizing particle (electron, proton, etc ) to pass through it, and the pes sage of an ionizing particle through the target producing 1omza- tion in rt may be spoken of as a Ast The typo of action we are studying ts caused by a single hit Now at 13 evident that the number of ints 1s simply proportional to the dose of radiation given If the dose given 1s such that only a small proportion of the targets are hit, no distinction need be made between the total number of hits and the number of targets hit The number of targets hit 1s then proportional to the dose, and a atraight hne 18 Obtained by plotting (asin Fig 54) the yield of the reaction against the dose If the dose used 1s larger so that the number of targets hit 1s a considerable proportion of the whole number, cases will occur of several hits being obtained 1n a single target The number of targets hit will thus be less than the total number of hits Although the total number of hits increases in strict proportionality to the dose, the number of targets hit increases more slowly, so that the yield plotted against the dose gives a curve which 1s convex upwards (curve B, Fig 5) tending asymptotically to 100% at large doses If one 1s following say, the lallng of bactena or of other single celled organisms, the EXPONENTIAL SURVIVAL CURVES 15 expermental points and reads off the dose giving the natural logarithm = —1 (E) If loganthms to base 10 have been used, one reads off the dose giving logy, 2/ng= ~1, and divides it by 23, because log, 10=23 Tare 24 Natural logarithms and exponentials nin, log, n/ng DID, ¢ Ue DID, ¢ Mile 095 949= —0 051 002 0990 20 01353 0390 895 —0 105 004 0961 22 01225 085 837-0163 006 09432 22 o}108 080 Wi -0223 003 = 0.923 23 01003 075 712 —0 2838 0310 = 0.905 24 00307 070 613 0357 012 0887 25 90322 965 569 —0 431 014 0 Br9 26 09743 066 489 —O511 016 8s 27 @0072 055 402 —0 593 O18 0.835 28 OHS 050 307 ~0693 020 ©0819 29° (00550 O45 1201 ~0 799 025 0779 30) 00198 040 Tost ~oo16 030 0741 31 00550 035 2950 —1050 035 ©0705 32 y 0408 $30 2796 —1 204 940 9670 330 UOt69 6 25 S614 —1386 045 «0.638 34 9.0334 020 391 —1609 050 0607 35 © 9.0302 O18 285° 1715 055 9 0577 36 00273 O16 167-1893 060 ©0549 37 00287 é if 034-1966 065 0528 38 0224 14 880 —2120 070 0497 39 9u202 010 697 —2 303 075 0472 40 0.0183 068 474-2596 080 860449 41 00166 066 Wt —2813 O85 0497 42 QO100 on 004 —2 996 080 0.407 43 00180 781-3219 095 0387 44 0.0123 603" 493-3507 10 0308 450 90111 8 025 311-3 689 V1 0 333 46 VOl01 9920 088 =~3912 12 0301 47 09091 ons 800 4 260 13 0273 48 00032 ° oon 395 ~4 605 14 0237 49 0 0074 122 ~4828 022 9007 028 ~4 962 I8 0 302 51 067 i bout o oes 834 5116 12 0183 52 00055 ae 702 ~5 298 18 0165 53 00030 479-5521 19 0.150 54 (0085 9003 8191 —5 809 0 002 2785 ~6215 8001 7092 —6 908 ra loganthmic graph paper has been used, one reads off the sen. *edueing the surviving fraction to 0 368, or else takes Tho Teducing the surviving fraction to 0 1 and divides by 23 strate Y various methods of plotting radiation data are ilu- in Fig 5, the (hypothetical) data on which they are 74 THE TARGET THEORY given by the formula —dn/n=dD/D,, where Dy 1s the dose re quired to score an average of one hit per organism This formula integrates to log. (n/m)= —D/D,, or n=ng o-Ps (WT 1) The logarithms are natural logarithms to a base e, a. table of which 19 given (Table 24), together with a table of the exponen- tial function e~2/% The easiest way of testing whether an experimental survival curve 18 exponential 1s to plot against the dose not the surviving fraction, but its logarithm, when a straight line will be obtained of the survival curve is exponential Either natural loganthms to base e (Fig 5D), or logarithms to base 10 (Fig 58), may be used for this test, or the surviving fractions may be plotted directly on to loganthmic graph paper (Fig 5F) Natural loga- tithms are to be preferred since they facilitate the subsequent calculations Having established that the survival curve 1s exponential we can most concisely report the sensitivity of the material to radiation by stating what we may define as the mean lethal dose or iactiation dose, or 37%, dose By this we mean D,, the dose which corresponds to an average of one hit per target It can be obtained from the expenmental results in the following alterna- tive ways, depending on which method of plotting has been adopted (A) If the experment has been hmited to dosages at which only a small proportion (eg less than 10%) of the organisms have shown the effect studied, and 1f the results have been exhibited by plottmg the proportion affected against dose, one extrapolates the straight line curve best fitting the results and reads off the dose corresponding to 100% effect (B) If the number of orgamsms affected has been plotted against dose, and the experiment has not been hmuted to a small proportion of organisms affected, the dose required is that affecting 63 2% of the organisms, because 1—e-*=0 632 (C) If the ber of org ffected,1e surviving the dose, has been plotted against dose, one reads off the dose giving 36 8% survival, because e-7=0 368 (Hence the name 37% dose ) (D) If the natural loganthm of the surviving fraction has been plotted against dose one draws the straight line best fitting the EXPONENTIAL SURVIVAL CURVES 75 expermental points and reads off the dose giving the natural loganthm = ~ i {E) If logarithms to base 10 have been used, one reads off the dose giving logyyn/ng= —1, and divides it hy 23, because log, 10=23 Tav.e 24 Natural loganthms and exponentials ning log, ning Dib, eh DID, e7 ht, 095 949= —0051 002 0990 2 0 1353 090 895 —0105 004 6961 21 01225 085 837-0163 006 =: 942 22 01108 080 TF -0223 008 60.92 23 01003 075 712-0283 010 0.905 24 00907 070 643° —0357 O12 0887 25 00821 065 569° 0 431 014 0869 26 00743 060 489 0511 016 0.852 27 W0672 055 402 0.598 018 6835 28 = 00603 050 307 —0 693 020 Osi 29 00550 045 201 -0 799 025 0779 30 0.0498 040 084 —0916 030 O74 34 00430 035 950 —1050 035 0705 32 00408 030 796-1201 940 0670 33° 079 025 2614 —1386 045 «60 638 34 00334 020 391 —1 609 050 0 607 35 00302 018 285-1715 055 0577 36 O16 2107-1833 060 0539 37 a4 034 —1 966 065 0522 38 O12 880 —2120 070 0497 39 010 697 —2 303 075 0472 40 008 474-2596 080 0449 41 696 187-2813 085 0427 42 H 05 003 —2 996 090 0407 43 04 781 =3219 095 0387 44 00123 003 493 ~3507 10 0 368 45 0011 bed 311 ~3 689 ll 0 333 46 vor 0 920 088 —3912 12 0301 47 90091 1S 800 ~4 200 13 0 273 48 0 0082 oon 395 ~4 605 14 0247 49 90074 0 1720 ~ 4828 15 0 223 50 oni nee 038 ~4 962 16 0 202 51 0 000i Hird 884 ~5 116 17 0 183 62 90055 008 702 —5 298 18 0165 53 00050 479 5521 19 0180 54 005 mt logarithmic graph paper has been used, one reads off the doce *enueing the surviving fraction to 0 368, or else takes Th ¢ reducing the surviving fraction to 0 1 and divides by 23 ese various methods of plotting radiation data are illu- strated in Fig 5, the (hypothetical) data on which they are 76 THE TARGET THEORY based being given in Table 26 In tho particular example chosen, the 37% dose 15 10x 10° r Thig, then, 18 the dose needed to produce sufficient 1omzation in the tissue to obtam an average of one hit per target: The deductionof the37 % dose 1s the first stage 1n calculating the size of the target from the experimental data Tante 25 Example of the method of working up expermental data for the single hit type of action Dose {x 10?) o o2 o4 oo 08 10) 2 Fraction lulled 000 002 00f 006 O08 010 O18 Fraction surviving 100 098 098 O9f 092 090 082 Log, surviving fraction 000 [9s [96 It I92 190 {£80 Dose {x 10" r} 3 4 5s 10 16 8 30 Fraction kulled 026 033 039 063 078 088 095 Fraction surviving O74 087 O81 037 022 O14 005 Log, surviving fraction t70 160 150 I00 850 £00 300 The dose of radiation that suffices to affect an appreciable proportion of the organisms irradiated will usually correspond to the passage of a large number of :onizing particles through each organism Thus, when irradiating bacteria with a-particles, to kill 50% ofthe org: a dose corresp g to the passag g i 1d A 8 to 3 £05 - FI wm - Dose Dose Fra 6 Survival curves expected for A cumulative type ofaction B, single 1onzation or single ionizing particle typo of action through each bacterium of about 100 « particles is required On the target theory the lethal action 13 due to a single one of these hundred a particles, which happens to go through the sensitive region or target A superficially more natural explanation would be that the death 1s the cumulative effect of the chemical change produced in the organism by the hundred @ particles If the effect were cumulative, however, one would expect a shape of EXPONENTIAL SURVIVAL CURVES V7 survival curve such as Fig 6a, m which a dose equal to the mean lethal dose resulted in the survival of 50% of the organisms, while a certain proportion of the organisms were lulled by some- what smaller doses or survived somewhat larger doses, depend- ing on individual resistance This segmord shape of survival curve 131n contrast to the exponential shape Fig 63 expected when the action 1s due to a single-untl action (1e a single 1omzation or a single 1onzing particle), and the experimental realization of an exponential curve instead of a sigmoid curve 1s an argument sug- gesting that the effect being studied 1s due to a single 1omzation or a single ionizing particle rather than the cumulative effect of many lonizing particles When a curve definitely of the type of Fig 64 1s obtained, it does of course rule out the possibihty of the cause of death bemg @ single-unit action, but it 1s not equally certam that an ex- ponential survival curve such as Fig 68 rules out a cumulative action For such a curve could be obtained with a cumulative action if the resistance of individual organisms to the radiation varied very widely There may be found in the hterature dis- cussion ad nauseam of whether the exponential survival curve obtained, e g with bacteria, proves the disinfection to be of the single-umt action type, opponents of this view preferrmg to ascnbe the exponential curve to an extremely skew distribution ofresistance As long as the argument 1s based only on the shape of the survival curve, the conclusion must be largely subjective, since 1b depends on whether one regards the @ prior: improba- bility of the target theory to be greater or less than the @ prion improbability of the distribution of resistance to radiation among the organisms being of the extremely skew type required Under these circumstances additional cmtena rather than further dis- cussions are called for One remark, however, we shall add before leaving the subject of shape of survival curve The argument for the target theory interpretation clearly m- creases in force the more exactly the survival curve 1s exponen- tial, since while the distribution of resistance may be shew, there seems no reason why it should approximat e closely to a simple mathematical function Hence the value of determinmg the survival curve ag accurately as possible The establishing of a small systematic departure from the exponential curve does not, however, necessanly exclude the target theory, unless certain 73 THE TARGET THEORY complicating factors capable of distorting an exponential sur vival curve have been excluded Thus if one 1s irradiating, say, bacteria, and some clumping occurs, then the survival curve will be thereby mado shghtly sigmoid, since several hits, each on a separate bacterium, will be needed to make the clump inenpable of generating a colony Or if the organisms irradiated are not quite uniform, the more sensiti 6 will be Ailled more rapidly than the more reastant, and the curve obtamed by plotting the logarithm of the surviving fraction against dose will be concave upwards Or if attempts to extend the experiment to very low survival aro made by the use of largo doses, then some addi honal lethal action may become :mportant, auch as the produc tion of poisons in the medium, which will make the loganthmic survival curve convex upwards Very often these complrestions do not occur When they do, they naturally weaken the force of the survival curve argument as an indication of the appheabibty of the target theory to the particular case, but a slight departure from exponential shape should not be regarded as ruling out 8 single unit action interpretation when other indications support this interpretation, until the possible existence of compheating factors has been investigated There are several examples in the hterature of the bactencidal action of radiations, im which sig- moid survival curves have been reported, but experrments in which special care has been taken to avoid disturbing factors such as clumping have yielded exponential survival curves Time-intensity factor The fact that the effect :n one organism, when it occurs 1s due to one ionization, and as not the cumulative effect of several 1omzations, means that the manner in which the romzations are distributed in tnne has no effect For each 1ontzation produced in the irradiated tissue has a certain probatihty of produemg the effect, but this probability 13 uninfluenced by the time elapsed since the Jast 1omzation occurred or before the next one will occur Hence, mn the class of action of radiation we are con- stdering, the effect of a given dose 1s independent of the intensity of radiation and the manner in which itis fractionated Naturally this statement supposes that the maternal irradiated remains of constant sensitivity during the period over which the irradiation 1s spread ION-DENSITS OF RADIATION 79 On the alternative view that the effect observed 1s not due to a single ionization or single 1omzing particle, but to the cumu- lative action of a large number of ronizing particles, we might expect to find that the effect of a given dose depended on the intensity There are numerous stances in which a biological effect of radiation, not of the single umt action type, 15 found to require a larger dose when the radiation 1s administered at low intensity than when admimstered at high tensity The natural explanation 1s that the organism 1s capable of recovery from the effects of rachation provided this 1s administered sufficiently slowly The expenment of spreading a given dose over vanous lengths of time should always be made when investigating an action of tadyation suspected to be of the single unit action type, since to find that the effect of a given dose did mdeed vary with the duration over which it was spread would provide a strong argu- ment against the action being of thistype But to find the effect of a given dose to be independent of this duration cannot be regarded as conclusive evidence for a single unit action, since even with cumulative actions there 1s usually found o range of durations over which the effect of a given dose 1s independent of the duration Dependence on type of radiation if the 1omizations produced in irradiated tissue were spatially distributed at random, the yield in a reaction, im which a single ionization in the target sufficed to produce the effect, would depend only on the number of 1omzations produced per unit volume in the tissue, and would not depend on the wase Jength or type of radiation used Actually the 1omzations produced by X rays or radioactive radiations are localized along the paths of ionizing particles, and ionization can only be produced in the target if it 1s traversed by an lomzmmg particle If the latter is densely ionizing, 1 produces a Jarge number of 1onizations per micron path, then it 1s found with targets of the size existing in Practice that several 1onizations are produced when the lonizing particle tray erses the target. Since one suffices to produce the effect being studied, the additional 1omzations which contribute to the dose, but not to the biological effect, reduce the yield per ronization Thus densely 1onizing radiation s are less effective in 80 THE TARGET THEORY this type of action for equal renization im the tiasue Thas is the pnnapal means by which actions due to n single ronizafion can be recognized, since in some others mt which many soniatrons are required (e p chromosome breakage, Chapter vit) exactly the reverse 18 true, the densely tormzing radiations bemg more effec- tive per ronization than the lees densely rotting radiations In the amgle ronization type of action no appreciable dif- ference 1a to be expected, however, between y mya and different wave longths of hard X-rays with targets of the sze range commonly found (4-40my diameter) This 1s true despite the fact that the difference of ion density between the 1omzng particles (electrons) produced by ay ray of OO1A and an X ray of O1A ss considerable The reason is that along the track of the ionizing particle successive primary ronizations are separated by distances greater than tho target diameter, so that more than one primary tomzation rarely falls im the target (Many of the primary ionizations are the centres of small clusters of secondary sonization, so thatit frequently happens that tw o or three 10niza- tions composing a single cluster fall in a target The mean nuober of secondary 1omzetions per primary soruzation is, how- ever, approximately the same for thiferent wave lengths of y raya and hard X-rays, so that thig compheation does nob miroduce any difference in effinency per ronization ) Thus the yield of the reaction atudied for a grven number of yonzations per unt volume of irradiated tissue 1a practically independent of wave length over the range of wave lengths most easily accessible, viz y rays and X-rays down to say 50kV It 1s usually only when soft X rays of wave lengths exceeding LA are used, or neutrons or a rays, that a significant vanation as found Relation between target size and inactivation dose Varsous biological actions of radiations have been interpreted on the target theory for s number of years, though st is only fawly recently that adequate tests for the vahdity of such mter- pretations have been apphed The usual am ts to deduce from the observations the size of the target, in the hope of identifying 1t with some known cell structure In the case of actions ut * hich a single 1onuzetion im the target suffices, knowledge of the size and shape of the target should CALCULATION OF TARGET SIZE 81 enable the 37%, dose to be predicted, since we know sufficiently well the number of 1onzations produced per umt volume in tissue by lr of any radiation, and the spatial distmbution of the iomzations (numerical information on these points 1s to be found in the tables of Chapter 1) The converse problem of deducing the size of the target from the measured value of the 37% dose can also be solved if we assume the target to have some ample shape, eg spherical If data are available for the 37% doses with several different radiations, then the constancy of the estsmates of target size obtamed from the several radiations affords a highly desirable check of whether the single-iomzation theory 1s applicable to the particular action being studied, and whether the target 1s sufficiently nearly apherical to be assumed so in the calculation As will have been realized from the account given in Chapter 1, the spatial distribution of :omzation im irradiated tissue 1s com Ppheated, and an exact caleulationis laborious If the 1onmzations were produced in the tissue singly and at random, the calcula tions would be much easier One approximate method of calcu- lation, which we desenbe as Method I,x 18 to treat the problem as if the 1ommzations were m fact produced ingly and at random, and we deseribe Lelow the results given by this method and indicate the conditions under which it may be approximate ly valid Another approximate method which has been widely used (Method 11); 1s to take account of the fact that 1omuzations are localized along the paths of 1onizing particles (electrons, protons or a-particles) so that 1omzation can only be produced in the target when an iomzng particle passes through at, by postu lating that the effect ocours whenever an 1omzing particle passes through the target Still another method (Method IV)s takes account of the fact that when tissue is wradiated by X-rays the ionizing particles (electrons) are berated by the absorption of X ray quanta, and regards a ‘hit’ as the absorptio n of a quantum in the target Each of these methods 1g valid for certain sizes of target and certain radiations, but each of them leads to serious error when t Crowther, J.A (1924) 2 Glocker, R (1932 RB of Mi Cote , OF ‘ea ger WV (1934) , Lea DE, Haine s 3 Lacaas: agne A & Holweck F (19294), Wyckoff ,RWG (1930a,5) 80 THE TARGFT THEORY this typo of action for equal ionization in the tissue This ts the principal means by which actions due to a emgle iomzation can bo recognized, since in some others in which many 1onizations aro required (e g chromosome breakage, Chapter vi) exactly the roverse 18 truo, the densely forszing radiations being more effec tive per 1omzation than the less densely 1onizing radiations In tho single iomzation type of action no appreciable dif ferenco 18 to be expected, however, between y-rays and different wave lengths of hard X-rays with targets of the size range commonly found (4~40my diameter) Tins 1s true despite the fact that tho difference of ion density between the ronizng particles (electrons) produced by ay ray of 001A and an X ray of 01A is considerable The reason 1s that along the trach of the ionizing particle successive primary 1omzations are separated by distances greater than the target diameter, so that more than one primary iomzation rarely falls in the target (Many of the primary 1omzations aro the centres of small clusters of secondary 1onization, so that it frequently happens that two or three onze tions composing a single cluster fall in a target The mean number of secondary tonizations per primary 1omzation 1s, how- ever, approximately the same for different wave lengths of y rays and hard X-rays, so that this complication does not introduce any difference in efficiency per 1onzation ) Thus the yield of the reaction studied for a given number of 1omzations per unit volume of irradiated tissue 1s practically independent of wave-length over the range of wave lengths most easly accessible, viz y rays and X-rays down to say 50kV It 1g usually only when soft X-rays of wave lengths exceeding 1A are used, or neutrons or @ rays, that a significant vanation 1s found Relation between target size and inactivation dose Various biological actions of radiations have been mterpreted on the target theory for a number of years, though 3t 18 only fairly recently that adequate tests for the validity of such mter- pretations have been apphed The usual aim 1s to deduce from the observations the size of the target, in the hope of :1dentifymg it with some known cell structure In the case of actions in which a single ionization in the target suffices, knowledge of the size and shape of the target should CALCULATION OF TARGET S17F 83 and will be in the direction of underestimating the target size, but the method will be correct for targets smaller than the separation of consecutive 1onizations i the trach of the 1onizing particle Method II A method which has also been commonly adopted 1s to suppose that a ‘hit’ 1s obtamed whenever the ronizing particle traverses the target. This method will clearly be correct if the target 1s sufficiently much larger than the mean separation of tonizations along the path of the 1omzing particles to make it improbable that the 1omzing particles should traverse the target without leaving any 1onization init This method of calculation will, however, be on error if the target is small, so that it 1s possible for the ionizing particle to traverse the target without leaving any ionization in it In such cases, application of this incorrect theory will underestimate the target size It 1s clear that of the two procedures, Method I 1s valid for very small targets and Method II for very large targets Attempts have been made to apply one or other of these methods of calculation to predict the variation of biological effect with wave length of X rays for targets of given size The weakness of these attempts les in the fact that 1t turns out that the targets of hological interest are often of a size intermediate between the small size for which the first method 1s vahd, and the large size for which the second 1s vad Thus when the mactiation of small viruses 1s being considered, the first method 1s approxi. mately valid for y rays and hard X-rays, while the second method 1s approximately valid for very soft X-rays For medium wave length X-rays the relation between the size of the target and the separation of successive 1onizations 13 such that neither method of calculation 1s satisfactoryIt 1s evident that any attempt to base a theory of wave length vanation upon one or other of the two methods of calculation would be maccurate Method IIIT The associated volume method "The present author: has given a method of calculation winch should be satis factory over the :mportant intermediate range of target size, while tending to the same results as Method I for small target size, and to the same result as Method II at large target ze The method 18 illustrated by Fig 7A, which represents diagram- matically the distribution of 1omzation produced by an electron 1 Lea, DE (1940, b) 82 THE TARGET THEORY apphed outside this range A method: of calculation may be devised (Method III) not subject to this lmatation, but valid for any sizo of target and 1omzing particles of any 10n-density As explained in an earlier chapter, less than half of the total ionization 18 produced by the pnmary ionizing particle (electron, proton or @ particle), the rest being secondary 1onmuzation pro duced by secondary electrons (8 rays) ejected by the prmary ionizing particle from some of the atoms it 1omzes All the methods so far catalogued either neglect the distinction between primary and secondary 1omzations completely, or else take 1b into account crudely by deringthe produced by a é ray to form a compact cluster 2 This procedure, while valid for the majority of secondary electrons which are of low energy, fails to allow adequately for the appreciable pro portion of the total 1omzation contained in the tracks of more energetic é rays of longer range A rather laborious calculation can be made to take these é-rays into account The details of ths calculation are relegated to the Appendix, but in Figs 8-11 of this chapter the results of the calculation are presented m a form enabling experimental data of mactiyation doses to be rmmedi- ately converted to target size We now outline briefly the various metheds we have referred to for calculating the target size from the 37% dose Method I Tf the ioruzations were produced in the tissue singly and at random, the dose needed to produce an average of 1 ionization per target of volume v (1e the 37% dose) would be that dose which produces 1/v 1onizations per umt volume in tissue This procedure for calculating the 37% dose would be vahd if 1omzations were distributed at random im the tissue Actually they are localized along the paths of romzing particles If no ionizing particle passes through the target then no effect 1s achieved If an 1omzing particle does pass through, then several 1omizations may be produced (in the event of the target dimen sions bemg several times greater than the separation of con- secutive 1onizations), and hence the dose requmred for a given biological effect w ull be proportionately greater than on this method of calculation The crror will be great for large targets, 1 Lea, DE (19404) A (1936) Jordan,P (19388), 2 Les DE Hames RB & Coulson,C Lea D.E (1940a) THE OVERLAPPING FACTOR 85 1s the necessary and sufficient condition for an ionization to be produced in the target On account of the overlapping the associated volume will be less than NV $273 in 143 of tissue The overlapping 1s most in the case of the spheres associated with the separate 1omzations of a cluster These 1onizations, in a cluster of up to five or sx 1omzations, are only separated by about Imp (10-7 cm), and since the diameters of targets found in examples studied to date range from about 4 to about 40my, the overlapping in emall clusters 1s practically complete, so that the volume associated with a cluster is only $ar* The calculation 1s smplified by assuming this to be true of all clusters, so that the picture simphfies to Fig 78, in which a sphere 1s shown for each primary romzation only Since the ratio of the total number of iomzations, N’, to the number of primary 1omzataons, 7, 18 approximately 3 1, there will be n=N/3 spheres per unit volume On account of the overlapping of the spheres belonging to consecutive pmmary ionizations the associated volume will be less than n 473 and will be, say, fn fate A gmt (III-2) , per cubic micron Expression (2) 1s the sum of the associated volumes of all the 1on1zing particles in 13 of tissue, and 1s equal to the mean number of hits per target The individual associated volumes partly overlap, and the probability p that there will be at least one hit per target, which 1s also equal to the proportion of targets Int, 1s given by the following expression p=l—exp (-7 n 3") (TIT-3) F may be calculated: im terms of the diameter of the target 2r and the mean separation (Z) of consecutive primary ionizations in the path of the 1omzing particle, and 1s found to be or r-3 /o —2(1 -e-t)/E + 20-F/8}, 2 (111-4) where §=2r/L This function F'1s tabulated agamst £ 1n Table 26 We can readily show that for large targets (1e £31), when the overlapping spheres fuse into a cylinder (2) reduces to nLnr?, 1 See Appendix, also Lea, DE (1940a} 84 THE, TARGET THEORY over a small portion of ita path Tho solid dots are 1omzations, somo clusters of secondary on beng indicated S' that to get. an effect, eg o gene mutation, it 13 necessary for an tonization to be produced within a sphencal target of radius r OQ Gd: OOO O@OO dy 00O 6 Oro (TRERERR G CORSON Fic 7 Mlustrating the associated volume method of calculation Whats the probability of this happening when a dose equivalent to the production of N romzations per cubie micron 1s given? The calculation may be effected by the following construction, indicated in Fig 74 Round each 1omzation aa centre a sphere of rads 71s imagmed These spheres, which 1n general overlap to a greater or lesser degree, will occupy a nett volume which may be described as the assoczated volume For mutation to occur, the centre of the target must he within this associated volume, this ASSOCIATED VOLUME METHOD 87 calculation listed above as I, II and III have been used For the reasons stated Method I 1s hkely to underestimate seriously the target size deduced from the a-ray data, and Method IT similarly to underestimate the target size deduced from the Taste 27 Target d ’, Jeduced from 1 data on the inactive tion of a b hage by raddhat: P of four hods of eatculation Radiation y Taps X rays (15A) a rays (4eMV ) Inactivation dose (x 10° r) 58 98 35 Target diameter 1n mjt Method 1 12 100 59 Method IL 38 113 244 Method III 163 162 a4 Improved method 165 159 163 y tay data The figures given 1n the table bear out these antiei- pations, and Method III which should be free from these errors @ves a more conustent set of estimates The principal remain ing discrepancy 1s nm the high value obtained with a rays An improved method of calculation discussed later in the chapter, which takes account of 5 rays, removes this discrepancy, as the last hne in the table shous Method IV If the target 1s rather large, and X rays of very long wave length giving very short photoelectran tracks are used, the photoelectron has an associated volume which 1s re- presented n Fig 7¢ In an extreme case this evidently 18 little bigger than a sphere 4mr? Thus for very large targets and very short photoelectron trachs the whole photoelectron tracks act asaunit This is the justification for considering a ‘hit’ to be the absorption of an X ray quantum within the sensitive volume, which 1s the assumption made in Method IV In practice 1t 1s doubtful if there are any cases of direct action of radiation, of the ty pe where a single ionization suffices, where the target 18 so large that the associated volume is practically a sphere The type of geometry illustrated in Fig 7c, where the photoelectron range 18 greater, but not many times greater, than the target diameter, 1s, however, known, and the associated volume m this case is Clearly 4ar?(14-n/F) af there are n primary lonzations in the photoelectron track, thus giving a shghtly greater yield per ionization than would be obtamed if the lonizing particle pro- duced the same number of 1onizations per micron path but was estricted Jength Clearly the associated volume method 86 THE TARGET THEORY 1e nr? times the total length of track per cubre micron This 18 the probability of obtaining a Int on the assumption that a hit is obtained whenever the 1omzing particle passes through the target area mr? Our method thus reduces, for large targets, to Tantr 26 The overlapping factor F for values of£ from 0 to 10 at intervals of 01 co 01 oz 03 o4 0s o6 07 08 08 SOBmHtBaOG 1000 1038 1077 1116 1156 1197 2239 1282 1325 1369 1414 1460 1506 1552 1599 1648 1696 1745 1795 1846 1897 1648 2000 2053 2106 2159 2213 22967 2322 2377 2433 2489 2535 2602 2659 2717 2774 2832 289) 3008 3007 3127 3187 3246 3307 3367 3428 3488 3549 oo 2 ° eo &3 res oo a 3 3 3705 9857 3019 3O8t 4043 4106 4168 4231 4293 4357 4420 $483 45146 4609 4673 4736 4800 4864 4928 4991 5055 6120 5184 5242 5312 6376 5440 5505 5569 6634 5699 5763 6828 5892 5957 6022 6162 6217 6282 6347 6412 6477 6542 GEOT 6672 6737 For values of £ outside the range covered by the table the following approu mations may be used: For large £ Fe $(E+2/) For small £ Folthawey what we have described as Method II For targetd so small that the spheres do not overlap, (2) tends to the value n 4ar? which 1s the same result as given by Method I, except for the modifica tion that we take the eluster rather than the individual 1omzation as the effective unit, thus countmg n=N/3 clusters per cubic micron instead of N iomzations We illustrate the three methods of calculation in Table 27 1n which expermmental data: on the mactivation of S13 Dysentery bacteriophage 18 analysed in terms of the target theory The experiments give the inactivation doses for three rad:atzons of widely differmg 1on density, namely, & rays, X-rays (1 5A ) and yrays These imactivation doses mcrease in the order of m creasing 10n-density, the survival curves are exponential, and the effect of a given dose 1s found to be independent of the in- tensity at which 1t 2s ad ed These derations suggest that we are dealing with a single 1onization type of action, and one proceeds to calculate the size of the target: supposing it to be spherical Each of the three radiat gives an independ estimate of the target size In Table 27 the three methods of 1 Lea DE & Salaman MH (unpublished) ,wo]Hog|SeuepJOV(4g)suosmsuurodayneUdtrFoy(UIyVeIPeyootSsevqzrSpusp€E{(q¥)6s1u0a)dysuatSoryUpt6es0asPiSvtoind0g1ew[ar_Ms¢VweiqodU/OMTwuEop810CoW3aTlI sturygo(virstex6(atid Canoe)ster2g(Aneg)SereL(ves) roy ELT(|+H ET fe a cr ook tS Tee rt 0di1z 88 THF TARGET THEORY tenda to the samo limit as the single quantum method in cases (if any) where the latter 18 appheable Secondary fonization Wo have not so far taken account of secondary 1onization except to point out that small clustera of secondary ron:zation will not add appreciably to the effect of primary 1onization with targets of the sizo found in practice This will not be'true, how- ever, for the more energetic secondary electrons or é rays, which produce tracks ofappreciablelength Their importance 1s greatest with @-rays, since with this radiation the total length of 3 ray tracks 1s greater than the length of the a ray track itself (Plate Ia and Table 15, p 28) Noglect of the secondary 10n1za tion will result in an estimate of target size being obtained 3n excess of the true size of target The proposal was made by Mohler and Taylor: and followed up by other authors,: that secondary ionization should be taken into account by regarding the a ray track not as a geometrical line but as a column of radius 6 If any part of this cylinder was inside the target of radius r a hit was considered to be scored The effective target area thus became 7(r+6)? in place of a7? The value of b was to be determined either from Wilson chamber photographss or from calculations based on the recombination of ions in an @ particle column in airs On further examination this method 1s seen to be untenable The size of column found by e:ther of these methods 1s de- termined mainly by the distance an electron too slow to 1omze travels before attachment to a neutral atom to form a negative zon Ag we have given reasons for believing that the negative tons make no appreciable contribution to the biological effect, their spatial distribution 1s not relevant to the problem There 1s obviously no difficulty in principle in making an exact allowance for the é rays by the method of associated volume The method in this case 18 sllustrated by Fig 7D Here the usual construction of describing about each 1onrzation as centre a sphere of radius 71s applied also to the é ray track A knowledge 1 Mohler FL & Taylor, LS (1934) 2 Lea DE Hames RB & Coulson CA (1936) Jordan, P (1938) 3 Klemperer O (1927) 4 Jaffé G (1913) ep Chapter 1m p 50 100 pao my hs eysaetda Tat ? S Tor +4 t Basy ans Mi gaheisra oevapaloKTtadieI!s Serta TM qe IF = Hha oye a4yt Thefoals 1 ‘ t t v 4 7 ¥ia 10 Dependence upon the target diameter of the ratio of the 37 °% dose With a given radiation to the 37%, dose with yrays 2 \ rays (016A), 3 Xrays(i5A) 4 neutrons (la+D) 5, X raya(41A) 6 X rays (83A) 7 arays(6eMV) 8 « rays (JeMV) = i f a fe 10° & tte HA Mwoelicuglahrt a 7 10% L { eReE tof 195 toé 37% dose in roentgens Ii 9 Relation between 37% doso in roentgens and molveular weight (density 135g /em*) SECONDAR’ IONIZATION 89 of the number and energies of the é rays 1s required, this being provided by the tables of Chapter 1 With the aid of this more exact method of calculation we have computed the apparent target diameters to a-rays which would be given by the simple method of calculation which neglects consideration of the secondary 1omzation, for each of a number of (true) sizes of target The results are given in Table 28, and as expected the ‘apparent’ target diameters are greater than the true target Taste 28 Apparent target diameters to a rays of 5eMV ‘True target diameter (mp) 4 10 20 40 30 Apparent diameter 56 161 309 628 126 2 Apparent —true 16 51 109 228 462 diameter The difference between apprrent and true target diameter 1s also tabulated, and 1s seen to be by no means con stant, revealing the madequacy of the notion that an a-ray trach 4s to be regarded as a column of ionization of defimte diameter 5 Rather than the difference of the apparent and true target diameters being constant, the ratio 13 seen to be more nearly constant In particular, there 1s no justification for the tdea: that with large targets the apparent target diameter 1s approva mately correct, while with small targets it 1s made up mainly of the column size and 1s greatly m excess of the true target size The calculation of the relation between target size and 37% dose for any gwen radiation, using the associated volume method and taking é rays ito account, 1s tedious to perform and tedious to deseribe We content ourselves therefore by giving m Figs 8 and 9 the results of the calculation in the form of curves for several different radiations, relating the 37% dose to target diameter or molecular weight An outlme of the method of calculation will be found in the Appendix The result depends on what density 1s assumed for the matenal of which the target is composed Figs 8a and 9 are calculated for a density of 135 g fom 3, this bung about the density of diy protein 2 i Mohler FL & Taylor, LS (1934) " oe) ws desired to assume some other value for the density, Fig 85 Im rent n this figure D/p* 1s plotted against 2rp, D bemg the 37 % dose Thee gens 2r the target diameter in my, and p the density in g /em ? curves are valid for any value of p hod val eget dem aos Same mt] Seneeuetn Wty Tufints Duo 10 2 Ratio Fra 11 Dependenco upon the target diameter of the ratio of the 37%, dose with a given radiation to the 37% dose with X rays(015A) 3 X rays(1 5A) 4, neutrons (Li+D) 5 “rays {41A) 6 Xrays{83A) 7 @ reve {6eMV) § a rays (3eMV} SECONDARY IONIZATION 89 of the number and energies of the é rays 1s required, this being provided by the tables of Chapter rt Wath the aid of this more exact method of calculation we have computed the apparent target diameters to a rays which would be given by the simple method of calculation which neglects considerition of the secondary ionization, for each of a number of (true) sizes of target The results are given in Table 28, and as expected the ‘apparent’ target diameters are greater than the true target Taste 28 Apparent target diameters to a rays of 5eMV ‘True target diameter (mp) + 10 20 40 80 Apparent diameter 66 151 309 628 1262 Apparent —true 16 51 109 228 462 diameter The difference between apparent and true target diameter 1s also tabulated, and 1s seen to be by no means con stant, revealing the inadequacy of the notion that an @ ray trach 1s to be regarded as a column of tomzation of definite diameter b Rather than the difference of the apprrent and true target diameters being constant, the ratio 1s seen to be more nearly constant In particular, there 1s no justification for the dear that with large targets the apparent target diameter 1s approxt mately correct, while with small targets 1t 13 made up mamly of the column size and 1s greatly in excess of the true target size The calculation of the relation between target size and 37% dose for any given radiation, using the associated + olume method and taling d-rays into account, 1s tedious to perform and tedious to describe We content oursels es therefore by giving m Figs 8 and 9 the results of the calculation in the form of curves for Several different radiations, relating the 37% dose to target dhameter or molecular weight An outhne of the method of calculation will be found in the Appendix The result depends on what density 1s assumed for the material of which the target 18 composcd Figs 8a and 9 are calculated for 1 density of 135 g Jom 3, this bemg about the density of dry protemn . I Mohler, FL & Taylor Lb (1934) 2 Ifitis desired to assume some other value for the density Fig 85 isused In this figure D/p? is plotted against 2rp, D being the 379% dose Trcentgens 2r the target diameter m mp, and p the density in g /em 3 es6 Curves are vahd for any value of p RHI it &: = Fe a 50 Fig 11 Dependence upon the target diameter of tho ratio of the 37% dose with a given redietion to the 37% dose with X rays(015A) 3 X rays(1 5A) 4 neutrons (Li+D) 5 \ rays (41A),6 X rays(83A) 7 a rays (6eMV) 8 a rays (3eMV ) @-RAYS AND Y-RAXS gt density The estimates obtained with densely 1onizing radiations (xrays or neutrons) exceed the estimates obtained with less densely ionizing radiations (y-rays or X rays) There are some actions of radiation, namely, the hulling of large viruses and bacteria (discussed in Chapter 1\), nw hich on biological grounds we have not much guide whether to expect a single or multi target model to be required The fact that inconsistent estimates of target size are obtamed when ex periments with different radiations are used, in the sense of the estimate of size being larger for « rays than for y-rays, strongly suggests the necessity for the multi-target theory, with w hich a@ consistent representation of the numerical data can be ob- tamed: In this manner radiation expenments are able to con- tnbute to our knowledge of the nature of viruses and bacteria @-Tays and y-rays The sizes of target which are found in practice for actions of radiation interpreted on the single ionization target theory range from about 4 to 40 myein diameter When a target of this size 1s traversed by an electron hav mg an energy of several hundred electron halovolts, such as 1s produced by y rays or hard X rays it Wall not often happen that more than one cluster of 1omzation will be left in the target Thus to a first approximation we can Tegard the action as due to a single cluster, and the 37% dose 18 that dose which produces an average of one cluster im 1 volume equal to the target \olume Thus y ray experrments determine the total volume of the sensitire region This remains true even if the target 1s not spherical or if there 1s a multiplicity of targets any one of which 1omized causes the effect If an « particle traverses a target of the size mentioned, a large number of ionizations are mevitably produced m the target Hence we can regard every passage of an a-particle through a target as effective, and the 37%, dose 1s that dose which corresponds to an as erage of one « particle crossing each area equal to the area of the sensitive region Thus a ray expert- ments determine the total cross sectional area of the sensitive region, tic A consistent representation could also be obtamed on the assump n ofa single target m the form of a filament of width small compared dumens,length with or of 3 lamina of thickness small compared with its other 90 TRE TARGET THEORY As will appear in Chapter 1x, it 13 sometimes necessary to deduce the targot size not from the absolute values of the 37% doses tor any one radiation, but from the ratto of the values of the 37% dose for two different radiations To facilitate this type of caleulation, Figs 10 and 11 are provided If one has expen mentally determined the ratio of the 37% doses for some densely ionizing radiation (soft X rays, neutrons or a rays) compared to y rays, then Fig 10 enables one to read off the target diameter corresponding to this ratio If the density of the target material 1s 1 g /em 3, the abscissae in Fig 10 are simply the diameters 2r in millimicrons If the density 1s p g /om °, the abscissac are values of 279 Fig 11 gives similar curves for experiments in which X-rays (015A ) have been used in place of y rays The multi-target theory The assumption that tnere 1s a single spherical target, one or more ionizations within which cause the effect being studied, 18 plausible as an interpretation of expermments on the inactivation of small vnuses, and 13 found to represent satisfactonly the de- pendence of inactivation dose on 10n density of radiation There 1s one class of action of radiation in which we should expect to find a multipheity of targets This 1s the production of lethal mutations When Drosophila males are irradiated (see Chapter V) and suitable breeding tests subsequently carried out, the pro- duction of sex-linked lethal mutations mn the irradiated sperm can be established There are, on genetical grounds, known to be numerous genes residing mn the 4 chromosome of the irradiated sperm, capable of showimg lethal mutation, and a model ac- cording to which there are NV’ targets each of radius r, 1omzation an any one of which causes the effect studied, 13 clearly required in this case The modification this mtroduces into the theory 1s smply that the 37% dose 1s N tumes smaller than 1f there were only one target of radius r Apart from its plausibility on biological grounds, evidence indicating that a multi target: model 1s required 3s grven by the radiation experiments themselves For, 1f1n the lethal mutation experiments we attempt to mterpret the experimental data on the single target model, we find that consistent estimates for the target size are not ot d with ra drat: of diffe ton- ASSUMPTIONS OF THE TARGET THEORY 93 tions entering into previous calculations In particular, account 1s taken of secondary 1omzation, and of the fact that the number of r1onizations per micron path of an electron increases towards the end of the path Its beheved that this calculation 1s fairly adequate as regards the manner im which it deals with the spatial distribution of 1omzation, though hable of course to re- vision when better numerical physical data become available The calculation is based on the assumption that an effect to which the single-ionization target theory applies 1s produced when one or more 1omzations occur inside a sphencal target of defimte radius r, and 1s not produced when 1omzations occur outside the target. While the notion of a sensitive region 1s of course basic in the target theory, the actual conditions may be more complicated than those allowed for in the calculation, in any of the following respects (2) The target may not be sphencal (6) When an ionization 1s produced inside the target, the probability of the effect studied ensuing may be less than unity (c) There may not be a sharp demarcation between the inside and the outside of the target in the manner envisaged by the sumple theory, according to which the probability of an 1oniza- tion causing the effect 1s unity 1f the 1onization occurs just inside the target, and zero if 1t occurs yust outside Instead, there may be a diminution of the probability as we move out of the target without any discontinuity marking a precise boundary It 1s not possible to make a quantitative calculation taking these three factors mto account, since we lack the necessary numerical data It 1s, however, possible to see qualitatively in what sense the deductions of the simple theory will be im error by neglect of these factors Further, by examining the extent to which the simple theory 1s capable ot giving a consistent mter- pretation of expermental results, 1¢ 13 possible to form an opmion of the extent to which the deductions of the simple theory will be m error by negiect of these factors We shall find that the error is not v ery great, so that the methed of calculation we have outhned, while admittedly a simplified model, gives results which are essentially correct There are, of course, precedents 1n all sciences for a model factor =simplification of the true state of affairs giving 2 satis- presentation of the essential expermental facts The 92 THE TARGFT THRORY this stat { again g true for non spherical targeta or fo. multiple targets In tho case of non sphencal targeta we mean by cross sectional arca the average area presented to the lonizing particles, allowing for the Presumably random orienta tion of the targets relative to the ronizing particles These stalierzed atatements are of course only approximat ions, and it 18 intended that not these statement s but the curves of Figs 8-11 should be rehed upon in interpreting expernments Using a-rays and y rays as well as radiations of intermediate 10n density Tho itaherzed statoments are, however, of value in that they bring out the physical principl es underlying, for example, the use of a-ray and y ray experiments to determine both the number and the size of the targets when the multi target model 1 being used For, suppose there are N’ sphencal targets, each of radius 7, and suppose that the « ray etpermment has determined their total cross sectional ares, to be A, and the ¥y ray expertment has determined their total volume to be 7 Then Narr=d, Ninr=l, (IL-5) whence, solving these equations, Qr=BVj2A, N=16A%9nrP2, (IIE 6) Since the formula for V mvolves the square and cube of ¥ and A, 1618 strongly sensitive to experimental error sn the determina- tion of the 37% doses The ptlons of the single-tlonization target theory: Calcuistions of the relation between target stze and 37% dose Sor any given type of yad mvolve approximations and smmphfications necessamly troduced to make the calculations manageable Some of these simplifications concern the spatial distrik of the calculation being made on the assumption of a spatial distribution which 19 less complex than the actual distnbution The method of calculation outhned in the Appendix, which leads to the curves of Figs 8~11 relating target size and 37%, dose, has at the expense of a considerable increase in the labour of computation, avoided most of the approxima- t These considerations are to some extent suggested by an article of Fano U (1942) ASSUMPTIONS OF THE TARGFT THEORY 95 in the raho of 0 87f# 1 For a filamentous target 10 times as long as broad this 1s 1 28 4, for a filamentous target 100 times as long as broad 14,15 187 1 It is evident that small deviations from a spherical shape will not prevent the calculation made on the assumption of a spherical target giving a fait representation of the dependence of the efheiency of different radiations on the target size and 1on-density of the radiations In certam applications of the target theory, particularly the production of lethal mutations (Chapter v), we consider the cell to have a large number of spherical targets and use the experi- mental data to calculate the number and size of theso targets We could fit the experimental values of 37% dose for different radiations equally well on the assumption of a single filamentous target, or a small number of filamentous targets On the one interpretation we use the experiments to determine the number of genes capable of showing lethal mutation, on the other we determine the length of chromosome they occupy These are of course related quantities To conclude af we have reason to beheve that a particular action of radiation 1s of the single 1onzation type, and yet find that when we work out the size of the target from expenments made with different radiations we fail to get consistent results, but the estimates increase in the order of increasing 10n density, then a possible explanation 1s that the target 1s filamentous, or else that there are a number of spherical targets, 1om1zation in any one of which causes the effect studied Examples wall be found in Chapter rx illustrating this point (0) Probability less than unity We umagine the target to be spherical and of definite radius r Only 1omzations produced within the target can cause the effect studied, but an 1onization withm the target 1s not certain of causing the effect but has e certain probability less than umty of doing so Clusters of dif- ferent size will have different probabihtes Thus if the proba- bilty ofa single ionization causing the effect is 0 5, the probability of a cluster of two isomzations doing so will be 075, of three ronizations will be 0 875, and so on Call p the average proba- bihty of causing the effect for one cluster in the target When a radiation 1s used which produces clusters of ionization widely Separated along the track of the 1omizing particle, eg y-rays, fadiation experments normally give us the target volume Here 94 THE TARGET THEORY assumption made im the kinetac theory of gases that molecules are hard spheres, and tho concept in genetics that phenotypical characters are determined by single Mendelian characters, are familar examples We proceed to examine the affects of factors (a), (b) and (c) separately (a) Non-spherical target Tho effect of shape of target can be illustrated by taking tho extreme case where the target, matead of having a spherical shape, 1s.1n the form of a long thin filament, 1e a cyhnder of Jength 26 and diameter 2a, the ratio f= 2b/2¢ of length to breadth much exceeding unity Instead of attempt- ing the general solution for a radiation of any 10n-density, 1t will suffice for our purposes to take two extremes, viz. an sonizing particle producing its 1oruzations (or 10n clusters) at such wide separations that two clusters practically never fall in the target, and an ionizing particle so densely 1onzing that 1omzation 13 certainty left in the target whenever the ionizing particle passes through 1t We shall for the present purpose neglect d raya The experiment with the densely ionizing radiation will m effect the area pr d to the radiation by the target For an ionizing particle parallel to the axis of the cylindncal target this 1s 7a?, tor a particle perpendicular to the axis itis 4ab For randomly orientated cylindrical targets of length much exceeding the breadth we can readily show that the mean area presented is mab If now we interpret experments made on a matenal m which the sensitive volume ts a long filament on the incorrect assumption that it 1s a sphere (which presents an area ar?), we shall deduce for the radius of the sphere a value r where ar? =710b, te r=afi Experiments with the radiation of low 10n density will in effect measure the volume of the target, 1¢ 2a’ If we m- correctly assume the target to be spherical, we shall deduce for = 270%, 1e r=a(3f/2)! This 13 smaller it a radius r, where $ar3 than the previous estimate in the ratio 1 0 87f* Thus the in- correct. assumption of a spherical target when the target 1s actually filamentous will lead to the estumate of target radius deduced from experiments made with different radhation being inconsistent The target radius deduced from the expernments made with a densely 1omzing radiation (eg % rays) will exceed that deduced from a radiation of low 1on-density (eg + rays) ASSUMPTIONS OF THE TARGET THEORY 97 ton clusters at wide separations (eg y-rays) the experiment normally determines the total volume V =4{nr? of the target On the present model it will determine rd, 1e the volume mtegral of the probability We have drawn the two curves A and B of Fig 12 80 that fpdv=4ar, 1e so that if in & particular matance there 1s no sharp boundary to the target, and the curve B represents the variation of probability p with distance from P 10 Fra 12 Target with indefimte boundary the centre, then the radius r marked by the square cornered curve A is the radius of target which will be deduced when the experiment with y-rays 1s mterpreted on the assumption of a target of defimte radius When more densely ionizing radiations are used, and the ex- P2rimental data interpreted in terms of the usual theory which supposes a defimte boundary for the target, the radius calculated for the target increases as the 10n density of the radiation in creases Thus the circles C, D, Em Fig 12 represent the sizes of target to be expected for y-rays (1e radius r), and two more densely lomzing radiations D 1s calculated for an 10n-density 06 TUE TARGET THEORY they wall give usp $183, where $n Ris tho target volume Ifwe incorrectly neglect the factor p wo shall calculate an estimate of the target radius r which will bo connected to tho real value R by the relation p 47f?= 4773, or r= Rpt A sufficiently densely tonizing particle pasaing through the target will be certain to couse the effect Hence expenments with auch radiations, © g @ rays, will yield a correct estimate for the target radius The cxustence of the probability p less than umty will thus lead to estimates of the target size being greater for a rays than for y rays, the a ray value being moro nearly correct For a radiation of intermediate 10n density, for which 1¢ 13 necessary to use the method of calculation involving the over- lapping function F (compare p 85), we find that the mtroduction of the probainihty p leads to the result that the 37% dose, 1¢ the dose required for an a. erago of one effective hit per target, 18 SS. where F(£) 1s the function of £ defined by equation (4) but £=2rp/E instead of 2r/L It follows that af we are investi- gating an action of radiation of the lethal mutation type, m which we have a large number of sphencal targets, and deter- mune their size not from the 37% dose determined for any ons radiation, but from the ratio of the doses of two radiations of different 1on-density, then our neglect of the factor p will lead to our estimate of the target diameter being too small im the ratio p 1, and our estimate of the number of targets too large in the ratio 1p? (c) No sharp boundary to the target . Instead of the probability of an 10n cluater causing the effect bemg unity inside the target and zero outside, we suppose that the probibilty dimimushes as we move outwards from the target without any discontinuity In Fig 12 we plot the probability p of an ion cluster being effective as a function of its distance x from the centre 0 of the target The normal hypothesis 1s that the probability 1s umty up to radius 7 and zero beyond, as dicated by the square cornered curve A Qur present, hypothesis can be represented by the smooth curve B The curve we have chosen for illustration 1s the normal error curve, because it has the night general shape and 19 amenable to calevlation With a radiation which produces its 1 Refer to the Appendix for fuller details VALIDITY OF SIMPLE MODEL 99 theory as fortuitous, and due to the different complicating factors happening to cancel each other out The complicating factors are evidently not of sufficient rmportance to invalidate the simple theory A number of other cases are known which, when interpreted in terms of a single spherical target, yield estimates of target size which ure greater for a-rays than for y-rays This discrepancy may be due either to a multiphcity of targets, or to any of the three complicating factors we have been discussing Among these cases 1g the production of lethal mutations mm Drosophila Sperm Here we hnow from genetical evidence that there are Numerous genes capable of showing a lethal mutation When we estimate ther number on the multi-target theory, neglecting complications, the estimate obtained 1s, on genetical grounds, entirely reasonable There 1s no suggestion from these data that neglect. of the comphcating factors has led to a gross over- estimate of the number of targets We are encouraged therefore to beheve that the complicating factors which, if they were of sufficent numerical importance, would cause the estimate of the number of targets on the multi-target theory to be exaggerated, do not mm fact cause gross error 98 THE TARGET THEORY such that an average of five 10n clusters are produced im path length 2r, and E for an ton denatv five times greater The more densely 1om71ng radiation of the two last mentionedis, per ionization, the less effective According to the simple theory with 4 target of fixed rddius r, rt 19 4 63 times less effective On the modol represented by the probabihty curve B of Fig 1211s 2 75 times less effective Thus the absence of a definite boundary to the target wall make the efficiency per 1omzation fall off less rapidly with increase of ion density than if the target has a defimte boundary If1n such a case the target size 1s calculated on the ample theory which assumes oa defimte boundary to the target, the calculated target size will be greater for a densely lonizing radiation than for a less densely ronizing radiation The validity of the simple model That a curve such as Fig 129 13 hikely to exist in practice 1s due to the possibility of the spread of the effect of an :omzation, such as wag discussed on p 67 If we hnew more about the mechanism of this spreading effect we would be in a position to decide whether a curve deviating considerably from the square cornered curve, such as curve B, o1 a curve diffenng from A only by a slight rounding of the corners, were needed to represent this spreading effect Again, if we had more information about the ionic yrelds for chemical change in large protein molecules we would be better able to yudge whether values of p much less than unity were to be expected,: and so cause the deviation from the simple theory discussed on p 95 In the absence of such informa- tion the following consideration 1s helpful All three of the com pheating factors we have considered act in the samo sense, namely, that when target sizes are calculated with different radiations, the estimate obtained from «-ray data exceeds that from y-ray data Or, 2f a Iethal mutation experiment 1s being mterpreted on the multi target theory, the number of targets will be overestimated Now cases are hnown where consistent estimates of target size are obtamed from a-ray and y ray data These cases are the mactz\ ation of the small viruses discussed in Chapter rv Since all the compheating factors act m the same direction, we cannot explain this agreement with the simple nat present. labl 1 The very lunited that p is approximately unity even for large protem molecules (p 38} / NATURE OF VIRUSES 101 other small viruses will be obtamable crystalline when methods have been devised for producing sufficiently pure and concen- trated suspensions, but it 1s very improbable that this will happen with the larger viruses The crystallme viruses are, chemically, nucleoprotein Nucleoprotein 1s a component of living cells, being the substance of which chromosomes are constituted The chromosomes, which are threads of nucleoprotein, constitute the genetical mechanism of the cell It1s because the sperm and the egg each contribute their quota of chromosomes to the fertilized egg that the organ- ism which develops from it inherits characters from both male and female parent In the chromosomes are located the genes, the physical entities corresponding to the Mendelian characters Changes 1n individual genes, or in therr number or arrangement in the chromosome, cause changes in the behaviour of the cell, and tt 1s the genes which largely determine the potentialities for development and behaviour of the cell At cell division the chromosomes split longitudinally, each gene exactly reproducing itself The two halves of each chromosome separate, one going into each daughter cell It 1s by this mechanism that each daughter cell obtains exactly the same complement of genes as the mother cell Evidently a gene has the capacity for synthe sizing from the cell fluids an exact replica of itself Now the viruses have this property of synthesizing exact tephicas of themselves from the cell fluids of the appropriate host plant oranimal Thesmallest, erystallizable, viruses are molecules of nucleoprotein, as are presumably the genes There 1s thus a strong analogy, chemically andain beha. tour, between these viruses and gees, and uf we regard them as iiving things, they are not to be thought of as minute cells so much as naked genes (This does not apply to the largest viruses which probably are mmute cells ) Radiation experiments are consistent with this view of the nature of the crystallizable viruses The smallest viruses can be mactivated by a single 1onization almost anywhere in the virus particleThere are some actions of radiation on higher cells which can also be produced by a single 1onization, providing that it oceurs in the mght part of the cell The best established of these actions 1s the induction of gene mutation Larger viruses show some internal structure when examined under the electron microscope, as shown m the case of vaccinia Chapter IV THE INACTIVATION OF VIRUSES BY RADIATION The viruses: The viruses are agents responsable for a number of infectious diseases in animals and plants In addition, the bactenophages, which attack bacteria, are also considered to be viruses The viruses are distinguished from bactena and other microbes by their small size and their purely parasitte nature Nearly all the viruses are too small to be seen by the ordinary microscope, and they pass through the filters of unglazed porcelain or kreselguhr commonly used to stenlize liquids by filternng off bactena and other small organisms They will only mult:ply on or in hving cells, and have not so far been made to multiply in a non living medium in the manner in which bacteria can be grown in broths of suitable composition, or the cells of higher organisms cultured outaide the tissue to which they normally belong The viruses may be described as occupying an mtermediate position between the obviously hving and the obviously non hving, since they have some properties which we ordinanly thik of as peculiar to life, and others which are m marked contrast to living organisms Their capacity for reproduction 1s the charac teristic in which they resemble hving organisms The fact that some of the smallest viruses are crystalhzable 1s the most striking difference Plate II p, for example, shows crystals of the virus responsible for the ‘bushy stunt’ disease of tomatoes? Tobacco necrosis virus has also been crystallized, while tobacco mosaic virus has been obtained in a pseudo crystalline state (differmg from a true crystil in that the molecules, which are rod shaped, are arranged regularly in two dimensions but with- out regularity in the thnrd dimension, which 1s the direction of the axes of the rods) The crystals are obtained from concen- trated and purified suspensions of the virus It 1s posable that 1 This brief mtrod 13 ed for the of the reader who 18 not B virus sp h It may be d by reading Smith KM (1940) The barus Life ¢ Enemy Bawden, F C (1943), Plant Viruses and Virus Diseases, the chapter on viruses in Northrop J H (1939) Crystalline Enzymes end the chapter on bacteriophage by Delbruck, M in Nord FF & Werkman CH (1941-3) Advances 1n Enzymology 2 smth KM & Markham R SIZES OF VIRUSES 103 low, and a factor of 0 83 18 suggested by Table 29 in which the filtration end-point 1s given for several viruses whose hydrated chameters are rebably determined by other methods When making use of the published results of filtration experi- ments to determme the sizes of viruses, we have recalculated the sizes on the basis of the factor 0 83 relating the diameter of the hydrated virus to the a pd of the filter membrane Taste 29 The filtration of viruses {Diameters un millumicrona} Hydrated Virus diameter apd Retio Phage 813 18 25 072 Bushy stunt 37 40 O92 Shope papilioma ch] 76 Lot Phage Staph K 73 110 066 Vacema 200 250 080 Mean ratio 083 A number of phages have been investigated by Schlesinger by the sedimentation method,: and the size calculated on the assumption of a phage density of 110 or 112 We have re- calculated these sizes using instead the density of 122 now believed to be more probably correct : For the purposes of the interpretation of the radiation expen- ments, the dry size 1s more significant than the wet mze This 1s obvious when the experment has been made on the dry virus It 18 also so when the experiment has been made in solution, since there are mdications (see p I11) that the ome yelds for inactivation by ionization of the virus protein itself 18 con- siderably greater than the ionic yield for indirect inactivation following iomzation of water When the size has been determined by & method, such as filtration, which gives the size of the hydrated particle, 1t 1s therefore necessary to deduce from this the swe of the unhydrated particle The phages have, when hydrated, a density of 122 We have arbitranly assumed that when dry they have a density of 1 35, this bemg a typical figure for the dry density of such plant and animal viruses as have been obtamed sufficiently pure for the determm ation of density It will be realized that there 1s some uncertainty attaching to the sizes of most of the viruses In the hst of sizes wncluded in 1 Elford, W.J mm Doerr R & Halleuer, C (1938) 102 INAOTIVATION oF VIRUSES BY RADIATION virus in Plate IVp,: and probably resemble higher cells in that the genetically :mportant nucleoprotem comprises only a part of the virus The sizes of the viruses Know ledge of the s:zes of the viruses 18 of some importance in the interpretation of oxpenments on their inactiy ation by radia fion Tho methods available for the estimation of the sizes of viruses? aro, in addition to ultra violet hght photography, which 1s only possible with the largest viruses, sedimentation on High speed centrifuges, diffusion, filtration through graded collodion membranes, electron mucrography, and X-ray diffraction Determination of the density 1s necessary in interpreting sedi- mentation results, and viscosity measurements are sometimes helpful There are a few viruses for which consistent estimates of size have been obtained by several different methods, and the sizes of these can probably be considered known to an accuracy of 10% or better These are the viruses of tomato bushy stunt, tobacco mosaic, vaccinia, and the Shope rebbit papilloma All four of these viruses appear to be hydrated in solution, 1e to take up water with resulting increase of size and diminution of density When dried, the water 1s lost without permanent loss of infectivity, the reduction of diameter on drying being from 15 to 30% It 1s a natural presumption that hydration of viruses in solution 1s & general phenomenon In the case of a number of viruses which have been used m radiation experiments, the sizes have so far only been estimated by filtration Filtration through Elford’s ‘gradocol’ membraness 1s capable of yielding consistent estimates of the average pore chameter (a p d ) of the filter which just stops all the virus The principal source of uncertainty attaching to this method 1s the ratio of virus diameter to apd Elford has recommended the use of the ratio 3-} for a p d’s of 10-100my, and }-} fora pd’s of 100-500myz It appears, however,« that these factors are teo 1 Green, RH, Anderson, TF & Smadel JE (1942) 2 For a review of the methods see Markham R Smith KM & Lea DE (1942 1944) 3 See an ariscle by Elford WJ in Doerr R & Hallauer C (1938) Handbuch der Virusforachung 4 Markham KR, Smth Kv & Lea DE (1942 1944) ESTIMATION OF VIRUS ACTIVITY 105 about twice the theoretical mintmum value When the sssess ment 1s made on the average count of three or four plates eaclt containing about a hundred plaques, an accuracy of about 10%, 1s commonly obtained The purely statistical error, determined by x}, may be re- duced in methods: in which a culture of bactena m a hquid nutrient medium 1s moculated with a quantity of phage suspen- sion containing from 104 to 10° phage particles The lysis of the bacterial culture 1s followed by observation of its diminishing opacity, and the time required for a standard culture to be lysed to a given degree 1s determined Exact standardization of all conditions 1s of course imperative in this method, and it 13 1n less general use than the plaque counting method Plant viruses are most easily assessed by the local lesion tech- nique In this method a drop of the virus suspension 1s rubbed on to a leaf of the susceptible plant with a glass spatula After a few days local lesions appear (as in Plate I1c),: these marhing the points where virus has entered the leaf The method 1s not universally applicable, since with some viruses and some host Plants the plant may become infected without the appearance of local lesions at the points of entry It appears that a local lesion can be caused by the entry of a single virus particle, but determination of the weight of a pure virus preparation required to be rubbed on toa leaf to give one local lesion makes it evident that the number of lesions obtaimed on a leaf 1s only a minute fraction of the number of virus particles rubbed on to the leaf The number of local lesions obtained with a given quantity of virus depends on factors such as age of plant, size of leaf, tech Muque of the operator, and whether or not carborundu m powder or sand has been used to merease, by abrasion, the number of Possible pomts of entrv on the leaf It will be realized, therefore, that while the lesion count 18 @ measure of the activity of a virus preparation, the assessment. 1 hable to considerable experime ntal error This 1s reduced to a minimum by use of suitable procedures One such 1s the Latm square method, in which, for example, if the activities of fie preparations are to be compared with the aid of five plants each bearing five leaves, the inocula tions are arranged so that each virus preparation 1s moculated once on to each plant and once in 1 Krueger, A P (1930) 2 Smith KM & Markham R 10£ INACTIVATION OF VIRUSES BY RADIATION Table 33 wo have prefixed by the sign ~ the sizea which are regarded ag least rehable, either ov mg to their being based ona single method of size determination, or on two methods which have not agreed well The estimation of virus activity For oxperments such as the investigation of the inactss ation of viruses by radiations, quantitative method ofassessing s1Tus Activity 19 needed Since viruses cannot be grown on artificial media, the test of activity necessanly mvolves inoculating the appropriate sensitive organism wath the virus under test, and the methods of test are thus different for plant viruses, for animal viruses, and for bacteriophages The quantitative assessment 18 most accurate m the case of the bactenophages The method used 1s a8 follons A plate of a solid nutnent medium suitable for the growth of the bacterum concerned ts inoculated with 2 drop of bactenal suspension contasning o few million organisms, and the together with a measured drop of the phage suspension, medium liquid umformly spread over the surface of the nutrient with o glass spreader On incubation of the plate, the heavy us film of bacterial bacterial moculum results im a continuofor growth on the surface of the plate, exceptclear areas caused by the multipheation of the mdividual phage particles causing lysis of the bacteria in their immediate vicinty These clear areas or ‘plaques’ are easily countable with the naked eye, and are proportional to the shown in Plate IIs The number of plaquesng1s an strength of the phage suspension (providi excessive phage moculum 1s not used, mn w hich case the plaquesualwillrun together), and 1s not far short of the number of individ phage particles the precision obtamable 1s Immited put on to the plate « As in any counting method estimation of a phage by the number of plaques counteofd7 The s 1s subject, on this concentration based on 8 count onplaque of 100/n?% In practice, account alone, to a standard deviatiby errors , g the dilution further uncertainties are introduced tration suitabl e for mocula- of the phage suspension to atheconcen size of drops delivered by the tion, and by variation im e used to measure the moculum, be 50 standardized dropping pipett practice 18 hkely to that the standard deviation obtained ant that the ratio 1s about 1 2 x1 Eilts, EL & Delbruck, M (1938) augges TECHNIQUE OF VIRUS IRRADIATION 107 and 1s also sampled at the beginning and end of the experiment to check the possibshty of spontaneous acti ation occurmng In this way a curve is obtained (eg Fig 13) of virus activity against dose, and from this may be read off the dose required for —— , : 7 0 * 0 A . ‘ : Te € g 2 & i=] 5 B 2 c z t 2 iT 4 i) ity pty 3 10"r Dore Fio 13 The mactiation of plant viruses by radiations @ Virus irradiated ary © virus a: d in aq ix A TM rays (1 5A ) on tobacco necrosis virus, B y rays on tomato bushy stunt virus (Lea & Smith) any percentage reduction of activity It 1s strongly to be recom- mended that this procedure be adopted, rather than the de- termination of the dose which completely inactivates a virus preparation Experiments on suspensions are hable to be complicated by an indirect mactiy ation following ionization in the water rather than m the virus particles, and if st 3s desired to study the direct effect, irradzation of the virus dry 1s to be recommende d This Procedure also has the merit of enabling soft X-rays and & particles and other easily absorbed radhations to be used Fortunately, many of the viruses can be dned without loss of activity The addition of 4% of lactose to the solution before drying may be advantageous in the ease of a virus lable to loss of activity on dry ing 106 INACTIVATION OF VIRUSES BY RADIATION cach leaf position A rather simpler procedure, when deter mining, for example, tho actinity rematmng after several dif- ferent exposures of radiation, 18 to inoculate half of every leaf with the unirradiated virus, and the other half with the srad) ated virus This method depends on the fact that the two halves of a leaf ore more nearly alkke in sensituvity than different leaves By tho use of about ten half leaves to each virus preparation, an accuracy of about 20% im the estumation of relative virus con- centration may be expected The estrmation of animal virus activity may be exemplified by describing the assessment of vaccmia virus When & suspen- sion of this virus 1s introduced into the skin of a rabbit by means of a hypodermic needle, a red swelling appears m the course of few days at the ate of the moculation To compare the actinities of a senes of virus preparations, the hair is clipped from the back and flanks of a rabbit and the shin marked out into squares, s3y fix rows of ght squares each Each row serves to test one Vitus preparation A senes of eight tonfold or threefold dilutions of the suspension are made and a measured volume of each dilution in jected The most dilute moculum will contain no vitus and give rise to no lesion, the higher concentrations will give lesions The highest dilution at which 2 Jesion appears 1s a measure of the strength of the virus preparation Plate IL shows the flank of a rabbit carrying two rows of squares : The highest concentration 1m each row 2s moculated at the head end of the animal, and pro duces the largest lesion The end-point in this particular expen ment was reached at the fifth dilution of the series A single virus particle suffices to produce a lesion,» and the degree of variation of the end-point obtaimed when the same virus preparation 18 inoculated in several mdependent dilution series 38 no greater than that statistically mevitable » Technique of virus irradiation experiments If the virus 1s to be srradtated wet, a tube of the suspension 18 exposed to the radiation and samples taken at intervals and the activity aasessed by the appropnate methods, as outlined in the Previous section A control preparation 13 maintamed under conditions as comparable as possble, apart from being irradiated, x Salaman MH 2 Parker RF {1938} 3 Lea DE & Salaman MH (1942) DIRECT AND INDIPECT ACTIONS 109 sion the mactivation is partly direct, presumably by the inter- mediary of the same activated water wiuch is responsible for the chemueal effects of radiation in solution, but that broth or gelatin protect the virus by competing for the actrvated water rodIeniaoctgsnovaretison Se Ss. 4 & 10 3 = 1 n n 2, n 107 yo 103 10-4 10> 197? tom 1 & protein per ml fig {4 Dependence of mactivation dose on protem concentration @ Mamly virus protem, x mainly extraneous protein A Tobacco mosaie virus (Lea Smith, Holmes & Markham) B_ rabbit pay virus (Fried aa Anderson) Somewhat similar results have been obtained: with a punficd Preparation of tobacco mosaic virus The preparation was irradi- ated in several different concentrations and also dry Yhe macti- vation dose was the sume dry and in concentrated solution, but was legs in dilute sohition The addition of gelatm tu the dilute solution resulted in the activation dose being rused almost to the value for the dry preparation ‘The results are eahibited an i Lea DYE Smith KML Holmes BOS Markham R (1944) 108 INACTIVATION OF VIRUBES BY RADIATION A method of working with dry viruses which has been found satisfactory 18 the follonsmg A drop of molten 5% gelatin 15 apread over n cover shp and dned off ona hot plate A drep of & Vitus suspension is put on to the cover slip with a calibrated dropping pipette and allowed to dry, leaving a circular deposit 6-8 mm indiameter After nradiation the cover slp 1s dropped ito a measured quantity of water at 40° when the gelatin film almost instantl dissolves and the virus 15 resuapended Pro viding the orgmal virus suspension contained not more than say 1% of solids the layer of dried varus on the cover slp has a thickness of only a fow microns and may be used with soft radiations Direct and indirect actions of radiation on viruses Ifa virua is irradiated in aqueous auspension, it 18 clearly of first importance in the interpretation of the results to deerde whether the action 1s direct and due to the romation of the virus particles by the passage of :omzing particles through them, or mehrect due to 1omztion or exeitation of the water molecules leading for example to the production of free radicals, which then affect the virus The tests for distinguishing between these types of action have been discussed in Chapter The principal test 1s that 1f mactr ation of the virusis mainly due to the forma tron of ‘activated water’ the inactrvation dose: should increase with increasing concentration of the virus, and should also be mereased by adding protective agents to the solution, % hich are capable of competing with the virus for the actuated water If the action 1s a direct action on the virus without the inter mediary of activated water the 37% dose should be mde pendent of the concentration of the virus or of the addition of protective agents Luna and Exner: found that when bactenophages were irradiated in aqueous or saline suspension the rate of mactivation was more rapid than in broth suspension Addition of gelatin to the aqueous suspension reduced the macta ation rate to the value found in broth The indication is that in aqueous suspen «Lea DE & Salaman WH (1942) 2 Defined aa the dose reducing activity to 37%% of the initial activity (see p 74} 3 Luna SE A Fxner FM (1041) IONIC LIELD FOR INDIR}IOT ACTION ly sufficient to make the indirect action of smaller importance than the direct action Fig 148 shows how the inactivation dose of the Shope rabbit papilloma virus radiated by X-rays depends on the concentra- tion of the solution which 1s irradiated : The general shape of the eurve resembles that obtained with tobacco mosaic viTus (Fig 14.4), and the explanation 1s presumably the sime There 1s some mdication with both viruses that the inactiva- tion dose does not continue to diminish indefinitely with diminu- tion of concentration, though the experimental evidence 18 at Present not conclusive on this point This may be due to the majonty of the active radicals in a very dilute solution re- combining instead of reacting with the solute, as discussed in Chapter m (p 57) On the other hand, since specially purified water was not used in these experiments, the effect may be due to the unmntentional presence nm the water of a minute quantity of an impunty having a protective effect The curves in Fig 14 are theoretical curves calculated accor ding to equation (II-9) (p 62) on the provisional assumption that the water contamed a concentration of :mpurity having a deactivating efficiency equivalent to 61% 10-5 g/ml of virus (Fig 144), or 25% 10-6 g fml of virus (Fig 143) The ratios of efficiencies for wndirect and direct action needed to make the calculated curves fit the observations are y/I"=26x 10-4 (tobacco mosaic varus), and y/'=46%10-* (rabbit papilloma virus) With both viruses the ionic yield 1s evidently very much smaller for the mdirect than for the direct action EVIDENCE THAT VIRUS INACTIVATION IS DUE TO A SINGLE IONIZATION ‘The remamung discussion of this chapter will be concerned with the direct action of radiation on viruses, and we shall therefore confine ourselves to experiments in which the virus was either utadiated dry or, 1f m solution, in the presence of a sufficient concentration of protem, whether virus protein or extraneous Protein, for the indirect cffect to be ummportant This actually includes the majonty of experiments, since it 13 only by careful Punfication of the viruses that the evperiments can be carned t Fnedewald, WF & Anderson RS (1940 1941) as worked up by a, DE, Smith, K MW Holmes, B & Markham R (1944) NO INACTIVATION OF VIRUSES BY RADIATION Table 30 and in Fig 144 The conclusion 1s that the indirect action can be largely inhibited by use of a sufficient concen tration of protein, whether virus protein or gelatin In dilute solution, however, the indirect action predominates The shape of the survival curve 1s not a test of whether the action 1s direct or indirect, exponential curves being obtamed in either event, as explamed on p 61 It appears from Table 30 that when a preparation of tobacco mosaic virus contains 0 00022 mg /ml the inactivation dose 18 half as great for combined direct and indirect action as for direct TABLE 30 Inactivation dosea of various preparations of tobacco mosaic Virus! Concentration in g/ml of aA Inactis ation dose Virus Protective agent Qn uruts 10? r) Solid — 25 014 _- 29 0022 =- 29 ; 0 00022 _ 15 0 000022 - 05 0 0000014 _ o6 0.000022 Glucose 005 Os 0 000022 Gelatin 0001 24 0 000022 Gelatin 0 01 24 action alone Comparing with equation (II-9) (p 62) we deduce that the ionic yields for indirect and direct action are in the ratio of y/['= 1/4000 apprommately It 1s not surprising to find that with these very large molecules an ionization inside the molecule 2s more hkely to produce inactivation than an 1onzation in the water outside, even in the absence of any competition from protective agents Less punfied virus suspensions, contamung foreign proteins and other 1mpurities capable of exerting a protective action, ore found to be mmactivated at the same rate in solution and dry The curves of Fig 18, obtamed with plant virus preparatzons,: illustrate this pot The explanation 1s that at any dilution at which virus activity 1s ngh enough for the purpose of estuna- tion, the total concentration of virus plus protective agent 1s 1 Lea, DE Smth, KM Holmes B & Markham R (1944) 2 Lea, DE & Smith KM (1940 1942) Gowen JW (1939) also finds that the rate of snactivatron of plant viruses 13 the same wet and EXPONENTIAL SURVIVAL CURVES 113 ated by various radiations: Additional examples may be found im the papers from which these curves are taken The loganthm of the surviving fraction has been plotted against dose, so that an exponential survival is shown by the points lying on a straight Seosnlty 7 frlascutorivgvnin,g ano©lf 1roaifl =} 1 n ° i 3 n10'r Dose Fia 16 Exponential survival curves of irradiated anumal vin NSE {154A ) on vaccinia virus (Gowen & Lucas), B, a raya on vecema,vureatees Sslaman), C, X rays on the Shope tabbit papillorna virus (Syverton ef al} line within the error of the expernment The error in the assess- ment of virus activity 1s sometimes rather large, but no syste- matic deviations from exponential survival, as distinct from vandom variations, are suggested by the curves 1 Gowen, J W & Lucas, AM (1939), Lea, DE & & 1942), Wollman, E & Lacassagn - » Lea, mith, K Mf (1! e, A (1940), Wollman E , Holweck, & Luna, S$ E (1940) § yverton, F JT, Berry,G P & Warre: Luna, pa SE & E e wnputlshes y (1941), Lea, DE FM D treat & Salaman, y MH {942 1120 INACTIVATION OF VIRUSES BY RADIATION out in a sufficiently low protein concentration for the indirect effect to show up All detailed investigations which have been made of the in activation of viruses by romizing radiations point to the inactiva- tion of a single virus particle being causable by a single romzation The teats by which actions of this type may be recognized have been hsted in Chapter 111 (p 72), and we proceed now to give the experimental results of the apphcation of these tests to the viruses Exponential survival curves In Figs 13 and 16-17 we give a selection of surviy al curves for various plant viruses, animal viruses, and bacteriophages, irradi- =o ¢ g 3 2 & wo e B z & 3s 2 3 4 5 6 7 x08 ergsiem* Incident enetgy Dose we Sec tate warts X B ultra violet hight (2536A ) on tobacco necrosis, J survival curves of mradated plant viruses (Lea & Smith} virus C y rays on tobacco mosaic virus INTENSITY AND ION-DENSITY 115 Taste 31 Independence of inactivation dose on intensity Taactation Intensity Virus Radiation Intensity dose ratio Referer Tobacco necrosis: Ultra violet 67x10 13x 104 120 1 r light {ergem ~*sec “!) (ergem ~*) (25364 ) 56x10? 19x 108 (ergem ~*sec -4} (ergem —3) Vacemis X rays 10x 104 104x108 r wt z (5A) {r/min ) 8 32x 10? 098x 10%r (r fin ) Staph K phage X rays 134% 10¢ 0935x lor 2s 1 3 (154) {r fn ) 472x103 0 822x 10Sr {r /mun } Coh C36 phage rays 1 54x 108 508x108 r 19 1 3 {(15A) {x /roin ) 813 x 107 r 417x108 {t fran } t Lea DE & Smith KM (1940) z Lea DE & Salaman MH (1942) 3 Lea DE & Salaman MH (unpublished) Taste 32 Dependence of inactivation dose on ton density of radiation (Ion density increases from left to mght in the table All doses in units of 10° ¢) Radiation Reference ——~u—— XA rays X rays X rays Virus OTA L5A 21a Strains of tobacco mosate virus Mosaic 007 025 050 I Aucubsa 009 015 045 A denvative oll 022 0 X rays X rays @ rays y rays A 83A ~4eMV Tobacco mosaic virus 037 043 149 190 2 Tobacco necrosis 067 Oo S15 — Tomato bushy stunt 045 062 310 256 Vacemia virus 0 080 0 104 _ O21 3 Dysentery phage $13 058 099 _ 350 Coh phage C36 O21 043 — 094 ‘ Staph phage K 0079 0109 ~ 045 X rays X rays O1BA YA a rays Dyeentery phage C16 0039 «= 4s, 030 5 1 Gowen JW (1940) virus irradiated in solution 2 Lea DE & Smith KM (1942) virus irradiated dry 3lea DE & Salaman MH (1942) virug wradiated dry 4Llea DE &S IH lished: ) virus d dry wos soman E Holweck F & Luna SE (1940) irradiations made in ll4 | INACTIVATION OF VIRUSES BY RADIATION Independence of inactivation dose on intensity Only a fow oxperiments havo so far been made to test whether the effect of a given doso of radiation ws independent of whether 1f 18 spread over a long time at low intensity or concentrated in ONfSOna=Molot oS flrasocurtvigovni,g wnlt rt 1. 1 2. 3 ae i Can a 6 t0'r Dosa Fie 17 Exponentral survival curves of irradiated bacteriophages A X rays and f rays on phage {0'7A} on phage S13 {Wollman & Lacassagne) B a ravs phage C16 (Wollman Holweck & Luria) © X rays (hard) on C16 (Lure & Exner) D rays on phage C36 (Lea & Salaman) E X rays (1 5A ) on phage Staph, K (Lea & Sataman) a short time at igh intensity The results of these expernments are collected in Table 31 They are considered to show that there VIRUS SIZE AND INACTIVATION DOSE ly Fig 18 exhibits the same data as a dot diagram relating 10 activation dose to virus diameter, and a correlation in the direc tron of small virus size being associated with large inactiy ation dose: 1s unmistakably visible rdoeIomnatcsgievnatsion = pe tl x 0 Pu 40 60 8 100 200 3m Virus diameter in milimicrens kia 18 Relation between virus diameter and activation dose (for NX and y rays) Curve Ais the calculated relation between target diameter and macti vation dose of A rays of0 ISA on the assumption that there 1s a single spherical target, one or more ionizations in which suffice for the activation of the virus @ Phage © plant virus x animal virus In Fig 1918 plotted im similar manner the inactivation dose for « rays agamst the virus diameter Only a few viruses have been irradiated with & rays, the mactivatron doses for the six points in Fig 19, which were measured by Wollman, Holwech and Luna, Lea and Smith, and Lea and Salaman, have been 1 The existence of a correlation of this sort has been pointed out by Wollman, E & Lacassagne A (1940) for bacteriophages x {IG INACTIVATION OF VIRUSFS BY RADIATION 18 no variation of inactivation dose with intensity outside the error of the experiments Dependence of inactivation dose on fon-dens{ty of radiation Tn Table 32 are tabulated the results of expernments designed to determine whether the inactivation dose ts different for dif- ferent wave lengths nnd types of radiation It 1s advisable in making experiments of this sort to carry out the treatment with the different radiations on samples prepared from the same bateh of varus treated and estimated as far as possible identically It 15 not advisable to attempt to compare the mactivation doses for different radiations when these have been determmned by thfferent authors Table 32 shows unmistakably that the mactryation doses in crease in the order y rays X rays, and @ rays, and that when different wave lengths of soft X-rays are employed, the inactiva tron doses increase with increase of wave Jength No apprenable difference is found if attention 13 confined to different wave lengths of hurd X-rays + This general increase of :nactivation dose with imnerease of 102 density of the radiation 1s to be expected for actians of radiation caused by a single tomzation (ep Chapter rm, p 72), and to gether with the indications provided by the shape of the survival curve and the mdependence of inactivation dose on intensity constitutes the experimental evidence for regarding virus 10 activation as an example of this type of action Relation between virus size and inactivation dose Having estabhshed that the mactivation of viruses 18 & type of action to which the single 1omzation target theory should be appleable, Fig 84 of Chapter mt may be used to determine the target size from the experimental inactivation dose Its of par- fonlar interest to compare the target size with the size of the yirus In Table 33 are given the activation doses of a number of viruses radiated with X- or y-rays, together with the sizes of the viruses 2 ry Jura, SP & Frner, FM (1941) 2 We omit from Table 34 and Fig 18 tobacco mosai¢ virus and potato virus 1 since these are rod shaped, and while of definite diameter, have very variable lengths The length of the mmmum infective unit 13 not fnown VIRUS SIZE AND INACTIVATION DOSE 119 sted in Table 32 The correlation-bety cen inactivation dose and ‘varus 8128 18 strongly marked In Fig 84 of Chapter 111 we gave curves for various radiations of the 37% dose to be expected for an action of radiation due to | u rdoIeinoanctsngvaetiosn 10° Sasa oe Oe sD L 0 20 40 60 BU 100 200 Varus diameter in millimerons Fie 19 Rel between virus A and inact dose {for a rays) Curve A 13 the calculated relation between target diameter and inactivation lose of 4eMV a rays on the assumption that there 19 @ single spherical target one af more iomzations in which suffice to cause the mactivation of the virus @ Phege © plant virus x animal virus the production of one or more 1omzations na sphencal target of Given chameter We have transcribed the appropriate curves from Fig 84 to Figs 18 and 19 It1s seen that these calculated curves show a relation between macti ation dose and target size very similar in trend to that found between mactivation dose and “arus size experimentally « If the mactivation dose were used to 1 The agreement was even ncloser in sey close Tin ne eral instances when the pub hished valine ar. 118 INACTIVATION OF VIRUSES RY RADIATION Tavie 33 Relation betwoon size of virus and inactivation dose (of \ or y rays) {All doses in umits of 10% r All virus diameters refer to the unhydrated virus) Diameter Inactivation Virus my dose Reference Phage 813 16 039 1 . 16 058 2 Tobacco ringspot w 048 3 Tobacco necroma 25 067 j Tomato bushy stunt 29 045 3 Phage C13 ~3l 012 4 Freephalitis (St Louis) ~ 37 ~Ol 5 Phage C36 ~42 010 i . ~42 021 2 Phage megathersum ~ 43 009 r Shope rabbit papilloma 48 O44 6 Phage P28 50 009 4 Phage Staph K GL 0 054 1 . 4 64 0015 4 , 3 64 0079 2 Phage C16 ~68 0054 1 . ~ 68 0040 4 ~68 0.039 7 Phage T1052 68 0059 1 Fow] plague ~80 ~Ol 8 Phage PC 80 004 9 Phage subtilis ~110 0044 1 Vacema 200 008 10 Phage Streptococcus B oad 020 pas ” c _ on un : D — 008 n Shope rabbit fibroma 170 0008 12 + Wollman E & Lacasaagne A (1940) From the tabulated data mven by these authors logarithmic survival curves have been drawn and the 37% doses read off The X ray dose rates given by the authors refer to the surface of 8 layer of broth lem deep and it 18 necessary to convert them to average dose tates in the hquid by multiplying by 052 which factor we have calculated to take into account diminution of intensity in the quid by absorption and by increasing distance from the target of the X ray tu 2 Lea DF & Salamen MH (unpublished) 3 Lea DE & Smith K.M (1942) 4 Luna,SE & Exner, FM (1941) 3 Moore HN & Kersten H {1937} The mactivation dose can only be m ferred in order of magnitude from the incomplete data given by the authors 6 Syverton JT Berry GP & Warren SL (1941) 7 Wollman E Holweck F & Luna SE (1940) & Levin BS & Lomniski I (1936) The imactivation dose 13 inferred in order of magnitude from the fact that a dose of 10* to 15x 10"r produced about the same effect as a dilution of § x 10* times 9 Luna SE & Anderson TF (1942) quoting Luna SE & Exner FM (unpublished) The inactisation dose 1s not grven explicitly but 1s inferred in the statement that the target size 1s the same as previously determmed for phage C16 1o Lea DE & Salaman WH (1942) ix: Exner FV & Luma SE (1947 1z Friedewald WF & Anderson RS (1943) VIRUS SIZE AND INACTIVATION DOF 121 example, 1s a reasonably rchable estimate of the actual size of a gene Having stressed the importance of the general measure of agreement between the experimental pomts and theoretical curves of Figs 18 and 19, we proceed to discuss the detatled discrepancies It. 1s notrceable that for the larger viruses the dis- crepancy is in the direction of the target size being less than the TABLE 34 Inactivation of phace S13: Radiation fraya Nrays(15A) 2 rays (4eM¥ ) Tnactivation dose 058 099 35x 10%r Target diameter 135 159 16 3mp Unhydrated virus diameter = ]6my (hydrated 18mp) virus size This tendency 1s, however, not noticeable for the smaller viruses The smallest virus so far studied 1s the dysentery phage $13, and it happens that the size of this virus 15 furly tehably known: In Table 34 we see that the estimates of the target size by three different radiations, read off for the Corresponding inactivation doses from the curves of Fig 84 (Chapter 1) are mutually consistent although the mactivation TABLE 35 Inactivation of Staph phage Kr X rays a rays a rays Radiation y rays (5A) (TeV) (4eMV5 Tnactix ation dose 6079 0109 O21 045x10%r Target diameter 31 40 58 50myz Unhydrated virus diameter =64mjt (hy drated 75 my) doses covered a range of & 1, and that they agree with the size of the virus deduced from filtration, sediment ation and diffusion studies As an example of the results obtained w ith a somewhat larger virus for which the calculated target size 1s a little smaller than the virus size we give in Table 35 figures tor Staph phage K It 1s seen that not only are the calculat ed target sizes rather X ra RE & Salaman, MH (unpubli shed) 2 the thods of size d used with phage pl s $13 and h Sctermine the hydrated sizes The unhydrated sizes given in a a5 are calculated from the Tables 34 hydrated sizes on the arbitra fon | ‘at on drying they ry assump- take the density 135g fem® whiel: 1s typical or dry plant viruse s and dry protein genera lly 120) INACTIVATION OF VIRUSES BL RADIATION calculate tho target size, the diameter obtained would be withm a factor of two of tho virus size for twenty out of twenty three points in Fig 18 (fifteen out of exghteen viruses), and within a factor of 1 & for five out of ax viruses shown in Fig 19 Thisis tho basis of the proposal: that determination of the inactivation doae can be used to estimate the size of a virus The correlation between the calculated target size and the size of the virus 13 too close to be regarded as accidental In many inatancea the difference between virus size and target size 1s within the range of possible uncertamties in the size of the virus, calculation of the theoretical curve, and etperimental determina tion of the mactivation dose This measure of agreement 18 of importance in the theory of the biological actions of radiat Tt happens that quantitative radiation studies of the viruses have been undertaken only fairly recently Results had, however, been obtained earlier, par ticularly in the study of radiation-induced gene mutations and the action of radiations on bacteria, which suggested the applica tion of the target theory From these results target sizes were calculated, but since no alternative means were available of determining the target size, the explanation was rather hypo thetical For many viruses, however, 1t appears that at any rate to a first approximation the target 13 identical with the virus itself, and that the method of calculation proposed for deducing the target size from the activation dose yields a figure im fair agreement with the size of the virus by non-radsation methods The estabhshing of the validity of the target theory for the viruses does not, of course, prove its applicability to other bio Jogical actions of radiation It does, however, show that the minute amount of energy represented by a single 1omzation can produce an observable effect, an assumption which had to mans workers seemed unplausible It shows further that when the target theory 1s applicable, the size calculated for the target 1s rather close to the real size of the biological entity concerned, suggesting that the target size calculated for gene mutation, for using Elford s factor relating virus 8126 to pore diameter of the membrane were used mstead of the somewhat larger sizes now thought more probable E (1940), 1 Gowen J W (1940), Wollman E Holweck F & Luna,S Lea DE (19406) Lums,SE & Exner FM (1941) STATUS OF THE VIRUSFS 123 in this phage no differentiation between radioinsensitie and radiosensitive regions 1s plausible enough With viruses of medium size such as Staph phage K which we have just been discussing, while the discrepancy between target gize and virus size suggests such a differentiation one 1s not pre- pared uncompromisingly to insist on it in view of the fact that over simplification in the mode) 18 a possible alternative ex- planation of a small discrepancy of this sort However, the position 1s different im the case of the largest virus so far studied, namely, vaccima virus which in Figs 18 and 19 1s represented by the experimental points farthest from the theoretical curves Tasie 36 Non applicability of the angle spherical target theory to a large virus (vaccima): Radiation y rays \ raya (1 5A) a@ rays (BeWY ) Inactivation dose 0080 0 104 O21 x ser Target diameter 1 41 TO mye Mean unhydrated virus diameter = 200my (hydrated 235m) The sive of this virus 1s known fairly rehably and there 1s No reason to suspect serious efror in the radiation data The discrepancy shown by Table 36 between the target sizes for different radiations and between the target sizts and the virus size, 18 too great to be ascribed either to expermmental error or to over simplification of the model There 1s therefore m this virus definitely a differentiation be- tween radiosensitive and insensitive maternal and the radio sensitive part amounts (in volume) to less thin 1% of the whole virus In this respect vaccinia resembles bactema more than the smallest viruses and we shall defer fuller discussion of it until Chapter 1x The status of the viruses In the troduction to the present chipter 1t was mentioned that ino typical cell the highly specific and genetically important nucleoprotein constituting the chromosomes comprised a rela- tively small part of the whole cell. In the small erystafhzable vituses there 1s no such differentiation between genetically im- portant chromatin and cytoplasm the virus bemg composed of 1 Lea, DF & Salaman, MH £1942) 122) INACTIVATION OF VIRUSFS BY RADIATION smaller than the virus size, but that they increase in the order y roya, X rays, a rays In Chapter nr(pp 92-08) we discussed possible over eimplications of the ample model on which target sizes are calculated, and in particular the posstbility that the probability of an somzation causing mactivation might in certain places have a valuc intermediate between the extremes nought and unity which alone are contemplated by the simple mode) Tt was shown that over-simplification of this sort could lead te the calculated target atze being less for y rays than for « rays, and it 1s posable that the results with Staph phage K are to be explained im this manner On tho other hand, one might expect the same over simphification to lead to a aumilar discrepancy 10 the caso of the dysentery phage, where it 13 not found Alternatively, calculated target sizes less than the virus size could be interpreted to mean that not the whole of the virus was tve By rad 1ve we would mean here parts 80 easential to the infectivity of the virus that chemical change produced in them folfowing ronization would lead to loss of in- fectavity By radtomsensitive we would mean parts in which ionization could be produced (and presumably cause chemical change) without loss of mfectivity In higher cells, of course, only a very small proportion of the cell material 1s so essenttal that a amgle ionization can lead to a detectable effect Interpreting the results with Staph phage K along the lines suggested on p 91 one would infer that about 12% of the virus was rad. tive, the rad. tive matertal not being concen- trated in a single spherical mass but being e:ther a lamina, or a filament, or being multiple + Electron merographs of the larger phages show 2 some internal structure, indicating that the phage particle 1s not of a unsform composition throughout and thus may well be differentiated mto tive and radi tive parts On the other hand, several of the small plant viruses have been crystallized and thesr particles are believed to be single molecules of nucleoprotein If this 1s true also of the even smaller phage S13, the deduction from the radiation experiments that there 1s 1 On the mult: target theory the best fit of the experimental inactiva tron doses ws given by the assumption of 14 targets each of diameter 12 5my 2 Lane SE & Anderson TF (1942) STATUS OF THE VIRUSES 123 in this phage no differentiation between radioinsensitive and radiosensitive regions 1s plausible enough With viruses of medium size such as Staph phage K which we have just been discussing, while the discrepancy between target size and virus size suggests such a differentiation one 1s not pre pared uncompromisingly to insist on it in view of the fact that over simplification in the model 1s a possible alternative ex- planation of 1 small discrepancy of this sort However, the position 1s different in the case of the largest virus so far studied, namely, vaccinia virus, which in Figs 1% and 19 is represented by the expenmental points farthest from the theoretical curves Taste 36 Non applicability of the single spherical target theory to 4 large virus (yaccrma)1 Radiation. y rays N rave {15A) a rays (5eMY } Inactivation dose 0.080 0104 O21 10tr Target diameter 31 41 TOmy Mean unhydrated virus diameter = 200mg: (hydrated 235 mj) The size of ths virus 1s hnown fairly reliably, and there 1s no reason to suspect serious error in the radiation dati The discrepancy shown by Table 36 between the target sizes for different radiations and between the target sizes and the virus Size, 18 too great. to be ascribed either to experimental error or to over simplification of the model There is therefore in this virus defimtely a differentiation be tween radiosensitive and insensitive mitenal, and the radio- sensitive part amounts (in volume) to less than 1% of the whole \irus_ In this respect vaccima resembles bacteria mote than the smallest viruses, and we shall defer fuller discussion of it until Chapter 1. The status of the viruses In the troduction to the present chapter it was mentioned that na typical cell the lughly specific and genetically important nucleoprotein constituting the chromosomes comprised 1 rela- tively small part of the whole cell In the small crystallizable ‘ituses there 1s no such differentiation between genetically im- Portant chromatin and eytoplism, the virus being composed of 1 Lea, DF & Salaman WH (1942) 126 INACTIVATION OF VIRUSES BX RADIATION nucleoprotemn alone Regarting the larger viruses, mformation by direct methods 18 scanty It appears that radiation expen ments are able to offer sone information on the nature of the vituses Tho radiosensitise material, a single tonzation in which leads to the inactivation of the virus, 13 to be identified with the genetically important nucleoprote:, since we cannot plausibly ueagine a single ionization in the less epecific cy toplasm leading to inactivation Determmation of the inactsy ation dose enables the volume of the sensitive material to be calculated, and com parison with the volunte of the yirus enables the proportion of radiosensitis e matertal in the virus to be estimated In this way wo are able to show that the smallest viruses are almost cont pletely rad tive, in agi t with the ch I finding that they are entirely nucleoprotein Vaccinia, ono of the largest viruses, shows 4 differentiation between radhosensitive and mn sensitive material comparable to that m higher cells Genet cally, the smallest viruses may be considered as ‘naked genes , while the largest viruses are more akin to single celled organisms Radiation experiments afford a means of deciding to which cate gory 8 given virus belongs Inactivation of viruses by ultra-violet ight Not much quantitative work has been done on the mactiva tion of viruses by ultra violet light: The survival curves are exponential and the effect of a given dose 1s independent of the time over which it 1s spread These results suggest that the absorption of a single ultra violet quantum 13 able to cause 1n activation, in the same way that the corresponding results with ionizing radiations suggest that a single ronization or single ton izing particle 1s able to cause inactivation With ultra violet ight no additronal test 1s avatlable such as that afforded for 1omzmng radiations by the mncrease of inactivation dose with increase of 1on density, and the adequacy of a single ultra-violet quantum to cause Inactivation cannot be considered established with the same certainty as 15 the adequacy of a single 1omzation If we accept, however, that inactivation by ultra violet ght 1s due to 1 See Hollaender, A & Duggar BM (1936), Price WC & Gowen JW (1937), Ravers, T.M & Gaes FL (1928), McKmley EB Fisher R & Holden MW (1926), Oltsky, PK & Gates FL (1927) Lea DE & Smuth KM (1940) INACTIVATION BLY ULTRA-VIOLET LIGHT 125 a single quantum, we can calculate the quantum yield of the reaction, 1e the probability that a smgle quantum absorbed in the virus particle will cause inactivation It comes out to be very low, about 0 025 for phage $13: and 7x 10-4 for tobacco mosaic virus,: each exposed to ultra-violet light In in- orgame gas reactions quantum yields of the order of umty are usual However, with large organic molecules lower quantum yields are obtained, and a very low value for the much larger molecule of tobacco mosaic virus 1s not impossibles It1s evident that 1 18 not possible to calculate virus sizes or target si7ea from experments on the imactivation of viruses by ultra-violet hght, since, while the postulate that the production of a single 10n1za- tion leads inevitably to inactsvation seems to be fairly closely verified, the assumption that a single ultra-violet’ quantum absorbed Jeads ynevitably to inact: ation 38 n patent contradic~ tion to the facts for tobacco mosiac virus 1 Latarjet, R & Wahl, R (1945, see also Lataryet (1946)), found that the activity of a dilute suspension of phage $13 in distilled water was reduced to $7% by an imeident energy of 147 x 10 ergs/em? If we assume that the absorption coefficient of this virus for ultra violet ght. of 2536A 13 of the same order (~ 10‘ cm ~1) as the measured absorption coeffinent of plant virus protem (cp Table 1, p 5), this corresponds to an energy absorption of 1 47 x 10° ergs/em* or about 40 quanta im the volume 2 2 x 10-16 em 3 of one phage particle (since 1 erg of this radiation corresponds to 1 277 10" quanta) Thus if we accept the deduction from the exponential shape of the survival curve, that inactivation 13 due to e single quantum, and if we accept the evidence of the expermments with ionizing radiations (Table 34, p 121) that im phage $13 there 1s no differentiation mto sensitive and non sensitive regions, then we must infer that though inactivation, when it occurs, 1s due to only one quantum, the Probability of any grven quantal absorption bemg effective 1s only about one fortieth There appears to be no cumulative effect of the absorption of ndave dually ineffective quanta since in this event one would expect, not an exponential but a sigmoid shape of survival curve 2 Uber, FM (1941) leulated using the m data of Les, DE & Smith KM (1940) 3 Eg 0017 for the inactivation of trypsin by 2536A , Uber, FM & McLaren AD (1941) 4 Cp Jordan, P (19385), Crowther, JA (1938) Chapter V GENLTICAL EFFECTS OF RADIATION The mechanism of heredity: Modern gonetics 1s usually considered to have begun about the year 1900, when the expernments made by Mende! nearly forty years eathor were confirmed and their significance realized, and the basic principles can best bo explained in terms of these often- quoted experiments Mende had two pure breeding strains of garden peas, tall and awarf When crossed, the offspring (te F, generatton) were tall When the F, generation were self fertilized thesr offepring (re the #, generation) were composed of tall and dwarf plants mn the ratio of 2 2 The dwarf plants were all pure breeding, and one third of the tall plants were pure breeding, but the remaining tall plants wero tike the F, generation, that 13, tall im appearance (re phenotypically tall) but. giving the 3 1 ratio on self fertilization, indicating that genotyprcally they were bybnds Mendel’s explanation, which has not needed madification, 1s that the gametes (germ cells) of the tall plants carry a factorT and the gametes of the dwarf plants a factor t The F, plents thus carry the factors Tt, and since phenotypically these plants are tall the tall factor T 1s said to be dominant to the dwarf factor t which ts recessive The gametes of the F, plants will carry the foctorsT or t with equal frequency, and on self fertilization the combina tions TT, Tt tT tt, will occur therefore with equal frequency Thus in the F, generation from self fertilization of the F, plants we shall find pure breeding tall (TT), plants resembling the Fy plants {Tt, tall but not pure breethng), and pure breeding dwarfs {tt} in the ratia 1 2 2 1 The condensed introduction to genetics given in the opening sections of ths chapter 18 mtended to provide the reader who 13 not a genetiowt with the h ledge of the ples and logy of the subject to follow the d of the J effects of rad given. fater m the chapter It may be supplemented by reading the following books Tumoféeff Ressovsky NW (2937¢), Muahonsforschung, Darhng ton CD (1937) Recent Advances in Cytology, Waddington CH (1939), Tntroduction fo Modern Genetics, Sturtevant, AH & Beadle G'S (4446) Introduchon to Genetics, Cold Spring Harbor Symposia vol 9 (1$$1) on Genes and Chromosomes’ MECHANISM OF HEREDITY 127 Other characters of the pea were found to be inhented in a amilar fashion Thus a strain with yellow seeds crossed to a strain with green seeds gave yellow seeds in the F,, and yellow and green in the ratio of 3. 1m the F, generation, the factor for green (y) evidently bemg recessive to the factor for yellow (Y) Similarly, the factor for wrinkled seeds (r) was recessive to the factor for round seeds (R) When strains of peas differmg m both these characters were crossed the characters were found to be independently assorted Thats to say, when pure breeding yellow round (YYRR) and pure breeding green wnnkled (yyrr) plants were crossed, grving F, plants of composition YyRr, the gametes of the F, were not only YR and yr, but also, with equal fre- quency, Yr and yR This independent assortment 1s not, however, found with all Pairs of characters For example, in man, haemophiha and colour blindness are linked In a family tree which contains colour bind and haemophilic members, 1f one member 1s colour- blind and haemophilic it will be rare to find other members who are colour blind without being haemophihe or vice versa If, on the other hand, two famihes, one of which has haemophilic members and the other colour-blind members, intermarry, 1t will be rare for a descendant to have both haemophika and colour-blindness In an organism such as maize, in which many Mendelian characters have been investigated, they can be catalogued into linkage groups—ten m the case of maize—such that characters im the same group are usually Imbhed (like haemophilia and colour blindness mn man) and characters in different groups are not linked (ke seed colour and seed shape im peas) The fact that the majority of inherited qualities, e g height mathematical abihty in man, do not appear to obey Mendehan ows 18 expheable on the ground that a great number of inde- tehatly assorted simple Mendelian characters probably con- eases eeu qualities The simplicity of the Mendehan laws in ton cane as those we have been describing when single charac- The he observed suggests a simple mechanism of heredity thread Me wwomes Provide this mechanism Chromosomes are found in th structures, chemically described as nucleoprotein, base € nuclei of practically all cells and readily stamed by 'yes when the cell 1s im division, though difficult to demon- 128 QENETICAL FEELCTS OF RADIATION strate at other times The germ cells of a plant or animal contain a number 2 of chromosomes which 1s widely variable for different Speties, ranging from one up to several hundred, but constant for a given species ¢ g 241n the case of msn When at ferttliza tion the sperm and egg cells fusc, a eell 1s formed with 2n chromosomes, « g 481m the case of man The chromosomes ina germ cell are (in general) all different and can m fax ourable cases be recogmzed microscopically by differences in size and shape Each chromosome contributed by 4 male germ cell 13, with one exception, essentially identical (omologous) with a corre sponding chromosome contributed by the female germ cell One pair of chr the sex chre —1n many animals, including man and the fruit fly, Drosophila, 1s exceptional in that ina fertilized egg (or zygote} which 18 going to develop into a male org: the two chr of the pur are distinguishable, the X chromosome which has been contributed by the egg cell and the } chromosome which has been contributed by the sperm cell being different A zygote which 1s going to develop into a female organism, on the other hand, has two X chromo somes The sperms are of two hinds, those carrying an X chromosome giving rise to female zygotes and those carrying a Y chromosome to male zygotes (In birds, moths and butter- flies the female 1s heterozygous (A3}) Seed plants and many lower animals do not usually have the 1} mechanism of sex determination } The fertihzed egg divides into two cells, and by repetition of this process the organism 1s formed At each cell division an elaborate cycle of operations (mitosis) occurs which results in the two daughter cells being :dentical as regards their chromosome complement During the first stage of mitosis (prophase) the nuclear membrane disappears, and the chr , which in the resting stage between divisions are long thin threads, usually unstainable, contract as a result of sprralization and become loided with desoxyribose nucleic acid, so that they have the appearance (at metaphase) of short thich rods, readily stammg with basic dyes by virtue of the nuclee acid content At this stage the chromosomes are oriented in the equatorial plane of the cell At an early stage in this condensation process (or some times 1n the enterphase or resting stage or even in the preceding mutosis), each chromosome thread has spht longitudinally, and MECHANISM OF HEREDITY 129 when the chromosomes have become fully condensed the two halves of each chromosome separate (anaphase) and move to opposite ends of the cell (telophase) The torces responsible tor the movement of the chromosomes to opposite poles of the cell at anaphase are not understood, but appear to be applied to a particular organ of the chromosome, the cenfromere If, as some times happens after irradiation, a chromosome Jacks a centro mere, it 13 hable to be left behind and fail to be tncluded in esther daughter nucleus A nuclear membrane now forms round each group of chromosomes and the cell divides, 80 that two daughter cells are formed, each having the same number of chromosomes as the mother cell, and being, as regards the chromosomes, a very exact copy of the mother cell The process of mitotic di vision thus ensures that every cell in the adult organism contains 2n chromosomes, n derived from the n chromosomes originally contributed by its male parent and n derived from those contri buted by its female parent As regards the sex chromosomes, every tissue cell of a female contains two A. chromosomes, and every tissue cell of a male contains an X and a Y chromo some The adult orgamsm produces germ cells by a process hnown as metosis in which two successive cell divisions occur with only one chromosome division, so that the chromosome number is halved Dunng the prophase of meiotic division the chromosomes come together in pairs, the homologues derived from male and female patent lying in close contact along their length (synapsis) At (first) anaphase the homologucs sepatate again and move to Opposite poles of the cell, and the cell divides Dunng sy napsis each homologue sphts longitudinally, and so when the first cell division has occurred each chromosome 1s already spht The half-chromosomes or chromatids later move apart m a second anaphase, and a second cell division follows The final result 13 thus four cells, each of 2 chromosomes, each chromoso me equally hkely to have derived from the male or female parent As re Bards the sex chromosomes, each of the four cells produced at Taeiosis in a female (only one of which however functions as a germ cell) has an X chromosome, while of four sperms produced at meiosis ina male, two carry an A chromosome (denved from the mother father) ) and two carry a } chromosome (derived from the 130 GFNITICAL FFFECTS OF RADIATION Tho chromosome mechanism affords o satisfactory explana tion of the Mendelian Jaws of inheritance The physical entity corresponding to a Mendelian character 1s a. gene, o small portion ofa chromosome Since each somatic cell (1 ¢ tissue cell in dis tinction from germ cell) contains 2n chromosomes, each gene 18 normally present in duplicate, one denved from each parent When strains differ in a Mendchian character, tho gene corre- sponding 15 1n some way different in the two strains, we refer to different. allelomorphs of a given gene Thus one of the genes affecting eye colour in the frutt fly Drosophila melanogaster exists mm several allelomorphs which when homozygous (1e one allelomorph present in duplicate) give various eye colours de sertbed as white, coral, eosm, cherry, buff, 1vory, and others, all of which are recessive to the weld type allelomorph which 1s more common and which gives dark red eyes When a cross 1g made between two strains the offspring will contain two different allelomorphs, 16 will be heterozygous for the particular gene In general we might expect an intermediate phenotypical effect, and this sometimes occurs, but in practice the phenoty pe is more often nearly identical with that produced by one of the allelomorphs in homozygous condition, so that the presence of the other allelomorph, which we describe as recessive, 18 not detectable except by breeding tests The behaviour of Mendehan characters m crosses 1s explained by the behaviour of chromosomes at menosis and m feriilization Characters which are not linked correspond to genes 1n different chromosomes, and characters which are linked ere localized in the same chr In Drosophila mel: ter and mm maize the number of linkage groups (four and ten respectively) genets- cally determined has been shown to be the same as the haplord chromosome number (1e 7, m distinction from 2n, the diplod number) microscopically observed Further, the phenomenon of plete linkage 18 explained m terms of chromosome be haviour As mi d already, h phiha and colour blind ness in man are Imked Cases have been observed in which Imkage fails, indicating that a woman having both recessive genes m one of her X chromosomes and both normal allelo morphs m her other X chromosome has produced a germ cell having an X chromosome with the recessive allelomorph of one gene and the normal allelomorph of the other The explanation MUTATIONS 131 for this incomplete linkage hes in the fact that dumng the stage of meiosis in which four chromosome strands he in juxtaposition (four strand pachytene) it 13 possible for two strands to break at corresponding points and change partners, thus separating genes on opposite sides of the break which were onginally in the same chromosome thread and bringing them into separate threads, and vice versa Thus incomplete linkage 1s explained by crossing- over The closer together two genes are im a chromosome the swaller 1s the chance of crossing over occurring between them Thus the cross over frequency between two genes 1s a measure of their distance apart in the chromosome, and in this way chromosome maps can be constructed in which the genes in the chromosomes are shown in their correct near order Genes localized in the sex chromosomes are described as sex- linked Since the Y chromosome 1s usually genetically mnert and carries very few genes, a sex-linked gene 1s normally one localized inthe X chromosome If a female 1s heterozygous for the wild- type allelomorph and a recessive allelomorph of a sex linked gene, the phenotymcal appearance will be wild type Ifa male, however, carries the recessive allelomorph of a sex linked gene it will show up phenotypically since, as there 1s only one X chromosome in the male, there 13 no wild type allelomorph Present so that the male 1s hemizygous for the gene in question It 1s for this reason that haemophila, for example, while trans- mitted by a heterozygous woman, 1s not shown by her but 1s shown by those of her sons who receive from her an X chromo- some carrying the recessive, instead of a normal, gene Mutations Strains of plants and animals differing in that: different allelo morphs of a particular gene are present in the different strains are found naturally Moreover, a pure breeding strain occa- sionally gives nse to an organism having a different allelomorph ofa particular gene from the rest of the strain Such changes are called gene wutations, and with individual genes occur spon- taneously with a frequency of the order of 10-5 or 10-6 per generation The rate may be somewhat speeded up by nise of temperature in the case of Drosophila, m which this procedure 13 practicable, and the production of mutatio ns by chemical treat- at has occasionally been reporte d X rays ond other lonizing 132 GUNFTICAL FREECTS OF RADIATION radiations cause mutations at a rate greatly in excess of the spontancous rate, and radiation induced mutations have, amnce the discovery of this effect by Mullor in 1927, been sntensively studied, especially in Drosophila melanogaster Mutatton can doubtless occur, spontaneously or by radiation, in any cell, but a mutation in a single cell of an adult organism would bo practically impossible to detect Ifa mutation occurs, however, 1n a sperm or egg cell, every cell of the adult organism growing from the germ cell will carry the mutant gene Ifa mutation occurs in a cell of the developing organism at some atago intermediate betw cen the zy gote and the adult, then 1n the adult a mosaic effect. may be noticeable, as a result of the mutant character appearing in the group of cells which have developed from the cel] in which the mutation occurred, the remaimung cells being unaffected A great deal of the work which has been done on the induction of mutations by radiation has been carned out by wradiating Drosophila sperm Male flies are irradiated and mated to un- treated females Ifa mutation has occurred in one of the sperms of the irradiated males, every cell of the F, fly which hes ansen from the egg fertilized by this sperm will carry the mutation If the mutant gene 1s a dominant, the fly will show the character If it 1s a recessive, suitable tests will enable 1t to be brought te light in @ subsequent generation Even with X rhy induced mutations, the rate at which any given mutational step occurs 1s very low, since the X-ray dosage which can be given without producing stenlity 1s hmited to a few thousand roentgens, and the yield of even the most frequently occurring mutations 18 oniy about 10-* per thousand roentgens To accumulate data on the mutation frequency of a particular gene 18 therefore very laborious, and it has been usual therefore to record the total number of mutations, or the total number ocourring 1n a given chromosome The study of sex-hnhed muta- tions, :e mutations locahzed in the X chromosome, is techni- eally most convenient, since a sex linked recessive mutation will show up when present in the male, while a recessive mutation in an {ie a chr other than a sex chromo- some) will not show up when heterozygous, and to obtain it homozygous requires an add. g tion in the breeding tests GENES AND MUTATIONS 133 Among the visible mutations in Drosophila, recessives are several times more frequent than dominants, and most work has therefore been done on them The mayonty of recessive visible mutations as well as pro- ducing, when homozygous, the vistble change by which they are recognized, reduce the viability of the organism Thuis 1s not sur- pnising, since a mutant form which was advantageous to the organism would by selection probably have become the wild type There are a great many recessive mutations which, when homozygous, are lethal, so that the adult stage 1s never attained The number of these recessive lethals 1s in fact several times greater than the number of visible mutations By means of convenient breeding test (the CIB method of Muller) :t can be arranged that a culture of flies m the F, generation from an irradiated grandfather 1s devoid of males whenever a sex-lnked lethal has been produced in a sperm of the grandfather The de- tection of Jethals can thus be made with greater ease and freedom from subjective error than the detection of visible mutations For these two reasons a great deal of the work on radtation- induced mutations, particularly quantitative work requiring the recording of a large number of mutations to attam statistical significance, has been made on sex-linked recessive lethals This 1s to some extent unfortunate, since there 1s evidence that lethals are not entirely analogous to the visible gene mutations The nature of genes and mutations Views on the nature of genes are, at present, to a considerable extent speculative The evidence of Mendelian genetics 1s that the genes are arranged in linear order in the chromosome When homologous chromosomes are synapsed, crossing over takes place without producing effects in the genes, showing that the chromo- somes can be broken between consecutive genes without changing the genes The picture suggested by these facts 1s of beads on a string, the genes bemg independent units connected together by genetically inert matenal which 1s more Teadily breakable than the genes themselves Microscopical examma- tion may be considered consistent with this simple model Chromosomes in the condensed state at metaphase are too short and tughtly coiled for there ta be any possibilt y of resolving individual genes, the number of these in a chromosome of, for 134 GENLTICAL EFFFCTS OF RADIATION example, Drosophila, being beheved to be of the order of a thousand The most extended state normally available for ob servation occurs during meiotic prophase, when a plant chromo some may have a length of 100 In some organisms at this stage the chromosomes have a beaded appearance, and dunng aynapsis ‘he beads or chromomeres, and not merely the chromo somes as a whole, are accurately paired Still more detailed structure can be seen in salivary gland chromosomes: In the larvae of Drosophila and other two winged flres the salivary glands grow during the development of the organism from the embryonic stage to the fully grown larva not by the normal process of cell multiplication, but by increase of cell size without cell multiphcation In conseq the cella attain a volume about a thousand t:mes greater than that of ordinary cells During this process the chromosomes apparently undergo repeated duplications without splitting, so that each salivary gland chromosome 13 a bundle several microns m diameter, made up of some hundreds of chromosome threads which are not individually resolvable under the microscope The chr are led, and homo} are paired as 1n meiotic prophase The salivary gland chromosomes are crossed by disks of more deeply staining material which are presumably made up by the juxtaposition of the chromomeres of the individual chromosome threads The bands in the salivary chromosomes form a suf- ficiently characteristic and recogmzable pattern for even very small internal losses and rearrang ts of the chr to be detected, and for maps to be made im which the individual ‘bands, to the of some th ds in the a are numbered Ultra violet hght absorption measurements indicate that the disks are made up of nucleoprotein (protein in combination with nucleic acid), and the intervening portions, less deeply staming and less strongly absorbent of ultra-violet light, of protein of globulin and protamin types The bands are mechanically less extensible than the mterband portions The number of bands—647 in the X chromosome—is of the order of the probable number of genes The obvious hypothesis that the bands mey mark the positions of genes recerves support 1 Plate lila 8 (Catchearde DG) SALIVARY GLAND CHROMOSOMES 135 from the fact that there have been established many instances where the absence of a band in the sahvary chromosome can be correlated with a genetical effect consistent with a defi y (ve complete absence) of a particular gene, absence of the band mvanably being accompamed by the genetic effect Further, certain parts of the chromosomes known as the heterochromane Tegions (recognizable cy tologically by their retention dunng the resting stage of the charge of nucleic acid which the ordinary or euchromatic regions of the chromosome possess only during cell division) are hnown to be genetically inert, very few genes having been recognized in these regions In the salivary chromosomes the heterochromatic regions are, relative to the euchromatic regions, much contracted and do not show a well defined band structure It appears then that im the salivary glands the genes are located in the nucleic acid loaded bands, and it 1s a reasonable presumption than m ordimary chromosomes the genes are mole- cules of nucleoprotein located sn, or connected by, a protein backbone Observationally an upper hmit can be set to the size of the gene from the consideration that a number probably of the order of 1000 genes are present in the X chromosome of Drosophila, the salivary gland Jength of which 1s about 200” Thus the length of chromosome thread available to one gene is about 200mpz An estimate of about 100my1s given by measurements of the length in the sahvary chromosome of a definency known from its genetic effect to involve at least two genes: The diameter of a single thread cannot be obtained from salivary gland observ a- tions, but the volume of a metaphase X chromosome can be Measured, and if 1t be assumed that this 1s a coiled-up thread of length (when fully extended) 200 it, the thread diameter 1s calen- lated by Muller: to be not more than 20my im diameter Thus an upper hmuit for the space taken up by a gene m a chromosome 1s about 100x 20m, the corresponding molecular weight being about 25 millon The upper mit obtamed for the molecular Weight of the gene, which may of course greatly exceed the actual value, is comparable with the molecular weight of the crystalluzable viruses (e g tomato bushy stunt virus, 11 million , 1 Muller, HJ & Prokofyeva AA (£035) am Malloe TY T sinor 136 GENETICAL EFFFOTS OF RADIATION tobacco mosaic virus, 42 millon) Chemucally viruses are also nucleoprotein, and like the genes they have the property of synthesizing exact copies of themselves from a suitable intra cellular substrate If significance 1a attached to these ana- logies, one 13 led, on the one hand, to regard a eryatalhzable virus a8, genetically, the most primitive form of life, namely,4 naked gene, and to regard the genes in higher organisms 23 aute rote molecules, reg’ d in chromo- somes to facilitate synchronization m division This picture of the nature of the gene may be taken to be one extreme of the range of views currently held regarding the nature of genes On this view different genes would differ in structure to a marked degree, as do presumably viruses which are sero- logically unrelated, while the various allelomorphs of a given gene would have smaller differences, such as distinguish sero logically related strains of viruses Mutation of a virus to a re- lated strain has been observed Considering different allelomorphs as different stable atates of tially the same molecule, there will presumably be an activation energy for transition from one state to another, and the frequency of transition and its temperature coefficient should be capable of treatment by the methods of chemical kmetics In particular, the fact that mutation rate 13, on the scale of ordinary I reactions, extremely slow, leads to the expec- tation ofa temperature coefficient larger than ordinarily found n 1 react: duis expectation 18 realized, a temperature rise of 10°C producing a fivefold increage in spontaneous muta- tion rate, compared with a two- or threefold increase in ordmary chemical reactions The activation energy corresponding 14 about 1 5eV Chromosome structural changes, and the position effect The picture as we have so far presented 1t, of genes as autono- mous units and of mutation as internal change 1n a gene, 15 over- simphfied in that 1t neglects mteraction between genes, and mutational effects produced by structural changes in chromo somes which are readily produced by radiation and which are discussed in greater detail in Chapters vi and vir When a chromosome 1s broken by radiation, the broken ends x Timoféeff Ressovsky, NW Zimmer KG & Delbruck, M (1935) CHROMOSOME STRUCTURAL CHANGES 137 are usually left in a yomable condition They may rejom resti- tutmg the original chromosome, or 1f a chromosome 18 broken in two places the four broken ends may rejom in new ways Such dlegrtemate union will clearly lead either to a single chromosome in which the portion between the breahage points has been taken out and remserted in reverse order (:ntersion), or to two chromo some bodies, one a ring formed by the joining of the ends of the segment between the breakage points, and the other a rod formed by the joining of the two remaining segments: One of these two chromosome bodies lacks a centromere, and 1s likely therefore to be lost at cell division If the cell 1s able to survive the loss, we shall then have a cell n which one of the chromosomes 1s deficrent of a certain number of genes, the phenomenon bemg called deletion or deficiency If two different chromosomes are broken, illegitimate union between the four broken ends leads to znter- change (also called receprocal translocation), with the production of two chromosome bodies each having a portion of one of the two original chromosomes If one new chromosome body has both centromeres (1e 18 dicentric) and the other has no centro- mere (re 18 aceniric), as happens as a result of asymmetrical interchange,z the acentric fragment 1s lhhely to be lost at cell division and the dicentne chromosome may be broken, or eventually lost, or cause breakdown of the cell, owing to mechanical difficulties (for example, to the two centromeres sometimes moving to opposite poles at cell division) However, if the two new chromosome bodies each contain a centromere, as happens in symmetrical interchange,s there 1s no mechanical dis- advantage attaching to the new formation as compared with the orginal, no portions of chromosome have been lost (unless a minute deficiency occurs at a breakage point, as occasionally happens) and cells contaming such chromosomes are likely to survive Inverstons also are hhely to survive Duplications, 1¢ the presence m a, chromosome of an additional piece without there being a corresponding loss of the same piece from its normal place in the same chromosome or 1n another chromosome, are also known, so that a certaim number of gene loci may be Present three times in the somatic cells instead of the normal twice 1 See Fig 305,c 0,£, p 194 2 Fig 300 (p 194) 3 Fig 30F (p 194) 138 GENETICAL EFFECTS OF RADIATION It 18 convenient to distinguish between gross structural changes and minute, tho latter being mveratons, duphcstions, deficiencies, or translocations in which the abnormality 1s for only a small Jength of chromosome Dupheations and hetero- zygous deficiencies in Drosophela aro usually viable, if amall, but not if largo A homozygous deficiency, even when minute, 18 usually lethal, and a proportion of recessive lethals are, n fact, minute deficrencies A fow instances are known when 8 de ficency produces a yistble mutant effect Thus a deficency for & portion of the region 1A in the salivary chromosome map behaves as a recesarve visible mutation, producing, when homo zygous, a fly with a yellow body in place of the wild type grey body A deficiency which cludes tho band 307 behaves as 8 de , produemg, when heterozygous, a notching of the wing veins It 19 lethal when homozygous In addition to the phenatypical effects obtained with duplica- hons and deficiencies and which are due to the absence of some genes or the presence of extra genes, phenotypical effects are sometimes obtamed accompanying imversions and eucentnc interchanges in which, as far as can be seen xn the salivary chromosomes, there hos been neither loss nor gain of chromo some maternal but merely rearrangement: The phenotypical effect produced 1s an alteration in the characters governed by the genes which are located next to, or close to, the loct of the breaks in the chr and the possible explanations are either that the 1omzing particle which causes the break simultaneously causes mutation in a gene close to the break, or that the effect of a gene 1s modified by the genes tn its immediate neighbourhood, and hence 1s changed when a structural rearrangement follows a break close to the gene, although no internal change has oceurred in any of the genes concerned (positron effect) The phenomena are different, depending on whether the two breaks taking part m the stractural change are both in euchro- matic regions of the chromosomes or whether one 1s in hetero chromatin In the latter case, the effect of the structural change 1s to cause a euchromatic region to be joned to a heterochro raatic region Genes in the euchromatin up to a distance of twenty salivary bands away from the place where it now joins the heterochromatin are liable to be affected The effect may take x Tn Drosophila not usually m plant material POSITION EFFECT 139 the form of an unstable mutation resulting in a mottled pheno- type, owing to some of the cells show mg the mutant and others the wild type character Thus if the 3C1 band of the X chromo- some 1s brought into proximity with heterochromatin in this way, the cyes of the male are white with red pitches (The wild- type fly has red eyes, a male carrying the commonest mutant allelomorph of this locus has white eyes ) Conversely, a gene normally located near a heterochromatic region of its chromo- some may show a position effect as a reault of a chromosome structural change which brings it instead adjacent to a euchro- matic region « There are several lesz of evidence which make it tolerably certain that the charactenstic effects shown by a gene trans- ferred to or from the neighbourhood of heterochromatin are due to position effect, and not to a simultaneous change induced an the gene by the ionizing particle which broke the chromosome near it Itis possible that the different nucleic acid metvbohsm 1n heterochromatin compared with euchromatin interferes with the reproduction of the genes located near a heterochromatic Tegion The phenomena are rather different, n the case when both breaks talang part in the structural change are in the enchro- matin In these cases the effect 1s usually hmuited to genes 1m- mediately adjacent to the break mstead of extending up to a distance amounting to several percent of the whole chromosome length This circumstance makes plausible the explanation, already mentioned, that the 1omzing particle which causes the break may simultaneously cause mutation of the gene adjacent to the break It happens further that the tests referred to, by which a number of changes involving heterochromatin have been shown unmistakably to be position effects, cannot be applied to eects existing only in the immediate proximity of the break 1 The example of this 1s cubstus wmlterruptus located in the fourth hi near the | of the centromere (Dubinin, NP, Sokolov NN & Timakov, GG 1935) An interchange which separates the ey locus from the centromere and brings it into a euchromatic region of another chromosome may cause the wild type allelomorph of the ¢2 locus to fose ats di over the Mel: ph Interchange which brings the ¢1 locus into a heterochromatic region chromosome haa of another no effect 2 Dubnn NP & Sidorov, BN (1935), Muller HJ (1942) 138 GENETIOAL EFFECTS OF RADIATION It 18 convonient to distinguish between gross structural changes and minute, tho latter beng inversions, duplications, deficiencies, or translocations in which the abnormality 1s for only a small longth of chromosome Duplications and hetero- zygous deficrencies in Drosophila are usually viable, 1f small, but not af large A homozygous deficiency, even when minute, 18 usually Iethal, and a proportion of recessive lethals are, in fact, minute deficiencies A few instances are known when a de- ficiency produces a visible mutant effect Thus a deficiency for a portion of the region 1A in the salivary chromosome map behaves as a recess:vo visible mutation, producing, when home- zygous, & fly with a yellow body in place of the wild type grey body A deficiency which includes the band 307 behaves as & dominant, producing, when heterozygous, a notching of the wing veins It 28 lethal when homozygous In addition to the phenotypical effects obtained with duplica- tions and definencies and which are due to the absence of some genes or the presence of extra genes, phenotypical effects are bt d panying inversions and eucentric interchanges in which, aa far as can be seen in the salivary chromosomes, there has been neither loss nor gain of chromo some matenal but merely rearrangement: The phenotypical effect produced 1s an alteration in the characters governed by the genes which are located next to, or close to, the loc: of the breaks in the chrome and the possible explanations are either that the 1omzmg particle which causes the break simultaneously causes mutation im a gene close to the break, or that the effect of & gene 1s modified by the genes im its immediate neighbourhood, and hence 1s changed when a structural rearrangement follows a break close to the gene, although no internal change has occurred in any of the genes concerned (position effect) The phenomena are different, depending on whether the two breaks taking part in the structural change are both in euchro- mate regions of the chromosomes, or whether one is 1n hetero chromatin In the latter case, the effect of the structural change 13 to cause a euchromatic region to be joined to a heterochro matic region Genes mm the euchromatin up to a distance of twenty salivary bands away from the place where 1t now joins the heterochromatin are lable to be affected The effect may take 1 In Drosophila not usually in plant material VISIBLE MUTATIONS 141 In the case of bactena: the expenment consists simply in plating out suitable dilutions of the irradiated bactena on e solid nutnent growth medium, and examimng large numbers of colonies Each colony having grown from a single organism, & mutation in an irradiated bacterrum should jead to a colony in which every individual hasthemutant character Mutationsin the bacteria which lead to differences in the 61ze, colour, or surface texture of the colony arerecogmizable by inspection of the colonies A major difficulty an the study of bacterral and virus mutation 1s that contammation of the preparations by bacteria or viruses from outside 1s difficult to prevent, and such contaminations may be recorded as mutations The number of different characters which can be recognized 1s, moreover, small compared with the number available in higher organisms For these reasons little work has been done on the mduction of mutation in bactena and viruses It does not appear, however, that the process 1s essen- tially different from that in higher orgamsms Much the fullest information on the induction of mutations by radiation 1s available in the case of the fruit fly Drosophila melanogaster The most usual procedure 1s to irradiate the male flies, to mate them to untreated females, and to look for muta- tions in their offspring The mutations mvestagated are thus produced im the sperm of the irradiated male Dominant muta- tions will be visible in the F, generation The number of domi- nant mutations 1s, however, much smaller than the number of recessive mutations, and the latter therefore are usually worked with A recessive mutation induced in the sperm will not be visible in the F, generation unless special means are adopted If mutations of a particular locus are bemg investigated, then the irradiated wild type male can be mated with a female homo- zygous for a recessive allelomorph of this locus The great majority of the offspring will be phenotypically wild type, but an occasional one will show the mutant character This is due either to mutation of the wild type to a recessive allelomorph, or to the complete loss of the locus concerned owing to a minute chromosome definency having occurred which includes the locus: Further tests will decide between these possibilities 1 Gowen JW (1941), using Phytomonas stewarty 2 According to Muller HJ (1940) about one third of thy ie mutants found by this method are minut e d Accordingig to P: JT (1932) seven eighths are minute deficiencies 140 GENETICAL EFFECTS OF RADIATION Thus the oxplanation of mutational changes accompanying chromosome structural change not involving heterochromatin cannot be considered certainly decided This 1s partreularly un fortunatesince a fair proportion of the sex-linked recessivelethals, on which a great deal of the expenmental work on the production of mutations by radiation has been dono, are accompanied by chromosome structural changes, and it 1s necessary for an under- standing of the mechanism of action of radiation to hnow whether these lethal mutations are to be attributed to position effect of 8 gene adjacent to the break due to its being separated from its normal neighbour and brought into proximity with another gene, or whether they are due to internal changes produced 1n the gene by the 1omzing particle which caused the break We shall, from the analysis of the radiation experiments, find support for the latter explanation, and shall interpret lethal mutations accompanying gross chromosome structural change 10 this way instead of invoking the position effect (Thus 1s not to deny the existence of position effect completely, but merely to conclude that tt is not usually the cause of lethal mutations associated with structural change There are a few well-esta bhshed cases of position effect not involving heterochromatin, such as Bar eye 1) The production of visible mutations by radiation Mutations have been induced by 1onizing radiations in 8 great many orgamams z The method of investigation 18 naturally auf- ferent for different organisms In the case of plant viruses, the method adopted 1s to moculate the leaves of a suitable test plant with the urradhated virus solution The test plant chosen 1s one which does not give, with the unchanged virus, local necrotic lesions at the points of entry of the virus into the leaves Ifsome of the virus has been changed by the radiation into a form which does give local necrotic lesions, these can be detected despite the great excess of unchanged virus The lesions can be cut out and the mutant virus stram isolated and the permanence of the change tested 1 Cp Sutton E (1943) 2 A hst of orgamsms investigated 1s given by Timoféeff Res NW (1937a) 3 Gowen, J W (1941), using tobacco mosaic virus VISIBLE MUTATIONS 143 Visible mutant types differing only slightly from the wild type are legs easily overlooked by this method than by tho attached-X method, and reduced viability of mutant as compared with wild type will not lead to a depressing of the apparent mutation rate A lethal mutation will be shown by the F, culture having no males x10 4 A , 15 c 6} . " . tor of* ne x ab do Wx oO 1 2 1. G 1 J. 1 5 10 tor 15 T 2 3 Wr D E sh . a i «19074 4 ob ‘ Hop . y ar sk oe a p 4 4 ‘1 1 . nm 2 4 © ir z 4 @ x10F Dose Dose Fro 20 Proportion of visible mutations induced by Tays as a function of dose A-B tobaeco mosaic virus {Gowen} A type to aucuba B, aucuba to type © Neurospora (Demeree et al) D-F, Drosophila melanogaster (Timofeeff Ressovsky & Delbruck) D a single mutation step (wild type to eosin eye colour) E all sex linked recessive mutations detected by CIB method F all sex linked d d by attached X method In Fig 20 are shown the proportions of visible mutations pro- duced by X-rays in different organisms, plotted against the dose It 1s seen that, within the error of the experiments, the yield of mutations 15 proportional to the dose Tt 1s beheved that the y1eld of visible mutations produced by & given dose is independent of the wave length of the radiation 142 GENETICAL EFFEOTS OF RADIATION It ts often desired to collect all mutations occurring in a given chromosome, and not only mutations at o eclected locus Muta tions in the X chromosome are more easily studied than muta tions in autosomes, since the male has only ono X chromosome Normally it inhents this from its mother, but af a special attached X atock of females 1s used in the-matings, then the male recetvea its A chromosome from its father ‘Thus, when anarradi ated wild-typo male is mated to an attached-A female, a sex inked mutation produced in an irradiated spernt wall be revealed by the male offepring which develops from the egg fertilized by that sperm Alf that 1s necessary therefore 1s to examune the F; fies for mutant males The proportion of mutations 18 hable to be underestimated by the attached A method, not only because xt 19 easy to overlook a single mutant fly a culture, but also because most mutants are of reduced viability, and the proportion reaching matunty ts amaller than in the case of the wild type flhes Muller’s ClB method 1s more accurate, but has the disadvantage of requnng an extra generation The irradiated males are mated to CLE femalea The peculianty of ClB females is that one of ther 3 ehr CAITIES & te duplication acting as a dominant mutation, and producing an easily recogmzed narrowmg of the eyes (Ber eye) In addition, there 1s a long mveraon which effectively proventa crossing-over between this chromosome aud the other X ch during an the female Finally, there 1s @ recessive lethal m the chromosome so that a mele zygote receiving a ClB chromosome will not survive The radiated males are mated with CIB females and the half of the female F, offepring showing Bar eye is picked out Each of these females has two X chromosomes, one 1s a CLB, the other has been derived from the X ch of the aradiated sperm, and therefore carries a mutation if one was induced in this sperm by the radiation Each of these females 1s mated 1m a separate vial with one of its brothers, and the male F, offspring examined Each of these F, males receives its X chromosome from its mother In view of the lethal factor carmed by the CIB chromosome, m all the F, males which survive the X cbromosome 1s derived from the orginal wradiated X chromo some of the grandfather Hence 1fa mutation occurred as a result of the sradsation, ail the males of this culture will show 1t RECESSIVE LETHALS 145 pletely devord of males 1s determined, this gives the proportion of (viable) sperm which after urradiation had one or more re- cessive lethal mutations induced in the X chromosome A small correction 1s required for the small proportion of spontaneous lethals, and 1s determmed by carrying out a similar breeding test on unirradiated flies Expenments of this sort have been carned out for a number of years by different workers The prneipal results are (c) The number of lethals obtained increases hnearly with increase of doso 20 lweotPihesratcflpeahnetargme ~ a T iJ qT es T = n 2 L 4 1 2 3 wr, 4 3 6 Dose Fie 21 Percentage of sperm in which sex linked recessive lethal mutation 1s mduced by X rays (Tumoféeff Ressovaky) (6) The effect of a given dose 1s independent of whether 1% 18 concentrated into a short exposure at high intensity or 1s spread over a prolonged trme by fractionation or by the use of low mn tensity (c) Different wave lengths of X- and y rays are equally effective except for a possible shght reduction of efficiency for wave-lengths exceeding 1A Neutrons are somewhat less effective than X-rays, for equal 1on:zation in the tissue In Fig 21 the proportion of viable sperm which curry sex linked recessrye lethals 1s plotted agamst the dose of X-rays (the spontaneous lethals having been subtracted ) These data, ac- cumulated by Timoféeff Ressoveky: over a period of years, are x Timoféeff Ressoveky NW (1039) 144 GFNETICAL EFFEOTS OF RADIATION over tho usable rango 0 01-14 , and of the intensity at wich it 1s dehvered This conclusion has been accurately established for lethal mutations (vide snfra) in Drosophila Statistically ade quate expermments of a similar typo using vimble mutations would bo very laborious owing to the ten to fifteen trmes amaller rate of induction of visiblo mutations, but the proportion of visible mutations to lethals appears to bo the same under dif ferent conditions of irradiation,: which suggests that probably tho results obtained for lethal mutations apply also to visible Tate 37 Btutation constanta (2) Organism Mutation a Reference Tobacco mosaic virus Type > aucubs 18x )0-° 1 Aucubs + type 0s Bacteria (PA stewart) Average of three mutations 37 r affecting colony appearance Drosophila melanogaster ae’ a 2 wOWroOOewDen ws ut wt am fe +>m mo + tos for + wot 2 3 ww os i) 1 Gowen JW (1941) 2 Tumoféeff Ressovaky, NW & Delbrick BY (1936) Symbols used +eawild type, w=white eye colour, eye colour, wings f=oforked bristles 3 Tumoféeff Ressovsky, NW (19335) w*teany other allelomorph at the white locus mutations In view of the proportionality of mutation rate to dose, the simplest way of expressing mutation rate 1s by atatzng the mutatzon constant defined: as the probability « that a gene should mutate for a dose of one roentgen In Table 37 a het of mutation constants 1s given The general agreement in order of magnitude between the tation const of org: as different ag bacteria and Drosophila 1s remarkable Recessive Jethal mutations in Drosophila The general method employed in the investigation of sex- linked recessrve lethals in Drosophila 1s as follows A batch of male flies are rradiated under the chosen conditions and mated to CIB females The proportion of F, cultures which are com 1 Tumoféeff Ressovaky NW & Delbruck, M (1936) INTENSITY AND WAVE-LENGTH 147 Experiments, m which the effect of a given dose split mto & senes of fractions with rest periods between has been compared with the effect of the same dose given as a single exposure, have shown no difference in y1eld produced by fractionation : It may be concluded that the effect of a given dose 1s independent of the manner m which it 1s distributed in time 5 3 4b 3 S or I s oe . | me) 32 & F2 e 2F Fs 8 & & IF L J t 1 ' 0 lL. 7 7H J lor? To" ' OF 1 Roentgens per minute lethalsatd: t Fro 22 Yieldofsex lnkedr @ Timoféeff Ressovaky, Zimmer & Delbruck (X rays) © Wilhelmy, Timofeeff Reasovsky & Zunmer (soft X rays) x Ray Chaudhuri (y rays) Experiments usmg X-rays excited at different kilovoltages down to about 10kV (1e wave-lengths up to about 1A ), y rays and f-rays have not shown any difference in the yield per roent- gen for these radiations, as shown in Fig 232 Experiments with still softer X-rays of wave-length exceeding 1A have given a mutations ts proportional to the dose and independent of the intensity values of P of 0993 and 0 9996 are ob d ind iB bly little statistical vanation Further, there appears to be some error in the description of the experimental arrangement employed, since with the amount and disp of radium d bed, the doses in the 1932 experi ments ould have been at least a hundred times smaller than are required to produce the yield of mutations obtained 1 See Timofeeff Ressovsky NW (1937a) for 6 summary of these expernments, 2 Timofteff Ressovaky, NW & Zimmer K.G (1939) 146 GFNFTICAL FFFFOTS OF RADIATION based on the examination of some 60,000 F, cultures A straight line satisfactonly fits these points, the 2° test of goodness of jitr gwing P=0 16 Howovor, if the argument of Chapter 1 (p 72) 1s recalled, 1 will bo reahzed that sinco the experiment gives the proportion of aperm which carry a lethal, and not the mean number of lothals per sperm, the curve cannot be atnictly linear, since at no dose, however high, could the yield exceed 100% Instead, wo should expects the yield to be proportional to 1—e-TMP, where Ds the dose and m 1s the imtial gradient of the curve of yreld against dose In Fig 21 B 1s the curve 1-e-TM? and A its imtial gradient, which 1s 2 80% per 1000 r The x? test gives P=0 34 for the fit of B to the oxpenmental points Fig 22 shows the yield of sex linked Iethals per 1000 r pro duced by different mtensities of radtations Expenments by different authors taken together extend over a very wide range of intensities, from 0009 to 2700 r/min It 1s evident from Fig 22 that the yreld does not vary by more than a few per cent, if at all, for nearly a milltonfold variation of intensity « 1 The x! test 19 @ statistical test to determine whether the departures between 8 set of observational values and the values predicted by 8 hypothesis under teat are greater or not than could reasonably be ex pected on stat: 1g) ds P.s the probability thata dep equal to or greater than that observed should occur by chance A value of P between 0 9 and 0 1 18 interpreted to mean that the data tested are con aistent with the hypothesis A low value of P,eg <0 05 indicates that the data are not consistent with the hypothesis being tested, or that some unsuspected source of error is present A high value of P, eg between 0 99 and 1 0, ind. that the t of the observations is unreasonably low and casts doubt on therr rehability 2 As pomted out by Ohver, CP (1932), Gowen JW & Gay, EH (1933) Zimmer, K G (1934) 3 Timof ky, NW, Zam KG & Delbruck, M (1938) using X rays Wilhelmy, E Tumoféeff Ressovsky, NW & Zimmer, KG (1936) using soft X rays, Ray Chaudhuri, § P (1944) using y rays 4 Attention should be directed to the mutual agreement of the yields obtained at the two or three different intensttres employed in each experi ment, rather than to the shght difference in average yield obtamed in the i D Widely wave lengths were used by the different authors, and in some experiments lethals only and in others total sex lnked recessive mutations were count Hanson, FB & Heys F (1929, 1932) have also reported that the yreld of lethals produced by 7 rays 1s mdependent of the intensity We have not however included their results nm Fig 22 on account of some anomalies in their data When a x? teat 1s made of the goodness of fit of their 1929 and 1932 data respectively to the hyvothesis that the yield of X-RAYS AND NEUTRONS 149 The first quantitative measurements, and still the most com- plete, are those of Timofeeff-Ressovsky and Zimmer,: which have been included in Fig 23, and which show that the yield for equal iomzation m the tissue 1s only about two thirds as great for neutrons as for X-rays In Table 38 are collected data by various authors on the number of sex-linked lethals per v-unit of neu- trons These data have been corrected for spontaneous lethals Tate 38 The relative efficiencies of fast neutrons and x rays in the Production of sex linked lethal Drosophil Lethals per 100 X chromosomes per Ratio of Authors Radiation 1000 r or vy units yields Tunoféeff Ressoveky & Xx reys 2894005 0664006 Zimmert Neutrons 19240 15 Giles? A rays 241407 073403 Neutrons 176405 Dempster3 X rays _- 075 Neutrons Fanot Neutrons 23 403 080401 Demerec, Kaufmann & X raya 0934012 0664019 Suttons Neutrons 0524007 1 Timofeeff Ressoveky NW & Zimmer, KG (1938, 1939) Zimmer, K G & Timoféeff Reasovskv (1938 2 Gilea, NH (1943) 3 Dempster, BR (19410) 4 kano U_ (19436) 5 Demerec M (1938}, Demeree M, Kaufmann, BP & Sutton E (1942) Kaufmann BP (19410) and fitted by the least squares method to the formula 1—-e-TM2, and in the last column of the table the neutron yields per 1000v are compared with the X-ray yields per 1000 r using the figure 289% per 1000 r as the X-ray yield m cases where 1t was not determined as part of the neutron experment The experiments of Demerec, Kaufmann and Sutton were made with a stoch (Oregon-B) of Drosophila having an unusually low mutation rate, but when the neutron yield 1s compared with the X-ray yield with the same stoch the ratio1s approximately the same agin the other experiments The standard devations attached to the ratios in the last column are based on the number of lethals counted in the various expenments, and take no account of possible systematic errors in, dosimetry There seems to be hitle doubt that for inducing sex linked recessive lethais in Drosophita 1 Timofceff Ressovshy, , NW & Zimmer, . KG 193 8), Zi & Timofceff Ressovsky, NW (1938) ‘ Dy Bramer IK G 148 GENETICAL EFYFFOTS OF RADIATION somewhat smaller yicld Dosago measurements with these long wave-lengths aro hablo to error, particularly on account of un cortainty in the correction required for absorption in the tissue overlying the testes of the irradiated fliesGowen and Gay: obtamed a yield of 12% per 1000 r at wave lengths 15 and 23A , which by companson with other data 19 probably too low Wilhelmy, Trmofteff-Ressovaky and Zimmer obtamed a yield of 223403% per 1000r wang X-rays of 2-3A » Powlersitcpefhtneahlrasgme > 5a os uw T x! =I n te 1 ‘ L ' 2 } ator 4 6 Dose Fia 23 Jnduction of sex linked recessive lethals by different radiations (Timoféeff Ressovsky & Zimmer) Curve A @ Xraya, x soft X rays O yraya + frays Curve B: , neutrons Experiments using fast neutrons have been made, but, owing to the fact that neutron dosimetry 1s not yet usually 80 accurate as X-ray dommetry, thew rehability 1s an the «hele not ao high ag in the case of X- and y-rays For the results to have value 1b 18 -y for the dosages to be expressed in a unit comparable to the unt of X-ray dosage, eg im terms of Gray’s ‘y-unit’ (Chapter, p 20), which produces tn water an 1on:zation equal to that produced by lr of X-or y-rays American authors usually measure in terms of ‘n unita’, and we have converted to vy units m Table 38 on the basis that 1 n-unit=2 5 v-units 1 Gowen JW & Gay, EH (1933) z Withelmy, FE Trnoféeff Ressoveky, NW & Zimmer, KG (1836) In view of the d ties of doses the authors were not convinced that the yield was really lower than the yield of 289% obtemed with shorter wave lengths of GROUPING OF MUTATIONS SL that reported by Nishina and Moriwahy, and 1s in close agreement with the number expected on the basis of a random distnbution of lethals in rrradiated chromosomes It appears probable there- fore that no significant grouping effect exists The idea that a grouping effect 1s to be expected theoretseally has its basis in the fact that when a tissue 15 irradiated by neu- trons, the energy 1s dissipated by a comparatively small number of 1omzing particles (protons), each of which produces a large number of 1onizations per micron path The notion 1s that the passage of a proton through the testes of an rradsated fly will be arare event, but that when it happens usually several sperm will be affected Similarly, the passage of a proton through a sperm will be a rare event, but when it happens often more than one mutation will be produced This idea does not, however, bear further exammmation In a tissue irradiated by 1000 v units of neutrons, about 4 protons cross each square micron of areca Taking the dimensions of the sperm head to be 7 4 0 37,, this dose therefore leads to about 11 protons traversing each sperm The proportion of sperm m which lethals are mduced by this dose 1s 0 02, so that the probability of a lethal being produced in a sperm by a proton which crosses it 1s less than 0 002 There 1s thus no theoretical foundation for the idea either that with doses of the order commonly used the passage of a proton through a sperm 1s a rare event, or that when such a passage occurs the probability of a mutation 1s gh enough for more than one fre- quently to oceur We conclude that there 1s no justification either on experimental or on theoretical grounds for 1 belief that a Grouping effect of the sort looked for occurs with neutrons ‘There 1s clearly quite 1 high probability that a proton which produces a mutation, and which therefore presumably tray erses the chromosome thread, should pass through two or more ad- jacent turns of the spiral chromosome There 1s thus the poss: bihty that lethals may occur m compact groups Several lethals very close together would not, however, be distinguished from a single lethal by the methods used im the experiments cited It would be of great interest to determine whether the 51eld of sex linked lethals for a given amount of ionization diminishes stil further when a still more densely ionizing radiation 15 em Ployed Experiments with o rays are techmeally difficult on account of the short range (less than 70) of the rays im tissue 160 OFNETIOAL FFFFOTS OF RADIATION neutrons are, for equal romzation in the tissue, less effective than X-rays The suggestion has been made that, apart from the quant tative difference in the y1eld of mutations for equal :omzation in the tissue, neutrons differ from X-rays in that there 1s 0 tendency for a grouping of mutations to occur, 1¢ a tendency for several sperm in a given irradiated male to be affected Experiments do not, however, dicate that the distribution of Iethals 1s other than random Nagai and Locher: obtained a total of 44 lethals in 69 irradiated male flies which were tested, 1e an average of 0 6377 lethal per male In the absence of any specific grouping effect we should expect the numbers of males with 0, 1, 2, or 2 3 lethals to be given by a Poisson distnbution with m= 0 6377 The numbers expected on this basis are calculated to be 36 5, 23 8, 74, 18, and the evpermmental numbers are 39, 18, 10, 2 Tho agreement 1s satisfactory (y?= 2 3,n=2, P=0 3), and Nagat and Locher’s experiments therefore show no special grouping effect, but are consistent with the lethals being produced at random Similar data published by Nishina and Moriwaka: are also consistent with the distribution of lethals between different males being randoms (x?=2 6, n=2, P=0 3) Another type of grouping effect has been suggested by Nishine and Moriwakis Thus 18 a tendency for more than one lethal to occur in a given X chr They irradiated male fles with various doses of neutrons which produced lethals m from 5 to 15% of the sperm, tested some of the lethal-bearmg chromo somes, and obtained evidence that in 4 chromosomes out of 16 the chromosome carried more than one lethal This 1s five trmes higher than 1s to be expected on the basis of a, random distnbu- tion of Iethals mn irradiated chromosomes, and suggests a group ing effect (P=001) However, Fanos failed to obtain any grouping effect of this sort Out of 998 X chromosomes tested, 60 were found to carry lethals, but only 2 chromosomes carned more than one lethal This 1s a much smaller proportion than x Nagai MA & Locher GL (1938) 2 Mishma Y & Morwak: D (1939) 3 Nagai & Locher and Nishina & Monmwakt, however incorrectly report ther exp as of a grouping effect 4 Nishne, Y & Monwak: D (1941) Tho figures we quote refer to their experments NI NIV, NV and NVI 5 Fano U (19438) MUTATION BY @-BALS 153 between 100 and 8% In this way one 1s able to deduce: that the radiation induces 7 lethals in about 1000 X chromosomes The final estimate obtamed for the yield of sev-linhed recessive mutations by a rays 1s 0 84+03% per 1000r This 3s smaller than the yields obtamed with X-rays (2 89% per 1000 r ) or with neutrons (19% per 1000r), and indicates that the yield, for equal 1omzation in tissue, decreases in the order of increasing 1on-density of the radiation It 1s, of course, rather unsatis factory to compare the mutation rate mduced in primordial erm cells in the polar cap of the egg by one radiation with the mutation rate mmduced by another radiation in mpe Sperm, and more strictly comparable expenments are urgently required However, provisionally accepting the data at their face value, we have drawn up Table 39 showing the yield of sex lmhed lethals per 1000 r by various radhations The 10n-density of the Taute 39 Yield of sex linked lethala per 1000r of various radiations Brava y rays Soft X ravs Radiation or X rays 2-3A Neutrons =a rays. Yield ofsex Lnkedlethals 2 89% 223% 190% 084° Per 1000r (or v unite) tadiations sted increases from left to mght1n the table, a regular dimmution of yield with increasing 1on-density 1s apparent The following regults have thus been estabhshed a (a) The yield of recessrve lethal mutations 1s proportional to loge (6) For a given dose the yield 15 independent of the time over which the wrradiation 13 extended (c) The yield for a given ionization in the tissue diminishes with increase of 1on density of the radiation Thus the principal tests by which one recognizes an action of Tadiation produced by a smgle ionization (ep Chapter mm, p 72) are satisfied The idea has been current for some yeara that 2 mutation can be produced by a single 1on1zation in or in the immediate vicinity of @ gene, and in fact result (c) was predicted before the expen- 1 The method of calculation outlined above differs from that employed by Ward who arrives, however, at the same quahtative result ‘ess effective per ionization that 2 rays than X rays 162 GENETICAL HFFEOTS OF RADIATION Zimmer and Timoféeft-Ressovsky have shown that mutations are in fact produced by « rays when the flica aro mado to breathe an atmosphere contaming radon, but they wore not able to evaluate the dose recened by the gonads The most satisfactory method would be to make use of artificial insemination, which would permit of the sperm being wradiated sn wivo In the absence of results by this method, an attempt can be made to deduce the yield of Iethals from consideration of an experiment by Ward: At 8 cortain early stage during the development of a fertilized egg which 18 destined to give mse to a male fly, the cells which after numerous divisions and meiosis will form the sperm of the adult fly are congregated at ono end of the egg sufficiently near tho surface to be irradiated by raya from a source outade the egg Ward sradiated tho eggs at a stage m which there were about thirty of these primordial germ cells in the polar cap of the egg Ibis evident that if the radiation produces a recessive lethal i an X chromosome in one of these thirty cella, then a certain proportion of the sperm of the adult fly (namely all those con taining an X chromosome derived from the one in which the mutation was mduced) will carry the lethal, provided that damage produced by the radiation does not cause breakdown at some stage between irradiation and the formation of the mature aperm An X chromosome in whieh a lethal 1s not induced will, again provided that damage produced by the radiation does not cause breakdown, give nse toa normalsperm If experimentally at. 1s found that one quarter of the aperm of an adult fly carry & lethal, we unfer that in this fly the radzation has induced a lethal m one X chromosome out of four radiated s In Ward's experi- ments, 149 fies were tested and 7 were found to carry lethals, the proportion of sperm carrying lethals varyzng m different flhes 1 Zimmer, K G & Timoféeff Ressovsky, NW (1936) 2 Ward, FD (1935) 3 Evidently we are assuming that the proportion of Jethals 1s the same in X chromosomes we are able to test aa it 18 in X chromosomes which we are unable to test, on account of breakdown occurring at some stage a of the p dial germ cell and maturation of the sperm A atmular assumption is implicit m ali work on induced mutation yb d by di Drosophila sperm, since the doses given are normully sufficrent to render non viable » large propertion of the sperm and the exp it consists In the prop of induced in those which remam viable LETHALS AND STRUCIURAL CHANGES 155 known Joc:, and here the inference 1s that either there are no viable allelomorphs of these genes other than the wild type, or else that they do not produce phenotypes recognizably different by inspection More recently the situation has been complicated by the reahzation that the recessive lethals do not form a homogeneous class, but may be subdivided into the following three ty pes (A) The chromosome contaimmg the lethal may be struc- turally unchanged as far as can be determined by an examina- tion of the salivary gland chromosomes This type may reasonably be regarded as mutation to a lethal allelomorph, or loss by the gene of the power to reproduce itself (B) There may be a minute deficiency, revealed by the ab- sence of one or more bands in the salivary chromosome, the deficiency including the locus at which the lethal occurs The smallest deficiencies, eg where a single band 19 deleted, may perhaps be caused by the destruction of the capacity for repro- duction of a single gene Somewhat larger deficiercies involving several bands are probably due to the simultaneous breakage of the chromosome at two places due to the passage of a single ionizing particle Stall larger deficiencies, up to 50 bands or nearly one tenth of the whole chromosome, are probably due to independent breahage of the chromosome in two places by separate ionizing particles: Deficiencies exceeding about 50 bands are not observed, doubtless because they behave as dommant lethals,1e are not viable even im the presence of a non deficient homologous chromosome (C) There may be a gross structuril change (inversion or interchange) not usualy involving any cytologically detectable deficiency, one of the breaks comcding with or being very close to the locus of the lethal As already mentioned, there are two possible explanations of this One 1s that the lethal mutation 1s % position effect due to the separation of the gene concerned from its usual neighbour or to its being brought into contact with a different gene The alternative explanation 1s that the lethal 1s an interna! change mm the gene (mutation to a lethal allelomorph or loss of the power to reproduce) w hich was caused by the 1omzing particle which broke the chromosome 1 This distinction between deficiencies caused by one or by two lonizing particles will be justified later lot GFNETICAL FFFECTS OF RADIATION ment had been performed + If mutation 1s o rather direct result of romzation, one may expect that tho yield of X ray induced mutations will be independent of the temperature at which the matenal 1s held during irradiation Stadler observed no influence of temperature on induced mutations in barley The evidence in the case of Drosophila is conflicting (See p 364) Tantr 40 Indepondence of 5 told of eex linked lethals on the temperstere (Timoféeif Ressovaky & Zimmer)* Tempera ho of X 9% ofX ture chromozomes ho of chromosomes Dose {r} *¢ tested lethals having lethals 3000 10 B02 ci) B48 33 736 60 815 2750 3 1821 158 846 33 1645 1st 815 1200 7? 916 25 236 32 921 29 315 Tost of bypotheers that yield 1 independent of temperature gives xf2 15 n=3, Po Relation between lethalss and cht str B A few years ago it was supposed that a recesstye lethal muta- tion, Ithe a visible geno mutation, was an mternal change in the gene, either giving an allelomorph which was lethal, or else destroying the capacity of the gene for reproducing tteelf, so that the gene was lost On ths basis a consistent picture was built ups on which the prinerpal experimental facts concerning both induced and spontaneous mutations were explained The view that Jethals are essentially similar to visible gene mutations 13 supported by the fact that many Iethals have been found to occur at Joc of the chromosomes at whych visible allelomorphs are known Thus if a female fly 1s known to carry a recessive lethal an one of 1ts X chromosomes, which 1s otherwise wild type, and the white-eye allelomorph in the other X chromosome, then sf the fly 1s phenotypically white one infers that the lethal mvolves the white locus Many other Jethals are not connected with 1 Timoféeff Ressovaky N W (1937) 2 Timoféeff Ressovsky, NW & Zimmer, KG (1939) 3 Inthtssection by lethal 1s to be understood eex Ianked receastee lethal 4 The section foliows the treatment given by Lea, D E & Catche side DG (19154) 5 Cp Iimofleff Ressovsky, NW Zimmer, KG & Deibruck, M (1935) DFPENDENCE OF YIELD ON DOSE 187 each dose determmed the proportion of the lethals which were associated with gross structural change His results are repro- duced in Fig 24 It 1s evident that the number of lethals associated with gross structural change mcreases more rapidly than dose (curve I 1s the curve (dose)!), but that 1t 1s the fotal number of mutations (curve IIT) not the number unassociated with gross structural change (curve IT) which increases hnearly with dose lwoePtiehrptacfelnh:tramge ae = 2 1. ’ 2 3 aw, 4 BT 6 Dose Fie 24 Analysis of sex linked recessive lethals (Ohver) I lethals associated with gross structural change II, lethals not associated with gross structural change, ITI, total lethala To make the test objective, we have assumed in turn (i) that the mean number of lethals per chromosome 18 proportional to dose, (u) that it 1s the sum of two terms, proportional to (dose)? and (dose)? respectively, and have determmed what 1s the maxi- mum proportion of the (dose) lethals which can be admitted, without disagreement with experiment The x test was used as & measure of agreement with experiment, spontaneous lethals Were allowed for, as also was the distinction (discussed on p 146) between the mean number of lethals per chromosome and the Proportion of chromosomes carryimg lethals The experimental data used were those shown mn Fig 21, obtasned by Timofeeff- Ressovsky: and based on some 60,000 cultures The results of the 3? tests are given im Table 41, m which the proportion of {dost} lethals postulated 1s indicated by stating in the first 1 Timoféeff Ressovshy, NW (1939) 156 OENFTICAL EFFEOTS OF RADIATION 4\ decision between these two possible explanations of a lethal associated with a gross structural change should be possible on tho basis of the oxpenmental data of the sariation of yield with dose Lethals of class (A), not associated sith structural change, should increase linearly with dogo, being produced by single sonizing particles Those Jethals of class (B) which are associated with very small deficiencies produced by a single iomzng particle should also increase nearly with dose, A minority of the claas (B) lethals are associated with larger deficiencies caused by two 1onizing particles, and these will crease with a Ingher power of the dose If tho association of a lethal with a gross structural change 13 an example of the position effect, then the lethals m class (C) form a separate type tho number of which 1s proportional to the number of gross structural changes produced Now this number increases moro rapidly than the first power of the dose, since grosa structural changes involve two or more breaks which (with X rays) are produced by separate :onizing particles Cytological observation shows that in the dose range 1000-4000r the num ber of gross structural changes 1s proportional to (dose)! On the Posstion effect explanation therefore the observed lethals should be the sum of two types, one type not associated with gross structural change, the number of which 1s proportional to dose, and the other type, associated with gross structural change, the number of which 1s proportional to (dose)! The total number should therefore increase more rapidly than the first power of the dose On the other hand, :f the lethals associated with gross struc tural change are not Sy different from other lethals, and the association with the structural change 1s due to the curcum- stance that the ronizing particle which caused the lethal alse caused a break which happened to take part in a chromosome rearrangement, then the total number of lethals of types A and C should be proportional to the dose The number associated with gross structural change will increase as (dose)?, the residual number not associated with gross structural change will mcrease less rapidly than the first power of the dose Olver: made some experiments in which he determmed the number of sex linked lethals as a function of the dose, and at x Ohver CP (1932) THREE TiPES OF RECESSIVE LETHALS 159 that since some of the breaks which take part in structural change are lethals, so also some of the breaks which restitute are lethals Such lethals will be recorded as type A lethals (p 155), 1e lethals without any cytologically detectable chromosome change We have no reason to suppose a@ prior: that a type A lethal cannot be produced without the chromosome at the same tame bemg broken However, admitting the necessity, on other grounds, for a considerable number of restitutional breaks, a large part of the type A lethals must be restitutional breaks, and we shall see how far a consistent picture can be obtained on the basis that all the ty pe A lethals are restitutional breaks As a begining we need to know the numbers of the three types of lethal produced by a given dose The experimental result 1s that 3000 r produce in 1000 X chromosomes 87 lethals, of which 29 are type A, 18 are type B, and 30 are type C: Now the number of breaks in the euchromatin of the X chromosome which take part in gross structural change when a dose of 3000r is given to the sperm 1s 80 per 1000 X chromosomes,: 30 of these carry lethals (16 the 30 type C lethals) Evidently the probability that a chromosome break shall cause a lethal 1s 30/80=0 38 There are 18 minute deficiencies (1€ the type B Iethals), which are lethal because one or more loci are deleted There 1s reason for belheving that when a chromosome 1s broken in two places, the probabilities are approximately equal that the segment be- tween the breaks shall be deleted and that 1t shall be inverted 3 We presume therefore that there are also 18 mmute mversions An inversion will not, on our view, behave as a lethal per se, but since it involves two breaks each of which has a probability 0 38 of bemg a lethal, the probabihty 1s 1—(1—0 38)?=0 62 that at least one of the breaks will be a lethal Thus of the 18 minute inversions 18 x 0 62 = 11 will bebave as lethals, and will therefore be included 1n the type A lethals, since a minute inversion will rarely be recognized cy tologically This leaves 28 type A lethals which are restituted breaks L fee Lea, DE & Catcheaide, DG (19452) for details 2 Bauer Huction by Fano U ( (1941) ) from sal: hvary (19590) 2 gland observations of 3 Demeree “I Kaufmann BP Sutton E & F Demeree, Mi & Fano,U (1941) sno, Gee, 158 GENETIOAL FFFECTS OF RADIATION column the proportion of the total number of Jethals which at 3000 r aro of this class It 18 clear that the data provide no evidence for any (dose)! component, that a proportion as high as 17 5% 15 improbable, and that © proportion as high as 22% can practically be ruled out Romombering that such of the deficiencies of type B as involve two independent 1onizing particles will account for some Taste 41° Analysis of nex linked lethals into (dose)! and (dose)! classes Percentage of lethals at 2000r which Degrees of belong to tho {dose)l class x freedom P 0 23 2 O32 125 36 2 018 175 64 2 oo 220 a6 2 0008 of the admissible (dose)! component, 1t appears improbable that assumption (11) of the previous page 1s correct To establish this conclusion we need to know the proportion of lethals which are, at 3000r, associated with gross structural change Expert mentally :t 13 found that this proportion 1s 0354004: This proportion being higher than the maximum proportion wluch can be reconciled with Table 41, we conclude that the lethals associated with gross structural change do not constitute an additional class of lethals caused by position effect and requiring two 1onuing particles for the expression of the lethal, but merely represent those cases where the 1onizng particle which caused the lethal caused also a break which took part in structural change We shall develop the subsequent discussion on this basis There 1s good reason to believe: that not all the chromosome breaks prmaniy produced take part in structural change, but that many ot the broken ends reyom and the restituted chromo some 38 cytologicalily mdistingushable from an unbroken chro’ Whether restit or structural change occurs appears not to depend on a diilerence m the breakage process, but mamly on whether other breaks are available with which interchange can occur It seems necessary to accept therefore 1 Based on data by Oliver, C P (1932) Demerec,M (1937) Demerec, M & Fano, U (1941), reviewed by Lea, DE & Catcheside, D G (19454) 2 See Chapter vir DOMINANT LETHALS lol Domunant lethals in Drosophila A recessive lethal mutation in a dsploid orgatusdt ts a gene change (or deletion) which m the homozygous or hemizygous condition results in the orgamsm being non-vinble When the cells of the organism are heterorygous for tho lethal, the 10 4 08F 06 S. 4 osf 4 a oa} O33 - = osuPervfigpvionrgtsion 02 olr + 008 4 O05 0 06F 1 0-04 . 003F 0 027,- 0 008 A 0 006 B 7)1 i J n wl 1 sued, 4 © ay, 8 10 12 14 Doso Fic 25 Proportion of eggs fertized by irradiated sperm which attain the stage of A, larvae B, adult flea (Catchosido & Lon) organism 1s viable Since there are among visible gene muta tions both dominant and recessive mutations, we may by analogy expect also dominant lethals to exist’ An cee furtilzed by a sperm carrying a dominant lethal will by defmition not give nse to an adult organism In consequence a domimant Icthal cannot be studied in successive generation s and cannot be ob tained ina salivary chromosome, and we can only infer the currence ofa dominant Icthal in irradiated Drosophila sperm, 10 GFNELTICAL FFFECTS OF RADIATION Since only 38 % of breaks aro lethals, the total number of rest: tuted breahs must be 28/0 38=74 The total number of breaks of all sorts produced by 3000r in the euchromatin of 1000 (viable) A chromosomes 13 therefore 226, made up of 36 mn minute doletions (2 breaks per deletion), 36 in minute inversions, 80 in gross structural changes, and 74 which restitute Thus of tho 226 breaks at 3000r a proportion 80/226=35% take part in structural change At greater doses the proportion will be higher, at smaller doses 1t will be lower It 1s to bo noted that 11 out of 38 or about 30% of the ty pe A lethals are expected to bo minute inversions Shzynski,« from observation of salyary chromosomea, suspected that some of the non-deficiency lethals were minute inversions In this way it 18 possible to build up a consistent pictures of the production of recessive Icthals To aummanze Breaks are caused in the chr by tho passage through them of lonizing particles Sometimes an ionizing particle causes two breaks close together, in which case deletion or inversion may occur of the segment of chromosome between the breakage points The remaming breaks are available for taking part in gross structural change, the probability of a break doing so being dependent on the availability of other breaks, and thus increasing with increase of dose Those which do not take part in gross structural change rejoin in the onginal formation 3 Any break rrrespective of its subsequent hustory may result in a lethal change in the gene at or adjacent to the brethage pomt, this change probably often being destruction of the power of reproduction The probability of a break causing a lethal in this way 13 038 Thus any type of chromosome structural change— munute deletion, minute mversion, or gross structural change— 1s hable to have a lethal change m the gene adjacent to a breakage pomt Apart from this, a deletion wall usually have a recessive lethal effect on account of the absent loc: x Shzynsba, BM (1938) 2 Some 1 evid ipp the picture ined 13 given by Lea DE & Catcheside DG (1945a) 3 Weare lmuting our discussion at present to viable changes A third yt ofa broken leada to a d lethal (ude wfra) DOMINANT LETHALS 163 inert. Finally, it will be found that a quantitative eaplanation of the curve relating the yield of dommant lethals to the dose can be given on the basis that the cause of dominant lethals 1s the breakage of chromosomes by the radiation It appears that dominant lethals are for the most part chromo some changes, rather than gene mutations proper Many dele hons, involving a few bands of the salivary chromosome and one or a few known loci, have been studied, and have mostly been found to behave as recessive lethals It appears therefore that deficiency for a single gene usually has a recessive lethal and not a dominant lethal effect It seems probable that a gene muta- tion should have a more strongly lethal effect than a complete deficiency for the gene concerned, ao that dominant lethal muta tions in the strict sense of intra gene changes are probably rare Larger definencies than about 50 sahvary bands are, howevet, not found even in the heterozygous condition, nor are dicentric chromosomes ever found in salivary chromosomes There 18 reason to believe, however, that deletions are produced about as frequently as inversions of the same size, and that asym- metrical mterchanges, producing dicentric and acentric chromo somes, are about as frequent as symmetrical interchanges pro- ducing monocentric chromosomes Larger deficiencies must therefore be presumed to behave as dominant lethals An acentrie chromosome, since it lacks a centromere which 1s re sponsible for the commencement of the migration of the chromo- some to the pole of the dividing cell, 1s hable to be left out of both daughter nucle: The two centromeres of a dicentric chromosome may attempt to migrate to opposite poles, or if they migrate towards the same pole the sister chromatids may interlock, in either event leading eventually to loss of the chromosome or breakdown of the dividing cell 2 There 1s ttle doubt therefore that the larger deletions, and asymmetnical interchanges, behave as dominant lethals However, calculations based on the fre- quencies found for the observ able types of aberration show that these aberrations are not frequent enough to account for the whole of the dominant lethals 3 Moreover, the shape of the sur- vival curve (Fig 25) 1s not consistent with the explanation that dicentric interchanges and larger deletions are entirely re i Demerec, M, Kaufmann BP, Sutton E & Fano, U (1941) 2 Pontecorvo, G (1942) 3 Fano U (1941) 162 QENITICAL FFFLUTS OF RADIATION , for oxample, by observing that a proportion of the eggs fertilized by such sperm fail to develop into adult flies A number of authors have made expenments to determine how the proportion of eggs which hatch, or which reach the pupal or adult stages, diminishes with smerease of the dose of Tadiation received by the sperm wluch fertihzed the eggs Fig 25 shows tho results: obtained im an experiment of this sort The close proximity of curves A and B, whieh refer to larval and adult survival respectively, shows that death caused by uradia tion of the sperm usually occurs in the embryonic state, and that those organisms which survive to the larval stage usually suc cesafully develop into adults Examination of the developing embryo in an egg fertilized by an irradiated sperm shows abnormal division figures with clumped and broken chromosomes 2 The question immediately arises whether there 13 any justr fication for the term di t lethal in d: if experiments of this ort, carrying as 1t does the implication that s genetical effect 13 concerned, or whether the effect may not be a physio logical effects on the sperm not specifically affecting the genes or chromosomes There aro a number of argumentss which indicate that the effect 18 a genetic one In the firat place the sperm 1s almost entirely composed of chromatin, the volume of the head being about equal to the combined volume of the chromosomes 1t contains (taking as the volume of the chromosomes the volume they occupy when in their most condensed state, meiotic metaphase) Thus the action of radiations on sperm can hardly be on cytoplasm or nuclear sap Secondly, the proportion of female flies hatchmg from eggs fertilized by irradiated sperm 13 reduced more than the proportion of male flies, showing that the X-bearing sperm are more sensitive than the ¥ bearing sperm If the action 1s a genetic one on the chromosomes, then this result 1s to be expected atnce fewer breaks are produced in the ¥ chromosome than in the X, and 1t 1s genetically practically 1 From an experiment of Catcheside DG & Lea DE (1945a) Asummary ofother work on this subject 1s given in this paper In Fig 25 correction has been made for deaths mm the controls 2 Sonnenbhck BP (1940) 3 This vague term 13 convemently employed to denote an effect other than a direct effect on @ single structure such as a gene or a chromosome 4 Muller, HJ (1940) CHROMOSOME LOSSES 165 unjomed single breaks, if they do occur, will usually behave as dominant lethals That chromosome losses induced by a single 1onizing particle do in fact occur has been shown in experiments: specifically de signed to study viable loss of the sex chromosomes, and in which the zygote which had sustained the loss could be distinguished from one winch had not The percentage of losses induced was found to be proportional to the dose, indicating that loss occurred by a single break process The number of wable losses obtained in these experiments was small, much smaller than the number of unjoined breaks ex- pected in the sex chromosomes on the basis of the number of unjoined breaks in the autosomes needed to explain dominant lethals It appears that though the complete absence of one sex chromosome from the zygote would have been viable, the pro- cess of loss often caused death The explanation 1s possibly to be sought in the mechanical upset of mitotic diviston caused by the chromosome bridge which 1s formed when a dicentric chromo some 18 present We now proceed to obtain a mathematical expression for the yield of dommant lethals as a function of the dose of radiation given to the sperm on the basis of the mechanisms we have been discussing, namely, unyoined single breaks and asymmetrical interchanges 1 To make the mathematical analysis manageable it 1s necessary to simplify the problem to some extent The prnerpal simphfying assumptions are that the possibility of more than one break occurring in a single chromosome arm 18 neglected, and joming between the vamous broken ends in the cell 1s supposed to be at random The first simplifying assump- 1 Muller HJ (1940), Pontecorso G (1941) The method an Ponte corvo’a expenment was aa follows The irradiated males had a special Y chromosome contaming the wild type allelomorph (y+) of the X chromo some gene y (yellow body) They were mated to attached X females homozygous for y The female offspring are normally of the constitution X X/¥, derning the attached X chromosome X X from the mother and the Y¥ chromosome from the father, and are grey bodied since the y+ in the Y masks the recessive ygenesinthe X Xchromosome If asaresult of irradiation of the sperm, the sex chromosome (either X or Y) 1s lost from the zygote a female of conatitution X X{O 1s formed which will have @ yellow body since the y genes in the ¥ X are no longer 2 The treatment given is due to Lea DE & Catcheade DG (1948a) 104 GENETICAL EFFEOTS OF RADIATION sponmblo for dominant Jethale The survival curves, which in Fig 25 are plotted on a logarithmic ecalo, aro initially linear As explained in Chapter i, a survival curvo which 13 linear on 6 fogarithmuc realo suggests that a single jomzng particle causes the effect studied Now an interchange or a deletion (other than a minuto deletion) involves the production of two chromosome breeka by two separate 1onizing particles We conclude that in addition to the production of asymmetrical interchanges and deletions, there 28 some other mechanism, involving 8 single somuzing particle, which has a domment Jethal effect At small doses this mechamism predommates, giving the approximately linear curve At higher dosea the two-break aberrations, the number of which increases more rapidly than the first power of the dose, will become increasingly umportant, thus explaimmg the mcreased gradient shown at higher doses in Fig 26 Chromosome loss, caused by a single chromosome break, has been plausibly suggested: as the mechamam in question When a chromosomes broken, the two broken ends usually Jom, exther with one another restituting the ongmal chromosomes, or with other broken ends which may be available in the cell grving one of the various famihar types of chromosome aberration (inter- changes, mvereions or deletions) It appears, however, that na small proportion of cases the two broken ends renam unjomed If atall unyomed when the chromosome aplita into two chro- matids, +t appears hkely: that sister-unton of the chromatids will ocour at the breakage pomts At anaphase therefore we have one acentrie fragment and one dicentric chromosome As already explaimed, such chr are likely to be lost at cell division Now loss of a chromosome 1s certainly lethal to Drosophila in the case of the large autosomes IT and IIT, though a fly can exist a the absence of one of ita very small IVth chromosomes Moreover, X/O males are viable, and such individuals may be considered as normal males which have lost a Y or as normal females which have lost an X chromosome Thus losses of the Vth chromosome, or of the sex chromosome as p result. of irradiating eperm, may be viable However, since about 80% in the chromosomes JJ and IH, of all the breaks produced occur I Muller H.J (1940), Pontecorvo G (1941, 1042) z From analogy with known types of aberration in plant material See Chapter vr CHROMOSOME LOSSES 165 unjomed single breaks, if they do occur, will usually behave a3 dommant lethals That chromosome losses induced by a single 1onizing particle do in fact oveur has been shown im experiments: spectfically de agned to study viable loss of the sex chromosomes, and in wluch the zygote which had sustained the loss could be distinguished from one which had not The percentage of Insses induced was found to be proportional to the dose, indicating that loss occurred by a single break process The number of viable losses obtained mm these experiments was small, much smaller than the number of unjomed breaks ex- pected in the sex chromosomes on the basis of the number of unjomed breaks in the autosomes needed to explain dominant lethals It appears that though the complete absence of one sex chromosome from the zygote would have been viable, the pro- cess of Joss often caused death The explanation 1s possibly to be sought in the mechanical upset of mitotic division caused by the chromosome bridge which 1s formed when a dicentric chromo- s0me 18 present We now proceed to obtain a mathematical expression for the yield of dommant lethals as a function of the dose of radiation gtven to the sperm on the basis of the mechanisms we have been discussmg, namely, unjomed smgle breaks and asymmetnical interchanges 2 To make the mathematical analysis manageable 1t 1s necessary to simplify the problem to some extent The pmncpal simplifying assumptions are that the possibility of more than one break occurnng mm a single chromosome arm 138 neglected, and yommng between the vanous broken ends m the cell 1s supposed to be at random The first simplifying assump- 1 Muller, HJ (1940) Pontecorio, G (1941) The method in Ponte- corvo 8 experiment was as follows The irradiated males had a special Y chromosome contaming the wild type allelomorph (y+) of the X chrome some gene y (yellow body) They were mated to attached X females homozygous for y The female offspring are normally of the constitution X X/Y¥, denying the attached X chromosome X X from the mother and the Y¥ chromosome from the father, and are grey bodied since the yt in the ¥ masks the recessive y genea in the X X chromosome If aaa result of srradiation of the sperm the sex chromosome (either X or ¥) 18 lost from the zygote s female of constitution XX; {0 xs formed which will havee a Sellow body since the y genes in the X¥ X sre no longer 2 The treatment givenis due to Lea DE & Catchende DG (19454) 168 QENETICAL EFFECTS OF RADIATION tron will not be a serious source of error 1 The second assumption 18 plauatble for Drosophila sperm though not for some other matensls (sce Chapter vst) We take p to be tho probability that a given break shall neither restitute nor take part in interchange, but shall lead to chromosome loss and therefore to a dominant lethal effect q=1—p 1 the probability that it shall either restitute or inter change If thero are r breaka in the cell, q” 15 taken to be the probability that all either restitute or interchange Suppose that with dose D the mean number of breaks per sperm 18 m=aD The proportion of sperm having r breaks per sperm 1s given by the Poison distribution, and 1s e-TM m'/r! The probabihty that a sporm shall have no breaks 1s e“TM The probability that it shall have one break 13 m e~" Sperm with one break will contribute (1—-g)m e-TM to the number of dom nant lethals, and gm e~TM to the number of viable nucle: without aberration Of the }m?e-TM sperm with tno breaks per sperm, (1—92)4m? e-” will be dommant lethals owing to failure of one or both breaks either to restitute or to interchange In jm? g? e-" sperm the four broken ends will all join Under the assumption of random joing, m one third of these aperm there will be restitution, giving viable sperm without aberration, 2 one third there will be aymmetneal mterchange giving viable sperm with chromosome aberration, and 1n one third there will be asymmetrical mterchange adding a further quota to the dom:- nant lethals Thus of the sperm with two breaks, jm? g* e~TM will be viable without aberration, $m? g? e~TM will be viable with aberration, and the remaming $i? e-” (1 —§g?) will carry domt- nant lethals In general there will be e~TM m‘/r! sperm having 7 breaks In e-" mf gt/r! sperm no breaks will remain unjyomed In a sperm of this class the r breaks can yom m 135 (2r— (7 #2") = (2r) 1) ways, of which one way 18 viable without aberratron, r!— 1 ways are viable with aberration, and the remainder are inviable z 1 It can be avoided at the cost of some complication of the calcula tions Haldane TBS & Lea DE (unpublished) 2 Catcheside DG (1938a) cp also Fano U (1943a) These results follow from the postulate of randorn joming of broken ends as MATHEMATICAL THEORY 167 Collecting the contnbutions from sperm with various numbers of breaks, and replacmg m by its value aD, we have Proportion of cells which are viable and without aberration 13 Nae, where S,=14aqD+}(agDR+ + Ce (V-1) Proportion of cells which are viable (with and without aberration) 8 Y=e2S,, where S,=l+aqgD+}(agD)?+ + ee (V-2) Total number of primary breaks formed in viable sperm, per total Sperm 1s Z= e-2DS,, (QagDy rr! where S,;=aqD+3(agD)?+ 9 + + (V-3) (2r)! The sums S,, 8, and S, of the imfimte series mm equations (1), (2) and (3) can be shown to be given by the followmg algebraic expressions S,=cosh ./(2agD), (V-4) S,= 14 4(dragD) et*9? erf /(tagD), (V-5) l+agD Jn podeaD [1+ Teas Sy=4haqD |1+ 77 XS elePerfi(jagD)}, videg (V6) where cosh x= 3(e*+e-7) 15 the hyperbolic cosine, and erf ane e-** dz 1s the error function In Table 42 values of §,, Sy and S, are tabulated for a suitable range of values of agD One observable quantity 1s the proportion of viable sperm which have chromosome structural changes The theoretical ex- Pression for this proportion 1s evidently (1— X/¥)=(1 — 51/8), and 1s listed in Table 42 as a function of aqgD InFig 26 we show experimental data of the proportion of viable sperm having chromosome structural changes as a function of the dose,: to- gether with the theoretical curve (1—S,/S,) which has been fitted to the data by taking ag=0 57 per 1000r ass ee DG (1938a) Bauer, H + Demerec, M & Kaufmann BP 166 GFNETICAL EFFECTS OF RADIATION tion will not be a scrious source of error + The second assumption 18 plausible for Drosophila sperm though not for some other materials (see Chapter vit) Woe tako p to be the probabihty that a given break shall neither restitute nor take part in interchango, but shall lead to chromosome loss and therefore to 2 dommant lethal effect q=1—p is tho probability that it shall either restitute or iter change If there are r breaks in the cell, g” 13 taken to be the probability that all either restitute or interchange Suppose that with dose D the mean number of breaks per sperm 1s m=a@D The proportion of sperm having r breaks per sperm 1s given by the Poisson distribution, and 1s e-TM m’/r! The probability that a sperm shall have no breaks 18 e-TM The probability that st shall have one breah 1s m e-TM Sperm with one breal, will contnbute (I—g)m e-TM to the number of dom nant lethals, and gm e~TM to the number of viable nuclei without aberration Of the }m?e-TM eperm with two breaks per sperm, (1—?}4m? 6-" wall be dominant lethals owing to failure of one or both breaks either to restitute or to mterchange In }m? q? e-TM sperm the four brohen ends will all yom Under the assumption of random yomng, i one third of these sperm there will be restitution, giving viable sperm without aberration, 2 one third there will be symmetrical interchange giving viable sperm with chromosome aberration, and m one third there will be asymmetrical mterchange adding a further quota to the domi- nant lethale Thus of the sperm with two breaks, 3m? gem will be viable without aberration, 4m? g? e-TM will be viable with aberration, and the remaining $m? e~TM (1 — §q?) will carry dom nant lethals In general there will be e~TM m*/r! sperm having r breaks In e-TM m’ gt/r! sperm no breaks will remain unjomed In a sperm of this class the r breaks can join m 135 = (2r) fr 127) (2r—1) ways, of which one way 18 viable without aberration, r t_] ways are viable with aberration, and the remainder are inviable « 1 It can be avoided at the cost of some compheation of the calcula tions Haldane TBS & Lea DE (unpubhshed} 2 Catcheside DG (1938a), ep also Fano U (1943a) These results follow from the postulate of random joining of broken ends MATHEMATICAL THEOR} 169 Asecond observable quantity 1s (1 — ¥ ), the proportion of total sperm which are non-viable In Fig 27 the expermmental ob- servations: on the proportion of eggs fertilized by madiated sperm which fail to attain the adult stage are plotted, together Taste 42 Functions involved in the dominant lethal theory agD Ss Sy dy (1-55) Syl; 032 1337 1358 0.3952 v 0140 02914 050 1543 1592 0 6942 0 0309 0 4360 072 1811 1920 1152 0 0572 0 5996 098 2161 2373 1849 0.0935 07793 128 2517 2996 2915 0 1396 09731 162 3107 3858 4553 0 1945 1180 200 3762 5 060 7090 0 2565 1401 288 5557 9172 17.29 03041 1 885 392 8 253 1778 43.23 0 5357 2432 512 12.29 36 82 lbz2 0 6663 3 046 648 1831 8157 304 6 0 7755 3 734 800 2731 193 6 870 8 0 8590 4497 with the theoretical curve In computing the formula for Y (see equation (2)) we already know that ag=0 57, which enables S, to be calculated for each dose with the aid of Table 42, a still Temains arbitrary The value 2=0 75 was found to give the best fit of the theoretical curve to the experimental points It follows that g=0 57/0 75=0 76, so that we have the figures a=0 751s the number of primary breahs produced per sperm per 1000r q=0 76 1s the probability that a break shall join, either in restitution or im interchange p=1—q=0 24 1s the probability that a break shall remam unjomed, and shall behave as a dommant lethal It 1s with these values of a, p and q that the theoretical curves in Figs 26 and 27 have been computed It 1s of interest to calculate the mean number of breaks pnmanily formed per viable sperm (which will be a little less than aD) Referring bach to equations (2) and (3), this 13 seen to be S,/S, 85/8, 13 tabulated agamst agD m Table 42 Using the value 2q=0 57 just found and mterpolatmg in Table 42, we obtain the estimates given in Table 43 of the mean number of primary breaks per viable sperm We can calculate from these figures the number of primary breaks m the euchromatin of the 1 Catcheside, DG & Lea, DE {1945a) 168 GENETIOCAL EFFECTS OF RADIATION sol. 7 40F- e x we . ar x 10}- x “ ry L L L L i L ! 2 > io, 4 ’ ® Dose Fra 26 Percentage of viable eperm having gross chromosome structural Curve theoretical points experiments of @ Bauer Demereo & euffoann x Catcheade toof- ’ sot 4 coh wL 2b ‘ A A 1 1 1 2 4 6 0 6 10 2 Dose Fria 27 Percentage asa function of dose Curve theoretical of dominant lethals e‘pomta experunents of Catchearde & Lea SEX-RATIO DISTORTION 171 stock in which the X chromosome, instead of bemg in the form of a rod, 1s ring shaped, then the sex-ratio distortion 1s much greater The reason for this 1s that not only does a break which fails to join have a dominant lethal effect, but also a proportion mfatoelRmealfsteiso fa —L L i 1 1 n 2 4 6 My 8 10 t2 14 Dore ne 28 Depression of the sex ratio in the progeny of trrad:ated males Curves eoretical pointe experiments of A rod 4 stock + Hanson © Muller 3 Goren & Gey, © Bauer x Catcheside & Lea B ring X stock e Bauer, of the breaks which restitute For, if the broken ends rotate through half a revolution relative to one another before re joining, then when the chromosome «phits into two chromatids. instead of these forming two separate rings, they will form 2 single rmg of twice the size Such a chromosome, being dicentric wil} be lost and thus behave as a dommant lethal It 1s known: that in approximately half of the cases restitution is inviable Thus the probabihty that a smgle break shall have » dommant 1 From a etudy of the relative fre quencs of mversions rod X chromosomes Catcheside DG & Ler DE (19468) X end 170 GENFTICAL EF} EOTS OF RADIATION X chromosome, by making use ofthe result that a fraction 0 162 of all observed brenks occur there : Thus at 3000 r 0 162x 1 2350 199 primary brenks occur in the euchromatin of the X chromosome Tautr 43° Mean number of primary breaks per viable sperm Dose (r} 1000 1500 2000 3000 4000 6000 215 viable sperm Pn reake per 049 O69 089 123 154 Mean number of primary bi The analysis wo havo just given of the dommant lethals and chromosome aberrations leads to anestimate 0 199 of thenumber of primary breaks produced by 3000 r in the euchromatin of the A chromosome in sperm which remain viable Our analysis of recessive lethals on the basis of the hypothesis that recessive lethals are rey d brenhs led indep dently to an estimate of this same quantity, the value obtained (p 160) being 226 per 1000 sperm, 1e 0 226 per sperm The agreement between 0 199 and O 226 1s satisfactory, and shows that the tuo theories are com patible We have mentioned that in the progeny of irradiated males there are more males than females, mdicating that fewer dom nant lethals are produced in a } bearmg sperm than m an A bearing sperm by a given dose This can be ascribed to the known facts that fewer breaks are produced by a given dose in @ Y chromosome than in an X chromosome, in the ratio of 79 100 The calculation we have just given can readily be extended to predict the ratio of females to males in the progeny of males which have received a given dose of X rays, and the predicted curve,s together with the experimental! ports, 1s given In Fig 28a It has been founds that 1f the wradiated males are of a special 1 Deduetion by Fano, U (1941) from the observations of Bauer, H (19394) 2 Deducible from the observations of Bauer, H Demerec, M & Kaufmann BP (1938) 3 Details of the calculation are given mn Lea DE & Catcheside DG 1945, J W & Gay EH ‘ 4 Henson ¥ B (1928) Muller HJ (1928) Gowen, (1933) Bauer H (19396) Catcheside DG & Lea DE (19450) The expersmental data are reviewed in the last of the references cited 5 Bauer H (19396) SIZE OF THE GENE 173 siderations For (a) to be of serious importance it 18 necessary for sufficient spread of the effect of an 1onization to occur for there to be appreciable probability of mutation in a gene when an ionization 1s produced at a distance of the order of a gene diameter or more outside it. The possibility of some spread of the effect of an :onization has been discussed in Chapter A spread over a range of the order of |}my can be understood Spread over a distance equal to the separation of two sister chromatids at prophase ( ~ 100m) has been shown expermmentally not to oceur : The notion that the effect of an ionization can spread appreciable distances has been adopted by some geneticists: because the yield of mmute chromosome aberrations has been found to be proportional to the dose of radiation (Chapter v1), and this has been taken to mean that a single 1omzation can produce two chromosome breaks an appreciable distance apart However, the proportionality to dose does not necessarily mean that a single tonization causes the two breaks, but that a single tonizing particle causes them As explamed in Chapter vir we should in any event expect that two breaks pnmarily produced at asepara tion of less than about 100my would be produced by a single lonwzing particle and not by two separate tonizing particles Thus the finding that the yield of mmute aberrations 1s proportional to dose confirms expectation, but has no bearing on the question of whether two breaks can be produced by a single 1omzation or not It 1s probably safe to neglect the possibility of gene mutation resulting from ionization outside the gene by comparison with the probably much higher chance that 1omzatron inside will be effective, though the pomt 1s one which requires more investiga- tion However, that considerations (a) and (b) together do not im practice prevent target theory calculations giving essentially correct results is made clear by the argument we now proceed to develop The obvious methou or testing the correctness of the method by which it 1s proposed to calculate the size of the gene from 1 Long wave length X rays, which dissipate their energy in tissue by means of photoelectrons of range less than the separation of sister chromatids are unable to break both chromatids simultaneously (Catche ade DG & Lea DE 1943, Lea DE & Catcheside DG 194658} 2 Muller, H.J (1940) 172 OFNFTICAL FFFLOTS OF RADIATION lethal offect 13 p+4q in the caso of a rng chromosome, against p1n a rod chromosome Taking t of this pt the sex ratio depression in ring X stock can be predicted, and the agreement of the ex perimental points and the calculated curves is shown in Fig 283 Further, we can, from the expermentally observed propor tions of the sex-ratio distortion in rod-A and mng A stocks, deduce the ratio of p+ }q to p, which leads to values for pand g, namely, p=0 26, g=0 742 These valucs are in good agreement with the values pe 0 24, 7=0 76 already obtamed (p 169) We conclude that dominant lethal production 1s satisfactorily expl d by the Sh and calculations we have given Deductions concerning the size of the genes At the tme when the induction of gene mutations by radiation was discovered, the ‘target theory’ of the biological action of radiations was already current When it appeared that the evidence on the production of mutations by radiation was con astent with the view that a single ionization could cause muta tion of a gene, 1t was natural that the radiation data should be used to calculate the target size for mutation, and that the target should be identified with the gene More recent wnters have usually adopted a more cautious attitude, and some have gone so far as to say that the size of the target has nothing to do with the size of the gene + The reasons for doubting that the size of the target 15 a fatr approximation to the size of the gene are principally (a) It 1s argued that 1onization outside the gene may perhaps cause mutation (6) Tomzation inside the gene may not always cause mutation, but may have a probabiltty of domg so considerably less than We have already discussed in Chapter mr the modifications unit, made in target theory calculations by mtroducing these con 1 See Lea, DE & Catcheside DG (1945a) for details of the calcula tion 2 Catcheside, DG & Lea DE (19455) 3 This section follows the treatment of Lea DE & Catcheside, D G ‘ 4 Ee Timoféef Ressovaky, NW & Delbruck,M (1936), Muller HJ 19458) (1940) SIZE OF THE CLNF 175 bonds uniting the atom to its neighbours mn the molecule We should expect therefore tonization of an atom to result in chemical change in the molecule The review of the chemical effects of radiation in Chapter 11 was consistent with this expectation, an rome yreld of the order of umty bemg the ty pical result obtained in radiochemical studies There are some cases in which a cham reaction 1s mstituted so that many more molecules react than there are 1omzations produced There are other cases where the rome yield 1s low, probably owing to recombination of the pro ducts of decomposition of the molecule However, the more complicated the molecule the less probable seems exact restitu tion, and we can probably safely assume that the gene wv ul suffer chemical change as a result of the 1omzation of an atom im it In attempting to picture the probable results of chemical change in a gene we shall make use of the now widely adopted view that genes have a good deal in common with the macro molecular viruses Iti1s hnown that a certain amount of chemical change can be tolerated by a virus without producmg any permanent inherited change in it: Thus the chemical change produced in a gene by an ionization may somctimes not produce any genetically important effect Sometimes the change produced by the 1onizition will be + permanent stable change, so that the gene reproduces itself in the changed form Thus clearly happens in the case of gene muta- tion, when the chermscal change 1n the gene 1s recognized by a detectable change in its behaviour 2 It must be realized that we cannot be sure of detecting every permanent change in the gene by a change in gene behaviours For example, in the white eye allelomorphs in Drosopiiia we have a large number of different states of the gene which are distinguishable because eye colour 1s a character in which many quantitatively shght differences can be recognized The different allelomorphs also affect the colour of the Malpighian tube in the larva, but if we had to rely 1 Miller GL & Stanley WW (1941) showed that 70 % of the amino Groups of tobacco mosaic virus protein could be acetylated by chemical treatment without reduemg the infectivity of the virus After mult: plication of the treated virus in the host plant, the virus had returned to ita origmal chemical composi tion % We are not accepting the extreme view that all mutations are posi tion effects and are supposin;g that in a typical point mutat chemical change in the gene , non there 1s 174 GINFTICAL }FEFOTS OF RADIATION radiation expernmenta 1s to apply the same method in the case of some object as nearly as possible resembling a gene but of known size Enzymes and viruses appear to satisfy the required conditions As shown in Chapter ni, a single sonization in an enzyme molecule leads to ita inactivation, and as shown in Chapter 1, there 1s, in the case of the macromolecular viruses, a close relation between the ‘target’ size for virus mactivation and the size of the virus particle itself It 1s true that the target diameter, although often closer, sometimes differs by a factor of 2 from the yirus diameter, but in view of the extreme paucity of our knowledge of the size of genes by other methods, an estt mate which was probably correct to a factor of 2 would consti tute much the most precise estimate at present available The fact that tho probability of producmg a gene mutation 1s proportional to the dose of radiation and mdependent of the intensity makes it hkely that} geno mutation 1s produced by the passage of a single sonizing particle through the gene In the case of y rays the ionizing particle will be a fast electron, successive ] of fre +e primary tonzations) being produced at intervals of the order of 1/ along its psth We can be guite sure that the diameter of the gene 1s considerabl} less than 1, so that with y rays there 1s little chance of more than one ion cluster falling in the gene From the fact that y rays are, per ionization, at least as effective as more densely somzing radiations, we infer that a single ton cluster can eause a gene mutation The majority of 10n clusters are single 1onizations,t and only a small proportion of the total number of 1onizations are located in clusters larger than about three xonzations Thus we can fairly safely deduce that a single 1onization, or at most & cluster of two or three, suffices to cause gene mutation, though we have, of course, not yet given any proof that the probability of ® single ionization in a gene causing mutation approaches unt; What changes may be expected to follow the xonzation of an atom in a gene? The energy given to an atom im the process of ionization exceeds the energy necessary to break the chemical 1 The various methods which have been proposed eg by Muller HJ (1935) only give upper lumuts to the size of the gene the p of a positive and a negative ion (see> Chapter 1) 2 SIZE OF THE GENE 177 enzyme and virus mactivation to be analogous to lethal mutation in these substances, the ratto of the size of the target to the size of the molecule will be equal to the probabihty that an 1on1za- tion in the molecule ghall cause lethal mutation In Table 44 ve lst the target size and the particle size for the mactiy ation of some enzymes and viruses If we are correct in arguing by analogy from the mactivation of viruses and enzymes to lethal mutations in genes, the figures in ‘lable 44 show that the target Tasie 44 Enzyme and virus inactivation Molecular Target Enzyme or virus weight weight Ratio Reference Ribonuclease 15x 108 32 x10 21 1 Phage $13 17x 108 15 x10¢ o9 2 Tobacco ringspot virus 34x 10° 227 x 108 067 3 72x 108 Tobseco necrosis virus 157x 10° 0 22 3 Bushy stunt virus 10 6 x 106 2:32 x 108 022 3 DE, Smth, KM Holmes,B & Markham R (1944) 1 Lea, 2 Lea DE & Salaman MH_ (unpublished) 3 Lea DE & Smuth, K M (1942) size agrees with the gene size within a factor of 5 in weight (which 2s a factor of 1 7m diameter) Paying principal attention to the virus data, the difference between target size and virus size 18 in the direction of target size being less than virus size, which 1s to be expected in view of the fact, already discussed, that some changes m the virus produced by an tonization leave it still infective To determine the size of a gene from radiation data we need to denve from the observ ations an estimate of the probability that, with a myven dose, 1on1zation shall be produced in the gene From the discussion which we have given, this means the sum of the probabilities that there shall be visible mutation to any other allelomorph, or lethal mutation (excluding chromosome deletion), plus the probability that there shall be change m the gene which 3s either not permanent or which gives no detected change in its Properties Studying any given locus we are usually only in a position to determine the probability that a recognizable viable mutation shall occur, which will be an underestimate of the total prob ability, and will thus lead to an underestimate of the suze of the gene The mutation frequencies in Table 37 (p 144) are of the 176 GENLTICAL EFFI CTS OF RADIATION only on the colour of the Malpighian tube we should recognize fewer allelomorptis and hence im radiation experiments often fail to recognize the occurrence of a mutation when mutation had in fact occurred Even using the more sensitive eye colour as a means of detecting mutation, changes in the gene can occur without being detected, aince it. 15 known that allelomorphs exist having the same eye colour but differnng in other properties (e g viability or fertility:) It 18 evident therefore that the experimentally determined routation frequency of 2 given locus will usually underestimate the frequency with which permanent and viable changes are produced in the gene: Ths consideration probably applies especially forably to the experiments described earlier in the chapter (p 140) on the induction of mutations in a plant virus Here mutation was recognized by the production of a local lesion on a leaf moculated with virus, in place of the usual mottle It would clearly be unreasonable to assume conversely that, if after wradistion the virus still produced a mottle on the leaf, there was therefore no permanent change induced in the virus molecule by the radiation The fact that an iomzation in the virus had rather a small chance (of the order of 10-4) of producing the particular offect atudied does not necessanly mean that an ionization in the virus has only this small probalility of producing a viable n herited change The complete deficiency of a gene, when homozy gous, 1s usually lethal in Drosophila If as a result of 1omzation a gene suffers & change which causes the Joss of the power of reproduction or alternatively the loss of all its charactenstic activity, a recessive lethal will usually be recorded Lethal mutation at a given locus (excluding deficiency caused by deletion of a portion of chromo- some) appears to be not much more frequent than visible muta tion,s which 3s perhaps surpnsmg The probability that an yonization produced im a gene shall cause a lethal mutation 18 2 quantity of which we can make some estimate by appeal to ex- periments on enzyme and virus inactivation, since, if we take 1 Timoféeff Ressovshy, N W (19338) 2 This pomt has been emphasized by Timoféeff Ressovsky NW ‘ 3 Data bearing on this question have been given by Patterson, J T 1937a) (1932), Demerec VM (1937) Muller, HJ (1940) TARGET DIAMETER FOR MUTATION 179 tion, and then consider the modification mtroduced by allowing for the probability of lethal mutation being less than unity As explamed in Chapter mt, knowledge of the relative effinencies of soft X rays, neutrons and a-rays compared to y rays or hard X rays enables an estimate to be made of the target diameter In Table 45 we give the inferred target diameters (assuming density 1 35 g /om 3) deduced from the relative efficiencies listed in Table 39 with the aid of Fig 10 The estimate of the target Tasie 45 Target diameter for mutatton Hard X rays Soft & rave Neutrons @ rays Radiation or y rays 2-38 (Ia+D) ~3eMV Relative dose for equal 100 130 145 344 yield of mutations 2rp (m mz) — 6 12 Target dhameter (in my) _ 44 9 66 diameter obtained from the relative efficiency of different radia tions 1s very sensitive to error in the determination of this rela tive efficency, and may also be appreciably affected by over- symplification of the calculation: It 1s satisfying, however, that the order of target diameter given by Table 45 (4-9my) 1s con sistent with that already obtamed on p 178 (2-6my) by an entirely different method We may make an estimate of the number of genes in the 4 chromosome (strictly the number of genes capable of showing a recessive lethal mutation) as follows The yreld of sex-linked lethals by X- or y rays 1s 289% per 1000r, so that 1000/0 0289 = 3 46 x 10¢r 1s the dose required for an average of one lethal mut ition per X chromosome For a given size of gene we can read off from Fig 84 the dose required for an average of one mutation per gene Taking the gene diameter to be 4inj, the smallest value which seems consistent with Table 45, we find that the dose of X rays (0 154 ) required for an average of one mutation per gene 1s 29x 107 r The number of genes nm the \ chromosome 1s there fore deduced to be 2 9 x 107/3 46 x 10¢= 838 Lurger sizes for the 1 Eg a target diameter of about 4m was deduced from essentially the same data when this calculation was first given (Lea DE 1940} owing to the neglect of ¢ rays and some other simplifications 178 GENT TICAL FFFEOTS OF RADIATION order 1-10 x 10-* per gene per roentgen, 1¢ a dose of the order 107-10 r 18 required for an average of one mutation per gene Consulting Figs 8 and 9 we deduco that the molecular weight of the geno 18 10,000-100,000, or ste diameter (if spherical) 13 2~ Sms As explained, theac estimates are hable to be under eatimates for the sizes of the genes to which the data of Table 37 refer, though they may not necessarily bo underestimates for the average siz of a gene 111 Drosophila, sinco the genes of which the mutation rates are known include those most frequently suffering mutation under irradiation Turning now to lethal mutations at random loci, we have the advantage of knowing the relative officiency of different radia tions (Tablo 39) As discussed already, Jethals do not constitute a singlo type However, according to the view put forward earlier in the chapter, types A and C Iethals do not differ in ongin Type B lethals, namely, those involving cytologically detected deficiencies, are, however, of different ongin At any rate, deletions for more than one band probably involve two breaks close together and the deletion of the part of the chromosome between them, and therefore do not necessanly involve the ionization of the gene responsible for the lethal effect We should therefore estimate the mutation yield after excluding the chromosome deletions, and calculate the relative effinency of different radiations also after their exclusion It1s at present not clear whether deficiencies for single sahvary chromosome bands should be excluded, o1 whether such deficiencies are due to ionization in a particular gene causing loss of the power of repro duction, in which case they should be retained The proportion of X-ray random lethals which are deficiencies for one or more bands 1s about 30% ,: the proportion which are deficiencies for more than one band 1s lower, there 1s as yet no information available regarding the proportion of random lethals produced by other radiations which are cytologically detectable de- ficencies Lacking at the present time the information necessary to correct Table 39 for the proportion of lethals due to cyto logically detectable deficiencies, we shall, for the purposes of the present calculation, have to neglect this correction We shall! first make the ealcul on the P that one or more 1on1zations anywhere im the gene leads to lethal muta x Shzynski, BM (1938 1942) TARGFT DIAMETER FOR MUTATION 1i9 tuon, and then consider the modification introduced by allowing for the probabthty of lethal mutation being Jess than unity As explained in Chapter 11, knowledge of the relative efficiencies of soft K-rays, neutrons and a-raya compared to y rays or hard X-rays enables an estimate to be made of the target diameter In Table 45 we give the inferred target diameters (assuming density 1 35 g /om 3) deduced from the relative efficiencies listed in Table 39 with the atd of Fig 10 The estimate of the target Taste 45 Target diameter for mutation Hard X raya Soft X rays Neutrons @ Tavs Radiation or ¥ rays C8 A (4D) ~3eMh Relative dose for equa! 100 130 145 344 sreld of mutations 2rp (in my) - 6 12 :) Target diameter (2n my) _ 44 9 66 diameter obtained from the relative efficiency of different radia tions 1s very sensitive to error in the determination of this rela tive efficiency, and may also be appreciably affected by over- amplification of the calculation « It 18 satisfying, however, that the order of target diameter given by Table 45 (4-9my) 1s con sistent with that already obtained on p 178 (2-Gmyt) by an entirely different method We may make an estimate of the number of genes in the X chromosome (strictly the number of genes capable of showing @ recessive lethal mutation) as follows The yield of sex hnked lethals by X- or y rays 1s 2 89% per 1000, so that 1000/0 0289 = 3 46 x 104 r 1s the dose required for an average of one lethal mutation per X chromosome For a given size of gene we can read off from Fig 8a the dose required for an average of one mutation per gene Taking the gene diameter to be 4m, the smallest value which seems consistent with Table 45, we find that the dose of X rays (0 154 ) required for an ay erage of one mutation per gene 1s 29x 107r The number of genes in the X chromosome 1s there fore deduced to be 2 9.x 107/83 46 x 104= 938 Larger sizes for the 1 Eg a target diameter of abi out 4m was deduced from essential the same data when tins cafeulation was first given (Lea DF 19400), ing to the neglect of ¢ rays and some other amplifications , 178 OFNFTICAL TEFFOTS OF RADIATION onder 1-10. % 10-9 per gene per roentgen, 16 a dose of the order 107-109 Fr, is required for an average of one mutation per gene Consulting Liga 8 and 0 we deduce that the molecular weight of the gene 1 10,000-100,000 or ita diameter (if aphencal) 1s 2- Smy Aa oxplained, these estimates are lable to be under catimates for tho sizes of the genes to which the data of Table 37 refer, though they may not necessanly be underestimates for the Average fiz0 of a gone in Drotophtla, since the genes of which the mutation rates are known include those most frequently suffenng mutation under irradiation Turning now to lethal mutations at random locr, we have the advantage of knowing the relative efficiency of different radia tions (Table 30) As discussed already, lethals do not constitute a single typo However, according to the view put fo carher in the chapter, ty pea A and Clethals donot differ none” Type B lethats, namely, those ins olving eytologically detect deficiencies, are, howe, er, of different ongin At any ne deletions for moro than one band probably involve two bree close together and the deletion of the part of the chromosome between them, and therefore do not necessarily involve ue donization of the gene responsible for the lethal effect We show therefore estimate the mutation yield after excluding t ‘ chromosome deletions, and calculate the relative efficiency ° different radiations also after theirexcluaion Its at present n0t clear whether deficiencies for single salivary chromosome bends should be excluded, o: whether such deficiencies are due t0 ionization in a particular gene causing loss of the power of repro duction, in which case they should be retained The proport ion of X ray random lethals which are deficienci es for one or more bands 1s about 30%, the proportion which are deficiencies fot more than one band is lower, there 1s as yet no mformatio n available regarding the proport ion of random lethals produce d by other radiations which are eytologica lly detectable de ficiencies Lacking at the present time the information necessary to correct Table 39 for the proportion of lethals due to cyto logically detectable deficiencies, we shall, for the purposes of the present calculation, have to neglect this correction We shall first make the calculation on the assumption that one or more 1onizations anywhere in the gene leads to lethal muta x Slizynskt, BM (1938, 1942) COSMIC RAYS AND MUTATIONS 18} atratang the proportionality of yield to dose do not, of course, extend to an intensity as low as that of natural radiation They do, however, cover a very wide range (Fig 22), and if the theo- retical interpretation that a single 1onmizing particle 1s responsible for a mutation 1s correct, we should expect the yield to be pro- portional to dose however low the intensity A generous eati- mate of the dose rate due to natural radiation 18 50 1onizattons per cm ? of air per second, or 2 5x 10-8 r /sec , which corresponds to about 0 05r in the lifetime ofa fly Thus the rate of produc- tion of sex-lnked lethals per generation by natural radiation should amount to 0 00015%, which 1s about a thousand times amaller than the observed rate of spontaneous sex-lnked Jethals This calculation, based on an estimate of the tensity of the natural radiation denved from measurements of the natural Jeak of 1onizatton chambers, 1s not entirely convincing since living organisms sometimes concentrate radioactive matter in their tissues Mott Smith and Muller,: however, made measurements of the radium content of flies and concluded that it was insuf- ficent to account for the spontaneous mutation rate A further argument comes from the fact that the rate of induc tion of mutations by radiation 1s independent of temperature (Table 40), while the rate of spontaneous mutation 1s increased markedly by nse of temperature It thus appears certain that spontaneous mutations in Droso- phila are not due to natural radzation 2 There 1s some evidence with less well-mvestigated organisms pointing in the opposite direction,s but further expermments are required here Genetical effects of ultra-violet light The induction of mutations by ultra-violet hght has been studied in Drosophila, m maize, in Antirrhinum, and m some lover plants The small penetrating power of the radiation complicates the experiments with Drosophila Some experiments have been made by uradiating fertilized eggs in the polar cap stage,s aa in x Muller, HJ (1930) 2 See Rajewsky, BN & Trmoféeff Ressovsky, NW (1939) for further discussion and experiments strengthening this conclusion 3 Rayewsky, BN, Krebs, A & 7icklor H (1936) 4 Altenburg E (1934, 1936) 180 GENETICAL FFPECTS OF RADIATION gene are permitted by Table 45 and would lead to amaller est- mates of the number of genes in the X chromosome Thus if the gene diameter is Gmyz wo obtain 281 as our estimate of the num ber of genes in the X chromosome These estimates are of the night order, 4 figure of about 500 or 1000 having been suggested by several authors: Tho calculation as we havo so far made it 18 based on the assumption that the probability of a lethal mutation occurnng when an romzation 1s produced in the gene 18 unity A priors tt may be Jess than unity, though from the evidence of Table 44 we do not expect it to be an order of magnitude less However, 1n Chapter 111 (p 95) we went into the question of tho effect upon calculations of this sort of the probability, p, being less than unity, and concluded that the effect was to cause the estimate of tho size of the gene to be too small in the ratio of p 1, and the estimate of the number of genes to be too great in the ratio of 1 p? However, it 18 clear that there 19 not room for our estimate of the number of genes to be too great by any large factor, cer~ tainly not by 0 factor of 10 Hence p? cannot be much less than umty, certainly not lower than 01 Hence p cannot be as low 88 03 We deduce that our estimate of 4-9my cannot, be greatly in errur on this account It is probable that the average gené diameter does not exceed 10my, and we shall take 4~8my, 28 suggested by Table 45, as the most probable value Cosmic rays and mutations Following Muller s discovery in 1927 that 10mzing radiations are able to cause mutations, the idea occurred to several workers that spontaneous mutations may be due to natural radiation, 1e cosmic rays from outside the earth, y rays due to the radio- active content of the floor and walls of the room, or a- and f rays due to the content of radioactive matter in the organism itself This attractive possibility, however, has been ely disproved m the case of Drosophila In the first place, the n- tensity of the natural radiation is msufficient > With X rays and y rays the rate of production of sex linked Jethals 1s roughly 3% per 1000r, and 1s proportional to dose Expemmenta demon- 1 Eg Muller, HJ (1029), Gowen TW & Gay EH (1933), Demeree M (1934) 2 Muller H.J (1930) ULTRA-VIOLET LIGHT 183 According to Shzynski, some ultra-violet-induced lethals involve minute deficiencies detectable m the salivary chromosomes, but other reports: suggest that ultra violet hght does not cause minute structural changes This important question 1s therefore not yet settled The small penetrating power of ultra-violet hght 1s of less im- portance when smail objects such as fungal spores are radiated Expenments in which the yield of mutations as a function of dose has been studied have shown that the yield 1s proporttonal to dose at low doses, but that at sufficiently high doses the pro- portion of the viable spores which carry mutants fais to mse further and may even diminish 3 This unexpected result appears at doses at which the fraction of radiated spores which survive xs less than 1% It1s probably due to the mutant spores having a lower viability than those which have not suffered mutation As the result of extensive work by Stadler and his co-workers, a considerable amount of information 1s available about the genetical effects caused by mradiatmg maize pollen with ultra- violet hght, and about the differences between the effects of X rays and ultra-violet: hght .« The maize pollen ts spread in a single layer and irradiated from above After irradiation it 18 used to pollinate a maize plant The maize seeds developing are examined, and, if desired, sown to obtam F, plants At the time of radiation the pollen grain contains two haploid sperm nucler When they enter the embryo sac (which pnor to fertihzation contains eight haploid nuclei as the result of three consecutive divisions of one of the products of meiosis), one of the sperm nuclei fuses with the egg nucleus to form a diploid zygote, which by multiphcation forms the germ or embryo of the seed and 1 Shzynski, BM (1942) In 21 lethal bearmg X chromosomes he found 5 deficiencies viz 1 case of 1 band deficient, 3 cases of 2 bands deficient, and 1 case of 14 bands deficient 2 Mackenzie K & Muller HJ (1940) Muller, HJ (1941 a) reporting the preliminary results of an experiment by Bndges PN & Muller, H.J 3 E CW & Hollaender A (19394 6) Hollaender A & Emmons CW (1941) using Trichophyton mentagrophytea Demerec, M Kaufmann BP Fano U Sutton E & Sansome ER (1942) using Neurospora crassa 4 Experiments with ultra violet light are described by Stadler, L J (1939 1941) Stadler, LJ & Sprague GF (19364 6 ¢), Stadler, L.J & Uber, FM (1938 1942} Comparable experiments with X Tays are desenbed be ues” LJ (19282 1930a b 1931) Stadler LJ & 182 GENETICAL EFFFCTS OF RADIATION Ward's a ray oxpenments alrendy described (p 162) More usually adult males have been srradiated The flies are held with their nbdomens compressed between quartz plates and their undersides irradiated + By this technique the testes arc brought nearer to the surface, and a suffictent intensity of ultra violet light reaches the sperm to give a few per cent of mutations with the largest doses which the fly 1s able to sustain Absorption by tho intervening tissue 13, however, large and vanable, and it 33 not practicable to determine, for example, whether the yield is accurately proportional to the dose, or to compare the doses to the sperm of radiations of different wave lengths required to produce mutation im equal percentages of the sperm In practice the greatest percentage yields are obtainable with a rather long wave length, 3130A , since the flres will tolerate greater mtenst ties of this wave length than of shorter wave lengths, and 4 greater fraction of the incident energy penetrates to the testes Expressed mn terms of mutation frequency per unit energy absorbed in the sperm, shorter wave lengths are, however, probably more efficient In X-ray experiments a practical I:mit 18 set to the percentage of irradiated sperm in which mutation can be induced by the fact that with large doses the proportion of sperm remaming viable 1s greatly reduced by the induction of domumant lethals In ultra violet hght experments the practical limit seems to be set by the damage to the flies by srradhation of their tissues rather than by the induction of dommant lethals m the sperm, and 1n occasional fies the percentage of viable sperm having eex lmbhed recessive lethals 1s as high as 50% 2 Of considerable interest 1s the question whether recesstve lethals induced by ultra violet hght are entirely gene mutations (type A) or whether as with X rays, some are minute deletions (type B) and some are associated with gross structural changes {type C) Gross structural changes mduced by ultra violet hght are very rare by comparison w3th recessive lethals if they occur at all,s so that the proportion of type C lethals 1s nepghgible ¥ Mackenzie K & Muller HJ (1940) Note that ultra violet light intensities stated in this paper are by error 1000 tres too great Also Demerec VM, Hollaender A Houlahan WB & Bishop M (1942) 2 Demerec Mo Hollaender A Houlahan UB & Bishop Ve (1942} 3 Isolated of chr zes in ultra violet wradiated matenal have been reported by Muller HJ (1941a) and by Demerec Wi Hollaender A Houlshan MB & Buhop M (1942) . DEFICIENCIES IN MAIZE 185 enables deficiencies at selected loci to be detected which are pro- duced im the sperm nucleus which fertilizes the egg nucleus In this case it 13 necessary to sow the seeds and examine the F, plants Deficiencies can also be detected without the use of marked. loc: by the fact that, while maize 1s fairly tolerant of deficiencies when they are heterozygous in dsploid or triploid tisaue,r after meiosis has occurred and haploid cells are produced, all except the smallest deficiencies are usually lethal in the male gameto- phyte Consequently half of the pollen produced by an F, plant which 1s heteroz3 gous for a deficiency will be defective, and can be recogmzed as such by inspection of the pollen Ifthe F, plant 13 heterozy gous for a chromosome interchange, then about halfof the pollen 1t produces will be defective, having a deficiency of one chromosome segment and a duplication of another It 1s possible to decide whether a plant producing de fective pollen 1s heterozygous for a deficiency or for an imter- change by examining meiotic figures in the anthers z The principal interest of the maize experiments hes in a num ber of differences between the effects of ultra violet hight and X-rays Since dosages of ultra violet ight and X rays are not measured in comparable umits,; absolute yields per unit dose of the two radiations cannot be compared, and the companion rests therefore upon qualhitati e differences and upon differences in the relative frequencies of different types of genetic effect These differences we now proceed to enumerate 1 Deficiencies of a large part of a chromosome arm (Singleton WR 1939, Singleton WR & Clark, F J 1940) or even of a whole chromosome (Stadler, L.J_ 1931) are sometimes found in heterozygous condition in the F, plants following pollen trradiation Such plants are visbly de fective in growth and may not flower 2 In meiosis homologous chromosomes are paired If an interchange between two non homologous chromosomes has occurred one group of ur chromosomes and eight groups of two will be seen at diakinesis instead of ten gtoups of two (Stadler L.J 1931) The larger deficiencies can also be seen in merosis at the pachytene stage (Singleton WR 1939 Singleton, WR & Clark FJ 1940) 3 It would, of course be possible in principle to express both ultra violet light And X ray dosages in terms of ergs per cubic micron diy Stpated in the ch but the exp I data at present do not Permit this Itis likely in siew of results with viruses (Chapter m) that mes of these units ultra violet hght would be found to be much Jess effective than ¥ rays im producing all types of genetic effects 184 GENETICAL EFFECTS OF RADIATION eventually the F, plant The other sperm nucleus fuses with tuo of the other haploid nucle: (the polar fusion nucles) to produce a tnploid nucleus, which by multiplication develops into the endo- sperm, the starchy tissue which constitutes the bulh of the seed but does not persist in the F, plant The radiation may produce genetical effects in either of the two sperm nucle: A dominant lethal in the sperm which fuses with the polar fusion nuclei will result in a minature seed having a normal embryo but defective endosperm A domunant lethal in the sperm which fuses with the egg nucleus will lead toa germ- leas seed, a condition which 1s detectable by examination of the seed Visible mutations induced in this sperm may be detected by sowing the seed, when any dominant visible mutations will be Tevealed by inspection of the F, plants and the (more frequent) recessive visible mutations wil be revealed by self fertilizing the F, plants and looking for mutants in the F’, generation A large part of Stadler's work was concerned with the produc- tion of endosperm deficiencies The pollen wradiated came from a stock having the dominant allelomorphs of a number of genes affecting the endosperm (eg modifying sts colour or surface texture) The pollen was used to poll a plant h ‘gous for the receasive allelomorphs of these genes The seeds deyelop- ing after fertilzation normally showed the dommant phenotype 4s a result of pollen iradiation, however, some of the seeds showed the recessive phenotype This Joss of effect of the domi nant gene could be explained as being due either to mutatron to the recessive allelomorph, or to removal of a portion of the chromosome contammg the locus concerned In practice the latter explanation (deficrency) 1s believed to be m almost all casés the correct one « Asunular genetical tech employing marker genes affecting q plant characters instead of (or as well as) endosperm characters x With @ given dose of radi ton are babl, not more f1 than are ) gene Since h Ives many genes a specified locus 1s more likely to beaffected bya deficieney than it 33 to suffer gene mutation ‘There 23 also some exp for the endosp effects bemg due to deficiencies rather than to gene mutations namely the observa tion that when a chromosome contains two marked loci these often lose their effects together, suggesting the deletion of a portion of the chromo some which mcludes both genes DEFICIENCIES IN MAIZE 185 enables deficiencies at selected loci to be detected which are pro duced m the sperm nucleus which fertilzes the egg nucleus In thos case it 15 necessary to sow the seeds and examme the F, plants Deficiencies can also be detected without the use of marked loc: by the fact that, while maize 1s fairly tolerant of deficiencies when they are heterozygous mm diploid or tnplod tissue, after meiosis has occurred and haploid cells are produced, all except the smallest deficiencies are usually lethal wn the male gameto- phyte Consequently half of the pollen produced by an F, plant which 1s heterozygous for a deficiency will be defective, and can be recognized as such by inspection of the pollen If the F, plant 1s heterozygous for a chromosome mterchange, then about half of the pollen st produces will be defective, having a deficiency of one chromosome segment and a dupheation of another It 1s possible to decide whether a plant producing de- fective pollen 1s heterozygous for a deficiency or for an inter- change by examining meiotic figures m the anthers « The principal interest of the maize experments hes m a num ber of differences between the effects of ultra-violet hght and X rays Since dosages of ultra-violet light and X rays are not measured in comparable units,3 absolute yields per unit dose of the two radiations cannot be compared, and the comparison rests therefore upon quahtative differences and upon differences in the relative frequencies of different types of genetic effect These differences we now proceed to enumerate 1 Deficiencies of a large part of a chromosome arm (Singleton WB 1839, Singleton, WR & Clark, F.J._ 1940) or even of a whole chromosome {Stadler LJ 193]) are sometumes found on heteros} gous condition im the F, plants following pollen wradiation Such plants are visibly de fective m growth and may not flower . 2 In meiosis homologous chromosomes are paired If an interchange four chrom ote wot has 1, one group of josomes and eight groups of two will be seen at diakinea:s instead of ten groups of two (Stadler, L.J 1931) The larger deficiencies can also be seen im meiosis at the pachytene stage (bngleto: Singleton WR &Clark FS 1940) ve Be (omBleton WR 1939 3 It would of course violet hght and X ray dosages in terms,of cae per be possible in principle to expr sipated in the ch abe mien : but the exp I data at present do nat Permattins Jt 1 likely, in view of results with viruses (Chapter mn) that in terms of these units ultra violet light would be found to effective than X Tays in producing all types be much Tes of genetic effects “= 186 GENFTICAL LFFFOTS OF RADIATION (2) The ratio of the number of viable gene mutations to chromosome deficiencies m the F, plants 1s higher with ultra violet light than with X rnyst (6) In the majonty of cases an endosperm deficiency produced by ultra violet irradiation of the pollen affects only about half of the endosperm This 1s taken to mean that the chromosomes in the pollcn grain are already spht into two chromatids at the tumo of irradiation and the ultra violet hght breaks only one of them: In the majority of cases endosperm deficiencies caused by irradiating pollen with X-rays affect the whole chromosome, indicating that both chromatids are usually broken This dif ference 1a readily understandable af it 1s remembered that 10m1za tions (but not ultra violet quantum absorptions) are localized on the paths of somzing particles The chromatids beng closely juxtaposed, an ionizing particle which passes through one will usually traverse both {c) As already mentioned, chromosome deficiencies mvolving selected loci may be detected either in the endosperm or in the F, plants, and with X rays they occur with approumately equal frequency as 1s to be expected, since there 14 no reason to antici pate a difference in sensitivity between the two apparently identical sperm nucler, one of which fuses with the egg nucleus and the other with the polar fusion nucle: With ultra violet Thists shown by the ratio of total visible mutations appesring in the = pared with total di inferred from the segreg tion of defective pollen by the F, plants It 1s also shown in the case of a selected gene (A) by the fact that among 200 cases where X ray treat ment caused loss of the dominant effect, all involved some loss of via bihty and were probably deficiencies, none was simply due to mutation to the recesaive allelomorph, while among a smaller number of cases where ultra violet ght treatment caused loss of the A effect, four were shown to be true ti toa h (Stadler, L.J 1941) 2 In experiments in which end d for the ti A Pr, Su were recorded, 79 8% of all deficiencies were fractional (figure obtaned by grouping all data given by Stadler LJ & Uber F M 1042) It 18 possible that the 20 % of entire deficiencies represent cases where, by chance both sister ch: ds have been independently broken That such chance a will not be q 13 d by the fact that in the same experiments 13 % of the seeds were observed to be simul taneously deficient for two or more of the characters. A Pr Su although some coincident deficiencies are not observable being phenotypically indistingutshable from single defi and others pi bly behave as dominant lethals DEFICIENCIES IN WAIZE 187 light, however, there 13 a great difference, deficiencies being much more frequent in the endosperm than in the F, plants The difference 1s presumably due to restitution of an ultra-violet- induced break beimg less probable in the endosperm than in the embryo: (d) Following X arradhation of pollen deficiencies and inter changes are found with comparable frequency in the F, plants But after ultra violet irradiation of pollen, mterchanges are much less frequent than deficiencies and such as have been found are incomplete z If1t 1s borne n nund that the ultra violet deficiencies observed in the embryo constitute o small fraction only of those primanly produced (according to the evidence im (c} above), it 1s clear that ultra-violet hght 1s able to produce breaks but that these have much less chance of combining to form interchanges than when the breaks are produced by X rays The Teason 1s not clear, the phenomenon appears to be general, being exhibited by Drosophila and Tradescantia as well as maize It 1s not to be explaimed on the single 1omzing particle argument used in (6), since it 1s known (in the cases of Drosophila and Trade- scantia) that the two breaks taking prt in an X ray induced interchange are usually produced by separate ionizing particles (e) Ultra-violet induced defiaencies appear to be entirely terminal,s while X ray-induced deficiencies are mamly or en- turely interstitial An interstitsal deficiency involves exchange between two breaks, so that we have here an additional imstance of the smaller probabihty of exchange occurring between two 1 McChntock, B (1939) has shown that if a (mechanically) broken h 18 present in endosperm or g phyte, sister union will occur at the breakage point when the chromosome aphits_ In the embryo however sister union at a breakage point does not occur Once sister union has occurred, restitution 13 clearly »mpossible and thus the lower probability of ion in endosperm than in embryo suggested by Stadler's ultra violet light expernments may be due to sister union occurring m endosperm and not im embryo The difference will not be shown by X ray deficiencies since the-e usually mvolve both chromatids of the sperm chromosome and sister union will therefore presumably occur before fertilization (But in an} case X ray deficiencies are not usually termmal ) 2 Te of the four breakage ends only twoumte Asaresult an acentric Fogment 1S RR umattached and . fost Such incomplete interchanges changes ce y rays but less frequently than complete inter 3 Singleton WR (1939) Singleton WR & Clark, FJ (1940) 188 GENETICAL EFFECTS OF RADIATION breaks produced by ultra violet light than between two breaks produced by X% rays Tho relative efficiency of different wave-lengths of ultra violet light in inducing genetical change have been compared with curve shows the relative absorption o! acid at dif ferent wave lengths several experimental matetials, and the results are given in Fig 29 It 18 interesting to note the sumlanty between the various curves of genetic effect and the absorption spectrum of nucleic acid The inference 1s that it 1s absorption of ultra violet light by the nucleoprote: of the ch which leads to the genetical effect Chapter VI THE PRODUCTION OF CHROMOSOME STRUCTURAL CHANGES BY RADIATION« Expenmental materials The production of structural changes in the chromosomes by the sradtation of Drosophila sperm and of maize pollen has been bnefly described m Chapter V in connexion with the genetical effects associated with these changes In the present chapter we continue this discussion, and describe also chromosome structural changes 1n other organisms In Drosophila melanogaster, which has been very extensively studied genetically, chromosome structural changes can be recognized by genetical means, based on the changes in the link age relations of genes which follow their rearrangement in the chromosome set and on numerous special methods In recent years, however, studies of chromosome changes in Drosophila have increasingly been made cy tologically by examination of the salivary chromosomes, and with other orgamsms which have been less extensively studied genetically the cytological method: is the normal one For the detailed study of structural changes it 18 necessary to use nuclei in which the chromosomes are large and few in number, and in relatively few species are these condi- tions satisfied In view of the almost complete universality of the chromosome mechanism, tt may reasonably be anticipated that the main conclusions derived from the study of chromosome changes in the relatively small number of favourable expen- mental matenals will be of wider application Some caution 1s, however, needed in proceeding in this fashion, as 1s evidenced by certam detailed differences in the mechanisms of production of structural changes shown by the different organisms which have been investigated Chromosomes are, in general, only observable during cell division, and observations of structural change are made by x This chapter as di dp pally to a stat of the experi mental results Detailed interpretation 1s deferred to Chapter vir on See Darlington CD & La Cour LI (1942), The Handing of for the exp cytological examination t que paring mat of preparing material for 190 STRUCTURAL CHANGES IN CHROMOSOMES examination of metaphase or anaphase figures an stained pre Parations Irradiation during these phases does not cause im mediate chromosome breakage, perhaps because of the existence of a matrix of protein and nuclete acid which binds together the coiled chromosome, so that even though the chromosome thread may be broken, the chromosome does not separate into two pieces For the production of structural changes it 18 necessary to irradiate during the resting stage, or early prophase : Thus, the general procedure 1s to trradiate, to fix the material some hours or days subsequently, and to e metaphase and anaphase figures in the fixed proparations: Aberrations seen, for example, n metaphaso are then due to radiation which was given to the cell at a stage prior to metaphase corresponding to the tame interval which elapsed between irradiation and fixation If the time seale of the life cyclo of the material 1s hnown, the stago at which the radiation was given is known Some un- certainty, however, 1s introduced by the fact that irradiation 13 lable to cause a lengthening of the time scale 3 Tins method has been adopted in studying chromosome aberrations in rapidly dividing root tips of seedlings of omon, bean, tomato and other species s Root tips have the ments of small size and ready availability, and also the ment that in the growing regions of the tip the proportion of cells in mitosis 18 Ingh Another method of studying chromosome aberrations mn plant material which has been found convement 1s to irradiate flower buds and to study division figures in the developing pollen Either the meiotic division of the pollen mother cell may be used,s 1 Trradtation during Ys hase may lead to aberrations bemg discoverable at a subsequent‘an sion ‘Thus af Serara oocytes are uradiated during meiotic first or jons can be detected in the salivary gland chromosomes of the F, larvae {Metz CW & Bozeman ML 1940, Reynolds J P 1941 Bozeman ML 1943) 2 In favourable materials 1t may be possible to observe breahs mn prophase chromosomes and in this event fixation may be carried out immediately after wradiation Thus Bishop DW (1942) was able to demonstrate the existence of breaks m chromatids at the pachytene or dialonesis stages in Orthoptera 3mm after wradiation In general how ever observations are made at metaphase or anaphase 3 bee Chapter vitr 4 Eg Marshak, A (1937) Marquardt H (1938) Sax K (1941a) 5 Eg Marshak A (1935) EXPLRIMENTAL MATERIALS Ig! or the first: or the second: haploid untozs In Swanson’s method of studying the second haploid mitosis, which in Trade- scantia occurs after germimation of the pollen, the pollen 1s germinated on a smear of artificial medium on a glass slide An hour or two after germination the generattve nucleus enters the polien-tube which has a diameters of only 6 Consequently the chromosomes are accessible to radiation even by werkly pene- trating radiations such as ultra-violet hghts or soft X-rays s The pollen-tubes are fixed and stamed about 24 hr after germmation and the metaphase chromosomes are exammed It 1s conventents to add acenaphthene or colchicine to the artifinal culture medium These chemicals prevent spmdle formation in the dividing cell 80 that the chromosomes are seen ly mg in tandem formation in the pollen-tube m a manner favourable to the observation of breaks When chromosome breaks in Drosopiila are bemg studied cytologically the method 1s to srradtate male flies to mate them to untreated females, and to collect the larvae from the culture The salary glands of the mature larvae are dissected ont Stained, smeared and mounted, and the giant salary chromo Somes examined s Since many divisions occur between srradia tion of the sperm and fixation of the silivary glands, it 1s evident that inviable chromosome changes will not be obsers able by this method This 1s a disadvantage of the use of salivary chromo Somes which 1s, however, for many purposes outweighed by the fact that. chromosome changes can be studied in a detail not Possible in other materials The chromosomes m the mature sperm are In an inactive state This results in a further difference between experiments on Drosophila sperm and experments on cclls which re either mn interphase 0: m prophase but in either event are developing Chromosome breaks which are induced in Drosophila sperm by irradiation do not take part in structural rearrangement until the sperm enters the egg On the other hand, mn developing cells rearrangement takes place during and smmednitel y following the uradiation UEg Sax K (1940) g& Swanson, C P 2E (1940) 3 Catcheside DG & Tea DE (1943) + manson CP (1940 1942 1943) 5 © g Cateheside DG (1938a) Cp Plate [lla 6 192 STRUCTURAL CHANGES IN CHROMOSOMES An animal with large chromosomes, which has been found convement for the study of chromosome atructural changes at the division following irradiation, is the grasshopper: Either mitoma and meiosis of the getm cells in the testis are observed, or mitosis of the neuroblasts of the embryo Structural changes and physiological changes in chromo- somes: The chromosome changes with which we are concemed in this chapter are breakages, and structural rearrangements resulting from the Jouning in various fashions of the several breakage ends present in a nucleus in which two or more chromosome breaks have occurred This action of radiation appears to be direct, n tho sense that breakage 1s caused by an 1omzing particle passing through or in the immediate vicinity of the chromosome at the point at which the breakage occurs Breakages of this sort and the resulting rearrangements are, however, not the only type of hange produced in chri by the irradiation of cells Another change consists in the alteration of the surface proper tiea of the chromosomes so that they tend to stick together Thic results in chromosomes at metaphase adhering where they happen to touch, and to sister chromatids failing to separate completely at anaphase, giving bndges In severe cases the chromosomes may remain clumped at metaphase so that no further division stages ensue, or the bndges at anaphase may fail to break so that separate daughter nuclei cannot he formed The changes of ths type do not appear to be due to locahzed damage to the chr at individual points, such as could be explained by the passage of g particles through these points, but to a general change in surface properties covering the whole surface of the ch We can convemently denote this change as a phystologrcal effect of radiation on the chromo somes, in contrast to the term structural change which we reserve for breakages, and rearrang ts by umion of breakage x White, MJD (1937) Carlson JG (19384 6 19412 5) Bishop DW (1942) 2 See Plate III for micrographs of division figures showing chromo some structural changes (Catcheside DG) and Plate IV for micrographs of division figures ‘h phy x 1 changes (L tzk I, Marshak A Carlson, JG) PHYSIOLOGICAL EFFECT 193 ends, which can be attributed to a locahzed action of the radsa- tion on the chromosome thread : The most definite hypothesis which has been suggested for the surface stickiness of the chromosomes characteristic of the physiological effect 1s that the matrix of nucleic acid deposited on the chromosome when it 15 in the condensed and apiralized form assumed im division 1s, after rradiation, in a fluid unpoly- merized state instead of in the polymerized non sticky form 2 The surface stickiness can be produced by means other than uradation, and appears to be a much less specific effect than the structural changes In favourable cytological matenal m which each cell contains a small number of large, and m some cases individually recog nuzable chromosomes, it 1s possible to distinguish with certainty between the two types of chromosome abnormahty 3 Thus when two chromosomes are attached at metaphase, it 1s possible to decide whether the cause 1s a chromatid interchange (F3, Fig 31), which 1s a structural change, or 18 due to the chromosome surfaces sticking together along a portion of their Iength as a result of the physiological effect When a chromosome which 1s normally V-shaped assumes the form of a ring 1t 18 possible to deeide whether a ring and fragment structural change (D4, Fig 30) 1s concerned, or whether the ends of the chromosome have simply stuck together as an expression of the physiological effect of radiation By experments on such favourable matenals it has been established that the physiological effect of radiation, resulting in @ stickiness of the matrix, 1s exhibited m cells which are already 1 The terms employed in the hterature are primary effect of radiation for what we describe as physiological effect and secondary effect for what big desenbe as structural change (Alberti, W & Politzer, G 1923, 1924, ;sCkarek J 1927 Marquardt, H 1938) We have preferred not to use wathterms, Since tt 1s rather confusing in & treatment which deals mainly eee @ mechanism of action of radiation to describe as a ‘secondary thee What 1s probably a rather direct action of radiation on the mosome thread and as a ‘primary effect’ what 1s possibly a less rect action of radia tion 2 Dathngton CD (1942) co Marquardt, H (1938), pollen grain mitosis in Bellevalua romana mitone G (19410) neuroblast mitosis in Chortophaga, Sax, K (19412) (1943) in Alhum root tips Sax, K & Swanson, C P (1941) Koller, PC + pollen grain mitosis and root tip mitosis in Tradescantia 104 STRUOTURAL CHANGES IN CHROMOSOMES in division at the timo of irradiation Such cells continue division, porhaps with some Jengthemng of the duration of the various stages These cells, particularly those in prophase at the SYMMETRICAL ASYMMETRICAL INTRACHANGES INTRACHANGES Br | secerace: | yce.atm f Unter arm | tne arm Hive i g Nu § £2 Q va | AIARIAD>. z ie) QA 414 g @ a 2 SoA { | Oa) Se 4 o= Fria 30 Structural changea mduced xn unspht chromosomes e time of uradiation, are hable to show the stichimess of the chromosome surface typical of the phymological effect, leading to clumped metaphases and bridges at anaphase With large TYPES OF STRUCTURAL OHANGE 195 doses excessive clumpmg may prevent mitosis being com- pleted 1 Cells not already in division at the time of arradiation, but IAA LUN Al Bl cl. Di El Fl NAIR XT Ag Be ce D2 i) “Fe | EaSURIK) As ENO cs bs Es 13 A << AN NN AWE UNY |adan| | O VV 2 a as cs os], Fa] £4], KARA Fra 31 VIVE, Structural changes induced in aplit chromosomes nearing the end of interphase, experience a delay, and enter mitosis some hours or days later, the duration of the delay 1 See Chapter rx for further details 2 Or in the intial stages of prophase, Carison, J G {1941a) 106 STRUCTURAL CHANGES IN CHROMOSOMES depending on tho matenal and the dose: By the time these cells enter division the cell has recovered from the physiological offect,s which appears to be a roversiblo process, for the chromo somes in the metaphase and anaphaso of these divisions do not exhibit the surface stickmess charactenstic of the physiological effect They may, however, show structural changes It has been concluded, therefore,s that the phystological effects, but not the structural changes, are exhibited by the cells already sn division at the time of irradiation, and that the structural changes, but not the physiological effects, are exhibited by the celle which enter division after the exmration of the period of reduced mitotic actinty which follows wradiation This generalization 1s based upon the study of a limited number of oytologically favourable materials (see p 193, footnote 3), which are however not limited to asingle type of tissue or class of organism Ina less favourable matenal, m which the cell contains a large number of small, not individually recognizable chre , 1b as not possible to dis tinguish between the two ty pes of chromosome abnormality by cytological examination It 1s not certain whether the generaliza tion we have just given applies also to these materials That the physiological effect (surface stickiness of chromo somes) 1s not exhibited in cells which enter division some hours or days after srradiation, requires no special explanation other than that recovery 1s possible from the physiological change con cerned That chromosome aberrations of the structural change type do not occur in cells already in division and past mid prophase at the time of radiation can reasonably be explamed on the basis that although an 1omzing particle passing through the thread may still cause a break, the nucleic acid deposited on the spiralized chromosome tends to hold it together and hinders the separation into two parts of a chromosome of which the thread has been broken If such separation oceurs, the mutual repulsion that appears to exist bet metaphase chr 8 presumably prevents exchange occurrmg between breaks m different chr mess In Drosophila sperm the chr i See Chapter vill 2 At any rate m the matenals mentioned in footnote 3,p 193 It 1s possible that recovery 15 not complete in the case of some animal tissues (see Chapter x) 3 Marquardt, H (1938) 4 Irradiation during metotre metaphase and anaphase does lead to structural changes m Scrara (Bozeman M.L 1943) Interchanges TYPES OF STRUCTURAL OHANGE 197 are believed to be in a highly condensed state similar to their state during mitosis, and there 1s clear evidence that the chromo- some thread can be broken by irradiation of the sperm but that breakage ends cannot join until the chromosomes assume a less condensed state x Fragmentation 1s sometimes found in cells ecammed at ana- phase m experiments in which, according to the generalwation given above, only the physiological effect of radhatron 1s ex- Pected Most authors: suppose these fragments to be due to mechanical breakage of chromosomes proceeding to opposite poles at anaphase, but stuck together as a result of the physio- logical effect Itis not impossible, however, that some breaks of the structural type are mduced mn chromosomes irradiated sufficiently late in prophase to escape the inhibition of division experienced by cells not so far advanced 3 Experiments such as those of Marshak in which root tips of various plants are fixed 800n after radiation, and the proportion of anaphases which show fragments or bridges recorded, probably deal with a mix- ture of changes of the physiological and structural types, and are consequently difficult to mterpret TYPES OF STRUCTURAL CHANGE The principal types of chromosome structural change produced by 1omzing radiations aro illustrated diagrammatically in Figs 30 and 31, Fig 30 applying to cells in which the chromosome s are unspht at the time of irradiation, and Fig 31 to cells m which the chromosomes are split At metaphase or anaphase, when the aberrations are observed, the two sister chromatids formed by the longitudinal splitting of each chromosome are separately visible The exact time prior to metaphase at which splittmg occurs has between ch are rare pared to structural rearrangements t defi Junt ns ) wrthin chr 1 Muller, HJ (1940) 2 Marshak A (1937) White, MJD {1937}, Marquardt, H (1938), Sax K (1941q),Sax K & Swanson, C P (1941) 3 Thus Bishop, D W (1942) was able to detect breaks at diakinesis m Chortophaga 3 mun after irradiation The chromosomes were not stuck together, and these breaks cannot be explamed as h 1 breal: of ig to sep but stuck t of the Physnological her as a reault effect 4 Marshak, A (1937 19396) Marshak, A & Hudson J Cc (1937) 198 STRUCTURAL CHANGES IN CHROMOSOMES been a tatter for dispute, opinions ranging from early prophase of tho same division to as far back as prophase of the preceding division X ray experiments themselves furnish some informa tion on the time of split: If, at motaphase, all aberrations are seen to involve both chromatids in the samo way, thisis evidence that at the time when tho structural rearrangement took place the chromosome was singlo If, on the other hand, it 1s observed Tanrz 46 Tr from oh ‘ to chroma d types of as P 8 ‘PP Tradescantia pollen grain mitosis? Hours before metaphase 34.¢«33—:=CO3Z 18088 BC t per 100cells Ch 1220 99 #99 111 81 51 Chromated 00 06 00 39 42 33 Hours before metaphase 28 27) «626 2588 Taterch per 100cells Ch 99 39 00 00 00 Chromatid 21 61 66 108 177 Chortophaga neuroblast mitoeis? Hours before metaphaso 72 43 36 a4 12 Interch per 100 cells Ct 2 826 615 8 0 Chromatid 0 { 3 12 Pr 1 Bax, K (19416) 160r Numbers given by Sax have been multzplied by 3 to convert from breaks per 100 chromosomes to aberrations per 100 cells 2 Carlson, JG (19425), 125r that one chromatid takes part in a structural rearrangement in which the other does not teke part, 1t 1s evidence that the chromosome was already split into chromatids at the time when the structural rearrangement occurred The evidence of radiation experments 18 not completely conclusive,: but we shall for the purpose of classification of types of aberration be content to deseribe as unspht a chromosome which behaves as such m radiation experiments In Table 46 data are given Ulustrating the manner in which the numbers of chromatid and chromosome aberrations per 100 metaphase figures vary with the time elapsing between mradia tron and fixation 3 It 1s seen that rf the radiation 1s given 26 hr I Mather, K & Stone LH A (1933), Riley, HP (1936) Sax K & Mather, K (1939) 2 Seep 204 3 Additional data for plant material are given by Newcombe HB (19424) CHROMOSOME BREAKS 199 or less before fixation, only chromatid aberrations are seen in Pradescanha pollen-grain metaphases If the radiation 1s given 32 hr or more before fixation, practically only chromosome aberrations are seen The mdication 1s that splitting mm these experiments occurred between 26 and 32 hr prior to metaphase Chromosome breaks The simplest type of aberration produced m an unsplit chromosome is a simple chromosome break or terminal deletion, as illustrated in column A of Fig 30 Terminal deletions can be ob- served in experiments in which the cell 1s fixed at metaphase or anaphase of the division next following uradiation At anaphase the chromatid fragments formed by the sphtting of the deleted Tasur 47 Number of fragmenta per cell at various times after irradiation! Days elapsing between 13 21 3 5 6 8 9 4 irradiation and fixation Mean munber of fragmenta 248 194 186 130 093 100 O55 005 per cel fragment lag behind the centric chromosomes which are migra- ting to the poles Lagging 1s due to the absence of the centro mere, and usually leads to failure of the two fragments to be meorporated in the daughter nuclei: Consequently, uf many divisions elapse between irradiation and fixation, acentne frag- ments will not be observed Table 47 shows how the number of fragments observed per cell im spermatogonial divisions of Locusta migratoria dimimahes with lapse of time between irradia- hon and fixation s The whole tame covered by the tables enougl. for about four successive division s In view of these results 1t 1s not surprising to find that m Drosophila expenments, m which sperm are wrachated and i White MJD (1935) 2 An observation of Carlson, J G (19388) on Chortophaga suggests that in this matenal one ch ad fi t 15 included :n each daughter cell in the majority of diviaons But im Chortophaga (and in other materials, °& Onion root tips Sax, K 1941¢) the chromatid fragmenta umally fail to be imeluded in the cell nucle: and form micronucle: which eventually degenerate 3 White Mp (1935) ‘The disappea rance of fragmenta in successiv e divisions has also b yb: Oy K 1941a). een observed in other materials, e & mM onion root tips 200 STRUCTURAL ONANGES IN CHROMOSOMES salivary gland chromosomes are examined, no acentne frag ments are found Tho two new onda formed by the breakage of an unsplit chromosomo usually join either with each other, restituting the orginal chromosome, or with other breakage ends in the nucleus giving various types of structural rearrangement The terminal deletions observed at metaphaso are thus only a small fraction be of those initially produced Tho magnitude of this fraction ean estimated, though only by rather indirect methods It 1s about one twentieth for breaks produced in Tradescantia microspores * Tho proportion of breakage ends produced by wradiation of Drosophila sperm which are still unjoined at the tume of chromo- some split 13 about one-quarter: In Tradescantia the sitet chromatids formed by tho splitting of a brohen chromosome do not fuse at the breakage point but remain separate,s as indicated in diagrams A3 and A5 of Fig 30 In some other materials however sister chromatids fuse at the breakage points giving & dicentno chromatid and a single acentric fragment (dragrams A4and A6, Fig 30) The dicentne chromatid forms a bridge at anaphase, which usually eventually breaks The breakage ap pears often to be at the point at which fusion occurred,« but this 1s not variable, so that duplications and deficiencies may result from the anaphase bndge If the onginal break occurred near the centromere, so that the two centromeres In the dicentric chromatid are close together, the dicentric chromatid ney remain on the metaphase plate and so be completely cell 1s left lost s When an anaphase bndge breaks, each daughterthat.n such with a chromosome witha brohenend It 1s possiblechromos ome cases sister unton again occurs when this broken ome splits, leading to further bndges,s until Joss of the chromos in which or death of the cell eventually occurs The manner e cell anaphase bridges are gradually eliminated in the successiv divisions, probably by death of the cells carrying the aberrant 1 Lea, DE & Catcheside, DG (1942) 2 Chapter v, p 169 3 Sax, K & Mather, K (1939) 4 Carlson, JG (19384) 5 Cp discussion by Pontecorvo, G (1942) 6 As dm maize gi shyt McChintoch, B (1938) CHROMATID BREAKS 201 chromosomes, 18 illustrated mm Table 48,: which shows how the proportion of abnormal anaphases diminishes during the develop- ment of an omon root growing from a bulb radiated in the dormant state and subsequently germinated 48 Gradual Taste of at 1 h {Onion root tips Sax 1941a) Root length (em ) 1 2-3 6-8 10-13 20-25 Abnormal anaphases (%) 46 33 21 11 fi) The gradual disappearance of abnormal mitotic figures after irradiation has been demonstrated in chich fibroblast cells grow- mg in culture 2 Chromatid breaks Irradiation at a stage im which the chromosomes are already aplit may produce a break in one only of the sister chromatids giving a chromatid break, as illustrated im column A of Fig 31, and Plate III d,e The broken fragment remains close to the unbroken sister chromatid fragment in metaphase, posstbly being held to it by a matrix which envelopes both chromatids 3 The chromatid breaks vary in distinctness, sometimes appearmg only as achromatic lestons in the chromatid, and are not easy to Score with accuracy In some materialss constrictions are seen in the chromatids at anaphase, and these may be half-chromatid breahs,1e breaks in one only of the two threads of the chromatid, which must (on this interpretation) be inferred to be already spht into two threads in anticipation of the next cell division As with chromosome breaks 1t 13 believed that the chromatid breaks observed at metaphase constitute only a minority of those mutually produced, the majonty restitutmg or uniting with other breakage ends to give the more complex structural changes 1 Sax K (1941a) The dicentrics recorded in this exper:ment are due not only to the sister union of broken chromosomes but also to asym metrical interchange between non homologous chromosomes 2 Lasnitzki I (1943a) 3 Swanson CP (1942) observing the pollen tube mitosis in Trade écantia 4 F g Chortophaga neuroblast mitosis, Carlson JG (19380) Trade canta pollen tube mitosis Swanson CP (1943) Tradescantia poll mother cell meiosis, Nebel BR (1937) vn 202 STRUCTURAL CHANGES IN ONROMOSOMES Ysochromatid breaks Tn addition to cases where a singlo chromatid 18 broken, there are instances w hero both chromatids are broken at the same level This typo of aberration 19 termed an teochromatid dreal, and 1s Mlustrated in column B of Fig 31 and Plate Ie,d Its beheved to be produced by o single romzing particle which passes through, and breaks, both of the chromatids of the already spht chromosome It 1s clear that the metaphase con- figurations (B3, Fig 31) are similar to tho metaphase configura- tions possible in tho case ef a chromosome which has suffered 6 terminal deletion in the unsphit stage (A3,4, Fig 30), and has subsequently split ‘Phe question obviously arses whether we are Justified in regarding isochromatid breaks as twin breaks pro duced at the atage when the chromosome 1s already spht, or whether they aro not, in fact, produced by the breakage and subsequent sphiting of a chromosome which is unsplit at the time of irradiation The existence of the sachromatid break aa a distinct type of aberration 1s conclusively established in the case of Tradescanita microspores, the argument runs as follows It ts known that mterchange between breaks in different chromosomes occurs within a few minutes of the break bemg formed This follows from the manner im which the yneld of mterchanges produced by a given dose depends on the intensity (see p 262) It 1s evident therefore that if 1t 1s observed that the interchanges produced when a microspore 1s urradiated are chromatid interchanges 1 volving one but not both sister chromatids (E, F, Fig 31), this shows that at this stage the chromosomes are split But 1so- chromatid breaks are obtamed when microspores are uradantet at this stage, and occasionally even m the same cells and the same chromosomes im which chromatid interchanges are also found The conclusion that isochromatid breaks are caused by a smgle 1onwing particle traversing and breaking both of the sister chro- matids of an already split chromosome 1s strengthened by tho fact that ultra violet ght does not produce isochromatid breaks, though 1t does produce chromatid breaks: Quantum absorp iD ted in Drade polfen tubes by Swanson CP (1940 1942) confirmed by Catcheside DG & Lea, DE (unpublished) Inferred algo in maize pollen (see Chapter v, p 186) ISOCHROMATID BREAKS 203 tions in an ultra-violet hight experment are not localized in ines in the manner in which ionizations are localized along the paths of ionizing particles in an X ray expernment, so we should not expect with ultra-violet light to obtain breaks in both sister chromatids at the same level (except by chance coincidence of independently produced breahs) The ratio of the number of isochromatid breaks to the number of chromatid breaks obtamed with a given dose of X-rays varies with the stage of prophase at which the chromosomes are uradi~ ated The ratio is higher in early prophase than in late prophase, asis shown in Table 49 At early prophase the chromatids are Taste 49 The ratio of the bers of isock d and ck d breaks induced at different stages of prophase Tradescantia prophase of pollen gram division? Hours before metaphase 24 12 6 Ratio isochromatid chromatid 243 147 079 Tradescantia prophase of pollen tube division? Hours after germination of the 0 1 2 3 4 5 6-15 pollen grain Ratio wsochromatid chromatid 250 065 041 O24 O28f O18 O14 1 Sax K (1941B) 2 Swanson CP (1943} closely associated An ionizing particle which passes through one has therefore a high probability of also passing through the other As prophase advances the chromatids spiralize mn separate spirals, and their distance apart increases The probability of an ionizing particle which passes through one chromatid thread also Passing through the other thus dimmshes The chromosomes ‘were visibly spit to two chromatids 2 hr after germmation m the Tradescantva pollen tube experiments: quoted im Table 49 In view of the smooth rather than discontinuous change from the stage at which most breaks affect both chromatids to the stage at which isochromatid breaks are much less frequent than chromatid breaks, it 1s evident that the X ray method cannot Bive conclusive evidence of a chromosome being unsplit An alternative view would always be that the chromosome 3s split, but the sister chromatids are still so closely juxtaposed that an lonizing particle which breaks one usually breaks both We hav e, however, already pointed out that the X ray mcthod can give 1 Swanson, CP (1943) 204 STRUOTURAL CHANGES IN CHROMOSOMES conclusive ovidonce of the chromosome being split We conclude thereforo that af the X-ray experiments sndicate that the chromo- somes are split, this can be relied upon, but X-ray erdence for a chromosome being unsplit 18 not final The typical appearance of an isochromatid break 13 that ulustrated in the uppermost of the four alternative forms shown in diagram B3 of Fig 31 After breakage, sister chromatids Tanur 50 Frequency of non union of sister chromatids at isoch id breaks in Trade {Irradiated at room temperature oxcept where otherwise stated } Types of isochromatid break % of ends Division —_—_—_— not showing Re- examined Radistion SU NUp NUd NUpd Total union ference Pollen gram =X rays -— - - =—- = 6 t X rays 240-28 23 7 308 138 2 Xrays 1° 1801 80 110 142005 ba 3 20° 1027 98 63 33° (1226 95 30° a 33 at one 362 91 Pollen tube Ximys = 48 tg 50S @ ra; 27 2 a rays 18 2 3 7 30 317 5 1 Catcheside, DG & Lea DE (1943) in distal fragments, proximal non umons not scored e ) Smon's, P 2 Kotval, J P (unpublished) 3 Catchesrdo DG, Lea DE & Thodsy J.M_(1986a) 4 Kotval, JP & Gray, LH (unpublished) I am grateful to these workers for accesa to their provisional results prior to publication 5 Catcheside, DG & Lea DE (1943) with additional unpublished data undergo umon (SU) at the breakage point Less frequently sister unton fais to occur, either on the side of the breal, nearer the centromere (16 no sister union proximally, NUp), or on the side further from the break (1¢ no sister union distally, NUd), or on both sides (NUpd) The frequency of non umon seems to depend on the type of radiation employed, being more common with a rays than with X rays, ass clearly apparent in Table 50 « The obvious explanation: 1s that the more densely ronizing radiation (a rays) does more damage to a chromosome that 3t I Thoday, JM (1942) did not heep a record of the frequency of non union zsochromatid breaks in his X ray and neutron experiments but states that they are rare It 13 to be presumed therefore that the fre quency of non union A with Pp tes more closely to the X ray than to the & ray frequencies listed in Table 50 2 Catchesrde, DG & Lea DE (1943) INTRAOHANGES 205 breaks, and leaves less readily joinable breakage ends than does the less densely somszing radiation, X-rays « Chromosome intrachanges When two breaks are formed in the same chromosome, the four breakage ends may (apatt from restitution) yom in either of two ways, one way (symmetrical infrachange) leading to the :n- version of the part of the chromosome between the breaks (columns B and C of Fig 30), and one way (asymmetrical sntra- change) leading to the removal of the portion of the chromosome between the breakage points (1 tntercalary deletion), and thus to a ring and a rod, one of which will be without a centromere: (columns D and E of Fig 30) The behaviour of the vanous aberrations 1s shown in Fig 30 When the rng chromosome formed m this way splits, the chromatids may freely separate (D5 of Fig 30), or interlock (D7), or form a single double-size dicentric rings (D6) depending on the amount of twist of the chromosome hich occurred between breakage (D2) and jommg of the breakage ends (D3) A dacentne rng, or a parr of inter- lJoched rings, will give mse to bridges at anaphase After break- age of these bridges, fusion of the two breakage ends mm each daughter nucleus will probably occur reforming a rng chromo some in each nucleus 4 Acentric rings or fragments will usually 1 Since 1t 13 known that radiation given in early prophase retards to gome extent the course of mitosis, and since different doses and. types of radiation mey retard to different extents tt 19 arguable that m the caso of observations made at the pollen gram mitosis the cells were not necessarily all in the same stage of prophase when uradiated though in all cases irradiated 24 hr before metaphase This mght explain the difference between X rays and « rays if the Jomability of breakage ends wero greatly affected by the exact stage at which the chromatids were broken Considerations of this sort should not, however, apply on th pollen tube experiments in which the stage at the time of irradiation . d by the tume el b of the pollen and irradiation, 2 Itis possible (McClintock, have a centromere when ge of the % tay induced breaks deat B 1938) for both the nu, centromere sentromnen itself into two func! tioning portions ‘Lhis 19 probably 4 very 3 Sax, K (1940 1941a) 4 Arguing by analogy with the behaviour of interlocked an id dice: tings at aomatic divisions in a tin: g§ chromosome stock in maze @MeCha tock, B 1938, 19415) 204 STRUOTURAL ONANGES IN CHROMOSOMES conclusive evidenco of the chromosome being aplit We conclude therefore that sf the A-ray experiments indicate that the chromo- somes are aplit, this can be reed upon, but X ray evidence for a chromosome being unaplit ta not final The typical appearance of an ssochromatid break 1s that illustrated in the uppermost of the four alternative forms shown in diagram B3 of Fig 31 After breakage, sister chromatids Tanre 50 Frequency of non union of sister chromatids at d breaks in Trad. {Irradiated at room temperature except where otherwise stated } Division Types of f isochromatid break wa % ons Pe. examined Radiation SU NUp NUd NUpd Total union ference Pollen grain X rays —-— =- =—- =— = 6 T X rays 2400 (28 23 Ww 308 138 2 Xraya 1° 1801 80 110 14-2005 54 3 20° 102: 88 68 330 122 95 30° 308 33 2 6 362 93 a rays 32 27 2868 16 75 4 Pollentube X rays 45 1 2 ° 50 70 3 a rays 18 A 3 7 30 SE? 5 1 wate not Catcheside, DG & Lea DE (2943) 1 distal fragments, pro: ximal non wmyons M43) in 2 Kotval, J P (unpublished) 3 Catchessde DG Lea DE & Thodsy JM (1946a) 4 Kotval, JP & Gray LH (unpublished) I am grateful to these workers for access to their provisional results prior to publication 5 Catoheside, DG & Lea DE (1943) with additional unpublished data undergo union (SU) at the breakage point Less frequently sister union fails to occur, either on the side of the break nearer the centromere (16 no sister union proximally, NUp), or on the side further from the break, (1e no sister umon distally, NUd), or on both sides (VUpd) The frequency of non umon seems to depend on the type of radiation employed, being more common in Table 50+ with a rays than with X-rays, as 1s clearly apparent. The obvious explanation: x3 that the more densely tomizing radiation (a-rays) does more damage to a chromosome that st I Thoday, JM (1942) did not keep a record of the frequency of non umn isochromatid breaks m his X ray and neutron experiments but atates that they are tare It 1s to be presumed therefore that the fre quency of non union obtamed with neutrons approwumates more closely to the X ray than to the @ ray frequencies listed in Table 50 2 Catcheside DG & Lea DE (1943) INTRACHANGES AND INTERCHANGES 207 grain mitosis as small bodies (tsod:ametric fragments), pr d to be rings, and most commonly of about 14 diameter Chromatid intrachanges The corresponding types of aberration occurnng in a chromo some after if 1s split, 1@ inversions and deletions involving a single chromatid only, are shown in columns C and D of Fig 31 and Plate IIIf To ayond excessive multiplication of the number of diagrams, only snter arm tntrachanges have been shown, and «ntra-arm «ntrachanges (mm which both breaks occur in the same chromosome arm) have not been illustrated, although they occur Taste 51 Relat: qi of land asy 1 types of chromatid intrachange at the pollen grain metaphase in Tradescantrat No of h Prop of intra Temp _ —A~—~ changes which are °C Symmetrical Asymmetrical Total asymmetrical I 321 206 527 0394002 20 141 129 270 0484003 30 32 24 56 0434007 Further, mtrachanges between two breaks, one of which 1s m one chromatid and the other of which 1s in the sister chromatid, have not beenshown Its not usually possible to decide by examina- tion of the metaphase chromosome whether the intrachange has occurred between breaks m the same chromatid or im sister chromatids Probably these alternatives are about equally frequent The interarm mmtrachanges can be classified observationally into symmetrical and asymmetnical types, though subject to some error In the event of the intrachange bemg between breaks in the same chromatid, asymmetrical intrachange means intercalary deletion (column D, Fig 31) and symmetrical mtra- change means inversion (column 0, Fig 31) Some expermental data of the relative frequencies of the symmetncal and asym- metrical inter arm intrachanges are given m Table 51 Interchanges If each of two (unspht) chromosomes 1s broken, Jong of the four breakage ends can give mise either to a pair of centre 1 Catchesde DG, Lea DE & Thoday JM (19468) A constant dose of 150r of & rays was delivered at various dose rates 206 STRUCTURAL CHANGFS IN CHROMOSOMES be lost as a result of failure to bo included in either daughter nuclous, tho eventual result bomg often death of the cell: By ispecting Fig 30 and comparing column B with colamn D and C with E, at will bo realized that inversion and deletion are alternative aberrations resulting from two breaks in a chro mosome It1s of somo interest to know whether the alternatives are equally probable, but it 13 not very easy to obtain informa tion on this point In plant matenal, sradiated before chromo some split and observed at metaphase or anaphase following irradiation, deletions can be observed but inversions are not usually distingwshable from unchanged chromosomes In Drosophila oxpemments in which sahvary chromosomes are observed, inversions can be recogmzed, but deletions, unless very small, are not found since they are inviable However, there 13 some information indicating that deletions and inversions of the same length are equally freq in the X chr of Drosophila In bdth Drosophila and Trad manute interstitial dele tions, 1¢ asymmetrical intrachanges between breaks produced close together mm the chromosome, ocour with high frequency In Drosophila they are usually identified by genetical meanss and thew extent then determined by exammation of salivary chr In Trad they are observed. at the pollen 1 Different organisms vary in their tolerance of heterozygous de fimencies 2 Fano, U (1941), quoting experiments of Bishop, M Normal males were radiated and mated to females having attached X chromosomes Zygot gan X ot from thew father and an attached X from thew mother are normally nviable However, if a large part of the X chromosome of the father haa been deleted by the action of the radie tion, @ viable female results, which may be distinguished from X X/¥ females by recessive characters for which the X X chromosome 18 h being supp by normal all hs of these Iocr carried in the remaming part of the deleted X chromosome In this way the fr with which del of & given range of sizes occur can be lated, and Pp with the freq with which inversions of the same range of 61ze are found in salivary gland chromosomes 3 Thus Demerec,M & Fano, U (1941) mated wradiated male flies and selected F, females showing the character Notch {notched wings) Many of these flies had small deficiencies involving the locus concerned {band 307), which were examined cytologieally m female larvee of the Notch atoc]ks 4 Rick CM (1940), Newcombe HB (1942a,b), Giles, NH (1943) INTRACHANGES AND INTERCHANGES 207 grain mitosis as small bodies (1sod:ametric fragments), presumed to be rings, and most commonly of about 1p diameter Chromatid intrachanges The corresponding types of aberration occurring in a chromo some after 1t 1s spht, 1e imversions and deletions involving & smgle chromatid only, are shown in columns C and D of ig 31 and Plate IIL f To ax oid excessive multiplication of the number of diagrams, only enter arm mitrachanges have been shown, and tntra-arm itrachanges (in which both breaks occur in the same chromosome arm) have not been llustrated, although they occur Taste 51 Rel frequency of Land 1 types of chromatid intrachange at the pollen grain metaphase in Tradescantta? No of intrachanges Proportion of intra Temp r ~ changes which are °c Symmetncal Asymmetncal Total asymmetrical L B21 206 527 0394002 20 141 129 270 048+003 30 32 24 56 043+007 Further, intrachanges between two breaks, one of which 1s m one chromatid and the other of ¥ hich 1s in the sister chromatid, have not been shown It 1s not usually possible to decide by examina- tion of the metaphase chromosome whether the mtrachange has occurred between breaks in the same chromatid or in sister chromatids Probably these alternatives are about equally frequent The mterarm intrachanges can be classified observationally into symmetrical and asymmetrical types, though subject to some error In the event of the mtrachange being between breaks in the same chromatid, asymmetrical intrachange means intercalary deletion (column D, Fig 31) and symmetrical intra~ change means inversion (column C, Fig 31) Some expenmental data of the relative frequencies of the symmetnical and asym- metrical inter arm intrachanges are given in Table 51 Interchanges f Tf exch of two (unspht) chromosomes 1s broken, Jomng of the ‘our breakage ends can give rise either to a pair of centre doe nn DG Lea DE & Thoday JM (19468) A constant of X rays was delivered at various dose rates 206 STRUCTURAL CHANGES IN CHROMOSOMES bo lost as a result of failuro to be included an either daughter nuolous, the eventual result bemg often death of the cell: By inspecting Fig 30 and companng column B with column D and C with E, it will bo realized that mversion and deletion are alternative aberrations resulting from two breaks in a chro mosomo It 1s of somo interest todnow whether the alternatives are equally probable, but it 1s not very easy to obtain informa tion on this point In plant matenal, srradiated before chromo somo spt and observed at metaphase or anaphase following irradiation, delotions can be observed but inversions are not usually distinguishable from unchanged chromosomes In Drosophila oxperiments in which salivary chromosomes are observed, inversions can be recognized, but deletrons, unless very small, are not found since they are inviable However, there 8 some information indicating that deletions and inversions of the same length are equally frequent in the X chromosome of Drosophila : In both Drosophila and Trad fia, minute interstitial dele hiona,1e asymmetrical intrachanges between breaks produced close together in the chromosome, accur with high frequency In Drosophila they are usually identified by genetical meanss and their extent then determined by examination of salivary chr In Trad they are observed. at the pollen- 1 Different organisms vary in their tolerance of heterozygous de fiorencies 2 Fano, U (1941), quoting experuments of Bishop, M1 Normal males were irradiated and rated to females having attached X chromosomes Zygotes receiving an X chromosome from their father and an attached X + from their mother are normally inviable However, if a large part of the X chromosome of the father haa been deleted by the action of the radia tion, a viable female results, which may be distinguished from X x/Y fornales by recessive characters for which the x X chromosome 13 bein by normal al el -phs of these loci carried this way the 10 the remaming“part of the deleted X chromosome In occur q y with which del can be of a given range of sizes Icnlated, and d with the freq with which inversions of the same range of size are found mn ashvary gland chromosomes 3 Thus Demerece, M & Fano, U (1941) mated wradsated male flies and y F, females sh the ck Notch hed wings) Many of these flies had small defi Iving the locus d (band 307), winch were exammed cytologically in female larvae of the Notch stocks 4 Rick CM (1940) Newcombe HB (1942c b), Giles NH (1943) INTRACHANGES AND INTERCHANGES 207 gram matosis as small bodhes (1sod:ametne fragments), presumed to be rings, and most commonly of about 1 diameter Chromatid intrachanges The corresponding types of aberration occurring in a chromo- some after 1t 18 split, 16 myersions and deletions involving a single chromatid only, are shown m columns C and D of Fig 31 and Plate III f To avoid excesstve multiplication of the number of diagrams, only inter arm sntrachanges have been shown, and tantra arm ttrachanges (in which both breaks occur in the same chromosome arm) have not been illustrated, although they occur Tare 51 x freq y of l and 1 types of chromatid intrachange at the pollen gram metaphase in TradescantiaTM No of intrachanges Proportion of intra Temp r + changes which are °C Symmetneal Asymmetncal =Total asymmetrical 1 321 206 627 0394002 20 M1 129 270 0 48+003 30 32 24 56 0434007 Further, intrachanges between two breaks, one of which 1s in one chromatid and the other of which 1s m the sister chromatid, have not been shown It 1s not usually possible to decide by examina- tion of the metaphase chromosome whether the intrachange has occurred between breaks in the same chromatid or im sister chromatids Probably these alternatives are about equally frequent The mterarm intrachanges can be classified observationally ito symmetrical and asymmetrical types, though subject to some error In the event of the intrachange being between breaks in the same chromatid, asymmetrical intrachange means intercalary deletion (column D, Fig 31) and symmetrical ntra- change means mversion (column C, Fig 31) Some expermental data of the relative frequencies of the symmetrical and asym metrical inter arm intrachanges are given in Table 51 Interchanges f Tfeach of two ({unsplt) chromosomes 1s broken, Joming of the ‘our breakage ends can give mse either to a pair of centne dove cy ghestde DG Lea DE & Thoday, JM (19465) A constant Of 150r of X rays was dehvered at various dose rates 208 BTRUOTURAL CHANGES IN CHROMOSOMES chr ( trical snterchange, also called eucentric wnter- change, column F of Fig 30), or to a dicentnio chromosome and an acontric fragment (asymmetrical snterchange, also called dys centrte or irte snterchang ! G of Fig 30 and Plate IIIs, 7) Tho behaviour of these aberrations at anaphase 13 shown in the diagrams Symmetrical mterchange does not lead to any mechanical difficulty, nor are the daughter cells defiaent for any portion of the chromosome Symmetrical mterchanges should therefore be viable, and they are frequently observed n salivary chromosomes They are not readily distinguished m mitosis from unchanged chromosomes Asymmetncal iter- changes result in the formation of an acentnic fragment, which usually fails to be included in either daught leus At ana phase the dicontric chromatids formed when the dicentrie chro mosome splits may either go to separate poles (G5 of Fig 30), or form a emss cross bridge (G6), or mterlock (G7) The relative frequencies with which these types are seen in onon root-tipt anaphases are 2 3 1, in grasshopp blasts ansph 9 65 1 These figures suggest that types G5 and G6 are ap- proximately equally frequent and that type G7 1s rarer Symmetrical and asymmetrical interchanges mvolving & single chromatid only are shown in columns E and F of Fig 31, and Plate Til g,h Asymmetncal mterchanges, whether chr or chro- matsd, since they uvolve the formation of acentric fragments, are hkely to be non-viable Apart from the deficiency caused by the losa of the fragment, the existence of dicentric chromosomes which sometimes form bridges also causes mechameal difficulties at division In consequence the proportion of cells carrying acentno fragments or dicentric chromosomes gradually di- munishes m successive divisions following wradistion The data we have already given in Tables 47 and 48 illustrate this The death of cells as a result of asymmetrical mterchange, OF of sister-union following a chr or 1sock tid break, 18 due not only to genic unbalance resulting from losses of the chromosomes affected (though nm many organisms this would be a sufficient cause for death) but sometimes also to other causes, perhaps the mechanical difficulties experienced by dividing cells im which bridges are formed at anaphese For nm Drosophila 1 Sex K (19414) 2 Carlson, JG (19414) INTERCHANGES 209 chromosome loss following either type of aberration 1s lethal in a large majority of cases, even in experiments where by use of special stocks 1t 13 contrived that the orgamsm could be viable without one or two of its chromosomes « It 1s of terest to compare the frequency with which sym metrical and asymmetrical interchange occurs In unspht chro mosomes this 1s difficult, since symmetrical interchanges often TaBLe 52 Rel: of ay and Lek a h (X rays at room temperature except where otherwise atated } Noa of mterchanges o_o Sym Asym Re Material irradiated Division d 1 metrical ference Tradescantia microspores pollen grain mitosis 26 66 1 182 402 2 re 956 798 3 20° 508 A472 30° 149 154 Tradescantia pollen grains pollen tube mitosis Ungerminated Pe 49 46 4 15 70 5 Germnated 53 60 5 12 9 6 Chortophaga embryos neuroblast mitosis 5 9 7 1 Sax K & Mather K (1939) 4 Newcombe HB (1942a) 2 Sax, K (1940) 5 Swanson CP (1942) 3 Catcherde 7 Carlson JG (19418) | (1943) DG Lea DE 6 Catcheside DG & Lea,D E Thoday, J.B (19462) & (1943 cannot be distinguished from unchanged chromosomes at meta phage, so that only asymmetrical interchanges can be scored with accuracy, while in salivary chromosomes, in which symmetrical interchange can be scored with certainty, asymmetrical inter changes are absent, beg non-viable In chromosomes which are 1 Pontecorve, G & Muller, HJ (1941) Irradiated wild type male Drosophila were mated to tnploid females homozygous for brown in chromozome II and ebony m chromosome III Loss of chromosomes II andH's the result of asymmetrical mterchange between them could be thee le heen #perm concerned fertilized an egg containing two each of browe Tmosomes, and the offspring formed would be recognized by the chromosoy ebony a otherwise supp d by the rare ah omes Nevertheless such brown ebony offspring were extremely » showing that the process of los was almost invariably lethal 210 STRUCTURAL CHANGES IN CHROMOSOMES split whon the interchange occurs, so that only one chromatid of each chromosome 1s involved, symmetnieal and asymmetnical interchanges can bo separately scored Tho data collected in Table 52 suggest that symmetrical and asymmetrical inter changes are approximately equally frequent Though in some of the data statistically significant departures from equality occur, 4t 1s doubtful af the rehability of the distinction between sym- metrical and asy mmetneal interchange 1s sufficient to make such a difference certain Tho indications afforded by tho data wo have rov iewed are that in both tntrachanges and interchanges symmetrical and asym- metrical types are equally probable These results are clearly related, they indicate that there 1s no evidence in the union of radiation mduced breaks of any polanzation im the chromosome such as would prevent random joing of breakage ends of a bar magnet broken into preces Muller: has pomted out that this fact makes 1t rmprobable that breakage and union occur in hnhages an a polypeptide chain, since such linkages are polarized In Tradescantia 1t 18 found that some interchanges are incom plete, only two of the four breakage ends joing The propor- tions of incomplete interchanges which have been found m various experiments are hsted in Table 53 The proportion of interchanges which are incomplete 1s greater for a rays than for X-rays The explanation 1s presumably the same as that offered m the case of non umon isochromatid breaks, namely, that the densely romuzing @ particle does more damage 1n traveraing the chromatid than does a less densely 1omizing electron or proton, so that the breakage end 1s less likely to be jomable There 1s 2 general similanty between the two sets of data as 1s, mdeed, only to be expected since sister union at an isochromatid break 1s 6 type of mterchange The ion density of the protons which traverse tissue irradiated by neutrons 1s much Ingher then the average 1on density of the electrons which traverse tissue uradiated by X rays It mght have been expected therefore that the proportion of complete interchanges and non-union 1sochromatid breaka would with neutrons have been mtermediate between the values found with X rays and « rays, instead of approximating to the X-ray value as appears to be the case However, the 1on density of an elec- % Muller, HJ (1041) RELATIVE FREQUENCIES 2ui tron varies considerably along tts path, and there 1g evidence: that 1t1s mamly the more densely 1onzing parts, where the 10n- density 1s not much less than that of a proton, which are effective Taste 63 Proportion of chromatid interchanges whirh ere incomplete in Tradescantia Proportion of interchanges Division examined Radiation which are incomplete Reference Pollen gram metaphase Ta: 22mm 34l= 65%, I o P vee B8in 444=13 1, 2 A rays 20in 350— 57% L X rays 12in 206= 58% 3 Xrays 1° 219in 1754=125% 4 20° 1321p 980=13 5% 30° 50m 303 =1659%op Neutrons 5lin T77= 65% 5 & Tays 39m 165: Sy 8 Pollen tube metaphase X raya Qin Qi 74% 7 1 Kotval J P (unpublished) 2 Catchesrde DG, Lea DE & Thoday JM (1946a) 3 Catcheside D.G, quoted by Thoday JM (1942) 4 Catchesido DG,Fea DE & Thoday, JM (19465) A constant dose of 150r_was given at dosa rates renging from § to 300 r/mm There are no significant differences in the proportiony of h which are th et different dose rates nor are the proportions significantly different at the three different temperatures 5 Thoday JM (1942) 6 Kotval J P & Gray,L H (1947) The proportions independent of the dose 7 Catchesnde, DG & Les DE (1943) in breaking Tradescantia chromosomes The fact that the pro portion of mcomplete interchanges and non union isochromatid breaks 1s approximately the same for X rays and neutrons may be regarded as supporting this vew Relative frequency of interchanges and intrachanges It1s obvious that exchange between two breaks can only occur if the breakage ends have opportunity to come into contact It is of interest in this connewion to compare the frequency with which exchange occurs when the two breaks are (A) in the same arm of a chromosome, or are (B) m opposite arms of the same chromosome, or are (C) an different. chromosomes The expenmental frequencies can be compared with fre- quencies calculated on the assumption that union between breaks israndom,1e that the chromosomes are sufficiently mtimately tle DE &C DG (1942),d d in Chapter var 212 STRUCTURAL CHANGES IN CHROMOSOMFS mingled during the time union 2s occurring for tho probability of exchango to be independent of whether the breaks are in the same or different chromosomes If thero aro in the cell m chromosomes having a centrally placed centromere (1¢ cach chromosome having two arms) and t chromosomes having a terminally placed centromere (1e each chromosome haying ono arm), then if breaks are produced equally frequently in all the arms, tho expected relative fre quency of the three types AB Cis readily shown to ber 1 2m dim+t Qm+t) (2m +42)? 2m+t}* (WI-1) = at Taste 51 Proportion of intrachanges in which both breaks are in the eame chromosome arm {\ rays at room temperature unless otherwise stated ) Proportion of intrachanges in eh both breaks sre ho of mayor in same arm Matenat Examined ee ¢ Tr Ex Re irradiated at chromatid om of Expenmental pected ference osophila Salivary Chromosome 2 i PTin 41-066 055 t $ seri glands ulip micro~ Pollen gran Chromosome 12 0 181m 332055 O50 2 Epores dinsion ftudescantia Pollen tube = Chromosome = 8 Oo Tin 12teOG1 050 a Ringertaunated) ollen grains division ‘radeseantia, Pollen tube = Chromatit 6 oO Zin 6=033 050 3 pollen grains diviston (germinated) tadescantia Pollen grain Chromatrd 6.6 4 ICPOSPOTes. division 1° 201 in $27=2039 © 050 29° 1011n 270-037 0.50 Boe foin 56 =I 050 1 Catebeside DG (19380) using ¥ beanng sperin 2 Newcombe JI 1 (19422) 3 Catcheside,DG & Lea, DE (1943) 4 Catcheude’ DG Lea, D1 & Thoday, JM (1948a) In Table 54 are collected experimentally determined propor tions of mtrachanges having both bres in the same arm, to- gether with the theoretical values z 1 Newcombe, HB (1942a) 2 When mtrachtnges are produced m chromosomes not yet aphit at the time of rrrachation 1t 18 not possible to observe both the symmetrical (Qnversion) type and the asymmetrical (deletion) type in the same organisms, and the figures in the case of Drosophila are based on the observation of versions only and in the case of plant material on the RELATIVE FREQUENCIES 213 There are some instances n Table 54 where departures from expectation are recorded which are statistically significant, but taking the table as 2 whole 1t appears that there 1s not a very marked preference for yntrachanges between breaks in the same, as compared with breaks in opposite arms, or vice versa How- ever, taking into account the fact that mtra arm mtrachanges are more hkely to be overlooked than mter-arm intrachanges, a certain amount of preference for mtra-arm intrachanges cannot be considered excluded Further, minute interstitial deletions have not been included mn the mtra-arm intrachanges listed in Table 54 If these are included, the proportion of (asymmetrical) chromosome intrachanges which have both breaks m the same arm (in T'radescantya microspores) 1s 0 84, according to Rick « In Table 55 are collected data on the proportion of the total number of exchanges m which both breaks are in the same chromosome + The majority of the experiments cited (viz 10 out of 12) indicate that the proportion of exchanges m which both breaks are i one chromosome 1s somewhat higher than 1s to be expected on the basis of random umson between breakage ends When unfertilized eggs of Sciaras or of Drosophila, are uradiated, the preference for exchange between breaks in the same chromosome as compared to breaks sn different chromo- somes 1s extreme, the number of mterchanges being very small compared to the number of mtrachanges This exceptional be- haviour 1s presumed to be due to the chromosomes in the un- fertuhzed eggs being sn meiosis when wradsated observation of deletions only In the case of chromatid mtrachangea (produced in split chromosomes), where both types are observable, the figures refer to total hanges, sy ] and asy trical 1 Rick CM (1940) 2 In Drosophila the observations ate hnuted tosymmetrical exchanges In plant material m which unsplit chromosomes are irradiated, the obsers ations are limited to asymmettical exchanges, and im some exper ments {noted in the footnotes to Table 55) intra arm yatrachanges are not recorded In plant maternal 2 which the chromosomes are split at the time of irradiation, all types of ch a shange and 1 are in tended to be included, but the possibility of some intra erm intrachenges Ig looked cannot be Suded 3 Bozeman ML (1943) 4 Glass, HB (1940) 212 STRUCTURAL CHANGES IN CHROMOSOUFS mingled dunng the time union is occurring for tho probabihty of exchango to bo independent of whether tho breahs aro in the same or differont chromosomes If thero aro m tho cell m chromosomes having a centrally placed centromere (1 cach chromosome having two arms) and ¢ chromosomes having a terminally placed centromere (1e each ebromosome having one arm), then if breaks are produced equally frequently in all the arms, tho oxpected relative fre quency of the threo types AB Cts readily shown to be: 1 2m 1 dm+t (vt) (2m+t) (2m+ Wg Qn +i? Tanty St Proportion of intrachanges in which both breaks are in the same chromosome sh {\ rays at room temperature unless otherwise stated} Proportion of intrachangea in which both breaks are. ho of major same erm (1 acsome chromosomes Material Examined or a Ex Re frradiated at chromatld m i Expenmental pected fe renee Drosophila Sahvary Chromosome 1 fTin 4t—068 = 055 1 soern glands Tulip micros Pollen Bin 33-055 050 oe grain Chromosome 9 spores Lradescaniia Yolen tube Chromosome, 6 0 Tin 1214061 050 ollen grains division lungerminated) Tradescantia Pollen tube Chromatil Zin B~033 050 Hen grains dimsion tgerminated) Pollen aa Trodescantia grain = Chromatid. 6 0 mucrospores: ae 201 in B2T=0:39 0-50 20° 101 in 270=0-37 050 30° 26in 56=046 050 x Catcheside DG (19384) using 1 bearing apenia 2 Newcombe 11 I (19820) 3 Catcheade DG & Les DE ¢ 1943) 4 Catchende DG, Lea, DI & Thoday, J.M_ (1946a) In Table 54 are collected experimentally determined propor- tions of mtrachanges having both breaks in the same arm, to gether with the theoretical values 2 1 Newcombe H B (1942) 2 When mntrachanges are produced im chromosomes not yet spht at the time of radiation, 1t 18 not posstble to observe both the symmetncal (inversion) type and the asymmetrical (deletion) type m the same organisms and the figures in the case of Drosephtla are based on the observation of inversions only and m the case of plant material on the LOCATION OF BREAKS 215 chromosomes, the observations are then hmuted to breaks which have taken part m viable types of structural change, namely inversion and symmetrical mterchange The firat pomt to be considered 1s the distribution of breaks between euchromatzn and heterochromatin The parts of the chromosomes nearest the centromeres (proximal heterochromatic regions) differ from the bulk of the chromosomes in being genetically mert, 1 im con- taining few genes (or at any rate few genes detectable by their producing sharply alternatave effects in different allelomorphs) They are also distinguishable cytologically by differences in their staining properties at mitosis and meiosis, believed to be due to a difference in the amplitude or timmg of the nucleic acid cycle These heterochromatic regions occupy an appreciable fraction of the whole length of the chromosomes at mitosis or meiosis (one- third im the case of the X chromosome), but only a minute fraction ofthelength of the sahvary chromosomes It has been found that the relative frequency with which breaks occur in the euchromatic and heterochromatic regions approximates to the relative lengths of these regions in the mitotre chromosomes, not to their relative Jengths in the salivary chromosomes Thus Kaufmann finds that about 30% of all breaks in the X chromosome occur in the proxrmal heterochromatin,: which occupies one third of the length of the chromosome as seen in mitosis The conclusion that the break frequency 1s proportional to the mitotic length rather than to the salivary length 1s confirmed by the fact that the break frequency in the Y chromosome, which 1s mainly heterochromatic, and very short in the salivary glands, is comparable with that in the X chromosome and 1n the four arms of the autosomes 2 The sahvary X chromosome map 1s divided into 120 lettered segments of approximately equal length Part of the last 81X represents the proximal heterochromatin Kaufmanns 1 Kaufmann, B P (1946a) Tho observed proportion of breaks in the heterochromatin was 25% and the figure 30% makes allowance for exchanges entirely within the chromocentre, which escape detection 2 Kaufmann BP & Demerec M (1937) Bauer, H Demerec,M & Kaufmann, B P (1938) The X and ¥ chromosomes, and the four auto some arms 22 2R,3L 3R are of comparable lengths in mitosia 3 Kaufmann B.P (1946) see also Prokofyeva AA and Khvostova VY (1939) Similar but leas extensive data are given for the autosomes y Bauer H Demerec M & Kaufmann BP (1938) Bauer, H (1939) nd for Drosophila pseudoobscura by Helfer RG (1940} 214 STRUCTURAL CHANGES IN CHROMOSOMES Tantx 55 Proportion of exchangra having both breaks in the same chromosome (X raya at room temperature except where otherwise stated) ‘No of major eeich both breaks are in tion of exchanges in hromo~ € hromosome Chromosome somes Matenal Examined or Ex Re irradiated chromatht m ¢ Experimental pected ference Drosophila Saheary Chromosome 2 1 dlin 8t—0506 038 =: melanogaster glands sperm Drotophila 5 Seura Bahr: pnt Chrosmosome 1 3 M3in al SélwO208 0:20 2 aperm Onion root tip Roobtip Chromosome 160 01 (0082 3 mitoas Tulp micro Pollen-graln Chromosome 12 9 S3in 191=0173 O03 4 spores dinsion Tradeseanta Pollen gran Chromoso 2106 Ha00% ol mucrospores dision me Sein aia pecosd oa 6 o6*-ge" iit in 417-0255 0-001 Neutrons 10din 337=0315 001 7 Tradescantia Pollen-grain Chromatid 6 90 8 microspores division Ie S8Tin 2e81—023 O67 oor Bin 22504022 0167 Sin 359-016 0-167 heutrons 68In Tii~0088 O16T 9 arays 18in 181—0:088 187 10 Tradescantea Pollen tube Chromosome 6 0 I2Fin S36—-0360 O67 4 ollen grains division lungermunated) Trodescantia Pollen tube Chromatul 6 Oo Gin 27-0222 0167 1 ollen grains division (germinated) 5 Catcheside, DG (1938) Sahvary glands of female laryse were examined 2 hollerBo & Aled? Aa) Ee ate eG {1D10)Sabrary glands of to any appreciable distortion of the ratio a8 only one of the four mayorcheorhasomea thas a median female Jarvae vere used Hn ter-nrm intrachanges were classed 23 interchanges, but thas will not leed centromere. ite) Gains ming chromosomes and dicentnc chromosomes were scored 3 Sax kh (1941 $ Sax h (1940) Gentne ning and dicentne were ecored ‘The propor ion of intrachanges was ind: Dendent ‘of the dose Sax, h & Lnzmann EV (1939) Tables 2 and 6 Centric ring chromosomes and dicentric chromosomes were ecored ? Thoday JM (1943) Centne nng ch r and dicentne were scornd The a Catebeside DG Lea DIE &Thoday J " ‘apie The proportion of intrachanges was lose independent, of the intensity 9 Theday JM (1942) To hotval JP & Gray LH (1947) rr Catchesde DG & Lea DE (1943) Location of breaks in chromosomes Its of interest to determine whether breaks occur at random in the chromosomes, or whether certam points are specially hable to be broken Breahs can be located with high precision in Drosophila melanogaster by observation of the salivary gland FREQUENCY RELATIONS 217 Frequency relations As well as the distribution of breaks in chromosomes, the distribution of numbers and types of aberrations in cells has been investigated If a single chromosome break 1s produced by the direct action of a single 1onizing particle on a chromosome and 1s unaffected by the presence or absence of other beaks 1n the cell, we may expect the relative frequency with which cells are found con- taming different numbers of such aberrations to be distributed according to the Poisson formula That is to say, if m 1s the mean number of aberrations per cell, e-TM m*/r!1s the proportion of cells expected to contam r aberrations This expectation has been confirmed for chromatid breaks produced by neutrons in Trade- scantia microspores, and for breaks produced in grasshopper neuroblast chromosomes The experimental figures, together with the expected figures, are set out in Table 56 The +? test shows that the agreement between the experimental and ex- pected frequencies 1s satisfactory Results included im the table also show that in Trade the freq of occurrence of different numbers of isochromatid breaks, of interstitial dele tons, and of exchanges are approximately mn accord with the Poisson distribution In expermmenta in which Drosophila sperm are wradiated and the say ary chromosomes examined, the relative frequency with which different numbers of exchanges, and also exchanges of different degrees of complexity, are found 1s affected by their viability, since only viable configurations purvive to be classified In attempting to interpret experimental data m Drosophila the usual procedure 1s to assume (on the basis of the expernments cited in Table 56) that the numbers of breaks prmarily produced 1n different sperm are distributed in a Poisson distnbution, and then to calculate, on the basis of certain assumptions regarding the conditions affecting the combination of primary breaks, the relative frequency of different types of viable aberration Com Parison with experiment then affords evidence on the extent to which the assumptions are correct All viable structural changes in Drosophil a are symmetrical exchanges of varying degrees of complication The simplest is an exchange between tno breaks, which we term type 2 216 STRUOTURAL CHANOFS In CHROMOSOMES dotormined tho distribution of 1048 breaks among 114 lettered segments An apparently high frequency of breaks in certain intercalary heterochromatic regions may be explained as due to tho known compression of heterochromatin relative to euchro- matin in salivary gland chromosomes We conclude that m Drosophila breaks are fairly umformly distributed along the longth of the chromosome, and that, providing that mitotic length and not salivary length 18 conatdered, euchromatic and heterochromatic regions are approximately equally breakable In other organisms the location of breaks cannot be deter mined to such fine mits In Tradescantia microspores the relative numbers of breaks induced by X-rays in successive fifths of a chromosome arm from the proximal fifth to the distal fifth have been determined: The data show a tendency for more breaks to occur per unit length near the centromere (which 1s in the centre of the chromosome) than near the free end When Tradescanta pollen tubes are wradiated, however, either by X-rays or by ultra violet hght, breaks are more frequent near the free ends than near the centromere + The differences in break frequenoy between the proximal fifth and the distal fifth found in either microspores or pollen tubes, though statistically signi- ficant, do not exceed a factor of 2 An expenment on T'radescantia microspores in which the fre- quency of production of breaks in ordinary centric chromosomes was compared with the frequency of production in acentric frag- ments showed that the frequency of breaks observed per unit length was only one ninth as great im acentne fragments as in centre chromosomes s The explanation suggested 1s that, since the centromere 1s largely responsible for movement of the chromosome durmg division, the strams would be Jess m an acentric fragment than in a centno chromosome, thus making restitution more probable K (1939) cp also Sax, K (1938) who shows that 1 Sax,K & Mather, the distribution 1s the same for single breaks and for breaks taking part m exchange 2 Swanson CP (1942) 3 Sax K (1942) Fragments were produced by @ prior irradiation with X rays before the chromosomes were split The aberrations pro duced then were chromosome estructural changes and could be distin guished from the chrometid structural changes induced by the second dose given three days later after the chromosomes had spht FREQUENCY RELATIONS 217 Frequency relations As well as the distribution of breaks in chromosomes, the distribution of numbers and types of aberrations im cells has been investigated Tfa single chromosome break 13 produced by the direct action of a single 1omzmg particle on a chromosome and 1s unaffected by the presence or absence of other breaks in the cell, we may expect the relative frequency with which cells are found con- taming different numbers of such aberrations to be distnbuted according to the Poisson formula That 1s to say, if m 1s the mean number of aberrations per cell, e~” m’/r'1s the proportion of cells expected to contain r aberrations This expectation has been confirmed for chromatid breaks produced by neutrons in Trade scantia microspores, and for breahs produced in gtasshopper neuroblast. chromosomes The experimental figures, together with the expected figures, are set out in Table 56 The 32 test shows that the agreement between the experimental and ex- pected frequencies 18 satisfactory Results included m the table also show that m Trade the freq of occurrence of different numbers of isochromatid breahs, of interstitial dele tions, and of exchanges are approximately in accord with the Poisson distribution In eaperments m which Drosophila sperm are irradiated and the sahvary chromosomes exammed, the relative frequency with which different numbers of exchanges, and also exchanges of different degrees of complexity, are found 1s affected by their viabihty, emee only viable configurations survive to be classified In attempting to interpret experimental data in Drosophila the usual procedure 1s to assume (on the basis of the experiments cited im Table 56) that the numbers of breaks primarily produced 1n different sperm are distnbuted in a Poisson distribution, and then to calculate, on the basis of certain assumptions regarding the conditions affecting the combmation of prmary breaks, the telative frequency of different types of viable aberration Com- Parison with experiment then affords evidence on the extent to which the assumptions are correct All viable structural changes in Drosophi la are aymmetnical exchanges of varymg degrees of complicat ion The simplest 1 exchange between two breahs, which we term type 2 218 STRUCTURAL CHANGES IN CHROMOSOMES The only way in which a nucleus in which three breaks persist can bo viable 1s for cyclic oxchange to occur (type 3) With four breaks persisting one may either have a cyclic ox change involving all four breaks (type 4), or two separate two break exchanges (designated 242) Similarly, with six breaks Tate 55 Numbers of cells containing O 1, 2, 3, ete. aberrations Aberrations per cell Maternal Aberration 0 1 2 30 od test Reference Chortophaga Chromatid + lrochro- 14 NZ ag 5.1 x=10 rt neuroblasts matid breaks N84 1012 WS 59 10 a=3 €25r A rays PuOB Tradesrantia Chromatid breaks 408 263 BH 82 w=60 microspores 457 2331 638 NS 18 and 46 Gv neutrons Pal6 Tsochromatid breaks 483 20h 89338) (10:1 Xan8d 2 4204 250 437 62 07 23s Chromatid exchanges 478 32 490 OT Q Malt 2 4758 2075 453 66 O8 ned P=07 o 1 2 23 Trade Ch: 64 219 3 3 ald 3 mucrospores deletions 6198 2008 355 43 eed 400r 4 rays Pa08 Chromosome asym. 665 192 12 o =62 3 metneal exchanges STR 1685 29 18 03 re Ch: tad 278 273 45 0 xfa08 4 microspores 2280-2 «892 «159 OF na? 150k A rays PHOT The upper figure is the expenmental number of cells with the stated number of aberrations, the lower figure is the number evpected on the Po:sson distnbutton 1 Carlson, JG (19416) fot these unpublished data. hich were obt tained z Tam indebted to Mr Thoday in he the course of the expenments he describes in $s 1942 papel t {Thoday JM 19 42) 3 Rick, CM (1950) 4 Catcheside DG Lea, DE & Thoday, JM (10a), irradiation at 30° persistimg there are four alternatives (6, 4+2, 3+3, 242+ 2) Expenmental data exist of the relative frequencies of these yanious types in aberrant nuclei, and are reproduced in Table 57 + The ‘expected’ frequencies listed m Table 57 for comparison with the experimental frequencies are calculated by an extension of the theory: described mn Chapter V in connexon with dom nant lethals The proportion of sperm irradiated by dose D in which 2 breaks are primanly produced which unite in yiable ' combinations was there found to be e-*?(2agD)" Bayt It can 1 The experimental figures in able 57 are tahen from the analysis by Fano U (1941) of data of Bauer H Demerec M & Kaufmann BP (1938), and Bauer H (1939a) Similar data are available for Drosophila pseudoobscura (Koller PC & Ahmed, I A 1942) 2 Lea DF & Catcheside, DG (1945a) FREQUENCY RELATIONS 219 be demonstrated that of such sperm a fraction 1/2 (n— 2)! wall show a type 2 exchange, 4 fraction 1/3(n~ 3)! will show a type 3 exchange, a fraction 1/4(n—4)! wall show a type 4 exchange, & fraction 1/8(n—4)! will show type 2+2 and so ont These formulae are based on the same assumptions 1s were used in Taste 57 PB of cells types of chromosome exchanges (Drosophila sperm irradiated by X raya Upper figures expernmental, lower figures calculated assuming random unior of breakage ends } Type of exchange % Dose More Total no roentgens 2 3 4 242 complicated of cells 1000 80 133 67 _ _ 15 935 60 03 02 _ 2000 34 125 ~ 125 16 64 873 108 11 06 02 4000 596 118 42 146 98 144 751 11 36 18 14 Chapter v, namely, random joming between breakage ends, and each break supposed in a separate chromosome arm The ex- pected frequencies calculated on this basis are seen to agree tolerably with the experimental frequencies for 2-, 3 , or 4 break cyche exchanges The serious discrepancies: he in the fact that & greater number of cells showing more complicated types of aberration 1s found experimentally at the higher doses, and in the fact that, expermentally, aberrations compnising four breaks are usually ty pe 2+ 2, while, theoretically, type 4 aberrations are expected to be twice as frequent as type 24+2 The cause of these discrepancies 1s not yet understood Among viable structural changes duced by irradiation of maize pollen, and detected at diahimesis in the pollen mother cells of the F, plants, 11 configurations of type 24+2 and 3 of 1 In general, the fraction of viable sperm of n primary breaks which will chow an aberration totalling r breaks, made up of a 2 break ex hanges f 3 break exch y 4 break exch ete , 13 1 (n-r)tatBty! 22 3f ay 2 As pointed out by Fano, U (1941) See also Kaufmann BP (1941 1943) Fano, U (19438) 218 STRUCTURAL ONANGES IN CHROMOSOMES Tho only way in which a nucleus in which three breaks persist can be viable 1s for cyche exchange to occur (type 3) With four breaks pormsting one may either have 6 cyclic ex change involving all four breaks (typo 4), or tno separate two break oxchanges (designated 2+2) Similarly, with atx breaks Taste 56 Numbers of cells containing 0 1, 2, 3, ete., aberrations Aberrations per cell Matenal Aberration Oo 1 2 3 od Mitest — Reference Chortaphaga Chromatid + brochro- M42 as 5 4 xXield x neuroblasts matil breaka 1784 1042 BS 59 10 am. €25r Ary Tradescantia — Chtomatid breaks 4060 «363-868 z microspores S57 231 638 116 16 46 Gi neutrons Isochromatid breaks 43 fo 38) at z 480-4 2050 437 62 OT Chromatid exchanges «478 «2302 49s 7) 4158 375 453 66 08 9 t 2 33 rf 61g 219 3 3 3 microspores deletions 696 206 855 43 4007 X rays Chromosome asym 665 192 1 oO 3 ‘metncal exchanges 678 «1685 «209 «18 Tradescantia Chromatid interchanges 2276 213 15 0 4 microspores 22802 2692 159 O7 450r A rays ‘The upper figure 1s the experimental number of cells with the stated number of aberrations the lower figure is the number expected on the Poisson distnbution t Cartson, IG (1941a' I Yarn miiebied to Me Thoday for these unpublished data, which were obtained in the course of the experiments he deseribes in fus 1942 paper (Thoday, J MI 1942) 3 Ruck, € ME (1940) 4 Catchende DG Lea DE & Thoday JM (1046a) wradiation at 30° persisting there are four alternatives (6, 442, 343, 2+2+2) Expermental data euust of the relative frequencies of these various types in aberrant nuclei, and are reproduced in Table 57 + The ‘expected’ frequencies listed in Table 57 for comparison with the expermmental frequencies are calculated by an extension of the theory: described m Chapter v in connexion with domi- nant lethals The proportion of sperm irradiated by dose D in which n breaks are primarily produced which umte in viable t combinations was there found to be e~*? (2agD)" ( anh j Tt can i The experimental figures in fable 57 are taken from the analysis by Fano U (1941) of data of Bauer H Demerec M & Kaufmann BP (1938), and Bauer H (1939a) Sumilar date are available for Drosophila pseudoobscura (Koller, PC & Ahmed IA 1942) 2 Lea DF & Catcheside DG (19452) FREQUENCY RELATIONS 219 be demonstrated that of such sperm a fraction 1/2(n—2)! wall show a type 2 exchange, a fraction 1/3(n—3)! will show a type 3 exchange, 4 fraction 1/4(n—4)! will show a type 4 exchange, a fraction 1/8(n—4)! will show type 2+2 and so on: These formulae are based on the same assumptions as were used in Taste 57 Pi of cells diff types of chromosome exchanges (Drosophua sperm irradiated by X rays Upper figures experimental, lower figures calculated assuming random unior of breakage ends ) Type of exchange % Dose More Total no roentgens 2 3 4 242 comphcated of cells 1000 80 133 67 _ _ 1 935 60 03 o2 _ 2000 734 125 _ 125 16 64 873 108 et 06 02 4000 596 118 42 146 98 144 751 181 36 18 14 Chapter v, namely, random joming between breahage ends, and each break supposed in a separate chromosome arm The ex- pected frequencies calculated on this basis are seen to agree tolerably with the experimental frequencies for 2-, 3 , or 4-break cyche exchanges The serious discrepancies? he in the fact that & greater number of cells showing more complicated types of aberration 1s found experimentally at the higher doses, and in the fact that, experimentally, aberrations comprising four breahs are usually type 2+ 2, while, theoretically , type 4 aberrations are expected to be twice as frequent as type 2+2 The cause of these discrepancies 1s not yet understood Among viable structural changes induced by uradiation of maize pollen, and detected at diahinesis in the pollen mother cells of the F, plants, 11 configurations of type 2+2 and 3 of 1 In general the fraction of vtable sperm of n primary breaks which se show an aberration totalling r breaks, made up of a 2 break ex changes, 8 3 break exchanges y 4 break excl ete 18 1 (n—r)latBly! 22 38 ay 2 As pointed out by Fano, U (1941) See also Kaufmann BP (19418 1943) Fano, U (19438) 218 STRUCTURAL CHANGFS IN CHROMOSOMES Tho only way in which a nucleus in which three breaks persist can be viablo is for oyelic exchange to occur (type 3) With four breaks persisting one may either have o cyclic ex chango involving all four breaks (type 4), or two separate two break exchanges (designated 24+2) Similarly, with etx breaks Tapre $6 Numbers of cells containing 0, 1,2 3 ete aberrations Aberrations per cell Matenal Aberration o I 2 br | xitest Reference Chertaphaga Chromatid + brochro- mm NN 3B 5 1 old 1 neuroblasts matid breake N84 1012 WS 59 10 ‘nad @25r A rays P08 Tradescantia Chromatid breaks 406 203 5H a 2 x =89 2 Iictospores. 457 DI 38 NG 18 nos 46 Gy neutrons Pad Taocbromatid breaks 483 OOH 33 G odd 2 4804 205-0 437 62 OF $r3s Chromatid exchanges 478) OO? 49 7D eeld 2 4158 MOIS 453 66 08 and PeO7 o 1 2 33 Trad Ch 614 219 3 3 xolO 3 macrospores deletions 6196 28 35 43 and 400r X rays P=06 Chromosome asym- 685 192 13 9 xyab2 3 metneal exchanges 6i78 #1685 NO 1B 3 =3 os Tradeseantia Chromatid interc! 2216 273 1 o m8 4 mmerespore hanes roa geet 189 OT at 1 150 4 rays The upper figure 13 the experrmentat number of cells with the stated number of aberrations the lower figure is the number expected on the Po:sson distribution 1 Carlson JG (19%1a) 2 Lam indebted to Mr Thoday for these unpubLsbed data, which were obtained in the courseft the experiments he describes in hus 1942 paper (Thoday J.M" 1942) 3 Ruck, CAE (1940) 4 Catchesde DG Lea DE & Thoday JM (1946a), wradsation at 30° persisting there arc four alternatives (6, 4+2, 3+3, 2+2+2) Experimental data exist of the relative frequencies of these various types in aberrant nuclei, and are reproduced in Table 57 + The ‘expected’ frequencies listed in Table 57 for comparison with the experimental frequencies are calculated by an extension of the theory deseribed in Chapter v m connexion with dom nant lethals The proportion of sperm mradiated by dose Din which » breaks are primanty produced which unite in viable combinations was there found to be e~*?(2eqgD)" a It can in 1 The experimental figures in fable 57 are taken from the analysis by Fano, U (1941) of data of Bauer H, Demerec, M & Kaufmann, BP (1938), and Bauer H (1939) Similar data are available for Drosophila pseudoobscura (Koller PC & Ahmed IA 1942) 2 Lea DI & Cateheside, DG (1945a) EFFECT OF TEMPERATURE 221 to the colchicine reducing the movement of the prophase chro- mosomes and so favouring restitution compared with re- arrangement The yields of various kinds of aberrations induced by @ given dose of radiation in Tradescantia microspores 1s influenced by the temperature, as shown in Fig 32,8: The flower buds remamed. at the given temperature during the irradiation and for about an ou sD v xP A 2 ar B tof T y u Zo ar P y[-*) ar ° z — ¢ 3 iL 2 a fr 7 al b ° L 3 0 € 4+ E Bish c ab 5 ee 2b a s}°————___ ib & fi) n L i 6 1 ian 0 to 20° xn” 49° ° 10° 20° 30° 40° Tradescantra microspores Drosophila sperm Fic 32 The influence of temperature on the yield of radiation mduced chromosome structural changes A rings and dicentrics in unsplit chromo es B, split ch somes C) hh d breaks (6) 1s0ch d breaks, {c} cl romatid interchanges _C, rings and dicentrics nm unspht chromosomes (a) et 3° then fe to 3 or 38°, (b) irradiated at 38°, then tranaferred to 3 or 38° DD ‘h between ch Ir and IIT a at d E | between ch Aland IIT fe at x The sources of the data are as follows A Sex K & Enzmann, E V (1939) The data of Tables 6, 7 and 8 of this paper have been grouped together Doses 300-360r B Catcheside DG ,Lea,DE & Thoday,J M (unpublished) Dose 150 r Rick CM (1940) has irradiated unsphit chromosomes at 3° and 33°, fi Sax and E: ’a results with mngs and d and further sh that a similar effect occurs also with minute interstitial deletions ° o 220 STRUCTURAL CHANGES IN CHROMOSOMES type 4 were observed, showing a similar departure from the oxpected ratio of 1 2 to that found in the Drosophila expen Ments 1 Modifying factors The probabihty of restitution of a break primarily induced by radiation can bo modified by other factors beades the position of the break. in the chr and the y or absence ofa centromere in the chr If Trad tra buds are centri fuged during trradtation, the yield of structural changes 13 10 creased, as shown in Tablo 58 + The explanation of this effect 13 presunably that the nereased stresses in the chromosome thread caused by centrifugation tend to separate the breakage ends at a newly formed break and thus reduce the probability of restt tution Taste 58 I: d yield of ch 1 changes owing to centrifugation during srradsation (Tradescantia microspores Sax 1943) I h and int Pp d by ~120r in unspht chromosomes Without centnfugation 66% With centrifugation 13.2% Isochromatid breaka produced by ~J50r in spht chromosomes Without centrifugation 276% With centrifugation 458% th: and i produced by ~160r im eplit chromosomes Without centnfugation 267% With centrifugation 363% What may be regarded as the complement of this experiment was performed on omon root trps 3 Two batches were exposed to the same dose of X-rays, 300r One batch had been treated with colchicine, the other had not The yield of chromatid aber rations m the colchicine-treated series was only one third as great as m the untreated series, which was believed to be owing 1 Experimental data of btadler LJ & Sprague, GF (1937) and of Catcheside, DG (29888) combined See Table 2 of Catchesides P P Sax, K. (1943) Centritugation alone did not produce aberrations aper Saxs figures have been converted from breaks per chromosome to pert cell by multiplying by six in the case of single break aberrations, and by three in the case of two break aberrations 3 Brumfield RT (1943) as as \ erat with 3 rays © Incr sing ion density towards end of elotron track D. Fast electron I Photoclectrons produced by N\ ravs of wavelength 154 10 y tissue Pratt I Distribution of romzation produced bs difterent radiations LE lates pryntert va Creat 2 rdfane 222 STRUOTURAL GHANGYS IN CKO MOSOME S hour following The yield of aberrations at the Ingher tempers tures 18 significantly lower than at the lover temperatures 1 If the vsow 33 correct that modifying factors such as tempers- ture affect the joming of breakage ends rather than the primary production of breaks, then at should be possible to reduce the yield of structural changes by raving the temperatu re during the joing process Sax and Enzmann carried out an experment of this sort, the reauita of which are shown in Fig 326 + The flower buds were irradiated at 3° for 2 min + and 2 min Iater half of thom were transferred to 38° for 1 hr ‘Tho yield of aberrations nm this batch waa less than in the batch which was kept at 3° dunng the hour following irradiation, but not so low a3 1n experiments in which the buds were at 38° during the irradiation as well a8 for the following hour The result of this experiment indicates that some joining takes place during the radiation, and that joing continues also for some time after the end of the wradia- tion This conc} ig.in complete ag with the result of experiments on the variation of dose rate (described Inter) The yields of structural changes induced by uradiating Droso- plila sperm at different temperatures aro shown in Fig 32ps ‘There 18 some indication that the yield is reduced by raising the temperaturo, but the expenments are not m good agreement and the effect 13 not certaisly established « x This has been bbshed both for ip diated 24 br before ph when the ch are epht (Fig 32z) and for rancrosporea arrachated 5 days before when the ct areunepht(Fig 32a, alsoFaberg,A 0, 1940a) Sax,K & Enzmann, EV (1939) made & triat in which microspores were mradiated 48 hr before metaphase at a time wher spl wea The of the structural ch wore of tha ch type but a mmority were chromatid changes The number of ch I chi ip increased at the higher iP in this In view of the eontrary results ot din in which the ‘p were arrachated exther 24 hr or 5 days before metaphase 1b seems Lhely that the effect of raismg the tensperature at 48 hr was to merease the propor tron of ch which. were y spht, rather than to raise the gsold of ab per spit ch: 2 Sax K & Enzmann EV (1939) Dose 160r 3 The data for curves (a), (5), (c} are taken from the following sources (a) Muller and Pontecorvo, reported m Muller, HJ (1946), (0) Mickey GH (1938) {c} Makhujam dose 2000 r , reported in Muller, HJ (1940) 4 Muller, RJ (1940), placing principal reliance on the data shown as curves (a) and (c) of Fig 32D concludes that the yield 13 independent of the temperature A aray witho rays IT Recoil electrons and photoelectrons pro daccd by \ rave of wavelength 1-1 B Praton D Tast clectron F_ Photoelectrons produced by rvs of wavelength 15 4 10 p tissue Pratr I Distribution of ronization produced by different radiations {I lates printed in Great Britain B Bacteriophage plaqacs (Local Iestons (tobacco n¢cross 41rus) a al a” O : ©: Pa ; . “ D_ Crystalline tomato bushy stunt virus Pratt II Viruses oa Bs Lo hat tet : arSilty rt OFF as | Same CS ee a gat Par, Fi ‘ ae Ts 22, Wh er wrest Ww aa, t oS TM Sacer. * A bee? ag ad a1 6 ~ *, roe 4 " > r - re ee re fv 50m.” a reek eB —TM 7, Ale ~ ta Pusat: TT Chromosome structural changes ub Feeble me tanageeter Salivary chromosomes a inveision 6 interchange chromatid beet Ve len tube cP romosames (accpaplithene metaphaso) © se aerow epee ghiommatd incall (upper aires) msochromatid brosk (lower Toit nde ena break fol Trade cantia pollen gram mitoses f chro h normal meta jnerachange (prophaso) gy chromatid interchange (prophase) two foci ¢ duck Non ‘2, asymm strical chromosome mtcrchange (same cc ll at Clrango (nat eachtte chromosome y ace ntric fragment) A chromatid mtot 6° (met sphaso) 2 snaphse brulgo (due to tsochromatid breat) (Photomtcrographs by DG. Catchosde) og u 5 A é ao ? “es 3 c : oo et ” oN, Pane ‘ eb! a4 . i Sn 2, Saws PS peed V4 ! ‘ No Meet cot . aornval ie ee, Thick gt EF Chick wa SoOEy nt Le ta re Bo tet cots tong, forma . H Chick i en ete fi gee ret Foo wees rete ih ret e —, er A ae payer j 4 bay - bd! ‘eal AD ©) Bact cots Internist ‘4 atructury & J oy f ae bt ¥ aS m , ‘ Reesa , ' we, | Vaccmin siras internal led — structure J Grasshopper K Onton Puare Iv Bactena Viruses, rbn ormal divisi on figures MODIFYING FACTORS 223 It 3s beheved: that the young m new arrangements of breaks induced by wradiating Drosophila sperm 1s delayed until fertihza- tion of an ovum by the wradiated sperm occurs In this event one mght expect that the effect of temperature, if it exists, would be shown by the yield obtained bemg dependent upon the temperature at the time of fertilization The data reproduced in Fig 3282 do not show any such dependence According to an expenment of Kaufmann and Hollaender,s uradiation of Drosophila male flies by either infra red or ultra- violet radiation between the administrutiun of two 2000 r X-ray treatments reduces considerably the yield of chromosome struc- tural changes induced by the X rays The infra red or ultra- violet radiation alone had no eficet These results suggest that some restitution of breaks pnmanly induced by the X rays 13 posble in the sperm, and 1s aided by the administration of ultra violet or mfra-red hght If the dimmution of joeld at Ingher temperatures suggested but not established by Fig 32p 1s confirmed, the explanation wall presumably be that restitution im the sperm 1s aided by rise of temperature Marshak, bas found that treatsng onion seedlings with a dilute solution of ammonia prior to irradiation reduces the proportion of abnormalities seen in anaphases 3 hr after uradiation It 15 not at all obvious what conclusion to draw from this observation The treatment with ammona delays the onset of prophase Thus cells seen 1m anaphase 8 hr after wradiation are not in the same stage at the time of irradiation in the ammoma treated and un- treated series, and this, rather than any more fundamental effect, may explain the observation In any case, the chromo some aberrations studied are probably largely phy siological changes (cp p 192) The yield of chromosome structural changes obtamed with a gen dose in a given species is affected by the state of the chromosomes at the time of irradiation This effect 1s best in- vestigated m material in which at 13 possible to determine with certainty the stage of the cells at the time of wradiation In the i Muller HS (1940) 2 Maller and Pentecors 9 report ed in Muller, HJ {1940} 3 Reported seen Euanin Demeree Af, Kaufi ann, »aufm B BP, Fano U Sutton E & 4 Marshak A {19384,by 224 STRUCTURAT, CHANGES IN CHROMOSOMES oxpenments of Swanson,: the resulta of which are reproduced in Table 69, Tradescantia pollen grains were germinated on an atti ficial medium and srradiated at various times after germination, representing various stages of prophase of the pollen tube mitosis Tho yteld of aberrations 13 at a maximum 4 hr after germina- tion, when the ec!l 15 1n mid prophase and the chromosomes are spraying Tho y1eld 1s small in the ungerminated pollen grain when the chromosomes are at rest The results are consistent with the experiments already desenbed in which chromosome movement was mereased by centnfugation or decreased by colchicine or removal of the centromere, and from which it was concluded that mos ement of the chromosomes during irradiation favours permanent structural changes by hindering reatitution Tavtz 69 ‘Lseld of chromatid breaks and chromatid interchanges 10 Tradescantia potlen tubes irradiated at vanous times after germination (Swanson 1943 3707) Houra after Ung dot 2 3 4 05 Chromatid breaks per 100 cells 20 256 283 204 380 310 Tnoterchanges per 100 cells 30 68 Ir let 152 60 Hours after germination 6 vi 8 10 5 Chromatid breaks per 100 cells 190 416 19 14 o8 Intorchanges per 100 cella 17 00 00 00 08 The almost complete disappearance of aberrations when the chromosomes are 1n the fully condensed state 15 hr after germ: nation 33 attributed to the formation of a matrix round each chromosome which holds the chromosome together although breaks may be induced m the chromosome threads In experi- ments on Scrara oocytes 1t has been established that srradiation during first me1otie metaphase and anaphase can product chro mosome structural changes (not detectable at the division con- cerned but observed in the salivanes of the F, larvae) actually with a greater frequency than during prophase 2 The changes are, however, nearly all intrachromosomal, exchange between breaks in different chromosomes hardly ever occurring 3 It 1s probable therefore that irradiation of metaphase and anaphase chromo somes can cause breaks which are not cytologically detectable 1 Swanson, C P (1943) 2 Reynolds J P (1942) 3 Bozeman, ML (1943) SENSITIVITX AT DIFFERENT STAGES 225 at the division durmg which irradiation takes place, and which are less lhhely to give mterchromosomal structural changes than are breaks induced im interphase or early prophase If sister- union occurs at the breakage ends when the chromosomes split, such breaks induced at metaphase and anaphase may have a lethal effect at a subsequent division Experiments on a variety of materials have been deseribed m which cells are irradiated, fixed after the Japse of varying inter- vals of tame, and metaphase or anaphase figures examined for chromosome changes These experiments therefore consist essen- tially in determining the sensitivity of chromosomes 1 various stages prior to metaphase Their mterpretation ts complicated by the fact that radiation delays division, so that even though the time scale of the cell cycle miy be hnown in the umrradiated matenal (which 1s not always the case), there 1s hable to be a doubt concerning the stage which has been reached at the time of irradiation by a cell which 1s found, for example, to be in metaphase 24 hr later The general result appears to be that cells become less sensitive as prophase advances,: mn agreement with the data shown nm Table 59 The sensitivity mm interphase, prior to chromosome split, 1s rather lower than in early prophase, so that the highest sensitivity 1s reached in prophase : Dependence of the yield of structural changes on radiation intensity Studies of the dependence of the yield of various types of structural change on the intensity, dose, and kind of radiation 1 See for ple, Sax, K & & CP (1941) on Tradescantia microspores, Marquardt H (1938) on Bellevalia microspores, Carlson TG (19414) on Chortophaga neuroblasts 2 Marshak, A (1937, 19395 1942a 19426) has irradiated rat and mouse tumours, and the root tops of a variety of plants and examined the proportion of anaphases which are abnormal at various times after iradiation The maximum effect is shown by cells which are in anaphase 3 hr after wradhation It. appears probable that the abnormali ties at 3 hr in these expermments are mainly physiological effects induced cells sufficiently ads anced in mitous in to escape the temporary inhibiti of division experienced by on cells less far advanced at the tion time of irradia When instead of ‘abnormal pl »an abi defi the ly of structural change’ type was scored, the maxim um yield was found in cells irraciated 18 hr before metaphase (Marsh minute delet ak A 19395 scoring ions in bean root. tips) 226 STRUCTURAL CHANGES IN CHROMOSOMES havo been of grent value in elucidating the mech of the induction of structural changes by radiation, and these expen mental results wo now proceed to review A number of authors havo investigated the manner in which tho yield of chromosome structural changes mduced in Trade écantia microspores by a given dose of radiation depends upon tho timo over which the irradiation 13 extended The principal results are given in Fig33: It may be seen that with both X-rays and noutrons the jreld of chromatid and ssochromatid breaka 15 independent of intensity This 18 the result to be expected on the view that theso aberrations are produced by the passage of a single 1omzing parttcle through one or both reapec tivoly of tho chromatids of a split chromosome It may be seen further that with both chromatid and chromo some interchanges, the y:eld produced by a given dose ofX rays diminishes with increase of the time over which the irradiation 1 oxtended This result 13 readily explained on the views that the to breaks which take part in an interchango are produced by separate ionizing particles If the irradiation is extended over a prolonged time, a break in one chromosome has time to reats tute before another break 1s produced in its vicinity with which exchange 1s possible Experrments have also been made in which a given dose 1s either given in one concentrated exposure, or divided into fractions with rest penods between s The yield 1s less with the fra ted dose, in ag) t with the results of the intensity vanation experiment In contrast to the X ray results, it 1s found that with neutrons the yreld of both chromosome exchanges (curve ¢, Fig 33) and of chromatid exchanges (curve g, Fig 33) 1s mdependent of the time over which a given dose 1s spreads This suggests that a x Derived from the experimenta of Sax, K (1939, 1940) Marmelh LD ,Nebe} BR, Giles NH & Charles, D R (1942), Giles, NH (1943), Catchende DG, Lea DE & Thoday JM (19465) The results of Giles and of Catcheside, Lea and Thoday shown m curves e and f of Fig 33 agree well Those of Sax and of Marmelh, Nebel, Giles and Charlea shown i curves @ and 8 agree badly quantitatively, but both show a reduction of yield with prolongation of the time of exposure 2 Sax, K (1939) A fuller discussion 1s given wm Chapter vit 3 Sax, K (1939 19418) 4 Giles NH (1943) It was also found that the effect ofa given dose ‘was independent of whether it was given as a eingle exposure or divided nto two fractions separated by an interval of 15 or $2 mn DEPENDENCE ON INTENSITY 227 single omg particle usually causes both the breaks m the neutron induced eachanges « Experiments have been made to test whether the yreld of structural changes induced in Drosophila sperm by a given dose of X rays or y rays 1s independent of the tume over which the dose 1s spread The experiments are made by itradiating male fles, or impregnated females, and detecting structural changes, 6 05 A 0 r 1 » 1 L : 16 '0 20 x 40, snutes? © = b4bd $ n 1d é i + ; t gab a g 10 B 2 ost £ E o6- 3 S O4b xp x ee eo rs — O22 4 i ‘ L L 1 L _ n 04 2 4 6 8 anutes!? \2 \4 46 03h c : Z 0 2F- or + ¥—& 0 n r 4 L L L a. ni 0 2 4 6 i 1 14 6 minutes Duration of exposure ho BD i upon duration of byexp of yield of ab percellin 1 ® constint dose A erhromosome exchanges a 300r X rays (Marinelli Nebel Giles & Charles) b 320r X rays Sex) ¢ 65y neutrons (Giles) B chromatid and isochromatid breaks ¢ chromatid breaks 150 X rays (Catcheside Lea & Thoday} e isochromatid en (m@ 1507 X rays (Catchesido Lea & Thoday) x 1307 X rays {Giles} © 2 y neutrons (Giles}) C chromatid exchange s f mI%Or X rays (Catcha ‘© & Thoday} x 130r X raya (Giles) g @ 26v neutrons (Giles) I Giles NE (1940) A full discussion 1s deferred to Chapter 11 228 STRUCTURAL CHANGES IN CHROMOSOMES either cytologically in the salivary chromosomes of theF, larvae, or genetically by breeding teats » Tho resulta are given in Table 60, and show on the whole no evidence for any effect of reduction of intensity or fractionation of dose In either method only viable changes, 16 symmetrical exchanges involving two or more breaks, aro investigated Tasty 60 Ind a of i ust ortthe yield of chromosome structural changes hie in Drosophila sperm Dose Intensity HII interchanges Radiation r timin per 100 sperm Reference \ reys 2 230 29403 I X rays 2 100 38405 1 y rays 2000 08 34409 1 y rays 2 005 42410 1 X rays 5000 187 172415 2 A» rays 5000 25 17-0313 2 Dose B per Rediation r How fractionated 100 sperm = Reference X mys 3000 Single dose 40.24 3 X raya 3000 3 fractions of 1000r 6217 3 at I day intervals X rays 4000 Single dose 83 76 3 X rays 4000 4 fractions of 1000r 8182 3 at 1 day intervals X rays 4000 2 fractions of 2000r 83 57 3 at 16 days interval 1 Muller, Hw (194 3 Kaufmann BP Dempster (19418) ER (19418) The independence of yield on sntenstty or fractionation found in these expenments contrasts with the results obtained when X ray mduced two break exchanges are studied in Tr Two alternative explanations for this difference suggest them- selves One 1s that in Drosophila the two breaks taking part m an mterchange are usually even with X-rays, produced by the same lonizing particle Tho other 1s that no union of breakage ends takes place m the sperm, and that the breaks accumulate until opportunity for umon occurs after fertuhzation The firat explanation would require the yreld of aberrations to be directly proportional to the dose, and 1s ruled out by the expermmental evidence (given below) that the yield mcreases more rapidly than 1 Kaufmann, BP (19416) 2 Expersments by Muller, HF (1940} 1 collaboration with Ray Cc and Ty Also Demp ER (19418) DEPENDENCE ON DOSE 229 the first power of the dose The second explanation: 1s the ac- cepted one It ss supported by the fact that cases have been Teported of structural changes which apparently involve ex- change between a paternal chromosome broken by irradiation of the sperm, and a maternal chromosome spontaneously broken, suggesting that yong of chromosomes brohen by irradiation of the sperm 1s sometimes at any rate deferred until the sperm and egg chromosomes come into contact during the first cleavage of the fertilized egg = Dependence of yield on dose The manner im which the yield of structural changes increases with increase of the dose of radiation has been extensively atudied, and the results of these studies form the maim basis on which theories of the mechanism of induction of these changes are built The curves in Figs 34 and 35 1ilustrate some of the principal results obtamed 3 In some matenals xt 1s possible to observe sample breaks ‘These may be ether chromosome breaks (affecting an unsplit chromosome) or chromatid breaks (affecting only one of the sister chromatids of a split chromosome) or isochromatid breaks (affecting both sister chromatids of a spht chromosome at approximately the same locus) The yreld of each of these types appears to increase linearly with increase of dose, as illustrated for X-rays and for neutrons in Fig 34 4 This result 13 consistent 1 Maller H.J (1940) 2 Sidky AR (1940) Helfer RG (1910) Experiments designed to secure change b a and p iV ch by irradiation of both egg and sperm prior to fertihzation have given negative results (Glass, HB 1940) Probably union of maternal broken chromosomes 1s not delayed until fertilization 3 Taken from the followmg papers Bauer H Demerec, M & Kaufmann B P (1938), modified by addition of later work by Kaufmann BP (19416) Beaver HW (1939 quoted by Kaufmann, BP 19416) Sax, HK (1940 19418), Rick CM (4940), Giles NH (1940 1943), Carlson, JG (194la} Thoday, JM (1942), Neweombe HB (19426) Mannelh, LD Nebel BR Giles NH & Charles DR (1942) Catcheade DG lea DE & Theday JM (19464), Kotval JP (unpublished) 4 In Fig 34m (soc: d breaka produced hy X rays) the results of Sax and of other workers do not agree exactly but each separately gives astraight line The estimates of dose given by Sax are probably too hugh (Giles NH 1943) The imeanty 19 not convincin g in Figs 34,5 (chromatid breaks produce d by X raysand neutrons) Chromatid breaks 228 STRUCTURAL CHANGES IN CHROMOSOMES cither cytologically in thesali ary chromosomesof the F, larvae, or genetically by breeding tests » The results are given in Table 60, and show on the whole no evidence for any effect of reduction of intenmty or fractionation of dose In either method only viable changes, 16 symmetneal exchanges involving two oF more breaks, aro investigated Tastx 60 Ind di t of the yreld of chromosomestructural nena noes ik Drosophila wperm Doe Intensity 1-11] interchanges Radiation r rimoa per 100 eperm Reference \ rays 2000 250 29403 1 Xv rays 2000 100 38+05 1 y reys 2000 oa 34400 r yrmys 2000 005 42410 r X rays 5000 167 172415 2 rays 5000 128 170413 2 Dose Breaks per Radiation r How fractionated 100 sperm Reference X rays 3000 Single dose 49 24 3 X rays 3000 3 fractions of 1000r 5217 3 at 1 day intervals X rays 4000 Single dose 83 76 3 X rays 4000 4 fractions of 1000r 8182 3 at I day intervals X rays 4000 2 fractions of 2000r 83 57 3 at 16 days interval x Muller HJ (1940) Dempster, ER (19418) 4 Kaufmenn BP (19418) The independence of y:eld on intensity or fractionation found in these experuments contrasts with the results obtamed when X ray mduced two break exchanges are studied in 77 Two alternative explanations for this difference suggest them selves One 1s that m Drosophila the two breaks taking part m an interchange are usually even with X-rays, produced by the same ionizing particle The other is that no union of breakage ends takes place in the sperm, and that the breaks accumulate until opportunity for union occurs after fertihzation The first explanation would require the yield of aberrations to be directly proportional to the dose, and 1s ruled out by the experimental evidence (given below) that the yield mcreases more rapidly than 1 Kaufmann, BP (19416) 2 Experiments by Muller HJ (1940) in collaboration with Ray C and Alo Demp: ER (19415) DEPENDENCE ON DOSE 231 15 x 5 15 a a Jor b 10F d - + ce (3 Os O5b i” 0 rn 1 iy 1 1 200 400 00 200 400 00 10) D o5F 0 , f 100 200 300 2000 4000 6000 2000 4000 000 X rays Neutrons Fia 35 Yield of two break aberrations as a function of dose (Abscissae are doses in roentgens (4 rays) or » units d per cell (Tradescantia) or breaks per eperm (Drosophila) A chromosome ex changes in Trade Pp timeof3mm _ constant intensity of 160r permin x constant intensitv of 203Yr per mn O constant intensity of 27 r per mn a microspores x Thoday (Li+deuterons of leV energy) + ‘Giles {Be+deu terons of lleMf¥ energy) © Giles (Be+deuterons of 3eMV energy) minute end mature pollen Marelli Nebel Giles & Charley G Newcombe vx Rick + Giles (Be+deuterons of LleMV energy) E F exchange breaks in Drosophila en Bauer Demerec & Kaufmann © Bauer, x Demerec Kaufmann & ur 230 STRUCTURAL OHANGES IN CHROMOSOMES 13 9 % 10 z ost = t —___— “ BE) 10 150 10 G x osL- % 70 0Ot«‘ 3004000 XX rays Neutrons Fia 34 Number of breaks per cell as a function of dose (X ray doses mn Toontgens neutron doses in yume) A chromatid and ssochromatid breaks d breaks in T micro spores mph DE breaks in T Cc ¥F,G chromo some breaks an Tradescantia microspores or mature pollen Sax A Carlson x Thoday []) N + Giles 0 C: & Thodey V7 Kotval Lea are difficult to see and the departure from hneanty may he subjective error (ep discussion by Lee DE & Catchesrde DG 1942) Newcombe H B (19425) found that the number of chromosome breaha produced by increased bly more rapidly than thefirst power of the dose. This result, obtamed with doses of 240— 960r he explams by supposing that the proportion of breaks primarily produced which restitute 1s reduced at high doses DEPENDENCE ON DOSE 233 The yield of chromosome and chromatid exchanges induced in Tradescantia by neutrons 1s found to merease in direct propor- tion to the dose, as illustrated (for chromosome exchanges) in Fig 353: This 1s believed to be due to both the breaks taking part in the exchange beng produced by the same ionizing particle am neutron experiments,2 a hypothesis which also explains the fact (mentioned on p 226) that the yield of exchanges 13, with neutrons, found to be independent of the tensity With @ rays also, the yield of chromatid exchanges has been found to be directly proportional to dose s Interstitial deletions form a special class of exchanges which have been separately studied mn Tradescantia As shown in Fig 35 c and p, the yield of interstitial deletions 13 proportional to dose 1n neutron experiments, but in X& ray expemnments increases with dose according to a power of the dose intermediate between the first and the second Extensive experiments have been made to study the yield of structural changes induced in Drosophila by wradhation of the sperm The exchanges are detected erther by examination of the salivary chromosomes of F, larvae, or by breeding tests In exther event study 1s limited to viable ty pes involving structural changes which are all exchanges involving two or more breaks The results by the cytological method are gnven n Fig 355 and F, and by breeding methods in Table 61 It 1s seen that m all expermments on the production of gross structural changes by X wradiation of Drosophila sperm, the yield increases In proportion to a power of the dose intermediate be- tween the first power and the square, and 1s generally stated to be proportional to the 3/2 power of the dose Mullers beheves the power to approach the square more nearly at the lower doses, but the evidence 1s not at present convincing on this pont When minute structural changes are studied, 1€ changes in- volving two breaks separated by a distance of the order of 1% of the length of the chromosome or less, the yield 1s found to be Proportional to the first power of the dose, as illustrated in 1 The difference in gradients between curves d and e in Fig 353 is believed by Giles NH (1943) to be real, and due to the fact that the neutrons used in d were somewhat less energetic than those used tn ¢ 2 Giles NH (1940) 3 Kotval JP & Gray, LH (1947) 4 Muller HJ (1940) 239 232 STRUCTURAL CHANGES IN CHROMOSOMES with a break being produced by @ single ionizing particle The yield of breaks in Tradescantia han also been found to be hnear with @ particles « More complicated types of structural change involve exchange bets con two or more breaks, and the number of these aberrations produced by X rays increases more rapidly than the first power of tho dose Accepting that the yield of breaks primaniy pro- duced by X rays 1s directly proportional to the dose (on the basis of Fig 34) wo should expect that structural changes involving two or moro independently produced breaks should increase more rapidly than the first power of the dose, at any rate when tho doso 1s such that the mean number of breaks per cell 1s Jess than one This expectation 1s borne out by expenment both on Drosophila sperm (Fig 358) and on Tradescantia microspores (Fig 35a) The first expenments with Tradescantia were made by varying the duration of exposure at constant intensity, and showed that the yield of chr hang das the 3/2 power of the dose s It will be remembered however that with Tradescantia the yield of aberrations produced by a given dose diminishes as the duration of irradiation 13 extended In con sequence the shape of a dose curve obtained by varying the duration of exposure at constant mtensity depends upon the intensity employed This 1s illustrated in Fig 35a, 1n which the curves a, 6 and ¢ apply to vely d f t In curve a, obtained at the highest intensity, the yield 1s practi cally proportional to the square of the dose A square law curve 1s also obtained if the dose is varied by varying the mtenaity at constant exposure time It 1s concluded therefore. that funda- mentally the yield of exchanges produced by X-rays in Trade- acantia mereases aa the square of the dose but that the results are distorted by restitution of breaks occurrmg unless it 18 arranged either that the irradiation 1s completed in a short time (making restitution neghgible during the exposure) or that the irradiation extends over the same time at all doses (s0 equalizing the effects of restitution at the different doses) 1 Kotval, JP & Gray, LH (1947) 2 Fig 35a refers to h unspht The yield of ch 1 h d by d: apht chromo somes also mecreases more rapidly than the first power of the dose (Sax K 1940 Thoday, JM 1942) 3 Sax, K (1938) 4 Sax, K (1940, 19418) DEPENDENCE ON DOSE 233 The yneld of chromosome and chromatid exchanges induced m Tradescantia by neutrons 18 found to increase mn direct propor- tion to the dose, as illustrated (for chromosome exchanges) in Rig 355: This 1s believed to be due to both the breaks taking part in the exchange being produced by the same 1onizing particle in neutron expermments,: a hypothests which also explains the fact (mentioned on p 226) that the yeld of exchanges 1s, with neutrons, found to be independent of the tensity With a rays also, the yield of chromatid exchanges has been found to be directly proportional to dose Interstitial deletions form a special class of exchanges which have been separately studied in Tradescantia As shown in Fig 35 c and p, the yneld of interstatial deletions 13 proportional to dose in neutron experiments, but in X. ray expermments increases with dose according to a power of the dose mtermediate between the first and the second Extensive experments have been made to study the yneld of structural changes induced in Drosophila by irradiation of the sperm The exchanges are detected either by exammation of the salivary chromosomes of F, larvae, or by breeding tests In either event study 1s limited to viable types involving structural changes which are all exchanges involving two or more breaks The results by the cytological method are given in Fig 355 and F, and by breeding methods in Table 61 Tt 1s seen that in ali expermments on the production of gross structural changes by X irradiation of Drosophila sperm, the yield increases in proportion to a power of the dose mtermediate be- tween the first power and the square, and 1s generally stated to be proportional to the 3/2 power of the dose Mullers beheves the power to approach the square more nearly at the lower doses, but the evidence 1s not at present convincing on this point When minute structural changes are studied, 1 e changes in volving two breaks separated by a distance of the order of 1y, of the length of the chromosome or less, the yield 1s found to be Proportional to the first power of the dose, as illustrated In 1 The difference in gradients between curves d and e n Fig 353 as bohteved by Giles NH. (1943) to bo real, and due to the fact that the Neutrons used in d were somewhat less energetic than those used in e 2 Giles NH (1940) 3 Kotwal JP & Gray, LH (1947) 4 Multer, HJ (1940) 234 STRUOTURAL CHANGFS IN CHROMOSOMES Table 61 Tins suggests that a single ronizing particle usually causes both breaks tn such rearrangements Tho evidence avaiable nt present on the yanation with dose of the yield of gross structural changes induced by neutrons cannot be considered adequate to determme the shape of the curse Itis givenin Fig 363 Taste G1 X ray induced structural changes in Drosophila chromosomes investigated by genetical methods Doso Aberrations Power Chango studied t per sperm of dose Reference Gross structural changes Chromosome exchanges in 1500 0 0058 +0002 188 v volving 1 chromosome 3000 «=—0 0214 40005 168 6000 «00735 +001 Chromosome interchanges bo 380) §=60. 0021 +0 0008 230 2 tween chromosomes IT and 1500 00252 £6003 pore 1600) OG OLLG +0001 142 3 4000 «00830 +0 008 * Chromosome ex-hangea in 1000 0000434000008 | 44 4 volving chromosome I) 7 4006 0.00318 4 0 0004 1000 00007 +0 0002 103 5 200 O00LS +0 0003 13. 4000 §=0.:0036 +0 0000 10. 6000 §=-:0054 +0 0010 Chromosome exchanyes 1900-0017) +0003 1 5 2000 001L $0008 = 137 4000 0105 $0008) = 359 6000 69200 +0016 Minute structural changes Minute rearrangements in \ 1000-0. 000434000007 yyy 6 chromosome? 4000 0 0016240 00022 1000 0 000874000014 9g 4 4000 0 003170 0004 1 Tumoféeff Ressov sky NW (1939} 2 Muller HJ (1940) The yield quoted at 380r 1s compounded of some X ray data at 375r and some + ray data at 400r 3 Muller, H J (1940) quoting experiments of Muller and Sidhy 4 Muller HJ (1940) quoting experiments of Muller and Makin 5 Khvostova V\ & Gavnlova AA (1933) 6 Belgoveky, ML (1939) Exchanges detected are those which transfer the [Vth chromosome gene wus interruptus to a region ofany Such have the effect of weakening the dommance of the gene and permit a fly heterozygous for the wild type and the recessive to show the phenotype of the latter 8 The ¥ chromosome used 19 one in which a large mversion (sc*} has brought the gene y* close to the h The app rate of the 4+ gene ya much greiter than in an ordinary 1 chromosome and 1s due mainly to minute hi J of the gene (possibly deletions) rathor than to gene mutation proper DEPENDENCE ON DOSE 235 Experiments are sometimes made in which a materml 1s wradiated, fixed some hours or days later, and anaphase figures examined and classified as normal or abnormal This procedure gives less insight into the mode of action of radiation than does the more labonous process of clasafying the chromosome struc tural changes into the various ty pes It may, however, be the only procedure practicable when dealing with cells with a large number of small chromosomes, and even with more favourable matenals may be adopted when 1t 1s desired to obtain rapidly a statistically significant amount of data As a guide to the mter Pretation of such experiments we have plotted m this manner the results of experiments: on the irradiation of Tradescantia microspores, irradiated either when the chromosomes were split (Fig 364,B) or when they were unspht (Fig 36c,p} Plotted on & loganthmie seale, the pomts representing the percentages of normal division figures le quite close to straight lines With neutrons the yields of all types of structur wl changes induced in Tradescaniia chromosomes were found to be pro- Portional to the dose, so that we should expect the percentage of normal division figures to be lmear agaist doe when plotted on aloganthmic scale 2 In the case of the X. Tay experiments where the yields of some of the structural changes aie proportional to dose, while the yields of others are more nearly proportional to the square of dose, we should expect the curve of the percentage of normal division figures against dose to be convex upwards when plotted ona logarithmic scale The square Jaw aberrations, however, bemg in a minority, the convexity 13 hardly noticeable 3 In Fig 36 5 and ¥ we show the percentage of normal anaphase figures in a mouse lymphoma fixed 12 hr after radiation « 1 Thoday, JM (1942) 2 Up Chapter 111 pp 72-75 3 InFig 36 Aand C approximate more closely, perhaps, to atraight lines than is typical In the case of Fig 36a (irradiation 24 hr before meta Phase) there 1s belev ed to be some obsers ational error leading to under- sstimation of the mumber of chromatid breaks at high doses (cp footnote 4,p 229) As regards the experiment in which micro~pores were irradiated 5 days before metaphas e (Fig 36c), American authors (bax Riel Giles) relativel find a y higher Proportion of two break types of aberration than does Thoday, and curves of normal division figures der ed from their maternal when plotted as im Fig 36 would probably depart straight line more markedly from a 4 Marshak A {194920) 234 STRUCTURAL CHANGFS IN CHROMOSOMES Table 61 ‘Thin suggcats that a single 1omzing particle usually causes both breaks in such rearrangements Tho ovidence available at present on the yaration with dose of the yield of gross structural changes induced by neutrons eannat be canaidered adequate to determine the shape of the curse It 1s givenin Fig 35¢ Tantr Of X my induced atructural changes in Drosophila chromosomes investigated! hy genetical methods Dow Aberrations Power Chango studied r per sperm of dose Reference Gross structural changes Chromosome exchanges in 1500 6.0038 +6002 188 r volving \ chromosome 3000 «00214 +0005 168 6000 «00-0735 +001 Chromosome interchanges be 380 «60-0021 +0 0008 1R0 2 tween chromosomes IL and 15000-0252 0.003 1600 BOIS £0001 142 3 4000 0.0830 +0 008 * Chromosome ex hanges on Jou = 0.00043 + 0 00008 144 4 volving chromosome I\ 7 4000 = 0 00318 = 0 0004 1000-00007 +0 0002 103 5 2000 OO WUI4 + 00003 132 4000 «0 0036 +0 0006 104 6000-00054 +0 0010 Chromosome exchanges 1000, G017 +0003 124 5 2000 OOH 40005 = 337 4000-0105 +0008) 15g 6000 «0200 «+0016 Minute structural changes Minute rearrangements in 1 1000 = -0. 0043+. 0 00007 096 6 chromosome® 4000 «0 00162 + 0 00022 1000 «=. 00087 + 0 00014 093 4 4000 «6.00317 + 0 0004 1 Tumoféeff Ressovaky NW (1939) 2 Muller, HJ (1940) The yneld quoted at 380r 13 compounded of some X ray data at 375r_and some y ray data at 4608 3 Muller HJ (1940) quotarig experiments of Muller and Sidky 4 Muller HJ (1940) quoting experiments of Muller and Makki 5 Khvostova V\ & Gavnlova AA (1938) 6 Belgovsky, ML (1939) 7 Exchanges detected are those v hich transfer the IVth chromosome gene cubstus interruptus to region of any ch ch have the effect of weakening the dommance of the gene and permit a fly heterozygous for the wild type and the recessive to show the phenotype of the latter 8 The X chromosome used 19 one in which a large mversion (sc*) has brought the gene yt close to the The apy rate of the y? gene 18 much greater than m an ordinary 1 chromosome and is due manly O mnute rearranzements mn the neighbourhood ofthe gene (possibly deletions) rather than to gene mutation proper NEUTRONS AND X-RAYS 237 some, or an isochromatid break in a split chromosome which are first-power-law processes The curve shown in Fig 368 suggests that the single break process is predommant in these exper ments, as in the Tradescantia experrments shown in Fig 36 4 and ¢ Marshak has also made a number of experiments on rapidly dividing tissues m which the percentage of abnormal anaphases was determined in material fixed three hours after wrradsation, at atime when the temporary reduction of mitotic activity caused by the radiation was most marked The results of experiments of this type: are shown in Fig 36 and H If, as suggested earlier, abnormalities observed at anaphase three hours after irradiation are a mixture of physiological changes and structural changes in the chromosomes, 1t appears from these results that the propor- tion of cells subject to the former type of effect as well as the latter mcreases approximately in near proportion to the dose The relative efficiencies of different wave-lengths and types of ionizing radiation Study of the relative efficiency of radiations of different, 10n- density in producing structural changes in chromosomes 1s an important method of attack on the mechanism of this process The efficiencies of X-rays and neutrons have been compared with anumber of materials Providing that X-ray doses are measured In roentgens, and neutron doses m v untts,: units which represent, approximately equal energy dissipations in tissue, the ratio of the yields for equal doses of the two radiations may be taken to be the ratio of the efficiencies per :onization of the densely ionizing particles (protons) 1n the neutron experiments, to the less densely 1onizmg particles (electrons) in the X ray experiments Some experiments have been made to compare the efficiencies of X rays and neutrons m producing structural changes by uradiation of Drosophila sperm The results are not at present conclusive the exper ts shown in Fig 35 & and F3 suggesting 1 Marshak A & Malloch, WS (1942) 2 In reporting American experiments in which the neutron doses are given m n units we have throughout converted to t units on the basis that ln unit=2 5 » units (cp p 20) a 3, ~ my experiments by Bauer, H , Demerec, Mo & Kaufmann BP. a » jauer H (1939), Kaufmunn, b P (1941) neutron experiments y Demeres M, Kaufmann, BP & Sutton, E (1942) 236 STRUCTURAL CHANGES IN CHROMOSOMES Division figures wore classified as abnormal when they showed logging (acentric) fragments, or bridges (dicentne chromatids) too, “Tt T T T a, A R ” Po ior if T 8.385 T ass a Lo 8 10}- aa TT Sele 100 200 100 200 X rays Neutrons Tia 36 ‘The percentage of normal division figures as a function of dose Abscissae are in roentgens (\ rays) or » unita (neutrons) A, B Pradescantia division 2thr before (Thoday) C D Trade scantia merospore division irradiation 5 days before metaphase (Thoday} Kk F mouse ly mutoais a 12 hr before hase (Marshak) G H bean root tip mitoss irradiation 3 hr before anaphase (Marshak & Malloch) Such fragments and bridges can be produced either by asym metrical interchange between two chromosomes, s process e\ pected to be approximately square law with X-rays on the basis of Tradescaniia experiments, or by a break m an unsplit chromo DIFFERENT WAVE-LENGTHS OF \-RAYS 239 of physio1ogical and structural changes in the chromosomes The lower value of the ratio of efficiencies of neutrons and X-rays then obtained suggests that there 1s less difference in efficiency between the two radiations in producing the phystological effects than m producing structural changes In experrments on Tradescantia microspores, the yields of chromatid and isochromatid breaks have been found to be greater with a rays than with neutrons, wlule the yield of chro- matid exchanges has been found to be less with e-rays than with Taste 63 Relative efficrencies of different wave lengths in mducing breaks Wave length Number of breaka per Aberration A 100 cells per r A Pradescantia pollen tube mitosis* Chromatid breaks ~O15 058 +004 15 O62 +012 41 110 +011 83 0065 +0015 Isochromatid breaks ~0 15 oo99 +0018 16 0092 +0 016 41 0158 +0040 83 0 003440 0034 B Tradescantia pollen grain rmtosis? Chromatid breaks ~015 050+0 015 ~0 015 0 47+0013 Isochromatid breaks ~015 02540011 ~0 015 02340009 I Catcheside DG & Lea DE (1943) It 1s doubtful if any wsochromatid breaks are produced by X rays of 83A The yield given 1s based on the one wochromatid break observed in the chromosomes iradtated by this wave length 1¢ was probably a sp b not caused by the radiation 2 Kotval JP (unpublished) neutrons: The explanation of these results 1s discussed in Chapter va An important difference between @ rays and other radiations 1s the fact (pomted out on p 210) that breakage ends produced in Tradescanina chromatids by & rays appear to be un- jomnable in a much higher proportion of cases than breakage ends produced by neutrons and X rays The efficiencies of X-rays and y rays im producmg structural changes by irradiation of Drosophila sperm have been compared by Muller and Ray Chaudhun : The efficiencies per roentgen of 1 Kotval,JP & Gray LH (1947) The ex ‘periments were made b: immersing the inflorescences in a solution of radon ony 2 Reported by Muller HJ (1940) 238 STRUCTURAL CHANGES IN CHROMOSOMES that neutrons are slightly less effective, while other experiments: suggest that they are slightly more effective With all other matenals which have been investigated, and for all types of chromosome aberrations, neutrons have been found to produce a greater yield of aberrations for the same dose than A-raya, as may be seen by comparing curves for the tno Tantr 62 Relative efticiencies of neutrons and \ ravs (A figure greater than 10 means that # greater yield of aberrations 1s pro- duced by 1 v umt of neutrons than by Ir of X raya) Hours between Material Type of irradiation Relative Re atudied aberration and fixation —efficieney ference Tradescantia Chromatid breaks a4 22 1 microspores Tsochromatid breaks 24 3é 1 4 2 Chromosome breake 120 43 t Bean root tips Abnormal anaphases 12 6o 3 Sfouse ly Ab t anaph 12 35 4 Abnormal anaphases 3 23 4 Seeding reot tips = Abnormal anaphases 3 Bean 26 3 Pea 26 $ Tomato 26 $ 1 Thoday J Bf (1042) as worked up by Los DE & Catcheside DG (1942) 2 Gilles NH (1943) 3 Marshak A (1912a) 4 Marshak A (19428) 5 Marshak, A (19395) radiations in Figs 34, 35 and 36 It 1s only possible to state o preciso figure for the ratio of efficiencies when the dosage curves are of the same shape for both radiations, otherwise the ratio varies with dose In Table 62 we give the ratios of efficiencies 1n cases where the yield of abcrratrons 1s a Imear function of dose with both radiations In addition to ratios of efficrencies for aberrations of known types, for which data are only available for Tradescantia, ratios obtamed by Marshak by recording abnormal anaphases in rapidly dividing tissues have also been mcluded In the cases where fixation was made 12 hr efter irradiation the anaphase abnor- malities are pr bly structural changes, probably mainly chromosonie or 1sochromatid breaks, and the ratios obtamed are comparable with the ratios for these types of aberrations ob- tamed. with Tradescantia’ Where fixation was made 3 hr after irradiation, the anaphase abnormalities are probably a mixture 1 Dempster ER (19412a) COEFFICIENTS OF ABERRATION PRODUCTION 241 given are the values of m obtamed by fitting the equation y= mx to the expenmental results by the least squares method y1s the number of aberrations per cell, and x the dose mm roentgens or Tasit 64 Coetiicents of pruduction of aberrttions im Tradeseania muctospores Chromatid — Isuchromatid = Chronatid exchanges per cell. breaks per reaks per —_oOlOO TF cell per r cell per r perr perr Splt chromosomes 107? x lu? « lw? x 10-*x — Reference \ rays (015A) 0-725 40-08 = 1R1+0-21 1 (15a) 0G2 40-12 = = 2 (la) 110 +011 _ _ 2 (893A) 0-005 40-015 _ _ 2 Neutrous6 (La+D) 158 +008 099 4003 090740408 ad 1 @ rays(Rn+RaA+Hal) 196 4009 210 3000 §86059 2006 _ 3 Minute 1 exch: Chromosome deletions per cell per cell Unspht chromezomes 113) . 10xrr “Ailpe ve] rr he x 1 Orrtx it r (0-8 x per te x rr? Reference \ rays (O15 A} 006 4.001 - _ _- 0520-08 1 O1-1A =~ - 040+ - = ‘ Neutrons6 (Lag D) 026 20-02 _ — 0824002 - 1 Be +D) = O9200 — — ~ 3 1 Thoday,J M_ (1942) as worked up by Lea, DE & Catchende, DG (1942) 2 Catcheside, DG & Le: a, DE (1943) he expenmenta with solt X rays were made with polley tubes and bave been converted to yields in mucrospores for the purpose of Table 64 The conversion is made posable by the fact that yields have been determined in both pollen tubes and nucrospores 5A )anda rays Fora given type of aberration, the ratio of the pears to be not very different for the two radiations, The m tubes and microspores are equal within the error of the expen ment The yield of ssochromatid breaks 1s defimtely less 40 pollen tubes the ratio of yields being 0-38. {for X rays) Thus is beleved to be due to the sister chromatids being farther apart wn the pollen tubes 3-3} br after sowing than in the microspores 21 br before metaphase The yield of chromatid inter « aiso is less inpollen tubes than in microspores the ratio of yields (for X rays) berng 0-267 Thus is beheved to be due to the ch being less f ‘disposed for interch, ayn the tube shaped pollen tube than in the spherical microspore The yield of sochromatid breaks with 83A X rays 18 described 28 0 014, £<0905 Newcombe,3 employing higher doses, found that the yield of 2 hit exchanges increased with a power of the dose intermediate between the first and the second At these gh doses f 1s no longer constant, as 1s shown by the fact that the yield of simple breaks mcreases more rapidly than the first power of the dose Apparently with high doses of X-rays the proportion of breakage ends which are Jomnable dimimishes Thus while the total number 1 Sax, K (1940, Table 2, and 19416 Table 5) Only ssymmetrical exchanges were scored (1¢ rings and dicentrics) The total number of exchanges 1g obtamed by doubling the ber of asy 1 ex changes 2 The method adopted was to assume a value for £ to fit formula (3) by the lesst equarea method and to test the goodness of fit by the x method By tnal the value of £ was found which gave a yalue of x? corresponding to P=005 Thus was teken to be the lowest value of £ consistent with Sax s data 3 Newcombe, HB (19426, Table 6) The time of srradiation was constant and equal to 12 min We take as the number of asymmetrical exchanges centre mngs +d t: Y +2x +3xt and double to allow for the unobserved symmetrical exchanges Acentric mnga are ormtted to make tho results comparable with Sax a, and to avoid the meluson of any aberrations which from our monute interetitial deletions re pomt of weware 266 STRUCTURAL ONANGES IN CHROMOSOMES number of chromatid breaks pnmanly produced per roentgen, which we denote by € The values obtained are £=0 086 for X rays, £0 200 for noutrons, and £=0 083 for a rays bome of the methods we are going to discuss lead directly to estimates of £ Evidently, by reversing the procedure just de senbed, wo can, by making uye of the coefficients cited in Table 64, derive estimates of f from these estimates of€ Method JI A socond method of estimating £ and f comes from the conarderation that it 18 only the fact that the mayonty of the primanly induced breaks restitute which permits the yield of 2 hit exchanges to increase in proportion to the square of the dose Clearly, if all the jomable breaks took part mn exchange, the number of exchanges would be proportional to dose We should expect therefore that st high doses the yield of ex changes will increase less rapidly than the square of the dose The following approximate caleulation will serve both as a demonstration of the (dose)? law at low doses, and as an indica tion of the manner in which the yield of exchanges may be expected to depart from this law at high doses £D(i—f) being the number of jomable primary breaks pro- duced 1n the nucleus of radius # by a dose D, the mean number (additional to the given break) in a sphere of radius A centre at a given break: will be £D(1 —f) 4°/R°, the actual number beng distributed m a Poisson distribution about this mean The probabihty of at least one break occurring in this aphere is there fore [-e~2a-, which may be expanded mm ascending powers as ED(1—f)hYRQ—-JEDQ—f)h/R+ } This ex pression is the probabilty that a given one of the ED(i~J) jomable breaks m the nucleus shall exchange, and we deduce that the ber of (complete) exch per nitcleus, which 3s one half of the number of breakage ends taking part 1m ev- changes, 18 $ (ED)? (1~f)? AYR? (1~4ED(L—f) B/RP+ —} For small doses all the terms 1n the bracket except the umity can be neglected, and we obtain for the yield of exchanges &D? per cell, where 1 = }£?(1—f)* 3/8 At higher doses the second term 1 Assuming that the breahs are uniformly distmbuted in the nucleus In view of this assuroption the exchanges covered by this formula do not welude the minute 1 del the ligh fr of which depends on the higher than random probability of finding a second break in the aame chromosome PROPORTION OF BREAKS WHICH RESTITUTE 257 im the bracket 15 not neghgible, and we obtain for the yield of exchanges LD? {1 ~wan| or approwmately xp ( _ Number of exchanges Total number of jomnable breaks primanly produced, (VII-3) Sax: has desenbed expersments, using large numbers of cells, im which the yield of exchanges was determmed as a function of dove at a time when the chromosomes were unspht, the time of wradiation beng kept constant The yield increased very shghtly less rapidly than the square of the dose the difference not being statistically sgmificint These experiments consequently do not enable € to be determined, they can, however, be used to fix a fower lumits for £, and hence an upper himat for f The results obtamed (for X rays on unsplit chromosomes) are that § > 0 014, $<005 Nevcombe,s employing higher doses, found that the yreid of 2 hit exchanges increased with a power of the dose intermediate between the first and the second At these fugh doses f 1s no longer constant, as 1s shown by the fact that the yield of sumple breaks increases more rapidly than the first power of the dose Apparently with high doses of X-rays the proportion of breakage ends which are jonable dimmmishes Thus while the total number 1 Sax, K (1940, Table 2, and 19418, Table 5) Only asymmetrical exchanges were scored (1e rings and dicentries) The total number of bs as obtamed by doubling the ber of asy Lex changes 2 The method adopted was to assume a value for £, to fit formula (3) by the least squares method and to test the goodness of fit by the x? method By tnal the value of £ was found which gave 4 value of x4 corresponding to P=005 This waa taken to be the lowest value of 3 constent with Sax’s deta 3 Newcombs HB (18428, Table 5) The tume of irradiation was constant and equal to 12 min We take as the number of asyrametrical exchangea centric rings + dicentric chromosomes + 2 x tricentric +3 tetracentnc, and double to allow for the unobserved symmetric al exchanges Acentric arene grastted to make the results comparable with Saxs and te 6 inclusion of any aberrations which rainute interstitial deletions from our point of Pp wewere 258 STRUCTURAL ORANGES IN CHROMOSOMES of primary breahs 18 proportional to the dose, the number of jomable breaks primarily produced increases less rapidly than the first power of the dose This effect, as woll os the fact that at high doses the number of breaks taking part in exchanges 33 no longer a negligible fraction of tf total number of primary breaks, can bo taken into account by a shght modnfication of the theory we have already given Defining 1’ = 4£4/3/R3, we obtain the following approximate expression for the yield of (complete) exchanges produced by a dose D vor{1-2 hanges +2x chr breaks at dose DI Number of primary breaks at dose D (VIT-4) This formula was found to fit fairly well the manner of vania- tion with dose of the yield of exchanges found by Newcombe at 240, 480 and 960 r when tho value £=0 010 was employed The value of f at low doses (where f 1s constant) deduced by com- bining this value of £ with the coeffinents of aberration produc- tion hated in Table 64 (p 241) 13 0 078 Two methods of estimating £ and f somewhat similar to the method just described may be mentioned, though 1t has not proved possible to apply them im practice They depend on the fact that the chromatid or chromosome breaks which are ob served constitute a certasn fraction of the primary breaks re- maiming after some have tahen part in exchanges Since the yield of X ray induced exchanges increases more rapidly than the first power of the dose when the dose 1s varied, we mught expect the number of chromatid or chromosome breaks observed to merease rather less rapidly than the first power of the dose The larger the number of breaks prrmanly produced relative to the number which take part im exchanges, the smaller will be the departure of the yield of breaks from 2 linear function of the dose, and analysis of the dose curve should thus enable the number of breaks primarily produced per roentgen to be de- termined The experimental dose curves at present available (Fig 34D,¥, p 230) are not sufficiently accurate to permut of this method bemg apphed In a sumilar manner, since the yield of X-ray mduced ex changes decreases when the duration of exposure is increased at constant dose, we should expect the yield of chromatid and PROPORTION OF BREAKS WHICH RESTITUTE 259 chromosome breaks to merease slightly when tne duration of exposure 1s mereased at constant dose The experimental points shown in Fig 33, curve d (p 227), do not indicate any increase of the yield of chromatid breaks with the duration of exposure at constant dose They do not, however, rule out an increase of up to 10% in the yield between the shortest and Jongest tume used in the experiments, and when the calculation 1s made the corresponding limit set for f turns out to be f< 0 75 This, though doubtless true, 13 not helpful The method 1s evidently in sensttive, and data of very high prectston would be needed to obtam a, useful result from it Method III On p 256 1t was shown that, apart from smaller terms which we need not now consider, the yreld of 2-ht ex- changes induced by X rays was AD*, where b= 462(1—f)?23/R3 Now R, the radius of the nucleus, 1s 6, and A, the distance within which exchange occurs, was found earlier in this chapter to be ly Thus from the expermmental values of the coefficient of exchange production per cell per roentgen squared fisted in Table 64, p 241, we can deduce Eandhencef A similar method can be apphed to the 1 Int exchanges induced by neutrons and «rays If dis the length of path of all the protons (or @ rays) which traverse the nucleus per roentgen, and (1 —f) the number of jomable breaks primarily produced per roentgen, then §(1—f)/d is the number of jomable breaks primarily produced per micron of path length Hence 26 (1—f)/d 1s the probabihty that a second jomable break shall he produced within a distance h of a given break by the samc 1omamg partide Thus £2(1~f)*AD/d 1s the mean number of (complete) exchanges per nucleus produced by dose D Again taking h=1y:, we are able to deduce &, and hence f, by compamng ths formulaz with the expersmental coefficients of exchange production listed in Table 64 (p 241) The values of £ and f obtained by the application of method III are Lsted in Table 67 1 Table 18 p 32 gives the path length per cubic micron Multiplica tion by the volume of the nucleus (905,43) gives d 2 When applying this formula, or the formula for 2 hit exchang es, to split chromosomes £& should be replaced by (E~2c) where c 15 the co efficient of production of isochrom atid breaks Per cell per roentgen the ground , on grou s that the breaks which constitut ons: hi e rsochromatid breaks ahs are as not 260 STRUCTURAL CHANGES IN CHROMOSOMES Afethod IV: Most of the interchanges observed in a nucleus uradiated at a time when the chromosomes are ephit are chro matid mterchanges, in which one chromatid of each of two chromosomes has broken and exchange has taken place between the two breaks A certain number of configurations are seen which can be diagnosed as interchanges between an 2sochro matid break and a chromatid break These c/s mterchanges have been found with X-rays to be 874 times less frequent than ordinary ¢/e mterchanges, which indicates that 17 48 pnmary chromatid breaks are produced by X-rays to every primary 180 chromatid break But the number of chromatid breaks scored in the fixed nuclei in these particular experiments was 3 843 times as great as the number of ssochromatid breaks Hence it 1s inferred that the proportion (f) of pnmary chromatid breaks which persist 15 only about 3 843/17 48=0 22 time as great as the proportion (z) of isochromatid breaks which persist Taking 2=0 6 (see footnote 1, p 254) we deduce f=0 11 Method V Estimates of £ and f can be derived from considera tion of the relative freqaences of chromatid and ssochromatid breake, 1f we are prepared to admit that the production of a pmmary chromatid break requires the passage of an 1onming particle through one of the chromatids, and the production of an wochromatid break requires the passage of the same 10mzIng particle through both of the chromatids Considering the euster chromatids as a pair of parallel cylmders of radius 1, separated by e distance (axis to axis) of 3, 1b can be shown that the prob ability that an romzing particle which passes through a specified chromatid shall pass also through the sister chromatid 18 a= ={p—z (1-cos 9}, where sin 6 = 2r/s, g taking the values given in Table 66, ranging from 0 363 for chromatids in contact to 0 053 for chromatids separated by a clear distance of 5 diameters We cannot be certain that an ionizing particle which passes through a chromatid mevitably breaks 1t, we denote by p the probability (<1) that 1b does so Denote by z the ratio of the number of ssochromatid breaks observed to the number of 10- 1 Catcheside, DG, Lea DE & Thoday, J.M (1946a) VaLUES OF £ ann f 261 chromatid breaks primamly produced (the remainder resta tuting) Then }zpg 1s the ratio of the number of isochromatid breaks observed to the number of chromatid breaks primanly produced (The factor } 1s required since an isochromatid break Tantz 66 The g trical factor for wsoch d breaks Clear separation of chro. yoatids in diameters 0 05 10 15 2 3 6 2r}le 10 087 05 04a 933) 025 0167 g 2 0363 0222 O163 O128 6106 GO78 0053 amphes two chromatids broken ) Taking z=0 5 aa before (cp footnote 1, p 254), and noting that p cannot exceed unity and g cannot exceed 0 363, 1t follows that this ratio <0 691 Now the number of sochromatid breaks per cell per y-unit obtamed mm neutron experiments 1s 0 99 x 10-3 (Table 64, p 241) Hence E>0 99x 107-0091, 18 E>011, whence we deduce f<0 22 and of the fraction of primary breaks which afo unjoweble ne ®h TABLE OT Estimates of the number ot primary breaks per cell per roentgent! X raya Neutrons o rays 3 f € f 4 f Spht chromosomes Method I 0088 O04 0206 0004 04089 O4T Method IT 0-003 0081 0213 0000 O14 O33 Method IV 0015 0110 _ ~ ~ ~ Method V _ — rAN <0-22 _ ~ Adopted values 00 0 O2 060 020 Os Unapht chromosomes Mathod IT Sax’a data >0-014 <0-050 _ ~ _ _ Neweombeadata 0010 0.078 = _ _ ~ Method HI 0066 0010 0150 OID _ - We do not apply this method to aray and X-ray-nduced wochromatid breaks owing to complications which we discuss later (p 277) The estamates of £ and f derived from the five methods we have discussed are collected mn Table 67 With unspht chromo- somes the estimates are too few in number and too davergent in numerical values to enable any precise quantitative conclusions to be drawn There appears to be no doubt, however, that the number of breaks pnmanly produced greatly exceeds the num- ber visible at the tume of fixation x Per vu unt in tho case of neutrons 262 STRUCTURAL OMANGFS IN CHROMOSOMES The agreement between the estimates of — obtained by the several methods when applied to split chromosomes 1s aufficrent to give us some confidence in the mean values of £ The principal results aro (2) Tho proportion of breaks which aro unjoiable (1¢ f)1s the samo for neutrons and X-rays, but considerably greater in the case of a ray induced breaks (2) The number of breaks primarily produced per unit dose Qe &) increasea in the order X-raya, & raya neutrons Thus the offictency per 1onzation increases With increase of 10n denmty 0 going from X-rays to neutrons, but decreases with further in crease of 10n density m pasmng from neutrons to « rays The significance of this result 1s discussed later The agreement between the results given by the diverse methods employed in estamating & and f 1s médarect confirmation of the general soundness of the basic postulates of the theory Method V only gives an upper hit for f and a lower lumt for £ owing to the fact that a priors one 1s only able to ascribe upper lumits to g (viz_ 0 363, Table 65) and to p (viz 10) The fact that the mut for £ given by method V 2s within a factor of 2 of the probable values indicates that the actual values of g and p do not depart by more than a factor of 2 from these upper hmits In other words, if sister chromatids are not touchmg 24 hr be fore metaphase they are not separated by more than about 1 diameter (which would make g=0 163), and if a proton passing through a chromatid does not inevitably produce a break, the probability of :ts domg so 1s not Jess than one half Dependence of the yield of aberrations in Tradescantia upon the duration of exposure In considering exchanges hitherto we have supposed that all the primary breaks coexist mm the nucleus This 1s so if the dose 1s administered over a short overall time If the dose 1s spread over a prolonged time, erther by dividing it mto fractions or by use of a low intensity, many of the breaks restitute before time has elapsed sufficient for other breaka with which they nught exchange to be produced in ther vicmity Consequently, the yield of exchanges produced by a given dose of X-rays dimumshes with increase of the duration of exposure If we make assump- tions about the tame for which a break remams free, we can work PEPENDENGE OF YIELD ON INTENSITY 263 out a theoretical formula: for the dependence of the yield of ex- changes on the time of exposure Conversely, by fittmg such a formula to the experimental data we are able to deduce the mean time for which a break remains free before restatution or ex- change occurs Since we have shown that the great majonty of the breaks prmanily produced in the nucleus by X-rays restitute, we can without great error make the simphfying assumption that the gradual diminution m the number of free breaks which occurs after the cessation of irradiation 1s solely due to restitution and neglect the fact that exchange accounts for a small part of this diminution Thus if x, breaks exist m the nucleus at time 1=0, at time t the number (in the absence of further srradiation) wall be nof(), where f(t) 1s a diminishing function of ¢ The probability of a given break recombining in any short interval of time wall be proportional to the number of other breaks within range (1e within distance h) The mean number within range 18 a certain fraction of the total number x of breaka existing in the nucleus at that time Thus the number of breaks combming per unit time 1s proportional to n?, = fn? say This result 13 analogous to the equation for a bimolecular reaction m chemistry, or the equation for the combmation of positive and negative ions im electricity The total number of exchanges formed may be wntten fpr dt=fne [Fwy dt, (VII 5) where the integration extends over the period for which breaks exist in the cell To proceed further requires a hnowledge of the function f(t) The simplest assumption would be that all the breaks remam open for a constant tame 7 after the moment of their formation, and then rejoin in the orginal formation if they have not already taken part in exchanges Such umformity of behaviour 1s, how- ever, not very plausible, and instead the calculation has been made assuming that njng=f(i)=e-, 1e dnjdt= —njr, m- plying an average time 7 elapsing between breakage and restitu- thon, with the actual times distribute d in a skew manner about 1 The formula given 19 that of Lea, DE & Catchestde, DG (1942) An NH alternative a cheetreatment ea ase13 given by iv Marmell: 1, LD, Nebel, BR, Giles, 264 STRUCTURAL CHANGES IN CHROMOSOMES tha mean Some sort of justification for the choice of this par- ticular function is provided by the chemical reaction analogy we have already d If union between ends from different breaks 15 analogous to a second-order reaction, union between the ends of s singlo break may perhaps be regarded as a first order reaction, which has an equation of the type chosen Suppose that while the cell 1s being irradiated at intensity J roentgens per minute the rate of formation of pnmary breaks: is £1 Allowing for the rate of reunion we have dnfdt=£I—njr, (VI 6a) whence nee fI7(l-e-*) At time t= suppose the radiation to cease, n will then dimmush according to the equation ne EI (1 eT) ett-mir (VII 68) Substituting these values of n 1n equation (5) and integrating we obtain as our estrmate of the total number of exchanges pnt dt=4Bpr (IT) G, where Geo 2(r/T) {T}r~1+e-7} (VI-7) Thus the ber of exchanges produced in the cell by dose D=IT roentgen 1s proportional to {dose}* @ (VIZ 8) The function G 1s tabulated in Table 68 and 23 seen to have the value unity at 7’=0 and to diminish as 7 mereases We see un mediately thet the theory predicts, in accordance with Sex’s experiments (cp p 232), that if the dose 1s vamed by varying the ty at + time,so keepmg 7’ and therefore¢ constant, the number of exchanges produced 1s proportional to (dose)? Further, we see that if at constant dose the mtensity 3s vaned, the yreld of exchanges should be sunply proportional to @ In other words, the curve of number of exchanges against time over which the wradiation 1s extended should be identical an shape with 2 plot of the function @ This affords the simplest method of determming 7 by companson with experment 1 Strictly tobe with our p) defi of £ weshould write £(1—f) in place of £ throughout this section The omission of the factor (1—f) has no influence on the function @ 265 THE FUNCTION @ 369009090 6 9010 $0560 BL O 808 9e02t0o9$09310os9a10B1IZ0F1L2U0O (Ze=s)5984sAyxo096SFLnTL022OEa2698eqF7S9r06t83s0t0oOO(7)o2m4UTs£096L8f/o09a916P39€d38U10—2¥E0x-N0O0a2}]o2Ox=yY(JzJo)0gBO6T$eFOp9609sE1u61aY12t8S8os0£r0o600uoifNxpe]eyF86Bz1£L3sO2£954O8u6EUB9S016oL00OsyusgrSxBFoI938S6i11FOIa2£6d46le=V39021€6L¥D0y060ejduexe10.4) 86960 60890 3190 Lo 6s0903i9$E&1l8O0o0L908eC082to8S£1rr9e0o0ds09sT063iO1¥1tL400o90F10$590g1100107O0G04I8LaOl918ro)6dsZ0oe Ywag2z+elz—1=(z)p—-ZiOt eo29lT0 B8OF9rH1LlT0O ¥(206900608600 Gto-a}Staelg ¥0F90 8 oe Seonsew WOroa 266 STRUOTURAL OHANGES IN CHROMOSOMES For Sax’s oxperiments: at room temperature we find that 7=4 min, both for chromatid and chromosome aberrations Thus Fig 384 shows that the theoretical curve fits Sax’s data (Fig 4 of lis 1940 paper) of the production of chromosome ex changes by a constant dose of 320r spread over various times 20 ne) minutes: 06. 5 10 15 ; T T | —— A O4F = 02; g & g i £0 2 a gs B * ® oto 0 O5- ¢ 0 : ' : : : 5 i0 5 2 Fy 3 mutes Pia 38 Diminution with increased duration of exposure of the yield of d by a t dose of X rays in Tradescantia microspores over the time indicated 3207 3 ehromstid exchanges 150 divided into three2 min fractions with rest fractions Curves theoretical, pomts ex periods of 0 3 @ or 27 min between peruments of Son by varying the intensity The agreement 3s seen to be satis- factory Repetitions of ths expenment made m different Inboratories have given different experimental curves and corre- spondingly different values of 7 (cp curves a and b of Fig 33, p 227) It also appears that 7 21s influenced by temperature > x Sax K (1939, 1940) We have multiplied the ordinate scale by 0 03 to convert tt to exchanges per cell and mserted estimates of error talang the standard deviation of a figure based on the recording of n exchanges to be @ fraction 1), 2 Sax hk & Enzmann E¥ (1939), Catcheside DG Lea, DE & Thodav J.M (1946a) FRACTIONATION OF DOSE 287 For chromatid exchanges Sax’s data refer to experiments of a shghtly different type, mm which the radiation was given at constant intensity, but aplt up mto three 2 min fractions with rest intervals between the fractions, which in the different ex- permments took the values 0, 3, 9 or 27 min The application of the theory to this type of experiment presents no difficulties, but 13 a httle tedious The first stage 1s to calculate the number of free breaks existing at any given time During the first fraction of duration 7, the number of breaks mereases according to equation (6a), during the rest period 7, 1t decreases according to equation (65), thus at time 7, + 7’, the number of free breahs 1s n= Ela (Len Tit) eT Dunng the next fraction T, the number of breaks again in- creases, following the differential equation dnf/dt = €7 —n/r, with initial value m,, the solution of the differential equation 1s n= Elt(l—e)+ nett Thus at the end of the second rest interval 7’, the number of free breaks is n= El (Len Pe") e- Ter $n, et THe, and soon Evaluating JAntdt stage by stage gives the number ofexchanges Fig 388 shows the agreement of theory and ex- pemment obtained using the same value r=4 mim as was used for chromosome exchanges There is no evidence in Sax’s expen- ments of different rates of reyoining of breaks in spht and unsplit chromosomes If the dose is vaned by varying the tame of exposure at con- stant intensity, then with increase of dose @ dimimashes and equation (8) shows that the yield of exchanges increases less rapidly than the square of the dose Sax showed experimentally : that the yield of chromosome exchanges obtained at a constant intensity of 25r /min increased as the 15 power of the dose Es results are shown in Fig 39, 1n which both dose and yield of exchanges are plotted on a logarithmic scale With this method of plotting, a y1eld proportional to a power of the dose 1s repre- sented by a straight hne ‘The experimental points he between hnes a, representing (dose)? and line d, representing (dose), and 18 sco K (1938) Centric Tings and dicentric chromosomes were 208 STRUCTURAL ORANGES IN CHROMOSOMES Approumate closely to hne 4, representing (dose)'$ Curve ¢18 the function (dose)? @, @ being calculated using the value r=4muin derved from Sax's experments on the dependence of yreld on duration of oxposure at constant dose (Fig 384} It is 20 TT Ls T Torey & Ecxephalengres ol 008 0-06 0-04 rn ‘ 1 ae o 00 150206 300-400 500 600 600 10001500 Dose in roentgens Fra 39 Yield of nT as a function of doze at a constant intensity of 25r per minute (X rays) Pomts experimental (Sax) curves a (dose)*, b (dose) ® ¢ (dose)? @ d (dose)* seen to represent the experimental results satisfactorily except for some deviation at the nghest doses (corresponding to ex posure times exceeding 30 min ) No special theoretical significance attaches to the power 15 im the (dose)! 5 law w hich fits these abservations Ifa lower in COMPARISON OF DIFFERENT RADIATIONS 269 tensity had been used over the same dose range, a lower power would have been obtamed Ifa higher intensity had been used, a higher power, approximatig to (dose)? at sufficiently high m- tensity, would have been obtained The experimental results shown in Fig 354 (p 231) lustrate the dependence of the shape of the dose curve on the mtensity With the aid of the @ function a satisfactory account can be given of the manner in which the yield of exchanges depends on dose and intensity in experiments having a duration up to about 30 mm In expenments extending beyond 30 min, the theo- retical curve falls below the expermental curve for the longer exposure times Fig 39 illustrates this for an experiment on the variation of yield with exposure time at constant mtensity, and it has also been demonstrated in experiments on the variation of yield with exposure time at constant dose : The explanation may be that the number of breaks m the nucleus falls off on account of restitution more gradually than mdicated by the formula ~dn/di=n/r, or 1t may be that an appreciable propor- tion of the exchanges produced by X-rays are 1-hit exchanges, the number of which produced by a given dose 13, of course, independent of exposure time This pomt has not yet been elucidated The relative efficiencies of different radiations By the methods described mm an earher section it was possible to deduce from the experimentally observed yields of aberrations the numbers (£) of chromatid breaks primarily produced per cell per roentgen (or v-unit) by X-rays, neutrons, and a-rays m Tradescantea microspores radiated at a time when the chromo- somes were split (1e 24 hr before metaphase of the first haploid mitosis) The average values of £ are reproduced m Table 69 In addition, values of £ are given for soft X-rays of wave-lengths 15,4 1and83A These figures are obtained on the assumption that the values of £ with different wave-lengths are proportional to the respective yields of chromatid breaks per roentgen given in Table 63 (p 239) Since at was found that the proportion of Jomable breaks was not detectably different for medium X-rays and neutrons (Table 67, p 261), 1t seems hkely that 1t will not 1 Catcheside DG Lea DE & Thoday J,M (19468) 270 STRUCTURAL CHANGES IN CHROMOSOMES be different for different wave lengths of X-rays,: and that the procedure adopted for deducing £ for soft X-rays will therefore be correct Tante 69 Numbers of primary chromatid breake per cell pee roentgen (£) produced in Tradescantia Radiation & maya (0 15 or 154) 009 Isa) O17 Neutrons iuito GeV deuterons) 021 } 0O10 @ rays (Rn+Ra A+RaC’) O10 Reasons have already been given for supposing that s chro matid 1s broken by a single 1onizing particle We are now mm & position to answer tho question whether a single tonization causes the break or whether 4 number of 1omzations are necessary The tests by which actions of radiation caused by a single 1omza- tion can be recognized were discussed at length in Chapter mI One test 1s that the efficiency of different radiations (1e yield per unit dose, here £) should decrease in the order of mncreasing ton density, 1 in the order X rays, neutrons, a rays Table 69 shows, however, that neutrons are more efficient, not less efficient, than X rays It 1s concluded, therefore, that », single 1onization 1s not able to cause a break and that a number of lonwations are necessary More exactly, if the more densely ionizing particles (protons) in neutron expenments produce 1 times as many 1onizations per micron as the less densely 1omzing particles (electrons) n X-ray experiments, the fact that neutrons are more efficient per ionization shows that the passage of a proton has a probability of causing a break more than n tames a8 great If we draw hypothetical curves (Fig 40) relating the probability of an 1omzing particle causmg a break to its 10n- density, a curve such as B and not such as C or D must be assumed The essential feature required by the fact that neutrons are more efficient per 1omization than X rays 1s that the curve must have a steeply msing portion in which the probability of 1 The 10n densities of the 1onizing particles which traverse a tissue wradiated by soft X rays are the x0n d of the 1onzing particles when the tissue 18 radiated by medrum X rays and neutrons respectively 2 Per v unit in the case of neutrons EFFECT OF ION-DENSITY 271 breakage 1s mereasing more rapidly than the first power of the ion density An extreme form of curve B 1s the square cornered curve A, and while this 1s less plausible than a smooth curve such as B, we shall for the sake of the simphfication thereby achieved, 4 a B g 10F 2 lor 8 A % c > B > = 3 D & 3 & a BS ja 0 0 Ionations per # Tomzations per jt Fic 40 Hypothetical relations between the on density of a particle, and the probability of its causing a break when traversing a chromatid assume the extreme form A in some calculations The approxi- mation involved 1s not an absurdly crude one as 1t would be if the true curve had a shape such as D, Pig 40 a rays, which have an 10n density still greater than that of protons, produce fewer primary breaks per ionization (Table 69) It follows that the pomt repregentmg « ray 10n-density 13 at a part of curve B where the probability of breakage 1s creasing less rapidly than the first power of the dose It 18 therefore be- yond the shoulder of the curve, and the probability of an «- particle brealing a chromatid through which rt passes is evi dently close to umty We can obtain further evidence leading to the same conclusion by taking account of the dimensions of the chromatids The haploid length of the chromosome thread in Tradescantia 1s 486 pe, and the diameter of the thread 1s said to be 0 1z1 The volume of all the 12 chromatids mm the prophase nucleus will therefore be 76343 Since the total length of « ray track produced per Toentgen per 1s 0 56x 10-5 (Table 18, p 32), 1t follows that the total length of a ray track contamed within the chromatid thread 1s 4 3 x 10-3 per roentgen, and that the number of inter- 1 Sax HJ & Sax, K (1935), Sax, K (1938) The dimensions are thosa orton at pachytene, ‘Observed ytene, a stage r4 when the chr 2 ‘omosomes are greatly 272 STRUCTURAL CHANGES IN OHROMOSOMES sections ot a rays with the chromatid thread 1s 0043 per toentgen If 8 rays are allowed for (data also from Table 18), tho total number of intersections of « raya and é rays with the chromatid thread 18 0 113 per roentgen The number of chromatid breaks pnmanly produced per roentgen 13 010 (Table 69), which 13 compatible with the view that tho probability of an a particle which traverses the chro- matid causing a breah 1s approximately unity Making a similar calculation for neutrons (physical data from Table 18) we find that the mean number of chromatid intersec- tions per y unit 18 0 31, allowing for proton intersections only, or 0 40 allowing also for é ray intersections Comparing with the value of £ given in Table 69, viz 0 21 primary chromatid breaks per vy umt, we infer that the probability of a proton causing a break 18 a little less than umty Actually the protons which traverse the tissue in these experiments cover a rather wide rango of energies, and consequently of ion densities It 18 hkely that the less energetic (1¢ more densely 1on:zing) protons have a probabibty of practically umty of breaking a chromatid through which they pass, and that the more energetic (1 less densely ron1zing) protons have considerably lower probability This 1s suggested by Giles’s experiment (Fig 353, p 231) m which a decrease of the mean neutron energy (1¢ merease in ion density) caused some increase in the yield of aberrations per ionization On these grounds it appears hkely that the rising part of curve B, Fig 40 corresponds roughly to the range of on densities of the protons in neutron experiments We have now to attempt to interpret the vanation with wave- length of the number of primary breaks produced per roentgen by X-rays For equal doses of X rays and neutrons many more chromatids are traversed by 1onizing particles in X ray experi ments than un neutron experiments (as may be seen by com paring the figures of the total range in the tissue of the ronizing particles given in Table 18, p 32) Yet X rays produce fewer breaks It follows that the probability of a chromatid bemg broken by the passage of an electron through it 1s rather low, a x If a straight hoe a long cylinder at rand the mean length of etraight hne lying within the cylinder 15 equal to the diameter of the cylnder, viz 0 ly for the chromatid thread X-RAY BREAKS 273 fact which 1s to be ascribed to the 10n density in an electron track bemg on the average considerably lower than in @ proton track The number of 1onizations per micron produced by an electron increases rapidly towards the end of the track,: and for the last few tenths of a micron 1t 1s of the same order as in a proton track of a few electron-megavolts energy If this part of an electron track traverses a chromatid, the probability of causing a break must be quite high However, the 1on density towards the be- gmmng of anelectron track of, forexample, 20ekV energy,1s some ten times lower than in the last tenth of a micron In view of the rapidity with which the probability of an ionizing particle ga break dur hes with d of 10n density (ep curve B, Fig 40), 1t 1s evident that if this part of the electron track traverses a chromatid, the probabihty of a break being caused 1s very small We are thus led to think that an electron 13 Practically ineffective in causing breakage unless the densely lonizing ‘tail’ of the track traverses the chromatid, mn which event the probability of causing a break may be quite high The following considerations support this view Soft X rays of wave lengths 1 5and 415A dissipate their energy by means of photo- electrons of 7 5 and 2 5ekV respectively For equal numbers of toentgens, the numbers of photoelectrons ejected by the two wave lengths are in the ratio of 1 3 (Table 18, p 32) The numbers of chromatid breaks primarily produced per roentgen are in the ratio of 1 19 (Table 69, p 270) It follows that the efficiencies per photoelectron are im the ratio 157 1 Now the last 25ekV of path of the 7 5ekV photoelectron (which has a range of 1 54)2 13 of course indistingwshable from the whole track of the 2 5ekV photoelectron (of range 0 232) It follows that the probability of a break bemg caused by the first 1 27ft of @ 75ekV electron 1s little more than half as great as the probabihty of a break being caused by the last 0 234 The notion of an effective tail at the end of an electron track which 1s other “use practically ineffective 1s thus supported by the expen mental determination of the relative efficiencies of wave-lengths lS5and41A As a simphfied model for the purpose of calculation, we shall Suppose the probability of a chromatid bemg broken to be unity 1 See Plate Ic 2 Ranges read off from Table 10, p 24 274 STRUCTURAL CHANGES IN CHROMOSOMES if the densely sonizing ‘tail’ of the electron track traverses it, and to be zero if the earher part of the track traverses it On the basis of this model we can calculate the length 1 of the ‘tail’ and prediet the variation of effiaency with wave length In Fig 41 wo represent diagrammatically the passage of an electron through a chromatid thread ‘The effective ‘tail’ 1s the portion PQ of the electron track, the remaining mmeflective por- tion being represented by the interrupted line It 3s clear that A B Fra 41 Tho passage of an electron through a chromatid or through sister chromatids for PQ to traverse the chr d, while g parallel to the direction in which it 18 drawn, P may occupy any pomt in & volume equal to A (!—2r), where lis the length PQ, A 1s the area presented by the chromatid to the electron, and 2r 1s the mean path of the electron im the chromatid of radius r The area pre sented by a chromatid to an electron which 1s perpendicular to the chromatid avis 1s length x diameter, but allowing for random melination of electron path and chromatid axis, this 1s reduced by the factor }r The total area of all the twelve chromatids (haploid length 486y, diameter 0 1) 1s thus 4=76 3y° If the X rays hberate n electrons per y* per roentgen, the expected yield of primary chromatid breaks per roentgen 13 thus f£=nA (l—2r), (VII-9) where A =76 3? and 27=0 ly In tha formula J 1s the length of the effective ‘tail’ of the electron track, or the whole length of the electron track if this is so short that there 1s no meffective portion For X-rays of 415A, +0 0201 electrons per #° per roentgen (Table 18, p 32),andZ=017 (Table 69) Inserting these numencal values im equation (9) we deduce that 1=0211z Now the photo- ‘TAIL’ OF ELECTRON TRACK 275 electrons projected by X-rays of this wave length have an energy of 25ekV and a range of 0 234 The approximate equahty of this figure and the value of J yust deduced suggests that probably the whole of a photoelectron track of this energy 1s effective The length of effective ‘tail’ of a more energetic photoelectron must therefore be at least 0 2p oR) ad g 02% ° Oth ny i { 1 rl a =) —L2. I 2 3 4 5 6 7 BA X ray wave length Fic 42 The number of pnmary ch d breaks per { yasaf of wave length in the soft X ray region Curve theoretical points based on experiment, The length of ‘tail’ may be deduced by malung use of the data available for X rays of shorter wave-length For X rays of wave- length 154A ,~0 0067 electron per y* per roentgen (Table 18 Pp 82) and £=0 09 (Table 69, p 270) Inserting these numerical values into equation (9) we deduce that the length J of the effective ‘tail’ 18 0 282, and ats energy (Table 10, p 24) 1s there fore 2 8ekV We are now able to use equation (9) to construct a theoretical curve of vanation of £ with wave length over the soft X ray region s This calculated curve 1s shown in Fig 42, together with the values of £ given nm Table 69, which are based on expen- tment Its evident that the theory outlined gives a good general Tepresentation of the experimental facts Taking the length of the effective ‘tail’ to be 0 2872, it cin be deduced with the ad of Table 10 (p 24) that the energy 1 Values of n are obtained by interpolation in Table 18 p 32 Jis 0 284, or tho length of the photoolect ron track, whichever 1s tho smaller For a wave length A we take the photoelect ron energy to be 12 4/A—0 5ekV (cp p 10) and read off the correspon ding range in Table 10 (p 24) 276 STRUCTURAL CHANGES IN CHROMOSOMES dissipated ina chromatid which intercepts a length 0 1, 0f theless donsoly ionizing ond of tho ‘tail’ 1s about 0 6ckV , corresponding to tho production of 15-20 This 18 therefore the minimum amount of energy which, dissipated in a chromatid, 18 sufficient for the probability of breakage to approach unity The continued rapid decreage of the number of pnmary breaks per roentgen with diminution of wave length shown by the theo retical curve 1s not borne out by experiment for wave lengths shorter than 1 5A: There aro two explanations for this One 18 that the higher energy cloctrons projected in tho tissue by the shorter wave length X rays have aide tracks (3 rays) branching off the main track Such of these ¢ rays as have a range ex ceeding 0 1 # will be able to cause breaks additional to the breaks caused by the ‘tails’ of the main electron tracks Thus the yield of breaks per roentgen tends to a limiting value and not to zero as the wave length 1s dimmished s However, a calculation based on the distmbution of 6 ray energies given in Table 16 (p 28) dicates that the contribution made by the é rays 1s msufficient to account for the whole of the yield of 0 09 prmary break per roentgen at a wave-length of 015A , which 1s the expermmental result (Table 69) A second factor must therefore enter, which may be the followmg The theoretical curve of Fig 4213 based on the rather crude model that parts of the electron track less densely ionizing than the ‘tail’ are completely meffective in causing breaks The adequate manner in which the calculated curve fits the observations down to a wave length of15A (1e a. photoelectron energy of 7 5ekV ) shows that the probability of a break being caused by the first 4 7ekV ofa 7 5ekV electron track 1s indeed neghgible by comparison with the probability of a break being caused by the ‘tail’,16 the last 28ekV It 1s, however, pushing the model to extremes to imagine that with, for example, a 50ekV electron track at 1s still true that the probabihty of a break beng produced by the first 47ekV 1s completely negligible compared with the probability of a break being produced by the last 2 8ekV of track The application of rt Some decrease with diminution of wave length has been found Thus X rays of 015A produce shghtly fewer breaks than X raye of 15A ond y rays produce shghtly fewer breaks than X rays of O15A (Table 63 p 239) But this decrease 1s much less rapid than shown by Fig 42 2 Cp acalculation by Fano U (1943c) ISOOHROMATID BREAKS 277 equation (9) should therefore be limited to the soft X-ray region (xe wave lengths exceeding 1A) A quantitative theory cover- ing shorter wave lengths 1s not yet avatlable To develop such a theory it will probably be necessary to make assumptions about the form at low 1on-densities of the curve relating probability of breakage to 10n density of 1omizing particle (curve B, Fig 40) We have been able to develop theories for soft X-rays without such assumptions, since for these wave-lengths it was satis- factory to use the square shaped curve A, Fig 40 Isochromatid breaks The values of £ derive mainly from the expermmental yields of chromatid breaks per roentgen, the yields of other types of aberration entering ito the calculation of € only 1n subsidiary degree The agreement between the theoretical curve and experl- mental pomts in Fig 42, and the correlation (discussed on pp 271 and 272) between the values of & for neutrons and «-rays TABLE 70 Yields of isochromatid breaks per cell per roentgen (or v unit) Isochromatid Primary chromatid breaks per cell breaks per cell per roentgen per roentgen Ratio Radiation c) (cf) X rays (015 or 1 5A) 0 0027 009 0.030 (415A) 0 0044 O17 0 026 (834) <0 00009 0010 <0 009 Neutrons (L1+D) 0 0099 O21 0 047 @ rays (Rn+RaA+Rac) 00210 010 021 and the numbers of ionizing particles crossing the chromatids, thus serve as tests of the capacity of the theory to expiam the experimental yields of chromatid breaks We turn now to a dis- cussion of isochromatid breaks In Table 70 we hst the numbers (c) of isochromatad breaka obtamed per cell per re in Trade fia microspores {taken from Table 64, p 241), and in the last column give the values of c/E c/E 1 z times the ratio of the numbers of 120- chromatid and chromatid breaks primarily produced,: and 1s thus a measure of the relative frequency of primary production of isochromatid and chromatid breaks We proceed to discuss I 2 beg the ratio of tne numbers of rs0chromatid breaks observed and breaks p ly produced (cp footnote 1,p 254) 276 STRUCTURAL CHANGES IN GHROMOSOMES dissipated ina chromatid which intercepts alength 0 1 of theless densely onizing end of the ‘tail’ is about 0 6ekV , corresponding to the production of 15-20 1onizations This 18 therefore the minimum amount of energy which, dissipated in a chromatid, 13 sufficicnt for the probability of breakage to approach unity The continued rapid decrease of the number of prrmary breaks per roentgen with diminution of wave length shown by the theo rotical curve 1s not borne out by experiment for wave lengths shorter than 1 5A + Thero aro two oxplanations for this One 1s that tho higher enorgy electrons projected 1n tho tissue by the shorter wave length X-rays have sido tracks (é rays) branching off tho main track Such of these é rays aa have a range ex ceceding 0 1 wall bo able to causo breaks additional to the breaks caused by the ‘tatls’ of the main electron tracks Thus the yield of breaks per roentgen tends to a muting value and not to zero as tho wave length 1s diminished # However, a calculation based on the distribution of 6 ray energies given im Table 16 (p 28) indicates that the contribution made by the é rays 18 insufficient to account for the whole of the yield of 0 09 pnmary break per roentgen at a wave length of 016A , which 1s the experimental result (Table 69) A second factor must therefore enter, which may be the following The theoretical curve of Fig 42 1s based on the rather crude model that parts of the electron track Jess densely ionizing than the ‘tail’ are completely ineffective n causing breaks The adequate manner in which the calculated curve fits the observations down to a wave length of 15A (1e a photoelectron energy of 7 5ekV) shows that the probabikty of a break being caused by the first 4 7ekV ofa 7 5ekV electron track 1s indeed neghgible by companson with the probability of a breal, being caused by the ‘tail’,1¢ the last 28ekV It 1s, however, pushing the model to extremes to imagine that with, for example, a 50ekV electron track 1¢ 1s still true that the probabihty of a break bemg produced by the firat 47ekV 1s completely negligible compared with the probability of a break bemg produced by the last 2 8ekV of track The apphcation of 1 Some decrease with dimmution of wave length has been found Thus X rays of 0 ISA produce shghtly fewer breaks than X rays of 15A and y rays produce shghtly fewer breaks than X rays of 015A (Table 63, p 239) But this decrease is much less rapid than shown by Fig 42 2 Cp acaiculation by Fano U (1943c) ISOCHROMATID BREAKS 277 equation (9) should therefore be hmuted to the soft X-ray region (1e wave-lengths exceeding 1A) A quantitative theory cover- ing shorter wave-lengths 13 not yet available To develop such a theory 1t will probably be necessary to make assumptions about the form at low 1on-densities of the curve relating probahihty of breakage to 10n density of ionizing particle (curve B, Fig 40) We have been able to develop theories for soft X-rays without such assumptions, since for these wave lengths it was satis- factory to use the square shaped curve A, Fig 40 Isochromatid breaks The values of £ derve mainly from the experimental yields of chromatid breaks per roentgen, the yields of other types of aberration entering into the calculation of £ only in subsidiary degree The agreement between the theoretical curve and experi- mental points m Fig 42, and the correlation (discussed on pp 271 and 272) between the values of £ for neutrons and a-rays Taste 70 ‘\uelds of sochromatid breaks per cell per roentgen (or v unt} Yeoch ad Primary d breaks per cell breaks per cell per roentgen per roentgen Ratio Radiation {c) (8) (e/8) X rays (01S or 1 5A) 0 0027 O09 0 030 416A 0 0044 017 0 026 (83A) <0 00009 0016 <0 009 Neutrons (Li+D) 0 0099 021 0 047 a rays (Rn+RaA+RaC) 00210 00 O21 and the numbers of 1onzing particles crossing the chromatids, thus serve as tests of the capacity of the theory to explam the experimental yields of chromatid breaks We turn now to a dis- cussion of isochromatid breaks In Table 70 we hst the numbers (c) of isochromatid breaks obtained per cell per roentgen m Tradescantia microspores {taken from Table 64, p 241), and in the last column give the values of c/& c/£ 1s z times the ratio of the numbers of 180- chromatid and chromatid breaks primanly produced,: and 3s thus a measure of the relative frequency of primary production of tsochromatid and chromatid breaks We proceed to discuss rt 2 being the ratio of tne numbers of sochromatid breaks observed and d breaks iy produced (cp footnote 1, p 254) 278 STRUCTUPAL CHANGFS IW CHROMOSOMES the explanation of the marked vanation of ef£ with different radiations According to the treatment developed on p 261, which applies to neutron induced ssochromatid breaks, c/E=4}zpg, where p 1a tho probability of an ionizing particle wluch traverses a chro matid canaing a break, and g 1s 0 g trical factor depending on the distance apart of the chromatids A theoretical expression for the number of isochromatid breaks per cell per roentgert (c) ean be derived for X rays by a combination of the arguments of pp 26] and 274 Itus t= dzpg nd (l—4r) (VII-10) Companson with equation (9) for the number of primary breaks per cell per roentgen, viz £=nA (1-27), shows that for X rays CfE= kspg (I~ 4r)(l—2r) This 1s less than the value of ¢/f for neutrons by the factor (l~4r)((l—2r) The term ({— 4r) m equation (10) takes the place of the term (!—2r) 1n equation (9) owmg to the presumption that to cause an isochromatid break it 1s necessary for the electron to traversa both chromatids and not merely one, as illustrated diagrammatically m Fig 4in or==0 Jy, the chromatid diameter, and for X rays of wave length less than 38A, 1=0 284 (the length ofthe effective tail) Thus the factor (2— 4r)/(I—2r) 1s 0 44 According to Table 70, the experimental ratio of the ¢/£ values for X rays of wave length less than 3 8A , and neutrons, 18 0 64 For X rays of wave length exceeding 3 8A , the photoelectrons projected by which have a range less than 0 28, the value of required 1s the photoelectron length (2-0 2)/(7—0 1) thus de- creases, eventually to zero, with imerease of wave length (and consequent d t of photoelectron range) beyond 3 8A This 1s 1n qualitative agreement with the behaviour of the ex- penmental values of e/£ m Table 70 off, and consequently dzpg, 1s considerably higher for « rays than for neutrons Three factors contribute to this Ones that the proportion of 1sochromatid breaks having unjomable break- age ends 1s higher in the case of a rays For such ssochromatid breaks z= 1, and 1s probably less for sochromatid breaks having jomnable breakage ends The second factor 1s that 1n neutron ex- penments p 1s probably less than unity for the higher energy RECAPITULATION 270 protons (cp p 272) p will therefore be higher for a rays than for protons The third factor invokes rays Numerous short electron tracks branch from an @ ray track (see Plate La, p 10, and numencal data m Table 174, p 30) It may happen some times that when an « ray passes through one chromatid but not through the sister chromatid, the latter 1s nevertheless broken owing to a d ray from the @ ray track passing through st Some mdheation of the probable magnitude of this effect 1s afforded by the fact (taken from Table 174) that the number of é rays emitted from a length of « ray track equal to a chromatid dia meter (0 1) and having a range exceeding a, chromatid diameter (ve an energy exceeding 1 SekV ) 1s about 03 The addition of 8 term of this order to g in the formula ¢/£ = }zpg will result n an appreciable increase in ¢/£, since g 13 between 018 and 0 363 (p 262) We conclude that the theory affords a qualitative explanation of the experimental result that the ratios of the frequencies of Pumary production of isochromatid and chromatid breaks in- Crease in the order soft X rays, harder X-rays, neutrons, & Tays Recapitulation We conclude the discussion of Tradescantia by recapitulating, in non mathematical language, the desemption of the process of Structural change which has been built up in the preceding sec tions When Tradescantia microspores are irradiated (in the stage in which the chromosomes are spht) by X rays neutrons or radioactive radiations, the number of chromatid breaks pri marily mduced greatly exceeds the total number of breaks ob served at the time of fixation The majority of the breaks restitute after bemg open for a period of a few minutes Exchange be- tween breaks 1s possible during the tame the breaks are open If the dose of radiation 1s given in « short overall time, so that all the prmary breaks are open in the nucleus at the same time, the yield of X ray mduced exchanges 1s proportional to the square of the dose If the dose 1s spread over a longer time, restatution of the breaks first produced can take place before the later breaks are formed and the yield of X ray induced exchanges under these conditions 1s reduced For there to be an appreciable probabilty of exchange 280 sTRUOTURAL CHANGES IN CHROMOSOMES occurring between two breaks, it 1a necessary for them to be produced at an initial separation of not moro than about ly In « ray and neutron expenments, in which a comparatively smell number of iontzing particles traverse the nucleus with the doses commonly given, the majonty of the exchanges are ex- changes between pairs of breaks produced s:multaneously by the same ionizing particle Consequently the yield of these ex- changes (unhke the 2 hit X ray mduced exchanges) 13 propor tional to the first power of the dose and independent of the intensity If the 1onwing particle which breaks a chromatid also breaks the sister chromatid at approximately the same locus, then an isochromatid break usually resulta A proportion of the chromatid breaks pnmanily produced have unjomnable breakage ends As a result a certain proportion of the interchanges are plete, a certain proportion of isochro- matid breaks fail to ahow sister union, and a certaim proportion of the breaks not taking part in exchanges or ssochromatid unions remain as visible chr tid breaks instead of tuting The proportion of breaks which are not joinable 1s the same for X-rays and neutrons, but higher for « rays The number of chromatid breaks pnmatily produced per cell per roentgen can be inferred from the experimental yields of aberrations by allowing for the chromatid breaks which restitute It 1s found that this number 1s different for different radiations Correlation of the number of pnmary breaks per cell with the numbers and renges of the 1onwing particles which traverse the nucleus, enables the following conclusions to be drawn A proton (of not too high energy) or an a-ray traversing e chromatid has a probability almost unity of causmga break An electron, how- ever, 18 only likely to cause a break if it 18 the ‘tal’ end, where the 10n density 1s highest, which traverses the chromatid The length of this effective ‘taal’ ss 0 3z,x and the minimum number of 1onizations necessary to be produced in the chromatid thread of diameter 0 ly for the probabihty of causing a break to ap- proach umty 1s 15-20 In this way the relative effiaencies of different wave lengths and types of radiation in breaking chro matids can be explained Among X-rays, a wave length of about 4A hase higher efficiency per roentgen than either longer 1 Cp Plate Ie RECAPITULATION 281 or shorter wave lengths, neutrons are more effective than the most effective X-rays, and « rays are less effective With materials other than Tradescantza, the experimental data are not adequate for an analysis of this sort to be carried out According to experments cited in Table 62 (p 238), neutrons are more effective than X rays in mnducing chromosome structural changes 1n bean roots and in mouse tumours If this result 1s confirmed it 1s evidence that in these materials, as in Trade scantia, several ronizations and not a single ionization are needed to cause a break To test the applicability to these materials of other of the conclusions drawn from the T'radescantia exper- ments, 1t will be necessary to have additional data of 1on density vanation (eg @ ray or soft X ray experments), and data on the dependence of the yreld of aberrations on mtensity It will be highly desirable to classify structural changes nto chromatid breaks, ssochromatid breaks, and eachanges, and to determine the dependence on dose and intensity of the yields of these mdi vidual types of aberration Observations classified simply as abnormal anaphases yield much less information Chapter VIII DELAYED DIVISION Introduction A temporary inhibition of division appears to be a general action of radiation, haymg been demonstrated in a great variety of cells The duration of the delay increases with increasing dose, so that this effect of radiation differs from those we have dis- cussed in previous chapters in being a graded action rather than an all or none action It 1s therefore usually more appropriate to consider the mean delay produced in a batch of cella, rather than the proportion of the cells which are delayed, as 8 measure of tho effect produced by a given dose In the actions of radiation which we have considered previ ously, to produce the effect studied (eg mutation or chromo- some breakage), 1t 18 necessary to produce a single sonization, or a certain concentration of ionization, in a very Jimited locality In consequence the effect 1s always produced by a single 1onizing particle,: since the chance 1s shght of more than one 1onizing particle passing through the same locahty The region withm which ionization must be produced to delay divis on 1s probably not so small, and many somzations are probably necessary to cause appreciable delay Consequently, in X ray experiments, many ionizing particles contribute to the effect Thus the non- random spatial distribution of 1omzation mm irradiated tissue, and the ‘target-theory’ type of calculation, which play a large part m the mterpretation of the actions of radiation considered in previous chapters, are much less rmportant here = The fact that it 1s a delay in division, and not a permanent inhibition of division which 1s bemg studied, means that the action of radiation concerned 1s one from which recovery occurs, and any interpretation of experiments on delayed division must mecorporate some mechanism of recovery It seems hkely that the effect of the radiation in inhibitmg division 1s due to some chemical change m the cell, but 1t 1s not 1 X ray induced chromosome exchanges are itis true caused by two ionizing particles but thus 1s simply because each exchange involves two breaks The initial effect of the radiation the production of a breal. 1s caused by a single tonizing particle 2 See however p 304 CUMULATIVE DOSE 283 yet clear whether the change consists essentrally in the de- struction of some component needed for division to occur, or the production of some substance having an inhibitory effect The recovery, which eventually permuts division to proceed, pre- sumably implies the re-formation of the component destroved by the radiation (on the former view), or the removal of the in- hibitory substance (on the latter view) The state of the cell at a given moment 1s thus determmed partly by the dose which it has recerved, and partly by the extent to which recovery has taken place If the chemical change we have postulated were understood we could doubtless express the state of the cell in terms of the concentrations of the compounds concerned It 1s, of course, lnghly desirable that the chemmcal reactions should be investigated: Pending the acquisition of this information, & certain amount of progress can be made m the formal interpreta- tion of the results by mtroducing the concept of cumulative dose The cumulatiy e dose 1s a measure of the state of the cell allowing for the radiation 1t has received and the recovery which has ccurred It can be defined for our purpose as that dose of radia- tion m roentgens which given instantaneously to the cell would bring it to the same state (as regards the chemical change which 1s responsible for the delay of division) as that in which the cell finds itself at the given moment The concept of cumulative dose has been used by several authors: in developing theories of actions of radiation from which recovery is possible, and in which the chemical changes involved are at present unknown Ht sHould ey entually appear that the chemical change mvolved ls the production by the radiation of some inhibitory substance, then the cumulative dose will be proporttonal to the con centration of this substance If, on the other hand, the essential change 1s the destruction of some component in the cell, and recovery 1s its re formation, then the cumulative dose will be Proportional to the deficit of this component existing at any Ben time T The suggestion has been made (Mitchell J4 1942) that the delay in division 1s due to an interference an the nucleic reid eycle resulting an @ failure of the conversion of ribonucleic acid to desoxyribonuclere acid Evidence in support of this view 1s provided by the observation that nbonucleotdes aceumulate in thecytopla sm of cells after srrachation 2 Cf Hoffmann JG & Remhard MC (1934), Qumby EH & MacComb WS (1937) Lea DE (19382,5) 284 DELAYED DIVISION Different aspects of the pl of delayed division have been studied in widely different oxpermmental materals, m cluding bacteria, invertebrate eggs, and various rapidly dividing plant and animal tissues, and in this chapter we give an account of some of theso researches It will bo realized that delay in division 13 not tho only effect of radiationonacell Ifappreciable delay can bo produced by doses which do not kull the cell, then delayed division can be studiedIf, however, the dose needed to cause appreciablo delay hills a large proportion of the cells irradiated before division occurs, then 1t may prove imposable to investigate delayed division It will be realized further that the term ‘recovery’ 1s used here to mean recovery from whatever change 1s inhibiting division, and does not imply that the ‘re covered’ cell 1s in all respects normal It may, for example, have chromosome structural changes or other changes which lead to its eventual death It appears that delayed division 1s & phe numenon sufficiently distinct from lethal action to justify ss separate discussion + The delay of first cleavage in sea-urchin eggs An illuminating series of researches has been carried out by Henshaw and his colleagues: on the delay of first cleavage in- duced by X rayain Arbacia The general procedure 1s as follows Separate suspensions are prepared of the sperm and the eggs When the suspensions are mixed, a sperm enters each egg, the male and female pronucle: fuse, the diploid nucleus undergoes mitosis, and a cleavage furrow appears across the cell The time of first cleavage can be determined by microscopic examination of the lying cells, and 3s very nearly umform among all the eggs ofa batch The time when 50 % of the eggs have cleaved 1s taken ag the time of cleavage, and can be determined to about I min, the whole time elapsing between the mixing of the egg and sperm suspensions and cleavage being about 45 min (at 25°) If either the egg or the sperm or both are irradsated before fertilization, or if the zygote 1s irradiated after fertxhzation, the time elar x This pount 1s further discussed in Chapter Ix, p 311 2 Henshaw, PS (1932) Henshaw PS, Henshaw, CT & Francs DS (1933) Henshaw, PS & Francs DS (1936) Henshaw PS (1938) Henshaw, PS (1940), Henshaw, PS & Cohen 1 (1940) Tho iterpretation of these experiments in terms of cumulative dose follows the treatment of Lea DE (19385) OLEAVAGE DELAY 285 between fertilization and cleavage 15 increased, the amount ot the crease over the controls bemg referred to as the cleavage delay The cleavage delay produced by a given dose to the sperm 1s independent of their concentration, and of the fluid m which the sperm are suspended, indicating that the effect 1s a direct action on the gamete and not an indirect action due to the srra- dation of the suspending fiund Henshaw’s experiments consisted in determing the cleavage delay under various conditions of wradiation Cleavage delay can be caused by wradiation of ether egg or sperm, and for a given dose is approximately the same whichever gamete 18 uradiated s In view of the very much greater quantity of cyto- plasmic material in the egg, this observation suggests that the action of the radiation in delaying division 1s on the nucleus and not on the cytoplasm This has been convincingly demonstrated by expenments on half eggs s If eggs are centmfuged at Ingh speed they break into two halves, one half contaiming the intact nucleus, the other half having no nucleus Either half when fertilized will eventually cleave, though the time required 1s abnormally long for the non nucleated half egg Irradiation of 8 nucleated half egg pmor to fertilization causes the same delay 88 irradiation of a whole egg, but no delay 1s caused by irradia tion of a non nucleated half egg The validity of the concept of cumulative dose 1s supported by the expenmental resultss shown in Fig 43 Prior to fertihzation either the eggs, or the sperm, or both, are irradiated by various doses of X-rays spread over a constant time of 40 mm The cleavage delays obtained when both eggs and sperm are 1 Evans TC, Slaughter,JC Little, EP & Failla,@ (1942) Some other actions of radiation on the sperm were found by these authors to be indirect 2 Henshaw s earher expermments (Henshaw, PS & Francais DS 1938), and also the experments of Mavor, J W & de Forest, D M (1924), Suggested that irradiation of the sperm produces a greater effect than radiation of the unfertilized eggs Henshaw subsequently showed (1940) that this ap p im y 18 really due to partial recovery occurring in the eggs prior to fertilization Ifa sufficiently short time elapses bet the of the ar ad. and fertiahza tion, so that recovery 1s avoided, there 1s little difference an sensitivity tween epg and sperm, the egg being shghtly more sensitive 3 Henshaw, P § (1938) 4 Henshaw, PS & Francis, DS {1936) 286 DELAYED DIVIsION irradiated (curve C) are greater than when exther eggs alone (curve A) oraperm alono (curve B) are uradtated, but less than the sum of these two delays (curve D) The delays are therefore not additive However, what we should expect to be additive 13 not the delay but the cumulative dose Inspection of curves A and B shows that x roentgens to the egg produces the same delay as }z roentgens to the sperm : On the cumulative dose bass we should b 50 Cc g r) i B 8 30 4 3 & = A 5 20 § cay 19 L oy = 7 3 x10r Dose in Arbacia caused by X raye (Henshaw & Francia) Haga ieindinted DB aporm irmdinted © eggn and sperm irradiated 43° Ch delay Fro therefore expect that x roentgens each to egg and sperm should produce the same delay as 32/2 roentgens to sperm alone Curve Cis calculated on this basis from the experimental curves A an B, and 1s seen to fit the observations satisfactorily Henshaw’s observations on fixed material: have shown that the delay in cleavage caused by wradiation 1s due maimly to a great prolongation of prophase The stages prior to prophase, x The duration of 40 min was Jong enough for appreciable recovery to occur durmg the irradiation of the eggs (cp footnote 2 p 285) Sperm do not recover 2 Henshaw, PS (1940) PROLONGATION OF PROPHASE 287 namely, fertihzation, the approach of sperm and egg pronuclei, and thew fusion, are not delayed, while the later stages of division (metaphase, anaphase and telophase) are somewhat pro- longed but to a much less marked degree than in the case of pro- phase : We presume therefore that the effect of the radiation 1s dmiCenmluetaevsayge A. 5 16 15 ao 3 30 6 Minutes after fertihzation Fia 44 eer eee Cleavage delay when the fertiuized egg Bg is 1s wradi radiated at various times eeeerti tion (prbacia Henshaw & Cohen} A Imm B 2Zmn C 4mn to hinder the condensation of the chromosomes which occurs Goring prophase, and that the completion of prophase signifies that recovery 1s nearing completion The experrmental results shown in Fig 442 are in agreement with this view In these experiments fertilized eggs were exposed to X-rays at various 1 For the purpose of this description we are meluding in early pro Phase the stage which m the notation of Fry HJ 2 Henshaw, PS & Cohen I (1940) 1936), * (898), as stage 4 286 DELALED DIVISION irradiated (curve C) are greate r than when (curve A) orsperm alono (curve B) are wradiatedeith othe tans sum of these two delays (curve D) The delays are therefore not additive Howover, what we should expect to be additive 1s not the delay but the lative dose Inspection of curves A and B shows that x roentgens to the egg produces the same delay as jz roentgens to the sperm + On the cumulative dose bans we should D $0} Cc Cdlemailnvuantgeys 40h B sob i3 L o D 2 3 xl0'r Dose Fig 43 Cleavage delay in Arbacia caused by X rays (Henshaw & Francis) A, tggs radiated B sperm mradated C, eggs and aperm uradiated therefore expect that x roentgens each to egg and sperm should produce the same delay as 32/2 roentgens to sperm alone Curve Cis calculated on this basis from the experimental curves A and B, and 3s seen to fit the observations satisfactorily ~ Henshaw’s observations on fixed material: have shown that the delay mm cleavage caused by irradiation 1s due mainly to a great prolongation of prophase The stages prior to prophase, 1 The duration of 40 mm was long enough for appreciable recovery to occur during the irradiation of the eggs {cp footnote 2, p 285) Sperm do not recover 2 Henshaw PS (1940) DECAY OF CUMULATIVE DOSE 289 min each time the dose 1s doubled: On our interpretation according to which the duration of the delay 1s the time required forrecovery, we deduce that, in the absence of further wrradiation, the cumulative dose decays to half value in 25 mim The decay being exponential, we represent 1t by the function e~* The half value time of 25 mm corresponds to 7, the characteristic tame being 36 min , and the rate of decay of the cumulative dose in the fertilized egg being a fraction 1/r=2 8% per min The fact, shown by Fig 44, that the cleavage delay caused by a given dose 1s greatest if the dose 1s given 10 min after fertiliza- tion 1s to be expected on the present view The cleavage delay depends on the cumulative dose ewsting immediately pnor to the commencement of prophase If the irradiation 1s made 1m- diately after fertilization, there 1s time for some decay of cumulative dose to occur during the time elapsing between the uradiation and the onset of prophase This will be shown by the effect of a given dose given immediately after fertilization bemg less than the effect of an equal dose given some minutes later The gradients of the curves of Fig 44 during the first 10 min are consistent with recovery occurrmg at about the rate (2 8%, per min ) already found, The decay of cumulative dose can be followed over a longer Period in experiments: in which unfertilized eggs are mrradiated, and are then left (an sea water) for varymg periods before fertihzation Under these conditions the cleavage delay pro- duced by a given dose 1s found to diminish with increase of the Test penod With the aid of an experimental curve showing cleavage delay as a function of dose to the unfertilized egg, we are able to derive from the experimental results, which show cleavage delay as a function of rest period, curves showing cumulative dose as a function of rest period 3 When the cumu lative dose 18 plotted on a loganthmic scale, a straight hne 1s obtained, as wWlustrated in Fig 468, where the four lines refer to four different doses The cumulative dose thus decays according to the formula e~"* The value of 7 at 20-25° 1s deduced from the tn The data of curve B, Fig 43, obtained some years earher, undicate otthe delay * woreased by about 10 min when the dose 13 doubled D& . (1933) 1932), Hensh ( ) PS,F Hensh » CT & Francs, 3 See Lea, DE (19385) for details 288 DELAYED DIVISIOW times ranging from 0 to 35 min after fertilization, thus irradia ting the eggs in various stages up to the end of prophase The maximum effect of a given doso 13 seen to be produced when the dose is given 10-15 min after fertilization, 1e when fusion of tho pronuocler 13 complete and :mmediately before the visible Minutes irradiation, curve A 0 $ to 15 2 as x” 5] dCmelinlauvntaesgye 1 1 L i ——— 1 2 4 8 16 3 Minutes irradiation curve B Fra 45 Cleavage delay as a function of the dose recerved by the sperm (7800r /min , Arbacra Henshaw) onset of prophase The effect 1s reduced 2f the srradzation 1s made after prophase has started, and 1s neghguble 1f made after the completion of prophase Fig 45, curve A,: shows how the cleavage delay increases with increase of the dose given to the sperm prior to fertihzation A straight line 1s obtained if the dose 1s plotted on a logarithmic ucale asin Fig 46, curve B, the cleavage delay mcreasmg by 25 1 Henshaw, PS (1940) RECOVER1 RATE AND OX\GEN UPTAKE 291 at 20-25°, and unfertilized eggs at 0°, and the differences m metabohe rate as measured by the rate of oxygen uptahe: It 1s seen in Table 71 that 100/r, which 1s the percentage decay of cumulative dose per minute, 1s approximately proportional to the rate of oxygen uptake TaBLe 71 Correlation of recovery rate with rate of ox} gen uptake Rate of decay of Relatwe cumulative dose rate of Temperature rT (100/r) oxygen System °C min % per min uptake Fertilzed egg 20-25 36 28 3-5 Unfertilized egg 20-25 104 10 10 Unfertihzed egg o 360 03 02 Sperm 20-25 Large 00 OOL In expermments in which sperm were radiated Henshaw found that a rest period mtervening between irradiation and fertihzation did not reduce the cleavage delay Thus the correla- tion between rate of recovery and rate of oxygen uptake extends also to sperm, since it 1s known that the rate of oxygen uptake by a sperm 1s neghgible compared with that by an egg The fact that sperm left in sea water after irradration do not show recovery makes 1t umprobable that the recovery of wradi- ated unfertilized eggs left in sea water 1s due to diffusion out of the egg of mhibitory substances The correlation between re- covery rate and oxygen uptake favours the interpretation that the effect of the radiation 1s to destroy some nuclear constituent necessary for the condensation of the chromosomes, and that Tecovery consists in the re-formation of this constituent as a result of the metabolte activity of the cell It would be unjustifiable to deduce from Table 71 that the Tecovery process 1s one which directly requires oxygen The rate of oxygen uptake is presumably an indication of the general level of metabolic activity, and in Arbacia eggs appears to vary in different states of the egg in much the same way as does what- ever reaction 1s responsible for recovery It does not necessarily follow that the rates of recovery in different organisms will be Proportional to their respective rates of oxy gen uptake No re 1 Experimental data on the rate of oxygen uptake are given by Loeb J (1910), , Loeb, A J & Was tenevs,H (1911) PS (1931) Whitaker DM (1933) Needham J (1931 (0991) “Tang 290 DELAYED DIVISIOW gradients in Fig 46n to be 104 min, and to be independent of the dose In an oxpenment.in which the unfertilized eggs were kept at 0° after irradiation: 1t was found that the recovery was slower than 30 0 30h. Cdo1u0mnuilsfa0tneir ar a a ee OO Cd Rest period in minutes Fie 46 Decay of cumulative dose in unfertilized Arbacut eggs tO B at 20-25° atroom temperature Converting cleavage delay nto cumulative dose in the same fashion as before, Fig 46A 1s obtained The gradient corresponds to 7== 360 min There appears to be a correlation between the differences m the 7 values found for fertihzed eggs at 20-25°, unfertilized eggs 1 Henshaw, PS (1940) DIVISION STAGE DELAYED 293 calculation 1s 14 min , m four agreement with the expermmental figure of 12 min It appears therefore that the dependence of the effect of a given dose upon the time over which it 18 spread can, in these expenments, be explained on the basis of essentially the same recovery process which permits the eventual completion of the division The stage of division which is subject to delay When division is delayed by wradiation, 1t 18 of interest to determine m which stage or stages the retardation occurs, and also at which stage the cells should be iradiated to give the greatest delay The answers to these questions cen most readily be obtamed m the case of matemals in which the cells develop synchronously, so that all the cells van be irradiated at the same Stage The wrathation bemg made at a chosen stage, samples are fixed at intervals and the stages reached determuned Com- Parson with a control series enables the delay suffered in each stage to be measured Henshaw: has made experiments in this fashion to find which stages are subject to delay in the cleavage division in Arbacia In many expermmental matenals cella are present at all stages of mitous and mterphase, so that 2 13 not possible to choose a single stage to be irradiated Expemments made by fixig such matenals at mtervaly after wradiation and determming the numbers of cells in the various stages are therefore more difficult to interpret’ The interpretation 1s facilitated if the maternal chosen is one m which it 1 possible to follow the course of mitosis in the hving cells A cell can be selected and wradiated when itis maknown stage, andits subsequent behaviour studied The exammation of hving cells m this way has been used to Supplement observations on fixed preparations in the case of embryonic chick tissue growing in culture,s and whole grass- hopper embryos s In the latter maternal it has been found that wradiation produces the greatest delay 1f it 13 made at a stage in prophase when the chromosomes are already discrete, but pmor to the breakdown of the nuclear membrane A dose of 10-20r 1 Henshaw, PS (1940) 2 Cant, RG & Donaldson, M (1926) 3 Carlson, JG (19415) The cells examined were neuroblasts 292 DELAYED DIVISION covery from the delay of cleavage produced by irradiation was found in the unfertilized eggs of the clam Cumsngra,: although the oxygen consumption: ts rather hugher than in the unfertihzed eggs of Arbacta In Arbacia recovery occurs if a reat period elapses between the irradiation of unfertilized eggs and fertihzation It 1s to be pre sumed that recovery will also be occurring dung the urradiation if this 1s extended over a prolonged time, so that the effect of given dose will be less 1f 1t 18 ad ed at low intensity than if it 18 administered at high antensity This has, in fact, been shown to be the cases These considerations can easily be put into a quantitative form + If the cumulative dose existing at any time ¢ 18 D, then the tate of decay due to the recovery process 1s D/r, and the rate of increase of cumulative dose due to the radiation being ad munustered 1 J (where J 1s the dose rate in roentgens per minute) Thus dDjat=1—Djr, (VIII-1) integrating to D=Ir(1-e-*) (VOI 2) Two batches of eggs were irradiated by equal doses spread over 30 and 150 min respectively by the use of intensities zn the ratio of 5 1 They were fertilized immediately after the completion of the irradiation and the cleavage delays were found to be 21 and 12 min respectively According to equation (2), the cumu- lative doses at the time of fertihzation should be in the ratio of 5(1 e808) Ta aeman 71 4 1, since r=104 mm for unfertilized eggs at room temperature Since the actual dose used in this experiment 2s not stated, one proceeds to calculate it from the fact that the 30 min exposure resulted in a cleavage delay of 21 mm , and thence calculates the delay to be expected for the 150 min exposure The result of this i Henshaw, PS, Henshaw, CT & Francis DS (1933) 2 Whitaker, DM (1931) 3 Henshaw, PS Henshaw,CT & Francis, DS (1933) In Cuming however, un which no recovery occurs when @ rest period intervenes b 2 tron and fer the delay d by a given dose was found to be independent of the mtensity 4 Lea, DE (19384, 5) RAPIDLY DIVIDING TISSUES 295 occupies half the time interval between the telophase of one division and the metaphase of the next In mammahan: and chich2 tissue, however, the stage described as prophase lasts only about one third as long as metaphase, anaphase and telophase combined Bearing this in mind, the observations of Carlson which we have described are consistent with the conclusion reached by previous authors investigating a vanety of tissuess that a dose of up toa few hundred roentgens temporarily prevents cells from entering mitosis, but does not prevent cells already im mitosis completing division Rapidly dividing tissues The manner in which the mitotic count in a rapidly dividing tissue vanes durmg the hours following sradiation 1s illustrated mn Fig 478, which shows the result of an experiments in which chick tissue growmg in culture was wradiated by y-rays at an intensity of 25 roentgens per minute for 14, 24, 9 or 30 min , and fixed at various intervals after exposure With small doses (cp the 2} min curve) the retardation of mitosis at a stage just Previous to that recognized as prophase results im an initial diminution of mitotic count When mitotic activ ity returns, there are two classes of cells entering division One class consists of those cells which were so far from division at the time of utadiation that they had recovered from the dose given by the time they had reached the stage in which ceils which are delayed accumulate These cells therefore divide at the normal time The Second class consists of those cells which were im early prophase at the time of irradiation, and whose further development was held Up for some hours As these cells recover (1e the cumu- lative dose decays) they slowly pass through prophase s In consequence ot the fact that these two classes of cells are 1 Tansley, K, Spear FG & Glucksmann A (1937) ‘using rat retina 2 Juul,J & Kemp T (1933) Lasmitzka, I (2940) a? Alberti W & Pohtzer, G (1923, 1924) using corneal epithelium of St larvae, Strangeways TSP & Oakley, HEH (1923) Nebr iiecdia TSP & Hopwood FL (1926) and later workers using chick tissues grown im culture, Mottram JC Scott, GM & Russ S (1926) using Jengen rat sarcoma 4 Cant, RG & Spear, FG (1929) 5 Cp Carlson, JG (19415) 204 DELAYED DIVISION at this entical stago causes the cell toremain unchanged for come hours Cells radiated at an earher stage of prophnse are also held up in middle prophase, but for a shorter tie, 80 that they may reach metaphose actually before the cclls which were irradiated in the erstical stage, although thoy were less advanced at the time of radiation A larger dose (250r } causes the cells to be held up in early prophase (a stage when the chromosome threads aro still barely visible} instead of un middle prophase, and cells somewhat more advanced but not yet passed the ontical stage appear to revert to the condition of early prophase, whore thoy remain some hours before resuming mitosis Irradiatio n after the breakdown of the nuclear membrane enusea no delay Irradiation ats stage intermediatebbetween the enitical atago and the breakdown of the Buses nucl a short delay, which as shorter the further removed the cell 2 from the entical stage These conclusions are in general agreement with and those of Henshaw on Arbacsa eggs, already mentioned (p 286), with observations on other matenals The main points that moderat e doses of radsation given late m prophaso or at e subseq’ t stage couse hitle retardation Somewhere m prophage, or shortly be fore the visible onset of prophase, there 13 astage18where the delay produced by a given doze us a maximum 'Yhns well shown 13 Fig 44 Inraduation at or diately prior to this enitical stage causes the cells to be held up ‘The duration of the delay depends upon the cumulative dose at ths tune If wradiation made aome time previously, the cumulati ve dose has partly decayed hence the effect 18 by the tune the ertical stage 18 reached, andnearer Jess than uf the same dose had been given the ertical the effect of a given dose 18 & stage This ertical stage, at whichwhose division 1s held up 8¢ maximum, and near which cells as bemgin prophase In other cumulate, 1s described by Carlson ed as late interphase (1e immedi matemals: 1t has been regard funds propctahase ately l,priobutr torefle ) This duffbet 18 af probabt ly notterials a a3 menta can be d hed from the ease with which early prophn's maten al (grasshopper neuro the restintheg stage Thasin Carlso blasta): stage described a8 prophase lasts twice ss long a8 h h and teloph Inned, and m fact 2 Carlson IG (19410) iP 1 Op Spear, FG (1935) P PEPENDENCE UPON INTENSITY 297 cells and not as dividing cells In consequence, with larger doses the mtotic count fails to exceed that m unmradiated material dunng the period of recovery Curves such as those shown in Fig 47 can be constructed theoretically if the relation between delay in division and dose 6atrmc0iehodDrivu%ietcntsecnyges 3 8 . 8 cs rn " L 19 2 » Roentgens per minute Fro 48 Depend upon the y of the dose required to produce e given delay of division Curve theoretical, points experiments of Cant: & Spear is hnown As explained in the discussion of cleavage delay in Arbacia, this 1s essentially the same as the relation between the cumulative dose remaining at a given moment and the time which has elapsed since wradiation But the rate of decay of cumulative dose can be deduced from experiments on the vara- tion of the effect of a given dose with the intensity at which itis given Canti and Spear: wrradiated chick tissue in culture and counted the number of cells in division 80 min after the sradzation y Cant: RO & Snear FO (10971 296 DELAYED DIVISION entering mitosis together, the mitotic activity during this period is higher than mn unuradiated matenal Since the cells of the second class are recovering as they pass through prophase, and therefore are spending an abnormally long time in this phage, the Maipocmteairnctiefsvirntocvitalcigyety 0 1 2 3 4 5 Hours after srradiation Frio 47 Mitotic activity at vanous times after wradation Exposures ofaA (Cant: & Spear y raya on chicktetas an culture) 2} 9 and 30mm at 26r /mm proportion of mitotic cells which are im prophase 1s higher than d curve 6 normal during the recovery wave * Larger doses of radiation cause the death of many of the cells entermg division, and such cells are recorded es degenerating 1 Demonstrated in bean root tips by Pekarek J (1927) im rat tissue by Tansloy K Spear, FG & Glucksmann, A (1937) 1m tadpole tissue by Spear, FG & Glucksmann, A (1938), m chick tissue in culture by Lasmtzki I (1940) DELAY AS FUNCTION OF DOSE 299 cells which degenerate when they enter division are not included in the experr:mental curves The duration of mitotic delay as a function of dose has been investigated in a number of tissues, and Fig 49 illustrates some of the results obtained: A precise figure cannot be given to the : CT § dr 4 3 s § at 1 Fd 600 700 800 A $50}- e $00 e sob Qo L i. 0 i 1000 2000 nO 4000 Dose in reentgens Pea 49 Division delay in hours as a function of dose in roentgens A, chick (Cartan Caeare (@ Canti & Spear x Lasnitzki) B phag bl (Albert mn & Pony (Tansley Spear & Glucksmann) D Triton comes & Pp Taken from the experiments of the following authors Alberti, W ‘olitzer, G (1924) using the cornea of the newt Triton, Cant, RG & pear FG (1929) Lasnitzia, I (1943a) using chick tissue in culture ‘ansley K, Spear FG & Glucksmann A (1937), using the retina of the Chertopnen JG (1938a), using neuroblasts of the grasshopper rat, 298 DILAXYED PIVISION finshed They determined the dose needed to reduce the mitotic count to half that in unirradiated cultures at several different intensities, obtaming the results shown m Fig 48 At low in tensities the doso required to produce a given effect 13 several times greater than at high intensities Suppose that D, 1s the cumulative dose ¥ hich must exist at the end of irradiation in order that the mitotic activity shall be re duced to 50% 80 min Iater ‘Then from equation (2), p 292, we have D=Ir(1—-e*"), where ¢ 1s the duration of wradiation at intensity J needed to produce the cumulative dose J, at the end of the swradiation Jt 1s therefore the dose required at mtensity J to produce the observed effect Thus wn) tfr. t= Dyae (VIE 3) Fig 48 shows that a theoretical curve constructed according to this equation, and with r=217 min, fits the experimental ob servations satisfactorily « A second datum required ts the manner in which the mitotic activity at 80 min after irradiation depends upon the dose (when the intensity 1s high), this beng provided by the expert ments of Spear and Grimmett « With these data the theoretical curves of Fig 474 have been constructed s Theso curves, which show the yanation of mitotic activity over a period of 8 hr after rradiation utilize only experimental data of mitotx counts made 80 min after wradia tion The general agreement between the calculated and experi- mental curves shows that, as m the case of Arbacea eggs, the recovery during irradiation which results in the effect of a given dose being diminished by spreading 1t over a prolonged time, 18 essentially the same process as the recovery after wradiation which permits the eventual 1eturn of mitotic activity That the experimental curves in Fig 47 show a lower recovery wave than the theoretical curves 1s due to the circumstance that 1 Cp Lea DF (29382) 2 Spear, FG & Gnmmett L G (1983) 3 bee Lea DE (19386) for details of the calculation and for a com parison of theoretical and expertmental curves obtamed when o second aurradiation 13 made 80 or 160 min after the first INHIBITION OF DIVISION OF BACTERIA 301 amhibitmg the division of all the orgamsms, the radiation kills some of them Meanwhile the length of the cells mereases very greatly (see Plate [Va,n) A measurement of the total length of all the cells in the culture would underestimate the growth rate, amce dead cells as well as growing ones would be included 4 4 ¥ E3 : 4 & $s 2 3 % ba 4 & : : 27 4 a TL \ f A r 1 L 2 4 6 2 4 6 Hours Fia 50 Inhibition of division in Bact colt (Lea, Hi famnes & Coulson) A, total ount in urradinted culture B, viable count in irradiated culture C’ total igth in umrradiated culture D length m irradiated culture -v rays at 35r /min Curve D therefore shows the manner in which the length of the longest orgamsm increases during the irradiation It 18 seen that the gradient of curve D,1¢ the rate of mcreaso of length of the non dividing cells in the wradiated oulture, 1s practically the ame aa the gradient of curve C,1¢ the rate of mcrease of total length of all the cells m the umrradiated culture Thus the growth Tate (meaning rate of volume imcrease) 28 the same for un- wraciated cells as for (living) irradrated cells, but in the former meepivision occurs so that the cells mamtain a nearly constant cobb increase am number, while in the latter case division 1s ther s 80 that the swe of the individual cells increases but mber remains constant of te 51 shows the resulta of an experiment on the mradiation ean root-tips: Normally about 10% of the cells in the 1 Pekarek, J (1927) 300 DELALED DIVISION duration of the dclay produced by stadiation of o rapidly dividing tissue, since cells aro wradiated at different stages and suffer different delays In constructing Fig 49 the delay has been taken to be tho period clapsing between the radiation and the commencement of the mise in mitotie activity which follows tho period of reduced mitotic activity Increase of cell size An imereaso in the size of cells, or of thew nuclei, following irradiation has been observed in a number of matenals : In some cases, though perhaps not in all, the effect requires no explana tion other than the inhibition of division which 13 known to occur If the cells are prevented from dividing, but continue to grow at the normal rate, then the szo of the cells will crease instead of their number The effect 2s most easily investigated 10 the case of bacteria Tho division rate of these organisms bemg hugh, 1t 13 possible to hold up division for a tame dunng which each cell would, if unirradiated, have divided several times Fig 60 allustrates the effect of irradiating a growing culture of Bacterium coli: In tho absence of wradsation, the number of cells im tho culture increases as an exponential function of the time, so that a straight line 1s obtained by plotting the loganthm of the cell count against the time Since the mean cell size re- mains nearly constant, and since practically all the cells are viable, there 13 no appreciable difference between the gradients obtained by plotting (on a logarithmre scale) the total count, the viable count, or the total length of all the organisms m the cul- ture Curve C of Fig 50 shows this gradient, corresponding to & tenfold increase in about 4} hr If the growing culture 1s placed over a radium source (continuous y wradation at 35r /min ), division ceases after a short time and the total count becomes practically constant as shown by curve A The viable count diminishes (curve B) owing to the fact that in addition to 1 Eg bactema Spencer RR (1935) Lea DE Hames RB & Coulson CA (1937) yeast Holweck F & Lucassagne A (1930), Wyckoff, RWG &Luyet, BJ (1931), Protozoa Mottram, J C (1926) Robertson, M (1932) fungal spores Luyet, BJ (1932), algae, Luyet, BJ (1934) bean root tips Pekareh J (1927) Mottram JC (1933a) mouse tumours Mottram JC (1927) Ludford RJ (1932) human tumours Gluchsmann, A (1941) 2 Lea, DE, Haines RB & Coulson CA (1937) a: COMPARISON OF DIFFERENT RADIATIONS 303 The relative efficiencies of different radiations in causing delay of division Probably the most convenient material to usein compamng the relative efficiencies of different radiations m causing delay in division would be marme invertebrate eggs, but expenments with radiations other than X rays have not yet been made using this matenal A few experments have however been made using rapidly dividing plant and animal tissues With a rapidly dividing tissue two methods of ecvperment can beused Ones to irradiate the tissue and to determine the mito- fic activity at intervals after irradiation, and to measure the duration of the delay as a function of the dose in the manner of Fig 49 The efficiencies of different radiations can then be ob- tained from comparison of the doses needed to cause equal delays The second method 1s to determine the mitotic count at a de- fimte time after irradiation, preferably when mitotic activity 18 at a mmmum (which 1s at a time after irradiation somewhat in excess of the duration of mitosis) The relative efficiencies of different radiations in inhibiting davision are then deduced from measurement of the doses needed to produce equal reduction of mutotic count, Of the few experments at present available, the best have been made on the bean root trp,: using the second method The results are shown mm Fig 52, m which the mitotic count 3 hours after uradiation 1s expressed as a fraction of the mitotic count m un- utadiated controls, and plotted on a logarithmic scale agamst the dose Plotted in this way, the pomts le approximately on straight lines, and the relative efficiencies can be taken to be the Tatios of the gradients The efficiencies of the three radiations, Neutrons, y rays, and o-rays for equal 1omzation im the tissne are approximately in the proportions 4 2 1 respectively The explanation suggested by the authors for their results 1s that for inhibition of division to occur a certain number of ionzations must be produced, not in the nucleus as a whole, but 1 Gray, LH, , Mottram JC, ’ Read, . J & S; pear, FG (1946) Gray UH, Mottram, JC & Read J (unpublished) I am indebted to Dr tay for communicating the resulta of these experrments, and his in terpretation of them Prior to publication . 302 DELAYED DIVISION dividing rogron of tho root tip are m mitosis at any given moment, and the average nuclear diameter 1s about 8 54 After uradiation (by about 1600r ) the mitotic activity 13 greatly re- duced as shown in Fig 615 Meanwhile the mean nuclear dia meter increases, rising to about 11 5 after 100 hr, the corre sponding merease in nuclear yolume being plotted sn Fig 51A 05 B ny n 4 4 n a 0 2 cy a 100 Ro 140 Houre after irradistion Fie 51 Inhibition of divigion in bean root tips (Pekarek) A nuclear volume relative to control nuclear volume B mutotic activity relative to control mutotio activity The fact that the nuclear volume doubles mn about 3 days 13 compatible with the rate of growth bemg unchanged by the wradiation, but resulting m increase in cell size instead of mn Grease in cell number The merease of cell or nuclear size in plant and animal tissues after irradiation cannot be expected to be so striking as the 1n crease of size of bacteria illustrated in Plate 1Va,p The mhbi- tion of division often persists for a time which 1s only a fraction of the intermitotic period Thus if the intermitotic period 1s 3 days, and the inhibition lasts for 1 day, we shall expect a 30% merease of cell or nuclear volume, which corresponds to an m crease of only 10% m diameter Careful measurement of many cells will be required to estabhsh such an mwcrease COMPARISON OF DIFFERENT RADIATIONS 305 ionizing than « rays) would be less effective, or at any rate not more effective, than y rays The authors have put forward an igemious mechamsm by which the higher efficiency of neutrons can be explained, but 1t volves some assumptions which appeat to be rather unplausible An alternative explanation of the lower efficiency of a-rays compared to less densely 1omzing radhations would be that the change produced in the nucleus leading to the mbubition of division 1s a chemical reaction involving ‘activated water a rays would then be less efficient for the same reason as that put forward m Chapter m to explain their low efficiency in de composing protems mm aqueous solution (ep p 60), namely, that owing to the bigh concentration of active radicals in the neigh bourhood of the a-ray track, some radicals disappear by re- combination before reacting with the solute concerned This mechanism would not provide an explanation of the fact that neutrons are more effective per 1omzation than y rays The relative efficiencies of the different radiations m causing mbuibition of division cannot therefore be considered entirely understood It 1s desirable that expernments should also be made by the firat method mentioned, namely, the determination of the doses of the different radiations needed to produce the same interval between the mutual fall of mitosis and its subsequent nise, since this experiment should decide between the alternative mechanisms which have been suggested for the lower efficiency ofa rays compared to y-rays No special mgnificance 1s attached to the approxmately exponential shape of the y ray curve of Fig 528 The cells, whose delay m division causes the reduction m mitotic count at fixation, three hours after wradiation, are the cells which are due to enter division any time between 0 and 3 hr after wradhation 2 1 As pointed out to me by Dr Gray If the low efficiency of « rays in reducing mitotic activity three hours after sradiation 1s dus to an @ ray Producmg more than the of an the sensitive region then & rays should be nc less effective than y rays when the doses needed to cause mbibition of division for an @qual time (longer than Shr) are compared If however the low efficiency of a rays 1s explained 88 & low ionic yreld in gome chemucal reaction, then the efficiency should be low by whichever ‘way it 19 measure d erat ns the duration of mitosis to be approximately 3 hr in the bean ap 304 DELALED DIVISION anywhere within a amaller volume estimated to be about 3 fem diameter: It 1s supposed that a singlo @ particle traversing this region produces several times moro than the minimum amount of 1onization (a fow thousand sonizations) needed for mhibition of division to occur a rays aro thus less efficient per ionization Fie 52 Reduction of mitotic count esa function of dose (Gray Mottram Read & Spear) uradiation) Mficortamciutnfiosutrcnoiltc arradiation) on oo o tt 30100 L 150 Dose sn energy untts A, neutrons B, y mys C a rays (bean root tips fixed 3 hr after D neutrons, E y rays (chick tissue in culture fixed 80 min after te 200 n 250 than less densely 1on1zing radiations, for the same reason as the case of chromosome breakage and virus inactivation, namely, that the extra 1omzation contributes to the dose without in- creasing the proportion of cells affected One would expect on the basis of this argument that neutrons, bemg more densely ionizing than y rays (though less densely 1 The authors pomt out that the nucleolus is of about this ize If the cause of the delayed division 1s interference with the nucleic ad cycle, it 18 not unp ible that the lus should be d since itis a reservoir for ribonucleic acid during the resting stage Cp also Mottram, TC (1932) COMPARISON OF DIFFERENT RADIATIONS 305 romzing than a-rays) would be less effective, or at any rate not more effective, than y-rays The authors have put forward an ingenious mechamsm by which the ngher efficiency of neutrons can be explamed, but 1t involves some assumptions which appear to be rather unplausible An alternative explanation of the lower efficiency of o-rays compared to less densely 1onizing radiations would be that the change produced m the nucleus leading to the mbhuibition of division is a chemical reaction involving ‘activated water « rays would then be less efficent for the same reason as that put forward in Chapter m to explain their low efficiency in de- composing proteims 1n aqueous solution (ep p 60), namely, that owing to the high concentration of active radicals m the neigh bourhood of the a ray track, some radicals disappear by re- combination before reacting with the solute concerned This mechamsm would not provide an explanation of the fact that neutrons are more effective per omzation than ‘y rays The relative efficiencies of the different radiations in causing inhibition of division t therefore be dered entirely understood It1s desirable that experiments should also be made by the first method mentioned, namely, the determmation of the doses of the different rathations needed to produce the same interval between the imtial fall of mitosis and 1ts subsequent ms@, since this experiment should dectde between the alternative mechanisms which have been suggested for the lower effinency of a rays compared to y-rays: No special significance 1s attached to the approxmately exponential shape of the y ray curve of Fig 528 The cells whose delay m division causes the reduction mn mitotic count at fixation, three hours after wradhation, are the cells which are due to enter division any time between 0 and 3 hr after wradiation : 1 As pomted out to me by Dr Gray If the low efficiency of rays im reducing mitotic activity three hours after irradiation 1s due to an a ray producing more than the of in the sensitive region, then & rays should be no leas effective than 7 raya when the doses needed a to cause hibition of division for an equal tyme (longer than ) are pared Ift the low effi ry of @ rays 13 explained a8 & low tonic yneld in some ch 1 tion, then the effi y should be low by whichever way it 1s measured :ont ‘eking the duration of mitosis to be Spproxmately 3 hr mn the bean ap 306 DELAYED DIVISION and the mimmum delays needed to prevent them from bemg in division at the time of fixation therefore range from 3 to 0 hr respectively In vsew of this heterogeneity m the stages of the cells concerned and in the delays necded to be induced in them, it 13 not surprising that the number of cells im division at the tume of fixation diminishes gradually with increase of dose Neutrons are also more effectivo per ronization than y rays 10 causing a reduction of mitotic count shortly after :rradiation mn the case of chick tissue grown im culture, as shown in Fig 52, curves D, Et In these experrments an exponential curve 1s obtained with noutrons (Fig 52D), but the y ray curve departs from the exponential form in having an mutual flat portion (Fig 652) Thus difference 13 mterpreted by the authors: to mean that a single ronizing particle causes the delay 1n the neutron expert ments and that many are required in the y ray experiments 1 Gray, LH, Mottram, JC, Read, J & Spear, FG (1940) The ¥ ray curve 15 quoted from Spear, FG & Grimmett, LG (1933) 2 Spear, FG, Gray, LH & Read, J (1038), Gray, LH, Mottram, J & Spear, FG (1940) JC, Read, Chapter IX. LETHAL EFFECTS Lethal effects of radiation have been studied by a great number of authors on a great variety of experimental matenals, partly because of the ease with which experiments can be made in which the criterion of effect 1s the death of an organism or of a cell, and partly because of the practical importance of the lethal action of radiations on cells in the treatment of cancer No attempt 1s made in this chapter to review the whole of this very considerable literature Attention 3s confined to the lulling of single celled orgamsms and of mdividual cells of multicellular organisms, and is directed mainly to lethal actions which may be understood in terms of the mechamsms described in the earher chapters The kalling of multicellular orgamsms as distinct from the lalng of theur mdividual cells 1s not discussed, nor 1s the lulhng of a cell discussed when this 1s due, a8 xt probably 13 in some circumstances, to a change produced by the radiation nm the surrounding tassues or fluids rather than to the diss:pation of energy by the radiation in the cell itself Death precipitated by division It has been shown in many experimental matenals that when ® cell 1s lulled as a result of wradiation, death does not occur immediately, but at or following the next division that the cell undergoes To killa cell immediately requires 4 much larger dose than to cause a cell to die at or following its next division Thus, 10,000r of X rays delivered to yeast does not cause immediate death The radiated cells divide once, but the daughter cells are usually unable to divide further and eventually die A much larger dose 1s required to kill the cells without division, 30,000r sufficmg only to kill 50% m this way = Sumilarly, when chick tissue growing in culture 1s irradiated,: doses of 2500r and upwards are required to cause the death of ne 4ppreciable proportion of resting cells without the mterven- aon of division, but a dose of 100 r suffices to cause the death of Layer BS tiene Lacassagne, A (1930) Cp also Wyckoff, RWG & 2 Leamtzki I (19434 6) 306 DPLAXYFD DIVISION and the minimum delays necded to prevent them from bemg in division at the time of fixation thereforo range from 3 to 0 br respectively In view of this heterogeneity mn the stages of the cells concerned and in the delays needed to bo induced im them, it 1s not surpnaing that the number of cells wn division at the time of fixation diminishes gradually wath increase of dose Neutrons are also more effective per tomzation than y rays causing a reduction of mitotic count shortly after wradiation n the ens of chich taste grown in culture, ss shown in Fig 82, curves D, Ex In these experiments an exponential curve 18 obtained with neutrons (Fig 62D), but the y ray curve departs from the exponential form in haying an intial flat portion (Fig 62k) This difference 1s mterpreted by the authors to mean that 8 singlo 1onizing particle causes tho delay an the neutron expert ments and that many are required in the y ray experiments 1 Gray, LH, Mottram, JC, Read, J & Spear, FG (1940) The y Tay curve 13 quoted from Spear, FG & Grimmett, LG (1933) 2 Spear, FG, Gray, LH & Read J (1938), Gray, LH, Mottram, JC, Read, J & Spear, FG (1940) DEATH PRECIPITATED BY DIVISION 309 embryos If, however, the eggs are hept for 7 weeks at 5° after irradiation, 80 preventing cell division during this tume, and are then restored to 25°, 40% of the eggs develop mto normal embryos + That cells wradiated by small doses during the resting stage break down at division has been demonstrated by Spear and his T T T “T 6 Xue ¥— 10 15 a 2 30 35 Hours after radiation Frio 54 Numbe: a ra of dividing and degenerating eclis at various times afte conciation (rat eye Tansley Spear & Glucksmann) . mitotic count as per, a3 ee grt Mitotic count x percentage of cells which are degenerate 18 F frmn (y raya) »C 6mm D 10ma E 12min F, 30mm exposure at 1 Cooh, FV (1939) 308 LYTHAL EPPEOCTS a considerable proportion of those cella which attempt division in tho haurs following irradiation Tig 53 shows tho percentago of the pollen of Tradescantia which germinates, the anthers having received a dose of 800r at various times before dehiscing: If the dose of radiation is 100 to & ,° g & 4 & 3 4 7 3 a g 2 x 1) TE “4 Days before dehiscing Fia 53 Germination of pollen irrsdisted at different stages by 8 constant dose of 800 r (Newcombe) given one to five days prtor to the time of dehisemg, the per- centage of pollen germination is as high asin unirradiated pollen If the same dose 1s given 6 days or more prior to dehiscing, only 3% of the pollen grains germmate The discontmulty 1s due to the occurrence of the pollen grain mitosis about 6 days before dehiscmg The radiation 1s evidently lethal 1f nuciear division mtervenes bet the wradsation and the ger tion test, but not 1f no nuclear division intervenes If the change whtch leads to death at division 1s one from which recovery can occur, 1t may be posstble to reduce the lethal effect of a given dose by adopting some means of mereasing the time interval between irradiation and the next division Thus if eggs of the parasitsc worm Ascaris are utadsated by 8000r and allowed to develop at 25°, only 1 or 2% develop 2nto normal 1 Newcombe, HB (1942a) See also expemmentsa by Poddubnaja Arnoldi V (1936) and Koller PC (1943) DEATH AND DELAYED DIVISION INDEPENDENT 311 The cause of death at division The fact that m many experimental materials radiation delays division, and that a proportion of the cells die when they enter division after this delay, suggests the possiblity that delay of division and death at division may be essentially sxmilar phe- nomena differmg mm degree rather than in kind Qn this supposi- tion a moderate amount of damage to the cell causes some delay in entering division or a prolongation of the early stages, while a greater amount of damage (produced by a larger dose of radia- tion) causes increased delay mm entering division and breakdown dunng division This interpretation 13 not however inevitable, and appears to be defimtely wrong in a number of instances, mn which evidence has been obtamed that delay of division, and death at the resumption of division, are independent phenomena The general method of testing whether they are independent or telated phenomena is to vary the conditions of the expenment insome way,e g the type of radiation, dose rate, or temperature, and to see whether the two effects vary with the conditions in the same Way or in different ways If marme invertebrate eggs are radiated and then fertihzed, it 1s found: in some species (e g in the sea-urchin Arbacia and the clam Cumingia) that a moderate dose causes a prolongation of the time elapsing between fertilization and the first cleavage of the fertihzed egg, while larger doses completely prevent Cleavage from occurnng This 1s m keepmg with the view that the mhibition of division and the lethal action are actions dif- fering in degree rather than m kind But in other species (the marine worms Chaetopterus and Nerets) the largest dose which can be given without completely preventing cleavage m all the ©88s causes ttle or no delay m cleavage m those which survive Again, if Arbacia sperm are irradiated and then mixed with unfertihved eggs, a moderate dose causes a prolongation of the ume elapsing between fertibzation and the first cleavage of the fertihzed egg, while larger doses completely prevent cleavage ore two effects are caused by quite different mechanisms t eavage delay 1s due to the dissipation of energy by the radia- ion in the sperm itself, and the effect of a given dose 18 ade Pendent of the concentration of the sperm suspension and of the 1 Henshaw PS, Henshaw CT & Francis, DS (1933) 310 LETHAL FFFFCTS co workers: in a number of rapidly dividing twsues Fig 54 illustrates the typo of result obtained » After mrradiation matotic activity 19 reduced, and may remain practically at zero for some hours after the larger doses (ig 54F,F) The return of mitotic activity 18 found to comeide with the appearance of degenerating colls in the tisaue, strongly suggesting that the degenerative pro cess begins at division With tho smaller doses (Fig 544,8) 8 minonty of the cells entenng divinon degenerate With the larger doses (Fig 541,+)1t appears that the majonty of the cells which attempt division die in the early stages of mitosis and are recorded as degenerating cells Tho | that the degenerating cells seen in experiments of this typo are cells which break down at division has been con firmed by experiments on frog tadpoles sn which the mitotic activity can be controlled by varying the temperature or the feeding conditions 3 Irradiation normally results in the appeat- ance of degenerate cells a few hours later If, however, the tad poles are chilled immediately following an irradiation at room temperature (a procedure known to reduce the rate of entry of cells ito division in unirradiated tadpoles), the degenerate cells fail to appear Conversely, in experiments in which tadpoles are uradiated at a tume of reduced mitotic activity (brought about by chilling) degenerate cells do not appear until mitotic activity 1s reatored by returning to room temperature 1 Tansley, K Spear FG & Glucksmann A (1937), using rat eye tissue, Spear FG & Glucksmann, A (1938 1941) Glicksmann, A & Spear FG (1939), using tadpole eye and brain tissue Lasmitzla, I (1940, 19434), using chick tissue growing in culture 2 Tansley, K Spear FG & Gluckamann A {1937) on developmg rat eye tissue The numbers of cells im the various stages of mutosis were counted in the germinative zone 1n a section of an Irradiated eye, and compared with the numbers in 8 section of an eve of an unirradiated rat from the same hiter The number of degenerate cells per 100 (undif d) cells was alao d din the diated eye In drawing Fig 54 we have taken the sum of the numbers of cells in metaphase phase and telopt asa of the mitotic activity For this purpose it 18 preferable to omit prophase owing to the hkelihood of the duration of prophase m irad d tissue bly that n unirradiated tissue in which event the numbers of cells n prophase will not be a valid measure of the rate of entry of cells nto division 3 Gluckemann, A & Spear FG (1939) Dividing and degenerating eells were counted in the eye and brain DEATH AND DELAYED DIVISION INDEPENDENT 311 The cause of death at division The fact that in many experimental materials radiation delays division, and that a proportion of the cells die when they enter division after this delay, suggests the possibility that delay of division and death at division may be essentially similar phe nomena differing in degree rather than in kind Qn this supposi- tion a moderate amount of damage to the cell causes some delay i entering division or a prolongation of the early stages, while 4 greater amount of damage (produced by a larger dose of radia- tion) causes increased delay 3n entering division and breakdown dunng division This interpretation 1s not however imevitable, and appears to be definitely wrong in a number of instances, m which evidence has been obtamed that delay of division, and death at the resumption of division, are independent phenomena The general method of testing whether they are mdependent or related phenomena 1s to vary the conditions of the experrment msome way,e g the type of radiation, dose rate, or temperature, and to see whether the two effects v ary with the conditions in the same Way or in different ways If marine nvertebrate eggs are wradiated and then fertilzed, it 1 found: in some species (@ g in the sea urchin Arbacea and. the clam Cumimgia) that a moderate dose causes a prolongation of the time elapsing between fertilization and the first cleavage of the fertilized egg, while larger doses completely prevent cleavage from occurmng This 1s mn keeping with the view that the minbition of division and the lethal action are actions dif fering in degree rather than in kind But m other species (the marine worms Chaetopterus and Nerews) the largest dose which can be given without completely preventing cleavage 3n all the ®8g8 causes little or no delay in cleavage mn those winch survive Again, of Arbaca sperm are irradiated and then mixed with unfertilized eggs, a moderate dose causes @ prolongation of the time elapsing between fertahzation and the first cleavage of the fertilized gg, while larger doses completely prevent cleavage These two effects are caused by quite different’ mechanisms Cleavage delay 1s due to the dissipation of energy by the radia ion in the Sperm itself, ind the effect of a piven dose 1s inde Pendent of the concentration of the sperm suspension and of the 1 Henshaw, PS Henshaw CT & Francis DS (1933) 312 LETHAL EFFECTS naturo of the suspending fluid Fatlure of the sperms to fertilize effectively 18, however, an indirect action due to the production of somo change 1n the suspending fluid, oa 18 shown by the fact that tho offect of a given dose 15 greater in dilute than m concen- trated suspensions + Az has already been mentioned, recovery from the lethal effect of radiation on tho eggs of the parasitic worm Ascaris occurs if tho eggs are stored for a prolonged time at 5° after radiation Tasty 72 Relative doses of y rays, neutrons, and a rays which produce equal effects in the bean root tip Criterion of effect y Tays Neutrons @ rays Delayed division 10 05 20 Lethal effect 10 O12 on Howover, this storage leads to no recovery from whatever change 1s responsible for the cleavage delay also caused by radiation, for on returning to 25° after prolonged storage at 5°, the cleavage delay induced by the radsation 1s undiminished + In the bean root, tho efficiencies of y rays, neutrons, and & rays have been compared in cauaing delay in division an the rapidly dividing cells of the root-tip s The efficiencies of the same radia tions have also been compared in causing cessation of growth of the bean root, a phenomenon presumably due to the death of cells in the proliferating region of the root-tip + In the upper le of Table 72 are hsted the relative doses of the three radiations needed to reduce mitotic activity to the same fraction of the normal initotie activity three hours after wradiation In the lower line are listed the relative doses needed to cause cessation of growth in 50% of the root tips It 1s seen that the relative efficiencies of the three radiatzons depend upon which entenon of effect 1s chosen, a result which strongly suggests that the delay of division and the lethal action are due to mdependent mechanisms ‘ 1 Evans, TC, Slaughter, JC, Littl, EP & Failla G (1942) Dilute suspensions can be protected by the addition of protems It appears Likely that an ‘activated water’ reaction 1s concerned as in the inactivation of viruses un dilute suspension (p 108) 2 Cook EV (1939) 3 Gray, LH, Mottram, JC, Read, J & Spear FG (1940) Gray LH Mottram JC & Read,J (unpublished) Cp Chapter vitr p 303 4 Gray, LH & Read, J (19420 b,cd) Gray LH Read J & Poynter, BY (1943) *- INACTIVATION OF VIRUSES 313 These examples show that it 1s not in general safe to assume that the same change in the cell 1s responsible for delay in division and for the breakdown of a cell which attempts to divide after the expiration of the delay, though the assumption may of course be correct in some materials To account for the fact that death does in many cases occur dumng or following division, we must seeh for some action of radiation which s lethal at or following division but not lethal before division Genetical changes, or changes m the chromo- somes, suggest themselves as possibly fulfilung these require- ments, and these types of lethal action of radiation we now pro- ceed to discuss LETHAL MUTATIONS The inactivation of viruses The mactivation of the viruses, which has been separately discussed m Chapter 1, 18 the simplest example of a lethal action Jt wall be recalled that in the case of the small, crystal- lizable (macromolecular) viruses, an ionization anywhere in the ¥irns particle suffices to cause inactivation In the case of the larger viruses, it appears that a single 1onization 1s sufficient to inactivate & virus particle, but that it 1s necessary for the 1omza- tion to be produced, not anywhere in the virus, but in the ‘radosensitive’ part, which constitutes only a fraction of the total ‘olume of the virus particle Reasons were given mn Chapter rv for believing that this radiosensitive part 1s to be identified with the genetically important part of the virus, the larger viruses being supposed to have genetical and non genetical parts dif- ferentiated as m the higher cells, while m the macromolecular viruses no such differentiation 1s suspected An estimate of the swe of the radiosensitive region can be deduced from the radiation experiments As explained in Chapter 1m, from the measurement of the mactivation dose with & rays, an estimate can be made of the area presented to the radiation by the sensitive matemalin the virus The activation dose of vaccma arradiated by a-rays of about SeMV was found to be 211x105r: Now for a dose of 1000r of a rays of this energy, 071 & particles cross each square micron of iradhated tissue (Table 18, p 32) Thus witha dose of2 11x 105r anay erage Passat D E & Salaman MH (1942) The inactisation curve 1s re “yw 6p, p H3 314 LETHAL EFPEOTS of 1 & particle crosses each area of 1/(211 x0 71)=0 0067p? This then, 1s our estimate of the area presented to ana ray beam by the sensitive part of vaccinia virus It 1s leas than the cross sectional area of the w holo virus, w hich 1s about 0-23 x 0 17=0 04? Electron micrographs have however shown internal structure in this virus The micrograph reproduced in Plate [Vp: shows @ centrally placed body of diameter ranging from 0 08 to 0 12#n different virus particles (and posstbly additional smailer bodies), the corresponding area being 0 005 to 0 011? These areas are in fa agreement with the figure 0 0067% deduced from the a ray inactivation experiments It 1s a plausible conjecture to identify the radiosensitive region, the ewstence of which 13 inferred from the radiation experments, with the mternal etructure (or structures) revealed by the electron microscope, and to suppose that the genetical apparatus of the virus 1s contained in this structure A first speculation would be that a single 1omzation anywhere within this structure of diameter about 0 1 causes inactivation of the virus However, this appears not to be the case For if the target diameter 1s calculated on the basis that there 18 a single sphencal target, 1onization anywhere within which leads to in- activation ofthe virus, we find values of 31 mz for y rays, 41 mje for X-rays, and 70mp for a rays (see Table 36, p 123) As ex- plained in Chapter 111, a systematic increase of calculated target diameter with of ion density of the radiation 1s evidence that theres not a single spherical target, but erther a target which 18 very far from spherical, or a multiplicity of spherical targets ionization in any one of which leads to mactivation of the virus Thus last (mult: target) model 1s plausible sf we are prepared to regard inactivation of vaccima virus as 8 lethal mutation, pro duced by the :omzation of any one of a large number of genes Since nothing 1s Lnown, as yet, about the genetical apparatus of viruses, this explanation 1s necessarily speculative We shall, however, pursue it to the extent of showing that it affords an explanation of the manner m which the mactivation doses of the different radiations vary with the 1on density of the radiations It 18 also possible to estimate the size and number of the genes im x Green RH Anderson TF & Smadel J E (1942) Sumilar in ternal structure 13 shown in electron micrographs obtamed by Salaman, MH & Preston GD (unpubhshed) VACOINIA VIRUS 315 vaccima m the same way that estimates can be obtained of the ae and number of the genes in Drosophila by study of radiation- induced lethal mutations (p 179) The calculation 1s facilitated by the graphs provided in Chapter mm, and 15 made as follows Expernmentally, the in- activation doses of « rays of SeMV and y raysare 2 11 x 108 and 080x105r, which are in the ratio 264 1 Interpolating in Fig 10(p 90) between the curves for deMV and 6eMV a rays, we deduce that this ratio of mactivation doses corresponds to the target diameter 2r and density p, satisfying the relation 2rp=8my Taking the density to be 135 (ths being about the density of virus protem) we deduce that the diameter of the target 1s 6my The inactivation doses of the three radiations to be expected for a single spherical target of diameter 6 my can be read off from Fig 8A, and are seen to be 100-120 times greater than the ex- Penmental mactivation doses It follows that there are about TaBLE 73 Vaccima virus cal and expe 1 tivation doses X rays a rays Radiation y Tays {L5A)} GeMV ) Expermental 080 104 211 Calculated 076 114 218 All x 108r 110 targets in one virus particle, any one of which 1onized leads to mactivation of the varus (since the mactivation dose with NV targets 15 1/N times the mactivation dose with one target (ep P 90)) We conclude that in vaccima there are 110 genes each of chameter (or stnetly target diameter) 6myz The theoretical inactivation doses of the three radiations calculated on this basis: are set out in Table 73, and are seen to be in good agree ment with the expermental inactivati on doses surg os 84 shows that the inactivation dose of y rays expected with a ome le target of 6mp diameter 1s 084x107r With 110 targets the hese inactivation dose 1s 0 84x 107/L10=0 76x 108r The calcula a 18 made similarly for other radiations For @ rays of 5eMV it 1s nee ry to interpolate between the curves given for 3 and 6eMV ‘The numb, r and size of the genes given here are somewhat different from the same id and size inferred by Lea DE & Salaman, iH (1942) from the bh ‘xperimental mactivation doses owmg to their use of less accurate Physical data and methods of calculation 316 LFTHAL BFFFCTS Undue weight should not be attached to the estimate of the number of genes, since this 1s very sensitive to any error m the oxperimental inactivation doses or in the physical data and mothod of calculation We can safely any, however, that af we are correct in interpreting the inactivation of vaccinia virus 08 a lethal gene mutation, then the number of genes 13 certainly greater than one, though & great deal smaller than in an organism such as Drosophila in which there are beloved to be ceveral thousand The killing of bacteria Tt 38 convement to consider the bactericidal action of radia- tions at this pomt, since this can also be interpreted as lethal mutation Many authors havo studied the bactericidal action of X-rays, neutrons, radioactive radsations, and ultra violet bght: The general method 1s to uwradiate the bacteria either dry, or 10 aqueous suspension, or spread on the surface of a nutrient agat medium If the last-mentioned procedure 18 adopted the ob x Xrays Lacassagne, A & Holweck, F (1928, 19294,6), Wyckoff, RWG (1930a,6), Ellinger, P & Gruhn, E (1930) Claus WD (1933), Levin, BS & Lominsk: I (1935), Pugsley, AT Oddie, TH & Bddy, CE (1935), Luna SE (1939), Zirkle, RE (1940), Lorenz, KP & Henshaw, PS (1941) Lea, DE, Haines RB & Brotscher E (1941) arays Chambers, H & Russ, 8 (1912), Herak, F (1983 1934a,5). Bruynoghe R & Mund, W (1935) Lea DE, Hames, R B & Coulson, CA (1936), Luna SE (1939), Bonét Maury, P & Olwier, HR (1939) Zirkle RE (1940) 8 rays and cathode rays Chambers, H & Russ, S (1912), Wyckoff, RWG & Rivers, TM (1930), Knorr M & Ruff, H (1934), Baker SL (1935), Spencer RR (1035), Lea, DE, Haines RB & Coulson, CA (1936) yrays Spencer RR (1935) Dozow KP, Ward, GE & Hachtel, ry (1936), Lea DE, Hanes RB & Coulson CA (1937) Neutrons Lea,DE Haines RB & Bretscher,E (1941), Spear FG 1944) t Ultra violet hght Coblentz, WW & Fulton HR (1924), Baker, 8 L & Nanavutty, SH (1929) Gates FL (192925 1930) Ehrsmenn O & Noethhng, W (1932), Wyckoff RWG (1932) Dushkm, MA & Bachem, A (1933), Duggar, BM & Hollaender, A (1944¢ 6), Dreyer, G & Campbell Renton, ML (1936) Hollaender, A & Claus WD (1936), Herak F (1936) Koller, R (1939) Lea DE & Hanes, RB (1940) Wells, WF (1940) Rentschler HC, Nagy R & Mouromseff, G (1941) IRRADIATION OF BAOTERIA 317 servation consists simply n incubating the plate for a few hours until the colomes reach a size allowing them to be counted by naked eye or under low magnification, and comparing the num- ber of colonies on the radiated area with the number of colonies on an equal unirradiated area If the bacteria have been mrradi- ated in the absence of nutriment, suitable dilutions are moculated. onto nutnent agar plates after 1radiation, and incubated until the colonies are countable by naked eye The cnterion of death in these experiments 1s thus inability to give mse to a colony visible to the naked eye or under the low power of the micro- scope The usual method of reporting the result of an experiment 1s to plot a survival curve showing the fraction of the organisms capable of giving nse to colomes as a function of the dose Tt has been shown that the fraction of the organisms 1n an Aqueous suspension which are hilled by a given dose of radiation 38 independent of the concentration of the orgamams in the Suspension,: indicating that the death of a bacterium 1s due to energy dissipated by the radzation m the bacterium itself, and 13 oe indirect action due to the dissipation of energy im the water Some caution 1s required in the use of the method 1n which bacteria are urradiated while spread on the surface of nutnent gar, since 1t has been found that large doses of radiation pro duce Poisons in the nutrient medium which lull bactena sown on its In quantitative experiments precautions should be taken to ensure that the organisms yn a suspension are thoroughly dis- Persed before being plated For if the organisms are clumped, each colony will be produced not from a aingle organism but fom a clump of orgamsms A dose of radiation which renders ta the organisms meapable of colony formstion will not, under 86 circumstances, reduce the number of colomes observed to olf, aince a clump of several orgamams half of which are killed will still give rise to a visible colony To avoid trouble of this sort the aqueous suspension should be shaken mechanically for about 7, Lea DE & Haines R B (unpublished) Bact colt was irradiated one Tay in ag F of from 10 to 10* es per ml Co2 gsley,‘ AT, . Oddie TH & Eddy, yy CE (1935), using blentz, W Ww & Fulton HR (1924) X rays, using ultra violet hght 318 LETHAL EFFEOTS half an hour before use, and organisms such as Staphylococes having a strong tendency to clump are best avoided Most workers studying the bactericidal action of radiations havo obtained oxponential survival curves, indicating that the p2o -Noa 2o9s0— NaoOa o909~OyAebD Sfuravcitvoing eogoe-e2-2-2 Pom ao 3 «10° ergsfem * Dose Fia_55 Exponential survival curves of irradiated bactota A X raya({1 5A) on& aertryke (Wyckoff) B arayson Bact colt (Zirkle) C, # rays on spores of B mesentericus (Lea Hames & Coulson) D y raya on spores of B mega thertum (Lea Hames & Coulson} | neutrons on Bact colt (Lea Haines & Bretacher) F ultra violet light (28034 ) on B megathertum {Horcik) fraction of the orgamsms lulled by a given mcrement of dose I t, throughout the irradiation When the aur- viving fraction 1s plotted on a logarithmic scale against the dose, BACTERIA SURVIVAL CURVES 319 a straight line 1s obtained: Examples of exponential survival curves obtamed with various organisms and radiations are plotted in this manner in Figs 55 and 57 Occasionally survival curves deviating systematically from the exponential shape have been reported The deviation takes the form of the fraction of orgamsms lulled by a given increment of dose bemg less at small doses than at large doses, so that a sg- mod survival curve 13 obtained A typical sigmoid survival curve, plotted on linear and logarithmic scales respectively, 13 shown in Fig 56, curves c and d, curves a and b being exponential survival curves It 1s probable that exponential survival 1s the typical result, and that the occasional finding of a sigmoid curve 1s due to some disturbing factor One such disturbing factor 1s clumping of the organisms If the proportion of organisms which survive a given dose 1s the exponential function e-*, where 2 18 proportional to the dose, then the probabhty that an individual organism shall be killed by this dose is 1-e-* If the organisms are sown in clumps of n dividuals, the probability of all n organisms of a clump being killed will be (l1—e-*)" Hence the proportion of the clumps which produce colomes after a dose proportional to = will be 1-(1—e-2)" Tins function represents, not an expo- nential but a sigmoid survival eurve Curves c and d of Fig 56 have been calculated by this formula with n=4, and ulustrate the survival curves to be expected when the organisms are in clumps of 4 Curves a and b of Fig 56 are the survival curves 7, 1e the survival curves to be expected for the same organ- isms when not clumped Sigmoid survival curves which have been obtamed with organisms such as Staphylococc: having a strong tendency to clump are probably to be explained in this fashion 2 A A second cisturbing factor resulting in a sigmoid survival wah being obtamed may be met with when the organisms are whe while spread on the surface of a nutrient medium in doses oe substances are produced by the radiation At small © concentrations of the poisons will be insufficient to hull son Chapter UI p 72 for a fuller discussion of methods of plotting 2 Gates be and the :mplications of exponential survival Gurene caf b (19294) obtained sigmoid survival curves with Staph lated by ultra violet light 320 LFTNAL FFFFCTS any bacteria, and tho rate of death observed will be that due to tho direct action of the radiation on the bactena With larger doses the concentration of poisons may be large enough to cause appreciable mortahty, and the rate of death will then be greater than the rate duc to the direct action of the radiation alone: Sfuravcitvoing Dose Tia 56 Survival curves (theoretical) andb exponential survival eurves e-* cand d sigmoid survival curves 1-(1—e-#)* r Ths mey be the explanation of the distinctly sigmoid survival curve F (1929q) irradiating 'Pyo \ eyamgue S* nh & Holweck A rays of wave A Lee DE Hames length 83A same RB & Coulson, CA (unp ) ting the with the BACTERIA EFFECT OF INTENSITY 321 The most convement way of summanzing the result of an experiment in which an exponential survival curve 1s obtamed 1s to state the dose which reduces the surviving fraction to e1=37%: This dose, which in the case of virus and enzyme mactivation we referred to as the mactrvation dose, 1s more appropnately referred to as the mean lethal dose when bactena are concerned Table 74 summanzes the results of experrments which show that the mean lethal dose 1s the same, within the error of the Tastz 74 Independence of mean lethal dose on intensity 7 Intensity MLD Radiation Organism rfémin r Reference @ rays Spores of B 9 84x 108 23x lot 1 mesentericus 6 12x 108 26x 104 X rays (8A) Spores of B 6 24x 10° 15x 10s x mesentericus 6 02x 10* 12x 105 4 70x 105 17x lo Xrays (015A) — Bact cols 63 56x 108 2 209 &7x10° ergs/cm */sec ergs/em ® Ultra violet ight {2536A Bact colt 12x10! 75x 10° 3 ) 31x 10s 85x 105 64x10 85x 10> } peo DE, Haines, RB & Bretscher E (1941) Les, DE & Haines RB (unpublished) tho m 2, DE & Hames, RB (1940) The exposure time required to deliver t ean lethal dose in these experiments ranged from about 1 min to about sec Rentschler HC Nagy R & Mouromseff G (1941) have shown that ordeeas tl dethal dose 1s still unchanged when it 1s delivered in a time of the expenment, whether the wradation 1s made at a low intensity and spread over a prolonged time, or at a high intensity and concentrated in a short time The effect of a given dose has also been found to be inde- Pendent of the temperature at which the bacteria are maintame d ring the irradiation Table 75 showss the fractions of spores of . ptenterscus which survive a constant dose of 2x10'r of Y8 given at vanous temperatures The mean lethal dose has Ba " me wav e length action of of X ra ys obtamed an exponential survival curve the the organisms surviving @ given dose being greater than in 1c gne and Holweck’s experiment, especially at large doses : Py Chapter m, p 74 3 re Table 86 p 374 for additional data B,Hames RB & Coulson, C A (1936) 322 LETHAL EFFECTS also been found to be indopendent of the temperature when spores of 2 mesentertcus are irradiated by f rayst at 41, 20 and —20°, when Bact cols 1s irradiated by y rays: at 0 and 37°, when B prodigiosus 1s irradiated by @ rayss at various tempera tures from 2 6 to 36°, and when Bact cols 13 srradsated by ultra violet hghts at & and 37° Tante 75 Independence of tempersture {a rays, 2x10'r on spores of B mesentericus) Temperature °C Fraction surviving +50 0514006 +20 0524003 0 0564003 —20 058+003 A limited number of researches have been made to compare the efficiencies of different radsations in kalhng bactena It 1s highly desirable that the experments with the different radia- tions should be made by a single group of workers under condi tions as nearly as possible alike Misleading conclusions are likely to be reached if results obtained with the different radia- tions by workers in different laboratories are collated, even though the orgamsms they are using are nominally the same Lacassagne and Holweck,s radiating ‘Pyocyantque S’ with two wave lengths of soft X rays, obtamned an exponential sur- vival curve with X-rays of 4A, and a sigmoid survival curve with X rays of 8A They interpreted the exponential survival curve to mean that a single quantum of the 4A radiation sufficed to lull the organism The sigmoid survival curve was interpreted on the mult: hit target theory (cp Chapter 1, p 71) to mean that four quanta of the soft radiation were needed to kill the organisms The sigmoid curve has however not been reproduced by other workers wradiating the same organisms with X-rays of the same wave length 6 so that this interpretation 3s probably incorrect x1 Lea DE Hames RB & Coulson CA (1936) 2 Lea DE, Hames, RB & Coulson CA (1937) 3 Heretk, F (19348) 4 Rentschler, HC Nagy R & Mouromseff G (1941) 5 Lacassagne,A & Holweck F (19294) 6 See p 320, footnote 2 BACTEBIA WAVE-LENGTH DEPENDENCE 323 Wychoff wrradiated Bact colt with soft X-rays of several wave- lengths, and obtamed the survival curves shown in Fig 57: The mean lethal doses read off from these curves: are given in Table 76, and are seen to mcrease on the whole as the wave-length of to 08 o6- A B O-4F- o2b FO ean 1 ale, L § ap 5 ite) 5 [e) 5 goer Cc B E ost i-4 & = z 3 a L i 10 15 L ‘ 3 10 6 Fa) 3B xe Dose Fig Si Kiting of Bott coh by sok Stays (Wrekos) A O64. BOTA. C,15A. D23A. E 408 the radiation increases, 1e as the 1on-density of the photo- electrons which dissipate the energy m the bacteria mcreases Results obtained by other workers studying the relative efficiency of different radiations in lolng Bacé cols are also given mTable76 Itisseen that, whule the results of the three series of &xpenments do not agree with each other numencally, in each T Wvchot, RWG {19308 ) 2 Except m the case of wave length O-T1A where the mean lethal 18Ose RivenaainTe.fable shown th 1s the mean of two expermments only one of which 322 LETHAL EFFECTS also been found to be indopendent of the temperature when spores of B mesentericua are irradiated by f raya: at 41, 20 and —20°, when Bact cols is uradiated by y rayss at 0 and 37°, when B prodigiosus 13 rrachated by a rayay at vanous tempera tures from 2 6 to 36°, and when Bact cols 1s irradiated by ultra violet lights at 6 and 37° Tanty 75 Independence of temperature {a rays, 2x10'r on spores of B mesenterseus ) Temperature °C Fraction surviving +50 05130 06 +20 0524003 oO 0564003 -20 0584603 A limited number of researches have been made to compare the efficiencies of different radiations in Jalling bactena It 1s highly desirable that the experments with the different radia tions should be made by # single group of workers under condi tions as nearly as possble alike Mislead } are likely to be reached of results obtamed with the different radia- tions by workers in different Jaboratones are collated, even though the orgamsme they are using sre nominally the same Lacassagne and Holweck,s wrachating ‘Pyocyantque S’ with two wave lengths of soft X rays, obtamed an exponential sur- vival curve with X rays of 4A, and a sigmoid survival curve with X-rays of 8A They interpreted the exponential survival curve to mean that a single quantum of the 4A radiation sufficed to all the organism The sigmoid survival curve was interpreted on the mult: nt target theory (cp Chapter 1m, PD 71) to mean that four quanta of the soft radiation were needed to lull the orgamsms The sigmoid curve has however not been reproduced by other workers radiating the same organisms with X rays of the same wave length,‘ so that this interpretation 3s probably incorrect x Lea, DE, Haines RB & Coulson CA (1936) 2 Lea DE, Hames, RB & Coulson, CA (1937) F (19346) 3 Hercik, 4 Rentschler, HC Nagy, R & Mouromseff, G (1941) 5 Lacassagne A & Holweck F (19292) 6 See p 320 footnote 1 LETHAL MUTATIONS IN BACTERIA 325 These results parallel those obtasned im the study of the in- activation of viruses and the production of mutations m Droso- phila, and suggest (cp Chapter mt) that a single 1omzation (in the nght place) suffices to lalla hactenum This conclusion 18 only plausible biologically if we suppose the effect to be on the genetical apparatus of the bactermmm For the typical effect of one tion 1s ch 1 change in one molecule, and it 18 un- hkely that the concentration of any cytoplasmic constituent 18 so neely balanced that change m a single molecule of it leads to the death of the bactenum On the other hand, in orgamsms which have been investigated genetically, lethal effects due to losses of, or changes in, single genes are common t The idea that the lullng of bactena by radiation 1s to be interpreted aa lethal mutation 1s on other grounds plaumble In the researches on which 1s based the conclusion that death ws due to a single 1omzation, the emterion of death has invanably been the inability of the wradzated bacterium to give rise to a colony visible to the naked eye or under low magmification The few microscopical studies which have been made of the behaviour of individual bacteria after wradiation have shown that a bac- tenum exposed to a dose sufficient to prevent its giving nse to ® colony does not suffer rapid disintegration, as happens for exemple when bacteria are killed by strong chemeal dism- fectants A motile bacterium retains its motility,: and though faihng to give nse to a visible colony, a bacterum which has been lulled? by radiation may divide once or twice when sown onto | nutnent medium 3,4 A spore whtch has received e dose large enough to prevent its giving mise to a visible colony will neverthe- less germmnate when sown onto a nutrient medium « These results Suggest that, as 1s often the case with higher cells, the death of an uredisted bacterium occurs at or following division The Cause may very well be an effect of radiation on the genetical teeeretus We shall continue the discussion on the assumption ton “e effect m question 1s of the nature ot a Jethal gene muta- nce practically nothing 1s hnown about the genetical pparatus of bactena, this standpormt 18 provisional We adopt 88 Tecesaive lethals sophia, where losses of single genes usually behave 1 At any rate m Drosoph i Rannoshe R & Mund, W (1935) now, CF & Lea, DE (unpublished) 3 Lune, SE (1939) 324 LFTIHAL EFFECTS series the mean lethal doses increase from left to mght m the table, which 18 the order of increasing 10n density A similar increase of mean Iethal dose with increase of ton density has been found with other vegetative bactena + With spores, however, 1t has been found that the mean lethal dose 1s Tantz 76 Moan lethal doses of various radiations an Bact cols (Doses in units of 1@r or 10°} Reference X ray wave length (A ) 058 ov 15 23 40 r Mean lethal doso 42 46 43°67 #84 Radiation X rays (03A) a raye(~SeMV ) a rays(~2eMV)} 2 MLD 39 o7 G4 X mys — Radiation £ rays y mye O15A 15A 83A Neutrons a-rays 3 MLD ‘4 5 60 65 75 71 24 2 Zirkle, RE (1940) This I uthor ore doses an units of eV fa" tissue For i Wyckoff RWG (19306) we purpose of Table 76 these dosea have been calculated back to roentgens, 7 Eee, bE,biees R.B & Brotecher,E (1941) the peal data quoted by Zirkle the same for y rays and different wave lengtha of X-rays, and 1s leas for a rays* This interesting difference in behaviour between apores and vegetative bacterra has not yet been suffinently investigated The killing of bacteria interpreted as lethal mutation The studies of the bactericidal action of radiations which we have reviewed im the preceding section have established the following results (1) The survival curves are exponential (n) The mean lethal dose 1s dependent of the ty and of the temperature at which the irradiation 1s made (u) In the case of vegetative bactena, the mean lethal doses of different in the order of increasing 10n density 1 Lea DE, Haines, RB & Coulson, CA (1936), using S aureus, Lea DE Hames, B pyo RB & Coulson, CA (unpublished), usmg cyaneus and B proteus 2 Lea, DE, Hames RB & Coulson, CA (1936), Lea, DE, Hases RB & Brotscher, BE (1941) usmg spores of B mesentericus LETHAL MUTATIONS IN BACTERIA 327 Studies of the killing of bactena by monochromatic ultra- violet hight have shown that the survival curves are exponentral and that the effect of a given dose 1s independent of the in- tensity « These results suggest that, as with omzing radiations, Taste 77 Bact cols calculated and experimental mean lethal doses A rays —_-—_— Rediation rays yrays 015A 115A 83A Neutrons a rays Expermental 4 52 60 65 75 71 a4 Celeulated 44 44 57 78 176 88 23 All x10°r or 102 the effect 1s caused by a single unit action, which with ultra- violet hght would mean the absorption of a single quantum No third test, corresponding to the ion-density vamation test with ionizing radiations, 1s applicable with ultra violet hght, so that the interpretation that the lethal effect of ultra-violet light 1s due to the absorption of a single quantum (in a particular region) 13 Tess securely established than is the interpretation that the lethal effect of an lonizing radiation 1s due to the production of a single ionization Quantitatne experiments usually give the incident energy in ergs/em ? necessary to reduce the survis ing fraction to a given fraction, conveniently chosen to be 37% The absorption co effiaent of bactenal protoplasm being hnown,: one can deduce the mean lethal dose in units of ergs per gram Doses of 1onzing, tadiations measured in roentgens can be converted to ergs per a by means of Table 2(p 8) In ths way it 1s founds that : a a hundred times as much energy (in ergs) 1s dissipated in t —_ lilled by ultra-violet, ght as in a bacterium hilled to. i, If one supposes the lethal effect to be a gene muta- absorbed quantum yield (1e the probalnhty that a quantum lese the Bee gene shall cause lethal mutation) 13 very much ds n the ionic yield A similar conclusion was reached in cussing the mactivation of viruses (p 125) 1 Eg Wyckoff RWG (1932), Lea, . 3 see F L (1930), guoved 2 Tbe Ls p 3 Haunes RB (1940) DE & Hanes RB (1940) for details 326 LETHAL FFFYOCTS it since a good representation of the radiation results can be given on the assumption that we are dealing with lethal gene mutation According to the bottom hino of Table 76, the imactivation dose of Bact col irradiated by a raysof about GeMV 19 24x ier With rays of thes energy, 071 & particles cross ench square micron of radiated tissue per 1600 r (Table 18, p 32) Thas with a dose of 2 4x 10's an average of 1 a-particle crosses each area of 1/(24x071)=000.? Now while tho ordmary hactero logical staining methods do nat show mmternal structure, by the use of stable methods internal structures have been demon- strated in bacteria Plate [Vc shows a micrograph of Bact colt: taken from an 18 hr culture such as was used in the radiation expennments Internal structures are clearly visible having & diameter of about 0 3: and an area therefore of about 0 077" In view of this comendence of areng, st ts plausible to suppose that for an & particle to produce a lethal mutation it 13 necessary for at to pass through this structure The mternal structures are believed on observational prounds to be chromosomes + Further calculation proceeds exactly as in the case of vaccum virus which was discussed eather (p 318) It1 apparent to begin with that the hypothesis that « single iomzation anythere mn the body of diameter 6 3c causes Jethal mutation 1s nat satisfactory For, 1f we make the assumption that there is a single spherical target 1omzation anywhere within w hich causes lethal mutation, we obtain a target diameter which with of 1on- density from 0 08, for y-rays to 0 2 fora rays We can however obtain a consstent representation of the deta by assuming that there are a large number of genes, tomzation of any one of which canses lethal mutation By proceeding as desersbed mn the case of vaccimta virus it 13 found that the assumption of 250 genes having an average diameter of 12mp satisfactorily fits the ex- perimental data The companson of experimental mean lethal doses with the values caloulateds on this phon 1s made 2 Table 77 1 Robinow, CE z Badian, J {1933}, Robinow, CF (1642, 1944) 3 Somewhat different estunates of the aze and number of the genes were obtaned by Lea DE Hames, RB & Bretscher EB (1941) + the same pert ; i data owing to the use of less accurate B i dats and of DROSOPHILA EGGS 329 death of the cells concerned at or following division It has already been pomted out that it 1s at this time that cells die which are killed by moderate doses of radiation It 1s evident therefore that lethal chromosome structural changes must ac- count for some of the cells which che at or following division, but it 1s not yet possible to say in general whether the majority, or only a mmority, are to be accounted for in this way One can attempt to answer the question in the case of the few materials, Particularly Drosophila sperm and Tradescantia microspores, in which chromosome structural changes have been sufficiently in- vestigated Drosophila melanogaster Changes in Drosophila sperm w hich result in the egg fertilized by an iwradhated sperm failing to develop to the adult stage have been discussed in detail an Chapter \ under the heading Dommant Lethals (p 161) The conclusion was reached that the whole lethal effect could be explained as being due to lethal types of chromosome structural change The demonstration, though strongly Suggestive, was not completely convincing since in Drosophila only viable types of structural change have been Studied In particular, the proportion of breahage ends which undergo sister union m preference to joining with other breakage ends has to be determined from expemments on dominant lethals and sex ratio distortion, since 1t cannot be determimed from a study of salivary chromosomes It 18 probable that the whole dommant lethal effect obtained by radiating Drosophila sperm can be explained an terma of chromosome structural change, but the evidence 1s at present circumstantial ten Drosophila eggs are wradiated before fertihzation, a taal Proportion of the zygotes fail to develop into adult fhes 20 when it 1s the sperm which are urradiated by the same dose : et many fewer chromosome interchanges are produced by uradiating unfertilized eggs than by nradiating sperm: with the Same dose t ‘has been remarked that this observation shows that while obtan be correct to ascribe the whole dominant lethal effect atry, ed by uradiating Drosophila sperm to chromosome etural change, some of the dommant lethal effect produced in : See ©g Sonnenblich, BP (1940) ee eg, Glass HB (1940) 3 Muller, HJ (1938) 328 LETHAL EFFECTS LETHAL CHROMOSOME BTRUCTURAL CHANGES Incidental reference to types of chromosome structural change which are probably lethal has been made in Chapters Vv and v1 Tho principal types likely to have a Iethal effect are simple breaks (Figs 304 and 31a,n), and asymmetrical exchanges (Figs 30D, E,G and 31p,F) Al] these types result in the production of acentric fragments, which will be lost at division gooner or later owing to their Incking the centromere which ordinanly ensures thew inclusion in a daughter nucleus afferent organisms are tolerant of the Iting genetic defi in diff degree In Drosophila melanogaster, the loss of even 6 %, of the X chromo some has a dommant lethal effect, while 2n maize the Joss of even 8 whole chromosome may be viable Most of the types of structural change mentioned above result also sn the production of dicentric ch and cl tid: Dicentrics probably often have a lethal effect apart from ther association with acentno fragments This may he explamed partly by the genetic unbalance resulting from unequal breabage of dicentric chromosomes which form bridges at anaphase, and partly by the mechanical difficulties experienced by a dividing cell in which ar, anaphase bridge forma (ep Chapter v, p 163) Bridge formation 1s however not lethal in all materials, smce the bridge may break, and the daughter cells may then survive if the genetic unbalance 18 not too severe « Simple breaks which do not restitute, and asymmetrical ex- changes (other than 1 delet which hehave as recessive Jethals rather than dominant lethals) are certainly lethal m Drosophsla sce evidence of them 1s never found in the sahvary chromosomes In plant matenal, which 1s more tolerant of genetic unbalance, a certain number of these aberrations may be viable though probably most are not Symmetneal exchanges are viable im all orgenisms investigated, but they result in partial stenhty (see p 338) Lethal types of chromosome structural change will lead to the i If the triploid zygote from which the endosperm tissue of maize develops contams a broken chromosome” sister umion of chromatids occurs at the kage point and an \p bridge results This bridge breaks, and in each daughter cell sister umon occurs at the breakage ends resulting m bridges at the next division ‘The process of bridge formation breakage and sister umon thus in cell in the endosperm (McChntock B 1941a) DROSOPHILA EGGS 329 death of the cells concerned at or following division, thes° that ce! already been pointed out that rt 1s at this time on It 18 eviden which are Jalled by moderate doses of radiati s must ac- therefore that lethal chromosome structural changedivision , but ng count for some of the cells which die at or followithe it 18 not yet possible to say im general whether majority, or only a mnority, are to be accounted for in this way One can few matenals, attempt to answer the question in the case of the in particularly Drosophila sperm and Tradescantia microspores, which chromosome structural changes have been sufficiently in- vestigated Drosophila melanogaster Changes in Drosophila sperm v hich result in the egg fertihzed by an srradhated sperm failing to develop to the adult stage have been discussed mm detail in Chapter + under the heading Dommant Lethals (p 161) The conclusion was reached that the whole lethal effect could be explained as bemg due to lethal types of chromosome structural change The demonstration, though strongly suggestive, was not completely convineing since in Drosophila only viable types of structural change have been studied In particular, the proportion of breakage ends which undergo sister union in preference to joing with other breakage ends has to be determmed from experiments on dominant lethals and sex ratio distortion, since 1t cannot be determined from a study of sahvary chromosomes It 1s probable that the whole dommant lethal effect obtamed by mrradiating Drosophila sperm can be explained in terms of chromosome structural change, but the evidence 1s at present circumstantial When Drosophila eggs are wradhated before fertilization, a lugher proportion of the zygotes fail to develop into adult flies than when it 13 the sperm which are irradiated by the same dose : Yet many fewer chromosome mterchanges are produced by uradating unfertilized eggs than by uraciatang sperm: with the same dose Tt has been remarkeds that this observation shows that while it may be correct to ascribe the whole dommant lethal effect obtained by irradiating Drosophila sperm to chromosome structural change, some of the dommant lethal effect produced m 1 See, eg, Sonnenblch BP (1940) 2 See eg Glass, HB (1940) 3 Muller, HJ (1838) 380 LYTHAL EFF? OTS unfertiized eggs must be due to some other cause (e g aneffectan tho cytoplasm) This conclusion, though superficially the obvious Reduction from the dats, docs not follow af chromosome etruc tural changes and dominant Jethals are interpreted as in Chapter Vip 166) Two constants o and g enter info the expression for the yields of chromosome atructural change and dominant Yothals a is the number of breaks primarily produced per nucleus per 1000r Tt 1 probably determined mainly by the ahmensions of the chromosome thread and ss therefore probably the same in the egg and the sperm q 1s the probalnlity that 8 breakage end primarily produced shall som with another break age end in pref tor g unjomed unt the time of chromosome split, and then undergoing aster umon g will be determmed by such factors as the time of spht and the freedom of movement of the chromosomes, and may sery well be different in the egg and sperm nucle: Tahing to be the same for egg and sperm nucle, the expert mental feet that many fewer viable chromosome structural changes are produced in the egg than mn the sperm by o given dose D means that ¢ 18 much smaller in the egg than in the sperm, wi which ot has the value 0762 Tho etpresnon for the proportion of the fertshaea eggs which develop to the adult stage when one of the gametes has been given a dose D 1s given by equation V-2 {p 167) as e-*7S; e~D depends on a but not on g, and rs less than unity S, de- pends on g as well as «, takes the value unity when g=6, and 13 greater than unity when gis greater than 0 Experimentally the proportion of fertihzed eggs wlnch hatch when the egg has been uradiated dimimishes approximately exponentially with the dose a Evidently for the egg which we have already deduced 25 emaller then g for the sperm, 19 sufficiently nearly zero for S, to be practically umty 3 z Mose premsely 2: terms of the theory of Chapter v, the proportion of viable gametes havmg structural change 2 1 -S,/S, (p_ 167), and ass seen in Table 42, this quantity mereases with merease of og a and D bemng the same tn egg and sperm g must therefore be smalier in the egg 2 Sonnenbick BP {3940} 3 The assumption that ¢ is nearly zero 15 equivaient to assuming that nearly all the breaks prunariy produced undergo aster union, and there fore behave as lethals m preference to reakttuting or exchangmg mith other breaks DROSOPHIL4 EGGS 331 Thus when it 1s the unfertihzed egg which 1s radiated, the Proportion of fertilized eggs which develop to the adult stage 13 eP This 1s smaller than when 1t 1s the sperm hich 1s radiated on account of the omission of the term 8, Making this argument quantitative, it can readily be shown that a dose which results in 50 % of dominant Icthals when given to the sperm will result in 86% of dominant lethals when given to the fertilized egg « This deduction may be compared with the experimental results of Sonnenblick, who found that a dose which gave 40%, of dommant lethals when del ered to the sperm gave 82% of dormant lethals when delivered to the unfertilized egg, while a dose which gave 56% of dominant lethals when delivered to the sperm gave 94% of dominant lethals when dehvered to the egg Its evident that dominant lethals in unfertilized eggs, as well 48 In sperm, can be explamed by lethal types of chromosome structural change Several authors: have studied the lethal action of X rays on fertuhzed eggs of Drosophila The general method employed 1s to Provide the flies with food on which to deposit their eggs, to col- lect a batch of eggs, and to irradiate them at a time after laying which vanes in different experiments from a few minutes to a few hours The proportion of eggs which hatch is plotted as a function of the dose, sigmoid survival curves being obtained, and the dose required to reduce the proportion hatching to 50% 1s determined The sensitivity of the eggs to radiation varies with the stage of development, the 50 % dose haying a minimum value of 160r 90-120 min after laying 3 The 50% dose for a batch of ©ee8 uradiated within 30 min of laying when usually not more than one or two nuclear divisions have occurred, 1s 290 r Direct cytological observations have not been made to de- termine whether chromosome structural changes account for the make ne = 0 76 for sperm, 50% survival is obtained at a dose which aD aos 195 whence agD=148 With these values of aD and aqD, fertilmod ee and S,=3 5 (interpolating in Table 42 for S,) For un We tak 683 radiated by the same dose we take aD = 1 95 as for sperm roe ©q~0 80 that S,=1 Thus the survival is now e~*2=0 142 which ‘ans 86% of dominant lethals ° kd Packard C (1926) and later papers Henshaw PS & Henshaw, (1933) and later papers 3 Packard © (1935) 332 LETHAL EPFECTS lethal effect The best one can do therefore at present 18 to at- tempt to make an estimate of the proportion of nuclei hkely to suffer lothal chr structural change on the bams of the dats arvnilabfo for sperm and unfertihzed eggs Presumably, for a diploid nucleus, a, the number of breahs pomanly produced per 1000r will havo o valuo twice os great as that for a haploid gamete,2e a=1 5 fora diploid nucleus No data are available from which to estimate the value of g If we assume 1t to be sniall, as appears to be the caso in the unfertilized egg, the pro portion of irradiated diploid nucle: which survive a dose D will be e-?P With a dose of 200r, aD=029x15=0435 and e250 65 Thus the dose of 200r, which 13 observed expen mentally to hall half of the eggs srradiated at an early stage of dev clopment, 1s sufficient to produce lethal chromosome struc tural changes in about one third of the nucle: in an egg This calculation suggests that lethal chromosome structural changes may play an important part in accounting for the kullng of Drosophila eggs in an early stage of development To sum up our discussion of Drosophila, we may say that there are strong indications that Jethal chre structural chang play © major part in accounting for the failure of eggs to hatch after the rrradiation of the sperm or egg prior to fertihzation, oF of the egg soon after fertilization The evidence 1s, however, at present circumstantial Tradescantia Chre structural changes visible at the metaphase of the first haploid mitosis (pollen gram division) m Tradescanha followmg irradiation of the microspore pnor to this division have been ex! vely investigated « The subsequent behaviour of the pollen grain has also been studied 7 At the pollen gra:n mitosis (in unirradiated material) nuclear division but not cell division occurs, so that the two daughter nucle: remain in the same cell Of the two nuclei one, the gene rative nucleus, will divide again after the germmation of the pollen gram and give mse to the sperm nucler, which take part im fertahzation The other nucl the ti leus, plays no role after germmation has occurred 1 See Chapters v1 and vit 2 Koller PC (1943) TRADESCANTIA POLLEN 333 Dunng the week which follows the pollen gram mitosis, the two nucle: become distinguishable, the generative nucleus de- veloping into a long crescent shaped body When at the end of this time the mature pollen grain falls onto the stigma of a plant or a smear of artificial medium on a micro- scope slide, germmation occurs A pollen tube grows out of the pollen grain, the elongated generative nucleus enters the pollen tube, and undergoes division (the second haploid mitosis) there ‘Tf the microspore has been irradiated prior to the pollen grain mitosis, after this division small nucler (mrcronuclet) additional to the generative and vegetative nucle: may be seen These are acentric chromosome fragments which have failed to be mcluded in either daughter nucleus at the pollen grain mitosis TaBre 78 Correl. of the of with failure to develop after irradiation? Micronuclea = Macronucle: present absent. Pollen grain differentiation Normal 60 165 Suppressed 217 15 Pollen tube Normal 34 92 Absent 330 100 A microspore which has received a dose of a few hundred Toentgens during early prophase of the pollen gram division, or the interphase preceding, completes this division In some cells subsequent development proceeds normally, in others the gene- Tative nucleus may fail to differentiate and the pollen grain fail to germinate, and so die without having been able to carry out its function of fertihzation It has been found that there 1s a marked correlation between failure to develop normally and the existence of micronuclei ,1 of acentric chromosome fragments This 1s illustrated in Table 78, which shows that of the pollen grams which fail to develop Pollen tubes, or in which the generative nucleus fails to dif- aaenate, the majority have micronuclei, while of those which levelop normally the majonty are without micronuclei the wee observations on Tradescantea give strong support for ae tef that chromosome structural changes resulting in entric fragments which fail to be included m the daughter nuclei are often lethal dehuseimg” 1 Koli PC (1948) A dose of 360r was given 8 days before 334 LETHAL EFIEOTS A further demonstration of the relation between chromosome structural changes and Jethal effect 1s provided by Tig 58 The curve shows the proportion of pollen grain metaphases showing lethal types of chromosome structural change (1¢ chromatrd 1 L L L 100 20 Buu 0 Doge in roentgens F1o 58 Relation betwoen chromosome structural changes and lethal effect 0 Tradescantia pollen grains Curve showa percentage of pollen grain metaphases showing lethal types of I change Exp 1 points (Koller) show percentage of pollen grains failing to germinate breaks, isochromatid breaks, and asymmetrical exchanges) 48 & function of the dose given 24 hr before pollen grain metaphase ' The points show the proportions of pollen grains which fail to germmate as a result of doses of 90 and 360r given at about this same stage + 1 Authora have not scored aberrations in this particular way The curve has been constructed in the following fashion, making use of the ffi of aberration production hsted im Table 64, p 2421 The yields of chromatid and 1sochromatid breaks per cell produced by a dose D roentgens are 0 725 x 10-* D and 0 271 x 10-* D respectively The yield of ch tid produced by dose Dig 1 81x 10-* @D? (for the factor G see equation (VII 8), p 264), and of these half are asymmetrical dose D ‘Thus the mean number of lethal aberrations duced per cell by 1s m= 0 996x 10-5D+0 905x 10-*D7@ The proportion of cells having one or more lethal sberrations 1s 1— e-" with this value of m 2 Koller, PC (1923) Kollers figures for irrachation made $9 days before dehiscing have been used The proportions of pollen grains failing to germmate are somewhat higher if the irradiation 1s made 10 days before dehiseing, somewhat lower if made 8 days before dehiscing ~ BEAN ROOT 335 There 33 fair agreement between the points and the curve, showing that chromosome structural changes are able to account for the failure of pollen grains to germinate The fact that at high doses the proportion of pollen grains failing to germinate 1s not so lugh as the proportion of pollen grain metaphases having lethal types of chromosome structural changes suggests that some of the ‘lethal’ types of chromosome structural change do not prevent pollen gram germination These may be the smaller deficiencies , or it may be that only half of the chromatid breaks are lethal, namely, those which cause deficiencies in the vege- tative nuclens + The bean root The lulling of bean roots by the radiation of the region near the root typ im which the cells are rapidly dividing has been extensively studied 2 In the umrradsated root-tip, half of the daughter cells formed in the dividing region remain there and undergo further division The remainder do not divide further but elongate It is the addition of these elongated cells to the root behind the dividing region which accounts for the increase of length of the root If the root trp 1s irradiated, the rate of increase of length of therootdummishes This 1s due to some of the cellsin the dividing region bemg killed Asin other rapidly dividing tissues wradiated by moderate doses, the cells breah down at division After small doses the rate of mcrease of length of the root eventually returns to normal, imdicating that a sufficient number of cells in the dividing region have survived to restore (by division) the normal cell population in this region With larger doses the growth of the Toot 1s not resumed, and it dies, presumably indicating that nearly all the cells in the dividing region have been hulled tare are a number of indirect lines of evidence suggesting ascribed pune of bean roots by radiation 1s largely to be doses. 0 lethal types of chromosome structural change The €s ol'y rays, X rays, neutrons, and a rays needed to kill 50% nuelonn ts the assumption suggested by Koller that it 1s the vegetative tween the not the generative I which 1. the resp germination gma or culture medium and pollen grain and inittates 2 Mottram JC (1933a,6 1935a 6) Grry, LH & Read J (1942a, be dy Gray, LH, Read, J & Poynter, VM (1943) 336 LETHAL EFFECTS of the bean roots irradiated have been found to be G51, 435, 75 and 72 onergy-units respectively, the ratios of efficiencies of the four radiations for equal ioruzations in the tissue thus being 10 16 87 90+ This 1s the samo sequence of efficiencies ag that found for the production of lethal types of chromosome atructural change in Trad. microspores, in which the relative effinencics aro 10 11 28 282 The fact that the sequence of efficiencies 18 the same suggests that the mechamsm involved may be the same in the two cases, while numerrcal differences may reasonably be expected for chromosomes of different s1zo Tt has also been found that Jow ering the temperature increases the proportion of bean roots lulled by « given dose s It1s known that in Tradescantsa the number of cells showing chromosome structural changes 1s increased by Jowenng the temperature (ep Fig 32, p 221) It has algo been pointed out that the effect of 8 given dose on the bean root 1s reduced by aphitting the dose into fractions, with rest intervals between, in » manner which 13 con sistent with the reduction of effect being due to there being & amaller number of chromosome exchanges produced im expert ments in which there are long rest periods, owing to breakage ends formed by the first fractional dose having all restituted by the time the second fractional dose 18 given + Marshaks has determined the proportion of anaphase figures in the bean root-tip which show chromosome fragments or bridges at various times ranging from 3 to 24 hr after wradis- tion The abnormal anaphases at 3 hr are probably mainly the Tesult of ‘physiological’ changes mn the chr the ab normal anaphasea at the later times are probably mamly ehr atr 1 changes s The ratios of efficiencies he i Gray, LH Read, J & Poynter, M (1943) 2 As before the yield of lethal types of chromosome structural change 13 taken to be the sum of ch: d breaks, fF 1 breaks, and half the chromatid interchanges at 100 r The yrelds for X rays, neutrons, and a rays are taken from Table 64, p 241, and the relative yields for X raya and y rays sre taken from Table 63, p 239 3 Mottram JO (19355) 4 Gray LH (19446) 5 Marshak, A (1942a) In interpreting hus resulta with neutrons, we have assumed, as usual that 1 umt<=2 5 v units 6 The terms ‘physiological’ change and ‘structural’ change are used. in the sense of Chapter vi, p 192 PHYSIOLOGICAL CHANGES IN CHROMOSOMES 337 finds for X rays and neutrons at these later times range from 1 32tol 67, which are consistent with the ratio] 5 8 found for the lethal effect on bean roots Marshak finds that the proportion of anaphase figures which show fragments or bridges can be represented by the formula 1-e-4D where D 1s the dose of X-rays m roentgens and 4 takes values ranging from 0011 at 3 hr after irradiation to 0 0022 24hr after radiation With a dose of 435 r, which 1s the dose luilng 50% of the bean roots in Gray and Read’s experiments, the proportion of anaphases which have bridges or fragments 1s deduced from this formula to range from 99 % 3 hr after wradia- tion to 62% 24 hr later The proportion of anaphases with bndges or fragments 1s thus ngh enough to account for the death of the root, presuming that the daughter cells from these ab normal anaphases usually die Lethal physiological changes in chromosomes As was mentioned in Chapter vi (pp 192-7), wradiation of cells already suffiaently advanced in division at the tzme of uradiation not to suffer the delay in division expertenced by cells not so far advanced may result in appearances at metaphase and anaphase suggestive of a surface stickmess of the chromosomes At metaphase the chromosomes clump together, and at anaphase the separation of sister chromatids 1s himdered Division figures of this sort obtained in chick tissue, mm grasa hopper neuroblasts,2 and m onion root-tips,s are Wlustrated in PlateIVe-x Similar appearances have been described 1n other tissues 5 ro Severe cases the bridge formed at anaphase may be too othe to break, and division will not then be completed In cases where division 1s completed, the daughter cells may 1 Lasnitzia, I (19435) Chick tissue m culture was irradiated for 25 also Ja St 100r fmm and fixed immedately after the exposure Cp : cn & Kemp T (1933) 2007 lson, JG (19418) Chortophaga neuroblasts were irradiated at 3 Marsh for 40 min and fixed 15 mm after exposure ate end Aca’ ie jiadson JC (1937) Onion root tips were given a & Amphibia (Alberti, Ay W & Politzer G 1924) grasshopper a (White, M J D 1937), plant microspores (Marquardt, H PC 1943), plant root taps (Pekarek, J. 1927, Marquardt, 1938, Sax, K 19414) pet we 348 LETHAL EFFECTS bo genctically unbalanced owing to the breakage of anaphase bridges Thus the physiologienl effect 18 probably often lethal Tho metaphase and anaphase figures suggesting a surface stickiness of tho chromosomes are typically seen soon after wradsation in cells too far advanced in divimon at the tume of uradiation to suffer the temporary inlibition of division Abnormal anaphases aro also acen among the cells which divide some hours later, after the end of the period of mhibited division Theso abnormal anaphases are supposed to be due to structural changes rather than to physiological changes im the chromo somes,: though tho distinction between the two types of ab- normal anaphaso can only be made in matenals favourable for cytological study In view of the evidence presented in the preceding sections it 1s likely that cells showing abnormal ana- phase configurations will eventually die and be included among the count of degenerate cells There 13 evidence: that m animal tissues many of the cells which die while attempting division after the cessation of the period of temporary inhibition do go at an early stage of division, and are recorded as degenerating cells without ever bemg m- cluded in the metaphase and ansphase counts Such casualties cannot plausibly be accounted for by chromosome structural changes since the genetic unbalance or mechanical difficulties at anaphase presumed to account for the lethal effect of chromo- some structural changes cannot take effect until the chromo somes divide They are not at present understood Hereditary partial-sterility fe): str 1 changes which do not lead to the pro- duction of acentnic fragments or dicentric chromosomes do not have a lethal effect on cells carrying them There are nexther lagging fragments nor bridges at division, and each cell has @ complete complement of genes of Tf, ag the result org radiating anadult ,asy trical interchange between two chromosomes 13 induced im one of the x According to Marquardt H (1938) usmg Bellevela romana, Carlson, J G (19410), usmg grasshopper neuroblasts, Sex, K (1941 a), using onion root tips Spear FG & 2 * ecording to the observations of Tansley, AK (1938) Glucksmann A (1937), Spear, FG & G! Lasmtza ZT (1940) HEREDITARY PARTIAL-STERILITY 339 gametes, the F, mdividual resulting from the fusion of this gamete with a normal egg or sperm, though phenotypically normal, 1s usually partially stenle The explanation of this phe- nomenon 1s made clear by Fig 59 In this figure the complete Gamete with Normal gamete interchange x Association of four chromosomes at meioms in the F, heterozygote | [ Spores or gametes Produced by the 1 heterozygote & or & Normal Carry Deficiency duplication interchange Viable Non viable Fia 59 Hereditary partial sterility caused by symmetrical chromosome interchange chromosome complements of the various gametes and zygotes ®re not shown, but only the two chromosom es which take part in the interchange noneraptor gametes, one carrying the interchange and one venta s are represented in the top lime The second line repre- meiotig © constitution of a (diploid) cell of the F, individual At homolo Prophase pairing normally occurs in twos between the When ous chromosomes denved from male and female parent some a interchange 1s present pairing of homologous chromo- insteaa ements requires the association of four chromosomes of tio, as represented m the second hne of Fig 59 At 340 LETHAL EFFECTS meiotic first anaphaso tho four chromosomes separate, two going to cach polo If the chromosomes which in the second hine of Fig 59 are drawn diagonally opposite to one another proceed to the same pole, then two nucle: result which each have a full complement of genes If they proceed to opposite poles, then nucler result which are deficient for one chromosome segment and have another chromosome segment present in duplicate These nucler undergo the second meiotre division, the haploid nucle: which result being the microspores or macrospores in the ease of a plant, or the gametes in the caso of an animal The alternative types of haploid nuclet aro shown in the bottom line of Tig 59 Thoso which have the deficiency-duphication con figuration will not give mse to a viable F, generation Thus the F, organism 13 partially sterile Of those gametes or spores which carry a full complement of genes, and which are therefore vinble, half are completely normal, and the other half carry the interchange and will therefore give rise to an F generation which while phenotypically normal 1s partially stenle, hhe the F, generation Thus the F, organism 1s partially sterile, and of 1ts offspring, halfare normal and half are partially stenle The partial stenhty 1a similarly transmitted through succeeding generations Radiation induced partial sterility has been investigated in some dctailin maize + A maize plants fertilized by pollen which has received a dose of a few hundred roentgens of X-rays, and the seed resulting collected and the F, plants rased Those w hich appear phenotypically norma]: are selected and their pollen examined Some of the pollen grams are visibly abnormal, fail to germmate, and are thus incapable of fertihzmg The genetic unbalance has resulted in breakdown, pr bly at the micro spore division In the case of ammals where no haplord mitosis takes place 1 Stadler LJ (19305 1931) That the explanation of the partial sterility 13 h 38 confirmed by the obser of rings of four chromosomes at diakinesis in the pollen mother cells 2 Some plants of stunted growth are obta:med in an experiment of this sort owing to simple breaks having been mduced in some of the rrradiated pollen The F, plants from thzs pollen are heterozygous for a deficiency If they flower they will be partially sterile owmg to deficiencies being present in half of the spores This partial stemlty 33 not transmitted to later generations PARTIAL-STERILITY IN MICE 341 after meiosis, but the products of meiosis are themselves the gametes, a deficiency-duplication does not render a sperm in- capable of fertizing The zygote however does not give nse to 4 normal embryo, but death occurs at some stage presumably dependent on the extent of the genetic unbalance Parhal sterthty hag been investigated in mice: A dose of a few hundred Toentgens 1s given to either a male or a female parent and the F, offspring are mated to normal animals Some of these F, females, mated to normal males, are found consistently to produce small htters In the same way, some of the J, males Mated to normal females are found consistently to father small hitters Investigation shows that the small hitters are due to the death of some of the embryos in the uterus Half of the hving tuce belonging to these small litters are completely normal, half, while apparently normal, m turn give nse to small litters when Mated to normal mice Recapitulation and application to rapidly dividing animal ues It 1s Probably true to say that a sufficiently large dose of Tachation given to any cell at any stage will cause its immediate death Such effects, produced by doses of radiation which cause fenous of ch 1 change 1n all parts of the cell, are of no great theoretical mterest or practical importance Much More sigmficant 13 the fact that 1t has been found in a great vanity of cells that moderate doses of radiation, of the order of 4 few hundred or a few thousand roentgens, which must produce & rather shght arsount of chemical change in the cell as a whole, are able to cause the death of a cell, not immediatel y, but only at or following nuclear division This behaviour suggests that the killng of cells by moderate doses of radiation may be due to an effect on the genes or chromosomes In the case of the vegetative bacteria and a large virus (vacema) the Proportion of the organisms which are Jolled 1 By Snell GD (1935, a 1939 a 1941), ' Snell GD & Ames qost FB the cause of the partial sterility 18 a chr 19: n confirmed cytologically t ‘ hes by Koller, PC & Auerbach who observ CA (i941), spormate genscn” : sAkociation of a mng or chain of four chromosomes at 342 LFTIAL EFFECTS depends upon the conditions of the irradiation (dose, intensity, temperature, and son density) in precisely the same manner as does the proportion of irradiated Drosophila sperm in which gene mutation 1s induced We have therefore interpreted the killing of bactena and of large viruses as lethal gene mutation Ths interpretation 1s provisional since at present practically nothing 3s hnown about the genetics of bacteria and viruses It seems, however, to be a plausible interpretation since not only can the radiation results be understood on this basis, but also the num ber of genes per cell inferred from the radiation experments 18 smaller for a bacterium than for Drosophila, and smaller for vaccinia virus than for a bactenum If the interpretation of lethal action as Iethal mutation 15 extended also to the small crystalbzable viruses, tho radiation results indicate that these viruses are single naked genes Thus the gene number per cell needed to explain the Iethal action of radiation as lethal muta tion increases from viruses to bactena, and from bacteria to Drosophila, a8 would be expected on general grounds There 18 no evidence to suggest that gene mutation plays & large part in the killing of organisms other than bacteria or viruses In Drosophila, recessive lethal gene mutations are pro duced by irradiation in the eggs and sperm and presumably also im diploid cells But recessive lethal mutation in 2 diploid cell will not be lethal unless it 13 in the X chromosome of the male, owing to the presence of a normal allelomorph mm the same cell In those orgamsms un which chromosome structural changes, as well as lethal actions, have been investigated, namely Droso phila sperm and eggs, Tradescantsa pollen, and bean root tips, fawly strong evidence, though at present circumstantial ev- dence, has been presented for the view that the main cause of the lethal effect 1s the production of types of chromosome structural change which lead to bridges at division or genetic unbalance after division The killing of the cells of rapidly dividing animal tissues by radiation 1s of practical importance in view of the use of radia trong in the treatment of cancer Unfortunately the cells of human tissues and of the tissues of the usual laboratory animal» have many and small chromosomes, and it 1s not possible with these cells to make a detailed analysis of the changes suffered by chromosomes as a result of irradiation, such as 1s possible in the RAPIDLi DIVIDING TISSUES 343 most favourable cytological materials In view of the remarkable simuarity of behaviour of the chromosomes of widely different species under normal conditions, 1t 18 reasonable to expect some similanty m their behaviour after irradiation It seems reason able therefore to take the results obtained with Drosophila and Tradescantia as a guide m interpreting expermmental observa- tions with rapidly dividing animal tissues In the first place there 13 ample evidence (detailed earlier m this chapter) that the cells which degenerate after the wrradiation of these tissues with moderate doses commence to do so only at dimsion This result 1s consistent with the idea that the cause of degeneration is some change in the chromosomes Two types of change in the chromosomes have been discussed, structural changes and physiological changes: Structural changes in these materials are likely to be recognizable, if at all, only at anaphase (by the production of bridges and fragments): and after division (by the presence of micronucle1) Chromosome structural changes may be produced at any stage m the hfe cycle of a cell, and wall only lead to degeneration after division Thus when a rapidly dividing tissue 18 uradiated, de Benerate cells may be expected to commence to appear when mitotic activity 18 resumed after the temporary mhibition They will continue to appear for a time at Jeast equal to the inter- mitotic period, smce each batch of cells entering division will Provide its quota of cells with lethal types of structural change Thus in fact 1s what is observed (cp Fig 54, p 309) The second type of change suffered by the chromosomes as a Tesult of wradiation (the ‘physiological’ effect) 1s a surface stickiness, possibly accounted for by the nucleic acid being de- Posited on the chromosomes in a fluid unpolymerized state 1 The phy Siological effect 1s typically exhibited by cells already in division at the time of irradration, and results in clumped meta- Phases and bridges at anaphase, such as are illustrated nm vary- ing degrees of severity in Plate [V E-K 4 The physiological effect Presumably accounts for the abnormal division figures observed meee terms are used throughout im the sense of Chapter vr p 192 uradiatio Tarshak A (1942) found that durmg the 24 hr following the 50% of thy of a mouse lymphoma by 600 roentgens of X rays at least 3 Da 1 © cells observed at anaphase showed bridges or fragments sungton, CD (1942) 4 See footnotes 1,2 3 on p 337 344 LETHAL EFFECTS in rapidly dividing animal twsues in the hours immediately following irradtation, before tho resumption of mitotic activity « Tf, as 18 said to bo the case,s recovery 13 complete from the physiological effect by the time mitotic activaty 1 resumed, then degencration of cells which takes place after the resumption of mutotio activity cannot be asenbed to the physiological effect In thia event the physiological effect can play only ¢ emall part in causing the death of a rapidly dividing tissue (at any rate a5 a result of 8 englo irradiation) since, even af all the cells in di vimion at the time of irradiation are affected, these titut only a amall fraction of the total number of cells in the tissue However, st must be remembered that the conclusion, that the physiological effect on the chromosomes 13 not exhtbited by cells which enter division after the resumption of mitotic activity, was reached by studying cells with a longer time scale and may not apply to rapidly dividing animal tissues In view of the difficultzes of direct cytological observation, one may hope to obtam evidence of whether chromosome structural changes play a large or a emal] part in accounting for the lolling of cella m rapidly dividing annmal tissues by atudying the manner in which the proportion of cells affected vanes with the dose, with the intensity, and with the type of radiation, and com paring with the known behaviour of chromosome structural changes 1n this respect 1 Eg by Lasnitzki, I (19430) 2 Cp Chapter vi, p 196 Appendix I SUPPLEMENTARY CALCULATIONS: The relation between dosage in air and energy dissipation in tissue: X-and y rays The roentgen 1s a unit based on the production of romzation m air Dosage expressed in roentgens can readily be converted to terms of energy dissipation per unit volume of au To convert. a dose in roentgens into terms of energy dissipa- ton in tissue, it therefore suffices to know the ratio of the energy cissipations per umt volume in tissue and in air for the particular radiation For X- or y rays this 1s the ratio of the values of r+a,,, where 7 1s the photoelectric coefficient, and o, 18 the part of the Compton scattering coefficient o which corresponds to the transference of energy to a recoil election a,, the part of the Scattering coefficient which corresponds to the energy of the scat tered radiation 1s not relevant here, since the radiation scattered ma small volume of irradiated air or tissue wall not usually contribute to the ionization in that small volume Where a large volume of tissue 1s being irradiated, the scattered radiation from the surrounding tissue does of course add to the ionization ina given cell, but since the scattered radiation 1s mcluded in the dose in Toentgens, providing the 1onization measureme nt has been made ma cavity in the tissue or in a suitable phantom, it 18 still correct to use 7+ 07, and not T+ Ogio, On the other hand, 7 15 the total photoelectrre absorption coefficient without subtraction of that part of 1t corresponding to energy of the excited charactenstic radiation of the absorbing atom, smce such charactenstic radzation from hght elements has a short range and will in fact often be absorbed mm the same cell in which it 18 produced eM, the scattermg absorption coefficient per electron, 1s 1 This Appendix compris es a number of calculat which have been ions, the results of Tequired in the text but the details of which are only of technical interest 2 Table 2 (p 8) and Table 3 (p 12) are based on this section 346 ATPFNDIX I independent of the composition of the senttercr, and 1s given by tho well known formula of Klein and Nishina + one fT 2(lta)® 1432 | (l4a) (1422-204) 17a" FRct| aF(14a) (+2) at(1+ 2x8 4a? ~aresap” (“Bez ta) steel], (apn Ita ot where amhu/me?, ¢=4 8026x 10-, m=9 1066~ 10-%, ¢=2 99770 x 10, AesG 624 x 10-7 For small values of« this may be expanded as T= 87* ta —4 2at4 14 7a3—AG aly mec! J e%q 18 tabulated in Table 80 for various wave lengths of radia- tion By multiplying by the number of electrons per gram m any given tissue, the value of ¢,/p, the scattermg absorption co- efficient per gram for tho tissue, may be calculated The number of electrons per gram 1s readily calculated from the el tary analysis of the tissue with the aid of the figures given in Table 79 of the number of electrons per gram in various elements hhely to occur in tissue 7, the photoelectric coeffinent, has to be obtamed from experi- mental values of the absorption coefficent z=r+o of the elements, by subtracting from these expermmental values the theoretical (Klem-Nishina) values of o, the scattering coefficient The scattering coefficient per electron 1s Om ae QretT eso Alta)? at log (1 + 2a) lta + gp log (1 +22)— 755 +3a | + (Ap -2) 1 Klem,O & Nishina Y (1929) Note that the formulae of Klem and Nisha are meorrectly quoted in several text books eg Rutherford, E, Chadwick J & Elhs, CD (1930), Kirchner F (1930) Rasetts F (1937) z For wave lengths d O2A the Ht observed exceeds the Kem Nishina scattermg owing to the coherent segttermg However, for wave lengths long enough for the difference to be the 13 ao much less than the photo electric coefficient that little error 1s made in the estimation of the latter by the use of the Klein Nishina formula COMPTON AND PHOTOELECTRIC ABSORPTION 347 or for small values of a, 87e " = Gee a ll ~2a+5 Qat—13 Ba3+32%4at | Tantze 79 Photoelectric absorption coefficients (1/p) Electrons Photoelectric Power ofA in per coefficient photoelectne Element Z a g xl0- = 0. 0089Z4 1A law x 1 1 008 5075 6 009 C 8 1201 3.009 1150 305 N 7 401 3010 1 854 0 8 16 00 3012 2.809 Na nu 23 00 2 881 7200 Mg 12 24 32 2972 974 Al 13 26 97 2903 12 20 Ss 4 28 06 3.005 15 88 P 15 3102 2912 19 06 8 18 3206 3.006 24 04 285 a 17 35 46 2 888 27 86 A 18 39 94 2714 31 29 K 19 39 10 2.927 39 8 fe 2 40 08 3 005 481 ‘e 26 55 84 2 804 1010 Air 1 Bs 3007 T/p=2 OBAT 4.0 41A2 0 Virus protein (p 7) 4343 ie 2 BOAP es 3216 T[p=1 SGA 84.0 B7A8 SF Wet tissuo (p 7) 3307 tho=2 26A8 8.40 27Ar Oe ef 18 tabulated m Table 80, wlule a convemient compilation of expenmental values of y/o for various wave lengths has been made by SJ M Allen: It 1s found that when the theoretical Values of o/p are subtracted from Allen’s Lip values, and the tle values 80 obtained converted into values of T4, the coefficient Per atom, they may be satisfactorily fitted by a formula of the usual type 7,,0c ZA", the values obtained for the coefficients m and nm being mdicated in the equations Tat= 148 x 10-8241 An, with n=3 05 for elements OC, N, O, n= 2 85 for elements Na~Ye Thus the photocleetnc coeffic ient per gram 18 t p70 0080 =) an, JAX (Ap 3) Zand A being the atomic number and atomic weight and 7 t In the appendix to Com pton AH & Allison SK (1935) 1 Theory and Experiment 23 ( ) X rays 346 APPLNDIN I independent of the compoution of the scattercr, and 1s given by the well known formula of Klein and Nishina + 2nelF 2(1+a)? 143 9 (l+a)(14+2%—22%) a= mic| ak(1-+2a) (14+2a)!* _al(¢ Sa) ~ grea (“B2-et ss) ret t2n)], (ap 9 where amhu/met, e=4 8025x 10-, m=9 1066x 10-, ¢=209776x 10, Aes 6 624 10-” For small values ofa this may bo expanded as Snes am Freya roe) +] 2 14 Ta 2 AOyfgat 4 Latt ¢q 18 tabulated in Table 80 for various wave Jengths of radia tion By multiplying by the number of electrons per gram in any given tissue, the value of o,/p, the scattering absorption co efficient per gram for the tissue, may be calculated The number of electrons per gram 15 readily calculated from the elementary analysis of the tissue with the aid of the figures given in Table 79 of the number of electrons per gram in various elements likely to occur 1n tissue 7, the photoelectric coefficient, has to be obtamed from exper! mental values of the absorption coeffiaent z=T+o of the elements, by subtracting from these experimental values the theoretical (Klein-Nishina) values of o, thescattering coefficient 7 The acattermg coefficient per eJectron 18 are 2Al+ey? lta met —z log (1+ 2a) e a (1420) a ++ 5g log (1422) — | » (Ap -2} x Klem O & Nishina Y (1929) Note that the formulae of Klem and Nishma are meorrectly quoted m several text books eg Rutherford E, Chadwick J & Ellis CD (1930), Kirchner, F (1930) Rasetts F (1937) 2 For wave lengths O2ZA the I; observed exceeds the Klem Nishina scattermg owmg to the coherent segttermg However for wave lengths long enough for the difference to be the 1s so much less than the photo electric coefficient that little error 1s made m the estimation of the latter by the use of the Klem Nishina formula COMPTON AND PHOTOELECTRIC ABNORITION 347 or for small values of cr, Sie c= anal] — 2a + 5 2a 2 ~13 32343280! 34 3924 ab TaBLeE 79 Ph lectric absorption coeffi s (tip) Electrons Photoelectric Power ofA in per coefficient photoelectne Element =Z A gxl0- 9 0089244 law H 1 1008 5975 0.009 Cc 6 1201 3 009 1150 305 N 7 1401 3.010 1 854 u 8 16 00 3012 2 809 No u 23 00 2.881 7200 Mg 12 2439 2972 O74 Al 13 2697 2003 12 20 & 14 28 06 3.005, 15 88 5 15 31 02 2912 19 06 a 16 32.06 3.006 2404 285 ‘A 7 35 46 2 888 27 86 K 18 39 94 2714 3120 é 1g 3910 202 398 Fe 20 40 08 3 005 481 ® 26 55 84 2804 1010 #5 3.007 Tip =2 O5A? S40 41At 8 vi 3343 T/p=2 50A3 Wen Pprotemn (p 7) 3216 tip=1 56A% 54.0 67A2 et tissue (p 7) 3307 T/p=2 26A¥ S40 OTA? Hs ef ls tabulated in Table 80, while a convenient compilation of xpermental values of Hip for various wave lengths has been made by SJ M Allen: It 1s found that when the theoretical Values of ofp are subtracted from Allen’s zip values, and the Tip Values so obtained converted into values of 7,,, the coefficient sed roan they may be satisfactomly fitted by a formula of the , ‘ type 7,,0¢c ZTMA", the values obtained for the coefficients m nd n being indicated in the equatio ns Tar=1 48 x 10-26 741 A”, with n=3 05 for elements C, N, O, n= 2 85 for elements Na-Ke Thus the Photoelectric coefficient per gram 1s 770 0089 (7) a, (Ap -3) T ZA 1 n Z and A bemg the atomic number and atomic weight and x TInt! m Th 8ppendix to Compton AH & Alhson, SK (1935), X rays 348 APPFNDIX I taking the values given above: A 2s the wave length in Ang stroms The values of 0 008924 1/A are tabulated for hght elements in Table 79 Wath tho aid of this table a formula for the photoelectric Tante 80 Nien Nishina coefficients Photoelectric Radation Coefficients? x 10% absorption in ¢ a — — > water @ A(A) Av(ek¥ ) wo Me % tip Ooh 24265 5108 6524 00638 6460 3733 002 = 12192 1022 6401 «6001296 «= 6.278 4.508 003 6 8088 1532 6284 01769 6 107 1309 00L 086066 2043 6172 02269 6945 0 5442 0-05 0 4853 2554 6068 02731 8793 02755 006 =6d.4084 3083) «B62 03188 5 647 0 1582 07 =—-0. 3466 3376 5862 03517 5511 0.09874 008 03033 4087 5773 03936 5380 0 06570 009 «©. 2606 4593 5681 04276 5256 © 03590 O10 92428 SI0B «659904605 138 0-03328 Or 6 2208 5619 «65526 §=60.4806 5 026 0 02488 012 0 2022 6130 6437 «05178 4919 0.01908 015 01618 7662 5218 O5914 4627 0.00966 018 01348 9195 5023 08497 4373 0 00553 021 01155 = 1073 4847 07031 4144 0 00346 O24 O1011 1226 4687 «07455 «3842 0 00230 027 00899 1379 4542 07812 3761 0 00161 030 00809 1532 4409 08218 3598 0.00117 033 00735 «1686 4287 08375 3450 0 00087 038 00674 1839 4176 08601 = 3.315 © 00067 039 gos22 1992 4070 08793 3191 0 00052 042 00578 «= 214 3973 98953 «3 078 0 00042 045 00539 229 3883 09091 2974 0.90034 048 00506 245 3798 09217 2877 0 00028 08 004044 30865 3507 09562 2552 @ 00014 08 003033 4087 3140 09820 «= 2:158 0 0006 19 = 0.02428 «5108 2865 09873 1870 9.90003 12 = 9 02022-6136 2650 99823 1668 0.90002 14 001733 7152 2474 «O97 «= 1503 0.00001 16 Oost? 8173 2328 09580 1368 9 00001 18 001348 9195 2200 09424 1258 0.00000 20 001213 1022 2090 o9zeo 1164 0 00000 1 Multiply these coefficients by 3 343 x 10" to obtaim coefficients per gram in water,1e multiply the actual numbers listed by 0 03343 absorption coefficient of a tissue can be calculated if sts elemen- tary analysis 1s available Formulae of this sort for air, water, virus protein and a wet tissue (for the composition assumed for the two last-named sec p 7) are included m Table 79 1 A more complicated empirical formula, covering all elements has been given by Victoreen J A (1943) &-RAYS, PROTONS AND ELECTRONS 349 The formulae may be compared with the empirical formulae given by Kustner: and Muller,z namely, Air t/p = 2 330333, Water 1/p=2 5445 #2 These formulae are based on actual absorption measurements on the two substances, the allowance for scattering be:ng made m & semi-empineal fashion which seems less satisfactory than the use of the Klem-Nishina formula, especially in the present crcumstances when the Klem-Nishina formula has in any case to be used to deduce +0, from 7 Kustner and Trubestein: five empincal formulae for various tissues based on actual absorption measurements with these tissues We have preferred however to calculate 7+ go, from the elementary analysis com- bined with the 7 coefficients for the separate elements, smee in this way a more consistent set of figures less affected by expen- mental error in any one experiment 1s hhely to be achieved The assumption implicit in this procedure that the photoelectric absorption of an atom 18 independent of its chemical combina tion 1s unhhely to be wrong since photoelect ne absorption 1s mainly by the unnermost electron shells Formula (3) 13 only valid on the short wave side of the K absorption edge For long waves, eg A>1A, it has been thought best not to employ an empincal formula, but instead to use Allen’s values of #/p and to subtract from them ,/p, 60 obtaming y/p~o,/p=T/p + o4/p The ratio of the absorption coefficients im tissue and aur of a Particular wave length depends on the elementary analysis of the tissue, and the data in Tables 79 and 80 suffice to calculate the ratio if the elementary analysis 1s known The calculation has Leen carned out for two compositions of tissue and water, selected wave lengths, and the results employed in Table 2 P 8) % rays, protons and electrons If the expermental matenal 18 wradiated im a thin layer by a beam of particles, eg a -Tays. s Protons or electrons, then the ratio of the energy dissipation an tissue and air is simply the ratio of the stopping powers of the two substances These are calculated by the Beth e formu la (cp Pp 350-2) 1 Kustner, H & Trubestern H (1937) 2 Muller,J (1938) 350 APPENDIX 1 Neutrons Tho unit of neutron dose used in Tablo 2 13 the ‘v’ (cp p 20), which 1 based upon the ronization produced in a amall oir cavity in irradiated water Ionization in the air cavity may bo converted into energy dissipation in air by 35eV per ion parr Nearly all the ronization in the air, and energy dissipation in tho water, being produced by the protons which are sot into motion by the neutrons, the energy dissipation in the water can be obtained from the energy dissipation in the aur by multiplying by the stopping power ratio of the two substances for protons of the appropriate energy We thus readily obtain the figures given in Table 2 for the energy dissipation m water corresponding to lv of neutrons To calculate now the ratio of the energy dissipation ine tissue of known composition to that of water wo note that the energy of recoil of an atom of atomic weight A for a given neutron energy 18 proportional to 4/({1+.A)%, while the number of recoil atoms per unit volume with o given neutron dose 1s proportional to po/A, where p is the fractional content, by weight, of the particular element in the tissue, and o 18 ats atornic cross section for neutron scattermg We proceed therefore by evaluating z t+4p ae for all the elements in the tissue, and compare the figure obtamed with the corresponding figure for H,O In the case of nitrogen, disintegration resulting in the emission of an a-particle or a proton occurs as well as nuclear recoil, and has te be taken mto account m the calculations « The values of Kikuchi and Aohiz have been used for the scattermg cross sections, and of Baldinger and Hubers for the disintegration cross sections Spatial distribution of ionization in irradiated tissue+ ‘Lheoretical formulae exist for computing data of the sort given m Tables 10-18, and have been conveniently summarized by Bethes A certain amount of experimental data also exists, usually for air or other gases Since 1t would im any case be 1 As pomted out by Gray, LH & Read, J (1939) 2 Kikuch § & Aohi, H (1939) 3 Baldinger, E & Huber, P (1939a 6) 4 This section describes the manner in which ‘Lables 10-18 of Chapter x have been obtained 5 Bethe, H.A (1933) SPATIAL DISTRIBUTION OF IONIZATION 351 necessary to appeal to theory to fill in the gaps in the experi- mental data, a more consistent set of figures can probably be obtamed by relying entirely on the theory, which has therefore been done in this book Calculated values of energy dissipation and primary 1omzation by electrons in gases agree with experi- ment within the accuracy of the experrments, which 1s not very great For « rays, energy dissipation 1s known experimentally rather accurately, and experimental values are here more re- hable than theory : As regards 6 ray production, expennmental data are rather meagre Experiments by Hornbeck and Howell: and Shearin and Pardue: indicate that the theory 1s correct for the production of fast 6 rays by fast pnmary electrons Expen- ments by Alpers agree fawly well with theory for fast «-rays, but give fewer 6 rays than theory for slow a-rays The experumental difficulties of the measurement of ranges of d rays of a few hundred volts must however be very great, and we have pre- ferred to rely on the theory The principal uncertainty in the appheation of the Bethe formulae 1s the modification of fotmulae derived for hydrogen- hke atoms to more complicated atoms and molecules A value has to be assigned to £, the effective mean ionization potential of the atom or molecule The value of £ 1s usually chosen for each element to give agreement between the theoretical stopping Power formula and the experimental stopping power of the ele ment for a rays Manos has given a table of values of # which includes, hydrogen 16, air 86, and oxygen 100eV, based on % particle range measurements in these gases, and suggests E=45eV for H,0, interpolated between the values for hydrogen and oxygen This value of £ we first used in calculating energy dissipation im tissue However, 1t appears that Mano obtained B= 45eV for H,O by considermg Z=(2+8)/3=333 as a ‘Weighted mean atomic number’ of the constituent atoms, and this procedure 13 probably unsound Gray+in arecent review has &lven evidence indicating that the Bragg additive law relating the « Tay stopping powers of molecules to those of their con- 1 Cp Livingston, 09. S & Bethe, H A (1937) 2 Hornbeck G & Howell, I (1941) 3 Sheann PE & Pardue, TE (1942) 4 Alper T (1932) 5 Mano G (1934) 6 Gray LH (1944a) 352 APPENDIX I stituent atoms 1s obeyed to one or two percent Ifwe accept the additive law, then it 18 evident from its position in the formula that the effective value of # should be calculated from the values 16 and 100eV of the hydrogen and oxygen atoms as a weighted geometric mean, viz (16? 100%°t=69eV It 1s unfortunate that the only direct measurements of a particle ranges m water: are the principal exceptions to the additive rule These expert ments give 324 for the range of a polonrum «-particle, and 60 fora Ra C’ « particle, considerably lower than the values given m Table 12, which are derived from the accurately known (mean) Tanges in ar with the aid of the theoretical stopping power ratio of water and arr, the latter calculated with E=69eV for water These experimental ranges would be more nearly in agreement with B=45eV for water However, we have accepted Gray’s conclusion that the weight of the evidence 1s in favour of the validity of the additive law We therefore recalculated the stop ping powers on the basis of £=69eV , and all stopping powers and ranges for « particles, protons and electrons m Chapter I are calculated on this bams We did not think it necessary, however, to recalculate some of the data in subsequent tables, and Table 15 of Chapter r and Tables 81, 82 and 83 of the Appendix are based on an energy range relation for electrons which has been computed with #=45eV imstead of £=69eV which we now prefer The difference 13 small, since £ has less influence on electron atopping power than on «-particle stopping power, and in any event there 1s no certainty that # values deduced from a particle stopping power experiments are the best values to use m computing electron stopping powers In calculating stopping powers all the extranuclear electrons, 10 per molecule for H,O, are considered effective In calculating primary 8p tion the contribution of the two mnermost electrons 13 1gnored, and 18eV taken as the effective :onization potential of the remaining 8 electrons Srmularly, in calculating S ray energy distributions, only the 8 outermost electrons are considered & rays of energy less than 100eV are not considered, the calculation of ther number 13 more difficult than for more energetic é rays, and in any event such short é rays are from our pomt of view better regarded as 10n clusters it Michl,W (1914) Philipp K (1923) TARGET WITH SHARP BOUNDAR} 353 TARGET THEORY CALCULATIONS: The overlapping factor F An 1onizing particle produces 10n clusters at a mean separa- tion of ZL along :ts path An effective hit 1s supposed scored when in its passage through the target, taken to be a sphere of radws r, one or more 1on clusters (1e primary ionizations) are produced in the target It 1s required to calculate the average number of effective hits per target when a dose of radiation corresponding to the production of 7 10n clusters per unit + clume 1s delivered The dose given corresponds to the production of an average of n4mr3 10n clusters in a sphere of radius r Since the mean path of an iomzing particle in a sphere 18 47/3, and the separation of consecutive ion clusters averages L, an average of {r/L 1on clusters are produced by each tonizing Particle passing through the sphere Hence n§n8f(dr/L)soL ar (Ap -4) lomzing particles cross the Sphere nL 2rydy will cross the sphere at distances be- tween y and y 4 dy from its centre, and such particles will have a path leng th of 2e=2(r2—y2)tin the sphere (sec Fig 60) In this path length the mean number of Fic 60 Hlustrating target calculation 1on clusters produced 18 2z/L, and since clusters are produced hot at equal intervals but according to the mean free path law, (le) 13 the probability that at least one cluster will be Produced Hence the mean number of hits per target is nb i (l-e-®2/L) 2nydy= dank. {” (1—e7*/L) xdz, r oO since 22 tyra? Sart nal {L—2(1—e-$)/E2-+2e-£/E}, where £=2r/L, =37 n/F, where F = (2£/3)/{1—2(1—e7#)/E2+ 26-/E}, (Ap -5) 1 This seetion supplements Chapter m1 354 APPENDIX I If the dose corresponds to the production of an average of one son cluster per volume $73, then 1/F 1s the mean number of hita per target As shown in curve A of Fig 61, this 28 unity fora radiation of low ion density (L>2r), and diminishes as the number of ronizations per micron path increases 10) 5 08 bb ® a &ool 06) 5 $ 2 Z out 3 02 A L 1 1 L L L 2 4 6 8 Jo t2 Relative :on denaty (2r/L) Fio 61 Relative efficiencies of sonuzing particles of different 10n densities A, for equal numbers of ion clusters per umit volume B, for equal numbers of ionizing particles per unat rea This 1s the proof of formula (III-2), p 85 F'1s tabulated as a function of § in Table 26, p 86 If the dose corresponds to the passage of an average of one ionizing particle per target, then the mean number of hits per target 3s easily shown to be 1—2(1 —e#)/f2-+ 20-€/E = 2E/3F (Ap -6) Tins tends to the value umty for a densely 1omzing particle (L<2r), smce such a particle passing through the target 38 certam to produce 1onzzation 1n it, and 1s less then umity for less densely 1omzing particles It 1s plotted as curve B of Fig 61 Fig 61 shows that for a given number of ronizing particles pet umt area, densely zoning particles are more effective than less densely ionizing particles, but that per tomzation, densely 1on- izing particles are lesa effective m this type of action TARGET WITHOUT SHARP BOUNDARY 355 Target without sharp boundary Suppose that an ion cluster produced at a distance p from the centre of the target has a probrbility e~/** of being an effectave ‘lut’ Then an ionizing particle which passes through the target at a distance y from the centre (cp Fig 60) will produce a num ber dz/Z of 1on clusters in any path length dx, and hence alto- gether will produce a mean number { +0 eV" dx/L of effective cy) huts in the target. ‘Ths integral ev aluates to +0 (evry) en Odes (bn/Lye VM = 2, say (Ap -7) Ths being the mean number of effective hits, the probability of there being at least one hit wall be (1—e-#) For a dose of radia- tion corresponding to the production of 7 10n clusters per unit Volume the number of ionizing particles passing at distances between y and y+dy from the centre of the target 1s nL 2mydy (aa before), and hence the mean number of Its per target for this dose 1s nL fro —e7*)} Inydy, mtegrating to 0 nb mb? {0 5772 +-log 25 + 2+ (z)}, (Ap 8) where tabJjn/L and E1(z) e| e~dz/z 1s the exponential a integral * For an ionizing particle of low 10n-density (L> 6), equation (8) tends to the value nLnb?z,=nntb3, which can be wntten in the form n$m(1 10b)3 Companng with the ordmary formula na? for the mean number of 1on-clusters produced in a target of definite radius r by a radiation (e g y rays) in which the 1on- clusters are widely separated, 1t 18 evident that for such a radia- ton our target. without sharp boundary will behave as a target of apparent radius r= 1 105 Tf we now use a densely iomzing radiation with which we should expect, with a target of definite boundary, every Jonizing particle Passing through the target to give an effective hit, and deduce the apparent target radius R on this basis, then from equation (8) we have TR? = mb? {0 5772 + log 29+ Hr (z9)}, 356 APPENDIX 1 whence Ress {0 6772 +log (0 8x 2r/L)-+Es (08x 2r/L)}\, (Ap -8) because ber/11 and zsbdn/barJn/(l 1L)=0 8x 2r/L It 1s by eubstituting 2r/L= 6 and 26 respectively in equation (9) that the radu of circlea D and E of Fig 12 (p 97) have been caloulated The dated volume Iculation: Secondary electrons of less than 100eV energy are assumed to produce clusters sufficiently compact to behave as units, and have an associated volume no bigger than that of an isolated primary ionization Secondary electrons of more than 100eV energy are regarded as drays Spheres are described around each of the n 1omzations in the é ray track as centres, as in Fig Tp (p 84), and the associated volume deduced, being 2 {7r5/F, where F'1s the usual overlapping factor (Table 26, p 86) calcu lated from the ratio (£) of the target diameter 2r to the mean separation L of primary ionizations in the path of the é ray This 18 the associated volume of the d ray additional to the associated volume of the primary 1onation at which the é rey originated This last associated volume 1s not included with the é ray since it 1s already mncluded in the calculation of the associ ated volume of the pmmary 1onizing particle The quantity n 4nr3/F 15 1n fact the term omitted from the cruder calculation of method III (p 83) which 1g the distinction bet a éray and a cluster Calculation of associated volume for any energy of electron The procedure will be made clearer by quoting a few figures Let us consider how we calculate the associated volume for a primary electron of any desired energy completely absorbed in the tissue, for a target of diameter say 10my, of the same density as water We start by considering the last lekV of the electron trach Sphttmng the lekV imto intervals 0-0 1, 0 1-02, 0 2-0 3ekV, ete , we obtain by differencing Table 10 (p 24) the ranges corre- sponding to these energy intervals, and hence, usmg Table 11 1 Figs 8 and 9 are based on the calculations described an this section ASSOCIATED VOLUME 357 {p 25), the number of primary tonizations produced im each interval Between 0 4 and 0 3ekV , for example, the prmary electron travels 0 0045j¢ producing primary iomzations at the Tate 584 per 4 Thus it produces 0 0045 x 584 = 2 63 primary ions, having @ € value of 001%x584=5 84, since 2r=O001f and I/L=584 per » From Table 26 (p 86) we read off the corre- sponding value F=4131 The associated volume between 0 4 and 0 3ekV 1s thus 473 (aa) Working throughout in units of volume of 473 we wnite this simply as 0637 Adding up nine similarly calculated contributions for the other intervals we obtain 5 851 as the associated volume for the primary electron between I and OekV , and similarly obtain associated volumes for the mtervals 09,08,07, ,OlekV to0 We have now to allow for the é rays Instead of calculating Separately for each 0 lekV interval, we consider it sufficient to calculate the 3 ray production per unit Jength of path by a %5ehV primary electron, and multiply it by 0 05344y, the Tange of the primary electron between 1 and OekV From Table 16 (p 28) we find that an 0 5ehV pnmary electron produces o rays of energy between 01 and 0 15ekV at the rate 9 12 per # path, and of energy between 0 15and 0 25ekV at the rate 7 38 Per # path, and none more energetic than this The actual num- ber of § Tays 1s thus 0488 between 01 and 0 15ekV (mean energy 0 125ekV ), and 0 394 between 0 15 and 0 25ckV (mean O2ekV ) The assoctated volumes for an electron of 0 125 and 0 2ekV we can interpolate i the table of associated volumes we have just calculated, they are 0 453 and 0738 Thus 6 rays add 0 453 x 0 4884.0 738 x 0 394=0 512 to the associated volume 5 851 This represents an addition of 8 74 % for the 6 ray contni- bution to the associated volume of a lekV electron Takang this to be sufficiently nearly constant, we correct by the same per- centage the table of associated volumes for smaller electron energies The next stage 1s to consider the range interval 2 to lekV of the primary election This we divide up into subranges 1 0-1 2, 12-14, (18-2 0ckV , and calculate the associated volume for the pnmary ions in each subrange as before We then read off the 8 ray production by a primary electron of 1 5ekV from Table 16, tabulating the numbers of & rays of energies 0 1-0 15, 0 15-0 25, 358 APPENDIX I 0 26-0 35ekV , ete , up to thomaximum é ray energy of0 75ekV The centres of these ranges are 0125, 02,03, ,07ekV, and we now know the associated volume for an electron of any energy up to lekV Thus we can deduce the associated volame for all the grays Tho é rays are found to add 930% to the associated volume of the primary electron between 2 and 1ekV , and 1¢ 1s assumed that the same correction percentage 13 vald for 18-1, 16-1, ete Thus we deduce the associated volumes, 4 my contributions included, for any electron energy up to e Building up in further steps of 2-4, 4-8, 8-16ekV , etc, we finally obtain tables of the associated volume for any electron energy The computation has been carned up to 480ekV for target diameters of 4, 10, 20, 40 and 80mz, and Table 81 gives these figures By this stepwise method of calculation if 18 seen that we tomatically allow (except in the first step) for ‘rays pro- duced by é rays’ a-rays and protons Table 81 1 used when the primary particles are electrons,1e in X-ray, # ray or y-Tay wradiations The early part of 1t 18 also used to make allowance for the é rays when the prmary particles are a rays or protons In this case we read off from Table 17 4 or 3 (pp 30, 31) the number of é-rays haying energies im the intervals 0 1-0 15, 0 15-0 25, 0 25- 0 35 ekV , ete , multiply each by the associated volume, given n Table 81, of an electron of energy corresponding, and so obtain the contribution due to the drays This 1s to be added to the associated volume for the primary 1onizations, which 3s smply 1/(LF) per micron path, where 1/Z 1s the number of pnmary 1onizations per micron and F' the overlapping factor, calculated ag usual In this manner we have drawn up Tables 82 and 83 of easociated volume per micron path or ekV energy loss by « rays and protons of the stated energies For the sake of interest we have shown separately the contnbutions of primary particle and of d raya of >100eV energy The totals, also given, are the figures used 1n calculation of the 37% dose If the target material 1s of density p higher than water, then the ionizations are correspondingly closer together and the over- lapping for a target diameter 2r and density p 18 as great as it would be for a target diameter 27p 1n tissue of umt density The ASSOCIATED VOLUME 359 TABLE 81 Associated volume per electron, for electrons of energies 0 1-480ekV Becton Target diameter 2rp in mp ekV 4 10 20 40 80 o1 0956 0 3867 0 1933 0.0965 0 0482 02 1933 07961 0 3988 0 1993 0 0997 03 2994 1277 0 6435 0 3222 01611 04 4129 1 832 0 9315 0 4678 0 2340 05 5318 2 464 1261 0 6350 0 3180 es 6 546 3142 1634 0 8236 oan 3 871 2040 1033 05 08 9 084 4643 2 480 1 262 0 6325 09 10 38 5472 2954 1511 6 7583 10 1169 6314 3 456 1778 0 8939 1446 8 104 4543 2361 1191 14 17.25 9 996 5728 3012 1525 16 2008 1195 7.000 3726 1894 8 22 88 13 97 8 348 4499 2 298 2571 16 04 9 762 5 342 2734 33 3296 21 42 13 55 7 622 3952 35 40 22 26 99 17 62 10 18 5355 1 aT 47 32 69 2192 12 99 6 932 358 2 7 $ 8417 62 53 45 64 29 69 16 99 ; 2 1 74 74 85 76 37 27 21 88 § 133 8701 66 10 45 22 2717 2 1280 99 53 7671 53 50 32 81 n 143 0 1123 87 67 62 15 38 82 B en 109 4 79 8 ul 1868 1501 1204 88 75 58 0 5 ee 1626 131 4 97 87 64 81 0 55 1751 1425 1071 7178 5 388 0 238 6 1990 1552 108 9 30 40 8 3017 2559 204 6 148 8 10 2 5 264 6 313 0 255 0 1905 so esate Sag Seek 07 3 $55 9 7449 6627 5707 4636 80 tis 1 8726 7810 678 8 5593 90 Lae 999 5 898 9 785 8 6560 100 Hie 1126 1016 8940 753 3 io 1252 1134 1002 8511 129 582 1380 1254 H13 9519 150 Pid 1505 1370 1221 1051 180 25a 1886 1725 1551 1352 210 ons 2263 2077 1880 1655 20 ue 2633 2497 2207 1957 300 2201 3012 2778 2535 2261 360 Sook 3768 3486 3198 2876 420 Bete 4518 4189 3857 3489 480 bers 5263 4887 4513 4101 Note 6012 6589 8173 ANT diameter arene, densities p other than 1g fem? the figures headed target 8Te In units of Sma regarded as values of 2rp not of 2r The associated volumes 3 360 APPENDIX I same tables suffico for different density tissues providing th rules given in tho footnotes to the tables are adhered to A complication anses in the caso of a rays not present for le: densely 1omzing radiations With rather large targets 1f ma Taptx 82 Associsted volumes for « rays {Per micron track, and per ekV energy dissipation ) @ ray Target diameter 2rp, 1m mp energy eMV 10 20 40 1 Due to primary jons Due to Viays 1499 9 7 37 50 16 08 Total per # track 5258 Total, por okV 0 1992 2 Due to prumary sons 3750 Due to ¢ rays 28 81 Tota) per y track 6641 Total, per ckV 03771 3 Due to prmary fons 3749 Dus to é rays Total per pz track Total per av 4 Due to primary ions Due to é raya Total per x track Total per ekV 5 Due to primary ions Due to é raya Total per 4 track Total per ekV 6 Due to prumary ions Due to é rays Total por ¢ track Total per ekV 7 Due to primary ions Due to é rays Total per p track Total per ekV 8 Due to prumary ions Due to é rays Tota} per 4 track 76 25 Total per ek¥ 1154 055 Note For tissue density other than 1g /em * the figures headed ‘target diame should be regarded ag values of 2rp instead of 2r Further the figures of associa volume per micron of @ particle track,1¢6 the first three figures of each group of { sre to bs multsphed byp The associated volumes are in units of $77? happen that the associated volumes constructed round two é ray tracks may merge, asin Fig 7F, p 84 It would be difficult to allow for this effect by a mgid calculation, but the following method, which has been adopted, should prevent any serious ASSOCIATED VOLUME 361 erroransing Instead of taking wto account all é rays of energy exceeding 100eV , account 1s taken only of érays of energy exceeding energy 1’, where HV 1s chosen so that the number of d rays of energy > W 1s 1 per distance 2r along the a ray track Taste 83 Associated volumes for protons {Per micron track, and per ekV energy diesipation } Proton Target diameter, 2p, in mu ene: an “+ 10 20 40 80 1 Due to primary ions =—-235 3 1328 72 64 37 20 wT Due too rays” 1083 528 28 33 1456 733 Total per # track 343 6 185 6 1010 5176 26 Of Total per ekV 1241 6 704 3 646 1869 0 9404 2 Due to primary ions 160 2 1094 67 59 3651 18 63 Dus to é rays 691 365 20 82 ui 572 Total per pi track 2294 1459 $841 4767-2438, Total, per ekV 13 78 8761 5310 2 863 1463 3 Due to pnmary ions = =—-:122. 2 9165 61 88 35 50 18 50 Due to é rays 621 2877 1706 947 496 Total per » track 17431204 189% 4498 2346 Total per ekV 14.29 9 870 8470 3 686 1922 4 Due to primary tons 9929 7873 5655 3429 1833 Due to é rays 4239 2408 14 68 838 448 Total, per # track 417 1028 7123 42 67 22 81 Total, per ekV 14 54 10 65 7312 4380 2342 8 Due to primary ions 72:87 6158 4783 3167 17.89 Dus to é rays 3136 18 50 1173 697 386 Total, per pz track 104 2 80 06 69 56 38 64 2175 Total, per ekV 1477 11 34 8 439 6474 3081 8 Dus to pnmary ions 57 95 6073 4134 29:15 17:36 Due to 6 raya 25:18 15 22 9 88 605 345 Total per u track 83 13 65 95 51 22 35 20 20 80 Total per ekV 1485 11:78 9 148 6 286 3716 10 Due to primary ions 4833 4326 36 41 26 88 1677 Due to é raya ais 13 02 g6L §52 315 Total per p# track 6951 5630 45 02 3240 19 92 Total per ekV 14 88 1205 9 637 6 936 4263 Note For tissue densities p other than Ig /em * the figures headed ‘target diemeter thould be regarded as values of 2rp instead of 2r Further, the figures of essociated Volume per micron of proton track 1¢ the first three figures m each group of four &re to be multip by p The d are in units of $71? The correction 1s small except for the larger sizes of target The correction 18 of course only apphed when, on working out the value of I, it 13 found to exceed 100eV Tables 82 and 83 refer to definite energies of @-particles and protons, not to complete absorption in the tissue They are thus directly suitable for use when thin films are wrradiated by @ rays 362 APPENDIX I or protons which do not suffer complete absorption in the film When complete absorption occurs in the tissue, ag in neutron expernments or @ ray experiments using dissolved radon, in the absence of a calculation made specifically for this case an average particle energy should bo used Table 81 refers to electrons completely absorbed in the tissue and 1s thus surtable for use with X rays and y rays Unity should be added to the figures of associated volume per electron to allow for the two ends of the electron track This has not been done already in this table since it 13 required to be useable also for 5 ray calculations for which the addition would be mncorrect (The figures in Table 81 give the associated volumes represented in Fig 7£,p 84, the addition of unity to the associated volume per electron is required to give the associated volumes repre sented in Fig 7¢ ) If an expermment 1s made using # rays irradiating thin films of matenal, in which the f rays are not completely absorbed, the table may be used by differencing Conversion of associated volumes to 37% dose The calculation of the 37 % dose for any given size of target,1e that dose which corresponds to an average of one hit per target, 1s now straight forward A dose corresponding to lekV energy dissipation per #8 gives an associated volume of Vx $77 per x*, where V 1s the figure for associated volume per ekV read off from Tables 82 or 83 or deduced from Table 81 for the appropnate radiation and target size 1—exp (— V x 4ar5) 1s thus the probabukty of secur- ing a hit with a dose of1 ekV per #° The 37% dose,1e¢ the dose giving an average of one hit per target, 18 thus 1/(V$zr*) ekV per #? We can now deduce the dose m roentgens by reference to Table 2 (p 8) If the density 18 not umty but pg /em , the inactivation dose is 1/(Vp$7r°) ekV per 10-” g As an example let us calculate the inactivation dose to be expected for a virus having target diameter 2r= 14my irradiated by a rays of 5eMV, the density bemg p=1 4 g /em * We have 2rp=14x14=19 6mp, and mterpolating m Table 82 between the columns 2rp = 20 and 2rp= 10 we find that for a 5eMV a ray the associated volume 1s V=1 78 (units of 47r?) per ekV Thus the mactivation dose 13 1 o 178xi 4x $n (0 007) =279x 10%ekV per 79x 105ekV 10° g per 10-12 37% Dose 363 In Table 2 (p 8) we read that 1 r 1s 66 65eV per 10-2 g in virus protein Thus the mactivation dose 18 279 x 108/66 65=4 19x 10%r In this manner the curves of Figs 8 and 9 and thence of Figs 10 and 11 of Chapter mr have been calculated 362 APPENDIX 1 or protons which do not suffer complete absorption in the film When complete absorption occurs in the tissue, ag in neutron experiments or & ray experiments using dissolved radon, in the absenco of a calculation made specifically for this case an average particle energy should bo used Table 81 refers to electrons completely absorbed in the tissue and 3s thus smtable for use with X rays and y rays Unity should bo added to the figures of associated volume per electron to allow for the two ends of the electron track This has not been done already in this table since it 15 required to be useable also for 3 ray calculations for which the addition would be meorrect (The figures in Table 81 give the associated volumes represented in Fig 7£,p 84, the addition of unity to the associated volume per electron 1s required to give the associated volumes repre sented in Fig 70) If an experiment 1s made using # rays irradiating thin films of maternal, n which the # rays are not completely absorbed, the table may be used by differencing Converston of associated volumes to 37% dose The calculation of the 37% dose for any given size of target, 1e that dose which corresponds to an average of one hit per target, 18 now straight- forward A dose corresponding to lekV energy dissrpation per # gives an associated volume of Vx $ar* per 23, where V 3s the figure for associated volume per ekV read off from Tables 82 or 83 or deduced from Table 81 for the appropriate rathation and target size 1—exp (~ V x ar) 1s thus the probability of secur ing 8 hit with a dose of lekV perp? The 37% dose,re the dose giving an average of one hit per target, 18 thus 1/(V$nr°) ekV per x? We can now deduce the dose in r by reference to Table 2 (p 8) If the density 2s not umty but pg /em %, the inactivation dose 1s 1/(Vp$ar3) ekV per 10-* g As on example let us caleulate the mactivation dose to be = l4myp radiated expected for a virus having target diameter Sr by a rays of 5eMV, the density bemg p=1 4 g /em* We have Qrp=2 14x 1 4=19 6mp, and mterpolating wt Table 82 between the columns 2rp= 20 and 2rp= 10 we find that for a SeMV a ray the associated volume is V=1 78 (units of $7r%) per ekV Thus the mactivation dose 15 i 107%? oo ng 279 Ta8x 1 Tx Fr (0 00798 V 105A Pper 8 DOMINANT LETHALS 365 cells ‘There 1s evidence: that the mutation rate 1s less in unmpe sperm (presumably spermatogonia) than mn ripe sperm, the ratios bemg 018 1 for sex linked lethals, 0 67 1 for sex-linked visibles, and 0 58 1 for second-chromosome lethals That the difference between mpe and unmpe sperm ts so much more marked in the case of sex linked lethals than in the case of second chromosome lethals 1s presumably due to the fact that the spermatogoma are hemizygous for these lethals but heterozj gous for the second chromosome Iethals, and strongly suggests that the apparently low yield of sex inked lethals in unmpe sperm 1s mamly due to germinal selection,1e the death of spermatogonia containing the Iethals 2 The yield of visible mutations m barley produced by & given dose 13 four times as great when germinating seeds are irradiated than when dormant seeds are irradiated 3 DOMINANT LETHALS Ip ty) A most convincing demonstration that dommant lethals are mdeed due to damage to the chromosomes and not to the cytoplasm 1s afforded by an experiment which has been made using the parasitic wasp Habrobracon 4 The eggs of this wasp, if fertilized, develop into diploid females, if they are not fertuhzed they develop into haploid (gynogenetic) males Sperm which do hot enter an egg are, of course, incapable of development An unfertihzed egg which receives a few thousand roentgens does not hatch, whether fert:hzed or not, subsequent to irradiation But an unfertilized egg which receives a much heavier dose (order 3x 10*r) and 13 then fertilized by an untreated sperm may develop into a haploid (androgenetic) male in which the chromosomes of the egg play no role Evidently the dose of 3x 10r leaves the cytoplasm still capable of supporting the development of the intact sperm nucleus This experment demonstrates that the dominant lethal effect obtained with smaller doses to the egg must therefore be due to damage to the nucleus of the egg 1 Reviewed by Timoféeff Ressovsky NW (19378) 2 Its known that most sex linked lethals are lethal to individual cells (when homozygous) and not merely to the whole orgamsm (Demerec M 936) 3 Stadler LJ (1930) 4 Whiting AR (1946) Appendix IT TEXTUAL REVISIONS AND ADDITIONS: MUTATIONS (p 1541 The influence of temperature Stadler: has shown that the yield of visible mutations induced by irradiation of barley seed 18 the same, per umst dose, at 10, 20, 30, 40 and 50°C Expermments by different workers recording sox linked recessive Jethals 1n Drosophila have given conflicting results The experiments summanzed in Table 40: show no change 1n the y:eld of mutations with a 30° change of tempera- ture More recently, Kings determined the yreld of sex inked lethala produced by doses of 600-3600 r, given either at room temperature (~25°C) or at 05°C His resulta can be sates factorily fitted by the usual formula 1—e~TM? (p 146), the values of m being at 0 6° 7 40+0 70 lethals per 100 sperm per 1000r, at ~25° 34340 29 lethals per 100 sperm per 1000T, showing a quite sig) fi of rate at low temperature Medvedevs also obtained a sigmficantly higher yreld at 0°C than at ~ 20°C, and at 19°C than at 37°C p 154] Mutation yields in different cells It 1s of considerable interest to know whether the probabilty of a given mutational step being duced by a given dose 1s & constant or 1s different for the same mutational step in different 1 Mainly in the form noted by the author 2 Stadler, LJ (1930a) 3 Timoféeff Ressovsky, NW & Zimmer, KG (1939) Makhysm, JK (1944), ep Muller a J (1940), also obtamed no difference m the 4 King ED (1947) “The author yields at ~5 and ~37° states that the presence of 1ce sur ding the vialthe flies in the lower temperature oxper! ment increased the dose by 56% which hes not been allowed for in calculating the yrelds 5 Medveder NN (1935 1938) CHROMOSOME STRUCTURAL DAMAGE 367 Modifying factors (p Whale exposure of male Drosophila to infra-red radiation alone does not cause chromosome structural changes, exposure to infra red before X raying results in the yield of chromosome structural changes being increased : Thus 24 hr exposure to infra red rays of wave length 8000-20,000 A increased the Percentage of sperm showing chromosome structural changes after 4000r from 3144 % to 47+ 4%, an increase equivalent to raising the X-ray dose to between 5000 and 6000r No sigm- ficant changes were detected in the yields of dominant lethals, or of sex-lnked recessive lethalsz when the X-irradiation was preceded by infra red There 1s probably also an increase in the yield of chromosome structural changes when the infra-red treatment follows the Xx Taying a Expenments have also been made to study the effect of a pnor or subsequent treatment with ultra-violet light upon the yield of chromosome structural changes produced by X rays Kaufmann and Holleenders irradiated Drosophila males with 4000r of X-rays, and subsequently exposed them to a (surface) dose of 18x 108 ergs/cm ? of ultra violet hght of wave length 2536A Sahvary gland chromosomes of F, larvae were exammed for ross chromosome structural changes Chromosome structural changes were produced in 23 2+ 2 5% of the sperm, compared to 30% produced by 4000r without ultra-violet treatment It appears that ultra-violet hght (which does not itself induce gross 1 Kaufmann BP, Holleender, A & Gay, EH (1946) Breaks were counted in the salivary gland chromosomes of F, larvae The infro red Tediation raised the body temperature of the flies by about 7° 2 Kaufmenn BP & Gay, EH (1947) 3 Experiments to determine whett bseq infra red rh difies the yield of ch structural changes produced by X rays &re complicated by the fact that mfra red accelerates the Tipening of immature sperm Since immature sperm are less sensttive to X rays than mature the result of prol d infra red t: of male flies after X raying and before mating 15 to d sh the y1eld of ch structural changes However, if the females which have been mated to X ray males are then exposed to infra red radiation dunng the penod of oviposition, @ higher proportion of the F, larvae have chromoso structural changes me than i the ab: of infra red treat: (37 543% agauist 29 $4250 after 4000r according to Kaufmann BP (19468)) 4 Kaufmann BP & Hollaender A (1946) 366 APPENDIX ti The experment with Habrobracon aleo demonstrates that damaged chromosomes are positively lethal to a cell and not meroly negatively ineffective, emcee a fertilized egg in which esther egg or sperm haa been damaged by a moderate dose of radiation prior to fertilization does not hatch, but an egg in which either egg or aperm chromosomes are absent, or else have been rendered completely ineffective by a very heavy dose of rada tion, hatches as a haploid onganiam CHROMOSOME STRUCTURAL DAMAGF fp 230) Location of breaks When the obserted distribution of breaks was compared with the distnbution to be expected on the hypothesis that the prob- abihty of & break being produced in a guven segment 3 simply proportional to the number of bands in the segment, the expert mental distributzon differed significantly from the distribution expected on this hypothesis, the difference lying not m any pro nounced gradient of breaksbility from onvend of the chromosome to the other, but in the existence of a number of regiona m which the mean number of breaks per band was three or four times the average It 1a beleved that these segments contam heterochro matin, ames in salivary gland cells pairing occura among these regions, and between them and the proximal heterochromatin + {fp 217} Frequency relations Instead of studying separately the distribution of chromahd breaks, wochromatid breaks and interchanges as has been done in Table 58, one can determine the proportzan of cells which conten r chromatid breaks, together with s isochromatid breaks, together with ¢ imterchanges If the three types of aberrations are distributed mdependently in Poisson distributiona with mean valtes m,, m, and m, respectively, the proportion expected 6 (or mifirt} (e-me mzfat} (o-m mb/t) The results of tests of ths sorts on Tradescanha microspores show agreement with the hypothesis that the three types of a berrat distr buted mdependently m Poisson distributions x For add J evidence d. and seo Kaufi BP 1044), also Shaynsia BM (1945) 2 Catcheade DG Ee DE & Thoday JM (10460) CHROMOSOME STRUCTURAL DAMAGE 369 interchange between breaks in different chromosomes, and also hindering separation of the two breakage ends to make a permanent break Sensitivity at different stages {p 2 If Drosophila males are wradiated, and then mated to succes- sive batches of ururradiated females, the offepring from these matings are derived from sperms which, in the case of the first batch, are mature, and which in the later batches are at succes- sively earlier stages of spermatogenesis at the time of wradiation The yield of chromosome interchanges obtamed mn the successive batches progressively dumuushes,: showing that the earlier stages of spermatogenems are leas sensitive to the production of inter- changes than are the mature sperm Whiting: compared the sensitivity to X-rays of the oocytes{p 2 of Habrobracon irradiated at first meiotic metaphase and at diplotene (first meiotic prophase) respectively Lethal types of chromosome structural change led to death of the embryo 1n the morula stage, and could also be detected by the appearance of bridges and fragments at meiotic first and second anaphase and cleavage divisions Metaphase was many times more sensitive than diplotene, and the lethal effect was due to sister-union isochromatid breaks, which occurred only between the centromeres and the proximal chiasmata (deduced from the fact that bridges are seen at second metotic anaphase, not at first anaphase) This part of the chr isin t , ance the ch ta are r g the Separation of the centromeres, and the high senmtivity 1s explained by no restitutaon of prrmary breaks occurrmg Diplotene 18 considerably less sensitive, and sister-umion 180- chromatid breaks occur between chiasmata, as well as between centromere and proximal chiasmata (deduced from the fact that bridges are seen at first anaphase as well as at second anaphase) Some interchanges between breakages in different chromosomes 1 From 12 to 0% with 4000r im the expenments of Catsch, A & Radu, G (1943) Irradiated males were mated to females homozygous for the é d-ch gene bar eye and the third chromosome gene spineless The F, mate offspring were tested for hnkage between these genes 2 Whiting, AR (1948) 368 APPPNDIX 0 chromosome structural changes) reduces the proportion of X ray induced breaks which tako part in viablo types of re arrangoment Thoro appeared to be no corresponding changes in the proportion of X-ray induced breaks which behaved as dominant lethals Swanson: amularly found that the yield of chromatid inter changes induced by X raya in T'radescanhia pollen tubes was reduced by ultra violet hight (which does not itself induce inter- changes) Table 84 shows tho offect of a 30. sec exposure to ultra violot hght of wave longth 2536A upon the yield of chromatid mterchanges produced by 247r of X rays The X-rays were given in every case 2 hr after the germination of the pollen Taatz 84 Modification by ultra violet hght of the y:eld of chromatid mter changes produced by 247¢ of X rays in Tradescantia pollen tubes (Swanson (1944)) Time of ultra violet exposure in relation Yield of chromst:d to X ray exposure interchanges per 100 cella No ultra violet oo4 Ultra violet 1 hr before X rays 008 Ultra violet ummediately before X rays 022 Ultra violot ummediately after X rays 000 Ultra violet $ hr after X rays 0 Te} average 0-94 Ultra violet Lhr after X raya ri These results show that ultra violet light given either before, or soon after, the X-rays 1s able drastically to reduce the proportion of X-ray induced breaks which take partin chromatid interchanges That no such effect 1s found when the ultra-violet light 1s given after } hr or more has elapsed after the X-irradsa- tion 18 evidence that interchanges in pollen tubes (as im micro spores) are completed within this time The yield of sochromatid breaks 1s not much affected by « prior or subsequent ultra violet irradiation The yreld of ch tid breaks ts reduced, despite the fact that ultra-violet light alone 1s able to produce chromatid breaks It appears therefore that the effect of ultra-violet hght 18 to make restitution of an X-ray induced break more probable, and to reduce the probability of two breaks exchanging, except m the case of breaks at the same loci in sister chromatids Swanson considers that the effect of the ultra violet light 1s to make the nucleic acid matmnx of the chromosome firmer, so hindering 1 Swanson, CP (1944) CHROMOSOME STRUCTURAL DAMAGE 371 not sigmficantly changed by change of exposure time, while the 2 hit component diminishes with prolongation of exposure time An tsochromatid break mvolves two breaks, and while it appears that, with all radiations, these are usually produced by the same ionizing partrcles which traverse both sister chromatids of a split chromosome, there 1s no reason to doubt that if sister chromatids were broken at about the same locus by separate ionizing particles an isochromatid break would result Thus we should expect the yield of isochromatid breaks to be made up of two terms, a ‘I-hit’ term proportional to the dose and inde- pendent of the intensity, and a ‘2 hit’ term proportional to the square of the dose and diminishing with prolongation of exposure ume at constant dose Of the data shown in Fig 348, the most complete are those of Sax, and while these may be fitted by a straight line the x? test gives P=0 07), the fit 1s considerably improved when they are fitted by the formula y=ax+ fx? (the x? test gives P=06, despite the loss of one degree of freedom) Relative eficiencies of different types of radiation (p 240) Comparing the yields of exchanges, Kotval: found y rays less efficient than X rays in the ratio 0 80+ 0 07, while Catcheside et al: found y rays less efficient than X-rays in the ratio 0 77 +0 05 Ey idently, any difference that exists between y rays and X-rays 18 shght, and in the direction of y rays being the less efficient The following provisional explanatson may be advanced for [p 243) the difference in relative frequencies of the different types of chromosome aberration in X-ray and ultra violet experiments In the first place, the fact that isochromatid breaks are not Produced by ultra violet light presents no difficulty, since \sochromatid breaks found m experiments with ionizing radia- tions are attributed to the passage of a single 1omizing particle through both chromatids Simcoe ultra violet quantal absorp tions are not locahzed along tracts in this way, we should not expect to get isochromatid breaks im ultra violet experiments The fact that exchanges between breaks in different chromo Somesdo notoccur multra violet expertments in which chromatid t Kotval (unpublished) 2 Catcheside DG Lea DF & Thoday, J M (19465) 370 APPENDIX lt occur at tho higher doses, and tho yield of these interchanges 13 reduced by fractionation of dose, which affords evidence that 4 considerable proportion of primary breaks restitute It seems probable that tho lower senmtivity at diplotene 18 due to the lower tension in the chromatid threads permitting restitution of a large proportion of the breaks primarily produced It 1s noteworthy that the sensitivity of Habrobracon sperm: 3 comparable to that of tho oocytes in first metaphase rather than diploteno, confirming tho view: that restitution does not occur in sperm, and that the condition of the chromosomes in sperm 18 comparable to that :n metaphase (p 233) Dose relations The yield of exchanges produced by neutrons 1s proportional to the dose and independent of intensity, while the yield of exchanges produced by X-rays 1s proportional to the square of the dose at high y and d hes at lower ti This difference 1s believed on theoretical grounds (see p 250) to be quantitative rather than qualitative, that 18 to say, 1b 18 believed that the yield of exchanges 1s, with both radrations, equal to the sum of two terms, a ‘1 hit’ term proportional to the dose and independent of intensity, and a‘2 hit’ term proportional to the square of the dose and dependent upon intensity It has been found possibles to detect the 1 hit component in X ray experiments The yield of interchanges per cell (y) produced by a given dose (x roentgens) was fitted by the formula y=ax+ x" In these experiments four doses (approximately 25, 50, 100 and 150r ) were given In a constant time, which was 251 mm 1m one series and 1 2 min in another The values of « and f obtamed are given in Table 85 It 1s seen that the I hit component (a) 18 Tanrz 85 Analysis of X ray induced interchanges into 1 Int and 2 hit components (ysax+fz" is the number of ¢/c interchanges per nucleus produced by & dose of x roentgens ) Duration of exposure (min ) @x 104 per r Bx 108 per r# 251 2664091 38940 92 12 185+1 04 13 064119 x Heidenthal G (1945) HJ (1941) 2 Maller, 3 Catcheade , Les DE & Thoday JM (19468) DG, BACTERIA 373 Teverse the argument and to use the values of & given by other methods to show that the probability of an exchange between two breaks produced at a separation not exceeding h=ly approaches umity Alternative derivation of Gr [p 2u The experimental fact that the jield of interchanges produced by a given dose dimmmshes with increase of the exposure time must mean that two breaks at a given position produced at different times have a smaller probability of exchanging than the same two breaks at the same position would have if produced amultaneously We may designate by f(t) the factor by which the probabihty is reduced with increase of t, f(t) diminishes from the value unity at ¢=0 Consider two breaks independently produced by an irradiation at umform intensity which extends over time 7 Each of them may occur with equal probability anywhere m the time 7, and it 1s readily shown that the probabibty of the two breaks being Separated by an interval between ¢ and ¢+dt1s 2(1—¢/T) (di/T) The ratio @ of the number of exchanges produced by the given dose spread over time T' to the number which would be produced by the same dose given in a very short time 1s evidently Tr 1 o~ [20 ~H2) (at/T) fl)=2] _ flTx) L—2) de To evaluate G we must assume some plausible form for the function f(t) The simplest function fulfillmg the condition of being unity at ¢=0 and diminishing gradually with increase of tis f(t)=e-" With thas form off(t) we obtain G=2(7/T)? {T/r-1+e-Th} BACTERIA Effect of intensity [p 321 Table 86 presents further data show ing how the mean lethal dose 1s the same, within the error of the experiment, whether the Mradiation 1s made at a low intensity and spread over a prolonged time, or at a high intensity and concentrated in a short time This table is thus supplementary to Table 74 (p 322) yy Mateheettea MO Ten AO BMW. TA fan mee 372 APPENDIX 11 breaka aro freely produced 13 moro difficult to understand, and at first glanco ascems to demand the assumption that ultra violet induced breaks are different in naturo from X ray- induced breaks Howover, wo have scen (p 368) that ultra violet light preceding or following X irradiation reduces the proportion of X ray induced breaks which take part in gross chromosome structural changes Presumably ultra-violet ght treatment simultancous with the X-irradiation would have the same effect Evidently, therefore, ultra-violet ight has, besides the property of producing breaks, the additional property of modifying the ehre or the nuclear sap in some way which reduces the proportion of breaks which take part m exchange : This would seem to ho a sufficient explanation of the absence of exchanges in ultra violet experiments on Tradescantia pollen tubes, and my apply also to Drosophila sperm and maize pollen The effinency of ultra violet hght in producing breaks 1s ex- tremely low Tho yteld of chromatid breaks obtainedin Swanson’s experiments, for example, corresponds to about 1 chromatid break observed per 10‘ quanta incident on the chromatids Up 29} Evaluation of breakage frequency It has been assumed above that when two breaks are produced (simultaneously) at a separation less than h=1y, exchange between them is certain It would perhaps be more plausible to suppose, not that exchange 1s certain, but that union 1s random between the four breakage ends, which would make the prob abihty of exchange recurring equal to two turds If this modification 1s adopted, the values of £ hsted for Methad IfI in Table 67 should be mereased by about 20%, and the values of f correspondingly reduced It 1s arguable that we have no nght to assume either that exchange 1s certain, or that exchange ts random between the four breakage ends, since the arguments of the last section which led to the conclusion that most pairs of breaks which exchange are initially separated by a distance not exceeding 1 did not give any estimate of the probability of exchange occurring under these circumstances If this view 1s accepted, Method IIT cannot be used In this event, however, it 1s permissible to 1 Swanson CP (1944) suggests that the action 1s on the matrix of the chromosomes BIBLIOGRAPHY Titles given are paraphrases of the original titles in many tnstances ArnERsotp, PC & Lawrence, JH (1942) Physiological effects of neutrons Ann Rev Physiol 4, 25 Atzert, W & Porrrzer, G (1923) Influence of X rays on cell division PartI Arch Mikr Anat 100, 83 Avpertr, W & Pourrzer, G (1924) Influence of X rays on cell division Part II Arch Mtkr Anat 103, 284 Atrsopr, CB {1944) Radiochemistry, a review of recent progress Trans Faraday Soc 40, 79 Axper, T (1932) Production of é rays by @ particles Z Phys 76,172 Atrensuna E (1934) Production of mutations by ultra violet ight Amer Nat 68, 491 ALrEenBurG, E (1936) Mutations by the polar cap method of treatment Bul Zh 5, 27 ANDERSON, RS & Harrison, B (1943) Effect of X rays on ascorbic acid J Gen Phystol 27 69 Atwoop, K & Rotuzrson, GK (1941) Efficiency of the primary ph 1 process in sol J Chem Phys 9, 506 Backstrom, HL J (1940) Filters for ultra violet hght Acta Radtol , Stockh , 11, 327 Bapian, J (1933) The chromatin and development cycle of bacteria Arch Mikrolnol 4, 409 Baer SL (1835) Action of # rays on bacteria and viruses Brit J Exp Path 16 148 Baxer SL & Nanavorry, § H (1929) Action of ultra violet hght on bacteria and bacteriophage Brit J Exp Path 10, 45 Batpincer, E & Houser, P (1939a@) Disintegration of nitrogen by fast neutrons Nature, Lond , 143, 894 Batpincrr, E & Huzer, P (19396) Duismntegration of mtrogen by fast neutrons Hely Phys Acta 12, 330 Baver H (1939a) Production by X rays of chromosome mutations in Drosophila Chromosoma 1, 343 Baver H (19398) X ray mduced chr in ring X h of Drosophila Natur haften, 27, 821 Baurr H, Demerec, M & Kavrmann, BP (1938) X ray induced ch Jt in Dr hal iy ae 23,610 Bawpen FC (1943) Plant Veruses and Virus Diseases Waltham, Massachusetts Bawpen FC & Pre, N W (1938) Chemical, physical, and serological Properties of plant viruses Tabul trol , Berl + 16, 355 Bru Govsky MLE (1939) Frequency of X ray induced minute chromo some rearrangem in Drosophila ents Bull Acad Sc. U RSS p 159 Bevrorp F (1936) Monochromator for ultra violet hght J Opt Soc Amer 26,99 374 APPENDIX 1 Taiz 86 Independence of mean lethal done on intensity Intenaty MAD Radiation Organi (+ fran) (r) Reference X reys (071A) Bactonophage, 57,500 468x110" 1 cis 1730 473 114 469 X rays (071A) B dysentersae, 40,000 51x10" Y 2,180 83 834 52 % raya (1 S4A} Yonat 157,000 10-1 x 10" 8& ellipsordeur 4,800 10-0 135 4103 Ultrs-viotet light (ergaem ~* nec} (erga erm —*) 2536A B dysenteriae, 43 000 245 Y6R 8 300 24g 4,800 241 830 245 260 264 83 260 xl 260 8 258 Yenst 140 000 152 S eispsndeus 14,800 796 2,800 780 805 770 280 778 224 315 28 776 1 Latarjet, R (1942) 2 Lataret, R (1944) {p 395) Metabolic disturbances A culture of bacteria rendered non infective by a heavy dose of radiation will nevertheless continue to respirer and will support a limited multiplication of bactenophage> These properties ate not retamed by bacteria killed by heat or chemical disinfectant x Bonét Maury, P Perault, R & Enchsen, ML (1944) 2 Rouyer, M & Lataryet, R (1946) BIBLIOGRAPHY Titles given are paraphrases of the original titles in many instances Arszrsorp, PC & Lawrence, JH (1942) Physiological effects of neutrons Ann Rey Physiol 4, 25 Atszatt W & Poxrrzer,G (1923) Influence of X rays on cell division PartI Arch Mtkr Anat 100, 83 Atseri, W & Porrrzer, G (1924) Influence of X rays on cell division Part II Arch Mikr Anat 103, 284 Attsorr, CB (1944) Radiochemistry, a review of recent progress Trans Faraday Soc 40, 79 ALrer, T (1932) Production ofé rays by a particles Z Phys 76, 172 Atrenzore, E (1934) Production of mutations by ultra violet light Amer Nat 68, 491 Atrenaura, E (1936) Mutations by thepolar cap method of treatment Bul Zh 5, 27 ANDrRson, RS & Hanpison, B (1943) Effect of X rays on ascorbic acid J Gen Physiol 27, 69 Arwoop, K & Rotierson GK (1941) Efficiency of the primary Photoch L process in sol J Chem Phys 9, 608 Bacustrom, HLJ (1940) Filters for ultra violet hght Acta Radsol Stockh , 11, 327 Bapian, J (1933) The chromatin and devel ys eyele of bact Arch Mikrobiol 4, 409 Bazer, § L (1935) Action of # rays on bacteria and viruses Br J Exp Path 16, 148 Barer, SL & Nanavurry §H (1929) Action of ultra violet light on bacteria and bacteriophage Brit J Exp Path 10 45 Batpivarr E & Huner P {1939a) Disintegration of nitrogen by fast neutrons Nature, Lond , 143, 894 Batowcer, E & Huser, P (19395) Disintegration of mtrogen by fast neutrons Helv Phys Acta 12, 330 Bauer H (1839a) Production by X rays of chromosome mutations in Drosophila Chromosoma 1 343 Baver H (19396) X ray induced chr t m rng X chr of Drosoph Natur haften, 27, $21 Baver, H, Demerec M & Kaurmarn, BP (1938) X ray induced h lalterat. in Drosophil he G 23 610 Bawoen, FC (1943) Plant Viruses and Verus Diseases Waltham Massachusetts Bawoen, FC & Prez, NW (1938) Ch 1 ph and serological Properties of plant viruses Tabul biol, Berl, 16, 355 BELcovsxy ML (1939) Frequency of X ray induced minute chromo some reatrangementain Drosophila Bull Acad Sa URS S »p 159 Bewrorn, F (1836) Monochromator for ultra violet hght J Opt Soc Amer 26,99 376 BIBLIOGRAPHY Bertie, H (1033) Quantum mechanics ofone and two electron problems Hands Phys 24 (1), 273 Brsnor, D W (1942) Cytological demonstration of chromosome breaka soon after X irradiation Genetica, 27, 132 Bonér Mauny,P & Ousvien, HR (1989) Utilization of tho biologicaleffect of radiation in the study of rmmumty Act Sci Ind No 725,p 492 Ponnenya (iil) Noautron encrgy from D4D reaction Phye Ree Bonven TW & Bnvunaxesn Wf (1935) Neutron energy from Li+-D duntegration Phys Rev 48, 742 Bovnrr, TW & Brupaxer, WM (1098) Noutron energy from dis integration of Boe, B, and C by douterons Phys Rev 50, 308 Bowry, Eat (1042) The Chemical Aapecte of Laight Oxford Borrsax, ML (1943) Effects of srradiation on oocytes of Stara Genetica, 28 71 Brarraws, kG (1938) Decomposition and synthesis of Hi by a particles J Phys Chem 42, 617 Brooa, E (1043) Chemical action of X rays on a non aqueous solution Nature, Lond, 153, 448, 530 Bromrterp, RT (1943) Effect of colchicine pretreatment on the fi 3 y 4 h 1 ab induced by X ursdiation Pree Nat Acad Sea, Vash, 29, 190 Bruynoonz, R & Munp W (1935) Effect of radon on bacteral motihty C.8 Soe Bil Paris, 92, 211 Bussz, WF & Dantezs, F (1928) Chemucal effects of cathode rays on oxygen, air, nitric oxide and carbon dioxide J Amer Chem Soc 30, 5271 Canvon,C V & Rice,O K (1942) A monochromator using a large water pnsm Re, Ses Instrum 13, 513 Cant, RG & Donarpson, M (1926) Effect of y rays on nutosis Proe Roy Soc B, 100, 413 Cant1 RG & Srear FG (1927) Effect of y rays on mitoais tn miro Proc Roy Soc B, 102 92 Cant, RG & Srran, FG (1929) Effect of y rays on rutosis sb vitre Proc Roy Soc B, 105 93 Carnsox, JG (1938a) Effecta of X rays on the nauroblast chromo somes of the grasshopper Genetws 23, 696 Carison JG (19385) Mitotic beh of ynduced fr lacking spindle attachments Pree Nai Acad Scr, Wash , 24, 500 Casrson, TG (lé4la} X cay mduced angle breaks im neurcblast s ofthe hopper Proc Nat Acad Ses Wash ,27,42 Canrson, JG (18416) X radiation on grasshopper chromo Effects of somes Cold Spr Harb Symp 9, 104 Catcaserps, DG (19380) Frequency of induced structural changes 1 the chromosomes of Drosophsla J Genet 36 307 Carcuesipe, X ray induced interchanges DG (19383) Frequencies of mmaize J Genet 36, 321 Carcuesiwe DG & Lea DE (1043) Effect of comzstion distribution on chromosome breakage by X rays J Genet 45 188 BIBLIOGRAPHY 377 Carcurstpe, DG & Lea, DE (1045a} Induction of dominant lethals in Drosophila aperm by XK rays J Genet 47,1 Catcursipe, DG & Lea, DE (19456) Domunant lethals and chromo some breaks mm ring X chromosomes of Drosophila J Genet 47, 25 Caucnors, Y (1932) Cathode ray tube J Phys Radium, 3, 512 Cuampers, H & Russ, S (1912) Bactericidal action of radon Proc Roy Soc Med 5, 198 Cuarz,GL & Coz, WS (1937) Photochermcal reduction with % rays and effect of additive agents J Chem Phys 5, 97 Curx, GL & Pickers, LW (1930) Chemical effects of X rays and energy relations involved J Amer Chem Soc 52, 465 Craus, WD (1933) Bactericidal action of X rays in the presence of heavy ions J Exp Med 57, 335 Costeyrz, WW & Fouron, HB (1924) Bactenmdal action of ultra Molet ght Se, Pap US Bur Stand 19, 641 Cold Spring Harbor Sympoma (1941) Vol 9, Genes and Chromosomes Courtoy, AH & Arzison, §K (1035) X rays wn Theory and Expert ment New York Coon, EV (1939) Recovery of Ascans oggs from X rays Radrology, » 289 Coormer, WD (1926) Cathode ray tube J Franklin Inst 202, 693 Coorgr, FS, Bucnwaup, CL, Haskins, CP & Evans, RD (1939) Sathode tay tube for biological experuments Rev Sei Instrum 0, 73 Gowri, JA (1924) Action of X rays on tissue cells Proc Roy Soc 1» 96, 207 owrner, J A (1926) Action of K rays on Colpidium colpoda Prac Roy Soc B, 100, 390 Caowrae, JA (1938) Bhological action of radiations Brit J Radiol 1, 132 Curnm, P (1935) Radiacimty Pans Daz, WM (1940) Effect of X rays on enzymes Buochem J 34, 1367 Dare, WM (1042) Effect of K rays on the conjugated protem d amno acidoxdase Biochem J 36 80 Dar WM (1943a) Effects of X rays on acetylcholine solutions J Physiol 102, 50 Date WM (19436) Effect of XK rays on aqueous solutions of bio logically active compounds Brit J Radiol 16 171 Datz, WM, Merevrra, WJ & Tweepm MCK (1943) Mode of action of a on aq 1 wature, Lond , 151, 281 Daruinaton, CD (1937) Recent Advances tn Cytology London Danuneton CD (1942) Chromosome chemistry and gene action Nature, Lond 149, 66 Dartineron, CD & La Coun LF (1942) The Handling of Chromo somes London Der, PI (1932) Attempts to detect the inte: "sjoctrans, Proc Roy Soe A 136,127 on OF neutrons with 378 BIBLIOGRAPHY Drunec, M (1934) The gene and its role in ontogeny Cold Spr Harb Symp 2,110 Demenrc, M (1037) | Relationsup between various chromosomal Drsrrec, M (1038) Heredaae effects of N rays Radiology, 30, 212 hanges in 2: loma, Fug jub vol p 125 Drvwenec, Io& Favo U 941) Deficiencies in Drosophila Proc Nat ftcad Ser, Wash , 27, 2 Demrnec, M, HottarNpen A, Houraay, MB & Uisvor, M (1942) T'ffects of monochromatic ultra ssolet ight on Drosophila Genetics 27, 139 Demenze, M, Kaurmaxrn BP & Svrron, L 1042) Genetic effects tb: m Drosophat 140 Demrnec, M, Kaurmans, BP, erro.ké& thax U (1941) The gene Yearb Carneg Instn, 40, 225 Demerro, M, Kaurmans, BP, Fano U, Surton, E & SANSOME, LR (1042) The gene Yearb Carneg Instn, 41 190 Drurstrr, ER (18fla) Ind: of Jethals, Isthals and ct h in Drosophila by neutrons Proe Nat Acad Ses, Wash, 27, 249 Desrster, F R (19416) Dependence upon intensity of the yield of ray induced chromosome structural changes om Drosophila amer Nat 75, 184 Dessaver, F (1023) Point heat theory Z Phys 20 288 Dresinson, RG (1935) Photochemical reactions in gases and solutions Chem Rev 17, 413 Dickson, RG (1938) Photochemical reactions in hquids and gases J Phys Chem 42,739 Doerr, R & Hauraver, C (1938) Handbook of Vsrus Research Vienne Dozors KP, Warp, GL & Hacuren, FW (1936) Effect of y rays on Bact coli Amer J Roentg 35, 392 Drexer, G & CaMPBELL Renton, ML (1936) Bactericidal action of ultra violet hght Proc Roy Soc B, 120, 447 Duaye, W & ScHEVER, O (1913) Decomposition of water by o rays Radtum 10, 33 Dusinmy, NP & biporov B.N (1938) The position effect of the hairy gene Biol Zh 4 555 Dusinry NP Soxorov, NN & Trntaxov, GG (1935) Cytogenetic study of the position effect Biol Zh 4 707 Duco, BM (Editor) (1936) Brelogrcal Effects of Radzation New Duacar 7 M & Hoxtsrnper A (1934c) Action of ultra violet hght Yor! on bacteria and viruses J Bact 27, 219 Puccar BM & Horzamnper A (1934) Action of ultra violet ght on bactena and viruses J Bact 27 241 Duranp E Hortaenper A & Hovutanan MB (1941) are violet absorption sf of the wall of Drosoph Hered 32 51 Dusunin, MA & Bacuew, A (1933) ‘lime factors concerned in the bactencidal action of ultra violet hght Proc Soc Exp Bol NY 30, 700 BIBLIOGRAPHY 379 Eurisuany, O & Noeruunc, W (1932) Bactericidal action of mono chromatic ultra violet hght Z Hyg InfektKr 113, 697 Exuincer, P & Gruuw, F (1930) Bactericidal action of secondary radiations from X rays Strahlentherame 38, 58 Ens, CD & Aston, GH (1930) Absolute intenstties and internal conversion coefficients of the y rays of RaB and Ra€ Proce Roy Soe A, 129, 180 Enus, EL & Detsrucr, M (1938) Growth of bacteriophage J Gen Phynol 22, 365 Exmons, CW & HounaEnprr, A (1939a) Production of variants in Trichophyton by ultra violet hight Genetics 24, 70 Exons CW & HornaENDER, A (19396) Production of mutations in Trichophyton by ultra violet ght Amer J Bot 24, 467 Evans, TC, Stravcuten JC Lirrte, EP & Faia, G (1942) Influence of the medium on radiation injury of sperm Radiology 39, 663 Exner, FM & Luria, & E (1941) Sizes of bacteriophages determined by X ray inactivation Scrence, 94, 394 Evxrme, H, Hirscurerver JO & Taytor, HS (1936a) Theoretical treatment of ortho para hydrogen conversion by «& particles J Chem Phys 4, 479 Eyric, H, Hinscuretper JO & Taxtor HS (19365) Radio chemucal synthesis and decomposition of HBr J Chem Phys 4 570 Fapercek AC (1940) Chromosome fragmentation by 1X rays J Genet 39 229 Fanerct AC (1940b) Equivalent effect of A rays of differont wave length on Tradescantia chromosomes J Genet 40 379 Fano, U (1941) Analysis and interpretation of chromosomal changes in Drosophila Cold Spr Harb Symp 9, 113 Fano, U (1942) On the interpretation of radiation experiments in genetics Quart Rev Biol 17, 244 Fano, U (1943a) Mech of ind: of gross chr 1 re arrangements in Drosophila sperms Proc Nat «cad Ser, Wash, 29, 12 Fano, U (19435) Neutron induced lethals in Drosophila Genetics, 28 14 Fano, U (1943¢) Production of 10n clusters by A rays Nature Lond 151, 698 and 152 186 Franck J & Ranrnowrtscu, E (1934) Free radicals and the Photo chemistry of solutions Trans Faraday Soc 30, 120 Fricke, H (19340) Reduction of O, to H,0, by the irradiation of its aqueous solution with A rays J Chem Phys 2 556 Fricke H (1934b) Chemuco physical actions of A rays Cold Spr Harb Symp 2 241 Fricke H (19354) Decomposition of H,O, by the irradiation of ats aqueous solution with X rays J Chem Phys 3 364 Fricke H (19356) Chermncal properties of\ ray activated mol Cold Spr Harb Symp 3 55 ” Y ofecules 380 BIBLIOGRAPHY Friczz, H (1038} Donaturation of proteins by tonfzing radiations Cold Spr Harb Symp 6, 164 Fricxy, H & Brownscomnr, ER (1033a) Tffect of X rays on chro mato solutions J Amer Chem Soc 55, 2358 Fricke, H & Brownscomnz, ER (19336) Inabibty of X rays to decomposo water Phys Rev 44, 240 Fricke, H & Hanr, EJ (1034) ‘Transformation of formic acid by d of ita aq lution with X raye J Chem Phys 2, 624 Fricke H & Hant, EJ (1935a) Oxidation of ferrous iron by irradia tion of its aqueous solution with X raya J Chem Phys 3, 60 Fricsee, HH & Hant, E.J (19355) Ox:datron of the nitrite to the mitrate ton by X rays J Chem Phys 3, 305 Fricxe, H & Hart, EJ (1935¢) Oxidation of the ferrocyanide arsenite, and selentte rons by X raya J Chem Phys 3, 596 Fricke H & Hart, EJ (1935d) Decomposition of water by X raya in the presence of tho iodide or bromide 1on J Chem Phys 3 596 Fricke, H & Haat EJ (1936) RB induced by ph ation of the water molecule J Chem Phys 4, 418 Fricke H, Harr, EJ & Surru,H P (1938) Chemical action of X ray activated water J Ohem Phys 6, 229 Fricke, H & Morse, § (1927) Action of X rays on ferrous sulphate Amer J Roentg 18, 426 Fricnz, H & Monse, § (1929) Action of X raya on ferrous sulphate solution Phil Mag 7, 129 Fricke, H & Perensen, BW (1927) Action of X rays on haemo globin Amer J Roentg 17, 611 F amp, WF & A RS (1940) Inactivation of rabbit papilloma virus by X raya Proc Soe Exp Bwl,NY,45 713 ip,WF &A RS (1941) Influence of protem and virus on the tivation of rabbit psepull virus by Xrays J Exp Med 74 463 Friepewaip, WF & Anprrson, RS (1943) X raya on cell Effects of virus associations J Exp Med 78, 285 Fry, HJ (1936) Time schedule of mitotic changes im developing Arbacw eggs Biol Bull Woods Hole, 70, 89 Garzs, FL (1929a) Bactencidal ection of ultra violet ight J Gen Physwol 13, 231 Gares FL (19298) Bactemecidal action of ultra violet hght J Gen Phystol 13, 249 Garces, FL (1930) Bactericidal action of ultra violet ght J Gen Physol 14 31 Gepye GR (1931) Decomposition of nitrous oxide by cathode rays J Chem Soe p 3016 Gepve GR & Atrmone, TE (1930) Decomposition of ammomsa by cathode rays Proc Roy Soc A, 130, 346 Gevve GR & Atumone, TE (1932) Synthems of hydrazine and ammonia by cathode rays J Chem Soc p 1158 BIBLIOGRAPHY 38k Gnzs, NH (1940) Induction of chromosome aberrations by neutrons in Tradescantia microspores Proc Nat Acad Ser, Wash , 26, 567 Guzs, N.H (1943) Comparative studies of the cytogenetic effocts of neutrons and X rays Genetics, 28, 398 Giass,H B (1940) Differential susceptibihty of the sexes an Drosophila } by. to the effects of X raya im pr chr a Genetics, 25, 117 Groczrn, R (1932) Quantum physics of the biological action of X rays Z Phys 77, 653 Grocuter, G & Linn, SC (1939) Electrochemistry of Gases and other Dielectncs New York Guiczsmann, A (1941) Quantitative examination of human biopsy I taken from diated car Brit J Radiol 14, 187 Giucxsmann, A & Srean, FG (1939) Effect of y rays on the cells of fasting tadpoles, and of tadpoles at low perature Brit J Radiol 12, 486 Gowen JW (1939) Effect of X rays of different wave lengths on viruses and genes Third Int Cancer Congr p 17 Gowen, JW (1940) Inactivation of tobacco mosaic virus by X rays Prog Nat Acad Sa, Wash 26 8 Gowen, JW (1941) Mutations in Drosophila, bacteria, and viruses Cold Spr Harb Symp 9, 187 Gowen, JW & Gay, EH (1933) Gene number, kind, and size in Drosophila Genetica, 18, 1 Gowen, JW & Lucas, AM (1939) Action of X rays on vaceimia virus Sctence 90 621 Gray, LH (1937} Radiation dosimetry Brit J Radiol 10, 600, 721 Gray, LH (1944a) I hod of rd energy Proce Camb Phil Soc 40, 72 Gray, LH (19446) Dosage rate mradiotherapy Brit J Radvol 17,327 Gray, LH, Morrram, JC, Reap, J & Srzar, FG (1940) Biological effects of fast neutrons Brit J Radiol 13, 371 Gray, LH & Reap, J (1989) Neutron dosimetry Nature, Lond, 144, 439 Gray,L H & Reap, J (1942a) Effect of ionizing radiations on the bean root Brit J Radiol 15, 11 Gray, LH & Reap, J (19428) Lethal action of y rays on the bean Toot Brit J Radiwl 15 39 Gray, LH & Reap, J (1942c) Lethal action of fast neutrons on the bean root Brit J Radiol 15, 72 Gray, LH & Reap J (1942d) Lethal action of « rays on the bean root Brit J Radwl 15 320 Gray, LH, Reap, J & Pornrer M (1943) Lethal action of X rays onthe bean root Brit J Radtwl 16, 125 Grrrn, RH Anperson, TF & Saver, JE (1942) Morphological structure of the virus of vaccinia J Exp Med 75, 651 Grote W (1937) Quantum yields of gas reactions induced by short wave ultra violet hght Z Phys Chem B, 37, 307 Gunruer P & Howzarrer, L (19392) Decom position of water vapour by A rays Z Phys Chem B 42 346 382 BIBLIOGRAPHY Giwrnen, P & Horzarren, L (19306) The X ray eensttivity of hquid water andico Z Phys Chem B, 44, 374 GOnruer, P & Letcutren, H (1936) Decomposition of HI and syn thesis of HBrby A rays Z Phys Chem B, 34, 443 Hanson, F B (1028) Effects of X raya on productivity and sex ratio in Drosophila Amer Nat 62, 352 Hanson, FB & Heya, F (1929) Analysis of effecta of different rays of radiaim im producing lethal mutations in Drosophila Amer Nat » 2 Hanson, FB & Hexs, F (1932) Radium and lethal mutations in Drosophila Amer Not 66 335 Hasxins CP (1938) Apparatus for atudying the biological effects of cathode rays J Appl Phys 9, 553 Heipr, LJ (1939) Morcury lamp for ultra violet light of wave Jength 2536A Science, 90, 473 Herren, RG (1940) X ray induced and naturally occurring chromo somal ¥ an Drosophila peeudoob Genetics, 26,1 Hevsuaw, P& (1932) Effect of X rays on time of first cleavage an Arbacta eggs Amer J Roentg 27, 800 Hensnaw, P'S (1038) Action of X rays on nucleated and non nucleated egg fragments Amer J Cancer, 33, 258 Hensuaw, P& (1940) Action of rays on the gametes of Arbacia punctulata Amer J Roentg 43, 899 Hensnaw, PS & Conzn, 1 {1940} Action of X rays on the gametes of Arbacia punctulata Amer J Roentg 43, 917 Hensuaw, PS & Francis, D§ (1996) Effect of X rays on cleavage in Arbana eggs Brol Bull Woods Hole, 70, 28 Hevsuaw, PS & Hensuaw, CT (1933) Changes in susceptibility of Drosophila eggs to particles Biol Bull Woods Hole 64 348 Hensuaw, PS Hensuaw, CT & Francis, DS (1933) Action of X rays on Arbacia eggs Radsology, 21, 533 Here, F (1933) Action of @ rayaon bacteria Strahlentherame, 47, 374 HErcrik Ff (1934a) B dalactionofa rays Alentherapre, 49, 438 Hencix, F (19346) Temperat ficient of the b dal action of arays Strahlentherapte, 49, 703 Hercix, F (1936)~ Bactericidal action of ultra violet light J Gen Phystol 20 589 Hinsyerwoop, C.N (1940) Kenetres of Chemical Change Oxford Hirescureiper, JO & Taytor HS§ (1938) a particle reactions in CO, O,, and CO, systems J Chem Phys 6 783 Horrmann, JG & Reinuarp, MC, (1934) Recovery from the effects of irradiation Radzology 23 738 Hotmay, E P (1936) Ab spectra of ty tryptop: and thew mixtures Brochem J 30, 1795 Hornaenper A & Craus WD (1936) Bactericidal action of ultra violet hght J Gen Physrol 19 753 Hortaenper A & Ducecar BM (1936) Action of ultra violet light on Bact cols and tobacco mosaic virus Proc Nat Acad Ser Wask 22 19 BIBLIOGRAPHY 383 Hontaenper, A & Emsrows, CW (1941) Production of mutations in fungi by ultra violet hght Cold Spr Harb Symp 9, 179 Houweck,F & LAcASsaGNe A (1930) Action on yeasts of softX rays C.R Soc Biol, Parts 103, 60 Hornseck, G & Howert, I (1941) Production of secondary electrons by Prays Proc Amer Phil Soc 84 33 Hovsvon, RA (1911) Absorption of hght by morganic salts Proc Roy See Edinb 31, 521 JarFé, G (1913) Recombination of sons in @ particle columns Ann Phys , Lpz , 42, 303 Jorpan, P (1938a) Physical structure of genes and viruses Natur wrasenschaften, 26, 693 P (19385) Jorpan, Biol 1 action of rad Radol 2 16 Jorpan, P (1938c) Methods and results of radio biology Radologtea, 3, 157 Suncers, JC (1932) Decomposition and synthesis of ammonia by arays Bull Soo chim Belg 41, 377 Juut, J & Kemp,T (1933) Effect of X rays y rays ultra violet ght and heat on chick tissue in culture Strahlentherame, 48, 31 Kanne WR (1937) Preparation of polonium sources Phys Rev 52 380 Kara Micnamova, E & Lea, DE (1940) Ionization measurements in gases at high pressure Proc Camb Phil Soc 36, 101 Kaurmann, BP (1939) Distribution of induced breaks along the X chromosome of Drosophila Proc Nat Acad Sa, Wash 25 571 Kaurmann, BP (1941a) Effect of intermittent exposure in the produc tion of chr b an Drosophila Proc Nat Acad Ser, Wash 27, 18 Kavrmann BP (19416) Induced chromosomal breaks in Drosophila Cold Spr Harb Symp 9, 82 Kaurmany, BP (1943) Comg duced rear of Droso phila chromosomes Proc Nat Acad Sct Wash, 29, 8 Kaurmann BP & Demerec M (1937) Hrequency of induced breaks am chromosomes of Drosophila Proc Nat Acad Scr, Wash 23,484 Kaye, G WC & Bmxs W (1937) Free air chamber for the measure ment of yrays Proc Roy Soc A 161 564 Kaye, G@WC & Binks W (1940) Emussion and transmission of & and yrays Brit J Radwl 13 193 KerneauM, M (1909) Decomposition of water by # rays and ultra violet light Radwum 6, 225 Kavostova, ¥ V & Gavritova AA (1938) Relation between number of translocations in, Drosophila and dosage Biol Zh 7 381 Krevcur, S & Aoxkr H (1939) Scattering of fast neutrons Proc Phys Math Soc Japan, 21 75 Kinsey, ¥ E (1935) Effects of X rays on glutathione J Biol Chem 210 551 Ruircuyer, 240) F (1930) General physics ph 0: fx rays Lapng efandb Exp Phys 382 BIBLIOGRAPHY GOstuzr, P & Horzarrry, L (19306) Tho X ray sensitivity of liquid water andice Z Phys Chem B, 44, 374 Gtaruer, P & Leicuten, H (1036) Docompoasition of HI and syn thos of HBr by A rays Z Phys Chem B,34 443 Hansoy, F B (1928) Effects of X rays on productivity and sex ratio in Drosophila Amer Nat 62, 352 Hansov, F B & Heys, F (1929) Analysis of offecta of different rays of radium in producing lethal in Drosophila Amer Nat 63, 201 Hanson, FB & Heys, F (1932) Radium and lethal mutations in Drosophila Amer Nat 66, 335 Hasxina, C P (1938) Apparatus for studying the biological effects of cathode rnys J Appl Phys 9, 553 Hexwr, L.J (1939) Mercury lamp for ultra violet ght of wave length 2536A Scsence, 90, 473 Heuren, RG (1940) 4 ray induced and naturally occurmng chromo somal variations in Drosophila pseudoobscura Genelsce 26, 1 Hevsuaw, P§ (1932) Effect of X rays on timo of first cleavage in Arbacia egga Amer J Roentg 27, 890 Hensuaw, P § (1938) Action of X rays on nucleated and non nucleated ogg fragments Amer J Cancer, 33, 258 Hensuaw, PS (1940) Action of X rays on the gametes of Arbacta punetulata Amer J Roentg 43, 899 Hensuaw, PS & Conen,! (1940) Action of X rays on the gametes of Arbacia punctulata Amer J Roentg 43, 917 Hensnaw, PS & Francis, DS (1936) Effect of X rays on cleavage in Arbacia eggs Biol Bull Woods Hole, 70 28 Hensuaw PS & Hensuaw, CT (1933) Changes in susceptibihty of Drosophila eggs to a particles Biol Bull Woods Hole, 64 348 Hensnaw PS, Hensuaw, CT & Francis DS (1933) Action of X rays on Arbacia eggs Radiology, 21, 533 Hexcrx, F (1933) Action ofa rayson bacteria Strahlentherapre, 47, 374 Hercik,F (1934a) Bactericidalactionofa rays Strahlentherapre 49,438 Hercix, F (19346) T of the t dal action of arays Strahlenthcrapre 49, 703 Hererx F (1936)7 Bactencidal action of ultra violet hght J Gen Phystol 20, 589 Hinsyetwoop C.N (1940) Arnetics of Chemical Change Oxford Himscureper, JO & Taynor HS (1938) « particle reactions in CO, O,, and CO, systems J Chem Phys 6, 783 Horrmann JG & Remnnarp MC (1934) Recovery from the effects of wradiation Radeology 23 738 Houway EP (1936) Absorption spectra of tyrosine, tryptophan and therr mixtures Brockem J 30, 1795 Hoxuaenper, A & Claus WD (1936) Bactericidal action of ultra violet hght J Gen Phystol 19, 753 Howaenper A & Duccar BM (1936) Action of ultra violet hght on Bact coly and tobacco mosaic virus Proc Nat Acad Scr Wash 22,19 BIBLIOGRAPHY 385 Lza, DE (19385) Delay in cellular division Brit J Radvwl 11, 554 Lea, DE (1940a) Radhat hod for det ber of genes m Drosophila J Genet 39, 181 Lea, DE (19405) Sizes of viruses and genes by radiation methods Nature, Lond , 146, 137 Lea, DE & Carcaesrpz, DG (1942) Induction by radiation of chro abe: in T' J Genet 44, 216 Lea, DE & Carcuesrpr, DG {1945a) Recessive lethals, dominant lethals and chr ab in Drosophila J Genet 47,10 Lea, DE & Carcrestpy, DG (19456) Bearing of radiation expert ments on the mze of the gene J Genet 47, 41 Iza, DE & Hares, RB (1940) Bacterscidal action of ultra violet hght J Hyg, Camb ,40 162 Lea, DE, Hames, RB & Bretscrer, E (1941) Bactencidal action of X rays, neut and radioactive radiat J Hyg ,Camb ,41,1 Lea, DE, Hares, RB & Coutson, CA (1936) Bacterrerdal action of radioactive radiations Proc Roy Soc B, 120, 47 Lea, DE, Hanes RB & Covtson, CA (1937) Actions of y rays on bacteria Proc Roy Soc B, 123, 1 Lea, DE & Saraman, MH (1942) Inactivation of vaccima virus by radiations Brit J Exp Path 23 27 Lea, DE & Surrx, KM (1940) Inactivation of plant viruses by radiations Parasitology, 32 405 lza, DE & Syn, KM (1942) Inactivation of plant viruses by y Taya, X rays and a rays Parasitology, 34 227 Lea,DE,Smra, KM Houmes,B & Marguam, R (1944, Direct and mdirect actions of radiation on viruses and enzymes Parasitology 36, 110 Lev BS & Losmsxr I (1935) Bactericidal action of soft X rays CR Acad Ser, Paris, 200, 863 Levin, BS & Losansgr I (1936) Inactivation of fowl plague virus by XKrays CR Acad Ser, Pans, 203 287, 350 Lewis, B (1928) Photochemical decomposition of HI J Phys Chem 32, 270 Lrvp,8 C (1928) Chemical Effects of « particles and Electrons New York Lup, SC & Barpwetx, DC (1928) Synthesis of ammonia by @ raya J Amer Chem Soc 50,745 Lip, SC & Banpwert DC (1929) Ozonization and interaction of oxygen with nitrogen, under a rays J Amer Chem Soc 51 2751 Linn, 8C, Banpwerz, DC & Perry JH (1926) Chemneal action of @ rays on J carbon pounds J Amer Chem Soc 48, 1556 Linn, SC & Lrvinesron, R (1932) Photoch a poly of acetylene J Amer Chem Soc 54 94 Linn SC & Lrvasroy, R {1936) Radiochemical synthesis and de composition of HBr J Amer Chem Soc 58 612 Linp, SC & Oac, EF (1931) 1 Pp ffi of the synth of HBr byarays Z Phys Chem Bodenstem festband, p 801 Livryeston, MS & Berne, HA (1937) Nuclear dynamz cs: mental Rev Mod Phys 9, 245 » “pert 384 BIBLIOGRAPHY Krem,0O & Nranma, ¥ (1926) Scatte of radiation ring by free electrons Z% Phys 82, 853 Kursrenen, © (1027) Dutnibution of ions in a ray tracks Z Phys Kuare, E & Scunrinen, H (1939) Mutat induced in ions Sphaerocarpue by ultra violet light Proc 70h Int Gongr Genet p 175 Kvonn, Wo & Rory, H (1934) Action of cathode raya on bacteria and bactoriophage Arch Hyg 113, 92 Korren, L R (1939) Bactericidal action ofultra violet light J Appl Phys 10, 624 Korie, PC (1943) Effects of radiation on pollen grain development, on and Proce Roy Soc Edinb B, 61, 398 Kottrr, PC & Auwep, IARS (1042) X ray induced structural h h gosin of Drosophil fool J Genet 44,53 ¥ Koutrr, PC & Aurrnacn, CA (1941) Chromosome breakage and sterility in the mouse. Nature Lond 148, 501 Kotusman, AD & Essex, H (1940) Effect of electric fields on the docompoaition of N,O by a rays J Chem Phys 8, 450 Kotvat JP (1944) Production of chromosome structural changes 10 Tradescantia microspores by radiations Theeis, Cambridge Knuzgen, AP (1930) Estimation of bacteriophage J Gen Phynol 13, 557 Kruczr PG (1940) Bhological effects of slow neutrons Proc Nat Acad Ses, Wash , 26, 181 Kusrnen, H & T N, H (1937) Analysis of X ray absorption into ph lectric Pp and Compt Ann Phys. Lpe , 28, 385 L A &F F (1928) Bi dal action of X rays OR Acad Sct, Paris, 186 1316, 1318 Lacassaonz, A & Hoxweck F (19290) Bactericidal action of soft XK rays CR Acad Sct Pans 188, 197, 200 Lacassagnz A & Hotwecr, F (19295) Action of soft X rays on B prodigiosus C.R Soc Biol Paris 100, 1101 Lacassaonz, A & Hotwecx, F (1930) Action of X raya a rays and ultra violet hight on yeast OR Acad Sa, Paris, 190, 524, 527 Lannie, FC & Law SC (1938) Chemical action of a particles on aqueous solutions J Phys Chem 42 1229 Lasnrrzrxr I (1940) Effects of X rays on chick tissue m culture Brit J Radiol 13 279 Lasnrrzgr I (1948a) Effects of X rays on chick tiesue in culture Brit J Radwl 16 61 Lasnrr7xr I (19435) Effects of X rays on chick tissue mn culture Brit J Radiol 16, 137 Lavin, GI (1943) ip ultra violet Rev Ses Instrum 14, 375 Lavin , GI, Tromeson RHS & Dusos RJ (1938) Ultra violet absorption spectra of fractions isolated from pneumococer J Bul Chem 125 75 Lea, DE (1938) Time intensity factor Brit J Radiol 11, 489 BIBLIOGRAPHY 385 Lea, DE (19885) Delay in cellular division Brit J Radwl 11, 554 Lea, DE (19402) Radiation method for determining number of genes in Drosophila J Genet 39, 181 Lea, DE (19406) Sizes of viruses and genes by radiation methods Nature, Lond , 146, 137 Iga, DE & Carcuesme, DG (1942) Induction by radiation of chro ab st in Trad J Genet 44, 216 Lza, DE & Carcuesmpe, DG (1945a) Receasive Iethals, dominant Jethals and chr al yn Drosophila J Genet 47,10 Lea, DE & Catcuesipz, DG (19455) Bearing of radiation experi ments on the size of the gene J Genet 47, 41 Lea, DE & Harnzs, RB (1940) Bactencidal action of ultra violet hght J Hyg, Camb, 40, 162 Lea, DE, Harnes, RB & B ER, E (1941) Bi dal actron of X rays, neutrons and radioactiveradiations J Hyg ,Camb ,41,1 Lea, DE, Haryes, RB & Courson, CA (1936) Bactericidal action of radioactive radiations Proc Roy Soc B 120 47 Lea, DE, Harnes, RB & Courson, CA (1937) Actions of y rays on bacteria Proce Roy Soc B, 123, 1 Lea, DE & Saraman, MH (1942) Inactivation of vaccima virus by radiations Brit J Exp Path 23, 27 Iga, DE & Smrre KM (1940) Inactivation of plant viruses by radiations Parasitology, 32 405 Lea, DE & Sure, KM (1942) Inactivation of plant viruses by y rays, X rays and @rays Parastology, 34, 227 Lza, DE, S»arn, K M, Hotmes, B & Manxuam, R (1944, Direct and indirect actions of radiation on viruses and enzymes Parasitology, 36, 110 Levin BS & Lommskr, I (1935) Bactericidal action of soft X raya C.R Acad Ser, Paris, 200, 863 Levin B§ & Lowunsar,I (1936) Inactivation of fowl plague virus by Xrays CR Acad Ser, Paris, 203, 287, 350 Lewis, B (1928) Photochemical decomposition of HI J Phys Chem 32 270 Lip SC (1928) Chemical Effects of « particles and Electrons New York Iap, §C & Banpwert, DC (1928) Syntheus of ammonia by 2 raya J Amer Chem Soc 50, 745 Lup, SC & Barpwett, DC (1929) Ozontzation and interaction of oxygen with mitrogen, under a rays J Amer Chem Soc 51, 2751 Low,SC Barpwett DC & Pzrry, J UH (1926) Chemical action of @ rays on gaseous ted carbon Pp ds J Amer Chem Soc 48, 1556 Linn, SC & Livicston R (1932) Photoch 1 polym of acetylene J Amer Chem Soc 54 94 Lip SC & Livinesron R (1936) Radiochemical synthesis and de composition of HBr J Amer Chem Soc 58 812 LinnSC , & Ooo EF (1931) T of the synth of HBr bya rays Z Phya Chem Bodensteiffin festband p ‘01 Livrvaston, MS & Bere HA (1937) Nuclear dynamics expen mental Re. Mod Phys 9, 245 384 BIBLIOGRAPHY Krem,0O on & Nisnma Phys 82, 653 , ¥ (1929) (1929) Scat tering of radiatio by free electrons n Kemerenrn, © (1927) Distribution of ions in a ray tracks Z Phys Knarr,E & Scunrinrn, H (1039) Mutations indu in Sphaerocarpus ced __by ultra violet light Proc 7th Int Congr Genet p 175 Knonn, M & Rory, H (1934) Action of cathode rave on bacteria and bacteriophnge Arch Hyg 113, 02 Kortrn, L R (1939) Bactericidal action of ultra violet hight J Appl Phys 10, 624 Korrxr, PC (1043) Effects of radiation on pollen grain development thi and i Proc Roy Soc Edinb B, 61, 398 Kottrn PC & Anwep, [ARS (1942) X ray induced structural . hangesin ch of Drosophila pseudoob. J Genet 44,53 Koutrr PC & Avurnnaci, CA (1941) Chromosome breakage and sterility in the mouse. Nature, Lond , 148, 801 Korumpax, AD & Essex H (1940) Effect of electric fields on the decomposition of N,O by a rays J Chem Phys 8, 450 Korvar, J P (1944) Production of chromosome structural changes in Trad pores by G Thesis Cambnd, Kucrocr, AP (1930) Estimation of bacteriophage J Gen Physol 857 Krvorr PG (1940) Bhological effects of slow neutrons Proc Nat Acad Sex, Wash 26, 182 Kuerner, H & Tronesrem, H (1937) Analyas of X ray absorption into photoelectric absorption and Compton acattering Ann Phys. Lpz , 28, 385 Lacassacne, A & Hi F (1928) B dat action of X rays CR Acad Ses, Pars, 186, 1316 1318 Lacassaane A & F F (1929e) Bi dal action of soft Xraye CR Acad Ses Paris 188, 197, 200 Lacassaone A & Hoxwece, F (19295) Action of soft X rays on B Prodigosuse CR Soc Brol, Parts, 100, 1101 Lacassaone, A & Hotwecx F (1930) Action of X rays @ rays and ultra violet hght on yeast OR Acad Scr Pars, 190 624, 527 Lannie, FC & Liyp, SC (1938) Chemical action of a particles on aqueous solutions J Phys Chem 42 1229 Lasnrrzxt, I (1940) Effecta of X rays on chick tissue mm culture Brit J Radyol 13 279 Lasnirzxr I (1943a) Effects of X rays on chick tissue in culture Brit J Radiol 16 61 Lasyrrvxt I (19435) Effects of X rays on chick tissue im culture Brit J Radzol 16, 137 Lavin GT (1943) ip fied ultra viol Rev Ser Instrum 14, 375 Lavin GI Txsomrsoxn, RHS & Dusos RJ (1938) Ultra violet absorption spectra of fract: isolated from p J Bol Chem 125, 76 Lea DE (19384) Time intensity factor Brit J Radiol 11, 489 BIBLIOGRAPHY 385 Lea, DE (19388) Delay in cellular divinon Brit J Radtol 11, 554 Lea, DE (1940a) Rad method for det ber of genes in Drosophila J Genet 39, 181 Lea, DE (19405) Sizes of viruses and genes by radiation methods Nature, Lond , 146, 137 Lea, DE & CatcHesipe, DG (1942) Induction by radiation of chro abi in T J Genet 44, 216 Lza, DE & Catcnestpr, DG (1945a) Recessive lethals, dommant lethals and chr. b in Drosophila J Genet 47,10 Lza, DE & Catcnesipy, DG (19455) Bearing of radiation experi ments on the size of the gene J Genet 47, 41 Lea, DE & Hames, RB (1940) Bactercidal action of ultra violet hght J Hyg,Camb 40 162 Lea, DE, Harnes, RB & Brerscuer, E (1941) Bactericidal action of X rays, neutrons and radioactive radiations J Hyg ,Camb ,41,1 Lea, DE, Hares, RB & Covrson, CA (1936) Bactericidal action of radioactive radiations Proc Roy Soc B, 120, 47 Lea, DE, Hanes, R B & Covtson, CA (1937) Actions of y rays on bacteria Proc Roy Soc B, 123, 1 Lea, DE & Sanaman, MH (1942) Inactivation of vaccima virus by radiations Brit J Exp Path 23 27 Iza, DE & Smrra KM (1940) Inactivation of plant viruses by radiations Parasttology, 32, 405 Iza, DE & Surg, KM (1942) Inactivation of plant viruses by y rays, X raysandarays Parasitology, 34, 227 Lea, DE,Surrx K M, Horses, B & Marxnam, R (1944, Direct and indirect actions of radiation on viruses and enzymes Parasitology, 36 110 Levin, BS & Lominsxr, I (1935) Bactericidal action of soft X rays CR Acad Set, Parris 200 863 Levin, BS & Lomsgr 1 (1936) Inactivation of fowl plague virus by Xrays C.R Acad Scr, Paris, 203 287, 350 Lewis, B (1928) Photochemical decompomtion of HI J Phys Chem 32, 270 Lin 8 C (1928) Chemical Effects of« particles and Electrons New York Lip, SC & Banpwetz, DC (1928) Synthesis of ammoma by @ rays J Amer Chem Soc 50 745 Lisxv SC & Barpwerz DC (1929) Ozomization, and interaction of oxygen with nitrogen, under « rays J Amer Chem Soc 51, 2751 Low, 8C, Barpwert, DC & Perry, JH (1926) Chemical action of @ rays on gaseous d carbon pounds J Amer Chem Soc 48, 1556 Linn, SC _& Lrvrnestox, R (1932) Photochemical polymerization of acetylene J Amer Chem Soc 54 94 Lixo SC & Livinaston, R {1936) Radiochem:cal synthesis and de composition of HBr J Amer Chem Soc 58 612 Lr 8C & Ooc EF (1931) T of the I of HBr by arays Z Phys Chem Bodenstein festband p 801 Livincsron MS & Berze, HA (1937) mentel Rev Mod Phys 9, mas } Nuclear d Nuclear dynemics expen 386 BINLIOGRAPHY Loeu, J (1910) Prevention of tho texte action of vanous ®gencies upon the fertihzed egg through supproasion of oxtdation in the cell Scrence, 32, 411 Lozn, J & Wastennys, H (1011) Respiration rata of manne mverte brate eggs Biochem Z 36, 345 Lorenz, KP & Hrevawaw, PS (1941) Bactericidal action of X rays Radiology, 36 471 Luprorp, R J (1932) Cyto! I changes after di of mal growths Imp Cancer Res Fund Rep 10, 125 Luria, SE (1939) Action of X raya and @ raya on bacteria CR dead Ser, Parrs, 209, 604 Luna, SF & Anpensoy, TF (1942) Electron mucrography of bacteriophages Proc Nat Acad Ses, Wash , 28, 127 Lonm,SC & Exner, FM (1941) Direct and indirect actions of X rays on bacteriophage Proc Nat Acad Sex Wash , 27, 370 Luyrr BJ (1932) Effects of ultra violet hght X rays and cathode rays on fungal spores Radiology, 18, 1019 Luyrt, Bd (1934) Effect ofA mys on slgne CR Soe Brot, Pars, 116 878 Macrenzre K & Mutter, HJ (1940) Mutation effects of ultra violet light in Drosophila Proe Roy Soc B, 129 491 bel: McCurnrocr, B (1938) Production of b of ring shaped chromosomes Genetics 23, 315 MecCimerocx B (1939) Behaviour in successive nuclear divisions of & chromosome broken at metous Proc Nat Acad Ses Wash 25, 405 McCrmrocx, B (1042a) Stability of broken ends of chromosomes in maize Genetics 26 23. McCuinroce B (19415) A of with hi YB! deficiencies in maize Genetics 26 542 McKintey, EB, Fisvzr R & Horpen, M (1926) Inactivation of bacteriophage and of animal viruses by ultra violet light Proce Sot Exp Bool, NY , 23, 408 Mano, G (1934) Absorption ofa rays Ann Phys, Paris 1, 407 Marmerzi1 LD, Neset, BR, Guzs NH & Cnances, DR (1942) Induetion by X rays of chromosomal aberrations in Tradescantea microspores Amer J Bot 29, 866 Marzuam R, Suara KM & Lea DE (1942) The sives of viruses Mannnae Swern KM &Lxa DE rasztol 34 315 (1944) Size of the Shope rabbit papiioma virus 35 17EParasitology Marquarpr, H (1938) Roentgen pathology of mitoss & Bot 32 401 Marsesax A (1935) Sensitive volume of chromosomes determined by Xrradiation Proc Nat Acad Scr Wash 21, 227 Marszax, A (1937) Effect of X rays on chromosomes in mitosa Proc Not Acad Sev Wash 23 362 X rays, and Marsuak, A (1938a) Chromosome aberrations induced by the effect of sammoma Proc Soc Exp Biol NY 38, 705 BIBLIOGRAPHY 387 Marszax, A (19386) Stage of mitosis at which chromosomes are rendered Jess sensitive to X rays by ammoma Proc Soc Exp Bul, NY ,39, 194 Marsnax, A (1939@) Effects of fast neutrons on chromosomes in mitosis Proc Soc Exp Bul,N Y,41 176 Marsnax, A (19396) Nature of chromosome division and duration of nuclear cycle Proc Nat Acad Scr 25, 502 Marsnax, A (1942a) Relative effects of X rays and neutrons on chromosomes in different parts of the resting stage Proc Nat Acad Ses, Wash, 28 29 Maxsuaxk, A (19425) Effects of X rays and neutrons on mouse lym phoma chromosomes in different stages of the nuclear cycle Radrology, 39, 621 Marsnax, A & Hupson, J C (1937) Effect of X rays on chromosomes Radtology, 29 669 Marswax, A & Maxrocn, WS (1942) Effect of fast neutrons on chromosomes in meiosis Genetics 27 576 Marner, K & Stone, LHA (1933) Effects of X rays on chromo somea J Genet 28,1 Mavor, JW & pe Forest DM (1924) Relative susceptibilities to X rays of eggs and sperm of Arbacia Proc Soc Exp Bol NY, 22 19 Mayngonn WV (1934) P" ysical basis of biological effects of romizing radiations Proc Roy Soc A, 146, 867 Mayngorp, W V (1940) Energy absorption of X and y rays im tisstte Brit J Radwl 13 235 Maynzorp, WV & Honeysurne, J (1938) Depth dose with y ray3 Bnt J Radiol 11, 741 Maynxorp, WV & Roperts JE (1937) Measurement of y rays in Toentgens Brit J Radiol 10, 365 Me.vittz HW (1936) Mercury lamp for wave length 2536A Trans Faraday Soc 32 1525 Merz,C W & Bozeman, ML (1940) Induced chr f in Sevara Proc Nat Acad Ser, Wash, 26 228 Micut, W (1914) Range of @ particles in liquids SB Akad Wiss Wren 123 1905 Micrey, GH (1938) Effect of temp on frequency of trans] tions produced by X rays in Drosophila Genetics 23 160 Mmerx, GL & Stanuzy, WM (1941) Acetyl and phenylureido derivatives of tobacco mosaic yirus J Biol Chem 141 905 Mircnett JS (1942) Disturbance of nuclesc acid metabolism by & and yrays Brt J Exp Path 23 285 Mortwyn Huauers EA {1933} Kinetics of Reactions an Solutron Oxford Monxuer FL & Tayror, LS (1934) Bactericidal effects of X rays Bur Stand J Res 13 677 Moore HN & Kersten, H {1937} Inactivation of encephalitis virus by soft Xrays J Bact 33 615 Mornrvostar O Evans, RD & Hasxrns CP {1941) Cathode ray tube for biological experiments Rey Ser Instrum 12, 358 380 HIBLIOGRAPIHY Lora, J (1910) Prosention of tho toxie action of vanaus agence the fertilized egg through supprosion of oxidation in th Sevenee, 32 41 Lorn, J & Wastrxryva, It (101f} Respiration rate of manne tw brate eggs Biochem 7 36 945 Lonevz KP & Hrysuwaw, PS C1941} Bactoriedal action of¥ Radiology, 36, 471 Lupronp, RJ (1932) Cytological changes after srrad. of mal growths Imp Cancer Res Fund Rep 10, 125 Lunia, SE (1039) Action of ¥ rayx and @ rays on bactena. Acad Ser, Pans, 209, 604 Lunta, $F & Anprnsoy TF {10%2) Fleetron micrograp bacteriophages Proe Nat Acad Sex Wash, 29, 127 Lunia, SP & Fxver FM (1041) Direct and mdirect actions ofJ on bacteriophage Proc Nat Acad Sex Wash , 27, 370 Luyrr BJ (1932) Effects of ultra violet light, X rays, and ce roys on fungal spores Hadeology, 18, 1019 Luvrr, BJ (1934) Effect ofX raya on algae CR Soe Biol, 16 878 Macxevzr, K & WMucrrn HJ (1940) Mutation effecta ofultra hghtin Drosophila Proe Roy Soe B 129, 491 McCurvrock, B (1938) Prod of defi by ab t bebe of ring shaped chromosomes Genetica 23, 315 McCuinrock B (1939) Behaviour in successive nuclear diviston chromosome broken at metosis Proc Nat Acad Sev, Was! 405 McCurtock B (1941a) Stabilty of broken ends of chromoson maize Genetics 26, 234 McCuiwtocs, B (10416) Agsocintron of mutants with homoz defi in maize Genetics, 26 542 McKintey EB Fisuer R & Hortpen, M (1926) Inactivat: bacteriophage and of animal viruses by ultra violet hght Prot Exp Bwol,N Y 23 408 Mano G (1934) Absorption ofa rays Ann Phys, Paris, 1 40 Maninetrr LD, Nenel, BR Gres NH & Cnarntes, DR (: Induction by X rays of chromosomal aberrations in Trades muctospores Amer J Bot 29 866 Manxnam, R, Satrx KM & Lua DF (1942) The sizes of vr Parastology 34 315 Marknam R Smurre KM & Lea, D E (1944) Size of the Shoper papilioms virus Parasitology 35 17€ Marquarpt, H (1938) Roentgen pathology of mitomus Z Hoi 4 ol Marswax A (1935) 8 volume of det. Xuradiation Proc Nat Acad So Wash 21, 227 Marsuax A (J937) Effect of X rays on chromosomes in mi Proc Nat Acad Sct Wash 23 362 Marsuaxk, A (1938¢) Chromosome aberrations induced by X raya the effect of ammonia Proc Soc Hep Bol NY 38 105 BIBLIOGRAPHY 387 Mansnax, A (19385) Stage of mitosis at which chromosomes are rendered less sensitive to X raya by ammonia Proc Soc Exp Bul, N Y , 39, 194 Mansnax, A (1939a) Effects of fast neutrons on chromosomes in mitosis Proc Soc Exp Bol,N Y,41 176 Marswax, A (19394) Nature of chromosome divition and duration of nuclear cycle Proc Nat Acad Ser 25 502 Mansnax, A (1942a) Relative effecta of X rays and neutrons on chromosomes m different parts of the resting stage Proc Nat Acad Ser, Wash , 28, 29 Mansuax, A (19426) Effects of X rays and neutrons on mouse lym phoma chromcsomes in different stages of the nuclear cycle Radiology 39, 621 Marsnax A & Hunson, JC (1937) Effect of X rays on chromosomes Radwlogy, 29 689 BAK, A & Matitocn, WS (1942) Effect of fast neutrons on chromosomes in meioms Genetics 27, 576 Marner, K & Stove, LH A (1933) Effects of A rays on chromo somes J Genet 28,1 Mayor, JW & pr Forest, DM (1924) Relative susceptibilities to X rays of eggs and sperm of Arbacia Proc Soc Lap Bul,NY, 22, 19 Mayneorp, WV (1934) P" ysical basis of biological effects of ionizing radiations Proc Roy Soc A, 146, 867 Maynzorn WV (1940) Energy absorption of X and y rays in tissue Bret J Radwl 13, 235 Maynzonp, WV & Honrysurne, J (1938) Depth dose with y rays Bnt J Radiol 11, 741 Mayygorp WV & Roserrs, JE (1937) Measurement of y rays in toentgens Bnt J Radzol 10, 365 Mexviriz, HW (1936) Mercury lamp for wave length 2536A Trans Faraday Soc 32, 525 Merz CW & Bozeman, ML (1940) Induced chromosome changes in Scvara Proc Nat Acad Ser, Wash , 26, 228 Micat, W (1914) Range of « particles in hquids SB Ahad Wass Wren 123 1965 Micrey, GH (1938) Effect of ternperature on frequency of transloca tions produced by X rays in Drosophila Genetics 23 160 Mrizr, GL & Sranney WM (1941) Acetyl and phenylureido denvatives of tobacco mosaic virus J Biol Chem 141 905 Mircuetz JS (1942) Disturbance of nucleic acid metabolism by A andy rays Bnt J Exp Path 23 285 Moriwyn Hucuxs, EA (1033) Kinetics of Reactions 1n Solution Oxford Moutzr FL & Taxror, LS (1934) Bactericidal effects of X rays Bur Stand J Res 13, 677 Moors HN & Kersren H (1937) Inactivation of encephalitis virus by soft X rays J Bact 33 615 Mornmostar 0, Evans RD & Hasxms CP (1941) Cathode ray tube for biological expermmenta Rev Scr Instrum 12, 358 386 BIBLIOGRAPHY Lor, J (1010) Provention of the toxic action of various agencies upon the fertilized egg through suppression of oxidation in the cell Scrence, 32, 411 Lorn J & Wastrnrys, H (1011) Respiration rate of manne inverte brate eggs Biochem Z 36, 246 Lorenz, K P & Hexsnaw, PS {1041) Bactericidal action of X rays Radiology 36 471 Luprorp RJ (1932) Cytological changes after ir ad: of malignant grouths Imp Cancer Res Fund Rep 10, 125 Lurra, SF (1939) Action of X raya and a rays on bactena CR Acad Ser, Pans 209, 00 Lunia, SF & Anpensow, TF (1942) Electron mucrography of bacteriophages Proc Nat Acad Ser, Wash , 28, 127 Lunta,SE & Exner, FM (1041) Direct and indirect actions of X rays on bacteriophage Proc Nat Acad Ser, Wash , 27, 370 Luyrt, BJ (1932) Effects of ultra violet ght X rays and cathode Teys on fungal spores Radrology, 18, 1019 Luyrr BJ (1934) Effect of X mys on algae CR Soc Brol, Pars, 116 878 Mackenzie, K & Mutitrn HJ (1940) Mutation effects ofultra violet hight in Drosophila Proc Roy Soc B, 129, 491 McCurvrock, B (1938) Prod ofd byab beh of ring shaped chromosomes Genetzes 23 315 McCutock B (1939) Bel an nuclear d ofa chromosome broken at merous Proc Nat Acad Scr, Tash, 25, 405 McCrmrocr, B (1941a) Stability of broken ends of chromosomes in maize Genetics, 26 234 McCurnrocx, B (19416) A of ta with hb deficiencies in maize Genetecs, 26 542 McKintzy, EB, Fisyer, R & Horpen, M (1926) Inactivation of bacteriophage and of animal viruses by ultra violet hght Proc See Exp Bwl,NY 23 408 Mano, G (1934) Absorption of a rays Ann Phys, Paris 1 407 Maninerzr LD Neser, BR, Gnes NH & Caartzs, DR (1942) Induction by X rays of chromosomal aberrations in Tradescantia microspores Amer J Bot 29 866 Manxuast, R Surry, KM & Lea DE (1942) The sizes of viruses Parastology 34, 315 Marxeam, R, Sura, KM & Lea DE (1944) Size of the Shope rabbit papilloma virus Parasttology 35 176 Marovarpr H (1938) Roentgen pathology of mitosis Z Bot 32 401 Marsax A (1935) volume of determined by X wradation Proce Nat Acad Sc. Wash, 21, 227 Marsnax, A (1933) Effect of K rays on chromosomes in mutome Proc Nat Acad Sea Wash 23 362 Maxnsnak A (19384) Chromosome aberrations induced X rays and by the effect of ammonia Proc Soc Exp Bol NY 38 705 BIBLIOGRAPRY 389 . "Drovophala by neutrons Scr Pap Inst Phys Chem Tokyo, 36, ismina, Y & Morrwaks, D (1939) Induction of sex Inked lethals in Narva, Y & Morrwaxz, D (1941) Induction of sex hnked lethals m Drosophila by neutrons Ses Pap Inst Phys Chem Tokyo, 38, 371 Norv, FF & Wenxman, CH (editors) (1941-3) Advances in Enzymo logy New York . Norraror,J H (1939) Crystalline Enzymes New York Noves, W.A (1937) Photoch Id tion of NO J Chem Phys 5, 807 Noawzrrcer, C.E (1934) Effecta of @ particles on water and on ferrous sulphate solution J Phys Chem 38, 47 Nopxprncrr 0 E {1936a) Decomposition of air free water by & rays J Phys Chem 41, 431 Noaxsename, CE (19366) Production of H,O, in water by & rays J Chem Phys 4 697 ag 1 Nurnpehorr, CE (1937) I theory and reac tions Proc Nat Acad Sex, Wash, 23 188 Otpensura, O (1924) A light filter for mercury me 2536A Z Phys 29, 328 Onnsry, PK & Gares, FL (1927) Effect of ultra violet ight on S aureus and on the virus of vesicular stomatitis Proc Soc Exp Bul NY ,, 24, 431 Oniven, C P (1932) The effect. of varying X ray dose on the frequency of mutation in Drosophila Z wndukt Abstamm u VererbLehre, 6, 447 One, WIC & Buruen, J AV (1935) Rate of diffusion of deuterrum hydroxide in water J Chem Soe p 1273 Packarn © (1926) Killing of Drosophila eggs by X rays J Cancer Res 10, 319 Paczanp, C (1938) Variation with ago of y of Drosophila eggs to Xrays Parxen, R cae (1938) Tho infectivity of vaccima virus J Exp Med Radiology, 25 223 67, 725 Patrerson JT (1932) Lethal mutations and deficiencies produced by X rays tn the X chromosome of Drosophil a Amer Nat 66, 193 Perr, GN (1939) Quartz mercury lamps Brit J Radiol 12, 99 KAREE, J (1927) Influence of X raya on nuclear and cell division mn bean root tips Planta 4 299 Prsxorr, N (1919) Gaseous filters for ultra violet light Photogr 18, 235 Z wis Parser, K (1923) Stoppmg of o particles in hquds Z Phys 17, 23 and vapours Povpvuawasa ARnorpr, V (1936) Germination of pollen on artuficr medium Planta, 25, 502 al Porrzconvo, G (1941) Induction of chromosome losses Sperm and thei 2n Drosophila Linear depend on dosages of rad 41, 195 m J Genet 388 DIDLIOGRAPHY Morrran, JC (1026) Effects of Araya on Colpdium J Roy Bier Soe 46, 127 Morrnast, JC (1927) Early chango an nuclous of cells of tumoura ex pod to Arays Znt J Radiol 32, 61 Morrnast, JC (1932) Lifo history of nucleus and nucleolus and effects of f raysuponthom J Roy Aficr Soe $2, 362 Morrnas, JC (10334) Increase in nuclear esze following irradiation J Roy Mier Soc 53, 213 Morrnast, JC (10336) Radio sensitivity of the nondinding cel] nt J Radiol 6, 615 Morrnas, JC (1995a) Al an tivity of cells to radiat, produced by cold and anncrobiosis Brit J Radiol 8, 32 Morrram JC (10356) Effect of chemicals on radiosenativity of bean roots Bri J, Radiol 8 643 Morrram, JC, Scorr, GM & Russ, § (1626) Effects of# rays upon division and growth of cancer celle Pree Roy Soe B, 100, 328 Motren, HJ (1928) Problem of genie modification Z sdukt Abstamm u VererbLehre Suppl t, 294 Motzer, HJ (1929) Gene oa basis of life Proc Int Congr Plant Ses 1, 807 Mutrer, HJ (1930) Radsation and genetics Amer Not 64, 220 Metrier, HJ (1935) D: of ch and genes in dipt salivary glands Amer Nat 69, 405 Mower, HJ (1938) Biological effects of rediation mth special re ference to mutation Act Sev Ind no 725, p 477 Mutrer, HJ (1940) Anslyas of process of structural change 0 chromosomes of Drosophila J Genet 40,1 Mower, HJ (1941) Induced in Drosophila Cold Spr Harb Symp 9 181 Muiur, HJ & Macrenzre K (1939) Discrumunatory effects of ultra siolet hight on mutéstion in Drosophila Nature, Lond 143, 83 Mutter, HJ & Proxoryeva, AA (1935) The individual geno in relation to the chromomere and chromasoms Proc Nat Acad Sev, Vash 21,18 Muxrer,I (1938) Mase absorption coefficients of water and of squecus solutions Ann Phys , Lpz , 32, 625 Munn, W & Junarus, JC (1031) Polymerzzation of acetylene by fitays Bull Soc chim Belg 40 158 Nacar MA & Locuer GL (1938) Induction of mutations in Droso- phtla by neutrons Genetics, 23, 179 Narpv R (1934) Ionization curves of a rays Ann Phys, Para, t, 72 Nepex, BR (1937) Effects of K and rays on Tradescantia chromo somes Amer J Bot 24 365 Negpuam J (1931) Chemteal Embryology Cambndgs X raya on chromosomes J Genet Newcostne HB (19424) Effects of 43 145 Newcomer HB (19426) Effects of X rays on chromosomes J Genet 43, 237 BIBLIOGRAPHY 391 Sax, K (19416) Typos and freq of chr I ab induced by X rays Cold Spr Harb Symp 9, 93 Sax, K (1942) Mechanisms of X ray effects on cella J Gen Physsol 25 633 Sax, K (1943) Effect of centrifuging upon production of chromosomal aberrations by X rays Proc Nat Acad Ser, Wash , 29, 18 Sax, K & Enzmann, EV (1939) Effect of temperature on X ray induced chromosome aberrations Proc Nat Acad Scr, Wash , 25, 397 Sax, K & Marner, K (1939) Analysis of progressive chromosome splitting J Genet 37, 483 Sax, K & Swanson, CP (1941) Dhfferential senmtivity of cells to X rays Amer J Bot 28 52 Scorr, CM (1937) Biological actions of X and y rays Spec Rep Ser Med Res Coun, Lond, no 223 Srarin, PE & Parpue, TE (1942) Production of 8 rays by fast electrons Proc Amer Phil Soc 85, 243 Smnxy, AR (1940) Translocation between sperm and egg chromosomes in Drosophila Amer Nat 74, 475 Stnoxxrox, WR (1939) Cytological observations on deficiencies pro duced by treating maize pollen with ultra violet hght Genetzcs, 24, 109 Smetzton, WR & Cran, FJ (1940) Cytological effects of treating maze pollen with ultra violet light G@eneties, 25 136 Sw00, GJ & Wniemsen H (1938) Absorption of radium ¥rays Physica 5 100 Suzynskr, BM (1938) Salary chromosome studies of lethals nm Drosophila Genetica, 23 283 Suzynsxr, BM (1942) Deficiencies induced by ultra violet ight mn Drosophila chromosomes Proc Roy Soc Edinb B, 61, 297 Surry, C & Essex, H (1938) Effect of electric fields on the decomposi tion of ammonia by a raya J Chem Phys 6, 188 Ssnrn, KM (1940) The Virus, Iafes Enemy Cambridge Syzty, GD (1935) Ind hy X rays ofk ditary ch im mice Genetics, 20, 545 Syetz GD (1939) Induction of hereditary sterity m mice by neutrons Proc Nat Acad Sa, Wash 25,11 Swetz, G D (1941) Production by X rays of hereditary changes in mace Radwwlogy, 36 189 Sneit, GD & Ames, FB (1939) Hereditary changes m the de scendants of female mice exposed to X rays Amer J Roentg 41, 248 Bonnensuice, BP (1940) Cytology and development of embryos of X rayed Drosophila Proc Nat Acad Scr Wash, 26, 373 Srzan FG (1935) Tissue culture and radiological research Bret J Radiol 8, 68, 280 Srean oe (1944) Action of neutrons on bacteria Brit J Radiol Srean FG & Grtckssann A (1938) Effect of y rays on cella of the tedpolo Brit J Radvol 11 533 " 390 BIDLIOGRAPHY Powrr convo, G (1942) D. Iethala in D; xp ila J Genet 43, 5 Pontrconvo, G & Muizen, HJ (1941) Lethality of dicentric chromo somes in Drosophila Genetica, 26, 105 Pricr WC & Gowers, J W (1037) Inactivation of tobacco mosaic virus by ultra violet ight Phytopathology 27, 267 Puostry, AT, Opprr TH & Fony, CF (1935) Bactericidal action of N rays Proe Roy Soe B, 118, 276 Quinny, EH & MacComm WS (1937) Rato of recovery of human skin from X or y irradiation Radtology 29, 305 Rasewsex, BN, Krrng A & Zicrren,H (1936) Mutations by coeme rays Naturwssenachaften, 24, 619 Rasewsky, BN & Timorérry Ressovsxy, NW (1939) Cosmic ravs and mutations Z andukt Abstamm wu VererbLehre, 77, 488 Rasetti, F (1037) Elements of Nuclear Physics London Ray Cuaupuuni, SP (1944) Prod of in Drosop hel by y raya at low intensity Proc Roy Soc Edinb B, 62, 66 Rentscurern, HC Naoy, R & Mi *, G (1041) Bi dal effect of ultra violet hght J Bact 41, 745 Reywotps JP (1941) Ind of ch b Isties in Sciara by \ raya Proc Nat Acad Sev, Wash , 27, 204 Rick CM (1940) X ray induced deletions in Tradescantia chromo somes Genetica, 25, 466 Ritey, H P (1936) Effects of X rays on chromosomes in meiosis and mutosis Cytologia, Tokyo, 7, 131 Risse O (1929) X ray photolysis of HO, Z Phye Chem A, 140, 133 Rivers, TM & Gares, FL (1928) Inactivation of vaccinia virus by ultra violet light J Exp Med 47,45 Rosertson, M (1932) Effect of y rays on Dodo caudatus Quart J Aficr Sex 75, 511 Rostwow C F (1942) Nuclear apparatus of bacteria Proc Roy Soc B 330 299 Ropivow CF (1944) Cytological observations on bactena J Hyg Camb 43 413 Ruruerrorp E, Cuapwicx, J & Erzis, CD (1930) Radsations from Radwoactie Substances Cambridge Sax HJ & Sax K (1935) Ch r and b in mutosis and meiosis J Arnold Arbor 16, 423 Sax K (1938) Induction by X rays of chromosome aberrations in Tradescantia microspores Genetics 23 494 Sax, K (1939) Time factor m X ray production of chromosome aberrations Proc Nat Acad Ser, Wash 25 225 Sax K (1940) X ray mduced ch VE inZ Genetics, 25 41 Sax, K (1941a) Behaviour of X ray induced chromosomal aberrations in omon root tips Genetics 26 418 BIBLIOGRAPHY 391 Sax, K (19415) Types and freq of chr al induced by X rays Cold Spr Harb Symp 9, 93 Sax, K (1942) Mechanisms of X ray effects on cella J Gen Phystol 25, 633 Sax, K (1943) Effect of centrifuging upon production of chromosomal aberrations hy K rays Proc Nat Acad Sa, Wash, 29, 18 Sax, K & Exzmany, EV (1939) Effect of temperature on X ray- induced chromosome aberrations Proc Nat Aced Se, Wash , 25, 397 Sax, K & Mater, K (1939) Analyas of progressive chrornosome aphtting J Genet 37, 483 Sax, K & Swanson, CP (1941) Dsfferential sensitivity of cells to Xrays Amer J Bot 28, 52 Scorr CM (1937) Brological actions of X and yrays Spec Rep Ser Med Res Coun, Lond , no -223 Sexanin, PE & Parpve, TE (1942) Production of d rays by fast electrons Proc Amer Phil Soe 85, 243 Stoxy, AR (1940) Translocation between sperm and egg chromosomes in Drosophila Amer Nat 74, 475 Stvanzron, WR (1989) Cytological observations on deficiencies pro duced by treating maize pollen with ultra violet hght Geneftcs, 24, 109 Stvoteron, WR & Crarx, FJ (1940) Cytological effects of treating maize pollen with ultra violet ight Genetics, 25, 136 Szoo GJ & Wrurmsen H (1938) Absorption of radium ¥rays Phystea 5 100 Suzynser BM (1938) Salary chromosome studies of lethals mn Drosophila Genetica, 23, 283 Suzynsxi, BM (1942) Defimencies induced by ultra violet bght m Drosophila chromosomes Proc Roy Soc Edinb B, 61 297 Surry C & Essex H (1938) Effect of electric fields on the decompoar tion of ammonia by a rays J Chem Phys 6 188 Surra, KM (1940) The Virus Lafes Enemy Cambndge Swerz, @D (1935) Induction by X rays of hereditary changes 1 mice Genetics 20 545 Syett, GD (1939) Induction of hereditary stemlty in mice by neutrons Proc Nat Acad Sev, Wash,25 11 Syxrz GD (1941) Production by X rays of hereditary changes mm mice Radiology, 36 189 Sneuz GD & Ames, FB (1939) Hereditary changes in the de scendants of fomale mice exposed to X rays Amer J Roentg 41, 248 Sonneweiick BP (1940) Cytology and development of embryos of X rayed Drosophila Proc Nat Acad Sc Wash, 26 373 Srrar, FG (1935) Tissue culture and rachological research Brit J Radwl 8 68, 280 Spear FG (1944) Action of neutrons on bactens Brit J Radiol 7 348 Sreazk FG & Grocksmann, A (1938) Effect of y rays on cells of the tadpole Brit J Radio l 11, 533 392 BIBLIOGRAPHY Srzan, FG & Grdcxsuann, A (1041) Effect of y rays on cells of the tadpolo, apnced irradistion Ent J Radiol 14, 65 Srean, i G, Gnay, LH & Reap, J (1938) Biological effects of faat neutrons Nature, Lond , 142, 1074 Srran, FQ & Gamierr, LG (1933) Dependence upon sntenaty of the offocts of y rays on chuck tisauein culture Brit J Radiol 6,387 Srexcen, RR (1935) Action of f and y rays on bactena U.S Publ Htth Rep $0, 1642 Sraprer LJ (1928) Genotic offects of A rays in maize Pree Nat Acad Sel, Wash, 14, 69 Sraprer, LJ (10302) Recovery following genetic deficiency in maize Proe Nat Acad Ses, Wash , 16, 714 Srapurr LJ (10305) Some genetic effects of X rays in plants J Hered 21,3 Srapien, LJ (1031) Experumental modifiestion of heredity m crop plants Se Agne 1, 557 Srapizn, L.J (1039) Genetic studies with ultra violet radiation Proc th Int Congr Genet p 260 Sraprer, LJ (1941) Companson of ultra violet and X ray effects on mutation Cold Spr Harb Symp 9, 168 Brapier, LJ & Srraocur, GF (1936a) Genetic effects of unfiltered ultra violet hght on marze Proc Nat Acad Sex, Wash, 22, 572 Srapizz LJ & Srracve GF (19365) Genetic effects of filtered ultra violet bght on maize Proc Nat Acad Ses, Wash , 22, 579 Srapren, LJ & Srraacuy GF (1936c) Genetic offects of nearly mouochromatic ultra violet light (2536A) on maize Proc Not Acad Sa, Wash, 22, 584 Sraprze, LJ & Srraave, G F (1937) Contrasts in the genetical effects of ultra violet ight and X rays Sctence, 85, 57 Srapizr, LJ & Uper F (1938) Genotic effects of ultra violet bght on maize Genetica, 23 171 Srapier, LJ & User, F (1942) Comparison of genetic effecta of different wave lengths of ultra violet hghton maize G@enetscs 27, 84 BSrauzL, E & Jouner, W (1934) Number of y ray quanta emitted by radium J Phys Radwm, 5, 97 Srraciz, EWR & Puniies N WF (1938) Mercury lamp for 25384 Canad J Rea B 16, 219 X rays on solutions Stenstrom, W & Loumann, A (1928) Effects of of tyrosine and cystine J Biol Chem 79 673 Srrancewars, TSP & Horwoop FL (1926) Effects ofX rays on mutosis Proc Roy Soc B, 100, 283 Srranozways, TSP & Oaxrry, HE H (1923) Effects of X rays on chick tissue in culture Proc Roy Sec B, 95, 373 Srurtzvant AH & Beapix, G W (1940) An Introduction to Genetres Philadelphia gurron E (1943) Cytologuel analyms of Bar eye in Drosophela Genetice, 28, 97 Svepsero, T & Pepersen, K O (1940) The Ultracentrifuge Oxford CP (1940) Ind fe by ultra violet light andX raysinTradescania Proc Not Acad Sex Wash 26,366 BIBLIOGRAPH} 393 Swanson, CP (1942) Effects of ultra violet ight and X rays on pollen tube ch of Trad Genetics, 27, 491 Swanson, CP (1943) Dufferentisl sensitivity of prophase pollon tubs toX raysand ultra violethght J Gen Physiol 26 485 chromosomes Syvzrron, JT, Berry, GP & Warren, SL (1941) X ray inactiva tion of Shope papilloma virus J Exp Med 74, 223 Tana PS (1931) Oxygen tension oxygen consumption curve of un fertilized Arbacia eggs Biol Bull Woods Hole, 60, 242 Tanstey, K, Spear FG & GiicKsmann, A (1937) Effect of y rays on cell division m the rat retina Brit J Ophthal 21, 273 Txopay, JM (1942) Fffects of neutrons and X rays on chromosomes of Tradescantia J Genet 43, 189 Tuomas, LB (1941) Monochromatic source of mercury resonance Tadiation Rev Sct Instrum 12, 309 Trrortrrr Ressovsa11, N W (1933a) Back mutation and gene muta bility m different directions Z induke Abstamm u J ererbLehre 64, 113 Timorézrr RessoyvsKy, NW (19336) Back mutation and gene muta Inhty in different directions Z indukt Abstamm u VererbLehre 65, 278 Timoréerr Ressovsky, NW (1933c) Back mutation and gene muta bihty im different directions Z tndukt Abstamm u VererbLehre, 66, 165 Timorterr Ressoysny, NW (1937) Mutateonsforschung Dresden Turorterr Ressovskx NW (1939) Relation between gene and chromosome mutation Chromosoma, 1, 310 Tmorterr Ressovsky, NW & Detnruck, M (1936) Radiation researches on visible mutations and the mutability of single genes in Drosophila Z induhki Abstamm u VererbLehre, 71 322 Timorterr Ressovsky, NW & Zimmer, KG (1938) Induction of t by in Drosophila Naturutssenschaften 26, 108 and 27, 362 Tmortery Ressovsry NW & Zimmer, KG (1939) Radiation genetics Strahlentherame 66 684 Trmorterr Ressovsky NW, ZimuMer KG & Detsruck, M (1935) Gene mutation and genestructure Nachr Ges 183 Gattungen, 1,189 User, FM (1939) Ultra y1olet transmission by maize pollen Amer J Bot 26 799 User FM (1940) Mercury lamp source for monochromators Ret See Instrum 11, 300 User FM (1941) Quantum y1eld of inactivation of tobacco mosaic virus by ultra violet hght Nature, Lond , 147, 148 User FM & Errs VR (1941) Ultra violet absorption spectrum of ribonuclease J Biol Chem 141 229 User FM Havasnr T & Enis VR (1941) Ultra violet trans mussion by vitelune membrane of hen’s egg Sctence 93 22 User FM & Jaconsoun S {1938) Large quartz monochromator for biophysical research Rev Scr Instrum 9 150 304 BIBI LOORAPH Unen,1] M A McLarry, AD (1941) Photochermea) yield for activa tron of trypsin J Lrol Chem 141, 231 Vavauan, WY & Noyes WA (1930) Quantum eflicrency of ozone forenntion by short wave ultra violet light J Amer Chem Soc 52, 550 Nicrom+s, JA (1943) Xray mass absorption coefficients J Appl Phys 14, 05 Wanprsoroy, CH (1939) Introduction to Modern Genetics. London Wanp, F D (1085) Ind of in Drosoplula by « rays Genefics 20, 230 Wess,J (1944) Radhoch ry in aq 1 Nature, Lond , 153, 748 Wretrs, Wi (1940) Bactericidal action of ultra violet light J Franklin Inst 229 347 Wanraxer, D Mi (1931) Rate of oxygen consumption by fertilized and unfertihzed eggs 7 Gen Physiol 15, 167 Wurranen, D M (1933) Rato of oxygen consumption by fertilized and unfertilized eggs J Gen Physiol 16, 474 Warraxetrn, MD, Bsorssteap,W & Mircuens. ACG (1934) Prepare tron of polommum suurces Phys Ret 46, 629 Winrt MJD (1935) Lffects of \ raya on mitosis in spermatogonia of Locusta Proc Roy Soc B, 119, 61 Wart, MJ D (1937) Effect of X rays on first meiotic division mn three species of Orthoptera Proc Roy Soc B, 124 183 Wie, LO (1935) Q yrold for di iF of hy ultra violet hght J .dmer Chem Soe 57, 1559 Wityetmy D, Timorterr Ressoysxy, NW & Zrmmer KG (1936) Induction of mutations in Drosophila by soft X raya Strahlen therapre, 37, 521 Witrtasus, EJ (1930) Rate of loss of energy by # rays in matter Proc Roy Soc A 130 310 Wuson, CTR (1923) Ionization by X rays and Srays Proc Roy Soc A, 104 I, 192 Woutman, E, Horwecs, F & Luna, SE (1940) Inactivation of bacteriophage by X rays and a rays Nature, Lond , 145, 935 Wortman E & La vE, A (1940) Eval of the of bacteriophages by means of K rays Ann Inst Pasteur, 64 5 Woorrze, L (1919) Cnemucal actions ofa rays Radium 11,289 332 Wxexorr, R WG (19304) Bactericidal action ofX rays J Exp Med 52, 435 Wyoxorr RWG (19305) Action of X rays of various wave Jengths on Bact colt J Exp Med 52, 769 Wrerorr, RWG (1932) Action of ultra violet Lght on Bact colt J Gen Phystol 15 351 Wrcnorr, RWG & Loyer BJ (1931) Effects of X rays cathode rays and ultra violet ight on yeast Radiology 17 1171 Wycrorr RWG & Rivers TM (1930) Bacteriadal action of cathode rays J Exp Med 51, 921 BIBLIOGRAPHY 395 Zant, PA & Coorer, FS (1941) Physical and biological considera tions in the use of slow neutrons for cancer therapy Radtology, 37, 673 Znocer, K G (1934) Dependence of mutation rate on dose ofradiation Strahlentherapre, 51, 179 Zoome, KG & Tisorferr Ressovskx, NW (1936) Production of by @rays Strahlentherame, Zoomer, KG & Timoréerr Ressovsex1, NW (1938) Production of mutations in Drosophila by neutrons Strahlentherapte, 63, 528 Zmutz, R.E (1935) Kalhng of fern apores by a rays Amer J Cancer, 23, 558 Zimute, RE (1940) Kalhng of bacteria mould spores, and yeast by arays J Cell Comp Phymol 16, 221 304 BIBLIOGRAPHY Unrn,} Mo& MeLaney, AD (1911) Photochernical yield for inactiva tion of trypan J Biol Chem 141, 231 Vavowan, WE & Nove, WA (1930) Quantum efficiency of ozone formmtion by short wavo ultra violet hght J Amer Chem Soc 52, 550 Vicronrrs, J A (143) ray mass absorption coefficients J Appl Phys 14, 95 Wappivato~x, CH (1930) Introduction to Modern Genetics. London. Wann, 1 D (1935) Induction of mutations in Drosophila by a rays Genetics, 20, 230 Weiss, J (1044) Radioch yan ag 1 Noture, Lond, 153, 748 Writs, Wi (1040) Bactere:dal action of ultra violet ight J Franklin Inst 229, 347 Wuautannn, DM (1031) Rate of oxygen consumption by fertihzed and unfertilzed eggs J Gen Physiol 15, 167 Wurrakrr, D.M (1933) Hate of oxygen conaumption by fertilized and unfertilized egys J Gen Physsol 16, 474 Warraxura, 0D, Brorasteap, W & Mirenert, AC G (1934) Prepara tion of polonium avurces Phys Rev 46 629 Wiurt MJD (1035) Effects of A rays on mitosis in spermatogonia of Locusta Proc Roy Soe B 119, 61 Wuatrrr, MJ D (1037) Lffect of X rays on first meictic division in three species of Orthoptera Proc Roy Soc B, 124 183 Wna, BO {19J5) ¢ yaeld for d p of by ultra violet hght J .dmer Chem Soe 57, 1559 Witnecuy E, Tisorkerr Ressovaxy, NW & Zmeser, KG (1938) Induction of mutations in Drosophila by soft X rays Strahlen therapie 57, 522 Wittrams EJ (1930) Rate of loss of energy by f rays in matter Proc Roy Soc A 130, 310 Wirrson CTR (1923) Iomzation by X rays and frays Proc Roy Soc A 104, 1 192 Woruman E Honwece, F & Luria, SE (1940) Inactivation of bacteriophage by X rays and @ rays Nature, Lond , 145, 935 Wortman E & Lacassacne, A (1940) Evaluation of the dimensions of bacteriophages by means of X rays Ann Inst Pasteur, 64 5 w BE (1919) © J actions ofa rays Radium, 11, 289 332 Wycerorr RWG (19302) Bactericidal action ofX rays J Exp Med 52 435 Wycexorr RWG (19305) Action of X rays of various wave lengths on Bact cola J Exp Med 52, 769 Wycxorr, RWG (1932) Action of ultra violet hght on Bact colt J Gen Phystol 15, 351 Wacxorr, RWG & Luyet, BJ (1931) Effects of X raya cathode rays and ultra violet hght on yeast Radiology, 17 W711 Wycxorr, RWG & Rivers TM (1930) Bactericidal action of cathode rays J Lap Med 51, 921 ADDITIONS TO BIBLIOGRAPHY 397 Korvat, JP & Gray, LH (1947) Structural changes produced in mucrospores of Tradescantia by @ radiation J Genet 48, 135 Latarer, R (1942) La lo: do récip 6 dans |’irradiat dun bacténophage avec les rayons X Ann Inst Pasteur, 68, 661 Latanser, R (1944) Etudo expérimentalo de Ia loi de réciprocité dans leffet biologique primaire des radiations CR Acad Sa, Parss, 218, 294 Lararset, R (1946) Leffet tiologique primaire des radiations et Ie structure des microorganismes Rev canad Biol 5,9 Latanset, R & Want R (1946) Précisiona sur I’inactivation des bacténophages par les rayons ultraviolets Ann Inst Pasteur, 71, 336 Maxuisant,J K {1944} The ineffectiveness of temperature in influencing the production of mutations by X rays J Untw Bombay, B, 13, (3), 1 Mepvengv, NN (1935) The contributory effect of cold with irradiation in the production of mutations CR Acad Sct UR SS 4 (1x) 283 Mepvepev, NN (1938) The contributory effect of heat with irradiation in the production of mutations CR Acad Sa URSS 19,301 Monten, HJ (1941) Induced mutations in Drosophila Cold Spr Harb Symp Quant Biol 11, 151 Prokorveva AA & Kuvosrova VV (1939) Distmbution of breaks in the X ch of Dr hal k R Acad Sa URSS 23, 270 Rovrer M & Lararnset, R (1946} Augmentation du nombre de bacténophages en présenco de Bactéries stémlsées par irradiation Ann Inst Pasteur, 72, 89 Stizynsar, BM (1945) ‘Ectopse pairing and the distribution of heterochromatin in the X chromosome of salivary gland nuclei of Drosophila melanogaster Proc Roy Soc Edinb 62, But, 114 Swanson, CP (1944) X tay and ultraviolet studies on pollen tube chromosomes I The effect of ultraviolet (2357A) on X ray induced ch lab ties, 29, 61 TIMOFEEFF Ressovsxy, NW (1937) Uber Mutationsraten in reifen und unreifen Spermen von Drosophila melanogaster Biol Zbl 57, 309 Viiars DS (1926) Tr of the Oldenb h} filter for A2537 J Amer Chem Soc 48, 1874 Watrma, AR (1945) Dominant lethality and correlate d chrornosome effects in Habrobracon eggs X rayed in diplotene and in late meta Phase I Biol Bull Woods Hole, 89, 61 Wirma at (1946) Motherless males from irradiated eggs Scrence ADDITIONS TO BIBLIOGRAPHY Bowrr Mauny, P, Penavisz, R & Entensev, ML (1044) L'eetion bactériostatique des rayons X ot ultra violets Ann de VInat Pasteur, 70, 250 Carenestpr, DG, Lea, DF & Tropay, IM (1946a) Types of chromosome structural change induced by tho uradiation of Tradescantia microsporea J Genet 47, 113 Carcnesipr, DG, Lra, DE & Tropay, JIM (19466) The production of ch al in 7 an relation to dosage, intensity and temperature J Genet 47, 137 Carsen, A & Rapvu, G , (1943) Uber dio Abhéngigkest der rontgen ten Transl vom R d der G he phil de 38 Natur h 31/32, 368 D: M (1936) Freq ry of ‘cell lethals* among | lethals obtamned m the X-ch: of Dr Proc Nal Acad Ser, Wash, 22, 350 Forssnere, A (1945) Action of X rays on catalase and ita biological significance Ark Kems Min Geol A,21,no 7,p 1 Fonsszena, A (1946) Action of réntgen rays on the enzyme catalase Acta radtol , Stockh , 27, 281 Hemenrnat, G@ (1945) The occurrence of X ray mduced dommant lethal mutations in Habrobracon Genetics 30 197 Kaurmany, BP (3944) Cytology (In Ann Rep Dept Genet) Year p 115 Carneg Instn no 43, Kavrmann, BP (19460) Org of the ch I Break snd b in Drosophila melano gaster J Exp Zool 102 293 Kavrmann, BP (10468) M of the freq of 1 se ontecy induced by X rays in Drosophila IIT Effect of at the time of ck Genetics, 31 49 Kaurmann, BP & Gay, H (1947) The influence of x rays and nesr infra red rays on Jethals in Drosoph Proc Nat Acad Ser Wash,33 366 Kaurmann, B z & Hotunarnper A {1946} Modification of the induced by X rays in taoY Use of ultraviolet radiation Genetics, 31 368 Kavrmann BP, HOULAENDER A & Gay H (1946) Modification of Genetics Drosophila I ole of near infra red radiationupon 31 349 of the freq induced by X rays in Kina, ED (1947) The effect of low the fr X ray induced mutations Genetics 32 161 AUTHOR INDEX Where the reference 1s to a figure, the page number 1s in italics (113), where the reference 18 to a table, the page number 1s un heavy type (118) Aebersold, PC, 20, 375 Brownscombe ER, 41, 46, 380 Ahmed IA, 214, 218, 384 Brubaker, WM, 20, 376 Albert: W 193 295 299 337, 375 Brumfield RT 220, 376 Allen SJ M, 347, 349 Bruynoghe, R , 316 325, 376 Alhbone, TE 37, 380 Buchwald, CE , 15, 377 Allson, § K , 347 377 Busse WF , 37, 376 Allsopp CB, 33 46 375 Butler, J AV, 50, 389 Alper T 351, 375 Altenburg E, 181 376 Campbell Renton ML , 316, 378 Ames, FB 341, 391 Cannon, CV, 4, 376 Anderson RS 46, 199, 111, 118 375, Canti, RG , 293 295, 296, 297 299, 3768 380 Carlson, JG, 192 193, 195 198 199, Anderson TF, 102, 118, 122 314, 200, 201 208, 209, 218 225, 229 381, 386 Plate IVp 230, 293 294, 295, 299, 337 338 Aoki H_, 350 383 376, Plate IVs Aston, GH, 14, 379 Catcheside DG 1534, 154 158 159 Atwood K 39, 375 160 161, 162,165 166 167, 168 169 Auerbach CA, 341, 384 170 171, 172 173, 191, 192, 200 Anger, P 10 202 204 207, 209 211, 212, 214 218, 220 221, 226 227, 229, 230 Bachem, A 316 378 238, 239, 240 241, 245 953 255 Bachstrom, H LJ, 5, 375 263 266 269 376 377,385 Plate IIT Badion J 326 376 Cauchois, Y , 15, 377 Baker, SL, 316 375 Chadwick J, 17, 346 390 Baldinger E350 378 Chambers H 316 377 Bardwell DC, 37, 385 Charles DR, 226 227, 229 231, 241, Bauer, H 159, 167 168 170 171, 216, 283 386 218, 229 231, 237, 375 Clark, FJ 185 187, 391 Bawden FC,5 100 375 Clark GL, 40 46, 377 Beadle,G W 126 392 Claus, WD 316 366 382 Belgovaky ML 234, 375 Coblent7, WW , 316 137, 377 Benford F 4, 375 Coe, WS 40,46 377 Berry GP, 113 118 393 Cohen I 284 287, 382 Bethe HA 349 350 351 376 Compton AH 11 345 347 377 Binks W 14 15 383 Cook EV 309, 312 377 Bishop DW, 190 192 197 376 Coolidge WD,15 377 Bishop M 182 206 242 378 Cooper FS 15 18 366 395 Byorksteadt, W 17 394 Coulson CA 71 81 82 88 300, 307 Bonet Maury, P 316 376 316 318 320 321 322 324 385 Bonner TW 20 376 Crowther JA 71, 81,125 377 Bowen E.J 5 376 Cune P 10 377 Bozeman ML 190 196 213 224, 365 7 Dale WM 42 44 45 46 54 55 56 Bragg WH 351 57, 61 63 65 377 Brattain KG 37 376 Daniels F 37 376 Bretecher,E 316 318 321 324, 326 Darlmgton CD 126 189 193 343 377 3385 Dee PI 10 377, Plate Is Bridges PN 183 Delbrick M 100 104 126 143 144 Broda E 63 376 146 147 164 172 379 393 AUTHOR INDEX 401 Howell I , 351, 383 179 191, 200 202, 204, 207, 209, Huber, P_ 350, 375 211, 212, 214, 218, 221, 226, 227, Hudson JC, 197, 337, 387, Plate IVE 229 230, 238, 239, 240, 241, 242, 245, 253, 255 263, 266, 269, 283, Jacobsonn 8 , 4, 393 284, 289, 292, 298 300 307 313, Jafié, G, 50, 52 88, 383 315, 316 317, 318, 320, 321, 322, Jensen CO, 295 324 325, 326, 327, 376, 377, 383 Johner, W , 14, 392 384, 385 Jordan, P, 2,3 67, 82, 88, 125, 383 Lerchter, H , 37, 382 Jongers JC , 37, 372, 388 Levin, BS, 118 316 385 Joul J, 295 337, 383 Lewss B , 37, 385 Iand, SC, 38, 34, 36 37, 40, 41, 46, Kailan, A, 37 49 381 384, 385 Kanne, W RB, 17, 383 Little E P , 285, 312, 379 Kara Michailova E , 50, 383 Livingston, MS , 351, 385 Kaufmann, BP, 143, 149, 159, 168, Livingston R37, 385 167, 168, 170 183, 215 218, 219, Locher, GL, 150, 388 223,228 229, 231, 237, 375, 378, 383 Loeb J, 291, 386 Kayo, GwWwe,l4 15 283 Lohmann, A, 46, 49 68, 59 392 Kemp, T , 295, 337 383 Lominski I 118, 316, 385 Kernbaum, M 41, 42 383 Lorenz K P, 316, 386 Kersten H 118 387 Lucas AM 113, 381 Khvostova VV 234, 283 Ludford, R.J , 300 386 Kakuchr, § , 350 383 Luna, SE 108, 113, 17¢ 115, 117, Kinsey, VE, 46,49 58 383 118 120,122,316 325, 379, 386 394 Kirchner F 346, 383 Luyet, BJ , 300 307, 386 394 Klen, 0, 14 346 348, 349 384 Klemperer O , 49, 88, 384 McChntock B, 187 200 205, 328 386 Knapp E 188, 384 MacComb WS, 283 390 Knorr, M , 316, 384 Mackenzie, K 182 183, 242, 386 388 Koller LR 316 384 McKunley EB, 124 386 Koller, PC, 193 214, 218 332, 333, MeLaren, AD 5 125 394 334, 335, 337 341 3384 Makhyan: JK , 227, 222, 228 Kolumban AD, 37, 384 Makki AI 234 Kotval JP, 204 211 214 229, 230 Malloch WS , 236 237, 387 232 233, 239, 240, 241, 255, 384 Mano G, 35] 386 Krebs, A, [81 390 Marmell LD, 226 227,229,232 241, Krueger AP 105 384 263 386 Kroger PG, 18, 384 Markham R, 38, 100, 102, 105 109, Kustner H, 349 384 110, 111 177 385 386 Plate IIc = Marquardt H 190,193 196 197 225, Lacassagne,A 71, 81 113, 124 117, 337, 338, 386 118, 300 307 316 320 321, 322 Marshak, A, 190 192, 197, 223 225, 383, 384, 394 235 236 237, 238 336 337, 343, Le Cour LF 189 377 386 387 Plate IVE Lanning FC 40, 41, 46, 49 384 Mather K 198 200 209, 216 391 Losnitzkt,I ,192 201,205,296 299,307 Mavor JW 285 387 310 337 338 344 384, Plate IV z-1 Mayneord WV 15 22,81 387 Lavin, GI 5 384 Melville HW,4 387 Lawrence JH 20 375 Mendel GJ, 126 Lea DE 38 46 50 71 81, 82 83 Meredith WJ,45 61, 377 85 86 88 102 108 107 108, 109 Metz,C W 190 387 1x0 111, 172, 113 174-145 117 Michi W 17, 352 387 148 120 121 823 194 195 154 Mickey GH 227 222 387 158 159 160 167, 162, 165 186 Miller, G@L,175 387 168 169 170, 172, 172" 173, 177, Mitchell ACG,17 3094 400 AUTHOR INDEX Demmeroc, M , 143,14 188, 159, 163, 9, 167, Glass, HTB, 213 229, 329, 381 165,170,176,180 162,183 206,215,218, Glocker, R, 71, 81, 331 223, 220, 234, 237, 242 376, 378, 383 Glockler, G , 36, 381 Dompater, FR, 149 228, 238, 978 Olickemann, A, 205, 296, 299, 309, Deaaauer, } , 3, 67, 378 310, 333 381, 391, 392, 393 Dickinson, RQ 39, 378 Gowen, JW, 110, 113, 115, 120, 124, Doctr Fo 102, 103 378 140, 141, 243, 144, 146, 148, 170, Donaldson, BM, 203 376 471, 180, 381, 390 Doros KY, 316 378 Gray, LH, 15, 18, 20, 22, 46, 67, 71, Dreyer, G, 316 378 204, 211, 214, 232, 233 239 241 Duane, W 37,40 378 255,303 304,305, 306,312,335 336, Dubinin NP, 189 378 351, 381, 392 Dubos, RI , 5, 384 Green RH, 102, 314, 381, Plate IVb Duggar, BM, 9, 124, 316 378, 332 Grimmett, L.G , 298, 306, 392 Dushkin, BEA, 316 378 Groth, W , 37, 381 Gruhn, E , 316 379 Eddy, CE , 316, 317, 300 Gunther, P37, 40 41, 381, 382 Ehnsmann O , 316, 379 Elford WJ, 102, 103 120 Hachtsl FW , 316, 378 Ellingor, P, 316 370 Haines, RB 71, 81, 82 88 300, J0L, Etla,CD 14 17, 346 379, 390 316 317, 318 320 321, 322, 324, Eliw, EL, 104, 37! 326 385, Plate IV a,5 Elts, VR 5, 393 Haldane, J BS , 166 Emmons CW 183,369 383 Hallauer C 102, 103 378 Enzmann, E V , 214, 227, 222 266,391 Hanson, FB, 146, 170, 172, 382 Essex H 35 37,991 Harnson B , 46, 375 Evans RD, 15 377, 387 Hart,E.J ,41,42,43,45 46 49 55 58,380 Evans, TC 285, 312 379 Haskins, C P 15, 377, 382 387 Exner FM, 108 113, 214, 118, 120 Hayashi T 5 303 379 386 Heidt LI, 4 5, 382 Eynng H, 34,36 379 Helfer, RG, 214, 215, 229 382 Henshaw CT , 284, 289, 292, 311, 331, Fabergs AC 222 240 253 379 382 Failla G , 285, 312 379 Henshaw, PS 284 285 286, 287, 288 Fano U 92 143 149 150, 158 159 289 290, 292,293 294 322,316 331, 163 166 170 183, 206, 228, 219, 229 382 3386 276 378, 370 Herak F, 316, 318 $22, 382 Fisher, R124 386 Hoys, F, 146 382 Foreat, DM de 285 387 Hinshelwood CN 52, 382 Francis, DS 284, 285, 286 289 292 Hirschfelder JQ 34, 36, 379 982 311, 382 Hoffmann, J G , 283 382 Franck J, 39, 379 Holden M, 124 386 Fricke, H, 40 42, 42 43 45,46 49 Hohdey EP § 382 55 58, 61 379, 380 Hollaender, A, 5 124, 182 183, 223, Fredewsld W e (it 311, 118, 380 242 316 378, 379 382 383 Helmes, B 38 46 109 220 111,177, Fulton HR 316 31% 377 Fry, HJ 3! 287 Halteaptel L 37, 40,41 381 Gates FL 4 5 124,316 319 327 380 Holweck F, 71 81, 113 114 11S, 389 390 117-118 120 300 307, 316 320, Gavniova A.A, 234, Gay EH, 46 148 “io,171,180 381 321, 322 383 384 394 Honeybume J 15 387 Gedye GB, 37, 380 Hopwood FL 295 392 Gin NH, 149 206 226 227, 229 Hornbeck, G 351 383 230, 231, 233 235 238 241, 263 Houlshan MB 5 182 242 378 272 381, 386 Houston R.A 5 383 Sidky AR 220, 224,597 §dorey BN, 173, 7 Singlecn, WEL ISI, 17 232 Sizce GJ 14, 551 Slaczk e+ JC, 285,312, 75 ‘Taeedied Feserscy, NW. Io 135 Stoves BML 167 , PS Te. ts 18 5, 3, eT, eT Smadel, JE, 2,314, Int, Place IVS 143 IS2 138 157 LES 2, IE Smtth, C35 37 202 82 Is RRL Smuth, HP, #2 45, 46, 47 in. Si, Teast GGLIR rs 30 Tote, Et dM Smith, KL, 3F oe ran ero Teeeds. SOOR 45,61 377 407 110 WW 172, UWS ue W18 I2¢ 125,177 395, Pace er Uter FOL, € & 125, 13 16 18% Snell, G.D., 241, 331 243 22,303, 4 Sokolov NIN 139 373 Sonnenblek, BP, 162,329 200 391, Vaceras, WE, 37 3u 391 Viecoreen, JA, 20 3 394 Spear FG., 22, 71 234,295 S97 2g7 293 292 393 308 fm 29 BIN Wadinorn, CH. 124 334 312 316 333 305 331 391 322 Ward, FD. 152 153 182 3% Spencer R-R., 300 316 3°2 Ward, GE, 316 375 Sprague GF. 183 229 302 Wares, S.L, 113 118 393 Stadler LJ. 154 183 185 186 yaa Wesene-a H., 291 356 220 243 340 392 Wern P46 Stabel, E, 14 399% Wen J_.47 395 Stanley WAL, 175 337 Wels W.F., 316 394 Steane EW.R., 4 392 Whiake- D.M., 291 292 394 Stenstrom, W., 46 49 5% 59 392 Whitaker M.D 17 394 Stone LH.A, 198 337 White MJ.D., 192 19° 199 337 394 Strangewava TS P., 295 392 Whz EO 37 394 Sturtevant AH 196 392 Wilhelmy E 146 147 143 394 Sutton E 140 143 149 159 163 Willemsen H., 14 391 183 223 231 237 378 392 Wilbams EJ., 24 394 Svedberg T 5 392 Wilson, CTR 10 27 49 88 Swanson CP 191 193 197 201 202 Plate Ic-r 203 209, 216 224 225 240 242 Wollman E 113 7/4 115 117 243, 391 392, 393 120 394 Syverton JT., 113 118 393 Wourtzel E 37 394 Wyckoff RWG 81 300 307 Tang PS 291 393 323 324 327, 394 Tansley K 295 296 299 309, 310 338 393 Zahl P.A 18 395 Taylor HS 34 36 379, 382 Zickler H 181 390 Taylor, LS 9 88 89 387 Zimmer KG 136 146 147, 148 149 Thoday, JM 204 207 209 211 152 154 393 395 212 214 218 227 226 227 229 éurkle RE 17 316 318 324 395 402 AUTHOR INDEX Mitchell JS 5, 283, 387 Philipps, NW F , 4, 392 Moclwyn Hughes BA 62 387 Pickett, LW , 46, 377 Mohler, F L, 88, 89, 987 Poddubnaja Arnolds, V , 308, 389 Moore, HN , 118, 387 Moriwaki, D, 180, 389 Poiwon, 85D, 150, 166 217, 218 248, 5 Morningstar, O, 15 387 Pohtzer, G , 193, 295 299, 337, 375 Morse, 8 , 45, 380 Pontecorvo, G, 163, 164, 165 200, Mottram, JC, 22 205, 900 303, Sot, 209, 22f, 222 223, 389 390 306, 312 335 336 381, 988 Poynter, M , 312, 335 336 381 Mott Smith, LM 18t Preeton,@ D314, Plate Ilz Mouromseff G 316 321, 322, 390 Price, WC, 124, 390 Muller HJ, 132 133, 135 139, 141, Prokofyova A.A, 135 338 162 164, 105, 270 172, 172 173, Pugaley, AT, 316 317 390 176 180 AE, 182 183 197, 209 210, 227, 222 293, 228 229, 239 Quimby, EH, 283 390 234 239, 242, 329, 386 388 Miler, 1,349 388 Rabinowitech, E , 39 379 Mund, W , 36,37, 316 325, 376, 388 Rajowsky, BN, 181, 390 Rasotti, F , 346, 390 Nogni MA, 150, 388 Ray Chaudhurt, 5P, 146 7247, 228 Nagy R, 316, 322, 322 390 239 390 Naidu, BR, 17, 388 23,18 22,71 303, 304,306 312, Nanavutty SH 316, 3975 335, 336, 381, 302 Nobel, BR 201 226 227 229 231, Reinhard, M C , 283, 382 241, 263 386, 388 Rentachler HC, 316 321, 322, 390 Neodham J 291, 388 Reynolds J P. 190 224, 390 Newcombe HB, 198, 206 209, 212, Rice, O.K. Rick CM be “ora 218 221, 229, 4, 214, 229, 230 231 241 257 258, 261 308, 388 231, 235, 241, 252, 390 Nuwhina Y 14 150 346, 348, 349, Riley HP, 198 390 384 389 Risse, O 41, 390 Noething W, 316 379 Rivers TM 124, 316, 390 394 Northrop TH 100 389 Roberts JE 15 387 Robinow CF, 325 326 390 PlateIVe Noyes W A 37, 389 394 Robertson, M 300, 390 Nurnberger, CE, 40 41 46 59, 389 Rollefson G K 39 375 Oskley HEH 205 392 Ruff H 316 384 Oddie TH 316 317, 390 Russ § 295 316 366 338 Ogg EF 37, 385 Rutherford E 17 346 390 Oldenberg 0 , 5, 389 Olitshy PK, 124 389 Salaman MH 86 106, 108 113, 114 Olver CP, 146 156 157, 158, 389 115 117, 118 £21, 123, 177, 313 Ohvier HR, 316 376 314 315, 385 Plate IIa Orr WIC 50 389 Sax HJ 252 271 390 Sax K 190 191, 193 197 198 199 Packard © 331 389 200 201 205 208 209 214 216, Pardue TE 351 391 220, 221 222 225 296 227 229 Parker RF 106 289 230 231 232 235 245 252 253 Patterson JT 141 176 889 257 261, 264 266 267, 268, 271, Pedersen KO 5 292 337 338 390 391 Pel GN 4 389 Scheuer O , 37, 40 378 Pekarek J 193 296 300 301 337 389 Schlesinger M_ 103 Perry JH 37 386 Schreiber H 188 384 Peskoff N , 389 Scott CM, 69 391 Petersen BW 45 46 380 Scott, GM 295, 388 Phiipp K 17 352 389 Shearin PE 351 391 AUTHOR INDEX 403 Sidky AR, 229 234 391 230 231, 232, 235 236 238 241 Siderov BN., 139 378 253 255 S€6 2€9 393 Singleton, W-R- 195 187 391 Thornes LB. 5 393 Sz00 GA, 14 391 Trkompson, RAS. 5 324 Slaughter JC., 285 312, 379 Trmcfief Ressovacy NW. 126 195 Suzynsia BM., 160 178 183 242, 392 0 143 EAE NES M46, 147, 143 Smadel, JE. 102 314 331 Plate IV 149 152 184 157, 158 172, 176 Smuth, C35 37 391 ¥81 234 333 394 335 Smith, LP. 43 45 46 49 55 54 Tmakoy GG,139 3°53 380 Trubes*eimn, H., 349 384 South KML, 39 100 102 105 107 Tweed.e MW.C.R., 45 61,377 109,140 Wt 77? 113 115 1 118 124 195 177,335 Plate Hcx Uber Ft, 4 5 125 183 186 18, Scell, G.D., ant 31 243 292 393 3s Sokolov, NN. 139 373 Scnnenblck, B-P., 162, 329 330 31 Vacghan, WEL, 37 3% 301 Victoreen J.A., 20 343 33¢ FG. 92 Tl 204,295 296 297 299 299 303 39% 306 309 310 Waddmzvron. C_H., 125 394 312 316 333 365 391 391 392 Ward, F.D., 152 153 182 394 Spencer P.P., 300 316 392 Ward, G.E_, 316, 37S Sprague GF, 183 220 392 Waren, SL. U3 118 393 Stadler LJ., 15 183 185 186 138 Waesteness H 291 395 220 243 340 392 Weigert F., 46 Stakel, B., 14 392 Wes J.,47 33 Stanley, WML, 175 397 Wells W.F_, 316 39 Steane EWR, 4 292 Whitaker DIL, 291 292 3M Stenstrom, W., 46 49 $9 59 392 Whitacer MD., 17 394 Stone LH.A., 193 337 White 3UF.D., 192 197 199 337 394 Strangeways TS.P., 295 392 Wiz £0.,37 394 Sturtevant A.H., 126 392 Wilkelmy E.. M45 2f7 146 34 Satton, E, M0 143 149 159, 163 Willemsen H. 14 391 183 293 937 937 378 398 Withams EJ.24 34 Svedberg T.,5 392 Wilson, CT-P.. 10 2° 49 &s 394 Swanson, C_P., 191 193 197 201 202 Plate Ir-r 203 209 216 224 2295 210 243, Wolkran, E., 3 iff 115 117 118 243 391 399, 293 12) 3M Syverton JT. 113 118 393 Wourtzel, E.. 37 30 Wreko® PWG. 8b 307 318 Tang PS 291 393 G22 324 327,394 Tonsley K., 295 296 299 309 310 333 393 Zabl PAIS 395 Taylor HS. 34 36 379 332 Zickler H..181 399 Taylor LS..9 63 £9 38- Zimmer K.G., 136 146 147 141 149 Thoday JM, 204 207 209 211 152, 154 393 395 212 214 218 927 236 227 237 239 Zirkle PE.17 316 374 324 395 SUBJECT INDEX Where the reference is to a figure, the page number is in italics (113), where the reference is to a table, the page number is in heavy type (118) An esterisk attached to a page number (127%) indicates that the term indexed is defined on that page @ rays or particles 10¢ Ale absorption of, 15, 17 electrons por gram m, 347 associated volume of, 358 360, 361 X ray absorption in 347, 349 bactericidal action of, 316, 378, 321, Alanine 56 322, 324, 326, 327 Algns 300 chemical effocts of, 37, 41, 42 46 47, Allelomorphs or alleles 130° 87 Allium cepa (onion) 190 193, 199, 201, chromosome changes by, 204 210 208 214, 220 223 337, 338, 211, 232 233, 239, 240 241, 254 Plate IV x 255, 256, 259, 261, 202, 260 270, Alloxazin adenine dinucleotide 55 56 271, 272 277, 278, 279, 280 281 Ammonis 37, 223 column of ions of, 27 40-52 88, 89 Amphitta 295, 296, 299, 310 337 Srays produced by 28 30, 32 Anaphase 129°, Plates III! IVx 88-80 272 Antirrhinum, 181 delayed division by, 303 30¢ 305 312 Arbacia (sea urchin} 284-95 297 298, dosimetry of, 18 22 311 efficiency compared to 4 rays, 47, Arsenite 46 59, 00, 72, 87, 115, 121 153 179, Ascaris {a parasitic worm) 308 312 241,270 304,312 315,324 327 Ascorbic acid 46 energy dissipation per micron path Associated volume 83-89 356-63 25, 351, 352 Asymmetncal exchange between chro energy dissipation perr 8, 349 mosomes 137* energy of 17 Attached X stock of Drosophila, 142 rautations by 152, 153 179 143 165 range of 17, 25,32 240 352 Aucubs virus 115 143 144 rapidly dividing tissues effects on Auger effect 10 312, 335 336 Autosome 132* sources of, 17 18 stopping powers additive law of Brays 16¢ 351, 352 absorption of 13 24 41 target ze for 88-92 97, 98,122 123 bactericidal action of 316 318, 322 viruses, effect on, JJ8, JJ4, 115, 324 327 146 117, 129 121, 123 313, 314, chemnal effects of 37, 41, 42 31 5 dosimetry of 16 22 ‘Wilson chamber photographs of 49, energy dissipation per mc radon 18 Plate Ta energy dissipation porr 8 Absorption spectra 35 788 energy distribution of 16 Acenephthene 191 mutation by 147 148 Acentne chromosome 137* sources of 15, 18 Acetone, 55 Bactena Acetylene, 37 chromosomes of 326 Plate IVo A lesion (in 1} 201" of 78, 317, 318 319 320 ‘Activated water, 44", 47-65, 109 305 mbibition of division of 300 302, 312 Plate IVB Activation energy for mutation 136 lalled by single 1omzation 69 71 78, Active deposit I 7 77 81, 120 325 Affimty of zolute for activated water 56 lalhng by rathation 316-27 SUBJEOT INDEX 405 Bacteria (cont } ion density, effect of, 57-60 motihty of 325 ionic yields, 33*, 34, 37, 46 mutation of, 141, 144 knetica of indzrect action 52-54 spores of, 318, 321 322, 324, 325 lhquids and sohds, 37-39 target size and number 120 326 protective action, 55-57 ultra violet absorption by, 5 recombination of active radicals Bactenophage 48-82 density of, 103 reduced yneld mm dilute solution estimation of 104, Plate IIs 57-60 inactivation by radiation, 86, 87, solutions, 39-64 108 174 115 117, 418, 119, 121, spatial distribution of active radicals 177 48-62 size of, 103 118 ultra violet hght, 2 36, 37, 39 structure of, 122 Chick, 201, 293 205, 296, 297, 304, Barley, 154 306, 307 310, 337, Plate IVz-r Be+D neutrons 20, 21 Chlorine 36 Bean, 71, 190, 225, 236 238, 281, 296 Chortophag Af (grasshopper), 300, 301, 362, 303, 304, 305 312, 192, 193, 198 199, 201, 208, 209, 335-37 342 217, 218, 225 230, 248 293 204, Belleval.a romana 193, 225, 338 299, 338, Plate IVs Bethe formula, 349-52 Chromatid breaks, 195, 201% 203, Birds, 128 {see aleo Chick) 218, 221,224 226, 227, 229, 230, Boron 19 21 238, 239, 240 241, 242 243, 258, Bragg additive law, 351 259, 334 Plate IlId,¢ Breakage of chromosomes (see Chromo Chromatid exchanges 195, 198, 207- some structural changes) 14 217, 218, 220, 227 224, 226, Bromide, 41 227, 232, 233, 240, 242 279, 280 Bromine 36 37 334 Plate IIL j,g & Broth 105 108 109 4 isoch, d 260 Butterfhes 128 Chromatids, 129* achromatic lesions in, 201 x? test of goodness of fit, 146 attraction between 201 Caging effect of solvent 39 half chromatid breaks 201 Cancer, 307, 342 independent breakage of 186 Caproic acid 65 separation of, 203 241 261, 262 Carbon dioxide 36, 37, 42 46 55 sister union and non union 164, 187, Carbon monoxide, 36 46 200 204 210 225 245 253 254 Carboxypeptidase 44 255, 280 Cathode rays, 15%, 37 Chromomeres 134* Centrifugation, 102 121, 220, 285 Chromosomes 127* Centromere 129* absorption of ultra violet ight by, 6, Cenc sulphate, 46 48 34 Chaetopterus 311 econtric, 129 137" 163 164, 197, Chain reactions 36 63 175 199, 205 206, 208, 216 236, 328, Chemical bond ! 333 336 337, 343, Plate Wy Chemical effects of radiations behaviour normal 127-31 biologieal effect due to 1, 64 bridges 137, 163 165 192 194, 195, 197, 200 201 205 208 236 328 37, 57-60 336 337, 342, 343, Plates IIT competition between solutes 55-57 IVaurx decomposition of water 40-42 clumping of, 192-97, 343, Plate direct and inthrect action 60-64 Ive-x free etoma and radicals 47-48 dicentric 137%, 163 164 171, 200 gas reactions 34-37 208 236 328, Plate IIIs mmdirect action in aqueous solution dimensions of 67, 134 135, 215 252 42-64 O71 974 ono 406 SURJECT INDEX Chromosomes (cont) 187, 208 207, 213 217, 218 221, duplications 1 137, 200 339, 340 237, 233 241, 242, 252, 256 328 loss of, 164 105 185 209 phenotypical effects 138 139 142 maps of, 131, 134, 158, 139 random of non random union 165 matrix of, 190° 193 201, 224 166, 219 245 250 251 252 y t effect of radiation on, of breaks 158, 159 160 192°-97, 223, 225 237, 239, 336- 17], 172 187, 200 201, 216 220, 38, 349 344, Plate IVr-c 22t, 223 224, £26, 232 246 253- polarization in, 210 69 279 280 repulsion between 196 separation of breaks which exchange ring, 197, 171, 172,193 194 195, 205 249-52 259 280 salivary 134%, 135 138 139, 155, spontaneous 239 241, 243 369, 160 161, 163 183, 189, 190 symmetrjcal and asymmetrical 137° JOT, 200 206 209, 212, 214 215 163 194 195, 207, 208, 209, 210 217, 224 298, 233 246, Plate transition from chromosome to chro Tila matid types 198, 222 spiralzation of, 128 203, 224 tranalocations {eee exchanges) atickiness of {eee physiological effect types of 136-38, 79 195 107-211, of radiation on) Plate III time of spht 128, 197, 198, 199, viability, effect on 138 163 166 203-4 185 209 225 328 329, 333 340 Chromosome atructura! changes Chromosome structural changes induc behaving as dommant lethals 164-72 tion by radiation behaving as recessive lethals 154-60 centnfugation effect of 220 224 behaviour at cell division 129, 137, colchicine effect of 220 221, 224 163, 164 365 171, 194 195 199, comparison of different radiations 200 205 206 208 Plate IIE? 204, 21 226 227 233 237-40 poperiour at meicais 185, 339, 340, 254 255 261, 262 269-81, 338, 337 breaks or termina! del 187, b of ab among 194 195 197 109-205 218 229, cells 217-20 230, 236 238 241, 242, 243, 258 distmbution of breaks in chromo cyclical exchanges 218-20 somes 162 170 214-16 exchange between paternal and dose dependence 156 157 158 167, maternal chromosomes 229 168 169,170 173 229-37 exchanges (intrachanges or inter energy needed to bresk chromosome changes) 155-72, 187 194 195 276 198, 205-14, 218 219 220 221, geometrical factor (g) for isochro 224 226 227 228 229 231, 232 matid breaks 260° 261 262 278 233, 234, 241 242 248 249 250 251 252 256 257 266 279 328, intensity dependence 211 214 338-41, Plate WIla bfgs7k BP5-29 37 232 239 24) 246 healing 246 247 248 262-69 279 hereditary partial sterility 338-41 mathematical theory of 166-69 ancomplete exchanges 187" 210 modifying factors 220-25 246 247 211 242 253, 254 255 280 number of iomzations needed to interval between breakage and join break chromosome 245 246 270 mg 191 197, 223 228 229 246 276 280 281 £63 279 probability of onizing particle break anvermons snd deletions 159 163 ing chromosome 260 261 262 194 195 205 206 207 212 270 27% 272 273 276, 277 278 jomabilty of breakage ends 204 279 230 205, 210 239 253 257, 258 262 produced by single 1onzing particle 262 278 280 11 66 69 71 155 164 173 202 mumute uuiterstitial deletions 141 226 227 228 232 233 234 2416- 155, 156 158 159 163 178 183 49 250 260 280 282 SUBJECT INDEX 407 Chromosome structural changes, induc Deficiency (of gene or genes), 137%, 141, tion by radiation (cont ) 155, 156 158 159, 163, 176, 198 produced by two 1omzing particles 183 184, 185 186 187 159, 158, 164 187 226 248, 250 Degenerate cells 309, 310 280, 282 Delayed division (see Division) relative frequency of diffe types Del (see under Ch struc 196, 197, 211-14 tural changes and Deficiency) nng chrornosomes, structural change Density (of viruses or proteins) 89, wn 171, 172 103. senativity at different stages 190 Deuterrum hydroxides, 50 196, 198 203, 213 223-25 Dicentric chromosome, 137° temperature effects of 207, 209 Dichromate, 46 211, 221, 222 223 246 247, 266 Diffusion theory of (summarized), 279-81 of radicals, 50 ultra violet hght 182 183 242-44 of deutermm hydroxide 50 yield of breaks per unit dose 159 Dilute solution, reduced yield in, 57-00 T60, 167 169 170 241, 256 261 Diploid 130* 262, 270, 275 280, 330 332 Direct and indirect actions of radhation, Clam 311 60-64, 107-11 317 ClB method in Drosophila 133 142°, Dissociation of a gas molecule, 3 143, 144 Dissolved oxygen affecting 4 m Cleavage delay in fertilized eggs 284- solution 41 42 46, 47 95 311 Distmbution of radiosensitisity among Cluster theory 36 cells, 77 78 Clusters of sons, 27, 80 8 85, 86, 95 Division, delay mbibition and pro 174 Plate Ip Tongation of Colchicine, 191 290 221 228 bacteria 300 301 Coihsions between cleavage delay in fertilized eggs 284- H and OH radicals 48-52 57-60 95 radicals and solute molecules 53, 54 of d diet solute and solvent molecules 39 303-6 312 Colour blindness 127 130 cumulative dose 283 285 286 289 Competition between solutes, 45 55-57 290 292 295 297 Compton effect, 11, 13, 345-49 direct or indirect effect 285 Concentration effect on dose dependence 282, 288 289 299, bacterinidal action 317 30f 305 306 chemeal reactions in solution 43 44 intensity and fractionation effect of 49 57-64 292 297 298 sperm inactivation nucleic amd 283 312 304 virus inactivation 108 109 110 nucleolus 304 bade number of 1on2zations required 304 Condensation of chromosomes 128¢ oxygen uptake relation with, 29% Cosmic rays and mutations 180-81 292 Crossing over 131* 133 142 rapidly dividing tissues 71,194 196 Cumingia (clam) 292 311 205 223 225, 237 295-300 Cumulative dose 283* 285 286 289 recovery 282 283 287 289 291, 292 290 292 295 297 recovery time (r} 289 290 291, 292 Cumulative type of action 76 77 79 size increase (of cells or nucles} 300-2 Cyclotron 19 20 stage most sensitive to radiation 288 289 293-95 drays 27*, 48-32 82 88-89 179 272 Stage subject to delay 286, 287, 276 279 351 352 356 357 358 293-95 36l Plate Ta temperature effect of 290 291 d amino acid oxidase 46 55 56 Dommant 126* D+D neutrons 8 20 21 Domunant lethal, 161-72 Deactivation efficiency 184 329-329 56 62 63 111 Dose rate (2ee In enaity) 406 SUBJEOT INDEX Chromosomes {cont } 187, 208 207, 213 217, 218 22! Gupheations un 137, 200, 339 S40 234, 233, 241, 242 252, 258 328 Joss of, 164 105 185 209 phenotypicel offects 138 139 142 mépa of, 131, 134, 138, 139 random or non randam union 183 matrix of 199" 163 201, 225 106 219 245 250 251 252 } 3 offect of radiation on of brenks 158, 159 160 192*-97 223 225 237, 230 336-~ 174, 172 187, 200 201 216 220 39, 143 344, Pato FV en 221, 223 224 296 292 26, 253- Ppolanzation in, 210 68 290 280 repulaion between 196 separation of breaks which exchange ring 137, 171, 172 103, 194, 193, 208 249-82 259 280 salary, HH 135 338 130 153 spontaneoits 239 341, 243 18D 100 1Gl, 163 383, 189, 390 aymmetrcal and asymmetrical 137°, 191, 200 206 209 Biz 214 215, 163 294 195 207, 208, 209, 210 SIT 228 228 234 348 Piste tranation from chromosome to chro ab matid types 198 222 spiratszation of, 128 209 224 translocations {see exchanges) atichunoss of (ace physiological effect types of 130-38 79% 195 397-218 of radiation on} Piste Tit time of eplt 128 107, 198 199, wubthty effect on 138 163 166 185 209 225 328 329 333 340 Chromosome structural chi Chromosome structural changes, induc behavi as dominant ng lethals 164-72 tion by radiation behaving as recessive lethals 154-60 centnfugation effect of 220, 224 behaviour at cell division 129 147 calcneme affect of 220 221 224 3 IGS 165 171 194 795 190 companson of different radsatons 200 205 260 20R Plate 1ILt 204, 211, 226 227 255 237-40 behaviour at meioa, 185 339 340 254 255 261, 262 269-81 336 341 27 breake or terminal del 187 ib of among 334 395 197 109-205, 218 299 cells 227-20 240 236 238 241, 242 243, 258 distnbution of breaka in chromo eycheal exchanges 218-20 somes 162, 170 214-16 exchange between paternal and dose dependence 156 157 158 187 maternal chromosomes 229 268 169, 170 173 229-37 exchanges (intrachanges or inter energy needed to break chromosome changes} 185-72 187 iff 195 278 198 205-14 218 289 220 221, geometrical factor {g) for ssochro 224 226 227 228 299 231, 232 matid broake 260* 261 262 278 233 234 244, 242 2ag 249 260 278 251, 252 256 257 266 279 928 intensity dependence 24% 214 338-41, Plate la 6 fg 13k 226-28 231 292 233 241 246 heahng 248 247 248 262-69 279 hereditary partial stemlity 338-41 mathematical theory of 166-69 mcomplote exchanges 187% 210 modifying factors 220-25 248 247 211, 242 253 254 255 280 number of sonsrations needed to mterval between breakage and jou break chromosome 245 246 270 amg 19} 197 223 228 229 246 2768 280 28 263 279 probatihty of romzmg particle break uiversions and delehona 159 183, ing chromosome 280 261 262 I9¢ 195 205 206 2OTF o1z 270 272 272 273 278 207 278 jomabisty of breakage ends 204 279 280 205 210 239 253 25%, 258 261 produced by aingle sonizing particle 282, 278 280 HE 66 69 71 155 364 £73 202 mimute iterethtal delethens 141, 226 227 228 232 233 234 246- 185 156 188 169 163 178 183 49 250 240 280 282 SUBJECT INDEX 409 Fhes (see Drosophila, Scrara) Gas free water 41 Formaldehyde, 46, 49, 55 Gas reactions 34-37 Formate 56 Gelatin 108, 109 110 Forme acid, #3, 49, 55 Generative nucleus (of pollen} 332* Four strand pachytene 131* Genes Fowl plague virus 118 nature of 101, 130 133-36 Fractional deficiency {in a chromo number of 90 134, 135 179 180 some) 186* 315 326 342 Fractionation of dase, effect of 78 147, reproduction of 139 226, 228, 266 267 size of 12] 135 172-80 315, 326 Free atoms and radicals viruses and, 101 121, 124, 136 174 collisions with solute molecules, 53, 54 175 117 concentration of, 50-52 Genetical effects of radiation (see also diffusion of, 50-52 under Mutations and under Chro energy to form 47 mosome structural changes) explaimng radiochemical actions in cosmic trays 180-81 solution 47-48 deductions concerning size of gene formation from H,O, 47 172-80 m gases 35 dominant lethals in Drosophila 1o1~ im water 47-54 72 recombination of 48-52 57-60, 305 recessivo lethals in Drosophila 144~ Spatial distribution of, 47, 48-52, 68 St Frog, 296 310 relation between recessive lethats and Fructose 56 chromosomal changes 154-60 Fungal spores 743, 183, 188 300 ultra violet hght 181-88 visible mutations 110-44 @ (function of tame intensity theory) Genotype 126* 264, 265 Germ (see Bacteria, Egg Frmbryo, 9 ( 1 factor for Sperm) breaks), 260 261 262 278 279 Globulin 134 Gametes 126* Glucose 56 Gametophyte 185* Glutathione 46, 49, 58, 59 Gamma (y ) rays, 13* Glycerine 63 absorption of 14 15 Glycine 56 bactericidal action of 316 317 318 Gram roentgen 22 322 324 326 327 Grasshopper 192 193 198 199 201 chemical effects, 37 208 209 217, 218, 225 230 248 chromosome structural changes by 293 294 299 337 338 Plate IVa 211 228 239 240 Gross etructural change (of chromo delayed division by 295 296 297 somes) 138* 300 302 304 305 306 309 312 dosimetry, 14 22 Haemoglobin 38, 45 46 electrons hberated by 12 13 14 15 Heemopluha 127 130, 131 energy dissipation perr 8 Helf eggs 285 filtration of 14 Haploid 130* intensity at 1 cm from 1 mg Healing (of chromosomes) 246 radium 15 Heruygous 13]* mutation by 147, 148 179 Heredity 126-31 rapidly dividing tissues effects on Heterochromatin 135* 138 139 215 309 312 325 336 216 234 Scattering of 14 Heterozy gous 130* sources of 13 Hexane 50 Spectra 14 Mippurate 56 target volume determined by 91-92 a3 log: {genes or ch: viruses effects on 172, 115 116 12s* 118 121 122 123 314 315 Hydration of viruses 102 103 408 SUBJECT INDEX Doaunetry no mater union in 187 @ raya, 18 restitution in, 187 frye 16 ¥ says, 14 interconversion of units 22 chromosome Aefictonees in, 14-88 neutrons, 19, 20 aay protons, 19 defective 184 vitms violet hght, 4 Mutations affecting 194 K rays 6 master union of chromatids in, 187, Drosophila (fruit By} 4 6, 00, 128, 120 200 328 131, 132 133 344, 135 138 139 Energy dissipation 140, 342, 245, 284-72 195 196,180 by lealsation and excitation, 1,2 35 18k, lea 283, 187, 188 191 186, by ultra violet ight, 3 6 197 109 200 206, 208, 209 212, in aolute and actvent, 30 44 B13 214 218 216 S17 218, 249, per ion paly 7 44 220 227, 222 223 227 228 £32, por micron path 24 25 26 351 352 233 234, 237 242 263, 248, 248 per toontgen or: unit 8 22 34 -50 247, 315,525 328 329-32 342 243 per unit volume 8, 13 Dupleations (of portiona of chromo Energy unt 8, 22 sotnes}, 137%, 200 338 340 Easymea 37, ‘38 42 44:45 46 OA 85 334, v6 477 By meen 353, 352 Buchromstia 135%, 138 130 S09 B18 Egg Rie 23¢ and sperm, rilstive pongibvity, 285 Exutaton ae biologreal affects of, 2 Aclayed cleavage of 284-03, 311 chemical otionta of 35, 47 in solute sud solvent 39,40 42 43 faluze ta hateh 162 168 169 303 energy of 2 2 309 329-33 half-eggs 285 Exponential dose relation, 38 45 f1 2-98 107, 110, 122 115, 114, 124 ta 258 308 306, JH8, 319 320 radiation of, 152 153, 181, 233, 229 264 285 286, 287 288 289 £90 Z9L 2G2 308, 309 329-83 384 327, 330 34t Exponential function table of, 75 nucleus (in peed plants) 183° Extinction coefficient (of ultra violet Electrons (see also £ rays} light) 4 sbeorptian of 24 assorted volumes of 356-50 F {overlapping function) 85* 86 Attachment to moleculea ions oF 385-54 radicats 2 35 26 47,48 89 Fy, (Gtst and seco Pyand geners Sie! nd chromosome breakage by, 210 21% thon) 126° 249 272 273 f {proportion of chromosome breske 8 raye produced by 28 29 32 which are unjomsbla) 253-82 energy dissipation by, 24 349 351 Ferre sulphate 46 362 Ferneyarude, oh ‘6 energy dutnbution of 22 Ferrocyanids mucrographs 122 314 Plates Iie Ferrous sulphate 45 46 47 48 00 Ivo Fibroblacts irradiation of 201 293 umber crossing a cell 249 295 296 297 30q 306 307 310 numbe r 32 346 347 per unit volume 337 Plate IVn-t promary fomeation by 23 381, 252 Sor Baers f tad 273 zie mS. 216 280 Plate To tatews ght 5 range of 24 37 Pawan of viruses 102 103 ng et volt (eV) t rays 9 Embryo {rn seed plants 183° absence of 184 Furst heptod muitos 19% 409 SUBJECT INDEX Fhes (see Drosophila Scrara) Gas free water 41 Formaldehyde 46, 49, 55 Gas reactions 34-37 Formate 56 Gelatin 108, 109, 110 Formic acid, 23, 49, 55 Generative nucleus {of pollen}, 332* Four strand pachytene 131* nes Fowl plague virus 118 nature of 101 130, 133-36 Fractional deficiency (im a chromo number of 90 134 135 179 180 some), 186* 335, 326 342 Fractionation of dose, effect of, 78 147, reproduction of, 139 226, 228, 266, 267 size of 121, 135 172-80 315 326 Free atoms and radicals viruses and 101, 121, 124, 136 174 collisions with solute molecules, 53 54 175197 Concentration of 60-52 Genetical offects of radiation (see also diffusion of, 50-52 under Mutations and under Chro energy to form 47 raosome structural changes) explammg rachochemmeal actions cosmic rays 180-81 solution, 47-48 deductions concerning size of gene formation from H,O, 47 172-80 in gases 35 dominant lethals in Dresophila 1bl~ mwater 47-64 72 recombination of 48-52 57-60 305 recessive lethals in Drosophila 144- spatial distribution of, 47 48-52 68 Frog 296 310 relation between recessive lethals and Fructose 56 chromosomal changes 154-80 Fungal sporea 143 183 188, 300 ultra violet hght 181-88 vistble mutations 140-44 @ (function of time intensity theory) Genotype 126* 264, 265 Germ (see Bacteria Egg Embryo 9 factor for isock d Sperm) breaks) 260, 261 262 278 279 Globubn 134 Gametes 126* Glucose 56 Gametophyte 185* Glutathione 46 49, 58 59 Gamma (y ) rays 13* Glycerine 63 absorption of 14 15 Glycine, 56 hactencidal action of 316 317 318 Gram roentgen 22 322 324 326 327 Grasshopper 192 193 198 199, 201 thermeal effects 37 208 209 217 218 225 230 248 chromosome structural changes by 293 294 299, 337, 338 Plate IVa 211 228, 239 240 Gross structural change (of chromo Aelayed division by 295 296 297 somes) 133* 300 301 304, 305 306 309 312 dosimetry 14 22 Haemoglobin 38 45 46 electrons hberated by 12 13 14 15 Haemophika 127 130, 131 energy dissipation perr 8 Half eggs, 285 filtration of 14 Haplosd 130* intensity at lo cm from 1 mg Healing (of chromosomes) 246 radium 16 Hemuzygous 131* mutation by 147 148 179 Horedity 120-31 rapidly dividing tissues effects on, Heterochromatin 135* 138 139 215 309 312 335, 336 216 234 scattering of 14 Heterozygous 130* sources of, 13 Hexane 50 spectra 14 Hippurate 56 target volume determined by Homologous {genes or chromosom 91-92 es) viruses effects on 11Z 1i5 39* 11S 418 121 122 123 314 315 Hydration of viruses 102 103 410 SUBJECT INDEX Hydrezino, 37 86 00, LIS, 116 183 179 204, Yydnodwe acid, 37, 30, 46 205 210, 212, 237-40, 254 255 Faeroe acid, 96, 37, 46 262, 270 272, 272 273, 27%, 309 gon, 36, 37 40 43, 49, 38 41, 42 JO¢, 304, 306 312, 323, 324 327, ‘ 1 46. 338, 337 atoms (e¢ under Free radicals) ato affecting chemical effecta, 57-60 some azul affecting probability of effective nueloi (#e¢ under Protons) *hit’, 354 peroxide 40, 41, 42, 43, 48 Tonle yield, 33%, 34, 36 37, 38 40, 41 mulphide, 26° 45 46 47, 57, 58, 62 63, 64 85, Hydrory! radical (eee under Free atoma 110, 111 and radicals) Tonuzation 1° chamber for neutron doamotry 19 Tea, 40 energy of I 34 35 TMogiti union of ch inair perr 7 137¢ in solute and solvent 39, 40 42, 43 Tmpuntion, effects of traces of, 41, 42, potentia! 351 352 single actions due to 38 71-72 Inectivation doso 61*, 74 108 LH-16 153 325 ond poli content of solution, 62 109, spread of offect of 67-63 89 173 Ionizing particles (se¢ also under a rays, and target size 80-88, 116-23, 362- drays Electrons Protons) number per unit volume, 32 y of virusce 109 161, 243 110, 116-23 number travermng celle ip ‘a 249, 250 bite 210 212, 242, 253, 264 255, range per umt volume 32 1 lone Incomplete linkage (of genes}, 130° negative 2 34 35 Tadependont assortment (of genes), postive 2,34 35 recombination of 35 50 Indirect ections of radiations spatial distribution of, 10 49, 350- on chernicals, 39, 40, 42-64 52 Plater on sperm 285 Teochromatid breaks 295 202*-5 210 on viruses 63 84 107-11 RIT 218 220, 221 227 220 230, Infra red radiation, 223, 247 237 238 239, 240, 241, 242 260 Intenmty (or dose rate) 7* 72, 78-70, 263 277 278 270, 280, 28 334 1M, 115, 116, 124, 144 145, 146 Plate le dt 147 183, 214 225-29 241 246, 247, 248 262-69, 279, 292 297, Jensen rat sarcoma 295 298 321, 327 Kinetics 52-37, 138 137¢ Kiem Nwhna formula 14 345-49 Interphase 128¢ Interstitial deletion (of a chromosome) Lactose 107 187* Latin square 105 Be (ofa Lethal effects of radiation ascribed to chromosome physiologseal Todide 41 change 337-38 343-44 Ton-clusters 27, 80 $¢ 85, 86 05 174 ascribed to chromosome structurst Plate Ip change 328-37 341-43 Ton-columns 27, 49-52 88-89 Plate ascribed to fethal mutstion 313-16, Ta,3 324-27, 342 Yon-density (or specific 23° bacteria 316-27 341 342 affecting apparent target size 97 98, division related to 307-13 341 345 356 256 eggs 308 31} 329-32 affecting biological effecta 72 79-80 hereditary partial sterility 338-41 411 SUBJECT INDEX Lethal effects of radiation (conf } inhibition of 295-300, 302 303-6 pollen 3808 332-35 309, 310 309 312 of radiosensitivity of different stages rapidly dividing tissues S434 190 198 293-25 287, 283 289 root tips 335-37 293-95 spermatozoa 312 Molecular viruses 100-25 313-16 341 342 diameter 52 Lethal mutation {sce Recessive lethal weight, 53,65 89 135 136 177 178 and Domtnant lethal) Mosare 132° Leucylglyeine 56 Moths 123 . Linear dose relation $8 43 45 60 72 Mothty of bacteria 325 M5 146 148 153 156 257 158 Mouse 225 235 236 238 281 300 165 (81 183 229 250 231, 232 B4i 343 933 235 237 938 241 943 244 Vfults hit target theory 31 322 248 249 280 Mult: target theory 90-92 122 314,326 Jankage 127* Mutations altered by ch I h Hy induced, 132 change 18S endocperm affecting 184 Group 12%* nature of 132 133-36 154-60 incomplete, 130* relation to chromosome structurat Liquids chemical effects in 37-39 change 136-40 154-60 Lithum douintegration by neutrons spontaneous 131 180 181 ff 19 2) Li+D neutrons 8 20, 21 rate 136, 18] Liverwort spores 18 vrabilty affected by 133 142, 176, 183 Local lesions (due to plant viruses) Mutations induction by radiation 105 140 176, Plate IIc caused by single :onizstion 66, 69, Locusta migratorna 199 WOr 153 4 Loganthms 7495 comparison of different radiations -P ¢ to) 190 345 147 748 149 153 179 238 cosmic rays 180 181 Lymphoms 235 236 238, 343 dominant lethals 161-72 184 329- 32 Maze, § 127 130 18: 183-88 200 dominant vsible mutations 133 Lid 202 205 219 242 243 328 340 Hi 184 Malpighisn tube 175 170 dose dependence 143 144 145 146 Mammaha (see Man Mouse Rat) 1586 157 158 167 168 169 181, Man 127, 123 130 300 183. Mass spectrograph 34 grouping effect 150-51 Matrix (of a chromosome) 190* 193 in bacteria 141, 144 324-07 201 22¢ im barley [54 Mean lethal dose ol 74 321 335 336, in Drosophila (see Drowephila) 337 im viruses 140 142 [43 144 176 Meionis 129* 313-16 Mercury lamp 4 5 intensity not affecting 145 L146 147 Metaphase 128° Plate IEA 1 7 & relative frequency gene mutation and Methaemoglobin 35 46 deficiency 141 159 176 178 183 Methyl alcoho) 49 55 59 59 18t 186 Mero moles per 1000 r 34 44 recessive visible mutations 141-44 Micronucle: 199 333 343 184 Mierucnores (2¢e Pollen) sex Inked recessive lethals 133 140 Milneure {me} 15 18 144-60 170 182-83 Minute structural changes (of chromo target ee for 90 120 178 179 189 sornes) 138+ Mitosis temperature not affecting 154 128*-29 yield per unst dose 132 144 177, duration of stages of 294 995 305 178 180 412 SUBJECT INDEX Motatt of Drosophila mel: Nucleus (of cell) Bar (eye), 140, 142 membrano of, 128, 293, 294 brown (eye), 209 teparation from cytoplaam 285 cubits interruptus (wing vein), 130, size Increased by irradiation, 300-2 sizo of, 249, 302 ebony (body), 209 forked (bristles), 144 Onion, 190, 193 199, 201, 208, 214 moimature (wings), 144 220, 223 337, 338, Plate IVx Notch (wings), 138, 206 Oocytes 190, 224 acute (bristles), 234, 242 Oregon R (stock of Drosophua melana white (oye} sores 130, 139, 143 144, gaster), 149 164 178 176 Organic acids, oxidation of, 46 yellow (body), 138, 165, 234 242 Overlapping function (F) 85*, 86, 353-54 v unit, 20° Oxalate, 56 energy dissipation per, 8 Oxaho acid 49 55 58 59 nm unit, 20° Oxidation by radiation 41 42 46, 46, Natural rad:ation and mutation, 180-81 48 Nerevws, 311 Oxygen, 38, 37, 40 41, 42, 48, 291 Neuroblasts, 192, 193 198, 201, 208 Oxyhsemoglobin, 46 209 217,228 230, 248 293 204, Ozone 37 299 337, 338, Plate IVs Neurospora crassa 143 183 p (probability of collmon removmg Neutrons, fast active radical) 54-55 56, 57, 59 bactericidal action of 316 318, 324, p (probability of 1omzation in target 327 being effective) 93 95-96 97 122 chromosome structural! changes by 172 174-275, 178, 180 355 358 204, 210, 211, 218, 226, 227, 230 p (probabihty of iomzang particle 231, 233, 234, 235, 236, 237, 238, brea! achromosome) 260 282 239, 241 248 249, 255, 266, 259, 262 270 271, 272, 273, 276 278 261 262 269 270 272 277 278 279 280 279 280 281, 338 p (probability of sister union at & division delay by, 303 204, 305, 308 chromosome bresk), 166 169 172 312 P (probability of solute reacting) 54, doametry, 19, 20, 22, 237 0 61 efficiency compared to X rays 72 pH affecting chemical actions of radia 145 148 149, 241, 270, 336 tion, 47 energy dissipation per v urut, 8, 350 Pachytene, 131° mutations by, 145 148 149 150 Peas, 126 127, 238 161, 153 179 Permanganate 46 47 49 nitrogen ciantegration by, 19 350 Persulphate 63 nuclear projection by, 19, 20, 21 350 Phenotype 126* rapidly dividing tissues effects on, Photochemical reactions (see under 204 312,335 836 337 Ultra-violet light) scattering of 20 450 Photoelectne absorption 10 345-49 sources of 20 Photoelectrons 10° Neutrons slow 19 21 associated volume of, 87 Nitrate 46, 56 energy of 10 12 273 275 Nitnte 46, 56 number of 12 32 249 273 274 Nitrogen, 19 350 275 Nitrous oxide, 37 range of 32,173 249 273 276 Nucleate 56 Physiological effect of radiation 162 Nuclewe acid § 128 139 1838 196 193 192-97, 223 225 237 239 336-38, 196, 215, 283 304 343 343, 344 Plate IVr-x Nucleoprotan 5 101 122 123 124 Plague virus (fowl) 118 127, 134 135, 136 188 Plaques bactenophage 104 Plate Iz SUBJECT INDEX 413 Point heat, 3, 67 Protozoa, 71, 300 Poiwons produced by radiation, 64, 78, Pyocyaneus, 71, 320 317, 319 320 Poisson distnbution, 150, 166, 217, 218, q (probability of chromosome break 248, 256 not undergoing sister union) 166- Polar cap (of egg), 152%, 153 181 12, 330-32 Polar fusion nucle:, 184* Quantum energy, 6 9,12 13 Pollen Quantum yield, 36, 37, 39, 125 defective, 185, 186 development of, 332, 333 Rabbit fibroma virus, 118 dimensions of, 249, 259 Rabbit papilloma virus, 102 103, 109 failure after d Md, 123, 118 308, 332-35, 342 Radioactive matter in organisms, 180 181 ficial d on 191, 242 radiations (seea , 8 , y rays) uradiation of, 183-88 189 193 198, sources (see Polomum Radium) 199, 200, 201, 202 203 205 207 Radiochetmeal reactions 33¢ (see 209, 211, 212, 213 214, 216 217 Chemical effects of radiations) 218, 219 220, 221, 222, 224, 225 Radium 226 227 230, 231, 232, 233, 235 emanation (see Radon) 236 238, 239, 240 241, 242 243, y ray intensity at lem trom 1 mg, 245-81 308, 332-35 337 340 15 mother cell 190%, 201, 219 Ra A B,C C, 16 16,17 18 32 tube, 191, 203 204 209, 211, 212, 34 241, 270, 277 214, 216, 224,239 240,241 243 Radon (radium emanation), 13, 17, 18 255 32 33, 34, 41 46, 152 239, 241 ultra violet absorption by, 5, 244 270 277 Polonium, 17 Range of Polypeptide, 210 a particles 17, 25,32 240 352 Position effect 138*40, 155-58, 175 electrons, 24, 27 32 Potato virus X, 122 116 protons 26 32 Prmery effect of radiation on chromo Rat 225 295, 298 299, 309, 310 somes 193* Recessive 126* Pnmary ionization, 25 26 85 352 Recessive lethals 133* Pnmordial germ cell 162* Reerprocal translocation, 137* Probabihty (see p P) Recoul electrons, 11*, 12,32 345-49 Prophase, 128* Recoil nucle: 18 34 prolongation of 286, 287 294 296 Recombination of Protamin 134 ions 60, 88 Protection against effect of rad in P d of ch ld solution 45 48 49 55-57 60-65 36, 39 60, 175 108-11 312 radicals 48-52 57-60, 305 Protein 38 45 46,55 56 89 98 109, Recovery from effects of radiation 79 110 111, 112, 134 135 190 312 196 282 283 287, 289 202 292 Protons 19* 308 309 312 associated volume of 358 361 h d ) by 40, biological effects {see Neutrons fast) 41, 42,45 46, 48 8 rays projected by 31 32 272 Resonance radiation & dosimetry of 19 22 Resting atage 128* energy dissipation perr 8, 349 Restitution (of chromosome break) number traversing & cell 151 248, 158 159 160 171, 172, 187 200 249 250 201 216, 220 221 293 224 projected by neutrons 296 19 20 21 232 246 253-69 279 280 32 Ribonuclease 5, 38 46 177 ranges of 26 32 . Ring chromosomes, 137, sources of 19 172 172, 193, 194 195 205 414 SUBJECT INDEX Roontgen (r) 6* Spores (see under Bacteria, Fungs? energy dissipation per, 8 Pollen} nurnber of sonizing particles per, 32 Spread of effect of fonization, 67-88 Root tips, 100, 193, 107, 201, 208, 225, 69 173 236, 238, 281, 298, 300, 301, 302 Square law doao relation 232, 233 235, 303 304,905 312, 935-37, 338 342 236 241, 248 256, 257, 279 Stenhty, hereditary partia), 328 338-41 o, Ge o, (Compton ig coat k of ch 192-97 clente), 345, 346, 947,349 349 Survival curves a (ene croaa section for neutrons) ofirradistedbactena 318,319 320 323 is of irradiated eggs, 330, 331 Sabvary gland chromosomes 134 135 of irradiated viruses 107, 112, 113, 188, 139, 155 159, 160, 161, 163, 414, 124 183 189, 190, 191, 200, 206, 209, significance of shape of 70,71 72-78, 212 214, 215, 217, 224, 228, 233 110 124, 305, 306, 324 246, Plate Ila & 8ymmetnical exchange (between chro Scrara, 190 198 213, 224 mosomes) 137° Soa urchin, 284-05, 297, 298 312 Synapms 129° Second haploid mitosis, 191° 8. dary effect (of radiati on chro toh 7 absorption ) Mosomes), 193¢ 345, 347, 348, 319 Secondary ionization (see also 2 rays), r (time constant for chromosome 26 80, 85 umon) 263-69 279 Belenite, 46 r (time constant for decay of curnula Sex chromosomes, 128° tive dose}, 289 290, 291, 292 Sex linked, 131° Tadpole (of frog), 296 310 Sex ratio distortion, 170, 171, 172 Tail of electron track 273 274 275, Shope rabbit fibroma virus, 118 Ie 278 280, Plate Shope rabbit papilloma virus 102, 103, Target 109, WAL, 113, 118 area, froma ray deta, 91 313 314 326 Sigmoid survival curves 76, 77, 78, 319 calculations 353-63 320, 331 indefimte boundary to, 93 96-8, Single unit acon, 77* 355-56 Sister union (see under Chromatida) mult: hut theory 71, 322 Solda, chemical effects of radiation on, multi target theory 90-92, 122 314, 37-39 326 1 effects of radsat: number, 90-82 122 179 180,318 326 Bolut on 39 42-64 of bacteria 322 326 327 Somatic cell 130° of genes 172-80 Spatial distribution of of viruses 116-23 177 H and OH radicals 48-52 68 overlapping factor 85* 86 353-54 xonizations 10 49 79-90 92 93, shape 91, 93, 94-95 350-52 Plate I aze from 37 % dose 80-90 Specific 1omzation (see Ion density) mze, from relative efficroncy of two Sperm (of animals} 129° radiations 90, 122 179 315 326 chromosomes in 162 19] 196, 187 validity of target theory 98-99 srradiation of, 132 141-83 189 199 Telophase, 129* 200 206 209 212 214, 217 218 Temperature 219 227 223 227 228 234 237 affecting action of radiation, 72 154 242, 245 246 247 284, 285 286, 181 246 247 266 290,291 309 288 291 311, 312 329 341 310 312 321 322 336 relative radiosensitivity of sperm and affecting recovery from radiation. eee 295 329 effects 299 291 309 312 size of 151 rise caused by radiation 2 Sperm nucle: (in pollen) 183%, 332 Tormunal defteiency (ofa chromosome), Bpiralzation (of chromosomes) 128 SUBJECT INDEX 415 Tertiary electrons, 27 bactericidal action of, 2, 316, 317, Thiocyanate, 56 318, 319, 321, 322, 327 Thirty seven percent dose, 61°, 74 chemical effects of, 37, 39 and associated volume, 362, 363 chromosome changes by 182 183, and target number, 90 185 216, 223, 242-44, 247 and target size 80-88 d with :onizing radiat: 5, of bactena, 321 8,. 36, 37, 39 124, 125 185-88, of chemical reaction 61-64 202 203 of viruses 109, 110, 116-23 genetical effects of, 181-88 242-44 Three halves power dose relation 156, physical properties of, 4 5, 6 167 158, 232 233 267, 268 cuantum yield 36 37 39 125 327 Time intensity factor, 78, 114 115 viruses, effect on, 112, 115 124-25 6 124 144 145 246, 147, 163 wave length dependence of biological 214, 225-29 241 246 247, 248, effect, 3 182, 188 62-69, 279, 292, 297, 298, 321, 327 Vaccima virus, 101, 106, 173, 115, 118, Tissue 123, 313-16 341, Plates IIa IVb culture 201, 293 295 296 297, 304, Vegetative nucleus (of pollen) 332*, 306 307, 310, 337, Plate _IVz-1 335 Yous faba (bean), 71, 190, 225, 236, electrons per gram in, 347 energy ofa molecule 39 elementary analysis of, 7, 11 energy dissipation in, 8, 22 238, 281, 296, 300, 301 302, 303, X ray absorption in 347, 349 304, 305, 312 335-37 342 Tobacco mosaic virus 100 102 109 Victoreen dosemeter 20 120, 111, 272, 115 116, 125 136, Viruses 140, 143, 144 175 chemical change tolerated by, 175 Tobacco necrosis virus 100, 107, 112 crystalline 100 122 Plate IIp 115, 118 177, Plate Ic = density of, 103 121 Tobacco mngapot virus 118, 177 drying of, 107 Tomsto, 190, 238 electrons per gram in 347 Tomato bushy stunt virus, 100 102, 103, elementary analysis of, 7 107,115, 118 135 177 Plate IID energy dissipation n perr, 8 Tradescantia 67 71 187, 191 193, estzmation of 104-6 Plate IIa B o 198, 199, 200 201, 202 203 204, filtration of 102 103 119 121 206 207,209 210 241 212 213 genes and 101, 121, 124, 136 174 214, 216 217 218 220, 227 224 175, 177 225 226 227 228, 230, 231, 232, hydration of 102, 103 121 233, 235 236 237, 238 239, 240 mutation of 140 141, 143 144 176 241 245-81 308 329 332-35 nature of 100-2 122-24 313 336 342 343 serologically related 136 Translocation of chromosomes 137* shape of Trichophyton mentagrophytes, 183 sizes of 102 103 104, 118 135 136 Tnplod structure of, 314 Plate ]Vp Drosophila 209 ultra violet absorption by 5 endosperm 184 328 X ray absorption by 347 Triton (newt) 299 Viruses mactivation by radiation Trypsn 5 125 caused by single ionization 69 98, Tryptophan 5 101, 111-24 313 Tubp 212 214 of d d Tumours 225 235 236 238, 281 295 115 116 12%, 122 123 314 315 300 343. direct: and indirect action, 63 64 Tyrosme 5 46 47, 49, 58 59 107-11 interpreted 8s lethal mutation 313- Ultra violet hght biol P y ultra violet hght 2 3 124-25 5,182 18% 244 Vitelline membrane (of egg) 5 416 SUBJECT INDEX Water bactericidal action of 216-27 content of, affecting ionlo yield, 62, chemical offecta of, 38-68 chromosome changes by, 189-281, decaraposition by radiation, 36, 37, 32840 delayed division caused by, 282-306 electrons liberated in, per gram, 12 dosimetry of, 6 22 electrons per gram in, fect length of 9 347 electrons libersted by, 10, 12, 12, 13 energy diaspation in, por r, 8 energy dissipation in tissues by 8, 9 X ray absorption coofficients in, 347, 34649 348, $49 genetical effects of, 140-88 Wave length of ultra violet hght, vans lethal offecta of, 307-44 tion of effect with, 3 182, 188 physical properties of, 6-13 Wave length of X rays, variation of relative efficiency of different wave offect with, 47, 72, 115, 116 144, lengths of, 47, 72, 115, 116 144 145, 147, 148, 239, 240, 241, 269, M45, 147, 248, 239, 240, 241 269 270, 273, £276, 276, 277, 280, 322, 270, 273, 276, 278, 277, 280 322 923, 324, 327 925, 324, 327 Wild type, 180° scattering of, 11 348-49 Wilson chamber, 10, 49, 88, Plate I wirus inactivation by, 106-23 wave length distribution 9 € (number of primary chromosome Xenon 37 breaks per r ), 256-62, 260, 270 £ (in target theory), 85, 353 Y-chromosome, 128° X chromosome, 128% Yeasts, 71, 300, 307 X{O males 164 X X/Y (attached X) femates 142 143 Zea mays (maize) 5, 127, 130, 181, 165 183-88, 200 202 205, 219 242 X& rays 243 328 340 absorption of, 8, 240 345-49 Zygote 123°