Changes in the mutational response of silkworm spermatozoa exposed to mono- and polyfunctional alkylating agents following storage

Mutation Research

425

Elsevier Publishing Company, A m s t e r d a m P r i n t e d in The N e t h e r l a n d s

CHANGES IN T H E M U T A T I O N A L RESPONSE OF S I L K W O R M SPERMATOZOA E X P O S E D TO MONO- AND P O L Y F U N C T I O N A L A L K Y L A T I N G AGENTS F O L L O W I N G STORAGE E I I C H I I N A G A K I * AND I R W I N I. O S T E R

Department of Biology, Bowling Green State University, Bowling Green, Ohio (U.S.A.) (Received April 3rd, 1969)

SUMMARY

Following exposure of silkworm spermatozoa to several alkylating agents, treated spermatozoa were stored for various lengths of time in males. The frequency of mutations induced by two monofunctional agents, ethyleneimine and ethyl methanesulfonate, at two specific loci, pe and re, increased after storage. On the other hand, a reverse effect was observed when the polyfunctional agents, triethylenemelamine and mitomycin C were used. The present findings tend to suggest that these results m a y be a reflection of an essential difference between the mechanisms involved in mutation induction by the two classes of agents. The features of the two types of storage effect are discussed in the light of this possibility.

INTRODUCTION

At present, perhaps the most significant findings for understanding the mode of action of alkylating agents on deoxyribonucleic acid in relation to biological effects seem to be derived from studies of their cytotoxic action in microorganisms. The general view17,18,2° gained from this field is that polyfunctional alkylating agents We feel privileged to be able to dedicate this p a p e r in h o n o r of Professor CHARLOTTE AUERBACH on the occasion of her official retirement. I t is fitting t h a t the m a n u s c r i p t concerns the alkylating agents since, in addition to her m a n y other o u t s t a n d i n g contributions, her original discovery and the s u b s e q u e n t opening up of the field of chemical nlutagenesis involved a p o t e n t alkylating agent, m u s t a r d gas or fl,fl'-dichlorodiethylsulfide 1-3. One of us (IRWIN I. OSTER) feels particularly f o r t u n a t e in t h a t he had the privilege to work in her l a b o r a t o r y on two occasions; for one year d u r i n g 1953 54 and for several m o n t h s during the s u m m e r of I96I. N o t only did this o p p o r t u n i t y provide him with m a n y useful suggestions and invaluable advice for his research, b u t also, it was m a r v e l o u s to observe her incisive reasoning ability during f r e q u e n t discussions which she h a d with her staff dealing with diverse genetical problems. * P r e s e u t address : D e p a r t m e n t of R a d i a t i o n Genetics, University of Leiden, VVassenaarsewcg 62, Leiden (The Netherlands). A b b r e v i a t i o n s : HI, ethyleneimine; EMS, ethyl m e t h a n e s u l f o n a t e ; MC, nlitomycin C; TEM, triethylenemelanline.

31utation l~es., 7 (1969) 425-432

426

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IRWIN

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have a markedly greater efficiency than monofunctional ones as measured by in ~,/lro inactivation experiments. It appears t h a t the mechanisnl of their cytotoxic action can be related to their ability to cross-link the double strands of DNA, thus interfering with replication and preventing the partitioning of D N A between daughter cells during cell division. In addition, it is also well established that practically all alkylatin~4 agents which possess effective carcinostatic properties contain two or more functional groups. However, the picture seelns to be less clear in cases where tile effects studied involve mutagenesis. While it has been suggested t h a t monofunctional agents are consistently better as Inutagens in microorganisms, there now exists ample evidence t h a t in higher organisms both mono- and polyfunctional compounds are capable of inducing both point mutations and chromosome breakage. In this connection, it should be mentioned t h a t it was AUERBACH and her colleagues who first showed t h a t monofunctional compounds can act as powerful mutagens a4. This indicates t h a t these changes are not directly related to the ability of particular compounds to crosslink biologically important macromolecules. As a m a t t e r of fact, the theoretical consideration of the possibility t h a t a polyfunctional c o m p o u n d might more easily affect inter-chromosomal events b y reason of its cross-linking capacity, led NAKAO AND AUERBACH"a to compare the ratio of sex-linked recessive lethal mutations to translocations induced b y a monofunctional agent, ethylene oxide, to that produced b y a polyfunctional agent, diepoxybutane in Drosophila. However, contrary to expectation, the ratio of lethals to translocations was essentially similar following t r e a t m e n t with each of the chemicals. Soon afterwards, NAKAO el al. ~'4 reported that the finding holds true for such other mono- and polyfunctional compounds as EI and TEM. These results seem to be readily understandable if we assume that in Drosophila both mono- and polyfunctional agents are capable of inducing gene nmtations and chromosome breaks v i a a similar mechanism. While there had been good indications t h a t the difunctional agent, nitrogen m u s t a r d ~I, and the polyfunctional agent, TEM ~a ~, exhibit a so-called storage effect, WATSON3~,as has presented the first clear-cut evidence of an essential difference between the genetical effects of a mono- and a polyfunetional alkylating agent. He showed t h a t when Drosophila sperm treated in the male with such polyfunctional agents as TEM or diepoxybutane, are stored in females, the frequency of translocations increases with time of storage. In contrast, sex-linked recessive lethal nmtations showed only a slight increase over the same period. However, neither ethylene oxide or E I , the monofunctional counterparts, appeared to exhibit a storage effect. It has been suggested t h a t the increase m a y be related to hydrolytic degradation of D N A which is expected to be greater for D N A alkylated b y a polyfunctional agent to a low extent, and improbable for a similarly low extent of monofunctionally alkylated D N A 9. Be t h a t as it may, these observations constitute clear-cut examples of a qualitatively different reaction to mono- and polyfunctional alkylating agents and also offer interesting evidence t h a t potential chromosome breaks and potential mutations m a y behave differently under conditions of storage. In view of this it was t h o u g h t desirable to broaden our knowledge of tile spectrum of genetical changes induced by various nmtagens in different organisms with special emphasis on the effects of sperm storage. This might yield some clues for increasing our understanding of the mode of action of mutagens as well as the Mutali~)~ hV~., 7 (1060'1 425-q3-'

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S I L K W O R M S P E R M A T O Z O A E X P O S E D TO A L K Y L A T I N G A G E N T S

nature of this mutational event. The present study was concerned with attempts to assess the mutational response of silkworm sperm which had been stored following treatment with mono- or polyfunctional agents. MATERIALS AND METHODS

Two monofunctional agents, E I and EMS, and two polyfunctional agents, TEM and MC, were utilized. For estimating mutation frequencies in the silkworm, Bombyx mori, the specific-locus method was employed; two recessive visible genes, pink (pe) and red (re), served as the markers. The two genes are located on chromosome V at o.o and 31.7, respectively. Each affects the color of the serosa of the egg and the eye color of the adult. In the experiments to be reported below, only changes in egg color were investigated. Whereas the normal egg color is black, pe/pe eggs are essentially white and re~re eggs are red. Newly-enclosed male moths of a wildtype strain of the silkworm were injected with approximately o.I ml of various concentrations of the four test chemicals dissolved in a 0.4% saline solution. After 20 h, the treated male moths were mated individually to virgin females carrying the two marker genes. 24 h after the first mating each of the male nloths was again mated to new virgin females. Subsequently, each group of eggs was examined in order to determine the frequencies of whole-body and mosaicallyexpressed mutations. RESULTS

The results are summarized in Table I. It is readily apparent that in the silkworm both mono- and polyfunctional alkylating agents are capable of inducing a relatively high frequency of mutations at the two marker loci, pe and re, and that the bulk of these are expressed as mosaics. This observation is consistent with the previous observation that in many forms chemical agents tend to be much more effective in producing mosaic mutations in comparison to whole-body changes than X-rays in post-meiotic germ cells~-L On the other hand, the results furnish evidence for a different kind of mutational

TABLE

I

THE EFFECTS OF STORAGE ON THE FREQUENCY OF RECOVERED VISIBLE MUTATIONS INDUCED IN SILK~VORM SPERM TREATED ~VITH THE MONOFUNCTIONAL AGENTS, E l AND E M S , AND THE POLYFUNCTIONAL AGENTS, T E M AND M E

Agents

EI (2. lO .-3 ~,V/) EMS (o.o1%) TEM ( 2 . 1 o ~ zV/) MC (25 / t g / m l )

S p e r m used 2 o h after treatment pe re Mutation Mosaic 31utation frequency % % frequency % O. l l 5 : 0 . 0 3 0 IOO.O ( 1 4 / 1 2 123) 0 . 4 o 4 :I: 0 . 0 4 3 9 5 . 3 (86/21240) 0 . 9 8 0 ~:: 0 . 0 9 4 91. 5 (lO6/lO81O) 2.731 -- o.121 97-9 (489/17899)

Mosaic °/'o

0 . 0 5 7 ;} o . o 2 1 IOO.O ( 7 / 1 2 123) O.lO8 ~ 0 . 0 2 2 91. 3 (23/2i 24o ) o . 3 1 4 ~_ 0 . 0 5 3 IOO.O (34/10810) 1.866 - o.ioi 9o.1 (334/17899)

S p e r m used 48 h after treatment pe rg Mutation Mosaic iVIutation frequency % °o frequency % 0 . 3 5 o :!: 0 . 0 5 5 9 7 . 5 (4o/11421) 0.880 ~ 0.069 96.8

(i57/i7822) 0.640 ~ 0.076 95.6 (69/10765) 1-771 }: 0 . 0 9 6 9 6 . 9 (332/18744)

i~Iosaic %

o - 1 1 3 ~ o . o 3 1 IOO.O (13/11421) 0 . 3 0 2 ~ o . o 4 1 IOO.O (54/17822) o.241 - 0.047 92.3 (26/10765) 1.189 : 0.079 92.8 (223/18744)

Mutation Res., 7 (1969) 4 2 5 - 4 3 2

428

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response to inono- and polyfunctional agents following storage of treated sperm. It appears that when silkworm sperm treated with the po134unctional agents, I'll and EMS, are stored for 4 8 h in the male, the frequencies of recovered mutations an' significantly higher than that of samples taken 2o h after treatment. In contrast to this, it should be noticed that the frequencies of nmtations induced by the two polyfunctional agents, TEM and MC, decrease as a consequence of sperm storage. These seemingly reverse effects between mono- and polyfunctional alkylating agents in the silkworm are strikingly obvious. DISCUSSION

Unlike what has been reported for Drosophila, the present study revealed that in the silkworm, storage of sperm treated with monofunctional agents seems to raise the nmtation rate while storage in sperm treated with polyfunctional agents tends to lower the nmtation rate. By attempting to determine the basis for this differential response between organisms and between agents in one organism some light may be thrown on our understanding of mutagenesis in multieellular forms. There are essentially two methods which are used for experimentally storing sperm: either it may be stored in the female reproductive tract following mating by prohibiting oviposition and therefore fertilization which takes place in the oviduct, or it may be stored in the male by prohibiting copulation. When sperm treated with chemical agents are stored, the latter procedure necessaril.y makes it more probable that the cells will be exposed for a relatively longer time to an appreciable amount of the chemical than tile former. Indeed, a recent paper by NECASEK e'{ al. ~6 has shown that prolonged treatment with EMS causes significantly higher mutagenic effects in C o r y n e b a c t e r i u m sp. and Bacillu.s cgfett$. Since in the present study with the silkworm the treated sperm had been stored in the male, the observed increase in the mutational yield after storage of sperm treated with the two monofunctional agents, EI and EMS, might be due to prolonged contact with the chemicals. Conceivably, this explanation can also be invoked to explain the observations following treatment with the polyfunctional agents. In this case tile yield of nmtations after storage of sperm treated with TEM and MC would lye expected to either increase or possibly remain the same. However, contrary to expectation, the observed effects were apparently different. On general grounds it might seem that there are difficulties in attempting qualitative and quantitative comparisons between different mutagens. For example, mutational vield can only be related to viable gametes and selection may o,:cur at many points along the pathway from induction to the detection of the changes. Therefore, it inay appear to be reasonable at first glance to assume that solection of sperm is responsible for the decrease in nmtational yield following storage of sperm which had been treated with the polyfunctional agents, TEM and MC However, recent observations by NAKAO AND MACHIDA25 have shown that in silkworms the frequencies of mutations induced bv a polyfunctional agent, diepoxybutane, increase linearly with dose, even when the doses are st) high am to induce extremely high nmtation frequencies. Taking their findings into consideration, sperm selection cannot be assigned even a contributory role in explaining tile decrease in mutational yield after storage of sperm treated with TEM and MC. ]~Iulalion Res., 7 (I909) q25--t3e

SILKWORM SPERMATOZOA EXPOSED TO ALKYLATING AGENTS

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Alternatively, it is well established~9,3~,37, 38 that in Drosophila the frequency of translocations increases manifold after storage of sperm treated with polyfunctional agents. Perhaps, dominant lethal mutations which are largely due to chromosome breaks would also be expected to increase after storage of sperm treated with polyfunctional agents. As a m a t t e r of fact, RATNAYAKE3° has recently shown that in Drosophila the frequency of dominant lethals increases by storage of TEM-treated sperm. Therefore, it might be assumed that the observed decrease in the mutational yield after storage of silkworm sperm treated with TEM and MC could be correlated with a possible increase of d o m i n a n t lethal mutations, with the latter tending to eliminate some of the specific locus changes. Surprisingly enough, however, our preliminary results (unpublished) have indicated that in the silkworm the yield of dominant lethal mutations also appeared to decrease after storage of sperm treated with MC. NORTH has reported a somewhat similar phenomenon using alkylating agents and their non-alkylating analogs on the house fly, Musca domesticate, ~. On the other hand, it has recently been reportedl°,19,~l, 36 that the damage to DNA b y polyfunctional agents is subject to repair which could proceed by a sequence of steps involving a special removal of alkylated groups from DNA. If such a serial repair process exists in silkworm sperm cells, the observed decrease in the mutational yield after storage of sperm treated with TEM and MC m a y be related to a sort of repair event involving disappearance of the cross-linkings formed in the complementary strands of DNA. Although this is mere speculation, the idea presently seems to be able to explain readily the mutational response following storage of sperm treated with TEM and MC. If this should be true in the silkworm, the initial process involved in the mutagenic effects of the two polyfunctional agents, TEM and MC, might be connected with the formation of cross-links between the double strands of DNA and their subsequent degradation with time. In this connection it should be pointed out that it has been suggested on mainly theoretical grounds that whole-body mutations m a y arise through the formation of cross-links between the twin strands of DNA. Since polyfunctional agents are decidedly more efficient in forming cross-links than monofunctional compounds 8, one would expect a much higher proportion of whole-body mutations produced by the former than by the latter. Actually, FAHMY AND IrAHMY12interpreted some of their results using several mesyloxy esters as purportedly showing that the proportion of recessive visible whole-body mutations which could be randomly detected in Drosophila was significantly higher for the polyfunctional than the monofunctional compounds. However, observations b y SNYDER AND OSTER aa, utilizing the more objective technique of scoring induced mutations at specific loci, have indicated that the number of alkylating groups present in a compound do not appear to play a significant role in influencing the relative proportions of mosaic alterations which are produced. Our results of the present study with the silkworm have also shown that both mono- and polyfunctional agents are equally capable of inducing a high proportion of mosaic mutations. Taken together, notwithstanding FAHMY'S claim to the contrary, there is compelling evidence to reject an interpretation of the storage effects in the silkworm based on the relative ability of compounds to form cross-links. However, our preliminary results showing that almost all the mutations induced by MC in silkworm spermatogonia are also mosaically-expressed, suggest that in the silkworm the sequence of events during mutagenesis m a y be Mutation t?es., 7 (1969) 425 432

430

EIICHI INA(iAKI, IRWIN 1. ()SIER

somewhat different froln ()tiler organisms, in other forms, such as I)rosot)hila l(,r instance, lnutations which are induced in spermatogonia generally tend to be expressed as whole-body changes. Recently, TAZlMAa~ has presented interesting evidence that in the silkworm the double strandedness of DNA may not be the only source ()f mosaic mutations. He found that the frequency of X-ray-induced mosaic mutations rose rapidly approximately with the square of the dose, while that of whole-body changes increased linearly with dose. TAZlMA has interpreted these results as indicating that phenotypically expressed mosaic nmtations induced by X-rays in silkworm sperm are largely due to chromosome aberrations such as small deletions. If this is true, then it follows that the strandedness of DNA is not an important factor in the formation of mosaic nmtations by X-rays and a polyfunctional agent such as MC. On the other hand, the picture of the frequency patterns of X-ray-indu(-ed mosaic mutations in Drosophila is quite different from that obtained in the silkworm. The recent papers of INAGAKI ANt) NAKAO in, and MATSUDAIRA Ct al. "2"-have shown that the frequency of X-ray-induced mosaic mutations in Drosophila may already reach a constant maximum level at a fairly low dose level, whereas that of wholebody mutations goes up with an increase in dose. Although the mechanism(s) involved in the formation of mosaic nmtations induced by X-rays in Drosophila is not fully understood, the sharp contrast between the silkworm and Drosophila results is most likely due to a difference in the mechanism involved in the formation of mosaic genetic changes in the two species. Carrying this assumption one step further, it is conceivable that the mechanism involved in the production of nmtations by alkylating agents in the silkworm may also be different from that of Drosophila. Studies are currently underway to investigate this possibility further. In any case, it is logical to assume that elucidation of the mechanism inw)lw'd in the effects observed following storage of chemically-treated silkworm spermatozoa will shed additional light on the nmtational process in multicellular animals. It should also be mentioned that storage effects in plants following treatment of seeds with mutagens have also been found to occur fairly extensively. In several instances, a somewhat parallel phenomenon to that observed in the silkworm has been reported for alkylating agents by DUBINtNA using Crepis capillaris'L Whether or not a similar mechanism constitutes the basis for these observations in seeds and spermatozoa is still subject to speculation. ACKNOWLEDGEMENT

This work was supported by the U.S. National Science Foundation Grant G24 261 for work of I. I. OSTER and Associates. REFERENCES I AUERBACH, C., AND J. M. ROBSON, E x p e r i m e n t s on the action of m u s t a r d gas in Drosophila, Production of sterility and of m u t a t i o n , Report to the Ministry of Supply, London, 1942, XN3979. 2 AUERBACH, C., AND J. M. ROBSON, E x p e r i m e n t s on the action of m u s t a r d gas in Drosophila, I I. Genetical differences in susceptibility, IV. The p r o d u c t i o n of visible nlutations, Report t{, the Ministry of Supply, London, 1942, W I I 831. 3 AUERBACH, C., Chemically-induced m u t a t i o n s and r e a r r a n g e m e n t s , Drosophila lnform. £'erv., 17 (1943) 48 5 TM

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4 AUERBACH, C., Chemically-induced mosaicism in Drosophila melanogaster, Proc. Roy. Soc. (Edinburgh), 62, I I (1946) 2 i i - 2 2 2 . 5 AUERBACH, C., J. M. ROBSON AND J. G. CARR, The chemical p r o d u c t i o n of m u t a t i o n s , Science, lO5 (1947) 243 2476 AUERBACH, C., The induction b y m u s t a r d gas of c h r o m o s o m a l instabilities in Drosophila melanogaster, Proc. Roy. Soc. (Edinburgh), 62, i I (1947) 307-320. 7 AUERBACH, C., The chemical p r o d u c t i o n of m u t a t i o n s , Science, 158 (1967) 1141-1147. 8 BROOKES, P., AND P. D. LAWLEY, The action of mono- and di-functional alkylating agents on nucleic acids, Biochem. J., 80 (1961) 469-503 . 9 BROOKES, P., AND P. D. LAVCLEY, Alkylating agents, Brit. Med. Bull., 20 (No. 2) (1964) 9I. IO CARTHON, A. R., AND J. J. ROBERTS, Mechanism of the cytotoxic action of alkylating agents in m a m m a l i a n ceils and evidence for the r e m o v a l of alkylated g r o u p s from deoxyribonucleic acid, Nature, 211 (1966) 15o-153. i I DUBININA, L. G., The effect of alkylating c o m p o u n d s on the c h r o m o s o m e s before the stage of DNA synthesis, Proc. i2th Intern. Congr. Genet., i (1968) 116. 12 FAHMY, O. G., AND M. J. FAHMY, Cytogenetic analysis of the action of carcinogens and t u m o r inhibitors in Drosophila melanogaster, X. The n a t u r e of m u t a t i o n s induced b y the m e s y l o x y esters in relation to molecular cross-linkage, Genetics, 46 (1961) 447-458 . 13 HERSKOWITZ, I. H., The m u t a g e n i c i t y of "triazine" administered in s p e r m baths, Drosophila Inform. Serv., 29 (1955) 124. 14 HERSKOWITZ, I. H., The p r o d u c t i o n of m u t a t i o n s in Drosophila melanogaster w i t h chemical s u b s t a n c e s administered in s p e r m b a t h s and vaginal douches, Genetics, 4 ° (1955) 76-89. 15 HERSKOWITZ, I. H., Mutagenesis in m a t u r e Drosophila spernlatozoa b y " t r i a z i n e " applied ill vaginal douches, Genetics, 41 (1956) 605-6o 9 . 16 INAGAKI, E., AND Y. NAKAO, Comparison of frequency p a t t e r n s between whole-body and fractional m u t a t i o n s induced b y X - r a y s in Drosophila melanogaster, Mutation Res., 3 (1966) 268 272. 17 LAWLEY, P. D., Effects of some chemical m u t a g e n s and carcinogens on nucleic acids, in J. N. DAVIDSON AND "vV. E. COHN (Eds.), Progress in Nucleic Acid Research and Molecular Biology, 5 (1966) 89-131. 18 LAWLEY, P. D., AND P. BROOKES, The action of alkylating agents on the deoxyribonucleic acid in relation to biological effects on the alkylating agents, Exptl. Cell Res., Suppl. 9 (1963) 512 520. 19 LA~,VLEY, P. D., AND P. I3ROOKES, Molecular m e c h a n i s m of the cytotoxic a c t i o n - - d i f u n c t i o n a l alkylating agents and resistance to this action, Nature, 206 (1965) 480-483. 20 LOVELESS, A., Genetic and Allied Effects of Alkylating Agents, B u t t e r w o r t h s , London, 1966. 21 LOVELESS, A., J. COOK AND V. V~?HEATLEY, Recovery from the " l e t h a l " effects of cross-linking alkylation, Nature, 205 (1965) 980-983. 22 .~'IATSUDAIRA, Y., T. ITO, T. YAMASAKI, S. ISHIZAKA AND M. DOMON, On the relationship between the f r e q u e n c y of t w o t y p e s of m u t a t i o n s and soft X - r a y doses in Drosophila, Mutation Res., 4 (1967) 469-472. 23 NAKAO, V., AND C. AUERBACH, Test of a possible correlation between cross-linking and chrom o s o m e breaking abilities of chemical mutagens, Z. Vererbungslehre, 92 (1961) 457-461. 24 NAKAO, Y., E. YAMAGUCHI AND I. MACHIDA, F u r t h e r test of possible correlation between cross-linking a n d c h r o m o s o m e breaking abilities of chemical mutagens, Japan. J. Genet., 39 (1964) 155-163. 25 NAKAO, Y., AND 1. MACHIDA, The differences in visible m u t a t i o n p a t t e r n s b y the n u m b e r of functional a r m s of alkylating substances, (Abstract), Japan. J. Genet., 4 ° (1968) 408. 26 NECASEK, J., P. PIKALEK AND J. DROBNIK, The genetic effect of prolonged t r e a t m e n t with ethyl m e t h a n e s u l f o n a t e , Mutation Res., 4 (1967) 4°9-413 . 27 NORTH, D. T., S p e r m storage in the house fly. Effect on the n u m b e r of d o m i n a n t lethal m u t a tions recovered after t r e a t m e n t with several alkylating agents or their non-alkylating analogues, Genetics, 54 (1966) 352 . 28 NORTH, D. T., S p e r m storage: modification of recovered d o m i n a n t lethal m u t a t i o n s induced by t e t r a m i n e and analogues, Mutation Res., 4 (1967) 225 228. 29 RATNAYAKE, W., C. STRACHAN AND C. AUERBACH, Genetical analysis of the storage effect of triethylene melamine (TEM) on c h r o m o s o m e breakage in Drosophila, Mutation Res., 4 (1967) 38o-381. 3 ° RATNAYAKE, V~r., Effects of storage on d o m i n a n t lethals induced by alkylating agents (triethylene nlelanline and ethylenimine), iVIutation Res., 5 (1968) 271-278. 31 SCHALET, h . , The relationship between the frequency of nitrogen m u s t a r d translocations in m a t u r e s p e r m of Drosophila and utilization of s p e r m b y females, Genetics, 4 ° (1955) 594. 3 2 SNYDER, L. A., Evidence of an essential difference between point m u t a t i o n s and chronlosome breakage induced b y triethylene melamine in Drosophila spermatozoa, Z. Vererbungslehre, 94 (1963) 182 189 .

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33 ~NYDER, ],. ,\., AND l. I. ()STP2R, C o m p a r i s o n of g e n e t i c c l m n g e s i n d u c e d b v ~1 i~l~ln~Huncti~ll;~t a n d a p o l y f u n c t i o n a l a l k y l a t i n g a g e n t in Drc~sopkila mclanogaster, 3 1 . t a t i . ~ h'~'x, ~ (i,#,t~ 437 445. 34 STEVENS, C. ~l., A. MYLROIE, C. AE;I~;RBACH, } l. MOSER, IX. A. j I'2NS]';N, 1. I
]~Iutalion l?es., 7 (t969) 425 432