Studies on mutagen-sensitive strains of Drosophila melanogaster

49

Mutation Research, 166 (1986) 49-57 DNA Repair Reports Elsevier

M T R 06154

Studies on mutagen-sensitive strains of

Drosophilamelanogaster

IX. Modification of genetic damage induced by X-irradiation of spermatozoa in N2, air or O2 by 4 autosomal repair-deficient mutants W. Ferro Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, P.O. Box 9503, 2300 RA Leiden, and J.A. Cohen Institute, Interuniversity Institute for Radiopathology and Radiation Protection, Leiden (The Netherlands) (Received 18 October 1985) (Revision received 13 December 1985) (Accepted 18 December 1985)

Summary The influence of defects in D N A repair on the recovery of X-ray-induced genetic damage in spermatozoa of Drosophila melanogaster was studied. Basc males were irradiated in N 2, air or 02 and mated to females of 4 repair-deficient mutant types: mus(2)201 D1 (excision repair deficient), mus(3)306 TM (excision repair deficient), mus(3)302 D1 and mus(3)308 D2 (both excision repair and post-replication repair deficient). The frequencies of sex-lined recessive lethals and of autosomal translocations in the F 1 progeny were determined following standard genetic procedures. The responses in the different crosses with repair-deficient females were compared to those with repair-proficient mei + females (maternal effects). The main findings are the following: (1) with excision repair-deficient females the frequencies of spontaneous recessive lethals tend to be higher than with mei + females; (2) with excision repair-deficient females the frequencies of recessive lethals induced in N 2 and air and often in 02 are higher than with mei + females; (3) with post-replication repair-deficient mutants a maternal effect is found for X-ray-induced translocations - - both increases and decreases occur depending on the specific mutant type. The data are explained as follows: excision repair deficiencies cause the processing of primary lesions to be diverted from the error-free excision repair to the error-prone post-replication repair pathways. This results in enhanced mutational yields. After irradiation in O: secondary effects cause selective elimination of potential recessive lethals in those mutants that exhibit lowered fertility (mei-9, mus-302). Therefore those mutants have no differential maternal effect on the recovery of recessive lethals after irradiation in 0 2.

The changes in translocation yield with post-replication repair deficiencies are thought to be the result of defects in the repair of D N A breaks. These defects might cause the post-replication repair deficiency too. The group of post-replication repair mutants is heterogenous. The mutants with low fertility seem to cause decreased translocation frequencies by selective elimination through dominant lethality. The other mutants increase the frequencies.

This paper is dedicated to the memory of my good friend and fellow scientist Dr. N o r m a n K. Todd (Exeter, Great Britain) who died unexpectedly in October 1985. 0167-8817/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

50

Several earlier studies have documented the utility of repair-deficient strains of Drosophila in the analysis of the role of maternal repair processes that act on damage induced in male germ cell stages (e.g. Graf et al., 1979; Eeken et al., 1982; Ferro, 1983; Ferro and Eeken, 1985; Sobels et al., 1984). Both meiotic ("mei") as well as the mutagen-sensitive ("mus") mutants with different repair defects (as characterized biochemically with respect to UV-induced damage in somatic cells) have been used in these investigations. The results that have so far emerged have shown that the relationships between repair defects identified at the biochemical level and their influence detected in "maternal effect" studies on genetic damage induced in post-meiotic male germ cells are complex and variable and do not, as yet, permit unified interpretations. Since, in most of these studies, only a limited number of mutants have been tested (i.c. excision repair-deficient mutant mei-9 and post-replication repair-deficient mutants mei-41 and mus-lO1 (see Table 1); all of these are X-linked mutants) it was thought useful to extend these studies to 4 additional mutants, namely, mus(2)201 TM, mus(3) 302 TM, mus(3)306 D1 and mus(3)308 °2. The results are reported in this paper. Materials and methods

All 4 mutants selected for the present study are autosomal ones and known to be deficient in excision repair of UV damage although to different extents (Table 1); besides, mus(3)302 TM and mus(3)308 D2 also show defects in post-replication repair after UV irradiation. They were originally isolated in EMS mutagenesis experiments in which, following their isolation, they were tested for hypersensitivity to different physical and chemical mutagens. In cn mus(2)201 D1; e the mutant allele is located on chromosome 2, at map position 23; and the strain shows hypersensitivity to killing in the larval stages by MMS, HN2, UV and slightly by X-rays (Smith et al., 1980; Boyd et al., 1982). In st rnus(3)302 TM, it is located on chromosome 3 at position 45 and this strain also shows hypersensitivity to larval killing by MMS, HN2, UV and X-rays (Boyd et al., 1981; Boyd and Harris, 1981).

TABLE 1 DNA REPAIR PROPERTIES OF SEVERAL MUTAGENSENSITIVE MUTANTS FOR U V - I N D U C E D DAMAGE After Boyd and Harris (1981) and Boyd et al. (1983). Mutant locus

Excision repair capacity (wild type - 100%)

Post-replication repair

mei-9 mei-41 mus-lO1 mus-201 mus-302 rnus-306 mus-308

0% Normal Normal 0% 72% 47% 24%

Normal Defective Intermediate Normal Defective Normal Intermediate

In rnus(3)306 TM the mutant allele is localized to map position 56 on chromosome 3; the larvae of this strains while hypersensitive to killing by MMS and X-rays, show normal sensitivity (relative to wild type) to HN2 and UV (Boyd et al., 1981: Boyd and Harris, 1981). Finally in mus(3)308D2, the mutation is at map position 55 on chromosome 3; the strain is hypersensitive to killing in the larval stages by HN2 and UV and not by MMS or X-rays (Boyd et al., 1981; Boyd and Harris, 1981). A repair-proficient strain with an X-chromosome marker yellow ( y ) served as control. For the experiments, strains were used in which (1) the X-chromosomes of these mutant strains were replaced by y-marked X-chromosomes from the wild-type (repair-proficient) control; (2) the markers bw and st from the stock y sc sl In-49 sc~; b w ; st pP were introduced into the mutant strains (and cn and e removed from mus(2)201) using standard genetic techniques; and (3) the presence of the respective mutant alleles confirmed in MMS or bis-2-(chloroethyl)amine, chloride (an analogue of HN2) larval sensitivity tests. The genetic backgrounds of all these derived strains are similar (except for the presence of the respective mutant alleles) to those used in earlier experiments (Ferro, 1983; Ferro and Eeken, 1985) although the strains are not isogenic. These substitution stocks are hereafter designated as mus-201, mus-302, mus-306 and mus-308 and the control (y-marked) stock, as mei +. The methods used to study maternal effects

51

were basically the same as those used in earlier experiments (Ferro, 1983; Ferro and Eeken, 1985). Briefly, 2-3-day-old Muller-5 males (genetic constitution: In(l) SCS1L S¢ 8R -~- S , s ¢ S1 s ¢ 8 w a B) were X-irradiated in N 2 (3000 R) or in air (1500 R) or in 02 (1500 R) following a 20-30 min pretreatment in the respective gaseous atmosphere. The rationale for irradiation in different gases is based on the finding that different degrees of oxygenation present during irradiation result in qualitatively different types of damage (e.g. Ferro, 1983; Sobels, 1964a, b, 1969). The irradiated males were mated en masse to repair-deficient and repair-proficient females. After 1 day the males were discarded and the females were subcultured for 2 - 3 days. The F~ females and males were used in standard sex-linked recessive lethal and translocation

tests, respectively. In translocation tests, the F~ males were mated to virgin females obtained "automatically" from a stock y+ 1 6 3 T / C ( 1 ) D X y f; b w ; s t p P , in which the males do not hatch if kept at 25°C. Air- and oxygen-enhancement ratios (AER and OER) were calculated for induction of sex-linked recessive lethals. On the assumption of linearity, the induction rates per 1000 R were used for the respective gaseous atmospheres to calculate these ratios. A E R = induction rate in air/induction rate in N 2. OER -- induction rate in O2/induction rate in N 2. X-Irradiations were administered by an ENR A F machine operated at about 110 kV and 12 mA at an exposure rate of approximately 40 R/see. All exposures were monitored with a PTW Dosimentor system.

TABLE 2 F R E Q U E N C I E S OF SEX-LINKED RECESSIVE LETHALS IN S P ER MA TO ZO A OF Muller-5 MALES I R R A D I A T E D IN N 2, A I R OR 0 2 A N D M A T E D TO mei+ OR M U T A N T FEM A LES P values given for H 0 of no difference between mei + and mutant females. Treatment of males

Number

Number

Number

Freq.

Number

N umbe r

Freq.

Gas

of repl.

of chr.

of leth.

(%)

of chr.

of leth.

(%)

X-Rays (R)

m e i + females

N2 Air O2

0 3000 1500 1 500

3 3 3 3

mus-201 females

1 444 1476 1482 l 329

11 80 56 70

0.8 5.4 3.8 5.3

mei + females a

N2 Air 02

0 3000 1 500 1 500

7 6 6 6

0 3 000 1500 1500

4 4 4 4

2537 2217 2238 2182

6 155 96 151

Air 02

1 242 1 345 1587 1 462

0.2 7.0 4.3 6.9

0 3000 1 500 1 500

4 4 4 4

1465 1415 1411 1 465

20 107 78 120

1.4 7.7 5.4 8.6

0.01 0.12 0.39 0.005

2345 1 139 1670 1400

11 83 88 96

0.5 7.3 5.3 6.2

N.S. 0.13 0.16 0.41

0.3 8.3 6.5 8.3

N.S. 0.0007 0.02 0.02

0.0 6.4 4.5 5.8

N.S. 0.15 0.34 0.54

mus-306 females

3 69 70 90

0.2 5.1 4.4 6.2

mei + females a

N2 Air 02

1409 1 396 1446 1400

mus-302 females

mei + females a

N2

P

1 527 1 549 1639 1 305

4 129 107 108

mus-308 females

1 74 52 90

0.1 5.2 3.7 6.1

1 306 1 469 1434 1424

0 94 64 83

a These data with mei +, concurrent with mus-302, mus-306 and mus-308, are partially the same as sometimes 2 mutant types were tested in 1 experiment.

52

Statistical comparisons were made by calculating standard normal T values according to Fleiss (1973) (see also Mendelson, 1974). The results of different experiments were combined to give overall P values (2-sided test) for differences in responses between mei + and mutant strains.

unirradiated controls, the frequencies of recessive lethals are significantly higher with mus-201 females; (2) after irradiation in N 2 the frequencies of recessive lethals tend to be higher with mus-201, mus-302, mus-306 and mus-308 females but the difference (relative to mei ÷ crosses) is statistically significant only in the case of mus-306; (3) after irradiation in air higher yields are obtained with all 4 types of repair-deficient females, although again, only in the case of mus-306 females the difference is statistically significant; and finally (4) irradiation of males in O: results in significantly higher yields of recessive lethals with mus-201 and mus-306 females, but not with mus-302 and rnus-

Results The results on maternal effects for recessive lethals induced in spermatozoa of Muller-5 males are summarized in Table 2 and those for translocations in Table 3. To facilitate easy visual comparisons, the rates of induction (together with the pooled rates in controls) are given in Figs. 1 and 2. Also presented in Fig. 1 are the comparable data for mei-9 and mei + from the earlier study of Ferro and Eeken (1985). Inspection of Table 2 will show that (1) in

308.

The translocation data (Table 3, Fig. 2) demonstrate that (1) in none of the controls or after irradiation of males in N:, there are significant differences in the yields of translocations with any

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TABLE 3 F R E Q U E N C I E S OF 2 - 3 T R A N S L O C A T I O N S IN S P E R M A T O Z O A OF Muller-5 MALES I R R A D I A T E D IN N 2, A IR OR O 2 A N D M A T E D TO mei ÷ OR M U T A N T FEMALES P values given for H 0 of no difference between mei + and mutant females. Treatment of males

Number

Number

Number

Freq.

Number

Number

Freq.

Gas

X-Rays (R)

of repl.

of gametes

of transl,

(%)

of gametes

of transl.

(%)

N2 Air O2

0 3000 1 500 1500

3 3 3 3

1 10 22 25

0.1 1.2 1.8 2.2

N2 Air Oz

0 3 000 1 500 1500

7 6 6 6

mei + females

1 244 820 1 208 1 160

mus-201 females

1 162 936 1037 1 337

mei + females a

2 050 1736 1661 1436

0 3 000 1 500 1 500

4 4 4 4

N2 Air 02

0 3000 1 500 1 500

4 4 4 4

875 1 130 1 027 937

2 46 37 65

0.1 2.7 2.2 4.5

2 050 1065 1478 1221

0.0 1.5 1.7 1.4

N.S. 0.59 0.93 0.17

1 22 20 28

0.05 2.1 1.4 2.3

N.S. 0.60 0.01 0.00004

0.0 3.0 2.8 3.3

N.S. 0.85 0.02 0.61

0.0 2.8 2.4 3.2

N.S. 0.20 0.007 0.26

mus-306 females

0 31 18 36

0.0 2.7 1.8 3.8

1033 1 102 1265 784

mei + females a

773 992 837 979

0 15 18 19

rnus-302 females

mei + females a

N2 Air 02

P

0 33 35 26

rnus-308 females

1 19 11 26

0.1 1.9 1.3 2.7

941 1 162 1205 888

0 32 29 28

a These data with mei +, concurrent with mus-302, mus-306 and mus-308, are partially the same as sometimes 2 mutant types were tested in one experiment.

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54

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A.I.R

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of the 4 kinds of mutant females relative to m e i + females; (2) after irradiation in air, there is a significant decrease in yield with m u s - 3 0 2 , and a significant increase in yields with m u s - 3 0 6 and m u s - 3 0 8 and no demonstrable effect with m u s - 2 0 1 ; and (3) after irradiation in O 2, the frequencies are significantly lower with m u s - 3 0 2 females and a tendency for a non-significant decrease exists with m u s - 2 0 1 females. The results of calculations on air-enhancement ratios (AERs) and oxygen-enhancement ratios (OERs) are presented in Fig. 3. Including the data from the papers of Ferro (1983) and Ferro and Eeken (1985), the AERs for m e i + are from 1.3 to 1.8 and for the mutants in the range from 1.2 to 1.7. The O E R for m e i + averages around 2.2 and for the mutants, encompasses a range from 1.3 to 2.3. A possible reason for the OER differences with the mutants will be considered in the discussion. Discussion

The results presented in this paper extend those published earlier (Ferro, 1983; Ferro and Eeken, 1985) in showing that the "maternal effects approach" can unravel certain interesting aspects of the relationship between D N A repair defects and the mutational response after X-irradiation of male germ cells. Considered together with the earlier

results, it is clear that the responses observed are diverse: increased yields of m u t a t i o n s / c h r o mosomal aberrations with some, decreased with others and no effect with some other mutants. In spite of this diversity of responses, it now appears possible to advance a unified interpretation for the observed results. Starting from the basic premise that the strains employed are genetically sufficiently similar to attribute the observed differences to the m e i and m u s mutations, the interpretation takes into account (1) the levels of both excision and post-replication repair in the different mutants, (2) the nature of the lesions induced by X-rays in the different gaseous atmospheres, and (3) how these repair processes may act in converting premutational lesions into mutations. Since the biochemistry of repair in Drosophila has primarily been studied in somatic cells after UV irradiation and since here one is attempting to use these concepts to explain the role of these repair processes in the genesis of X-ray-induced mutations in germ cells, a number of assumptions are necessary. The most important assumption is that these two repair processes (excision repair and post-replication repair) also play an important role in handling X-ray-induced genetic damage. As it is conceptually difficult to envisage how excision repair or post-replication repair can act on D N A strand breaks (presumably the principal lesions involved in the formation of chromosomal aberrations) the other assumptions, outlined below, refer primarily to the data on mutations measured as sex-linked recessive lethals: (1) The principal (but not the only) premutational lesions, on which these repair processes operate, and likely to be relevant in this context, are damaged bases in the DNA. Although in general the types and relative proportions of different kinds of damage are known to be different after X-irradiation in N 2 relative to that under air or 02, the broad group of base damage should be the important type of lesions in either case. (2) A defect in one repair pathway leads to "shunting" of presumably all or some of the induced lesions normally processed by that repair pathway into the other pathway. The proportion of lesions thus channelled from one to the other depends on the magnitude of the repair defect in the first pathway.

55 (3) Excision repair is error-free whilst post-replication repair is error-prone. (4) A depression of O E R or A E R for recessive lethals in the different experiments with mutants relative to those with the repair-proficient control is suggestive of selective elimination of misrepaired or unrepaired lesions, induced under aerobic conditions, via dominant lethality. This phenomenon can be taken to reflect a qualitative difference between lesions induced under aerobic or unaerobic circumstances (see Ferro, 1983; Ferro and Eeken, 1985). (5) Meiotic abnormalities and low fertility of females (as is the case with some mutants) can account for some of the discrepancies between predicted outcome and actual observations (see below). With rnei-9 females which have no excision repair capacity (see Table 1) and are totally proficient in post-replication repair, one would expect that presumably all the induced lesions are channelled into the post-replication repair pathway. Since the latter pathway is error-prone, higher yields of mutations relative to rnei ÷ would be expected. This is in fact found in the N 2 and air series (for spontaneous lesions the same holds, see the increased frequencies in the unirradiated sampies), but not in the 02 series. With the mei-9 mutant the O E R is lower than that with mei + while the A E R is roughly similar to that with rnei ÷ (see Fig. 3). This finding suggests that a greater proportion of the induced lesions are eliminated in mei-9 via dominant lethality and not realized as mutations in the 02 series. Furthermore mei-9 females have meiotic abnormalities and low fertility. Consequently we believe that the lack of significant difference in mutation frequencies in the 02 series can be explained in this way. The response with mus-201 females which also totally lack excision repair (Table 1) would be expected in first instance to be similar to that with mei-9 in the unirradiated series, the N 2 and the air series; although the differences in induced frequencies between the mutant and the repair-proficient strain are not statistically significant, they are in the expected direction. After irradiation in 02 , however, the recessive lethal frequencies are significantly higher relative to mei ÷ (and in contrast to mei-9). This is, again, not unexpected as

the O E R is not depressed in mus-201 which means that no differential non-random elimination occurs relative to mei ÷ as no meiotic or fertility abnormalities do occur. The mutant mus-306 has 47% of the excision repair capacity of the wild type. Therefore more lesions than in rnei + but less than in mei-9 or mus-201 will be channelled into the post-replication repair pathway. The presence of an intact post-replication repair, the absence of serious fertility problems and an insignificant depression of the O E R together lead to the expectation of increased yields of mutations with mus-306 relative to mei + irrespective of the gaseous atmosphere in which the lesions were induced. This is in fact what is found. The rnus-308 mutant has only 24% of the excision repair capacity of the wild type. Thus more lesions than in mus-306 are expected to be shunted to the post-replication repair pathway; however this pathway's efficiency has been characterized as intermediate, which means that the chance for much error-prone repair is small. This coupled with the finding of a small depression of O E R would mitigate against increased yields of mutations with mus-308 females, as is borne out by the data. With mus-302, with 72% excision repair capacity compared to wild type, relatively less lesions than in mei-9, rnus-201, mus-306 or rnus-308 are expected to be channelled into the post-replication repair pathway. Further, the post-replication repair is defective and one would therefore expect no or a slight differential recovery of mutations relative to mei ÷ in the N 2 and air series, and this is what is observed. The depression in O E R relative to mei÷(1.7 versus 2.1) suggests elimination of a proportion of the lesions induced in 02 via dominant lethality. Either this factor alone, or in combination with the low fertility of the mutant would lead one to expect no significant differences in yields after irradiation in 02 (mus-302 versus mei ÷) and again this is consistent with our observations. These lines of reasoning can be extended to rnei-41 and rnus-101, the mutants used in our earlier work (Ferro and Eeken, 1985). As may be recalled, with mei-41 no differential recovery of mutations relative to mei + was found irrespective

56 of the type of atmosphere during irradiation of the males, mei-41 is deficient in post-replication repair and also has meiotic abnormalities. The lack of differences in mutational yields can be explained as the end results, of the inefficient operation of this repair pathway and meiotic abnormalities together. The strain employed did have a normal fertility, which leaves relatively little possibility for selective elimination after a specific treatment, and consequently the OER is not significantly changed with mei-41 females as compared to mei+. With mus-lO1, which is also post-replication repair-deficient and impaired in D N A synthesis, the mutational yields are lower than with mei +. Again these are consistent with the expectations and with the finding of a very drastic reduction in OER, which might link to the low fertility and non-random elimination of potential mutations through dominant lethality. While these postulated mechanisms and lines of reasoning can satisfactorily account for the differential recovery of mutations, it is difficult to invoke similar mechanisms to explain the differential recovery of translocations with the different mutants. It is clear, however, that the repair processes do play a role. A priori, one would not expect excision repair deficiency to affect the yield of translocations, since the primary lesions for these aberrations are double-strand breaks in the D N A and not base damage. This means that the observed responses must bear some relationship to post-replication repair or defects thereof. In fact none of the tested mutants with a defect only in excision repair (mei9, rnus-201 and mus-306) affect differentially the yields of translocations. In contrast all the mutants with a (partial) defect in post-replication repair do have such an effect. However, it is difficult to explain the disparate results obtained with mutants which have either a defective or an intermediate post-replication repair capacity. As will be recalled, both the partially defective mus-308 and totally defective mei-41 cause increased yields of translocations compared to mei +. In contrast, both mus-lO1 (partial defect) and mus-302 (total defect) cause decreased yields of translocations. It is worth noting that mus-lO1 and mus-302 on one hand and mei-41 and mus-308 on the other differ with respect to fertility. Both the first two

mutants have a low fertility. It is therefore not unreasonable to assume that potential translocations are eliminated as dominant lethals in crosses with mus-lO1 and mus-302 females, while with mei-41 and mus-308 more translocations are formed than with repair-proficient females. In our view these findings indicate that post-replication repair deficiency has no direct effect on translocation recovery. It seems likely that the maternal effects of the mutants on translocations indicate defects in the rejoining of DNA strand breaks. Similarly a defect in post-replication repair might better be called a defect in daughter-strand gap repair (Hanawalt et al., 1979) which suggests that the ultimate defects in post-replication repair-deficient mutants could also be relevant to strand break repair. Heterogeneity within the group of post-replication repair-deficient mutants is suggested by the different maternal effects found for these mutants. This idea is supported by the specific biochemical data on repair of UV-induced damage (Brown and Boy& 1981a, b) and the differences in mutagen sensitivity of the larvae, e.g. mus-308 is not hypersensitive to MMS or X-rays contrary to mus-302, mus-lO1 and mei-41 (Boyd et al., 1981). This last type of finding indicates that the ability to synthesize D N A across different types of lesions in the parental strand can be selectively modified in different post-replication repair-deficient mutants. For mutants mus-lO1 and mus-302 this modification could result in increased misrepair or non-repair leading to dominant lethality. In mutants rnus-308 and mei-41 poor coordination of break repair might result in increased numbers of open breaks at any one time leading to the formation of more translocations than in mei +. However, in the absence of a more precise knowledge of the actual biochemical defects that are responsible for the maternal effects on translocations in such mutants, this interpretation for the translocation data must remain tentative at present.

Acknowledgements I wish to express my gratitude to Professor F.H. Sobels for his continuous interest and to Professor K. Sankaranarayanan for many profitable discussions, support and guidance in the preparation of the manuscript. I also wish to thank Dr. J.C.J.

57

Eeken for useful discussions. I thank Ms. E.C. Klink for skillful technical assistance. This work was in part supported by Euratom Contract No. BIO-E-406-81-NL with the University of Leiden (The Netherlands). References Boyd, J.B., and P. Harris (1981) Mutants partially defective in excision repair in five autosomal loci in Drosophila melanogaster, Chromosoma, 82, 249-257. Boyd, J.B., M.D. Golino, K.E.S. Shaw, C.J. Osgood and M.M. Green (1981) Third chromosome mutagen-sensitive mutants of Drosophila melanogaster, Genetics, 97, 607-623. Boyd, J.B. et al. (1982) Identification of a second locus in Drosophila melanogaster required for excision repair, Genetics, 100, 239-257. Boyd, J.B., V. Harris, J.M. Presley and M. Narachi (1983) Drosophila melanogaster: a model eukaryote for the study of DNA repair, in: Cellular Responses to DNA Damage, Liss, New York, pp. 107-123. Brown, T.C., and J.B. Boyd (1981a) Postreplication repair-defective mutants of Drosophila melanogaster fall into two classes, Mol. Gen. Genet., 183, 356-362. Brown, T.C., and J.B. Boyd (1981b) Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair, Mol. Gen. Genet., 183, 363-368. Eeken, J.C.J., W. Ferro, H. Frei, B. Leigh, K. Sankaranarayanan and F.H. Sobels (1982) Three repair-deficient mutants of Drosophila; their effect in oocytes on the recovery of Xray-induced paternal genetic damage, in: A.T. Natarajan et al. (Eds.), Progress in Mutation Research, Vol. 4, Elsevier, Amsterdam, pp. 99-102. Ferro, W. (1983) Studies on mutagen-sensitive strains of Drosophila melanogaster, II. Detection of qualitative differences between genetic damage induced by X-irradiation of mature spermatozoa in oxygenated and anoxic atmospheres through the use of the repair-deficient mutant mei9 ~, Mutation Res., 107, 79-92.

Ferro, W., and J.C.J. Eeken (1985) Studies on mutagen-sensitive strains of Drosophila melanogaster, IV. Modification of genetic damage induced by X-irradiation of spermatozoa and spermatids in N 2 or 02 by mei-9 a, mei-41 D5 and mus(1)101TM, Mutation Res., 149, 385-398. Fleiss, J.L. (1973) Statistical Methods for Rates and Proportions, Wiley, New York. Graf, U., M.M. Green and F.E. Wi~rgler (1979) Mutagen sensitive mutants in Drosophila melanogaster; effects on premutational damage, Mutation Res., 63, 101-112. Hanawalt, P.C., P.K. Cooper, A.K. Ganesan and C.A. Smith (1979) DNA repair in bacteria and mammalian cells, Annu. Rev. Biochem., 48, 783-836. Mendelson, D. (1974) The effect of caffeine on repair systems in oocytes of Drosophila melanogaster, Mutation Res., 22 145-156. Smith, P.D., R.D. Snyder and R.L. Dusenbery (1980) Isolation and characterization of repair-deficient mutants of Drosophila melanogaster, in: W.M. Generoso et al. (Eds.), DNA Repair and Mutagenesis in Eukaryotes, Plenum, New York, pp. 175-188. Sobels, F.H. (1964a) Radiosensitivity and repair in different germ cell stages of Drosophila, in: S.J. Geerts (Ed.), Proc. l l t h Int. Congr. Genet., The Hague 1963, Vol. 2, Pergamon, Oxford, pp. 235-255. Sobels, F.H. (1964b) Post radiation reduction of genetic damage in mature Drosophila sperm by nitrogen, Mutation Res., 1, 472-477. Sobels, F.H. (1969) A study of the causes underlying the differences in radiosensitivity between mature spermatozoa and late spermatids in Drosophila, Mutation Res., 8, 111-125. Sobels, F.H., J.C.J. Eeken, W. Ferro, H. Frei, B. Leigh, K. Sankaranarayanan and E.W. Vogel (1984) Studies of the mutation process in Drosophila by means of repair-deficient mutants, in: Genetics: New Frontiers, Proceedings of the 15th International Congress of Genetics, New Delhi, India, December 1983, Oxford and IBH Publishing, New Delhi, pp. 317-328.