T H E EFFECT OF MATING INTENSITY ON MUTATION FREQUENCY PATTERNS DETECTED AFTER IRRADIATION O F
DROSOPHILA MELANOGASTER MALES1
G. LEFEVRE, JR., AND ULLA-BRITT JONSSON
The Biological Laboratories, Haruard Uniuersity, Cambridge, Massachusetts
Received June 12, 1964
M A N Y geneticists have dedicated themselves to elucidating the relationship between the radiation dose applied to germ cells and the frequency of muta- tions induced by the irradiation. At the heart of the problem has been the endur- ing hope that mutational responses under normal circumstances will always prove to be proportional to dose, so that the amount of genetic damage anticipated from any exposure can be accurately predicted. Stimulated by this philosophy, many estimates have been made of the number of mutations to be expected following exposure of human populations to low levels of radiation, even though the requi- site experimental data are necessarily derived from studies of such organisms as Drosophila and mice. The security of these extrapolations depends in large meas- ure on the validity of the basic data and, further, on the correctness of the pre- sumption that the detection of induced mutations is significantly aff ecteci neither by experimental design nor by variables peculiar to the organism chosen f o r investigation.
Quantitative predictions of genetic damage are sufficiently unreliable to cast doubt on their general usefulness. I n Drosophila, the mutation frequency detected following exposure to a given dose has been reported to be affected by such factors as the age of the male at the time of irradiation, the stage of maturity of the germ cell irradiated, and the sex of the germ cell irradiated. Moreover, ex- ternal conditions, such as oxygen tension, prevailing at the time of the irradiation greatly influence the yield of detectable mutations. (All of these factors are thoroughly discussed in the excellent review of GLASS [ 19551 )
.
A further diffi- culty is that mutation frequencies recorded by different investigators, who pur- port to have studied the same germ cell stages but who have used different procedures, can differ significantly. (Compare, f o r example, Table 3 of OSTER 1961, with Table 1 of TRAUT 1963.) In view of these difficulties, we may well wonder whether standardized experimental procedures will become widely ac- cepted, with the variables recognized and controlled, so that mutation frequencies resulting from a given exposure can be uniformly reproduced by different in- vestigators. Yet, until this occurs, extrapolations of mutational response to radia- tion from one organism to another will remain hazardous at best.This investigation was aided by research grants f" the Public Health Service (GM-07667) and from the National Science Foundation (G-20743).
880 G. LEFEVRE, JR. A N D U.-B. J O N S S O N
It is now our duty to call attention to still one more variable, hitherto largely ignored (see, however, TRAUT 1960), which must be taken into account in Dro- sophila radiation experiments before mutation frequencies detected at successive intervals after irradiation can be properly interpreted. This variable is the fre- quency with which irradiated males mate after exposure.
In a recent paper ( LEFEVRE and JONSSON 1964), the X-ray induced mutability of fully mature, motile sperm irradiated in the male was described as being unsuspectedly high. Nonetheless, when 3-day-old males were irradiated with 4,00Or, high mutability was observed only in sperm samples derived from the first mating after irradiation. The mutation frequency detected among sperm used in the second mating was already noticeably decreased, and by the third successive mating after irradiation, the mutation frequency had dropped to a value only half that observed in the initial mating. By contrast, when 7-day-old males were irradiated, a high mutation frequency persisted through three con- secutive matings after irradiation. (See also MOSSIGE 1963).
Two explanations seem possible for the different mutation frequency patterns observed following the irradiation of 3- and 7-day-old males. First, the intrinsic mutability of fully mature, motile sperm might be approximately twice that of immature, immotile stages and 7-day-old males might have a considerably greater preexisting store of motile sperm than do 3-day-old males. Thus, 7-day-old males, irradiated as virgins, would not utilize sperm that had been immature at the time of irradiation until they had mated more than three times; whereas, 3-day- old males, having only enough fully mature sperm to accommodate one or two inseminations, would necessarily have to use in the second or third mating sperm which were in less mutable, immature stages at the time of irradiation. Alterna- tively, mature sperm might possess a capacity to repair induced genetic damage, but this capacity diminishes with time. Thus, the bulk of the mature sperm in 7-day-old males, unlike those in 3-day-old males, would have become incapable of repairing radiation damage.
In our earlier experiments, following the irradiation of 3-day-old males, three successive matings were obtained as rapidly as possible, so that by the second mating insufficient time may have elapsed to permit all possible recovery from reparable genetic damage. If, instead, the matings had been spaced out after irradiation, then the results would have allowed us to distinguish between the two alternative explanations for the rapid drop in mutation frequency seen after the irradiation of 3-day-old males.
An experiment has now been undertaken in which 3-day-old males, exposed to 4,000r7 were allowed to mate but once each day after irradiation. The sex-linked recessive lethal mutation frequency was determined following each daily mating. The resulting pattern of mutation frequencies differed markedly from that ob- served when irradiated males of similar age were subjected to more accelerated mating programs.
MATERIALS A N D METHODS
MUTATION FREQUENCY P A T T E R N S 881
tion. The second stock, which provided females for mating with irradiated wild-type males, was y2 sc8 f dl-49 U Wa (yellow-&, inversion scute-8, forked, inversion delta-49, vermilion, apricot). All of these markers are sex-linked, and are fully described by BRIDGES and BREHME (19M).
Males to be irradiated were initially collected over a period of 4 to 6 hours from uncrowded cultures. Before irradiation, they were allowed to age for three days in isolation from females. Virgin females used for mating with irradiated males were collected in the same fashion and were also aged three days before use. All flies were raised in an insectary maintained at a
temperature of 25°C and 50 percent relative humidity.
The irradiations were carried out with a Keleket constant-potential X ray machine, operated a t 250 kv and 15 ma and provided with a 1 mm aluminum filter. At a target distance of 46 cm, the dose-rate was approximately 300r per minute. All exposures were timed to deliver 4,000r, and the exposures were monitored before and after irradiation with a standard Victoreen dosim- eter. The flies were placed in perforated size 000 gelatin capsules for exposure, which was carried out at room temperature.
Immediately after irradiation each male was placed in a mating chamber together with a virgin female. Each chamber was observed until the mating was consummated, after which the inseminated female was removed to a culture vial. In the course of the first week after mating, the inseminated females were individually subjected to a series of three subcultures. After mating, each male was left in a culture vial overnight without females, and the next day a single observed mating was again obtained. This procedure was repeated on the third, fourth, and fifth days after irradiation.
TWO additional experiments were carried out in which 3-day-old males were exposed to the same irradiation dose, but afterwards were stimulated to mate at more frequent intervals. In the first of these experiments, each male, following irradiation, was confined with a harem of
four virgin females for a period of 24 hours, and new harems were provided on the second, third, fourth. and fifth day after irradiation. The females that constituted a harem were separated from each other after removal of the male, and each female was thereafter subcultured individ- ually. In this manner, it was possible to determine how many females had actually been in- seminated by each male. In the second experiment, each male was successively provided with virgin females in order to obtain, as quickly as possible, a series of observed matings after irra- diation. Males that successfully accomplished three consecutive matings (which ordinarily re- required about 2 hours) were then left in a culture vial with a harem of four virgin females for the remainder of the first 24 hours after irradiation. On the second day, the males were again provided, consecutively, with three virgin females for observed matings, but then they were stored overnight without females. On the third and fourth days after irradiation, this procedure was repeated. Rather than being subjected to observed matings on the fifth day, however, the males were left with a group of five females f o r a final three-day mating period.
We should emphasize that the flies were not etherized in any of the manipulations, including the irradiations and matings. Further. all of the resulting F, female progeny from each sub- culture were tested, insofar as possible, by the usual technique f o r the presence of newly induced sex-linked recessive lethal mutations.
RESULTS
882 G . LEFEVRE, J R . A N D U.-B. J O N S S O N
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MUTATION FREQUENCY PATTERNS 883
16 14
12
10 8
6
1 2 3 4 5
Days a f t e r I r r a d i a t i o n
1 2 3 H 1 2 3 1 2 3 1 2 3
----
1 2 3 4 5 t o 7 1 2 3 4 5 t o 7
Days a f t e r I r r a d i a t i o n
FIGURE 1 .-Experiment 1 : Sex-linked recessive lethal mutation frequencies detected in single daily observed matings after irradiation of 3-day-old males with 4,000r. FIGURE 2.-Experiment 2: Sex-linked recessive lethal mutation frequencies detected following daily matings with harems of four virgin females of 3-day-old males irradiated with 4,000r. FIGURE 3.-Experiment 3: Sex- linked recessive lethal mutation frequencies detected following successive observed matings each day of 3-day-old males irradiated with 4,000r. Irradiated males were left with harems of four females overnight (H) following the first three successive matings on Day 1, and were left with harems of five females for a final three-day mating period beginning o n Day 5. FIGURE 4.- Patterns of mutation frequencies detected on successive days following irradiation with 4.000r of 3-day-old males subjected to three different intensities of mating after irradiation.
884 G. LEFEVRE, J R . A N D U.-B. J O N S S O N
TABLE 2
Sex-linked recessiue lethal mutation frequencies detected following obserued matings each day
of 3-day-old males irradiated with 4,000r (Experiment I l l )
Davs after irradiation
1" 2 3 4 5-79
31 109 60
166
1149 507 1553 717
107 8 65 39
965 119 1064 525
15
267 27 40 1 138
57 174
467 21 93
465 41 202 114 57
ffi7 - 8.4%
lstmating -=14.4% -- -6.1% -= 7.0% -- . . .
- 7.4%
2ndmating - = 11.1% -- -6.7%
--
-6.1%--
. . .3rdmating -- . . .
Overnight --
Total -=10.2% --
- 7.0% -= 10.9%
2 28
-6.7% -= 7.4% -- 18
-_
- 7.9% . . . . . . . . . - 12.2x.f
- 6.7% - = 8.3% - = 12.2% -6.3% --
45 74 653 3018 1380
879
Total all days: - = 8.71 %
*
0.28% 10092* Data for Days 1 and 5-7 taken from LEFEVRE and JONSSON (1964)
+
3-day mating period.DISCUSSION
1. Are mature sperm capable of repairing induced genetic damage? In all three of our experiments, described above, virgin 3-day-old males were exposed to an X-ray dose of 4,000r. Thus, we might well have expected the same result from each experiment. Yet, as Figure 4 clearly indicates, quite different patterns of mutation frequencies appeared. That mutation frequencies can be strikingly affected merely by the intensity of mating demonstrates that differential radio- sensitivity of germ cells in different stages of maturity, not recovery from the genetic effects of irradiation, is responsible for the inconstancy of mutation fre- quencies detected at various intervals after irradiation. If repair of genetic damage had produced the rapid decline in mutation frequency observed when 3-day-old males mated in quick succession after irradiation (see Figure 3 ) , then, when males were permitted to mate but once a day, a minimum mutation frequency would have occurred in the second mating after irradiation. But this was not observed: when the second mating took place 24 hours after irradiation, the muta- tion frequency was at an intermediate value, not at the low value reached only on the third day (see Figure 1 )
.
Obviously, the mutation frequency found at any particular time after irradiation is more closely correlated with the number of sperm that have been discharged since the exposure than with the number of hours that have elapsed.MUTATION FREQUENCY PATTERNS 885
inseminations, but caution must be used before equating the mutation frequency detected in a given sperm sample with the mutability of a particular stage of spermatogenesis. I n the male, sperm are neither conveniently pre-packaged in quantities appropriate for individual insemination, nor lined up in order of maturity so that they will be used in precise sequence in successive matings. Rather, as may be seen in dissections of males of different ages, sperm mature in elongated bundles of 64 (COOPER 1950) that lie in large numbers side-by-side i n the testes. As the terminal step of their maturation, the sperm become motile; then they wriggle loose from their respective bundles and intermingle in one writhing mass in the seminal vesicle to await the moment of discharge. AS a result, the “unit” of sperm transfer during mating (upwards of 4,000 sperm by previously unmated 6-day-old males, according to KAUFMANN and DEMEREC 1942) is a composite of many “units” of sperm maturation (bundles of 64). However, the female can store only 600 or 700 sperm, no matter how many are transferred by the male ( LEFEVRE and
JONSSON
1962).The number of sperm collected in the vesicles of an unmated male progres- sively increases with time, beginning shortly after emergence. Thus, maturation must proceed at a relatively steady pace. Even though 3-day-old males have accumulated several thousand motile sperm in the vesicles, augmented by many more in the proximal ends of the testes, the ability to transfer sperm to a female rapidly declines after two successive matings (LEFEVRE and JONSSON 1962), at which time the seminal vesicles are seen to be noticeably contracted. Still, in dissections, sperm can always be found passing into the vesicles, with their long tails extending back through the testicular ducts into the testes. Thus, the popu- lation of sperm in the vesicles slowly but steadily increases by the addition of newly matured sperm, but intermittently the accumulated population is deci- mated by ejaculation.
If irradiation could be accomplished instantaneously and if irradiated males would inseminate females immediately after exposure, then the mutation fre- quency obtained from the first sperm sample would reflect the mutability of fully mature, motile sperm. This ideal situation can only be approached in practice. I n our experiments, the 4,000r exposures required approximately 13 minutes, and placing all of the irradiated males in individual mating chambers with virgin females took nearly half an hour. Then, the males, even though they had not been etherized, required time to acquaint themselves with the opportunities put before them, some being more opportunistic than others. Finally, the time actually spent in mating averaged about 20 minutes. Without doubt, even in the first mating after irradiation, some sperm samples contained cells that reached full maturity only after the time of irradiation.
886 G. LEFEVRE, JR. A N D U.-B. J O N S S O N
effect should be less pronounced, but still demonstrable. By contrast, the accumu- lation of fully mature sperm in 7-day-old males is so great that dilution of the initial sperm sample by cells that were immature at the time of irradiation would be scarcely noticeable.
Depending on the magnitude of the initial store of mature sperm, which in turn depends on the age of the male, continued matings after irradiation will result in the eventual depletion of all sperm that had been fully mature at the time of irradiation. Thereafter, matings will provide sperm samples derived only from cells that had been immature at the time of irradiation. Judging by the shape of the mutation frequency curves in our experiments, we believe that two matings are sufficient to exhaust virtually all of the mature sperm present at the time of irradiation of previously unmated 3-day-old males. As earlier experi- ments showed, (LEFEVRE and JONSSON 1964), two days of mating are required to use up all of the mature sperm initially present in irradiated 7-day-old males. Further mating will produce, in due course, samples of sperm that were in even earlier stages of spermatogenesis at the time of irradiation. Beginning on the fourth day after irradiation of 3-day-old males, unless they have mated at a very slow place, cells that had been in the early spermatid stage at the time of irradiation will be sampled. Early spermatids, which should not be confused with the immature, immotile sperm stages described above (which are, in fact, late spermatids)
,
have long been known to be especially mutable (BONNIER and LUNING 1950; LUNING 1952a,b; AUEREACH 1954). When, on the fourth day after irradiation, sperm derived from early spermatids become available, once again nonhomogeneous sperm samples appear in which some of the sperm in a single insemination will be derived from less mutable immotile sperm stages and others from more mutable early spermatid stages.Following irradiation of an adult male, homogeneous sperm samples derived exclusively from irradiated early spermatids are as difficult to obtain as are homogeneous samples of sperm that were fully mature at the time of irradiation. Were the problem merely that of obtaining homogeneous samples of irradiated early spermatids, then pupal stages rather than adults should be exposed, as OSTER (1958, 1959) has pointed out; and uncontaminated samples of irradiated fully mature sperm could be conveniently obtained by exposing inseminated females. However, mutation frequencies that are obtained with such procedures may not provide the best evidence for the existence of intrinsic differences in the mutability of germ cells at different stages in their maturation because the pupal testis and the seminal receptacle of an adult female do not provide the same environment for irradiation as the adult testis. Thus, the best comparison of relative mutability results from the simultaneous irradiation of all germ cell stages in a common environment, as occurs when adult males old enough to have accumulated mature sperm are irradiated. Even then, the mating schedule must be nicely adjusted to the rate of sperm maturation if sperm collected from succes- sive inseminations are to consist of relatively pwre samples of, first, mature sperm, then immotile late spermatid stages, and finally early spermatids.
M U T A T I O N FREQUENCY PATTERNS 887
ments, the mutation frequencies observed in the initial mating after irradiation were 12.0, 12.9, and 14.4 percent, respectively. We should note that TRAUT
(1963), using a procedure similar to that of our second experiment, reported a first-day sex-linked lethal mutation frequency of 12.3 percent, following the irradiation of 3- to 4-day-old males with 4,000r. In all probability, none of these values reflect the mutability of completely homogeneous samples of sperm that were fully mature a t the time of irradiation. This belief is supported by the fact that in our earlier experiment, in which 7-day-old males were irradiated (LE-
FEVRE and JONSSON 1964), the mutation frequency found in the first mating after
irradiation with 4,OOOr was 16.5 percent, and in the second, 16.9 percent. We suggest that values of this magnitude provide a better estimate of the mutability of fully mature sperm than do values found immediately after the irradiation of 3-day-old or younger males.
I n order to determine whether the mutability of fully mature sperm irradiated in the male is directly proportional to the irradiation dose, we undertook a sup- plementary experiment in which 7-day-old males were exposed to 2,500 and to 5,000r. The incidence of sex-linked recessive lethal mutations was determined from only the first and second matings carried out immediately following irra- diation, and the resulting mutation frequencies were compared with that observed in earlier experiments using 4,000r. The data are presented in Figure 5. The linearity of the mutational response of mature sperm to irradiation dose is clearly evident, and indeed shows no evidence of the saturation effect that might well be expected at higher dose levels (MULLER 1954). Neither does it show an elevation
20
; 10
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D o s e in k r
888 G. LEFEVRE, J R . A N D U.-B. JONSSON
above a direct proportion to dose that might be expected from the production of two-hit lethals as higher doses. Presumably, the two effects, acting in opposite directions, balance one another. We might note that earlier investigators, who failed to take precautions to obtain homogeneous sperm samples, have on occasion obtained dose curves that departed from linearity (see, for example, EDINGTON 1956). Our results indicate that following exposure of adult males, the frequency of sex-linked recessive lethal mutations induced in motile sperm is approximately 4 percent per 1,000r. This value is appreciably higher than has previously been attributed to mature sperm by TIMOFEEFF-RESSOVSKY and ZIMMER ( 1939), SPENCER and STERN (1948), MULLER (1950), and IVES (1959), all of whom recorded values of less than 3 percent per 1,000r. On the other hand, mutation frequencies detected following the irradiation of inseminated females are high (OSTER 1958, 1959, 1961; TRAUT 1963), but no higher than those characteristic of the first mating after the irradiation of adult males three or more days old. The mutability of mature, motile sperm is in fact not affected by their location at the time of irradiation, but an accurate estimation of their mutability, follow- ing irradiation of adult males, can be made only if care is taken to obtain “pure” samples of mature sperm, uncontaminated with sperm derived from less mature stages.
4. What is the mutability of immature, immotile sperm stages? An estimate of the mutability of immature, immotile sperm stages can be derived from the mutation frequency exhibited at the low point of the U-shaped muation curve produced after irradiation. In our third experiment, this occurred on the second day after irradiation, but not until the third day in the other two experiments in which the males mated less frequently on the first day. We consider that a muta- tion frequency of approximately 6.5 percent represents a fair estimate of the mutability of immature sperm stages exposed to 4,000r. This, of course, indicates a mutability of only about 1.6 percent per I,OOOr, which is a remarkably low value for the mutability of sperm that in the older literature have usually not been separately distinguished from sperm that we have described above as “fully mature.” We may be sure that, except when inseminated females were irradiated, earlier values relating to the mutability of “mature” sperm reflect the mutability of nonhomogeneous mixtures of motile and immotile sperm stages, which actually have quite different mutabilities.
5. What is the mutability of early spermatids? I n our experiments, the mu- tability of early spermatids, as reflected by the mutation frequencies obtained five to seven days after irradiation did not exceed that of fully mature sperm. I n contrast, TRAUT (1963) recorded a mutation frequency of more than 18 per- cent on Day 5 , following an exposure of 3- to 4-day-old males to only 2,000r. In all probability, a dose of 4,000r destroys the earliest, most mutable spermatid stages. Thus, no one value can reflect the mutability of all early spermatids. There must be, in fact, a spectrum of stages, which have been illustrated by COOPER
M U T A T I O N F R E Q U E N C Y P A T T E R N S 889
Even then, doses of 1,000r or less would be required to assure the survival of the most sensitive early spermatids.
6. Is the overall frequency of detected mutations modified by mating intensity? Despite the fact that the mutation frequency patterns differed according to the intensity with which the males mated after irradiation, the total mutation fre- quency over the entire five to seven day mating period was remarkably similar in our three experiments, being 8.9,9.6, and 8.7 percent. A test for independence of these three values demonstrates their homogeneity
(x'
= 2.3, d.f. = 2, p = 0.3). Obviously, the total initial population of irradiated germ cells, both mature and immature, will eventually be sampled whether the males mate rapidly or slowly. If all progeny produced during the first week after irradiation are tested for the presence of newly induced mutations, the overall mutation frequency should be unaffected by the mating intensity. This would not be true if males were mated for only a limited time after irradiation, for then the entire post- meiotic population of germ cells would not be sampled. In such a case, the result- ing overall mutation frequency could be appreciably different from that found when matings were continued throughout the entire first week after irradiation. Because different investigators have not followed uniform procedures in con- ducting their mutation experiments, it is not surprising to find in the literature quantitatively different values for the incidence of mutations induced by given irradiation doses.PFABODY, and MRS. DOROTHY M. PARKER.
We wish to acknowledge the expert technical assistance of MISS BERIT NILSSON, MRS. ANN
S U M M A R Y
890 G . LEFEVRE, JR. A N D U.-B. J O N S S O N
higher than) that characteristic of mature sperm as germ cells irradiated in the highly mutable early spermatid stage come to be sampled.
Although the pattern of mutation frequencies is strongly affected by mating intensity following irradiation of adult males, the overall mutation frequency is not, SO long as the entire population of postmeiotic germ cells is sampled.
LITERATURE CITED
AUERBACH, C., 1954 Sensitivity of the Drosophila testis to the mutagenic action of X-rays. Ind. Abst. Vererb. 86: 113-125.
BONNIER, G., and K. G. LUNING, 1950 X-ray induced dominant lethals in Drosophila m e h o - gaster. Hereditas 36 : 4 4 5 4 6 .
BRIDGES, C. B., and K. S. BREHME, 1944 The mutants of Drosophila melanogaster. Carne& Inst. Wash. Publ. 552.
COOPER, K. W., 1950 Normal spermatogenesis in Drosophila. pp. 1-61. Biology of Drosophila.
Edited by M. DEMEREC. Wiley, New York.
EDINGTON, C. W., 1956 The induction of recessive lethals in Drosophila melanogaster by radia- tions of different ion density. Genetics 41 : 814-821.
GLASS, B., 1955 Differences in mutability during different stages of gametogenesis in Drosophila. Brookhaven Symp. Biol. 8 : I#-1 70.
IVES, P. T.. 1959 The mutation rate in Drosophila after high doses of gamma radiation. Proc. Natl. Acad. Sci. U.S. 45: 188-192.
KAUFMNN. B. P., and M. DEMEREC, 1942 Utilization of sperm by the female Drosophila melanogaster. Am. Naturalist 76: 445-469.
LEFEVRE, G., and U.-B. JONSSON, 1962 Sperm transfer, storage. displacement and utilization in
Drosophila melanogaster. Genetics 47: 1719-1736. -- 1964 X-ray induced mutability in male germ cells of Drosophila melanogaster. Mutation Research 1 : 231-246.
X-ray induced dominant lethals in different stages of spermatogenesis in Drosophila. Hereditas 38: 91-107. - 1952b X-ray induced chromosome breaks in
Drosophila melanogaster. Hereditas 38: 321-338.
Differential yields of mutations from the first and second matings after irradiation of mature sperm in Drosophila melanogaster. Repair from Genetic Radiation Damage. pp. 253-274. Edited by F. H. SOBELS. Pergamon Press, New York.
Radiation damage to the genetic material. Part I. Effects manifested 1954 The manner of mainly in the descendants. Am. Scientist 38: 33-59, 126.
production of mutations by radiation. Radiation Biology. Vol. 1 : 475-626. Edited by A. HOLLAENDER. McGraw-Hill, New York.
MULLER, H. J., I. H. HERSKOWITZ, S. ABRAHAMSON, and I. I. OSTER, 1954 A nonlinear relation between X-ray dose and recovered lethal mutations in Drosophila. Genetics 39: 741-749. OSTER, I. I., 1958 Radiosensitivity. Genen en Phaenen 3: 53-66. ~ 1959 The spectrum
of sensitivity of Drosophila germ cell stages to X irradiation, pp. 253-267. Radiation Biology.
Proc. 2nd Australasian Conf. Rad. Biol. Edited by J. H. MARTIN. Butterworths Scientific Publications, London. - 1961 On recovery in X-irradiated germ cells. J. Cell. Comp. Physiol. 58 (suppl. 1) : 203-207.
SPENCER, W. P., and C. STERN, 194.8 Experiments to test the validity of the linear r-dosehuta- tion frequency relation in Drosophila at low dosage. Genetics 33 : 43-74.
TIMOFEEFF-RESSOVSKY, N. W., and K. G. ZIMMER, 1939 Mutationauslosung durch Rontgenbe- strahlung unter verschiedener Temperatur bei Drosophila melanogaster. Biol. Zbl. 59 :
358-362.
TRAUT, H., 1960 Uber die Abhangigkeit der Rate Strahleninduzierter Translokationen und rezessiv Geschlechtsgebundener Letalfaktoren vom Stadium der Spermatogenese bei Dro- sophila melanogaster. Z. Vererb. 91: 201-205. ~ 1963 Dose-dependance of the fre-
quency of radiation-induced recessive sex-linked lethals in Drosophila melanogaster, with special consideration of the stage sensitivity of the irradiated germ cells. pp. 359-372.
Repair from Genetic Radiation Damage. Edited by F. H. SOBELS. Pergamon Press, New York. LUNING, K. G., 1952a
MOSSIGE, J. C., 1963
MULLER, H. J., 1950
