Analysis of mutagen specificity in Drosophila melanogaster

Mutation Research

Elsevier Publishing Company, Amsterdam Printed in The Netherlands

363

ANALYSIS OF MUTAGEN S P E C I F I C I T Y IN D R O S O P H I L A

MELANOGASTER

P. T. SHUKLA* Institute of Animal Genetics, Edinburgh (Great Britain)

(Received March 7th, 1972) (Revision received June 26th, 1972)

SUMMARY The previously reported difference between the mutational spectra of hydrazine (HZ) and hydroxylamine (HA) was confirmed for one selected locus (miniature) at which hydrazine produces no mutations in treated late larval spermatogonia or premeiotic spermatocytes sampled by 3 days' progeny. The genetically effective dose was measured in most experiments by the production of v mutants, and in a few by the production of sex-linked lethals. In a total of over 37000 X-chromosomes (16o0o from previous, and over 21000 from present experiments) treatment with HZ yielded no m mutation, but 9° v mutations. After treatment with genetically equivalent doses of HA, m and v mutations were about equally frequent. The ratio of visible mutations at the v locus to lethals on the X-chromosomes was exceptionally high after either treatment. So was the ratio of m mutations to lethals after treatment with HA. The hypothesis was tested that the lack of mutations to m after treatment with HZ was due to discrimination against a spermatogonium carrying an m mutation in the metabolic environment created by the treatment. Support for this hypothesis came from experiments in which the postulated damaging effect of m could have been wholly or partially suppressed by the presence of a second normal allele. HZ treatment of females or of males carrying a duplication for the m locus on the Y produced four independent mutations to m in 14ooo chromosomes altogether. When sampling of spermatozoa from HZ-treated males without the duplication was carried out 2I days after eclosion, two m mutations were obtained. This suggests that discrimination against m spermatogonia may take the form of delayed development into spermatozoa.

INTRODUCTION Preliminary tests have indicated that the mutation spectra of sex-linked visible mutations in Drosophila melanogaster m a y differ between mutagens~ a. The present * Present address: Division of Genetics, Indian Agricultural Research Institute, New Delhi (India). Abbreviations: car, carnation; cm, carmine; EMS, ethyl methanesulphonate; f, forked; HA, hydroxylamine; HZ, hydrazine; M-5, Muller-5 chromosome; m, miniature; N, aorm~ medium; v, vermilion; y, yellow. Mutation Res. , x6. ~I~7~t~ 3~t~ 3 7 t

364

P . T . SHUKLA

experiments were undertaken to confirm and analyse these observations. The procedure differs from that previously used in various respects. I. The comparison was restricted to HA and HZ, which had given the most striking disparity of mutational spectra. II. Whereas in the preliminary experiments all striking sex-linked visibles had been scored, the present experiments were restricted to two loci, one of which (m) had shown a very high degree of specific response, whereas the other (v) had responded equally well to both mutagens. The response of m to the two mutagens could therefore be standardized in reference to v. The other loci, cm and car, which resemble m in specificity, were tested only on a small scale to confirm the previous results. III. To avoid personal bias, all treated cultures and all mutants to be tested for allelism were coded by a laboratory technician, whereas all scoring of mutants was carried out by myself under standardized conditions of magnification and lighting. MATERIAL AND METHODS

Treatment was given throughout to third-instar larvae (about 70-80 h after egg-laying). The females providing the eggs had been kept overnight on yeast-enriched food, so that no mature eggs would be withheld. The treatment medium consisted of IO g baker's yeast, IO g glucose and 3 g agar in xoo ml water. This medium was mixed thoroughly with 0.03 M HA in phosphate buffer (pH 7) or with o.oi M HZ in borate buffer (pH 8.5). The vials or bottles used for treatment were stocked with only few larvae (IO-I 5 for vials, about 25 for bottles). They were kept at 25 ° until eclosion. The males (in a few experiments the females) were then kept for x more day before mating. Mating was done either in vials (I male with 2 females) or in bottles (xo males with 2o females). The parents were discarded after 3 days. With the exception of one experiment (last section of RESULTS), progeny were sampled for 3 days 0nly. Males for treatment came from an Or-K stock; in a few experiments, males carrying a duplication for m on the Y-chromosome were used. Females for treatment came from the same Or-K stock. The stocks used for testing the treated flies were (z) an attached-X stock, carrying yvf, for the recovery of m and v mutants from treated males, (2) a Muller-5 stock for the recovery of m and v mutants and of sexlinked lethals from treated males and for providing mates for treated females, and (3) a stock homozygous for v and m for the recovery of these two mutations by the specifc-locus method. All suspected m and v mutants were tested for allelism by mating them to standard alleles; only mutants allelic to these were recorded. RESULTS

Attached-X experiments The purpose of these experiments was to confirm the finding that HZ yields only v mutants, whereas HA yields both v and m. The results of three experiments, presented in Table I, confirm the absence of miniature mutants after HZ treatment, in contrast to a i : i ratio of m and v after HA treatment. No test of significance for this observation was considered necessary, since a difference between the expected number of about 4o m and the observed one of o is clearly significant. Mutation Res., 16 (1972) 363-371

365

MUTAGEN SPECIFICITY IN DROSOPHILA TABLE

I

EXPERIMENTS WITH ATTACHED X ' S

Number of chromosomes tested

Experiment

Treatment

Mutants

I

HA HZ

13 IO

6 o

6538 1 798

II

HA HZ

Ii 24

14 o

4968 6124

III

HA HZ

2 7

7 o

3541 3725

Pooled data

HA HZ

26 41

27 o

15o41 11647

Muller-5 tests In the attached-X experiments the v locus was used as a measure of the genetically effective dose. One might, however, consider the possibility that this locus is especially sensitive to HZ, so that at comparable frequencies of v the dose of HZ had indeed been much lower than that of HA. Expressed differently, the observed locus specificity might be due to a specifically high response of the v locus to HZ rather than to the lack of response of the m locus. To provide a broader basis for the measurement of the genetically effective dose, two Muller-5 tests were carried out, so that the doses could be measured in terms of sex-linked lethals. This test has the added advantage that each visible mutation appears as a whole culture and none is likely to be missed accidentally. On the other hand, the numbers that can be tested are small. Although the results, presented in Table If, are too small for a meaningful statistical test, the essential feature is the same as in the attached-X experiments: absence of m in the HZ cultures and a I : I ratio of v : m in the HA cultures. After both treatments, the frequencies of sex-linked lethals lay between 0.8 and 0.9%. These are low values, but they lie outside the range of spontaneous mutation frequency in this strain, which varies around 0.3o/0. The close similarity between the lethal frequencies produced by HA and HZ shows that mutagenically equivalent doses had been used. The ratio of lethal mutations to visible mutations at only two loci was surprisingly low, namely 2 : i for HA and 3:1 for HZ. Since many lethals are connected with deficiencies, this may have been due to genetical selection against sex-linked deficiencies or to an inability of these two substances to produce deficiencies in spermatogonia and premeiotic spermatocytes. TABLE

II

EXPERIMENTS WITH MULLER-5 FEMALES

Experiment

Treatment

Mutants v m

Lethals

Number of chromosomes tested

I

HA I-IZ

i 2

2 o

7 9

988 lO51

II

HA HZ

2 3

3 o

9 8

959 921

Pooled data

HA HZ

3 5

5 o

16 17

1947 1972

Mutation Res., 16 (1972) 3 6 3 - 3 7 1

366

v.T. SHUKLA

Specific locus tests After the experiments with attached-X and Muller-5 had confirmed the inability of HZ to induce m mutations, the question arose whether this lack of response was restricted to the m locus itself or was typical of the chromosomal region surrounding it. In the latter event, HZ would also fail to produce deficiencies in this region. As the m and v loci are separated by only 3 crossover units, these deficiencies could only be small. To test this hypothesis, two specific-locus tests were carried out b y m a t i n g treated males to homozygous vm females and scoring v and m in the F1 females. The results are presented in Table I I I . The ratio of v to m after H A treatment was 12:24, whereas after HZ t r e a t m e n t it was 12:o, again a complete absence of m in the HZ series. However, the original object of the experiments could not be achieved since all m u t a n t chromosomes turned out to be viable in the hemizygous males, and therefore were not considered to be deficiencies. This agrees with the previously given interpretation of the low (lethal: visible) ratio in the M - 5 tests. TABLE

III

SPECIFIC LOCUS TESTS

Experiment

Treatment

Mutants v m

Number of chromosomes tested

I

HA HZ

9 5

18 o

4178 4547

II

HA HZ

3 7

6 o

3136 2942

Pooled data

HA HZ

12 I2

24 o

7314 7489

Reconstruction experiments At this stage, possible causes of the observed specificity were considered. One of them was that, as in similar experiments with micro-organisms, the absence of m mutations after HZ t r e a t m e n t was due to selection against their appearance rather t h a n to failure of the t r e a t m e n t to produce them. It seemed possible that, in the environment of HZ-treated larvae, a m u t a t i o n to m might, by some pleiotropic effect, prevent the spermatogonium in which it had occurred from developing into a m a t u r e spermatozoon. I n micro-organisms, such questions can be answered b y reconstruction experiments. Unfortunately, this is not possible in Drosophila, because we cannot, b y reconstruction, imitate a condition in which a single m u t a t e d spermatogonium has to compete with a m a j o r i t y of n o n - m u t a t e d ones. As a rather poor substitute for such a reconstruction test, the following experiment was carried out. HA-induced m males were m a t e d to attached-X females, and the developing larvae were grown on food treated with either H A or HZ ; growth on normal food was used as a control. The same procedure was followed for males carrying an HZ-induced or HA-induced v. Thus there were altogether nine series : HAor HZ-induced v larvae on three media, and HA-induced m larvae on the same three media. In order to correct for generalized effects of the treated media on development of sperm, the frequency of Y-bearing spermatozoa was taken as a s t a n d a r d against which the frequency of m u t a n t spermatozoa was measured. Selection against mMutation Res., i 6 ( r 9 7 2 ) 3 6 3 - 3 7 1

367

M U T A G E N S P E C I F I C I T Y IN D R O S O P H I L A

spermatozoa on HZ might have resulted in a relatively reduced frequency of Xbearing spermatozoa, i.e. a decreased sex ratio. The results in Table IV show that the prediction was not fulfilled, for the sex ratio was the same in all series. In view of the very poor imitation of a reconstruction experiment, this was not unexpected. Other possibilities for testing the hypothesis were therefore sought. They were based on the consideration that such a pleiotropic effect, should it exist, would probably be recessive or, at best, semi-dominant, and might not be equally strong in germ cells in which the m mutation is present in the heterozygous condition. Two means of testing this were tried. One was the treatment of females, the other was the treatment of males carrying a duplication for the miniature locus. TABLE

IV

RECONSTRUCTION EXPERIMENT: SEX-RATIOS IN THE PROGENY OF MUTANT LARVAE REARED ON TREATED AND CONTROL FOOD (see text)

Genotype of larvaea

Medium

Progeny Male

Female

Sex-ratio (male[female)

vHZ vHZ vHZ

HA HZ N

358 241 193

336 167 I54

I.o6 1.44 1.25

vHA VHA vHA

HA HZ N

297 21o 157

239 188 126

1.24 1.12 1.25

mHA mHA mHA

HA HZ N

405 267 215

290 2o 5 136

1.39 1.3 ° 1.58

T h e source of t h e m u t a n t s is given as prefix; e.g. v H A m e a n s an H A - i n d u c e d v.

Treatment of female larvae After the usual larval treatments, females were tested by mating them to M- 5 males and scoring m and v in their sons. The results (Table V) are in agreement with the hypothesis: m mutations occurred in two out of three experiments with HZ. Although the data are too few for a test of significance, the finding that the ratio of v to m was much lower after treatment with HZ than after HA suggests that the postulated pleiotropic effect of m on the development of a germ cell in an HZ environment is partially dominant. TABLE

V

TREATMENT OF FEMALE LARVAE

Experiment

Treatment

Mutants v

m

Number o f chromosomes tested

I

HA HZ

o 4

5 2

1359 1303

II

HA HZ

4 3

4 o

3075 3616

III

HA HZ

3 3

3 I

1279 1621

Pooled data

HA HZ

7 io

12 3

5713 6540

Mutation R e s . , 16 ( i 9 7 2 ) 3 6 3 - 3 7 1

368

1,. T. SHUKLA

Treatment of male larvae carrying a miniature duplication

An alternative explanation of the results obtained by female larvae is, however, possible. Metabolism in female larvae might convert HZ to a m u t a g e n t h a t has a different action from t h a t of HZ in male larvae. To test for this possibility, treatment was given to male larvae t h a t carried on their Y a duplication of a short region of tile X including the m locus. The emerging males were m a t e d to attached-X females. The results are presented in Table VI. One m did indeed occur after HZ t r e a t m e n t in 7545 chromosomes. Admittedly, a single m u t a t i o n is weak evidence, but is has to be contrasted with the complete absence of m in altogether 37000 HZ chromosomes treated in normal males (over 16000 from the previous experiments and over 2I ooo from the present ones). TABLE VI TREATMENT

OF MALE LARVAE

Experiment

Treatment

Mutants v m

Number of chromosomes tested

I

HA HZ HA HZ HA HZ

4 5 3 4 7 9

35oo 37°o 399o 3845 749o 7545

II Pooled data

CARRYING

A DUPLICATION

3 ~ 3 o 6 I

FOR THE

MINIATURE

LOCUS

A n a l y s i s of a late brood

Since the results of the last two experiments suggested that, in the environment of normal larvae on HZ medium, a spermatogonium carrying an m m u t a t i o n is in some w a y discriminated against, I tested the possibility t h a t this discrimination might take the form of delayed development into a spermatozoon. Were this true, m m u t a tions might be found in late broods from HZ-treated larvae without the duplication. Treated males were m a t e d every three days to fresh virgin females of the attached-X stock, and mutations were scored in the seventh brood, produced 3 weeks after eclosion of the fathers. In a total of 6000 spermatozoa two m mutations (possibly a cluster) did in fact occur. Surprisingly, there were also two mutations to white, which had never been obtained in the previous tests. DISCUSSION The term specificity has been applied in this paper to differences between m u t a tional spectra produced b y different mutagens, independent of the cause of this difference. The particular specificity analyzed here refers to forward mutations at two loci; it is therefore locus specificity, not allele specificity. Whereas allele specificity m a y be due to specific reactions between m u t a g e n s and m u t a t e d sites, locus specificity involves reactions with thousands of nucleotides, m a n y of which, when deleted or altered, are likely to produce mutations. I t is difficult to explain this kind of specificity. Nevertheless, several clear examples have been reported. I n the fungus Ophiostoma, FRIES ANn KIHLMAN 1° found t h a t inositol requirers formed a very much larger proportion a m o n g auxotrophs induced b y purines than a m o n g radiation-induced auxoMutation Res., z6 (i972) 363-37 t

MUTAGEN SPECIFICITY IN DROSOPHILA

369

trophs. In the same fungus, UV failed to produce histidine requirers, although these did appear fairly frequently after treatment with nitrosomethylurethane 17. In this case, the origin of the specificity was found to lie in an inhibitory effect of complete plating medium on spores that had been treated with UV; chemically treated spores seemed to be resistant to this type of inhibition. In bacteria treated in the chemostat, NovlcK 15 observed that the ratio between mutations conferring resistance to either phage T5 or T6 was quite different after UV treatment and after treatment with purines. More recently, JAIN RAUT, AND KHAMANKARTM have reported a surprising case of mutagen specificity in the tomato, using HA, HZ or EMS. Preliminary data that also showed strong specificity of these mutagens in Drosophila have been published13; they form the basis of the present investigation. An early suggestion of mutagen specificity in Drosophila derives from HADORN'S work with phenol 11. Autosomal lethals that had been induced by treating explanted and subsequently reimplanted ovaries with phenol, showed an unexpectedly high number of cases in which lethals from different ovaries were allelie to each other. FAHMY AND FAHMY6 have reported the preferential production of new visible mutations in Drosophila by certain chemicals, but their evidence has been criticized 2. The present results--together with their attempted analysis--are a confirmation of some of the previously reported observations on the specific action of HZ and HA. They confirm the absence of rn and c a r mutations after treatment with HZ, and the absence of c m mutations after treatment with HA. Further analysis was restricted to mutations at the rn locus. As a standard of comparison, v mutations have been used, because these are produced by both treatments. Doses of HA and HZ that produced similar frequencies of v have been considered to be of comparable mutagenic strength. This procedure was justified by Muller-5 tests in which the ratios between sex-linked lethals and v mutations were similar for the two treatments, while m mutations were again absent after HZ treatment. The ratios of sex-linked lethals to visibles at only two loci were strikingly low in these experiments: 2:1 after HA and 3:1 after HZ. The lowest published ratio of lethals to visibles after treatment with a chemical has been reported by FAHMY AND FAHMY~ for phenylalanine mustard, which in their experiments yielded a ratio of IO lethals to 3 visibles on the X. Since their ratio refers to visibles on the whole X-chromosome, it would be expected to be much lower than that referring to visibles at one locus only. Moreover, as has been pointed out b y BROWNING AND ALTENBURG 4, their value is an underestimate because they included gonadic mosaics among the visibles but not among the lethals. The very low ratio obtained in the present experiments m a y suggest that neither HA nor HZ can produce deficiencies. Alternatively, deficiencies m a y have been eliminated by germinM selection. Deficiencies were also absent in the specific-locus tests, and this made it impossible to decide whether the absence of m after HZ treatment is due to refractoriness of only the miniature locus or involves a small chromosomal region surrounding it. The observation to be explained therefore, is the following. When mutations were scored in the first three-day broods of males that, as late larvae, had been reared on food that contained either HZ or HA in doses yielding comparable frequencies of sex-linked lethals and of v mutations, m were found only in the HA cultures; they were completely absent from the progeny of HZ-treated males. An interpretation in terms of molecular reactions between HZ or HA and the DNA of the miniature locus seems highly improbable. While HA is mutagenic in a variety of organisms, HZ produced Mutation Res., 16 (1972) 363-371

370

P.T. SHUKLA

auxotrophs in E. colilL but only few mutations 9 in T 4. Studies in vitro 8 have shown that HZ acts specifically on pyrimidines and, among these, selects uracil or thymine for its special target. HA, although also pyrimidine-specific, is known to act almost exclusively on cytosine s. Even if one makes the hazardous assumption that in the germ cells of Drosophila these mutagens also retain their molecular specificities, it is difficult to imagine that the DNA of the m locus has no AT pairs in mutationally vulnerable positions. Our experiments have shown that this is not so, since under the right conditions HZ can produce mutations in this locus. In a search for alternative explanations, one may consider AUERBACH'Semphasis on the role of cellular processes in tile creation of mutagen specificity 1. In one of the above-mentioned cases of mutagen specificity, the interaction between the plating medium and the mutagenically treated cell was responsible for specificitylL The same has been found in a case of mutagen specificity for reverse mutation from auxotrophy to prototrophy in Schizosaccharo~Lvces pombe ~. In Drosophila, the testis and the rest of the larva can be considered as the plating medium of a spermatogonium. In the experiments with HZ, this environment is contaminated with HZ or some of its metabolic products. In this environment, a spermatogonium containing a mutated gene will have to grow, divide and develop into a spermatozoon. It seemed possible that, in analogy with the experiments on micro-organisms, these processes are inhibited for a spermatogonium that carries a newly arisen m mutation. This would imply a pleiotropic effect of a morphological mutation, m, on metabolic processes acting in the spermatogonium. The recent finding that rudimentary has a deficiency for pyrimidine provides an example of such a situation a. Moreover, since m is sexlinked, the mutant locus is haploid so that recessive pleiotropic effects of this type would be expressed. Various approaches were used to test this hypothesis. Reconstruction experiments are, unfortunately, not possible in Drosophila. A comparison of larvae with wholly m or wholly v testes on HZ and HA showed no difference in the development of the X-bearing spermatozoa. More conclusive results were obtained when the m locus was treated in the diploid condition either in females or in males carrying a duplication for this region. If we suppose complete or partial recessivity of the postulated pleiotropic effect of m, some m mutations might be expected from HZ treatment of these larvae. This was, indeed, observed. Three independent mutations to m occurred in 65oo chromosomes from treated female larvae, and one in about 75oo chromosomes from duplication-carrying male larvae. It might be objected that these few mutants were of spontaneous origin; but this objection applies equally to at least some of the m mutations obtained on HA medium. The fact remains that 210oo male chromosomes in the present investigation and about 16 ooo in the previous one xa have not yielded a single mutation to m, whether induced or spontaneous. On the contrary, four independent m mutations were obtained after HZ treatment of only 14ooo germ cells that were diploid for the m locus. The finding that, even in these circumstances, the frequency of m was considerably below that of v suggests semi-dominance of the postulated pleiotropic effects: moreover, in male germ cells, discrimination might start when the m region has again become haploid through segregation. The finding of two--possibly not independent--m mutations in a very late brood (21 days after eclosion) of HZ-treated males suggests that discrimination against m-bearing spermatogonia may take the form of delayed development into 3futation Res., i6 (1972) 363-37I

MUTAGEN SPECIFICITY IN DROSOPHILA

371

spermatozoa. Alternatively, the action of HZ might not be the same in early and late spermatogonia. This will be tested by treating younger larvae. Although further tests of the hypothesis are necessary and will be carried out (e.g. treatment of females with a deficiency for the miniature locus; rearing of X-rayed larvae on HZ medium), the results so far obtained are in agreement with it. ACKNOWLEDGEMENTS

I am highly indebted to Professor C. AUERBACH, ~'. R. S., for her interest in this work, ever-ready help, useful suggestions and critical reading of the manuscript. Thanks are also due to Dr. SLIZYNSKA for useful discussions, to Mr. D. RAMSAY for his unfailing help in coding the cultures and to Dr. A. SCHALETfor kindly supplying the Y r n + stock. This work was carried out during the tenure of a research award from the Wellcome Trust, which is gratefully acknowledged. REFERENCES

1 AUERBACH, C., Mutagen specificity, Trans. Kansas Acad. Sci., 72 (1969) 273-295. 2 AUERBACH, C., AND B. WOOLF, Alpha and beta loci in Drosophila, Genetics, 45 (196o) 169117o3. 3 BAHN, E., S. NORBY AND ]~. SICK, Interallelic complementation for pyrimidine requirement in rudimentary mutants of Drosophila, Hereditas, 69 (1971) 229. 4 BROWNING, L. S., AND E. ALTENBURG, Gonadal mosaicism as a factor in determining the ratio of visible to lethal mutations in Drosophila, Genetics, 46 (1961) 1317-1322. 5 CLARKE, C. H., A case of mutagen specificity attributable to a plating medium effect, Z. Vererbungslehre, 93 (1962) 435-44 o. 6 FAHMY, O. G., AND M. J. FAHMY, Differential gene response to mutagens in Drosophila melanogaster, Genetics, 44 (1959) 1149-II7I. 7 FAHMY, O. G., AND M. J. FAHMY, Differential genetic response to the alkylating mutagens and X-radiation, J. Genet., 54 (1956) 146-164. 8 FREESE, F., Molecular mechanisms of mutations, in J. H. TAYLOR (Ed.), Molecular Genetics, P a r t I, Academic Press, New York, 1963, pp. 2o7-27o. 9 FREESE, E., E. BAUTZ AND E. B. FREESE, The chemical and rnutagenic specificity of hydroxylamine, Proc. Natl. Acad. Sci. (U.S.), 47 (1961) 845-855. IO FRIES, N., AND B. KlltLMAN, Fungal mutations obtained with methylxanthines, Nature, 162 (1948 ) 573. II HADORN, E., Weitere Ergebnisse der "in vitro Behandlung" yon Drosophila Ovarien mit Phenol, Pubbl. Staz. Zool. Napoli, 23 (Suppl. 195 o) 1-18. 12 JAIN, H. K., R. N. RAUT AND Y. G. I{HAMANKAR,Base specific chemicals and mutation analysis in Lycopersicon, Heredity, 23 (1968) 247-256. 13 JAIN, H. If., AND P. T. SHUKLA, LOCUSspecificity of mutagens in Drosophila, Mutation Res., 14 (1972 ) 440-442 . 14 LINGENS, P. VON, Erzeugung biochemischer Mangel-Mutanten yon E. coli mit Hilfe von Hydrazin und Hydrazinderivaten, Naturwissenschaften, I9b (1964) 151-156. 15 NoVlCK, A., Mutagens and antimutagens, Broohhaven Symp. Biol., 8 (I955) 2Ol-215. 16 TESSMAN, I., H. ISHIWA AND S. I~UMAR, Mutagenic effects of hydroxylamine in vivo, Science, 148 (1965) 5o7-5o8. 17 ZETTERBERG, G., O11 the specific mutagenic effects of N-nitroso-N-methylurethane in Ophiostoma, Hereditas, 48 (1962) 371-389.

2~utation Res., 16 (1972) 363-371