THE EFFECTS OF THE AGING OF X-RAYED MALE GERM CELLS UPON THE FREQUENCY OF SEX-LINKED LETHALS IN DROSOPHILA MELANOGASTER

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THE EFFECTS OF THE AGING OF X-RAYED MALE GERM CELLS UPON THE FREQUENCY OF SEX-LINKED DETHALS IN DROSOPHILA MELANOGASTER

THESIS

Presented to the Faculty of the Graduate School of The University of Texas in Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY

By Benjamin Bee Harris, 8.5., M.Sc. (Denton, Texas) Austin, Texas June, 1529

INTRODUCTION

Among the problems brought forth by Muller’s discovery (1926-*27) of the production of gene-mutations and chromosome abnormalities by X-rays, was the question of the relative genetic effectiveness of the rays when applied at different stages of the germ cell history, and the related question of the possible influence of aging the mature germ cells, upon the production or detection of these variations. Conclusive data regarding both these questions in so far as they concern sex-linked lethal changes in the germ cells of the adult male Drosophila, have been secured by

the present author, in a series of experiments conducted during the past sixteen months in the genetics laboratory of the University of Texas, at the suggestion and under the direction of Dr. H. J. Muller* The author acknowledges with full appreciation the many helpful suggestions, the patience, the interest and enthusiasm he contributed in making this treatise possible.

TABLE OF CONTENTS

PAGE The Problem • 1 Method •••••••••••. ... . 3 Dosage or Treatment ... ••••••••• 4 Age of Flies •••••••••• .. 4 Mating and Subsequent Handling of the Flies ..... 5 The P Flie s .... J The Flies . 5 The F Flies ••••••••••••• 6 2 Testing for the Loci 7 Technique Used in Production of Lethals from Treated Germ Cells Aged a Specific Length of Time 10 Sterility in the Individual Treated Males ......... 12 Sterility in the Males Grouped in 4 Day Periods ... 14 Sterility in the Matings 14 Sterility in the P and the F Controls 16 1 1 Primary Non-Disj unct ion * 17 Visible Mutations 18 The Individuals Affected Males 19

PAGE The Results by Four Day Periods . . 22 First Four Days .. ♦ 22 Second Four Days ..... 24 Third Four Days .............. 26 Fourth Four Days . ... . 28 Fifth Four Days 30 Sixth Four Days . 31 Induced Lethals as Indices of the ”Age w (Gametogenetic) of the Germ Cells at the Time of X-Raying Them... Group Lethals . 35 The Distribution of Lethal Frequencies in the Affected X-Chromo some of the Treated Group 40 Variation in the Lethal Frequency with Age after Treatment 42 The Time Element in Spermatogenesis of X-Rayed Drosophila (2-4 Days Old) 44 Recapitulation of the Mutant Effects from the 418 Treated Male s 46 Summary of Conclusions 49 Bibliography 72

The Problem

THS EFFECTS OF THE AGING OF X-RAYED MALE GERM CELLS UPON THE FREQUENCY OF SEX-LINKED LET HAI. S IN DROSOPHILA MSLANOGASTER

Investigations on the genetic effects of X-rays upon the germ cells of Drosophila have received more or less attention during the last twenty years, but more particularly since 1915* During the fall of 1926 Muller proved conclusively and repeatedly through his discoveries that X-rays do produce permanent heritable effects in the germ cells of certain treated adult male and female flies.

Many genetic problems have been given birth through X-raying the germ cells of Drosophila. These heritable effects, produced in treated flies, naturally raise questions in regard to the "age" or "stage" of the affected germ cells at the time of X-raying them. Obviously, if all "stages" of the germ cells are present during gametogenesis in adult Drosophila and if all stages are equally responsive to the

induced, effects of the rays and are equally viable later it can be seen that any one affected primordial germ cell would give rise by its division to the greater number of "duplicate" mutations, while the affected mature germ cells would produce one mutation each; however, the latter would be of a greater variety. In either case the frequency would remain uniform so long as the treated sperm were being used in fertilization.

During the fall of *27 and the spring of ’2B, under the direction of H. J. Muller, Oliver and Harris made investigations relative to the mutation frequency of sex-linked lethals in treated flies and the "aging" of the germ cells after X-raying them* The results obtained from this investigation were highly suggestive that the "stage" of the germ cells being treated with X-rays affects the mutation frequency in the case of sex-linked lethals.

The present problem "The Effects of the Aging of Xrayed Male Germ Cells Upon the Frequency of Sex-linked Lethals in Drosophila melanogaster" is the result of the preliminary work done during ’27-*2B in the genetics laboratory of the University of Texas*

Method

The technique used in this study is known as w the CIB method.* Briefly stated it is as follows': Treated male flies are crossed with non-treated *CLB* females; the CIB daughters, now having the treated chromosome of their fathers, are then mated with the brothers. Diagrammatically the scheme is as follows:

GIB ._____ P female X male 1 ' — 1 Sc v f

CIB Sc v f CIB Sc v f p 7 '• Sex ratio 1 Treated" ch. Treated ch. dies because 2 females : „ _ , of *l* male 1 male female female

CIB Sc v f F Q ‘ -4 fernale X < -4 male (the F brother) Treated ch. 1

CIB , CIB Treated ch. F c--~ * dies because dies 2 Sc v f Treated ch. of rt l w ment effects an *l* factor female female

Sex ratio 2 females : 0 males if treatment produced a lethal in P males.

The sex ratio in untreated GIB flies is 2 females : 1 males* However, when CIB females carry a lethal chromosome, received from their treated fathers, and are mated to their P brothers or any other male, they produce no sons, and the sex ratio is 2 females : 0 males.

Dosage or Treatment

The X-ray dosage given the male flies (wild type N ) used in this problem is designated as w TB. nl This treatment consisted of an exposure for 3° minutes at a target distance of 13 centimeters with 1 millimeter aluminum and a current JO K. V. peak, the amperage being 10. The dosage expression is

50 KV - 10 MA 13 CM - 30 Min. 1 MM. aluminum filter

Age of P1 Flies

Throughout this experiment the females had an average age of four days. The males were all between 2 and 4 days old (i. e. after hatching from the puparium) initially, but since they were used throughout the 24 day period of the test and were given a new 4 day old virgin every succeeding 4 days, they were always 4,8, 12, 16 or

20 days older respectively than their female mates.

1 Probably not more than the treatment described in the early experiments of Muller.

Mating and Subsequent Handling of the Flies

The P1 files

The treated males were immediately etherized and mated with non-treated CIB virgins, in pairs and were placed in 1* x 4* shell glass vials, serially numbered and provided with the necessary food. These flies were then called “treated stocks,” and were kept for four days in a constant temperature of 27° 0. At the end of this 4 day period these stocks were removed from the constant temperature incubator. Their F progeny developed at room temperature. The stock GIB females were discarded and the stock “treated males* were given new non-treated virgin CIB females and returned to the constant temperature, 27° C.» under the identical conditions as are described for the cultures of the previous 4 % days. This process was then repeated every 4 days, until six such 4-day periods had been gone through.

The F1 flies

days after the appearance of the first F progeny, from “treated stocks 11 , in the case of each of the four day series, the mating of the CIB females by their (Sc v f

brothers was effected. In each case this progeny was etherized one vial at a time and in every case possible four sister-brother matings were made from each production P -F n vial in the series. This group of four vials, XX £ with the initial serially numbered parent stock vial was then kept at room temperature. At least two days before any of the progeny appeared, the sister-brother parents were discarded by "flipping* them from the vial, thereby preventing the appearance of any parental-type male fly in a vial, except in cases of non-disjunction. j£ach series of the matings was handled in like manner.

The F2 files

The male flies of this groirp, due to the mechanism of sex linked inheritance in the CIB strain of Drosophila revealed (by their absence) any induced lethal X-ray effects because they "inherited* the treated chromosome of their grandfather, which came to them through their CIB mother.

These flies, after a sufficient number had emerged* were carefully examined to see if males were present* These males were noticed particularly to see whether there were few or many. If many males were present the vials were discarded; if few males (one or two) the flies were etherized to see whether they were non-disjunctional (Sc v f like their

N fathers) or plus types like their treated grandfathers). Where these males proved to be plus, the vial was then discarded; where the males all proved to be non-disjunctional, however, the vial was listed as "lethal,” because there were no plus males present. In the case of all the vials showing no plus males they were classed as "lethals” and were placed in a special group of that series. This group of flies was now called "temporary 0. K.’s*, because they showed lethal X-ray effects; these were held at room temperature so long as further "hatching* proceeded. There was no danger of a second generation in these cases. At the end of this time, usually about one week, these vials were "re-examined”; if, still, no males were present then the "testing for the loci” of the lethals was proceeded with.

Testing for the Loci

The locus of a lethal is determined by the identical principle as that used for a visible mutation, namely, by taking the mutant female fly, in which crossing over occurs, and back crossing her. The "recombination value" of the lethal with two known members of its group places it. The GIB females were not used in testing for the locus of the lethals because their "C" factor (inversion) prevents cross- ing over. The wild-type females present with the Bar females in the lethal culture had any lethal chromosome which came from treated males and their composition fulfilled the necessary requirements for studying crossing over. These were mated with scute-vermillion-forked males. Pour pairs of such flies were placed in half pint milk bottles, provided with food of a special composition that allowed the progeny to develop in greater numbers. They were kept at room temperature. Two days before the progeny emerged the parents were removed from the bottles.

Between five and seven days after the emergence of these flies they were identified and recorded, in their respective phenotypic groups.

The following diagrammatic illustration represents complete chronological account of the steps involved in mating and handling the flies after X-raying them®

Mating

After 4 days discard female and mate male to new virgin; and again each 4 Mate Bar females to Scvf days continue the process shown at Remove from culture after 4 days* left with progeny of the second and Inspect and discard non-lethal cultures, subsequent females. Mate plus females from lethal cultures to Scvf males v Remove from culture after 4 days. etherize progeny, classify and record the phenotypes. v Determine the location of the lethal.

Technique Used in Production of Lethals from Treated Germ Cells Aged a Specific Length of Time

Immediately following treatment the males were mated with non-treated virgins, in single pairs. These flies were placed in glass vials where they remained for four days. The female progeny which developed in these vials resulted from the treated X chromosomes of their fathers. Necessarily, then, the eggs which produced these females were fertilized at some period between one and four days after the flies copulated.

All the lethals which resulted from these matings (called the tt A* matings) then were from treated germ cells aged a known length of time, one to four days.

The surviving males (a few were either lost in transfer to the second vials or had died) of the first one to four day period were, immediately upon being removed from the one to four day vials, mated as before with new nontreated virgins, in pairs, for a second four day period. The lethals which resulted from the matings of this second one to four day group were from treated germ cells aged four to eight days (called the matings). The above method applies in arriving at the tt age” of the treated germ cells in the case of the X-ray effects produced in each of the

six series of flies reported in this paper ( W A to P* inclusive, corresponding with the P matings from which they were derived). The following diagrammatic representation gives the exact aging method.



Diagram of Successive Transfers of P Males in Aging Process The letter with the subscript on the vial denotes the identity of the male for that series; the notation under it gives the age of the gem cells after treatment.

Sterility in the Individual P1 Treated Males

Of the 306 treated males surviving the 24 days test twelve proved to be sterile throughout the six (1-4 day) periods. Seven became sterile after the first period, seven after the second, and seven after the third. Seventeen were sterile after the fifth and sixteen were sterile during the sixth (1-4 day) period.

The sterility due to males in the sixth period could not be determined, with certainty, for in half the cases, at least, the females could have been the cause. Eighty-two males, then, or 26.8 per cent of those surviving the experiment, were sterile the whole or a part of the twenty four days. Table I shows the number of sterile males and the duration of their sterility.

The greater frequencies in sterility with the individual males occurred during the first and the last two four day periods# There was a gradual increase in the frequencies between the first and the sixth periods.

Duration No. sterile % sterile No. males surviv- ing the period 1st, 2nd, 3 r <i> 4th, Jth, Sth periods (24 days) 12 2.9 408 2nd, 3rd, 4th, 5th, Sth periods (20 days) 7 1.76 395 3rd, 4th, 5th, Sth periods (16 days) 7 1.85 378 4 th, 5th 6th periods (12 days) 7 1-9 _ _ 359 _ 5th, 6th periods (8 days) 17 5*1 . . 33* Sth period (including female sterility) (4 days) 32 .... 20.8 306

TABLS I

Sterility in the Treated P1 Males Grouped in 4 day Periods

With the exception of the twelve to sixteen day period, during which time there was 1.2% less sterilitjr than in the eight to twelve day period, there was a consistent increase of sterility in each successive period. Ten per cent occurred the first four days and 37 per cent the last four days. Luring the twenty four days (6 periods) the average sterility was 21.3•

Table II shows the sterility frequency of the male 'group" in each four day period.

Per iods 1st 4 day 2nd 4 day 3rd 4 day 4th 4 day 5th 4 day 6th 4 day No. 408 39? 378 359 334 306 No. sterile 43 64 75 65 92 112 X sterile 10 16 19 18 27 37

TABLE II

Sterility in the F1 Matings

Sterility averaged in the matings. The frequency of the sterility showed a marked decrease in all the 1-4 day periods except the third and fourth (the 8-12 and 12-16 day groups). The maximum was attained during the third period.

The source of this sterility— whether mainly due to females or males-- is uncertain* Moreover, since virgins were not used and the amount of virginity may have varied somewhat in the different series (A to F) sterility of the males would have had a different amount of expression as here measured. The frequency of the sterility in the groups in respective 4 day periods follows in table 111.

Periods 1st 4 days 2nd 4 days 3rd 4 days 4th 4 days 5th 4 days 6th 4 days No* vials flies 1355 964 _ __ .996 1070 883 756 No. sterile vials in #2 group 149 78 121 110 60 16 Percent sterile in ?2 group 11.0 8.0 12.0 10 6.7 2.0

TABLE 111

Sterility in the P1 and the F1 Controls

Two hundred and nine pairs were used as controls. Thirty five of them, or 16«7%> produced no offspring. Since they were not further tested for sterility one can not be positive as to the accuracy of their indicated sterility percentage, or whether most of it was due to the males or the females.

Seven hundred and eleven pairs (the females being not necessarily virgin) were used in the experimental control group. Forty-five of these, or 6.3% produced no offspring.

Table IV gives the apparent sterility data for the two control groups.

The control pairs, as was the case with the treated pairs in this experiment, were kept in a constant temperature of 27°&during the + ime they were being used as "control stocks. **

Ho. males No. sterile % sterile 209 35 16.7 Ho. males No. sterile % sterile 711 .. 6.3.

TABLE IV

Primary Non-Disjunction

Porty-two cases of non-disjunctional males occurred in the 2180 vials yielding flies from the treated male parents. These vials yielded, on the average, about 15 flies each, giving a total of flies. The ratio of the exceptional males is about 1:779 or This is somewhat higher than the usual percent of non-disjunction as the CIB females are known to have more non-disjunction than normal females.

There were 6 cases in the first and 6 in the second four day periods. A maximum of 14 was attained during the fourth four day period and a minimum of 2 cases occurred in the sixth, or last, four day period. Table V shows the frequency of non-disjunction in the separate groups.

?_ e _ r _iod 1-4 days 4-8 days 8-12 days 12-16 days 16-20 days 20-24 days No. F, 6020 5925 5670 5385 5010 4590 Ko. non- dis j ♦ 6 6 4 14 10 2 Ratio 1:1020 _ 1:987 1:1417 1:384 l:?01 1:2295 % -09 .11 •07 CM • xO CM • .04

TABLE V

Visible Mutations

V/hile visible mutations were not looked for in this stuuy, due to the great amount of work involved in the technique used for lethals, eight cases were observed. Some of them have been tested for crossover values and their loci approximated. The visible mutation "spectacled eyes” vzas the first case discovered. It is a sex-linked character, an allelomorph of lozenge, which has its locus at 27*7« The eyes of this mutant, judging from the appearance they give under the highest power of a binocular scope, are free of distinct facets, are of a glazed texture, and are of a bright orange color, with a darker border. The visible "spectacled" was observed in the progeny of a CIB female X to a treated normal male.

The second kind of visible mutation found in the F progeny derived from treated males was "white." This mutation occurred three different times in the cultures. Crossing tests proved these cases to be the same white as the sex-linked white from untreated stock; the locus is placed at I.J.

"Bleached", later tested out by Dr. Muller and found to be an autosomal dominant, was the third kind of visible mutation detected. This character affects the eyes of the flies; it gives the eyes a mottled appearance, due to the absence of an even distribution of pigment to the facets.

The fourth, and last, kind of visible mutation detected was "rudimentary wing", another sex-linked character similar to the "rudimentary" long familiar to Drosophila workers. It causes the wing to be about two thirds the length of normal wing. The locus of this fourth "visible" has not yet been determined. Three cultures, with a total of forty-seven such mutant males, "rudimentary wing, were observed#

All the "visibles" found in this study, except possibly Bleached, have also been found by at least one other workex' in the same laboratory experimenting with different groups of treated flies, ana in each of the instances these mutants also had their origin in the effects of Xrays upon the germ cells of their treated progenitors.

The Individual Affected Males

Of the 418 treated males 212 produced the 292 mutants reported in this study. (The mutants are referred to in the course of this paper as lethals, chromosome abnormalities, semi-lethals, and visibles)#

Considering now the males that produced only one mutant in the 24 day period: Fifty seven such males produced their mutant in the first 4 day period, 41 in the second, 26 in the third, 7 in the fourth, 2 in the fifth, and 4 in the sixth 4 day periods respectively. These total one hundred and thirty seven males which produced a single mutant effect.

Thirty-eight males produced only two mutant effects each during the 24 day period* Eighteen of these gave rise to “doubles* (i.e* two mutants each the same 4 day period) as follows: Five the first 4 day period, 5 the second, 6 the third, 1 the fourth, and 1 the fifth periods respectively. Twenty of these two mutant producing males developed theirs as “singles” (i.e. one in either of two 4 day periods) and in the following period sequence: three in the first and second 4 day periods; 6 in the first and third, 1 in the first and fourth, 4 in the second and third, 3 in the second and fourth, 1 the second and sixth, and 1 the fifth and sixth periods respectively.

Seventeen males produced 3 mutants each during the period (24 days) and as follows: Six gave rise to a single and a double respectively in the first and second 4 day periods, 2 effected a double the first and a single the second, 1 a double the second and a single the fourth, 1 a single the second and a double the sixth 4 day period* The remaining three mutant producing males gave rise to them singly as follows: Five in the first, second, and third 4 day periods; 2 in the first, third and fourth; and 1 in the second, fifth, and sixth 4 day periods respectively.

Only 3 males produced four mutants each in the following manner: One gave rise to a single mutant the first, a double the second, and a single the third 4 day periods respectively; another produced a "triple* (i.e. three mutants in one 4 day period) in the first, and a single in the second period; the third male gave rise to a double in the first and a single in each of the two succeeding four day periods.

Two hundred and twelve treated males produced one or more mutant effects during the 24 days (i.e. the 2?2 lethals, JO chromosome abnormalities, 12 semi-lethals, and 8 visibles reported in this treatise)• These are summarized (tables 6 to 11 inclusive) in the discussion of the four day periods following this section.

The Results by Four Day Periods

First Four Days

Ninety-nine lethals or of the total were produced during the first four days of the experiment. Eighty of them are ‘’single cases**; sixteen are “doubles’* and three are “triples.** Eighty-nine males produced lethals during this period. Sixty-two of these males did not produce lethals subsequently; ten of them did. The lethal frequency for the period follows in table VI.

TABLK VI

Periods in Days after X-raying No. of male s 1-4 4-a b-12 12-16 16-20 20-24 57 1 5 2 2 2 1 1 2 1 1 1 3 1 3 1 1 5 1 1 1 3 1 2 1 1 1 2 1 2 1 1 2 1 6 1 1 2 1 1 1 4 1 si 11 1 ChA

Number of lethals produced in each period by all males producing one or more lethals in first period. (Eleven of the lethals are chromosome abnormalities (Gh. A.); four semilethals (si) occurred in this period).

Second Four Days

The percentage of the total lethals is somewhat lower for the second four days because there was a somewhat smaller total production of offspring. Sighty-one lethals were produced during the period, sixty-one of which are "singles* and ten "doubles.*

Seventy-one males produced lethals during the period. Sixteen of these had lethals assigned them during the previous period; three both previously and subsequently; six this period and a later one, and forty-five males produced lethals for their first and last time in the 24 days during the four days of this period.

Table VII gives the summary for this period.

Periods in Days after X-raying Ko. of male s 1-4 4-8 8-12 12-16 16-20 20-24 40 1 3 1 1 4 1 1 5 111 3 1 1 3 1 2 1 - 3 . 1 . . . 1 1 1 1 1 2 1 1 12 1 2 2 1 . 5 2 1 1 2 1 2 11 3 1 (si) 18 1 (ChA) 1 1 (V)

TABLE VII Number of lethals produced in each period by all males producing one or more lethals in the second period. (The visibles (V) are hereafter included in tables).

Third Four Days

Eighty-three X-ray effects were verified during the third four day period. Sixty-two of them were lethals, forty-six of which were "single cases" and eight "doubles." Twenty-one cases represent eighteen chromosome abnormalities and three semi-lethals. The lethals of the periods are 22*3% of the total for the twenty-four day period*

Fifty-four males produced the lethals of this period* Seven had produced lethals previously and one both previously and later. One male produced its first lethal, and a later one, during the period and thirty-one males produced their only lethals for the twenty-four day period.

A summarized frequency of the lethals and other Xray effects follow in Table VIII for this period*

Periods in Days after X-raying No. of male s 1-4 4-8 8-12 12-16 16-20 20-24 25 1 4 1 1 5 1 1 1 2 1 2 1 1 6 1 1 6 2 1 2 1 1 1 1 1 1 1 1 2 1 1 1 4 1 (si) 18 1 (ChA) 1 1 (V)

TABLE VIII Number of lethal s produced in each period by all males producing one or more lethals in the third period*

The Fourth Four Days

Not nearly so many lethal effects occurred during this period as in the previous ones. Only seventeen lethals (6.3% of the total number of lethals) were produced during the third four days; fifteen of them are "singles’* and one is a

Two chromosome abnormalities and one semi-lethal was effected during the period.

Sixteen males produced the lethals of the period. Five of the males had produced lethals in former periods. Kight males produced their first and only lethals for the 24 days during this period. None of the sixteen males which produced lethals in this period did so subsequently.

Table IX summarizes the X-ray effects for the period.

Periods in Days after X-raying No. of male s 1-4 4-3 3-12 12 -16 16-20 20-24 7 1 3 1 1 1 1 1 1 2 1 1 2 1 1 1 2 1 1 1 1 1 (si) 2 1 (ChA)

TABLE IX Number of lethals produced in each period by all males producing one or more lethals in the fourth period.

The Fifth Four Days

Five lethals, three of which are "singles" and one "double", were produced during this period and, in addition, one chromosome abnormality. The lethals which occurred in this period are only 1*9% of the total for the 24 day period. Four males produced them. One male produced a lethal previous to and after the period; two produced their first lethals in this period and subsequent ones in the last, and one male effected its only lethal during this period.

Table X shows a summary of the X-ray effects for the period.

Periods in Days after X-raying No* of males 1-4 4-8 8-12 12-16 16-20 20-24 1 2 1 1 2 1 1 1 1 1 1 1 1 1 (ChA) 1 1 (V)

TABLE X Number of lethals produced in each period by all the males producing one or more lethals in the fifth period.

The Sixth (and Last) Pour Day Period

Due to the fact that two males produced ’‘doubles" in this period the percentage is higher than for the previous period. Hight lethals were produced; two "doubles” and four "singles. "

Six males produced the lethals of this period. Pive of them had previously produced lethals and one had not.

Table XI gives the lethal frequency for the period.

Periods in Bays after X-raying No* of males 1-4 4-8 8-12 12-16 16-20 20-24 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 2 3 I (V)

TABL2 XI Number of lethals produced in each period by all the males producing one or more lethals in the sixth period.

Induced Lethals as Indices of the "Age" (Gametogene tic) of the Germ Cells at the Time of X-raying Them

That mature gametes do not divide and that the early antecedent germ cells which produce them do are well known cytological facts. With this in mind the geneticist is able to approximate fairly accurately, after noting the "grouping and variety" of mutations occurring in the progeny, the probable “stage or age" of the germ cells at the time the mutations had their origin. If the mutations are "duplicated" with great frequency they probably originated in the primordial cells; if they do not have a tendency to be grouped more than a random distribution would lead to, they are of several "varieties," and they probably originated in the mature gametes.

In the 12-16, 16-20, 20-24 day periods there is a much greater tendency for a given male to produce more than one lethal than would be expected in the case of "random assortment;" considering how few males (16 out of 343 the 12-16 day period, 4 out of 328 the 16-20 day period, and 6 out of 300 males the 20-24 day period) give any lethals in these periods* Why is this true? The only plausible explanation seems to be that it is division of the affected

cell (producing the lethals) was therefore immature when X-rayed. By referring to Tables VI-XI it is seen that there is not in the 1-4, 4-8, 8-12 day periods any greater tendency for a given male to produce groups of lethals than would be expected in the case of random assortment. This is true because of the failure to divide of the affected cells (producing the lethals) which were therefore mature.

We shall first consider the loci of all lethals from males which produced groups of lethals subsequent to twelve days, in periods D, K and I*. (See Table XII). Among the offspring of male #22s none were lethal except those present in the 12-16 day period (P) when it is seen that a •‘duplicate“ group of two lethal cultures developed. The loci of these two lethals is 66. This male produced no non-lethal offspring in period H and no offspring in period J.

Male #334 produced a single lethal culture, among his offspring during the 4-8 day period and a "duplicate" group-lethal culture during the 20-24 day period, twelve days later. The locus of the former is 67» and of the latter both were at locus 16.

One male, #2%, produced one lethal in period £ and two in r. The locus of the former is 33 and the loci of the latter are 36 and 33 J it is evident that these are identical (within the range of error of our counts)*

Male produced a lethal in the 16-20 day period and one in the 20-24 day period. The locus of the former is 44 and of the latter 47 (again a sensible identity).

Male #3° produced group lethals the 16-20 day period. The loci of these are 38 and 4? respectively; these were probably identical also as a difference as wide as this is sometimes to be expected with the numbers counted. In the periods A, B, and 0, on the contrary, there is no more identity in the loci of the group lethals produced by individual males than to be expected by random distribution (see Table XII).

The treated males contained both many mature and a few immature germ cells. Probably in both testes together there were (for the males which produced lethals in periods 16-20, 20-24 days) only "four" affected immature germ cells that produced all the sperm of periods D-E, since when there was a lethal, then, on the average one fourth of the treated offspring were lethal* This is borne out by the following facts: There were two "duplicate” group lethals in eight tested offspring from each of 3 males (Nos. 22?, 2JI, 362), three duplicate group-lethals in eight tested offspring from one male (No. 2JI) and one duplicate grouplethal in eight tested offspring from each of two males

(Nos. 362, 251).

Similar "grouping effects”— the duplicate group from an affected immature cell and the variety group from mature cells, — have been reported (by Patterson *2B) for the somatic cells affecting eye facets when Certain Drosophila were treated in the larval stage, and by Muller (*2B) for the mature egg and for the oogonial stage from certain treated adult female flies in his earlier X-ray experiments (1926). Stadler (*2B) found analogous group effects from barley X-rayed in the seedling stage* Grouping effects had also been known before in untreated material (e.g. Baur, 1918; Muller, 1920; Muller and Altenburg, 1921).

Group Lethals

Any group of lethals which have the same locus, and which have come from the same fly, are identified as having had their origin in a primary germ cell and are ’♦duplicates" which resulted from subsequent divisions of that cell, while the several "group* lethals (i. e. lethals from the same male) each having a different locus are identified as having had their origin in that identical number of different germ cells, presumably at a more mature stage* The latter are of a "variety" because their duplication in the

cells which produced them did not occur through subsequent divisions.

An analysis of the loci data assigned to the "group* lethals from certain treated male flies in this study reveals that their loci are identical* An analysis of the loci data assigned the "group* lethals produced by certain other treated males reveals a "different locus* for each lethal in that group. These data when compared then bring to light the fact that the loci of the "group* lethals are identical in the group of one class because they resulted from a treated primordial germ cell and were thereafter duplicated during gametogenesis, and that not one locus in the other "group* is identical to that of any other member of its group, because each of the latter developed from a mature gamete, each of vhich was affected by the X-rays at a different point on the respective X chromosomes, whose division in gametogenesis had been completed. Therefore one concludes that lethals, as well as visible mutations, are reliable indices with which to approximate the gametogenetic stage of the germ cells which produced them, at the time they were X-rayed•

Chromosome abnormality. Sterile.

(The numbers by each male Culture No. of Male give the loci of the group lethals produced in each period). 12-16 days 16-20 20-24 days days 1-4 days 4 Day 4-8 days Periods 8-12 days 7A 49 Ch 8a st - 52 9B Ch 41 19C 1-38 2 2D 20-63 26c Ch-Ch 30E 37-44 35a 33 64-47 42C Ch-. 7 47A 0-42 Uh 48A 15 65 Ch 55a 64-46 19 59 64A st -56 78a St 56 1.4 82B 9-6-34 26 86a 65 ch-58 56 93A 8 49-14

TABLE XII Group Lethals

Culture No. of Male Locus 1-4 Periods 4-8 in Days 8-12 12-16 16-20 20-24 97A 32-50 50 109B 56 Ch 0.0 117A 44 40-st 121A 45 24 143B Ch 40 146a 65 ■ 26 152A 24 6 0 162A 0 Ch 167B 56-5.3 178A 43 2 181B 7-56 191A 59 1 55 195A 19 28 206A 2-0 34 • 27 214A 58-1-46 1-3 216b 60 Ch 219B St 44 221A 23 Ch-64 225B 64-64 240A st Ch 243B 56-57

Group Lethals

Culture No. of Male 1-4 Locus Periods in Days 16-20 20-24 4-8 &12 12-16 24JA 25 12 246A 10 Ch 251il 33 36-3- 2 56a Ch 18 258a 49- Ch 261c 60-7.7 269A Ch Ch £1 289A 71 Ch - 302A 0-34 321A 2-22 s • 329A 2 26 54 18 333c - 20-44 334B - 67 15.6-16.5 335b .Ch ' 24.7 35oa . St-24 70 353B 49 2.2 361c 8 47 362E 44 47 370A 51-2.5 385A Ch 33-40 410A 57 Ch

Group Lethals

The Distribution of Lethal Frequencies in the Affected X-Chromosome of the Treated Group

The distribution frequency of the lethals in the affected treated chromosomes of the group for the twentyfour day period is shown in Figure 1.

The two hundred and one lethals produced on as many treated X chromosomes, after grouping them in the form of a graph, show a variable distribution frequency for the chromosomes. The "peak frequencies" occurred at units 1, 33, and 57; each have sixteen, and twenty-five lethals respectively. These latter places, then, seem to be based upon the number of lethal effects observed, the more responsive points to the effects of X-rays along the X chromosome in Drosophila. The peak at 33 is doubtful, however, as it is lower and with such small numbers there is a tendency to group at loci of markers®

It is clearly seen that all regions of the chromosome are affected. When the induced frequencies are grouped on basis of five units for the entire length of the chromosome, and are plotted in the form of a graph, it is interesting to note that the lowest "induced mutant density" occurs in the left half of the chromosome between ten and fifteen

units from the zero end. The right half of the chromosome shows two low densities (one between 35-40, the other between 50-55) either of which, however, are two and a half times greater than the low in the left half. (Fig. 2).

As a whole, the frequency distribution of the 201 cases is in full agreement with that of the 93 cases reported by Muller (*2B). figures I, A, B, C gives the manner of the distribution of the 201 lethals (whose loci were approximated) reported in this study and that of the 93 cases reported by Muller (*2B), both being recorded in the same fashion here.

Variation in the Lethal Frequency with Age after Treatment

Of the 272 lethals detected during the 24 days 240 occurred the first twelve. The latter number represents 89 percent of the total lethals produced, leaving only 11 percent for the last twelve days, although 46 percent of the offspring tested were produced then. The percentage lethal frequency based on the number of fertile vials offspring for the first twelve days, in four day periods, was 3,9, and 7 percents in the first, second, and third 4 day periods respectively. At the end of the first twelve days there was a decided and significant drop, 7 percent, in the lethal frequency, due no doubt to the explanation that there was used during the first twelve days all but a few of the mature sperm which were present at the time of treatment, and which obviously have a much higher mutation rate than the immature male germ cells. The above explanation is supported by the fact that 12 days, at least, is the minimum time required for affected immature germ cells (primordial cells) to become mature and take part in fertilization.

During the last twelve days, in which 30 lethals only, were detected, the frequency is quite different to that of

the first twelve days. Only 1.7 percent lethals appeared during the first 4 of the last 12 days, 0.6 the second four, and 1.0 percent the third four days, the average percentage frequency being 1.1 percent throughout the last 12 days. As will be pointed out in detail, in a later section, percent of the lethal frequency for this last 12 day period was contributed by only 5 males.

The Time Element in Spermatogenesis of X-rayed Adult Drosophila (2-4 Days Old)

Some cytological investigators state that some of the "early* 1 stages of the germ cells are present and multiply in the testes of adult Drosophila (Stevens, N. M. (1907, 1908))* However, until the data of the present study had been compiled (and disclosed the point now being discussed) from progeny records of individual adult irradiated male flies nothing accurate was known in regard to the "time span’* between an early germ cell stage and its definitive mature spermatozoa. As has been pointed out previously (in this treatise) "duplicate" group-lethals from certain treated individual males appeared as early as, but not previously to, 12 days after treatment, and the majority of such groups appear 16 days (and some 20 days) after the males producing them were treated.

It can now be approximated with certainty ifihich rt early stage (the primordial, the definitive spermatogonial, or the spermatocytic) is responsible for the duplicate mutants that appeared during the 12-24 day period. One may conclude (1) that each of the duplicate groups in question had origin in as many respective affected primordial germ cells because (a) the mutants composing either of the duplicate groups agree in identity; (b) an "early stage" primordium one now in consideration was affected by X-rays) thereafter undergoes many divisions in coming to maturity and develops a great number of spermatozoa-- a number sufficient to give the duplicate group expression, observed in this study.

(2) Since a primary spermatocyte, completing its growth stages, can give rise to only 4 mature sperm and each secondary spermatocyte to only one half the latter number, if either of these stages were affected by X-rays they would not produce a sufficient number of sperm (consider ing the chance they would have in "remaining together" during ejaculation and fertilization) to give significant group effects, like those obtained in this study.

(3) The experimental data obtained in this study thus places 12 days for the probable minimum "time span 11 required by the primordia studied in this way to differentiate into spermatozoa, and for these sperm to be ejaculated, in adult X-rayed flies (2-4 days old)*

Recapitulation of the Mutant Effects from the 418 Treated Males

During the course of this study the "individual" progeny record of 4-18 wild type treated males has been made. Of these, 30& survived a 24 day period test; 212 produced one or more mutants and 4 per cent of them were sterile the whole or part of the time . One hundred and twelve were lost at some time before the last period (due to accident or death)* Four hundred and six of the males produced offspring in one or more of the 4 day periods; 246 of those surviving the 24 day period produced offspring in each 4 day period, while 12 did not give progeny at any time. One hundred and fifty-six produced a single mutant and only 57 produced two or more.

Two hundred and ten males (untreated) were used as controls. One only produced a mutant among the 617 ‘’fertile” F 2 vials belonging to that group*

Five thousand four hundred and ninety fertile F cultures resulted from the treated males, and 210 mutant cultures were obtained therefrom* The crossover values of the latter made possible the determination of each of the 292 mutants described in this treatise.

The mutants observed were chiefly lethals, there being 272 of them. I'ifty chromosome abnormalities, 12 semi-lethals, and 8 visibles account for the total X-ray effects observed. A pronounced majority of these effects appeared during the first twelve days of the 24 day period. The mutants in the experiment as a whole were produced singly or in groups, the latter being of tw-o "kinds*--"variety * groups or "duplicate" groups.

Fifty-five variety groups and five duplicate groups were produced. A majority of the members of the variety groups appeared auring the first twelve days; in only 8 instances did a single mutant belonging to such group appear the 12-16 day period. The duplicate groups appear during the last twelve days. In only one instance did such group appear previously to 16 days after the males producing them were treated with X-rays.

The time of appearance of the two groups is explained on the basis of the stage (gametogenetic) the germ cells responsible for them were in at the time of treatment.

Duplicate groups result from the affected early germ cell stages (which thereafter undergo division in coming to maturity 12-24 days later), while the variety groups result from the germ cells affected in the mature stage (which had finished division)♦

The "kind" of grouping and the "time" the germ cells (responsible for it) give the effect is correlated with the specific "time span" (12-16 days) involved in the germ cell cycle of adult Drosophila.

The first conclusive experimental evidence setting forth the spermatogenetic time span (in the history of Drosophila) in adult flies was obtained through the use of X-rays on the "living organism" (a rather abrupt departure from the usual "killing" and fixing methods used by cytologists) .

SUMMARY OF CONCLUSIONS

The following facts emerge from a careful study of the data concerning the effects of "aging" the treated male germ cells on the frequency of sex-linked lethals:

1. The percentages of mutations found are sensibly identical when the treated mature spermatozoa have been aged zero to four days, four to eight days, or sixteen to twenty days (Table XIII). There is therefore (within these limits no correlation between the production of mutations in spermatozoa and their viability or the duration of their functional capacity. From the above it also follows that the mutational effect of X-rays upon the mature sperm probably is permanent during the life of the sperm.

2. The testes of the adult male Drosophila contain not only mature spermatozoa, but also immature germ cells, some of which undergo repeated divisions in the adult testis before becoming mature. (a) This is proved by the grouping of mutant progeny (Table XII) from the treated males in the case of all the periods subsequent to twelve days, there being much more of a tendency for plural mutant progeny from these periods than a random distribution of mutants among the progeny of the different males would allow.

(b) It is further proved by the identity of locus of the lethals in any given batch in these periods. (c) The figures concerning this grouping show that a single primordial germ cell, present in the adult testis at the time of treatment, multiplies during the ensuing two or three weeks to form, on the average, about half of all the mature spermatozoa then to be found in that testis. Therefore the proliferation of germ cells in the testis probably occurs through a system of one or a very few indefinitely reproducing cells functioning like apical cells, in that they give off, at each division, one cell like themselves and one other daughter-cell which is destined to redivide only a limited number of times.

3. (a) X-rays are far more effective in producing transmissible gene mutations and also chromosome abnormalities in the mature spermatozoa than in the immature male germ cells. This is illustrated by the fact that the frequency of mutations is five to tan times as great in offspring produced during the first twelve days after treatment than during the second twelve days, in the case of males which are allowed to reproduce from day to day (Tables XIV and XVI )• (b) The drop is very sharp just between these two periods, i. e. after twelve days (at 27° C.) (c) After 36

days the absence of significant effects of the rays seems especially pronounced, there having been no demonstrable effect with the numbers used (Table XVI ). (d) There are tvzo possible explanations for the relative ineffectiveness of X-rays applied to the immature male germ cells; it is not yet possible to say which of these (if not both) is correct. On the one hand, it may be that the mature spermatozoa are especially susceptible to the X-ray action, their chromatin being in a more easily affected condition. On the other hand, it may be that immature germ cells in which zygotelethal changes have been produced tend to be killed or to multiply much less rapidly than the genetically normal cells in the same testis (a kind of **germinal selection*), whereas in the case of mature sperm mutations and chromosome abnormalities do not affect the cell viability or functional capacity* (Table XIII)

4* The distribution of mutations in different regions of the X chromosome was found to have the same features as those reported by Muller for the mutations in his experiments* These phenomena therefore depend upon some real and relatively permanent structural peculiarities of the X chromosome. (I’ig* IB and C)

A significant drop in the lethal frequency occurs twelve days after irradiation.

"Time* or aging after irradiation effects a quantitative change in the manner of production of the mutants due to subsequent divisions upon a spermatogonium or earlier germ cell stage*

Twelve to sixteen days is the approximate time span required in the spermatogenetic process in X-rayed adult male Drosophila (2-4 days old)*

FIGURE I. A, B, G

A. Mutations per chromosome unit (spring 1929) Data for each unit recorded separately (Abs* total ® 201 lethals)

B. Mutations per chromosome unit Data for each 5 units grouped together for loci 5 to 70; data for each units grouped together for loci 0 to J. Solid block - No. lethals with JO or more male counts Broken line - No. lethals with fewer than JO male counts (Abs. total = 19J cases (201 less 6 duplicates)

C. Mutations per chromosome unit Muller f s results (spring 1927) Method of recording similar to that in B (Abs. total = 93 cases)

LETHALS COUNTED BUT NOT LOCATED

Due to the presence of parasites no offspring were produced by the parents from lethal culture numbers 40A and 638.

Sterility prevented F offspring in culture numbers 5,8, 641, 78, 240 283, 3501 A, 117HB, 219 and 2J4C. Due to accident, normal bar females were used in the and thereby prevented the determination of the loci of the lethals in culture numbers 117> 207» 238, 249» 259, 265, 279, 3801 and 38OIIA.

1 Right half of chromosome is affected. 2 Right half and left half of chromosome is affected.







Age of germ cells in days after X-raying No. of F -F 2 vials % Lethals Male removed after a single copulation within 1-10 hours after treatment 220 10.0 Males held 4 days without females before mating- 4-8 days 189 9-o Males held 16 days without females before mating- 16-20 days 183 9-o

TABLE XIII Winter 1929 (Males not allowed to copulate before period indicated).

Age of germ cells in days after X-raying Ko . of vials of flies % Lethals 1-4 1206 8.6 4.8 886 9-7 _. 8-12 875 7.3 12-16 960 1-7 16-20 823 0.6 20-24 740 0.8 Controls 617 0.2

TABLE XIV Spring 1929 (Same males mated with successive females every four aays).

Age of germ cells in days after X-raying Ko. of vials of fifes % Lethals 1-4 538 7-0 8-12 377 4.5 Controls 369. 0-3 Age of germ cells in days after X-raying No. of vials orflfes % Lethals 1-12 210? 4-75 12-24 1130 0.85 28-36 208 0.96 36-48 499 0.0

TABLB XV Spring 1928 (Same males mated with successive females every four days)* TABLE XVI Fall 1928 (Same males mated with successive females every four days)*

Culture Number S n vf w Normal S C Vf S C V f S f v Total c Locus 11A 11 3 6 1 21 0 41A 48 8 5 2 63 0 47IA 70 19 19 2 110 0 92A 55 61 1 10 12 139 0 162A 35 13 7 1 56 0 197A 9 2 2 13 0 2061IA 43 3 29 12 1 10 98 0 277A 43 7 7 57 0 302 IA 35 28 13 5 81 0 81 IB 25 7 14 1 47 0 100B 47 37 11 3 98 0 30 9B 30 14 4 4 52 0 103D 44 29 13 2 88 0 1J2B 74 53 32 7 166 0 109IF 15 18 16 1 51 0 1721* 44 26 11 4 85 0 6ob 27 21 10 1 59 0.5 33OD 66 44 1 25 12 148 0.6 181IB 61 40 1 14 4 120 0.7 421 IC 64 38 1 20 3 126 0.7 173A 53 6 35 1 11 4 110 1 19IB 72 38 1 10 121 1

TABLE XVII (This table gives the culture number and series identification; the phenotypic groups and the number of males in each with total; *and the approximate location of the lethal and semi-lethal X-ray effects of the males involved).

LETHALS Culture Number S_vf G Normal S c vf s c v f s f c V Total Locus 214B 22 11 5 1 2 41 1 19IC 34 2 36 12 2 5 91 1 78c 34 19 16 1 3 73 1 49B 57 36 1 9 10 114 1-3 2141IA 110 76 4 30 12 232 1.5 75b 76 20 1 10 1 108 1-5 77B 33 27 1 8 8 1 78 2 178c 68 1 9 11 4 93 2 329A 70 3 45 2 20 12 2 154 2.1 353c 64 2 53 3 12 7 142 2.2 14A 62 3 50 2 20 4 2 143 2-3 206IA 51 37 3 11 15 1 118 2.3 321 IB 55 30 3 15 6 109 2.6 20 9B 65 18 1 8 1 91 3 249C 33 1 10 1 20 1 66 3 175b 63 18 2 21 1 105 3-2 326B 74 4 26 4 25 3 1 137 4.6 393C 45 18 3 20 2 88 4 16711B 43 3 26 3 8 2 85 5 185B 26 2 9 6 3 46 5 39OC 40 19 4 16 1 2 1 83 6

LSTHALS Cult Lire S Q vf Normal S vf Sv f S f v Total Locus Number 1J2C 43 1 22 5 12 2 1 86 7 187c 47 5 17 3 8 3 2 85 7 361c 50 3 14 4 10 1 1 83 8 26111c 27 12 2 9 1 2 53 8 93A 78 2 37 12 28 3 2 162 8.5 821B 48 1 20 8 17 1 2 97 9 54B 60 1 2 41 1 18 5 128 9.6 93IIB 34 1 ,16 11 12 3 3 80 14 140C 60 2 15 10 21 4 4 116 14 48a 51 1 18 14 13 12 100 15 186b 54 12 8 14 14 93 15 3341? 43 4 18 10 16 28 101 15.6 116a 5 232 1 13 16 288c 47 1 16 12 23 1 3 103 16 334II? 43 4 16 14 14 3 2 5 101 16.5 218a 26 769 2 50 18 401B 12 5 5 3 1 26 18 329D 55 4 16 21 25 45 130 18.7 356c 51 16 18 12 2 6 105 18-8 195a 8 12 1 1 13 19

LETHALS Culture Number S vf c Normal s c vf s c v f s c f V Total Locus 24C 61 13 13 24 3 9 123 19*2 55B 37 1 21 28 11 1 2 101 19*4 22IIA 43 1 5 10 8 2 1 70 20 153A 55 10 16 14 11 2 99 20 340A 46 1 13 18 12 3 93 20 96b 37 3 13 18 13 2 1 5 92 20 324C 73 1 13 16 25 1 7 136 20. J 33310 59 1 10 14 33 3 120 20.8 408a 55 3 ii 19 17 1 2 108 21 28b 47 1 ii 20 15 2 96 22 321B 61 3 13 21 10 5 113 22 17D 42 1 6 14 13 1 2 79 23 115A 35 2 6 7 50 24 119A 43 10 25 7 1 5 91 24 152A 28 11 25 7 1 8 8o 24 35oiia 85 11 25 16 3 6 146 24.4 287B 6o 11 22 19 1 1 12 126 24.4 3350 58 3 6 25 10 1 4 5 112 24.7 123 D 73 2 8 21 22 1 2 10 141 24.9 24JA 34 4 14 7 1 6o 25 301A 47 6 8 21 6 5 93 25 8233 65 4 9 39 15 2 6 140 26.J

LETHALS Culture S c vf Normal S c vf S c Y f S c f V Total Locus Number 36a 16 1 5 4 26 27 206c 22 2 4 14 19 4 65 27 382A 43 2 20 12 1 1 2 81 28 195c 53 8 33 22 2 11 129 28 138b 39 1 6 30 20 5 101 28.1 136b 65 1 2 24 17 1 5 115 29.1 341A 82 2 5 35 13 7 144 29.5 176a 53 2 31 7 3 96 31 241D 64 1 2 34 9 1 9 120 31.5 174A 37 2 1 23 9 4 76 32 208IC 59 2 53 17 8 139 32 97IA 48 1 38 12 7 106 32.2 351A 14 7 2 23 33 70B 18 13 12 2 45 33 72B 36 6 4 1 6 53 33 76b 40 9 8 2 59 33 3851B 40 31 3 1 80 33 251a 67 33 14 7 121 33 251IIB 19 1 11 5 4 40 33 821 IB 4-3 24 21 1 1 90 34 206B 44 29 13 1 3 90 34

LETHALS Cult ure Number S_vf Normal S vf Sv f S_f v Total Locus c c c c 302HA 32 1 23 6 1 6 69 35 137c 48 1 29 14 1 1 1 95 36 251U' 54 13 13 1 2 4 87 36 102B 30 23 1 5 59 37 114B 22 10 2 6 2 2 44 37 19IIC 41 21 14 4 3 84 38 30ia 27 21 6 1 1 2 58 38 157B 67 46 14 5 3 135 38.4 385IIB 57 39 3 21 5 8 133 *0 117IB 80 47 3 16 1 1 148 40.3 143D 62 1 1 24 15 8 5 8 122 40.6 9D 56 44 7 5 2 6 120 41.2 4711A 61 29 5 4 5 8 112 42.6 325B 40 32 2 9 3 3 89 43 112A 60 1 26 4 3 1 1 96 44 178A 38 1 25 3 3 1 2 73 44 362a 60 45 4 6 1 4 120 44 117A 37 25 3 4 1 67 45 121A 29 27 1 3 2 4 66 45 333IIC 28 1 4 4 5 1 43 45 30IIE 29 16 5 6 1 57 45 219s 56 — 28 17 11 94 45

LSTHALS Culture Number S c vf Normal s c vf s c v f S c f V Total Locus 55IIA 67 12 30 7 14 4 134 46.2 7A 50 17 2 1 2 72 47 2A 50 1 32 2 7 3 5 100 47 2141IIA 16 13 2 3 1 1 36 47 35HC 50 1 40 2 5 3 3 104 47-3 361I> 57 42 6 8 3 7 123 47.3 3621’ 80 1 41 9 11 6 2 IJO 47.3 355a 61 1 32 5 16 6 7 129 48.2 2J8 IA 9 4 1 2 16 49 93 IB 11 1 10 2 5 1 1 31 49 144C 57 2? 3 12 2 99 49 128D 90 1 36 6 16 3 2 154 49.5 353B 54 1 41 4 12 3 2 117 49.7 97IIA 38 30 3 13 1 2 87 50 97B 50 1 32 4 11 3 101 5o-5 154A 17 7 1 2 1 28 51 370 IA 95 45 1 18 3 4 166 52 8c 52 3 1 36 4 16 5 1 118 52 85ft 55 30 4 16 1 106 52.2 357A 42 1 22 1 7 4 77 55 252B 68 8 4 80 55

LETHALS Culture Number S vf c Normal vf S c V f S„f c V Total Locus 329C 28 1 20 2 10 9 70 55 191C 55 29 1 16 6 107 55*6 64IIA 27 15 5 1 48 56*5 2 84A 9 6 5 2 22 56 • 5 267A 14 9 3 26 56*5 28?A 42 21 9 1 73 56 • 5 323a 29 23 4 2 78 58.5 344A 11 13 5 4 33 56.5 109B 106 60 5 171 56.5 167IB 44 1 24 3 3 75 56*5 181IIB 32 1 16 9 3 61 56.5 243IIB 69 46 5 1 121 56*5 293B 45 37 12 7 101 56.5 320 39 16 4 59 56.5 86c 54 13 8 8 83 56*5 392C 25 20 3 1 49 56.5 159$ 66 47 27 9 149 56*5 78b 75 1 40 14 1 131 56-8 2431B 49 1 44 17 2 113 57.4 126B 52 1 45 14 6 118 57-5 299B 50 2 41 22 8 123 58 196B 55 1 1 28 18 5 108 58-3

LBTHALS Culture Number S c vf Normal S c vf S v c f y V Total Locus 861 IB 56 1 1 29 13 4 104 58.4 2141A 95 1 3 48 22 10 179 58.7 41OA 38 1 16 13 2 70 59 376b 51 3 32 18 8 112 59.1 191A 58 2 30 1 12 3 106 59-2 3*3A 52 3 12 26 2 95 59*7 55c 43 1 1 20 1 15 6 87 59-7 261 IC 48 1 2 39 1 12 9 112 60 198a 59 1 3 24 1 21 3 112 60.9 105b 65 4 1 20 17 2 109 61.2 263A 63 7 1 49 28 9 157 61.5 269c 46 4 2 42 12 6 112 61.8 57A 75 6 1 59 2 16 2 2 163 62.5 345A 69 5 2 26 22 4 1 129 63 22IA 221IIB 35IC 49 53 80 5 4 10 2 3 2 31 22 46 1 1 10 12 9 16 3 5 114 97 152 63-5 64 64.4 225IB 29 3 1 13 1 8 6 61 65 225IIB 25 4 1 21 1 13 7 72 65 86A 79 7 1 30 3 18 4 2 144 65-5 146a 55 9 2 26 20 8 120 65*6 48B 59 5 5 26 11 1 107 65.8

LETHALS Culture Number S Q vf Normal s c vf s c v f V v Total Locus 387A 64 7 3 33 3 21 7 138 65-9 334B 57 9 7 49 13 18 153 66.9 134B 61 6 5 35 5 1 113 67.I 286b 31 6 2 35 2 11 87 68 62A 74 14 4 60 1 5 158 68-6 37C 59 8 6 30 3 26 11 143 68.9 35OB 59 16 10 29 6 1 121 70 289A 16 2 3 9 2 18 1 1 52 71

LETHALS (CHROMOSOME ABNORMALITIES) Culture S vf Normal vf S v f S f v Total Locus Number 0 c c c 35A 33 1 24 4 2 64 R 1 2 5oa 34 1 1 1 37 L & R 202A 20 12 23 L & R 2O5A 124 12 1 12 2 151 1 & R 247A 24 2 1 3 30 L & R 248a 45 1 46 L & R 256A 35 2 3 40 L & R 258IIA 20 1 1 22 L & R 269A 32 3 35 L & R 308A 7 7 14 . R 385A 100 27 2 129 R 7B 64 27 91 R 9B 55 29 64 r 86IB 13 1 2 16 L & R 88b 42 13 2 12 60 L & R 130B 85 3 1 7 96 L & R 143B 4 4 8 R

LETHALS (CHROMO SOO ABNORMALITIES) Culture S vf Normal S vf S v f S f v Total Locus Number c C c 148B 90 1 1 13 105 L & R 201B 46 4 50 R 216B 66 1 1 3 39 110 L 221IB ?8 23 2 83 R 227B 59 17 76 R 240IIB 32 10 1 43 R 268b 17 6 23 R 269B 50 1 51 R 280B 115 2 2 119 L & R 289B 54 6 2 62 L & R 335b 120 111 6 129 L & R 410B 80 24 86 L & R 6c 53 20 73 L 26IC 42 111 3 48 L & R 2611c 103 6 109 1 & R 34C 75 75 L & R 42IC 45 20 1 66 R 47 c 47 1 13 61 R 48c 71 4 4 1 80 L & R 52c 10 2 12 L

LETHALS (CHROMO SOME ABNORMAL IT IE S) Culture Number S n vf w Normal S vf c S v c f s c f v Total Locus ?6C 44 24 1 69 R 120C 78 37 115 R 148C 19 4 23 R 162C 58 1 4 1 1 65 L & R 217C 45 15 2 62 R 246C 25 15 40 R 271C 65 1 2 68 L & R 295C 93 2 1 1 97 L & R 3O5C 56 1 57 R 378C 91 91 L & R 94D 39 1 40 L 21 6b 45 45 L & R 10 9E 27 12 1 40 L

SEMI LETHALS Culture Humber S c vf Normal s c vf S v c f s c f n Total Locus 37OIIA 37 20 2 12 4 75 2 131D 49 8 33 7 16 11 6 4 125 7 246A 65 27 11 6 7 117 9 106C 90 8 24 14 24 5 1 166 11 245C 58 2 22 10 20 1 2 4 119 12 20C 64 4 14 7 11 3 4 107 14 221A 46 6 5 12 12 4 1 3 89 24 32 9B 56 2 10 31 17 5 6 127 26 146C 37 2 9 29 17 1 9 104 27 380B ±7 2 41 10 7 107 58-3 551A 51 3 4 26 1 9 93 65 2°1B 35 2 8 40 4 10 10 1 110 70 Control 79 40 14 11 7 72 0.0

BIBLIOGRAPHY

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Muller, H. J.: "Further Changes in the V/hite-ilye Series of Drosophila and their Bearing on the Manner of Occurrence of Mutation.” Jour, dxp. Zool., 31 > 1920, November, pp. 443-472.

Muller, 11. J.: "The Production of Mutations by X-rays, Proc. Nat. Acad. Sci., 14, 1928, September, pp. 714-724.

Muller, H. J.: "The Artificial Transmutation of the Gene.” Science, 46, 1927, July, pp. 84-87*

Muller, H. J. and Altenburg, N.; "A Study of the Character and Mode of Origin of Eighteen Mutations in the X Chromosome of Drosophila•" Anat. Rec., 20, 1921, December, pp. 213*

Muller, H. J. and Settles, F.: "The Non-Functioning of the Gene in Spermatozoa.” Seitschr. indukt. Ab st* Vererb., 43, 1927? December, pp. 3s9*

Patterson, J. T.: "The Effects of X-rays in Producing Mutations in the Somatic Gells of Drosophila.” Science, 48, 1928, July, pp. 1-7•

Stadler, L. J.: "Genetic Effects of X-Rays in Maize and Barley.” Anat. Rec., 37, 1928, December, pp. 176.