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A GENETIC ANALYSIS OF MUTAGEN-SENSITIVE MUTATIONS ON THE SECOND CHROMOSOME OF DROSOPHILA MELANOGASTER By DARYL STEWART HENDERSON B . S c , The U n i v e r s i t y o f B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1987 © D a r y l Stewart Henderson In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1 Y 3 Date OCT" \ D E - 6 ( 3 / 8 1 ) ABSTRACT Mutagen-sensitive (mus) mutations i n Drosophila  melanogaster render developing f l i e s hypersensitive to the l e t h a l e f f e c t s of DNA-damaging agents. In general, mus mutations i d e n t i f y DNA r e p a i r - r e l a t e d genes. In t h i s study, 5 new second chromosome mus mutations (mus2_05B1, mus208 B 1. mus209 B 1. mus210 B 1 and mus211 B 1), selected on the basis of s e n s i t i v i t y to methyl methanesulfonate (MMS), were characterized using a v a r i e t y of genetic t e s t s . One t e s t measured the MMS-s e n s i t i v i t y of double mutant mus s t r a i n s compared to t h e i r component single mutants. Mutant interactions were examined i n 8 double mus and i n 2 t r i p l e mus s t r a i n s containing combinations of mus201 D 1. mus205 B 1. mus208 B 1, mus210 B 1 and mus211 B 1 (or mus211 B 2). These analyses have revealed predominantly s y n e r g i s t i c and e p i s t a t i c responses to MMS. Taken together with the findings of previous genetic and biochemical studies of Drosophila mus s t r a i n s , these r e s u l t s suggest that 3 major repa i r pathways may operate i n f l i e s to correct damage caused by MMS. Mutagen c r o s s - s e n s i t i v i t y data and the r e s u l t s of the i n t e r a c t i o n studies suggest that mus mutations might serve as rapid and s e n s i t i v e bioassays of somatic genotoxicity caused by mutagens and carcinogens. To explore t h i s p o s s i b i l i t y , a simple mutagen t e s t system was devised employing t r i p l e mutant mus s t r a i n s . One s t r a i n (mus208 B 1 mus210 B 1 mus2ll B 2) was tested for s e n s i t i v i t y to 14 mutagens/carcinogens and 2 non-carcinogens. i i Eleven of the mutagens/carcinogens were r e a d i l y detected as genotoxic. Both non-carcinogens were non-genotoxic. These preliminary r e s u l t s demonstrate the f e a s i b i l i t y (and some limit a t i o n s ) of the proposed somatic genotoxicity assay and emphasize the need for further t e s t v a l i d a t i o n using a larger chemical data base. The temperature-sensitive l e t h a l mutation mus209 B 1 was subjected to extensive genetic analyses and to temperature s h i f t experiments during development. This locus was found to encode a product(s) that (1) i s esse n t i a l for v i a b i l i t y at v i r t u a l l y a l l pre-imaginal developmental stages (the l a t t e r h a l f of pupation appears to be an exception), (2) i s necessary for wildtype l e v e l s of resistance to the genotoxic e f f e c t s of MMS and i o n i z i n g radiation, and (3) i s required for female f e r t i l i t y . Confirmation of the p l e i o t r o p i c nature of t h i s mutation was obtained by meiotic and cytogenetic mapping studies and by complementation t e s t s with a series of a l l e l i c mutations. The mus209 B 1 phenotypes are s i m i l a r to ones conferred by mutations i n Drosophila and yeast that disrupt various aspects of chromosome metabolism. In t h i s context, some possible roles for mus209 B 1 are discussed. i i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x CHAPTER 1. GENERAL INTRODUCTION TO CELLULAR RESPONSES TO DNA DAMAGE I. Perspectives 2 II . C e l l u l a r Responses to DNA Damage 4 A. Enzyme-catalyzed photoreversal of pyrimidine dimers 5 B. DNA excision repair 8 C. Post r e p l i c a t i o n repair 20 D. Inducible repair responses 23 I I I . Mutagen-sensitive mutations i n Drosophila melanogaster 30 CHAPTER 2. INTERACTIONS BETWEEN MMS-SENSITIVE MUTATIONS I. Introduction 3 6 I I . Materials and Methods 39 I I I . Results 44 IV. Discussion 69 iv CHAPTER 3. MUTAGEN-SENSITIVE STRAINS AS GENOTOXICITY INDICATORS I. Introduction 79 II . Materials and Methods 82 I I I . Results 88 IV. Discussion 94 CHAPTER 4. A GENETIC AND DEVELOPMENTAL ANALYSIS OF mus209 B 1 I. Introduction 100 II . Materials and Methods 104 II I . Results 112 IV. Discussion 130 REFERENCES 138 APPENDIX A. ISOLATION OF MUTAGEN-SENSITIVE STRAINS 159 APPENDIX B. AN EXAMINATION OF THE INFLUENCE OF MATERNAL GENOTYPE ON THE SENSITIVITY OF mus OFFSPRING TO MMS 163 v LIST OF TABLES T a b l e Page 1- 1 Summary of properties of mus mutants of the second chromosome 33 2- 1 Relative v i a b i l i t y of mus homozygotes and heterozygotes i n untreated cultures 45 2- 2 A compilation of previously reported interactions i n double mutant stra i n s 71 3- 1 S e n s i t i v i t y of mus208 B 1 mus210 B 1 mus211 B 2 to simple a l k y l a t i n g agents 89 3-2 S e n s i t i v i t y of mus205 B 1 mus208 B 1 mus210 B 1 to simple a l k y l a t i n g agents 91 3- 3 S e n s i t i v i t y of mus208 B 1 mus210 B 1 mus211 B 2 to miscellaneous chemicals 92 4- 1 Cosegregation of female s t e r i l i t y and temperature-sensitive l e t h a l i t y i n mus209 B 1 114 4-2 The maternal-effect l e t h a l i t y of mus209 B 1/mus209 B 2 h e t e r o a l l e l i c females 125 4-3 The e f f e c t s of io n i z i n g radiation on the r e l a t i v e s u r v i v a l of mus209 B 1 homozygotes at various times i n development 128 v i LIST OF FIGURES Figure Page 1- 1 A schematic diagram of DNA repair pathways 7 2- 1 Procedure used to construct multiply-mutant mus st r a i n s 42 2-2 MMS s e n s i t i v i t y of mus201 D 1, mus205 A 1, mus205 B 1 and mus201 D 1 mus205 B 1 48 2-3 MMS s e n s i t i v i t y of mus205 B 1 mus208 B 1 compared to i t s component single mutants 51 2-4 MMS s e n s i t i v i t y of mus205 B 1 mus210 B 1 compared to i t s component single mutants 53 2-5 MMS s e n s i t i v i t y of mus208 B 1 mus211 B 1 compared to i t s component single mutants 56 2-6 MMS s e n s i t i v i t y of mus210 B 1 mus211 B 1 compared to i t s component single mutants 58 2-7 MMS s e n s i t i v i t y of mus_208B1 mus210 B 1 compared to i t s component single mutants 60 2-8 MMS s e n s i t i v i t y of mus201 D 1 mus208 B 1 compared to i t s component single mutants 63 2-9 MMS s e n s i t i v i t y of rous201D1 mus210 B 1 compared to i t s component single mutants 66 2-10 MMS s e n s i t i v i t y of mus205 B 1 mus208 B 1 mus210 B 1 and mus208 B 1 mus210 B 1 mus211 b 2 68 v i i 2-11 A model of DNA repair pathways i n Drosophila based on interactions between second chromosome mus mutations 74 4-1 Procedures used to analyze the cosegregation of the mus. ts l e t h a l , and female s t e r i l e phenes of mus209 B 1 107 4-2 Complementation maps of l e t h a l mutations uncovered by the M(2) 017 deficiency 117 4-3 S e n s i t i v i t y of mus209 B 1 homozygotes to heat or radi a t i o n treatments during development 120 4-4 S e n s i t i v i t y of mus209 B 1 homozygotes to heat pulses (29 DC) during development 123 A-1 Mating and sel e c t i o n protocol used to i s o l a t e second chromosome mus mutations 161 B-l MMS s e n s i t i v i t y and maternal e f f e c t s i n mus205 B 1. mus2Q8 B 1 and mus210 B 1 166 v i i i ACKNOWLEDGEMENTS I would l i k e to thank Tom G r i g l i a t t i , my thesis supervisor, for h i s u n f a i l i n g support, understanding and encouragement. For his advice on matters s c i e n t i f i c and otherwise, I am most appreciative. Don S i n c l a i r and Bob Devlin offered encouragement and made many valuable suggestions about t h i s research. I have benefited immeasurably from t h e i r knowledge and expertise. Many others i n the f l y lab made the day-to-day drudgery of f l y -f l i c k i n g sufferable, i f not enjoyable — I would e s p e c i a l l y l i k e to thank Murray Richter, Annette Bailey, Jo-Ann Brock and Vivian Ngan for t h e i r friendships. I am also g r a t e f u l to Vivian for typing some of the f i r s t drafts of t h i s thesis and for getting me started on the wordprocessor. Last but not l e a s t I wish to thank my parents Anne and Ken Henderson for t h e i r support. ix CHAPTER ONE GENERAL INTRODUCTION TO CELLULAR RESPONSES TO DNA DAMAGE 1 I. PERSPECTIVES In an open system, such as our bodies represent, compounded of unstable material and subjected continually to disturbing conditions, constancy i s i n i t s e l f evidence that agencies are acting or ready to act, to maintain t h i s constancy. Cannon, 1939 Deoxyribonucleic acid (DNA) i s not a stable molecule. For example, i n mammalian c e l l s the loss of bases due to spontaneous depurination and depyrimidation may be as high as 10,000 residues per genome per day (Lindahl, 1982). At t h i s rate, Thielmann (1984) has calculated that over the l i f e t i m e of a post-mitotic human c e l l (e.g., a nerve c e l l ) , 300 m i l l i o n bases are spontaneously l o s t from DNA. This represents about 2% of the t o t a l number of bases present. Additionally, each day about 100 cytosine residues and a smaller number of adenine and guanine bases may undergo spontaneous deamination (Lindahl, 1982). In p a r t i c u l a r , deamination of 5-methylcytosine produces thymine, i t s e l f a normal base i n DNA. Thus, GT mismatchs l e f t unrepaired are p o t e n t i a l l y mutagenic. In Escherichia c o l i . 5-methylcytosine residues appear to be "hotspots" f o r spontaneous GC to AT t r a n s i t i o n mutations (Duncan and M i l l e r , 1980). The i n t r i n s i c chemical i n s t a b i l i t y of DNA i s but one po t e n t i a l source of injury to the genome. Numerous other deleterious forces also impact upon the genetic material. For 2 example, chemicals of both i n t r a - and e x t r a c e l l u l a r o r i g i n , and radiations, both natural and man-made, continually disrupt the s t r u c t u r a l i n t e g r i t y of the chromosome (e.g., Rydberg and Lindahl, 1982; Bartsch and Montesano, 1984; Hutchinson, 1985). Yet despite these genomic assaults, the informational content of the DNA i s preserved with remarkable f i d e l i t y from one c e l l generation to the next. This preservation i s achieved by an extraordinary c e l l u l a r arsenal of DNA damage-surveillance and repair functions. The remainder of t h i s chapter provides a series of b r i e f accounts of some of the major c e l l u l a r responses to nuclear DNA damage. (The r e l a t i v e l y uncharted area of mitochondrial DNA repair i s not addressed (see Foury and Lahaye, 1987).) Many of these topics are further discussed i n relevant sections of the th e s i s . Unavoidably, a substantial portion of the material i n t h i s introduction has been gleaned from work ca r r i e d out i n E. c o l i • This enteric bacterium i s by far the best characterized organism i n terms of DNA repair. Discoveries of repair processes i n E. c o l i spawned many successful searches for analogous repair responses i n other prokaryotes and i n eukaryotes. Apparently, many of the major DNA repair processes are f u n c t i o n a l l y analogous i f not mechanistically s i m i l a r over a vast phylogenetic range (Hanawalt et a l . , 1979; Lindahl, 1982; Friedberg, 1985). Indeed, cloned E. c o l i DNA repair genes have recently been shown to complement repair defects i n both human 3 and hamster c e l l l i n e s (Samson et a l . , 1986; Margison et a l . , 1987) . Despite i t s usefulness, however, extrapolating from prokaryotes to eukaryotes has c e r t a i n l i m i t a t i o n s (Walker et a l . , 1985). There are aspects of eukaryotic DNA repair which have no counterparts i n prokaryotic repair (e.g., chromatin). S i m i l a r l y , there are aspects of DNA processing and repair i n metazoans which have no counterparts i n u n i c e l l u l a r organisms (e.g., poly(ADP-ribosylation)). For p r e c i s e l y these reasons DNA repair processes are being studied i n organisms other than bacteria. The yeast Saccharomyces cerevisiae and the f r u i t f l y Drosophila melanogaster are prominent examples. Accordingly, these two eukaryotic organisms are emphasized i n the following review. At t h i s juncture, those readers having a basic knowledge of DNA repair mechanisms can proceed to section III of t h i s chapter. For an account of the mechanisms of DNA repair more det a i l e d than that provided here, the reader i s referred to the comprehensive monograph by Friedberg (1985) which served as the primary source of t h i s information. I I . CELLULAR RESPONSES TO DNA DAMAGE In 1949 Kelner reported that the s u r v i v a l of UV-irradiated Streptomyces griseus spores could be improved dramatically i f , following UV treatment, the conidia were exposed to v i s i b l e 4 l i g h t . T h i s o b s e r v a t i o n marks the b e g i n n i n g of the study of DNA r e p a i r phenomena. Subsequently, l i g h t - i n d e p e n d e n t r e p a i r phenomena were d i s c o v e r e d , and these are c o l l e c t i v e l y r e f e r r e d t o as dark r e p a i r p r o c e s s e s s . The b i o c h e m i c a l b a s i s of the l i g h t - m e d i a t e d r e c o v e r y r e a c t i o n ( p h o t o r e a c t i v a t i o n ) i s c o n s i d e r e d i n s e c t i o n A. S e c t i o n s B through D d e s c r i b e a v a r i e t y of dark r e p a i r responses t o DNA damage. A schematic overview of c e r t a i n of these r e p a i r pathways i s i l l u s t r a t e d i n F i g u r e 1. A . Enzyme-catalyzed p h o t o r e v e r s a l o f p y r i m i d i n e dimers When exposed t o s u n l i g h t i n the 254 nm wavelength range ( u l t r a v i o l e t , UV), DNA s u f f e r s a c o n s t e l l a t i o n of base a l t e r a t i o n s , the most abundant u s u a l l y being the cyclobutane p y r i m i d i n e dimers (TT, CT, or CC) ( F r i e d b e r g , 1985). T h i s c l a s s o f photoproduct l i k e l y r e p r e s e n t s the major c y t o t o x i c (but not mutagenic) l e s i o n i n U V - i r r a d i a t e d DNA ( F r a n k l i n and H a s e l t i n e , 1986). Given the b i o l o g i c a l r e l e v a n c e of these DNA l e s i o n s , i t i s not s u r p r i s i n g t h a t most, i f not a l l , organisms possess enzymes (photolyases) which s p e c i f i c a l l y a c t t o r e p a i r p y r i m i d i n e dimers. Photolyases b i n d t o d i m e r - c o n t a i n i n g DNA and, i n the presence of v i s i b l e l i g h t , monomerize the j o i n e d bases. T h i s UV-damage r e v e r s a l phenomenon i s termed p h o t o r e a c t i v a t i o n (see F r i e d b e r g , 1985). E v i d e n t l y , c e l l s have taken advantage of the f a c t t h a t i n nature, v i s i b l e l i g h t accompanies UV. 5 F i g u r e 1. A schematic diagram of DNA repair pathways (modified from Hanawalt et a l . (1979)). 6 SPONTANEOUS BASE LOSS BASE DEFECT g l y c o s y l a s e AP SITE AP endonuclease ( i n c i s i o n ) i n s e r t a s e HELIX DISTORTION (e.g., UV dimer) damage-specific endonuclease ( i n c i s i o n ) a l k y l -t r a n s f e r a s e exonuclease / polymerase / l i g a s e p h o t o l y a s e I N T A C T D N A 7 Recent f indings i n E. c o l i impl icate the photorepair gene, phr. not only i n photoreact ivat ion but i n exc i s ion repa i r as wel l (Sancar et a l . , 1984; Hays et a l . , 1985). Photolyase st imulates the UvrABC exc i s ion endonuclease to remove pyrimidine dimers (but not other bulky les ions) from DNA. The nature of t h i s cooperative i n t e r a c t i o n i s not known. I t has been suggested that photolyase might promote UvrABC endonuclease turnover, or that i t might make the dimer-containing DNA a bet ter substrate for the exc i s ion endonuclease (Sancar et a l . , 1984). In Drosophi la . a mutation defect ive i n photoreact ivat ion has been l o c a l i z e d to the second chromosome at meiot ic map p o s i t i o n 56.8. The Drosophila phr mutant a lso appears to inf luence exc i s ion repa i r (Boyd and H a r r i s , 1987). B. DNA Excison Repair The exc i s ion removal of damaged or inappropriate bases from DNA const i tu tes the major mode of DNA repa i r i n any c e l l (Friedberg, 1985). However, the term exc i s ion repa i r does not re fe r s p e c i f i c a l l y to a s ing le enzymatic mechanism. Rather, a number of d i s t i n c t but interconnected routes and a c t i v i t i e s comprise the exc i s ion repa i r pathway (Figure l ) . 1. DNA q lycosy la ses : Mediators of base exc i s ion r e p a i r DNA glycosylases (reviewed by L indahl , 1982, 1986) are prote ins of r e l a t i v e l y low molecular weight, and genera l ly of 8 narrow substrate s p e c i f i c i t y . They appear to have neither subunit structure nor cofactor requirements. Although o r i g i n a l l y i d e n t i f i e d i n E. c o l i . most DNA glycosylase a c t i v i t i e s are ubiquitous. These enzymes cleave modified or inappropriate bases at the N-glycosyl bond to create apurinic or apyrimidinic (AP) s i t e s while leaving i n t a c t the phosphodiester backbone. For example, u r a c i l DNA glcosylase and hypoxanthine DNA glycosylase s p e c i f i c a l l y catalyze the removal from DNA of u r a c i l and hypoxanthine, the deamination products of cytosine and adenine, respectively. A major cytotoxic l e s i o n introduced into DNA by simple methylating agents (e.g., methyl methanesulfonate, MMS) i s 3-methyladenine. Under normal circumstances t h i s modified base i s ra p i d l y released from DNA by DNA glycosylases found i n both b a c t e r i a l and mammalian c e l l s (Lindahl, 1982) . In E_j_ c o l i . the enzyme l a r g e l y responsible for t h i s reaction i s 3-methyladenine DNA glycosylase I (encoded by the tagA gene) (Karran et a l . , 1982). This enzyme i s c o n s t i t u t i v e l y expressed and has a stringent substrate s p e c i f i c i t y (although 3-ethyladenine i s also a weak substrate). tagA mutants are hypersensitive to k i l l i n g by MMS (Evensen and Seeberg, 1982). The observation that tagA mutants are not completely devoid of 3-methyladenine DNA glycosylase a c t i v i t y (they r e t a i n 5-10% of the wildtype level) led to the i d e n t i f i c a t i o n of a second 3-methyladenine DNA glycoslase i n E_j_ c o l i (Evensen and Seeberg, 1982; Karran et a l . , 1982). 3-methyladenine DNA glycosylase II, 9 the product of the alkA gene, i s an inducible enzyme that has a much broader substrate s p e c i f i c i t y than i t s counterpart. In addition to removing 3-methyladenine, the alkA product excises 3-methylguanine and two minor pyrimidine a l k y l a t i o n lesions, 0 2-methylcytosine and 0 2-methylthymine (Lindahl, 1986). When induced as part of the adaptive response to a l k y l a t i o n damage (see section II.D.2), 3-methyladenine DNA glycosylase II accounts for as much as 50-70% of the t o t a l 3-methyladenine DNA glycosylase a c t i v i t y i n E^. c o l i (Karran et a l . , 1982). In contrast to E_j_ c o l i , mammalian c e l l s (from human placenta and c a l f thymus) appear to have only a single 3-methyladenine DNA glycosylase (Lindahl,1986). The mammalian enzyme i s c o n s t i t u t i v e l y expressed and has a broad substrate s p e c i f i c i t y , but i t does not recognize p r e c i s e l y the same lesions as 3-methyladenine DNA glycosylase II from E_j. c o l i . In addition to releasing 3-methyladenine, the mammalian enzyme catalyzes the removal of 3-methylguanine and 7-methylguanine, but not the 0 2-methylpyrimidines. Ionizing radi a t i o n and cert a i n radiomimetic chemicals (e.g., dimethylsulfate) may cause base a l t e r a t i o n s which, under appropriate conditions, lead to purines with opened imidazole rings. In the case of 7-methylguanine (the most abundant les i o n i n methylated DNA (Singer and Kusmierek, 1982)), cleavage of the imidazole r i n g produces a substituted 5-formamidopyrimidine. A DNA glycosylase which catalyzes the release of t h i s altered base from DNA has been i d e n t i f i e d i n extracts of E. c o l i . rodent 10 l i v e r , c a l f thymus, and human f i b r o b l a s t s (Lindahl, 1982; Friedberg, 1985). Several other examples of DNA glycosylases can be found i n Lindahl (1982) and i n Friedberg (1985). The extent to which base excision repair i s u t i l i z e d i n Drosophila i s d i f f i c u l t to assess. Attempts to detect Drosophila DNA glycosylases that catalyze the removal from DNA of u r a c i l , 3-methyladenine, or 7-methylguanine have been unsuccessful (Friedberg et a l . , 1978; Deutsch and Spiering, 1982; Green and Deutsch, 1983). I t i s possible that Drosophila r e l i e s on mechanisms other than base excision to remove altered or inappropriate bases from DNA (Green and Deutsch, 1983). However, f l i e s are not completely lacking i n a l l DNA glycosylases as a DNA glycosylase which excises oxidatively damaged thymine residues has recently been detected i n Drosophila embryos (Breimer, 1986). 2 . AP endonucleases AP s i t e s , whether spontaneously or enzymatically produced, are substrates for a var i e t y of d i f f e r e n t enzymes. For example, there i s some i n d i r e c t evidence that enzymes termed DNA purine insertases may r e i n s e r t the appropriate missing base d i r e c t l y into apurinic s i t e s i n duplex DNA (Friedberg, 1985). A c t i v i t i e s of t h i s sort have been i d e n t i f i e d i n human c e l l extracts, i n crude extracts from E. c o l i . and more recently i n Drosophila embryos (Deutsch and Spiering, 1985). However, insertase defective mutants have yet to be i d e n t i f i e d i n any organism (Friedberg, 1985). 11 Most AP s i t e s are probably acted upon by enzymes known as AP endonucleases (see F r i e d b e r g , 1985). These DNases c l e a v e the phosphodiester bond e i t h e r 3' or 5' t o the AP s i t e r e n d e r i n g i t s u s c e p t i b l e t o the d e g r a d a t i v e a c t i o n of an exonuclease. AP endonuclease a c t i v i t i e s are u b i q u i t o u s i n nature, and c e l l s appear t o possess m u l t i p l e s p e c i e s of these enzymes. There does not appear t o be a p r o t o t y p i c AP endonuclease form, however. They are u s u a l l y monomeric p r o t e i n s r a n g i n g i n s i z e from 20 t o 40 kDa (Loeb and Preston, 1986). Very r a r e l y the AP endonuclease f u n c t i o n may be p h y s i c a l l y a s s o c i a t e d w i t h a DNA g l y c o s y l a s e (e.g., the p y r i m i d i n e dimer DNA g l y c o s y l a s e s from phage T4 (denV gene) and Micrococcus l u t e u s have a s s o c i a t e d AP endonuclease a c t i v i t i e s ) . The major AP endonuclease i n E. c o l i i s but one of s e v e r a l c a t a l y t i c a c t i v i t i e s of the xthA gene product, exonuclease I I I (Weiss, 1981). T h i s r e l a t i v e l y s m a l l p r o t e i n (28 kDa) has a 5'-a c t i n g AP endonuclease a c t i v i t y , a 3' t o 5' exonuclease f u n c t i o n , a 3' phosphatase a c t i v i t y and a r i b o n u c l e a s e H a c t i v i t y . xthA mutants are h y p e r s e n s i t i v e t o k i l l i n g by hydrogen p e r o x i d e , but are o n l y s l i g h t l y s e n s i t i v e t o a l k y l a t i n g agents such as MMS. Most c e l l s , p r o k a r y o t i c and e u k a r y o t i c a l i k e , possess numerous exonuclease s p e c i e s t h a t v a r y i n s u b s t r a t e s p e c i f i c i t y and/or d i r e c t i o n of h y d r o l y s i s ( F r i e d b e r g , 1985). E x o n u c l e o l y t i c d e g r a d a t i o n i s f o l l o w e d by p o l y m e r i z a t i o n (these events are not n e c e s s a r i l y uncoupled or uncoordinated) and 12 l i g a t i o n (Figure 1). 3. Nucleotide excision r e p a i r The removal of damaged bases by DNA glycosylases represents one of at l e a s t two alternative routes i n the excision repair pathway (see Figure 1). However, in view of the multitude of d i f f e r e n t base al t e r a t i o n s which are known to e x i s t , i t i s u n l i k e l y that c e l l s possess a DNA glycosylase unique to each and every type of DNA l e s i o n . Instead, most forms of base damage ( p a r t i c u l a r l y bulky adducts and pyrimidine dimers) are probably removed by a general nucleotide excision repair mechanism. The UvrABC endonuclease found i n E. c o l i i s the best characterized example (see Friedberg, 1985). The products of three E. c o l i genes, uvrA, uvrB, and uvrC. are absolutely required for nucleotide (or oligonucleotide) excision repair. Mutations i n any of these genes render c e l l s hypersensitive to k i l l i n g by UV (and a v a r i e t y of other mutagens), and severely defective i n dimer excision. uvrA and uvrB mutants are completely defective i n the i n i t i a l i n c i s i o n event of the excision repair process. uvrC mutants carry out the i n c i s i o n reaction, a l b e i t more slowly than wildtype c e l l s do. A l l three uvr genes have been cloned and t h e i r products p u r i f i e d , advances which have led to the elucidation of the excision repair mechanism i n E. c o l i . The UvrA, B, and C proteins are believed to i n t e r a c t during nucleotide excision repair as follows. F i r s t , the UvrA protein 13 binds weakly at a nonspecific s i t e i n DNA containing bulky les i o n s . Next, the UvrB protein associates with the bound UvrA protein i n an inte r a c t i o n that increases the a f f i n i t y of the l a t t e r f or DNA. Then, by a mechanism that i s poorly understood, the UvrAB complex translocates along the DNA duplex u n t i l the s i t e of damage i s encountered. This translocation i s dependent on the ATPase a c t i v i t y of the UvrA subunit. The UvrAB complex forms a stable association with DNA i n the v i c i n i t y of the le s i o n , and i n the presence of the UvrC protein, the UvrABC endonuclease complex ("repairosome") i n c i s e s the same DNA strand at s i t e s which flank the damage. Thus, the i n c i s i o n reaction r e s u l t s i n two c l o s e l y spaced endonucleolytic strand breaks. In the case of pyrimidine dimers and psoralen monoadducts, one of the strand s c i s s i o n s occurs 7 nucleotides upstream of the 5' member of the dimer; the other nick occurs 3 or 4 nucleotides downstream of the 3' member. Thus, a p o t e n t i a l l y excisable dimer-containing oligomer some 12 to 13 nucleotides long i s generated (Sancar and Rupp, 1983; Yeung et a l . , 1987). This distance represents about one turn of the DNA h e l i x . The i n c i s i o n events represent only the f i r s t c a t a l y t i c steps i n the o v e r a l l excision repair process. P o s t i n c i s i o n events such as fragment release and gap f i l l i n g (repair synthesis) require the products of at lea s t two additional genes, uvrD (DNA helicase II) and polA (DNA polymerase I) (Caron et al.,1985; Husain et a l . , 1985). Beginning at the 5' nick, and i n the presence of the unwinding protein DNA helicase II, 14 the 5' to 3' exonuclease function of DNA polymerase I excises the oligonucleotide fragment. In a coordinated fashion, the DNA strand being degraded i s resynthesized by the polymerase function of DNA polymerase I, which uses the undamaged opposite strand as a template. F i n a l l y , the r e s u l t i n g 3' nick i s sealed by DNA l i g a s e . Most lesions y i e l d short repair patches ranging i n s i z e from 13 to 30 nucleotides (Hanawalt et a l . , 1979). Evidently, many of the gaps created by the excision removal of the 12 to 13 nucleotide fragment are lengthened by exonucleolytic degradation (Friedberg, 1985). While the molecular basis of nucleotide excision repair i n E. c o l i i s understood i n some d e t a i l , comparatively l i t t l e i s known about the enzymology of t h i s process i n eukaryotes. For example, genes encoding products analogous to the UvrA, B, and C proteins of E. c o l i have not been found i n any eukaryote. Information concerning eukaryotic excision repair has issued mostly from work carried out i n humans, i n rodents, and i n the yeast Saccharomyces cerevisiae. These studies reveal a genetic complexity to eukaryotic excision repair that i s unparalleled. For example, i n S. cerevisiae. at le a s t 10 genes, a l l of them members of the RAD3 e p i s t a s i s group, are required for nucleotide excision repair (Haynes and Kunz, 1981). S i m i l a r l y , studies of repair defects among individuals a f f l i c t e d with xeroderma pigmentosum (XP) indicate that nucleotide excision repair i n humans may involve at least 8 or 9 d i f f e r e n t genes (e.g., Hanawalt and Sarasin, 1986). Other studies suggest that as many 15 as 15 genes may p a r t i c i p a t e i n t h i s process i n humans (see Cleaver and Karentz, 1987). Moreover, i n both man and yeast, no fewer than 5 genes p a r t i c i p a t e at the i n c i s i o n step alone (Friedberg, 1985). The basis for t h i s complexity almost c e r t a i n l y resides i n the f a c t that eukaryotic DNA, unlike b a c t e r i a l DNA, i s intimately associated with histones and non-histone chromosomal proteins i n the form of chromatin (Friedberg et a l . , 1986). Thus, a subset of these excision repair genes might encode products f o r recognizing damaged DNA i n chromatin, and/or for making otherwise inaccessible lesions available to the actual repair enzymes. Support for t h i s idea comes from experiments conducted i n both humans and yeast. To summarize b r i e f l y , c e l l -free extracts from human XP c e l l s (complementation groups A, C and G) catalyze the removal of pyrimidine dimers from deproteinized p u r i f i e d exogenous DNA, but not from endogenous chromatin. On the other hand, extracts from normal human c e l l s carry out both reactions. Furthermore, the l a t t e r extracts excise dimers from XP chromatin as e f f e c t i v e l y as they do from t h e i r native chromatin (Mortelmans et a l . , 1976; Fujiwara and Kano, 1983). These findings suggest that none of the aforementioned XP mutants are defective i n the actual c a t a l y t i c a c t i v i t i e s of i n c i s i o n and excision, but rather i n events preparatory to the i n c i s i o n reaction, e.g., at steps which render the chromatin-ensconced dimer accessible to a DNA damage-s p e c i f i c endonuclease. Similar approaches applied to 5 i n c i s i o n defective yeast mutants (radl, rad2, rad3 f rad4 and radio) have yielded i d e n t i c a l r e s u l t s and conclusions (Bekker et a l . , 1980; Reynolds et a l . , 1981). These i n v i t r o findings are further supported by more recent i n vivo experiments i n yeast. Pyrimidine dimers i n UV-irradiated plasmid DNA transformed into a rad3 mutant were repaired with wildtype proficiency, whereas dimers i n i d e n t i c a l plasmids transformed into an E. c o l i endonuclease-defective uvrA mutant were not excised at a l l (Dominski and Jachymczyk, 1984). Apparently, chromatin i s a c r u c i a l determinant of the excision repair response i n eukaryotes. A f u l l appreciation of the mechanism of nucleotide excision repair i n E. c o l i was achieved only a f t e r the p a r t i c i p a n t genes had been cloned and t h e i r products reconstituted i n v i t r o . A s i m i l a r strategy i s being adopted i n a number of eukaryotes, a l b e i t with varying degrees of success. For example, attempts to clone human repair genes using DNA t r a n s f e c t i o n to rescue the mutagen s e n s i t v i t y of XP c e l l s have been frustrated by technical problems (Lehmann, 1985; Schultz et a l . , 1985). More successful approaches have been those which introduce human DNA into repair-defective hamster c e l l s (e.g., Westerveld et a l . , 1984; Cleaver and Karentz, 1987). The r e l a t i v e ease with which DNA can be manipulated i n S. cerevisiae has enabled most of the yeast RAD genes involved i n excision repair to be cloned (see Friedberg et a l . , 1986; Perozzi and Prakash, 1986 and references therein). Nucleotide 17 sequence data reveal that RAD1, RAD2 and RAD3 encode proteins of comparable si z e (estimated at 110 kDa, 111 kDa and 90 kDa, resp e c t i v e l y ) , whereas the RADIO gene and i t s predicted product (22.6 kDa) are considerably smaller (Friedberg et a l . , 1986). The RAD1 and RAD2 polypeptides share only three small regions of amino acid sequence homology despite t h e i r nearly i d e n t i c a l s i z e . The RAD1, RAD3 and RADIO polypeptides have i n common only a single l i m i t e d region of homology. However, t h i s sequence i s also homologous to one found i n prokaryotic and eukaryotic proteins that bind and/or hydrolyze purine nucleotides. The RAD3 putative nucleotide-binding domain shows p a r t i c u l a r l y good amino acid sequence homology with the nucleotide-binding region of the UvrA protein. Somewhat less homology ex i s t s between t h i s RAD3 sequence and the ATP-binding region of the UvrD protein (Friedberg et a l . , 1986). The RAD3 protein does not appear to be fu n c t i o n a l l y analogous to either E. c o l i protein, however, since i t f a i l s to complement the UV s e n s i t i v i t y of both uvrA and uvrD mutants (Naumovski and Friedberg, 1986). The RAD3 protein also contains a putative DNA-binding domain (Naumovski and Friedberg, 1986). Codon usage data suggest that RAD1, RAD2. RAD3 and RADIO are weakly expressed genes (Friedberg et a l . , 1986). This has been confirmed experimentally for both RAD2 and RAD3. Naumovski et a l . (1985) estimate that i n exponentially growing c e l l s there are less than 5 copies of RAD3 t r a n s c r i p t per c e l l . RAD3 (and RAD1) gene expression i s neither c e l l - c y c l e - r e g u l a t e d nor 18 altered by DNA damage (Nagpal et a l . , 1985). Estimates of the amount of RAD2 mRNA are even lower at less than 1 copy per c e l l (Naumovski and Friedberg, 1984). A completely unexpected property of RAD3 i s that i t encodes a function e s s e n t i a l for v i a b i l i t y (Higgins et a l . , 1983; Naumovski and Friedberg, 1983). In t h i s respect RAD3 i s unique among excision repair genes i n S. cerevisiae (Friedberg et a l . , 1986). This aspect of RAD3 i s discussed b r i e f l y i n Chapter 4. In Drosophila, 8 n o n - a l l e l i c mutations p a r t i a l l y or completely disrupt excision repair (see section I I I ) . At least 2 l o c i , mei-9 and mus201. are absolutely required for t h i s process, and both genes encode products that function at or p r i o r to i n c i s i o n (Boyd et a l . , 1987). C e l l s derived from mus201 D 1 embryonic or l a r v a l tissue exhibit reduced l e v e l s of an AP endonuclease a c t i v i t y (Osgood and Boyd, 1982) and f a i l to carry out unscheduled DNA synthesis following treatment with UV or a l k y l a t i n g agents (Dusenbery et a l . , 1983). Several mei-9 a l l e l e s are also defective i n these parameters of excision repair (Osgood and Boyd, 1982; Dusenbery et a l . , 1983). However, whereas the mus2 01 mutations phenotypically resemble members of the RAD3 e p i s t a s i s group i n yeast and the c l a s s i c a l forms of XP i n humans, the mei-9 mutations are unique. Unlike excision defective mutants i n any organism, mei-9 mutations are highly s e n s i t i v e to io n i z i n g r a d i a t i o n and exhibit strong meiotic e f f e c t s (Boyd et a l . , 1987). Based on phenotypic s i m i l a r i t i e s between mei-9 mutants and uvrD mutations i n E. 19 c o l i . Smith et a l . (1983) have speculated that mei-9 + may encode a DNA unwinding protein. Harris and Boyd (1987) have recently provided evidence for a p r e i n c i s i o n chromatin remodeling process i n Drosophila which exposes previously inaccessible pyrimidine dimers to excision repair. C. P o s t r e p l i c a t i o n Repair Damage to the template strands i n r e p l i c a t i n g DNA may hinder, or block completely, the progress of the r e p l i c a t i v e machinery. Moreover, excision repair i s rendered i n e f f e c t u a l i n single-stranded regions of the chromosome (e.g., i n the v i c i n i t y of the r e p l i c a t i o n fork). P o streplication repair mechanisms are those which allow replisomes to resume DNA synthesis on templates containing blocks to r e p l i c a t i o n . Operationally, t h i s c a p a b i l i t y i s assessed by quantifying the molecular weight of pulse-labeled DNA at various times following mutagen exposure (e.g., see Brown and Boyd, 1981a,b). Having encountered a r e p l i c a t i v e block (e.g., a pyrimidine dimer), any of several mechanisms could permit the s t a l l e d polymerase to continue synthesizing DNA on the damaged template. For example, one model posits that the replicase, altered i n some way as a r e s u l t of i t s forced stoppage (e.g., relaxed i n i t s r e p l i c a t i v e f i d e l i t y ) , simply resumes DNA synthesis across the non-coding l e s i o n and beyond. This mechanism, termed tra n s l e s i o n DNA synthesis, may be the basis of the mutagenic component of the SOS response i n E. c o l i (see section I I . D . l . ) . A second mechanism used by c e l l s to circumvent blocks to DNA r e p l i c a t i o n i s termed daughter strand gap repair (Hanawalt et a l . , 1979). The biochemistry of t h i s process i s reasonably well understood i n E. c o l i , the organism i n which i t was f i r s t discovered (see Howard-Flanders, 1981). This model posits that gaps are generated i n the nascent DNA strand as the polymerase stops strand elongation opposite the template damage, and then r e i n i t i a t e s DNA synthesis at points downstream. As the name daughter strand gap repair indicates, the gaps i n the nascent strands are the actual substrates for t h i s repair response. They range i n s i z e from 1 to 40 kilobases (Hanawalt et a l . , 1979). These secondary DNA lesions are eliminated by a s e r i e s of recA-mediated recombinational events as described below. (Other genes have also been implicated i n daughter strand gap repair, but the extent and manner of t h e i r involvement i s , for the most part, unclear (see Hanawalt et a l . , 1979; Friedberg, 1985).) RecA proteins cooperatively bind to gaps i n duplex DNA and a l i g n them with the homologous portions of the undamaged s i s t e r duplexes. Once paired, an exchange event f i l l s each gap with i n t a c t complementary DNA from the isopolar parental strand. The parental strand d i s c o n t i n u i t i e s so produced are eliminated by repair synthesis using the undamaged portions of the complementary strands as templates. At t h i s stage, the primary l e s i o n can be removed by excision repair. These events 21 constitute the c l a s s i c a l mechanism of p o s t r e p l i c a t i o n repair. The importance of t h i s pathway for conferring UV resistance to E. c o l i i s emphasized by the observation that uvr recA double mutants are some 50 times more sensi t i v e to k i l l i n g by UV r a d i a t i o n than either single mutant alone (Howard-Flanders, 1981). Pos t r e p l i c a t i o n repair, as i t i s operationally defined, has been demonstrated i n a variety of eukaryotes, including barley (Veleminsky et a l . , 1980), yeast (Resnick et a l . , 1981), Neurospora (Calza and Schroeder, 1982), Drosophila (Boyd et a l . , 1983) and mammals (e.g., Hanawalt et a l . , 1979; Lehmann and Karran, 1981). However, the mechanisms by which eukaryotic c e l l s overcome blocks to DNA r e p l i c a t i o n i s unclear. This uncertainty i s due mainly to two factors: 1) the large genome s i z e coupled with the greater complexity of DNA r e p l i c a t i o n i n eukaryotes presents technical and i n t e r p r e t a t i v e d i f f i c u l t i e s (see Hanawalt et a l . , 1979; Lehmann and Karran, 1981), and 2) a paucity of suitable repair defective mutants. Drosophila boasts probably the largest c o l l e c t i o n of p o s t r e p l i c a t i o n repair mutants (Brown and Boyd, 1981a; Boyd and Shaw, 1982). Ten n o n - a l l e l i c mutations exhibit complete or p a r t i a l defects i n t h i s repair pathway. The 4 strongest mutants have been grouped into two classes (Brown and Boyd, 1981a). Mutations i n one group (mus302 D 1 and mus310 D 1) appear to h a l t DNA synthesis at s i t e s opposite pyrimidine dimers, whereas those i n the other group (mei-41 D 5 and mus20_5A1) allow synthesis to 22 resume beyond the lesion, creating gaps i n the process. In contrast to bacteria, a recombinational mode of po s t r e p l i c a t i o n repair does not appear to operate to any appreciable extent i n Drosophila (Boyd et a l . , 1983). In t h i s respect the Drosophila and mammalian mechanisms are s i m i l a r (Lehmann and Karran, 1981; Boyd et a l . , 1983). However, elucidation of t h i s repair pathway i n eukaryotes awaits further study. D. Inducible Repair Responses The preceeding sections have dealt mainly with the mechanistic aspects of con s t i t u t i v e DNA repair processes. This section reviews how the expression of some rep a i r - r e l a t e d genes i s altered following exposure to DNA-damaging agents. 1. The SOS regulatory network of E. c o l i When exposed to conditions that i n t e r f e r e with DNA r e p l i c a t i o n (e.g., DNA damaging agents, n a l i d i x i c acid, thymine deprivation e t c . ) , E. c o l i undergo a series of p h y s i o l o g i c a l l y diverse changes c o l l e c t i v e l y termed the SOS response (reviewed by Witkin, 1976; L i t t l e and Mount, 1982; Kenyon, 1983; Walker, 1984; Ossanna et a l . , 1986). The SOS phenotype includes an enhanced capacity f o r DNA repair (both excision repair and recombinational r e p a i r ) , enhanced mutagenesis, delayed c e l l d i v i s i o n , prophage induction, and respiratory arrest. Most of these changes presumably serve to enhance the s u r v i v a b i l i t y of the damaged c e l l . (Prophage induction i s l i k e l y an adaptation 23 of the v i r u s for escaping the imperiled c e l l . ) The products of two genes, recA and lexA, regulate the SOS response. The LexA protein i s a repressor of at l e a s t 17 separate genes i n the SOS regulon. These include: recA, and lexA i t s e l f ; the excision repair genes uvrA. B, C and D; umuC,D. an operon necessary for UV mutagenesis; sulA, a gene involved i n c e l l d i v i s i o n i n h i b i t i o n ; and at 4 four "damage inducible" (din) genes whose functions are not known. In exponentially growing c e l l s the LexA protein represses i t s target genes by binding at one or two s i m i l a r upstream operator sequences (SOS boxes). In the uninduced state, many of the SOS genes, including lexA, recA, and uvr A, B, C, and D are c o n s t i t u t i v e l y expressed at low but b i o l o g i c a l l y s i g n i f i c a n t l e v e l s . Obviously, i n the absence of r e p l i c a t i o n arrest, c e l l s must maintain a steady state concentration of LexA repressor to prevent gratuitous induction of SOS functions. S i m i l a r l y , c o n s t i t u t i v e expression of recA apparently provides s u f f i c i e n t RecA protein for i t s dual roles i n SOS regulation and genetic recombination. The need for c o n s t i t u t i v e expression of the uvr genes i s understandable i n view of t h e i r r o l e i n excision repair. Most of the general features of SOS regulation are f a i r l y well understood at the molecular l e v e l . Blocked DNA r e p l i c a t i o n generates an as yet undetermined inducing signal which activates the protease function of the RecA protein. The RecA protease cleaves and thus inactivates the LexA repressors. This r e s u l t s i n derepression of the genes of the SOS regulon and 24 e x p r e s s i o n o f the SOS phenotype. As the DNA damage i s r e p a i r e d , the i n d u c i n g s i g n a l d i m i n i s h e s , and the RecA p r o t e i n s become p r o t e o l y t i c a l l y i n a c t i v e . As the l e v e l o f LexA r e p r e s s o r r i s e s , the c e l l i s r e t u r n e d t o the uninduced s t a t e . The d i s c o v e r y of the SOS response i n E. c o l i prompted a sea r c h f o r an analogous s t r e s s response mechanism i n mammalian c e l l s . A number of f i n d i n g s , i n c l u d i n g enhanced DNA r e p a i r and mutagenesis, v i r u s i n d u c t i o n , and induced c e l l u l a r d i f f e r e n t i a t i o n , a l l f o l l o w i n g treatments t h a t damage DNA or a r r e s t i t s r e p l i c a t i o n , are s u g g e s t i v e of an SOS-like response i n mammalian c e l l s ( H e r r l i c h e t a l . , 1984). Furthermore, a R e c A - l i k e p r o t e i n t h a t promotes homologous recombination i n human c e l l s has r e c e n t l y been i d e n t i f i e d (Cassuto e t a l . , 1987). However, t h e r e i s as y e t no evidence t o suggest t h a t e u k a r y o t i c c e l l s respond t o these treatments by engaging a p r e v i o u s l y r e p r e s s e d b a t t e r y of genes whose e x p r e s s i o n i s c o n t r o l l e d by a common r e g u l a t o r y system ( F r i e d b e r g , 1985). 2 . The adaptive response to a l k y l a t i o n damage E. c o l i exposed t o low, n o n l e t h a l l e v e l s o f simple a l k y l a t i n g agents (e.g., N-methyl-N'-nitro-N-nitrosoguanidine, MNNG) soon become c o n s i d e r a b l y more r e s i s t a n t t o the mutagenic and the c y t o t o x i c e f f e c t s o f subsequent h i g h e r doses of these same chemicals (Samson and C a i r n s , 1977). T h i s phenomenon i s termed the a d a p t i v e response t o a l k y l a t i o n damage (reviewed by 25 Walker, 1984; Demple, 1987). The adaptive response i s an inducible DNA repair pathway that operates independently of the SOS response. The mutagenic and c e l l - k i l l i n g lesions are repaired by separate components of t h i s pathway. Thus, c e l l death i s not simply a consequence of mutations i n e s s e n t i a l genes. Two DNA repair functions have been i d e n t i f i e d as the p r i n c i p a l antagonists of the a l k y l a t i o n damage. DNA glycosylase II, the alkA gene product (see section I I . B . l ) , excises the p o t e n t i a l l y l e t h a l 3-alkylpurines and 0 2-alkylpyrimidines from duplex DNA. This component of the adaptive response requires DNA polymerase I to repair the r e s u l t i n g AP s i t e s . The mutagenic lesions (0 6-methylguanine and the O 4-methylpyrimidines) are repaired i n s i t u by 0 6-methylguanine methyltransferase, a product of the ada gene (McCarthy et a l . , 1984). The l a t t e r reactions are unusual i n that the covalent transfer of a single methyl group from the alkylated base i n DNA to a s p e c i f i c cysteine residue i n the carboxy terminus of the methyltransferase protein, i r r e v e r s i b l y inactivates the methyltransferase function. This novel a c t i v i t y has been described as suicide repair (see Demple and Karran, 1983). Two other genes, alkB and aidB, are also induced as part of the adaptive response. The alkB gene forms a small operon with ada. I t s product may be involved i n the excision repair of cytotoxic lesions (Kataoka and Sekiguchi, 1985). The function of the aidB gene i s unknown (Volkert and Ngyuen, 1984). 26 Control of the adaptive response resides within the ada locus i t s e l f (Teo et a l . , 1984). The 39 kDa Ada protein, l i k e the RecA protein, has multiple functions. A second cysteine residue, t h i s one at the amino terminal end of the Ada protein, serves as an alkyl-group receptor i n a d i f f e r e n t suicide methyltransferase reaction i n which methyl phosphotriesters (MeP) of the DNA backbone, and not alkylated bases, are the substrates. The MeP methyltransferase a c t i v i t y does not appear to confer resistance to c y t o t o x i c i t y or mutagenicity (McCarthy and Lindahl, 1985), but rather serves as a p o s i t i v e regulator of t r a n s c r i p t i o n i n a manner described below. Unadapted c e l l s each contain about 20 molecules of the Ada protein. Some of the MePs generated following exposure to methylating agents are repaired by the MeP methyltransferase a c t i v i t y of the Ada protein. This p o s t - t r a n s l a t i o n a l modification converts the Ada protein into an e f f i c i e n t t r a n s c r i p t i o n a l activator of at least ada and alkA. The modified Ada protein then binds to s i m i l a r sequences i n the ada and alkA promoters at s i t e s upstream of the RNA polymerase binding domain (Teo et a l . , 1986). The r e s u l t i n g increased l e v e l s of Ada (150-fold) and AlkA protein are resposible for the a l k y l a t i o n resistance phenotype. The alkyltransferase may undergo further processing to y i e l d an 18 kDa polypeptide that repairs O-alkylated bases, and a 13 kDa polypeptide that repairs MePs. While the transferase a c t i v i t i e s are retained upon p r o t e o l y t i c cleavage, the regulatory a c t i v i t y of the Ada protein i s l o s t (Teo et a l . , 1986). Teo et a l . (1986) speculate that t h i s may be one way by which the adaptive response i s switched o f f when the c e l l i s no longer exposed to a l k y l a t i n g agents. An adaptive response s i m i l a r to that i n E. c o l i has been detected i n B a c i l l u s s u b t i l i s and i n M. luteus. but not i n Salmonella typhimurium or i n S. cerevisiae (see Yarosh, 1985). Mammalian c e l l s have 0 6-methylguanine methyltransferases with physical properties s t r i k i n g l y s i m i l a r to those of the 18 kDa E. c o l i protein (Samson, 1986). Whether or not these are induced as part of a mammalian adaptive response remains a contentious issue. The much higher c o n s t i t u t i v e l e v e l s of methyltransferase molecules i n mammalian c e l l s (Friedberg, 1985) may obviate the need for an inducible system. An adaptive response to oxidation damage that may involve as many as 30 proteins has recently been described i n E. c o l i and i n S. typhimurium. The k i n e t i c s of protein a c t i v a t i o n and the fact that some of these genes are under heat shock control indicate that the regulation of t h i s response i s l i k e l y to be complex (see Demple, 1987). 3 . Molecular approaches to the question of damage- inducible functions i n eukaryotes Using random yeast gene : E. c o l i lacZ contructs, Ruby and Szostak (1985) i d e n t i f i e d 6 fusions representing at l e a s t 4 d i f f e r e n t DNA damage-inducible (din) genes i n S. cerevisiae. Five of these are responsive to a v a r i e t y of agents including 28 UV, 4-nitrocjuinoline-N-oxide (4NQ0) , gamma rays, MMS, EMS, MNNG, and methotrexate. A six t h din-lacZ fusion i s induced only by UV and methotrexate. Depending on the fusion construct and the mutagen employed, beta-galactosidase a c t i v i t y was increased over the basal l e v e l by as much as 300 times. R e s t r i c t i o n enzyme patterns of 4 of the corresponding chromosomal DIN genes indicate that they are not RAD1. RAD2, RAD3, RAD6, RADIO. RAD50, RAD51. RAD52, RAD54. or RAD55. Using a d i f f e r e n t approach to the same question, McClanahan and McEntee (1986) isol a t e d cDNA that d i f f e r e n t i a l l y hybridized to RNA from mutagen-treated and untreated yeast c e l l s . They i d e n t i f i e d two 4NQO-inducible genes, termed DDR (DNA damage regulation), neither of which i s a l l e l i c to any of the DIN genes i s o l a t e d by Ruby and Szostak (1985). Interestingly, t r a n s c r i p t i o n of both DDR genes i s also induced by heat shock, and at l e v e l s comparable to those effected by 4NQO. Heat shock/DNA damage-responsive genes have also been found i n E. c o l i (Krueger and Walker, 1984) and more recently i n Drosophila (Vivino et a l . , 1986). The Drosophila gene encodes a 1 kilobase polyadenylated t r a n s c r i p t that i s induced by UV or heat shock. Its r e s t r i c t i o n enzyme pattern d i f f e r s from those of the known Drosophila heat shock genes, none of which are induced by UV (Vivino et a l . , 1986). Although presumed, i n none of these studies has i t been demonstrated that DNA damage per se i s the actual inducing s i g n a l . In p a r t i c u l a r , the observation that the din genes are sen s i t i v e to the antimetabolite methotrexate would 29 more l i k e l y suggest that they are responding to stress caused by nucleotide pool imbalances rather than DNA damage. I I I . MTJTAGEN-SENSITIVE MUTATIONS IN DROSOPHILA MELANOGASTER As evidenced by the many examples above, the recovery and characterization of mutagen-sensitive microbial s t r a i n s has provided considerable insight into the mechanisms of DNA metabolism i n these u n i c e l l u l a r organisms. These r e s u l t s provided the rationale and the impetus to search for analogous mutations i n the m u l t i c e l l u l a r eukaryote Drosophila  melanogaster. Mutations induced at more than 30 d i s t i n c t mutagen-se n s i t i v e (mus) l o c i i n D. melanogaster render developing f l i e s abnormally s e n s i t i v e to the genotoxic e f f e c t s of DNA-damaging agents (reviewed by Smith et a l . , 1980; Boyd et a l . , 1980, 1983, 1987; Wurgler et a l . , 1986). The f i r s t mus mutations i n Drosophila were is o l a t e d i n several systematic screens for MMS-and X ray-sensitive mutants of the X chromosome. (Smith, 1973, 1976; Boyd et a l . , 1976a; Nguyen et a l . , 1978). The nearly 100 mus mutants recovered i n those screens f a l l into 9 discrete complementation groups (muslOl. mus!02. mus_105, musl06, musl08. musiog, m u s l l l . mei-9. and mei-41) (Boyd et a l . , 1987). Some mutations were found to be a l l e l e s of two previously described recombination-defective meiotic mutants, mei-9 and mei-41 (Baker and Carpenter, 1972). For these str a i n s the mei designations were retained. In view of the frequency of redundant a l l e l e s , 30 i t i s doubtful that any more MMS-sensitive l o c i w i l l be discovered on the X chromosome. I f t h i s r e s u l t can be extrapolated to the rest of the genome, then as many as 50 to 60 MMS-sensitive genes may exi s t i n Drosophila. Analogous screening procedures were subsequently employed to s e l e c t for mus mutations on both of the major autosomes. Boyd et a l (1981) recovered 34 t h i r d chromosome mutations that confer s e n s i t i v i t y to MMS and/or to the b i f u n c t i o n a l a l k y l a t i n g agent nitrogen mustard (HN2). These mutations define 11 complementation groups (mus3_01, mus302. mus3 04-mus312) (Boyd et a l . , 1987). On the second chromosome, 8 mus s t r a i n s , representing 7 separate l o c i (mus2 01-mus207), were recovered i n two MMS screens (Boyd et a l . , 1981; Snyder and Smith, 1982). Two other second chromosome mus mutants had e a r l i e r been described by Khromykh and Zakharov (1978). One of these i s s e n s i t i v e to MMS (mus201 G 1), the other i s s e n s i t i v e to i o n i z i n g r a d i a t i o n (rad20lGlj ^  More recently, 32 additional second chromosome mus mutants were i s o l a t e d by Henderson et a l . (1987). Each s t r a i n i s s e n s i t i v e to one or more of the following mutagens: MMS, HN2, the bulky adduct-forming procarcinogen N-acetyl-2-aminofluorene (AAF), and gamma radiation. Their screening protocol d i f f e r e d from e a r l i e r s e l e c t i o n schemes i n that i t permitted the recovery of temperature-conditional mus mutants. They i d e n t i f i e d 5 temperature-sensitive (ts) str a i n s i n a l l . One of them, mus209 B 1, i s a ts l e t h a l mutation (see Chapter 4). The 7 strongest MMS-sensitive s t r a i n s i n that c o l l e c t i o n were characterized extensively (Henderson et a l . , 1987). Standard genetic mapping and complementation analyses showed that these mutations i d e n t i f y 4 new second chromosome mus l o c i (mus208, mus209, mus210, and mus21l). Two mus l o c i (mus208 and mus211) are each represented by two a l l e l e s . One mutant (mus205 B 1) i s a l l e l i c to a previously characterized mus locus (Snyder and Smith, 1982). A summary of t h e i r properties i s presented i n Table 1. Although these mutations were i n i t i a l l y selected on the basis of s e n s i t i v i t y to MMS, a l l of them (or t h e i r a l l e l e s ) exhibit s e n s i t i v i t y to at least one other mutagen, including some form of radiation. Furthermore, i n most cases the patterns of mutagen c r o s s - s e n s i t i v i t y vary from locus to locus, but are s i m i l a r between a l l e l e s . The former observation (especially as i t applies to the radiation s e n s i t i v i t y ) suggests that these mutations i d e n t i f y bona f i d e DNA r e p a i r - r e l a t e d genes as opposed to genes involved simply i n the uptake and/or metabolism of exogenously applied MMS. The l a t t e r observation suggests that most of these mus s t r a i n s represent genes whose products operate at d i f f e r e n t steps i n one of several d i f f e r e n t repair pathways. The notion that mus mutations are defective i n some aspect of DNA repair has i n fact been confirmed biochemically for many of the o r i g i n a l mus i s o l a t e s . Thus, of the 18 d i f f e r e n t mus l o c i which have been examined for repair abnormalities, 4 are defective i n the excision repair of UV-damaged DNA (Boyd et a l . , Table 1. SUMMARY OF PROPERTIES OF mus MUTANTS OF THE SECOND CHROMOSOME Strain Mutagen c r o s s - s e n s i t i v i t y a Map po s i t i o n MMS HN2 AAF BP UV io n i z i n g radiation 2 0 1 D 1 + + + 23 205 A 1 + - + - 64 205 B 1 + - - + - 54.9+1.6 208 B 1 + - + + - 89.8+3.3 209 B 1 + - - - + 92.8+2.6 210 B 1 + + + + - 69.1±3.1 211 B 1 + + - - + 50.4+3.1 a + = sen s i t i v e , - = not sensitive, blank = not tested. Although not indicated here, the degree of mutagen s e n s i t i v i t y may vary considerably between mutants (e.g., see Henderson et a l . , 1987). Data for mus201 D 1 and mus2_05A^ were compiled from Boyd et a l . (1982, 1987) and Snyder and Smith (1982). b Map positions and 95% confidence i n t e r v a l s for the "B" series mutants were determined as described i n Chapter 4 MATERIALS AND METHODS. Map positions for mus201 D 1 and mus205 A 1 were obtained from Boyd et a l . (1987). 33 1976b, 1982; Boyd and Harris, 1981; Luchkina et a l . , 1982), 6 are defective i n p o s t r e p l i c a t i o n repair (Boyd and Setlow, 1976; Brown and Boyd, 1981a; Boyd and Shaw, 1982), and 4 are at least p a r t i a l l y d e f i c i e n t i n both processes (Boyd and Harris, 1981; Brown and Boyd, 1981a). Mutations at 4 other mus l o c i express neither excision nor p o s t r e p l i c a t i o n repair abnormalities. However, some of these str a i n s do exhibit minor defects i n DNA synthesis following mutagen treatment (Brown and Boyd, 1981b). The remaining chapters in t h i s thesis characterize further the newest members of the Drosophila mus gene repertoire (Henderson et a l . , 1987). Chapter 2 describes, using genetic t e s t s , the manner i n which the new second chromosome mus genes are f u n c t i o n a l l y i n t e r r e l a t e d . Chapter 3, using observations made i n Chapter 2, explores the p r a c t i c a l p o s s i b i l i t y that multiply-mutant mus s t r a i n s may be useful as rapid and s e n s i t i v e indicators of chemical genotoxicity. Chapter 4 singles out for further genetic and developmental analysis the i n t r i g u i n g mutagen-sensitive locus mus209. 34 CHAPTER TWO INTERACTIONS BETWEEN MMS-SENSITIVE MUTATIONS 35 INTRODUCTION The o v e r a l l c e l l u l a r response to genome damage i s a complex molecular tour de force that i s poorly understood. For example, i n Drosophila melanogaster nearly 30 d i f f e r e n t genes control s e n s i t i v i t y to the DNA a l k y l a t i n g agent methyl methanesulfonate (MMS), and to add to the complicacy, numerous other r e p a i r -related genes have been i d e n t i f i e d (reviewed by Smith et a l . , 1980; Boyd et a l . , 1983, 1987; Wurgler et a l . , 1986). A simple yet powerful genetic approach to the problem of determining the functional i n t e r r e l a t i o n s h i p s among mutagen-sensitive (mus) genes i s double mutant analysis (Brendel and Haynes, 1973; Cox and Game, 1974). This technique, pioneered i n the bakers' yeast Saccharomyces cerevisiae. has been instrumental i n shaping the conceptual framework which underlies the current view of the eukaryotic response to genome damage. Mutations i n double mutants may interact i n one of three ways: e p i s t a t i c a l l y , a d d i t i v e l y , or s y n e r g i s t i c a l l y (reviewed by Haynes and Kunz, 1981; Game, 1983). In an e p i s t a t i c i n t e r a c t i o n , the double mutant s t r a i n i s no more sen s i t i v e to k i l l i n g by a mutagen than the most sensi t i v e single mutant. E p i s t a s i s implies that the gene products (conceptualized as DNA repair enzymes) operate at sequential steps i n a l i n e a r repair pathway. In an additive interaction, the s e n s i t i v i t y of the double mutant i s simply the sum of the s e n s i t i v i t i e s of the single mutants. A d d i t i v i t y i s thought to ar i s e when d i s t i n c t gene products use as substrates d i f f e r e n t DNA lesions produced 36 by a single mutagen. In a synergistic i n t e r a c t i o n , the s e n s i t i v i t y of the double mutant exceeds the l e v e l of s e n s i t i v i t y expected for an additive i n t e r a c t i o n . Synergism implies that the gene products, as components of d i f f e r e n t repair pathways, compete for the same DNA l e s i o n . The r e s u l t s of numerous multiple mutant analyses i n yeast indicate the existence of 3 e p i s t a s i s groups: RAD3. RAD6, and RAD52 (see Haynes and Kunz (1981) and references therein). Although some overlap exists (e.g., see Eckardt-Schupp et a l . , 1987), i t i s widely held that these groups define discrete c e l l u l a r (dark repair) responses to radiation-damaged DNA, and that c o l l e c t i v e l y , they constitute a complete set of DNA repair pathways. These ideas have emerged from the following observations. Mutant members within a group generally exhibit group-specific patterns of mutagen-sensitivity (for exceptions see Haynes and Kunz, 1981; Hoekstra et a l . , 1986), and display common, biochemically-defined repair defects (see Haynes and Kunz, 1981; Game, 1983). Furthermore, haploid t r i p l e mutant st r a i n s , composed of one mutant from each e p i s t a s i s group, exhibit " s i n g l e - h i t " s u r v i v a l responses at UV doses that produce only 1-2 pyrimidine dimers per genome (Cox and Game, 1974). F i n a l l y , mutants is o l a t e d on the basis of s e n s i t i v i t y to mutagens other than radiation (including MMS) almost invariably f a l l into one of the known ep i s t a s i s groups (e.g., Prakash and Prakash, 1977; Henriques and Moustacchi, 1980; Siede and Brendel, 1982). Taken together, these observations suggest that 37 no other major repair processes (apart from photoreactivation) e x i s t i n S. cerevisiae. Whether t h i s s i t u a t i o n t y p i f i e s the eukaryotic repair response remains to be determined. However, data from a number of other eukaryotic sources, including the f i s s i o n yeast Schizosaccharomyces pombe (Phipps et a l . , 1985), the bread mold Neurospora crassa (Kafer, 1983), and the nematode Caenorhabditis  eleaans (Hartman, 1985) are consistent with a t r i p a r t i t e response to DNA damage. In Drosophila. the scope of double mutant analyses has been r e s t r i c t e d l a r g e l y to the study of interactions among mutagen-se n s i t i v e mutants of the X chromosome (Baker et a l . , 1976, 1982; Smith, 1978; Nguyen et a l . , 1979; Smith et a l . , 1980). The types of interactions seen p a r a l l e l those observed i n yeast. However, too few mutant combinations have been characterized to provide a view as comprehensive as that i n S. cerevisiae . The present study i s an extension of previous double mutant analyses i n Drosophila. I t represents the f i r s t analysis of interactions among autosomal mus l o c i , s p e c i f i c a l l y those on the second chromosome. A t o t a l of 10 d i f f e r e n t multiple mutant mus str a i n s were tested for s e n s i t i v i t y to MMS. The r e s u l t s of these experiments p a r a l l e l the e a r l i e r findings i n Drosophila and suggest that DNA repair pathways i n t h i s complex eukaryote may be organized s i m i l a r l y to those i n yeast. 38 MATERIALS AND METHODS Strains Detailed descriptions of the v i s i b l e mutations and special chromosomes used can be found i n Lindsley and G r e l l (1968). Eight double mus stra i n s and two t r i p l e mus st r a i n s were analyzed i n t h i s study. They are as follows: mus201 D 1 mus205 B 1. mus201 D 1 mus208 B 1. mus201 D 1 mus210 B 1. mus205 B 1 mus208 B 1. mus205 B 1 mus210 B 1, mus208 B 1 mus210 B 1. mus208 B 1 mus211 B 1. mus210 B 1 mus211 B 1. mus205 B 1 mus208 B 1 mus2JL0B1, and mus208 B 1 mus210 B 1 m u s l l 6 2 . The mus mutations bearing the alphanumeric superscript BI or B2 were is o l a t e d as described (Henderson et a l . , 1987; see also Appendix A). A l l single and multiple mutant mus stra i n s carry the recessive mutations b p_r cn except cn mus201 D 1 (Boyd et a l . , 1982) and cn bw mus205 A 1 (Snyder and Smith, 1982), kindly provided by Dr. J . B. Boyd, and mus201 G 1 (Luchkina et a l . , 1982), obtained through the generosity of Dr. I. A. Zakharov. Mutant chromosomes were kept i n stock over the multiply-inverted balancer chromosomes In(2LR)CyO ( a l l single mutants except mus201 D 1) or In(2LR)SM5 (mus201 D 1 and a l l multiple mutants). Both inversions (hereafter referred to as CyO and SM5. respectively) are i d e n t i f i e d by the dominant marker Curly wings (Cy.) . Culture conditions were as described (Henderson et a l . , 1987). Methyl methanesulfonate (MMS) (CAS No. 66-27-3) was obtained from Sigma Chemical Company. 39 S t r a i n Construction I n i t i a l l y , double mutant st r a i n s were constructed that combined second and t h i r d chromosomal mus mutations. These suffered both v i a b i l i t y and fecundity problems, probably owing to the presence of the two balancer chromosomes. These early e f f o r t s were abandoned i n favor of constructing double mutant st r a i n s composed only of mus l o c i from the second chromosome. Double mus st r a i n s were constructed as diagrammed i n Figure 1. Females, trans-heterozygous for the two mus l o c i of in t e r e s t (e.g. A and B) were mated en masse to Gla / SM5 males. Putative double mus recombinant F j males (balanced over SM5 or Gla) were i n d i v i d u a l l y mated i n v i a l s to 3 or 4 Gla / SM5 females. cn Cy ( i . e . Gla"1") F2 females were c o l l e c t e d from each l i n e and mated to phenotypically s i m i l a r male sib s . Each putative double mus l i n e was tested for mutagen s e n s i t i v i t y and f a i l u r e to complement appropriate single mus s t r a i n s . A t o t a l of 13 d i f f e r e n t double mus stra i n s were constructed (although only 8 were analyzed i n t h i s study). Ten of these represent a l l possible pairwise combinations of 5 discrete second chromosomal mus l o c i (Henderson et a l . , 1987). The other 3 include the mus201 D 1 mutation. In addition, 2 t r i p l e mus s t r a i n s were synthesized (from appropriate single and double mus strains) by following procedures s i m i l a r to those used to make the double mutants. 40 F i g u r e 1. Procedure used t o c o n s t r u c t multiply-mutant mus s t r a i n s . 41 mus A musB Gla SM5 musA musB SM5 Gla cfcT mus A musB SM5 or Gla putative recombinants cr mus A musB SMS cfd 1 viable homozygotes ? 1. mutagen test 2. complementation test with single mus strains A and B 42 M M S Dose Response Curves For these t e s t s , 5 t o 10 yeast fed, mus / Cy. females were mated i n v i a l s t o about h a l f as many homozygous mus males. I n i t i a l l y , p a r ents were t r a n s f e r r e d t o new v i a l s each day f o r 2 or 3 days. These c u l t u r e s were l e f t u n t r e a t e d and served as c o n t r o l s (developmental temperature = 25°C). In the t e s t s e r i e s , females were allowed t o o v i p o s i t f o r 12+1 hours a t 22°C. C u l t u r e s were t r e a t e d by t o p i c a l l y a p p l y i n g 0.25 mL MMS at 24+2 hours f o l l o w i n g the s t a r t of o v i p o s i t i o n ( i . e . , p r i o r t o h a t c h i n g ) . Approximately 12 hours l a t e r , t r e a t e d c u l t u r e s were s h i f t e d t o 25°C where they remained f o r the d u r a t i o n of development. S u r v i v a l v a l u e s are presented as a r a t i o o f mus homozygotes t o mus / Cy_ hete r o z y g o t e s . These v a l u e s were normalized t o the homozygote:heterozygote r a t i o o b t a i n e d i n the c o n t r o l s . 43 RESULTS In t o t a l , 13 double mus st r a i n s were constructed (although interactions were analyzed i n 8 st r a i n s only). Ten of these represent a l l possible pairwise combinations of 5 d i s c r e t e second chromosome mus l o c i (Henderson et a l . , 1987). The other 3 include mus201 D 1 together with mus2_05B1, mus2_08B1 or mus210 B 1. In addition, 2 t r i p l e mus st r a i n s were synthesized: mus205 B 1  mus208 B 1 mus210 B 1 and mus208 B 1 mus210 B 1 mus211 B 2. The p o s s i b i l i t y that the progenitor mutations, although v i a b l e sing l y , might intera c t to cause l e t h a l i t y i n the double mutant was i n i t i a l l y a concern. Such synthetic l e t h a l interactions may be r i f e among combinations of mutants at l o c i involved i n chromosome metabolism (for examples i n Drosophila. Neurospora and Saccharomyces see Smith et al.(1980), Kafer (1983) and Malone and Hoekstra (1984), r e s p e c t i v e l y ) . However, none of the 15 multiply-mutant mus s t r a i n s described above turned out to be l e t h a l . Moreover, with the exception of s t r a i n s bearing mus2_09B1 (which are not included i n t h i s analysis - see below), none of the homozygous mus individuals i n any s i n g l e or double mutant s t r a i n appears to s u f f e r from v i a b i l i t y problems (Table 1). The r a t i o of homozygotes to heterozygotes i s very close to the expected value of 1 for a l l but one s t r a i n . In the case of mus201 D 1 mus210 B 1, the survival r a t i o i s skewed dramatically i n favor of the mus homozygotes. The reason for t h i s deviation i s unknown. The t r i p l e mutant st r a i n s exhibit only a s l i g h t reduction i n v i a b i l i t y . This Table 1. RELATIVE VIABILITY OF mus HOMOZYGOTES AND HETEROZYGOTES IN UNTREATED CULTURES Strai n Relative v i a b i l i t y of homozygous mutants a'k b pr cn 201 D 1 205 A 1 205 B 1 208 210 211 BI BI BI 201 201 201 205 DI DI DI BI 205 B 1 208 B 1 208 B 1 210 BI 205 208 210 208 BI BI BI BI 210 B 1 210 B 1 211 B 1 211 BI 205 BI 208 BI 210 BI 208 B 1 210 B 1 211 B 2 0.96 0.94 0.93 1.00 0.98 0.97 0.98 1.05 0.93 1.61 0.99 1.03 1.07 1.02 0.96 [3869) 1668) [3990) [5073) 1710) 1534) ;i932) [6115) [3576) [3998) ;3883) [5858) [4233) [3433) [3320) 0.89 0.88 [2814) ;i3944) In a l l cases, progeny were derived from matings between heterozygous mus / Cy females and homozygous mus males (see MATERIALS AND METHODS). Cultures were maintained at 25°C. a Relative v i a b i l i t y = no. of mus/mus adults no. of mus/Cy adults Total number of progeny are given i n parentheses. 45 s i t u a t i o n c o n t r a s t s w i t h t h a t i n Neurospora where the poor v i a b i l i t y of double mutant s t r a i n s p r e c l u d e s the s y n t h e s i s of t r i p l e mutant mutagen-sensitive l i n e s (Kafer, 1983). For those double mutant s t r a i n s b e a r i n g mus209 B 1. MMS s e n s i t i v i t i e s w i l l have t o be examined a t 22°C. mus209 B 1 i s a t e m p e r a t u r e - s e n s i t i v e l e t h a l mutation t h a t i s n e a r l y completely l e t h a l , even a t 25°C (see Chapter 4 ) . S i n g l e Mutant Versus Double Mutant MMS S e n s i t i v i t i e s Among p r e v i o u s l y recovered second chromosomal mus mutations, mus201 D 1 and mus205 A 1 are the most s e n s i t i v e t o MMS (Boyd e t a l . , 1982; Snyder and Smith, 1982). For t h i s reason, and because both mutations e x h i b i t c a t e g o r i c a l d e f e c t s i n DNA r e p a i r , they are i n c l u d e d here t o serve as y a r d s t i c k s a g a i n s t which members of the newest c o l l e c t i o n o f second chromosomal mus mutants can be compared. F i g u r e 2 shows the MMS-survival curves t h a t were generated f o r the a l l e l i c mutations mus205 A 1 and mus205 B 1. I t can be seen t h a t mus205 B 1 i s by f a r the more s e n s i t i v e a l l e l e . S i n c e the weaker mus205 A 1 s t r a i n i s completely d e f e c t i v e i n p o s t r e p l i c a t i o n r e p a i r (Brown and Boyd, 1981a; Boyd and Shaw, 1982) and p a r t i a l l y d e f e c t i v e i n e x c i s i o n r e p a i r (Boyd and H a r r i s , 1981), i t i s i n f e r r e d t h a t mus205 B 1 has r e p a i r d e f e c t s t h a t are s i m i l a r t o , and a t l e a s t as severe as those i n mus205 A 1. 46 Figure 2. MMS s e n s i t i v i t y of mus201 D 1 f mus205 A 1. mus205 B 1 and mus201 D 1 mus205 B 1. S o l i d squares, b p_r cn (3575, 2838); t r i a n g l e s , mus201 D 1 (1718, 969); open c i r c l e s mus205 A 1 (1905, 989); s o l i d c i r c l e s , mus205 B 1 (2310, 1591); open squares, mus201 D 1 mus205 B 1. The numbers i n parentheses indicate the average, and the minimum number of f l i e s scored per non-zero dose point, respectively. The error bars indicate 1 standard deviation of the mean calculated from at lea s t 3 separate treatments (data for mus205 A 1 at 0.04% MMS were obtained from 2 treatments). 47 0.01 0.02 0.03 0.04 % MMS 48 E p i s t a t i c Interactions The biochemical pleiotropy of mus2 0 5 A 1 suggests that mus205+ encodes a product that i s common to both the excision and po s t r e p l i c a t i o n repair pathways. In order to determine the e f f e c t of a mus205+ d e f i c i t on other known or suspected DNA repair defective mutants, 3 d i f f e r e n t mus20J5 B 1-containing double mutant mus st r a i n s were examined for s e n s i t i v i t y to MMS. Tests of double mutants involving mus205 B 1 and any of mus201 D 1. mus208 B 1, or mus210 B 1, have revealed e p i s t a t i c interactions; that i s , each double mutant s t r a i n i s no more sen s i t i v e to MMS than the most sensi t i v e single mutant, mus205 B 1 (Figures 2, 3, and 4 ) . These re s u l t s are s i m i l a r to those of Smith et a l . (1980) who found that mus205 A 1 and mei-9 a interact e p i s t a t i c a l l y . Based on these findings, i t would appear that mus_2j05+ and the other MMS-sensitive l o c i described here act sequentially i n a common l i n e a r repair pathway. The excision repair defects assigned to mei-9 a (Boyd et a l . , 1976b), mus201 D 1 (Boyd et a l . , 1982), and mus210 B 1 (see below) are consistent with t h i s notion (mus208 B 1 has not been characterized biochemically). However, t h i s interpretation i s complicated by the fact that mus205+ has a pos t r e p l i c a t i o n repair function as well. Moreover, the interactions observed i n the paired combinations of mus201 D 1. mus208 B 1 and mus210 B 1 (see below) suggest a complexity that i s not revealed by the e p i s t a t i c interactions j u s t described. 49 F i g u r e 3. MMS s e n s i t i v i t y of mus205 B 1 mus208 B 1 compared t o i t s component s i n g l e mutants. C i r c l e s , mus205 B 1 (2310, 1591); squares, mus208 B 1 (2632, 1411); t r i a n g l e s mus205 B 1 mus208 B 1 (2087, 1632). For a d d i t i o n a l i n f o r m a t i o n see legend t o F i g u r e 2. 50 51 F i g u r e 4. MMS s e n s i t i v i t y of mus205 B 1 mus210 B 1 compared t o i t s component s i n g l e mutants. T r i a n g l e s , mus205 B 1 (2310, 1591); squares, mus210 B 1 (1675, 926); c i r c l e s mus205 B 1  mus210 B 1 (2751, 2261). For a d d i t i o n a l i n f o r m a t i o n see the legend t o F i g u r e 2. 52 0.01 0.02 0.03 0.04 % MMS 53 An Additive Interaction The mutations mus208 B 1 and mus211 B 1 appear to in t e r a c t a d d i t i v e l y (Figure 5). This r e s u l t suggests that these mus l o c i operate i n disparate repair pathways, each capable of removing a d i f f e r e n t type of DNA l e s i o n caused by MMS. Consistent with t h i s hypothesis i s the observation that the mutagen cross-s e n s i t i v i t i e s of these two single mutants d i f f e r (see Table 1 i n Chapter 1). Synergistic Interactions Several instances of synergism were observed. For example, the combination of mus210 B 1 and mus211 B 1 exhibits a f a i r l y strong synergism (Figure 6). This i n t e r a c t i o n i s rather s t r i k i n g since mus211 B 1. by i t s e l f , i s not sen s i t i v e to MMS unless extremely high doses (0.12 to 0.15 %) are applied (Henderson et a l . , 1987). The synergism implies that mus210 B 1 and mus211 B 1 i d e n t i f y defects i n d i f f e r e n t repair pathways that normally compete for the same type of MMS-induced l e s i o n . Furthermore, i t can be inferred that the pathway i d e n t i f i e d by mus211 B 1. although usually of comparatively minor importance for the repair of damage caused by MMS, assumes a more prominent r o l e i n c e l l s d e f i c i e n t i n the mus210+ function. In other words, i n the absence of normal l e v e l s of mus210+ a c t i v i t y , DNA lesions are "channelled" into the mus211+ pathway (see von Borstel and Hastings, 1985). A second example of synergism i s provided by the combination of mus208 B 1 and mus210 B 1 (Figure 7). The heightened MMS-Figure 5. MMS s e n s i t i v i t y of mus208 B 1 mus211 B 1 compared to i t s component single mutants. Squares, mus208 B 1 (2632, 1411); t r i a n g l e s , mus211 B 1 (2182, 1244); c i r c l e s , mus208 B 1 mus211 B 1 (3712, 3053). For additional information see legend to Figure 2. 55 20 H I 1 1 1 • 0.01 0.02 0.03 0.04 % MMS 56 F i g u r e 6 . MMS s e n s i t i v i t y o f mus210 B 1 mus211 B 1 compared t o i t s component s i n g l e mutants. Squares, mus210 B 1 (1675, 926); t r i a n g l e s mus211 B 1 (2182, 1244); c i r c l e s , mus210 B 1 mus211 B 1 (3520, 996). For a d d i t i o n a l i n f o r m a t i o n see the legend t o F i g u r e 2. 57 0.01 0.02 0.03 0.04 % MMS 58 F i g u r e 7. MMS s e n s i t i v i t y of mus208 B 1 mus210 B 1 compared t o i t s component s i n g l e mutants. Squares, mus208 B 1 (2632, 1411); t r i a n g l e s , mus210 B 1 (1675, 926); c i r c l e s , mus208 B 1 mus210 B 1 (2735, 1701). For a d d i t i o n a l i n f o r m a t i o n see legend t o F i g u r e 2. 59 0.01 0.02 0.03 0.04 % MMS 60 s e n s i t i v i t y of the mus208 B 1 inus210 B 1 homozygotes i s es p e c i a l l y evident at 0.02 % MMS. A very s i m i l a r pattern of s e n s i t i v i t y i s seen i n the combination of mus201 D 1 and mus208 B 1 (Figure 8). As mus201 D 1 and mus210 B 1 both i d e n t i f y genes required for excision repair (see below), t h e i r s y n e r g i s t i c interactions with mus208 B 1 suggest that the l a t t e r locus encodes an a c t i v i t y that functions i n some alternate repair pathway, for example, i n some aspect of the p o s t r e p l i c a t i o n repair process. The e p i s t a s i s between mus205 B 1 and mus208 B 1 i s consistent with t h i s i n t e r p r e t a t i o n . The single mutants mus201 D 1 and mus210 B 1 are v i r t u a l l y indistinguishable i n t h e i r responses to mutagens; each displays a pattern of mutagen c r o s s - s e n s i t i v i t y that i s c h a r a c t e r i s t i c of excision repair-defective mutations generally. Indeed, mus201 D 1 i d e n t i f i e s a gene whose product normally functions at or p r i o r to the i n i t i a l i n c i s i o n step of the excision repair response (Boyd et a l . , 1982). While the mus210 B 1 s t r a i n has not been characterized biochemically, i t f a i l s to complement the MMS s e n s i t i v i t y (data not shown) of an independently i s o l a t e d mus mutation that i s defective i n excision repair (Luchkina et a l . , 1982) . Thus, an excision repair defect almost c e r t a i n l y forms the basis for the mutagen s e n s i t i v i t y of mus210 B 1 as well. Accordingly, mus201 D 1 and mus210 B 1 both are assumed to be members of the same e p i s t a s i s group, a Drosophila equivalent of the RAD3 group (excision repair) of yeast. The yeast model predicts that members of the same e p i s t a s i s group should inte r a c t e p i s t a t i c a l l y . To te s t t h i s i n regard to 61 Figure 8. MMS s e n s i t i v i t y of mus201 D 1 mus208 B 1 compared to i t s component single mutants. Triangles, mus201 D 1 (1718, 969); squares, mus208 B 1 (2632, 1411); c i r c l e s , mus201 D 1 mus208 B 1 (2839, 491). For additional information see legend to Figure 2. 62 0.01 0.02 0.03 0.04 % MMS 63 mus201L,x and mus210BJ-. an MMS dose-response curve was generated for the mus201 D 1 mus210 B 1 s t r a i n . Instead of the expected e p i s t a s i s , a very strong synergism was observed (Figure 9). MMS S e n s i t i v i t i e s of T r i p l e Mutant Strains Any pairwise combination of mus208 B 1. mus210 B 1, and mus211 B 1 exhibits a s e n s i t i v i t y to MMS that i s greater than that of any component single mutant (see above). In view of previous examples from yeast (e.g., see Cox and Game, 1974), these findings suggest that a t r i p l e mutant s t r a i n composed of these mutations might exhibit a s e n s i t i v i t y to MMS that surpasses that of each double mutant combination. To explore t h i s p o s s i b i l i t y , the s t r a i n mus208 B 1 mus210 B 1 mus211 B 2 was synthesized. Somewhat su r p r i s i n g l y , mus208 B 1 mus210 B 1 mus211 B 2 i s no more sen s i t i v e to MMS than i s the most sensi t i v e double mutant s t r a i n , mus208 B 1 mus21Q B 1 (Figure 10). A second t r i p l e mutant s t r a i n was s i m i l a r l y tested. mus205 B 1 mus208 B 1 mus210 B 1 i s no more sen s i t i v e to MMS than i s the highly s e n s i t i v e single mutant, mus205 B 1 (Figure 10). 64 F i g u r e 9. MMS s e n s i t i v i t y of mus201 D 1 mus210 B 1 compared t o i t s component s i n g l e mutants. T r i a n g l e s , mus201 D 1 (1718, 969); c i r c l e s , mus210 B 1 (1675, 926); squares, mus201 D 1 mus210 B 1 (1196, 946). Data f o r 0.02% mus201 D 1 mus208 B 1 were obtained from 2 treatments o n l y . For a d d i t i o n a l i n f o r m a t i o n see legend t o F i g u r e 2. 65 0.01 0.02 0.03 0.04 % MMS 66 Figure 10. MMS s e n s i t i v i t y of mus205 B 1 inus208 mus210B-L and mus208 B 1 mus210 B 1 mus211 B 2. C i r c l e s , mus205 B 1 (1905, 989); s o l i d squares, mus208 B 1 mus210 B 1 (2735, 1701); open squares, mus205 B 1 mus208 B 1 mus210 B 1 (698, 497); t r i a n g l e s , mus208 B 1 mus210 B 1 mus211 B 2 (3719, 572). For additional information see legend to Figure 2. 67 0.01 0.02 0.03 0.04 % MMS 68 D I S C U S S I O N In p r i n c i p l e , functional i n t e r r e l a t i o n s h i p s among mutagen-sen s i t i v e l o c i can be ascertained by analyzing mus mutant interactions i n double mutant s t r a i n s . Using t h i s approach, i t should be possible to organize mus genes of related function into common pathways of DNA repair. In practice, however, the v a l i d i t y of a p a r t i c u l a r assignment i s la r g e l y dependent upon the uncertain nature of both the mutant a l l e l e (e.g., leakiness) and the biochemistry of the gene product (e.g., monomer versus component of a multimer). Thus, for s i m p l i c i t y , the following assumptions are made i n i t i a l l y . F i r s t , i t i s assumed that mus l o c i i d e n t i f y discrete, DNA rep a i r - r e l a t e d a c t i v i t i e s which operate at successive steps i n l i n e a r pathways. Second, i n the absence of additional data, mutagen c r o s s - s e n s i t i v i t y differences are assumed to r e f l e c t fundamental functional differences between mus gene products. (However, the c o r o l l a r y , that mutations having s i m i l a r patterns of mutagen c r o s s - s e n s i t i v i t y are d e f i c i e n t i n s i m i l a r functions, cannot always be assumed to be true (cf mei-9 and mei-41).) F i n a l l y , mus mutations are assumed to be amorphic (see Muller, 1932), at lea s t i n terms of t h e i r DNA repair functions. The v a l i d i t y of these assumptions, and additional l i m i t a t i o n s of the methodology are considered below i n reference to s p e c i f i c mutant-mutant interactions. The most severe form of mutant inte r a c t i o n i s that which r e s u l t s i n unconditional l e t h a l i t y . In Drosophila. for example, 69 4 of 11 d i f f e r e n t double mus s t r a i n s , constructed with a l l e l e s of 7 X-linked l o c i , e xhibit synthetic l e t h a l i t y (Table 2). Perhaps s i g n i f i c a n t l y , these l e t h a l interactions are r e s t r i c t e d to combinations of mutants obtained from four l o c i , of which at l e a s t three specify essential functions (Baker et a l . , 1982; G a t t i et a l . , 1983). In contrast to e a r l i e r studies (Table 2), synthetic l e t h a l interactions are not represented i n t h i s work. However, the pattern of nonlethal interactions seen previously c l o s e l y p a r a l l e l s the d i s t r i b u t i o n of interactions observed here. Thus, s y n e r g i s t i c interactions are most numerous, followed i n turn by e p i s t a t i c and additive interactions. This r e s u l t may not be s u r p r i s i n g since, on the basis of simple p r o b a b i l i t y , one would expect s y n e r g i s t i c interactions to be more prevalent than e p i s t a t i c ones i f indeed mus mutations are aligned into discrete repair pathways. The strong s y n e r g i s t i c i n t e r a c t i o n between mus201 D 1 and mus210 B 1 was s u r p r i s i n g . Conventionally, synergism implies that the i n t e r a c t i n g genes encode products of d i f f e r e n t repair pathways (epistasis groups) which compete for the same kind of DNA l e s i o n . However, t h i s interpretation i s d i f f i c u l t to reconcile with the i d e n t i c a l phenotypic properties of the mutants. Although the t r i v i a l explanation of hypomorphy cannot be discounted, a provocative al t e r n a t i v e i s that mus201+ and mus210"*" encode components of a multimeric excision repair complex analogous to the UvrABC excision complex i n E. c o l i Table 2. A COMPILATION OF PREVIOUSLY REPORTED INTERACTIONS IN DOUBLE MUTANT STRAINS Double mutant a I n t e r a c t i o n * 5 R e f e r e n c e 0 1 0 1 A 1 1 0 5 A 1 l e t h a l 6 1 0 1 A 1 m e i - 4 1 A 3 6 1 0 5 A 1 1 0 9 D 1 2 1 0 5 A 1 m e i - 4 1 A 3 6 102 mei-9 e p i s t a t i c 5 2 0 1 D 1 m e i - 9 A T 1 3 2 0 5 A 1 mei-9 a 6 mei-9 a m e i - 4 1 A 3 a d d i t i v e 6 1 0 1 D 1 mei-9 a s y n e r g i s t i c 4 102 105 5 1 0 2 A 1 m e i - 4 1 A 1 1 1 0 3 D 1 mei-9 a 4 1 0 5 A 1 mei-9 a 6 mei-9 a m e i - 4 1 D 5 4 mei-9 a mei-41 1 a For some mutations a l l e l i c d e s i g n a t i o n s were not p r o v i d e d , k In most cases the i n t e r a c t i o n was assessed based on s e n s i t i v i t y t o MMS. c (1) Baker e t a l . , 1976; (2) Baker e t a l . , 1982; (3) Dusenbery e t a l . , 1983; (4) Nguyen e t a l . , 1979; (5) Smith, 1978; (6) Smith e t a l . , 1980. 71 (Seeberg and Steinum, 1983; Yeung et a l . , 1983; Grossman et a l . , 1986). At present, there i s no d i r e c t biochemical evidence to substantiate the existence of a multimeric complex f o r nucleotide excision repair i n eukaryotes. I t s existence i s inf e r r e d l a r g e l y through circumstantial genetic evidence and from the paradigm provided by studies i n E. c o l i . For example, i n S. cerevisiae at least 5 genes orchestrate the i n i t i a l i n c i s i o n event, and no fewer than 10 l o c i p a r t i c i p a t e i n the ov e r a l l excision repair process i t s e l f . Likewise, the existence of 9 d i s t i n c t complementation groups among indivi d u a l s with xeroderma pigmentosum suggests that excision repair i n humans may involve a comparable number of genes and thus perhaps a complex i n t e r a c t i o n of gene products (Friedberg, 1985). I f a s i m i l a r l y complex excision repair mechanism ex i s t s i n Drosophila. t h i s may explain the apparent anomalous in t e r a c t i o n between mus201 D 1 and mus210 B 1. In support of t h i s notion, Perozzi and Prakash (1986) recently reported that two amorphic members of the RAD3 (excision repair) e p i s t a s i s group i n yeast i n t e r a c t s y n e r g i s t i c a l l y . Based on the preceding arguments, mus201 D 1 and mus210 B 1 have been assigned to the same pathway, namely that responsible for excision repair. I f the remaining double mutant interactions are taken at face value, then one possible organization of repa i r pathways i s i l l u s t r a t e d i n Figure 11. For s i m p l i c i t y , substrates S T and S 2 represent two d i f f e r e n t types of MMS-72 F i g u r e 11 . A model of DNA r e p a i r pathways i n D r o s o p h i l a based on i n t e r a c t i o n s between second chromosome mus mutations. 73 74 induced DNA l e s i o n . The excision repair pathway acts upon both substrates, i n keeping with i t s major role i n DNA repair. mus210 B 1 and mus211 B 1 have been assigned to d i f f e r e n t repair pathways based on t h e i r d i f f e r e n t mutagen c r o s s - s e n s i t i v i t i e s , and s y n e r g i s t i c interaction i n mus210 B 1 mus211 B 1. Both the excision repair pathway and that i d e n t i f i e d by mus211 compete for the same DNA lesion, S 2 . The notion that p a r t i c u l a r types of DNA l e s i o n can be "channelled" through d i f f e r e n t repair pathways has important implications for mutagenesis. Certain repair pathways are error-prone or mutagenic (e.g. RAD6 i n S . c e r e v i s i a e ) f whereas others are error-free (e.g. RAD3). This implies that blocks i n an error-free pathway could force a greater proportion of DNA lesions into a pathway that i s mutagenic. Conversely, blocks i n an error-prone pathway could r e s u l t i n more lesions being channelled into an error-free pathway. Although other explanations are tenable, the channelling hypothesis accounts for the observations that at least one rad3 mutant i s a mutator, and that rev3 mutants (rev3 i s a member of the RAD6 e p i s t a s i s group) are antimutators (see von Borstel and Hastings, 1985). mus208 B 1 has been assigned to a unique pathway based on two l i n e s of evidence. F i r s t , mus208 B 1 and mus210 B 1 (or mus201 D 1) int e r a c t s y n e r g i s t i c a l l y . Furthermore, the double mutant mus208 B 1 mus211 B 1 exhibits an additive i n t e r a c t i o n . The former r e s u l t implies that mus208+ and mus210+ control steps i n alternate pathways that compete for the same l e s i o n ( S i ) ; the l a t t e r r e s u l t suggests that the pathways i d e n t i f i e d by mus208 and mus211 B 1 operate independently to correct d i f f e r e n t classes of DNA l e s i o n . mus205 B 1 exhibits e p i s t a s i s with mus201 D 1 and rous210B1. as well as with mus208 B 1. These r e s u l t s can be explained i f disparate repair pathways share, at some point, a common step. In Figure 11 t h i s i s represented by the convergence of the pathways near t h e i r termini. This hypothesis i s consistent with the observation that mus205 A 1 i s d e f i c i e n t i n two kinds of repair, excision and p o s t r e p l i c a t i o n (Boyd and Harris, 1981; Boyd and Shaw, 1982). However, t h i s key p o s i t i o n occupied by mus205 seems somewhat at odds with i t s li m i t e d range of mutagen c r o s s - s e n s i t i v i t y (see Table 1 i n Chapter 1). Apparently, mus205+ i s required only to repair damage caused by monofunctional a l k y l a t i n g agents and lesions r e s u l t i n g from UV ra d i a t i o n . Damage a r i s i n g from bulky adduct-forming chemicals l i k e N-acetyl-2-aminofluorene, or from the b i f u n c t i o n a l a l k y l a t i n g agent nitrogen mustard, must be repaired by mechanisms that bypass the requirement for mus205+. Obviously, new pathways, i n addition to those already depicted i n Figure 11, are necessary to accommodate these observations. A further complication stems from the observation that the t r i p l e mutant, mus208 B 1 mus210 B 1 mus211 B 2. i s no more sensi t i v e to MMS than the most sensi t i v e double mutant, mus208 B 1 mus210 B 1. However, since d i f f e r e n t mus21l a l l e l e s were used i n making the multiple mutants, i n t e r s t r a i n comparisons may not be v a l i d even 76 though mus211 B 1 and mus211 B 2 exhibit very s i m i l a r mutagen s e n s i t i v i t i e s . Strain v a r i a b i l i t y and a l l e l i c differences were major sources of anomalous interactions i n S. cerevisiae (Game and Cox, 1972; 1973). This p o s s i b i l i t y can be tested by constructing the appropriate mus211 B 1-containing t r i p l e mutant s t r a i n . In the absence of biochemical data concerning the r o l e of these mus gene products i n DNA repair, the contributions of additi o n a l pathway analyses are somewhat lim i t e d . Ultimately, a model of DNA repair i n Drosophila that more accurately r e f l e c t s the true repair response w i l l come from a combination of genetic, biochemical, and molecular b i o l o g i c a l characterizations of mus and other rep a i r - r e l a t e d mutations. 77 C H A P T E R T H R E E M U T A G E N - S E N S I T I V E S T R A I N S A S G E N O T O X I C I T Y I N D I C A T O R S : A P I L O T S T U D Y 78 INTRODUCTION A recognition of the p o t e n t i a l l y hazardous e f f e c t s of DNA-damaging compounds, es p e c i a l l y i n r e l a t i o n to human carcinogenesis and heritable and somatic disease (e.g., Ames, 1979, 1983; Hartman, 1983), together with the knowledge that as many as 80% of a l l cancers are caused by factors i n our environment (Doll and Peto, 1981), has i n recent years made the task of i d e n t i f y i n g environmental genotoxins a public health p r i o r i t y . Because both l i f e s t y l e and occupation appear to be important determinants of the carcinogenic process (Doll and Peto, 1981), recent e f f o r t s have concentrated on i d e n t i f y i n g mutagens/carcinogens among the chemical constituents found i n foods and i n drinks (e.g., chemicals d e l i b e r a t e l y added, nat u r a l l y occurring or r e s u l t i n g from preparation), i n non-food products (e.g., cosmetics, medicinals, p e s t i c i d e s ) , and i n the work environment (Nagao et a l . , 1978). Current estimates place the number of chemicals i n commercial use at about 70,000, with 700 to 3000 new ones being introduced each year (Hoffmann, 1982). However, epidemiological studies have i d e n t i f i e d only 22 chemicals, industries, or i n d u s t r i a l processes for which there i s s u f f i c i e n t evidence to support a causal association with cancer occurrence i n man (Bartsch et a l . , 1982). Another 18 chemicals are highly suspect as human carcinogens based on data derived from both epidemiological and animal studies (Bartsch et a l . , 1982). A l l t o l d , the human carcinogen data base i s d i s t r e s s i n g l y small. For reasons of p r a c t i c a l i t y , carcinogen t e s t i n g i s necessarily c a r r i e d out i n rats and mice (and very r a r e l y i n other mammalian species). Yet even so, to t e s t j u s t a single chemical i n a rodent bioassay may take 2-4 years and cost as much as 1 m i l l i o n d o l l a r s (Lave and Omenn, 1986). These factors preclude the use of animals i n any mass screening programs for carcinogenic agents, and have spurred the development of more than 100 short-term bioassays of genotoxic a c t i v i t y (usually mutagenicity) using a vari e t y of prokaryotic and eukaryotic materials (e.g., H o l l s t e i n et a l . , 1979; de Serres and Ashby, 1981) . The Salmonella/microsome mutagenicity assay ( i . e . , the Ames test) i s the most widely used and the most extensively validated short-term t e s t (e.g., Haroun and Ames, 1981). However, the Ames t e s t by i t s e l f does not appear to be a r e l i a b l e enough predictor of ei t h e r rodent or human carcinogens (e.g., Brusick, 1983) or of mammalian germ c e l l mutagens (Bridges and Mendelsohn, 1986). Thus, a p a r t i c u l a r thrust of current environmental mutagenesis research i s to i d e n t i f y a small number of eukaryotic t e s t s that can complement the already entrenched Ames t e s t (Ashby et a l . , 1985). In princple, these complementary assays would be capable of detecting rodent mutagens and carcinogens that are d i f f i c u l t or impossible to detect as p o s i t i v e i n the Ames t e s t . Drosophila has been for years, and continues to be, an important organism for use i n genetic toxicology. I t s numerous 80 advantages have been w e l l d e s c r i b e d (Vogel and Sobels, 1976; Baars, 1980; Vogel, 1981; Graf e t a l . , 1984; V a l e n c i a e t a l . , 1984; Wurgler e t a l . , 1984, 1985; Wurgler and Vogel, 1986). The o b s e r v a t i o n r e p o r t e d i n Chapter 2, t h a t c e r t a i n multiply-mutant mus s t r a i n s are h y p e r s e n s i t i v e t o MMS, may form the b a s i s of a r a p i d , somatic g e n o t o x i t y assay i n D r o s o p h i l a . In p r i n c i p l e , i t may be p o s s i b l e t o i d e n t i f y combinations of mus mutations t h a t a r e extremely s e n s i t i v e t o a wide spectrum of d i f f e r e n t l y a c t i n g g e n o t o x i c agents (e.g., Nguyen e t a l . , 1979). To pursue t h i s p o s s i b i l i t y , two t r i p l e mutant mus l i n e s were c o n s t r u c t e d as t e s t e r s t r a i n s . T h i s chapter p r o v i d e s a p r e l i m i n a r y e v a l u a t i o n o f the proposed somatic g e n o t o x i c i t y assay. The r e s u l t s o b t a i n e d f o r 16 chemicals are r e p o r t e d . 81 MATERIALS AND METHODS Strains For a description of the v i s i b l e mutations and sp e c i a l chromosomes used i n t h i s study, consult Lindsley and G r e l l (1968) . The t r i p l e mus mutant s t r a i n s , b p_r cn mus205 B 1  mus208 B 1 mus210 B 1 and b p_r cn mus208 B 1 mus210 B 1 mus211 B 2. were constructed as described i n Chapter 2. The mutations mus208 B 1. mus210 B 1 and mus211 B 2 were selected as constituents of one t e s t e r s t r a i n because of t h e i r unique mutagen cross-s e n s i t i v i t y and demonstrated pairwise interactions. This combination of mutations i s p o t e n t i a l l y extremely s e n s i t i v e to a wide spectrum of d i f f e r e n t l y acting mutagens. The second tester s t r a i n incorporated mus205 B 1 i n place of mus211 B 2. Although mus205 B 1 d i d not int e r a c t other than e p i s t a t i c a l l y with any other mus mutation (see Chapter 2), previous unpublished observations showed that i t was highly s e n s i t i v e to a number of monofunctional a l k y l a t i n g agents. Thus, mus205 B 1 mus208 B 1  mus210 B 1 may be a p a r t i c u l a r l y good indicator of t h i s important mutagen c l a s s . Cultures were maintained as described previously (Henderson et a l . , 1987). F l i e s were raised at 22°C or 25°C. Chemicals Benzo(a)pyrene (B(a)P, 50-32-8), benzo(e)pyrene (B(e)P, 192-97-2), cyclophosphamide (CP, 6055-19-2), 1,2,3,4-diepoxybutane (DEB, 298-18-0), diethylnitrosamine (DEN, 55-18-5), dimethylnitrosamine (DMN, 62-75-9), hexamethylphosphoramide 82 (HMPA, 680-31-9), methyl methanesulfonate (MMS, 66-27-3), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 70-25-7), methylnitrosourea (MNU, 684-93-5) and safr o l e (SAF, 94-59-7) were obtained from Sigma Chemical Company. Caprolactam (CAP, 105-60-2), d i e t h y l s u l f a t e (DES, 64-67-5) and dimethyl s u l f a t e (DMS, 77-78-1) were purchased from A l d r i c h Chemical Company. Ethyl methanesulfonate (EMS, 62-50-0) was obtained from Eastman Kodak. Formaldehyde (FA, 50-00-00), as formalin, was obtained from Fisher S c i e n t i f i c . C r i t e r i a f o r Chemical s e l e c t i o n MMS, EMS, DMS, DES, MNU, MNNG, DMN and DEN are a l l monofunctional a l k y l a t i n g agents. MMS, EMS, DMS, DES and MNU were selected as a series of al k y l a t i n g agents each having a d i f f e r e n t propensity to enter into unimolecular (S^l) or bimolecular (S N2) reactions (Hoffmann, 1980). S N1 al k y l a t i n g agents (e.g., MNU) are comparatively more e f f e c t i v e at al k y l a t i n g oxygens i n nucleic acids, and consequently are better mutagens than S N2 a l k y l a t i n g agents (e.g., MMS, DMS). On the other hand, Sjj2 a l k y l a t i n g agents react with highly nucleophilic s i t e s (e.g., ri n g nitrogens) and are much more e f f e c t i v e at producing chromosomal aberrations than t h e i r S N1 counterparts (see Vogel and Natarajan, 1979). Chemicals such as EMS and DES react both unimolecularly and bimolecularly (Hoffmannn, 1980) . MNU, MNNG, DMN and DEN belong to the N-nitroso chemical c l a s s . N-nitroso compounds are widely occurring (e.g., i n tobacco smoke, nitrite-preserved f i s h products, and cer t a i n 83 cosmetics) potent carcinogens (Scanian, 1984; Bartsch and Montesano, 1984; Preussmann, 1984). Both DMN and DEN are promutagens/procarcinogens. The s t r u c t u r a l isomers B(a)P and B(e)P were chosen for study as representatives of the unsubstituted p o l y c y c l i c aromatic hydrocarbons. Both compounds occur ubiquitously i n products of incomplete combustion (e.g., cigarette smoke, gasoline and d i e s e l exhaust, b r o i l e d or smoked foods, roasted coffee e t c . ) , as well as i n unburned f o s s i l f u e l s , i n vegetables and i n vegetable o i l s etc. (IARC, 1983); i . e . , they are major environmental pollutants to which i t i s v i r t u a l l y impossible to avoid exposure. B(a)P (a bulky adduct-forming promutagen/procarcinogen) i s active i n short-term tests ( i t i s often used as a p o s i t i v e control) and i s carcinogenic to experimental animals (IARC, 1983). B(a)P may be a causative factor i n several human cancers (e.g., skin, lung and colon). Many aspects of B(a)P have been reviewed by P h i l l i p s (1983). There i s li m i t e d evidence that B(e)P i s active i n short-term t e s t s ( i t i s , however, mutagenic i n the Ames t e s t ) , and there i s i n s u f f i c i e n t data to allow an evaluation of the carcinogenicity of B(e)P — i n some studies i t i s considered a noncarcinogenic analog of B(a)P (IARC, 1983). DEB i s a potent genotoxin i n a v a r i e t y of organisms (reviewed by Ehrenberg and Hussain, 1981). I t i s an a l k y l a t i n g agent than can produce both mono- and b i f u n c t i o n a l a l k y l a t i o n 84 products. The l a t t e r adducts are i n t e r - and intrastrand cross-l i n k s . In Drosophila. genetic evidence suggests that DEB may act by producing deletions (Shukla and Auerbach, 1980; Olsen and Green, 1982). CP, a human carcinogen (Bartsch et a l . , 1982), i s a widely used chemotherapeutic a l k y l a t i n g agent which, l i k e DEB, can enter into both mono- and bif u n c t i o n a l a l k y l a t i o n reactions. However, for the l a t t e r reactions to occur, CP must f i r s t be metabolically activated (Brendel and Ruhland, 1984). FA i s both mutagenic and carcinogenic (d'A. Heck and Casanova-Schmitz, 1984). The basis for FA genotoxicity i s unclear, although FA i s known to react r e v e r s i b l y with amino groups of nucleic acids and to form crosslinks (Singer and Kusmierek, 1982). HMPA and SAF were selected as chemicals that are inactive or d i f f i c u l t to detect i n the Ames assay (de Serres and Ashby, 1981). The mechanism by which HMPA exerts i t s genotoxic e f f e c t s i s unknown. HMPA does not appear to be a simple methylating agent (Vogel et a l . , 1985). SAF i s a natural carcinogen of plant o r i g i n . I t i s a major constituent (85%) of o i l of sassafras, and i s found i n minor or trace quantities i n cocoa, mace, nutmeg, black pepper and a number of other plant products (Hall, 1973). The ultimate reactive product of SAF appears to be sa f r o l e - 1 ' - s u l f a t e , which reacts with 0 6-guanine i n DNA (Hathway, 1986). Singer and Kusmierek (1982) have reported additional reactions. 85 CAP i s a chemical which shows no evidence of carcinogen-i c i t y i n long-term rodent bioassays (Shelby and Stasiewicz, 1984). I t i s used here as a negative cont r o l . Solvents Appropriate amounts of B(a)P and B(e)P were f i r s t dissolved i n a small volume (< 1 mL) of dimethyl sulfoxide and di l u t e d accordingly with ethanol. MNNG was s i m i l a r l y dissolved i n DMSO but d i l u t e d with 70% ethanol. DMS, DES and MNU were dissolved i n ethanol. SAF was dissolved i n 70% ethanol. Water was used for a l l other chemicals. The various solvents did not a l t e r the r e l a t i v e v i a b l i t y of the genotypes. Mutagen S e n s i t i v i t y Test Protocol The p r i n c i p a l t e s t e r s t r a i n used i n t h i s study was h ET. cn mus208 B 1 mus210 B 1 mus211 B 2. A second s t r a i n , b p_r cn mus205 B 1 mus208 B 1 mus210 B 1. was used to te s t only a few se l e c t a l k y l a t i n g agents. The chemical t e s t i n g protocol used here i s e s s e n t i a l l y the same as that used i n Chapter 2 to detect mus mutant int e r a c t i o n s . Yeast fed mus / SM5 females (where "mus" indicates the b fjr cn - marked t r i p l e mus chromosome) were mated i n bot t l e s to homozygous mus males. Several days l a t e r these adults were placed into fresh v i a l s (5 pair s per v i a l ) and l e f t to lay eggs. After 24 hr the parents were transferred to new v i a l s and the o r i g i n a l v i a l cultures (eggs) were treated with 86 0.25 mL o f an a p p r o p r i a t e c o n c e n t r a t i o n of a t e s t c h emical. (In g e n e r a l , l i t e r a t u r e v a l u e s were used as guides i n s e l e c t i n g dose l e v e l s ) . The same parents were t r a n s f e r r e d d a i l y t o f r e s h v i a l s f o r up t o 5 days t o e s t a b l i s h new c u l t u r e s f o r treatment. a d u l t progeny were counted and c l a s s i f i e d w i t h i n 2-3 weeks of treatment (FA delayed development by as much as 2 weeks). S u r v i v a l v a l u e s are presented as a r a t i o o f mus homozygotes t o mus / Cy_ h e t e r o z y g o t e s . These v a l u e s were normalized u s i n g the s u r v i v a l v a l u e o b t a i n e d f o r the u n t r e a t e d c o n t r o l s (see Table 1 i n Chapter 2). P r i n c i p l e o f the Method T h i s system measures the developmental s e n s i t i v i t y of mus homozygotes r e l a t i v e t o p h e n o t y p i c a l l y d i s t i n g u i s h a b l e , r e p a i r -p r o f i c i e n t rous/Cy (mus"*") h e t e r o z y g o t e s . The h e t e r o z y g o t e s serve as a r e l a t i v e l y m u t a g e n - i n s e n s i t i v e i n t e r n a l c o n t r o l . In t h i s way, g e n o t o x i c doses can be d i s t i n g u i s h e d from those t h a t are s y s t e m i c a l l y t o x i c , and t h e r e f o r e not n e c e s s a r i l y DNA-damaging. 87 RESULTS The r e s u l t s of t r e a t i n g rous208B1 rous210B1 mus211 B 2 to varying concentrations of 8 d i f f e r e n t simple a l k y l a t i n g compounds are presented i n Table 1. In a l l but two cases (DMS and DES) the genotoxic e f f e c t s of these compounds are r e a d i l y apparent. For example, MMS k i l l e d v i r t u a l l y a l l homozygous f l i e s at a concentration of 2.5 mM. Indeed, a l l of the methylating agents (except DMS) produced very s i m i l a r l e v e l s of l e t h a l i t y at t h i s concentration. By contrast, a 30 mM concentration of EMS was required to e f f e c t nearly complete developmental l e t h a l i t y of the mus homozygotes. In t h i s respect, a q u a l i t a t i v e l y s i m i l a r trend i s also evident for the dialkylnitrosamines, DMN and DEN; i . e . , the mus homozygotes are s u b s t a n t i a l l y more se n s i t i v e (by at l e a s t a factor of 4) to DMN than to DEN at equimolar concentrations. Although other explanations are tenable, these l a t t e r findings may r e f l e c t the fa c t that, on average, methylating agents are about 20 times more reactive than t h e i r ethylating homologs (Singer and Kusmierek, 1982). Neither of the d i a l k y l sulfates DMS and DES i s obviously genotoxic to mus208 B 1 mus210 B 1 mus211 B 2 despite a wealth of information from other t e s t systems to the contrary (reviewed by Hoffmann, 1980). However, upon closer inspection, DMS appears to be weakly genotoxic at the comparatively high concentration of 30 mM. The unadjusted r e l a t i v e s u r v i v a l value at t h i s dose i s 0.66 (432/659), a value too low to be due simply to 88 Table 1. ALKYLATING SENSITIVITY OF mus2 0 8 B 1 AGENTS mus210 B 1 mus211 B 2 TO SIMPLE Chemical C o n c e n t r a t i o n (mM) Normalized r e l a t i v e s u r v i v a l a MMS 1.0 2.5 0.69 0.02 (754) (309) EMS 10.0 20.0 30.0 0.90 0.20 0.03 (1122) (1649) (1331) DMS 10.0 20.0 30.0 0.91 0.91 0.75 (985) (1632) (1091) DES 20.0 30.0 40.0 0.95 0.88 0.89 (689) (817) (892) MNU 1.0 2.5 5.0 0.41 0.06 0.00 (1772) (1496) (1412) MNNG 1.0 2.5 0.51 0.03 (1771) (1302) DMN 2.5 0.00 (787) DEN 2.5 5.0 10.0 0.61 0.11 0.01 (1627) (1604) (1157) Normalized r e l a t i v e s u r v i v a l i s the homozygoterheterozygote r a t i o from the t r e a t e d c u l t u r e s d i v i d e d by the homozygoterheterozygote r a t i o from the u n t r e a t e d c o n t r o l c u l t u r e s . The l a t t e r v a l u e i s 0.88. The numbers i n parentheses i n d i c a t e the number of f l i e s s c o r e d per c o n c e n t r a t i o n . 89 v a r i a b i l i t y . (The r e l a t i v e s urvival values from untreated control repeats range between 0.76 and 0 .96, averaging 0.88). A dose-response r e l a t i o n s h i p may well be demonstrable for DMS using concentrations above 3 0 mM. The response of the second t e s t e r s t r a i n , mus205 B 1 rous208B1  mus210 B 1. to MMS, EMS, DMN and DEN i s presented i n Table 2. I t can be seen that mus205 B 1 mus208 B 1 mus210 B 1 i s extremely s e n s i t i v e to a l l four chemicals, even more so than mus208 B 1  mus210 B 1 mus211 B 2 i s . This hy p e r s e n s i t i v i t y i s attributed to the presence of mus205 B 1 which, by i t s e l f , i s highly sens i t i v e to a l l four of these monofunctional a l k y l a t i n g agents (unpublished observations). The remaining 8 chemicals were tested using mus208 B 1 musJ210B1 mus211 B 2 only. The r e s u l t s of these experiments are given i n Table 3, and they are r e l a t i v e l y straightforward. The r e s u l t s obtained for FA indicate that i t i s weakly genotoxic ( i . e . , comparatively high concentrations of FA are required to e l i c i t a detectable response). Similar conclusions about FA were reached by others using the Ames assay (Connor et a l . , 1983) and two mutagenicity tests i n Drosophila (Szabad et a l . , 1983). DEB k i l l e d a l l mus208 B 1 mus210 B 1 mus211 B 2 homozygotes when applied at concentrations of 2.5 mM and above. A 1.0 mM dose f a i l e d to e f f e c t a response. These r e s u l t s compare favorably with the l e v e l s of DEB-induced l a r v a l k i l l i n g observed for a number of other Drosophila mus stra i n s (Olsen and Green, 1982). 90 T a b l e 2. SENSITIVITY OF mus205 B 1 mus208 B 1 mus210 B 1 TO SIMPLE ALKYLATING AGENTS Chemical C o n c e n t r a t i o n (mM) Normalized r e l a t i v e s u r v i v a l 3 MMS 1.0 2.5 <0.01 0.00 (206) (198) EMS 10.0 20.0 0.09 0.00 (505) (584) DMN 2.5 0.00 (708) DEN 2.5 0.02 (835) Normalized r e l a t i v e s u r v i v a l i s the homozygoterheterozygote r a t i o from the t r e a t e d c u l t u r e s d i v i d e d by the homozygoterheterozygote r a t i o from the u n t r e a t e d c o n t r o l c u l t u r e s . The l a t t e r v a l u e i s 0.89. The numbers i n parentheses i n d i c a t e the t o t a l number of f l i e s s c o r e d per c o n c e n t r a t i o n . 91 T a b l e 3 . SENSITIVITY OF mus208 B 1 mus210 B 1 mus211 B 2 TO MISCELLANEOUS CHEMICALS Chemical C o n c e n t r a t i o n 3 Normalized r e l a t i v e (mM) s u r v i v a l * 3 FA 2 0.73 (1541) 4 0.31 (1243) 6 0.05 (1180) CP 2.5 0.00 (1464) DEB 1.0 1.01 (765) 2.5 0.00 (1419) 5.0 0.00 (988) B(a)P 0.05 0.42 (1933) 0. 10 0.02 (1568) B(e)P 0.10 1.01 (2548) HMPA 5.0 0. 35 (981) 10.0 <0.01 (1056) SAF 2.5 1.01 (937) 5.0 0.93 (663) 10.0 t o x i c CAP 10.0 0.91 (2185) 20.0 1.05 (897) 30.0 1.09 (1058) FA c o n c e n t r a t i o n i s expressed as % v/v f o r m a l i n :H20 Normalized r e l a t i v e s u r v i v a l i s the homozygoterheterozygote r a t i o from the t r e a t e d c u l t u r e s d i v i d e d by the homozygoterheterozygote r a t i o from the u n t r e a t e d c o n t r o l c u l t u r e s . The l a t t e r v a l u e i s 0.88. The numbers i n parentheses i n d i c a t e the t o t a l numbers of f l i e s s c o r e d per c o n c e n t r a t i o n . 92 The procarcinogen B(a)P i s decidedly genotoxic at the comparatively low doses of 0.05 and 0.10 mM. Previous i n v i t r o studies have demonstrated that Drosophila microsomal preparations are capable of metabolizing a v a r i e t y of procarcinogens, including B(a)P (Baars et a l . , 1977; Hallstrom and Grafstrom, 1981), to t h e i r reactive forms. The findings reported here, and those of Boyd et a l . (1976a) and Nguyen et a l . (1979), suggest that l a r v a l somatic tissues can e f f e c t a si m i l a r a c t i v a t i o n of B(a)P i n vivo. In contrast to the strong genotoxic e f f e c t of B(a)P, B(e)P was not genotoxic at the concentration used. HMPA and SAF are d i f f i c u l t or impossible to detect as mutagens i n the Ames t e s t (see de Serres and Ashby, 1981). Of these, only HMPA i s genotoxic to mus208 B 1 mus210 B 1 mus211 B 2. While SAF f a i l e d to e f f e c t a response at either 2.5 or 5.0 mM, the 10 mM concentration k i l l e d a l l developing f l i e s i r r e s p e c t i v e of t h e i r genotype. This l a t t e r r e s u l t i s the only example of a to x i c reaction i n t h i s study. However, i t points out the importance of the int e r n a l control ( i . e . , the heterozygous f l i e s ) i n t h i s t e s t system. The non-carcinogen CAP did not e f f e c t any detectable l e t h a l i t y i n mus208 B 1 mus210 B 1 mus211 B 2. CAP tested as marginally p o s i t i v e i n the somatic mutation and recombination t e s t i n Drosophila (Wurgler et a l . , 1985). 93 DISCUSSION To date, mus str a i n s have been used mainly to elucidate c e l l u l a r mechanisms of DNA repair, recombination and mutagenesis. Their application to chemical genotoxicity testing has p r i n c i p a l l y resided i n e f f o r t s to enhance the s e n s i t i v t y of both t r a d i t i o n a l and recently-developed mutagenicity assays i n Drosophila (e.g., Zimmering, 1982, 1983; Vogel et a l . , 1983; Fujikawa et a l . , 1985; Wurgler et a l . , 1985). The p o s s i b i l i t y that mus s t r a i n s themselves might serve d i r e c t l y as genotoxicity in d i c a t o r s has remained v i r t u a l l y unexplored since Nguyen et a l . (1979) f i r s t demonstrated the potential of t h i s approach using X-linked mus and mei mutations. The present study follows up on t h i s idea. However, the t e s t protocol employed here d i f f e r s from that of Nguyen et a l . (1979) i n that mus mutations of the second chromosome are used. The mus t e s t described here i s fast, easy to do, and inexpensive. Thus, a large number of chemicals can be screened e f f i c i e n t l y . Moreover, a considerable advantage of t h i s t e s t over comparable d i f f e r e n t i a l k i l l i n g assays that u t i l i z e r epair-d e f i c i e n t mammalian c e l l s (e.g., Hoy et a l . , 1984), i s that phenotypically d i f f e r e n t mus mutations can be combined r e l a t i v e l y e a s i l y into single tester s t r a i n s , thus p o t e n t i a l l y broadening the t e s t ' s detection c a p a b i l i t y . A t o t a l of 16 chemicals were tested i n t h i s p i l o t study. Of the 14 chemicals generally considered genotoxic ( a l l but B(e)P and CAP), 11 tested as p o s i t i v e . While t h i s l e v e l of 94 concordance i s respectable, i t s si g n i f i c a n c e i s somewhat tempered by the fac t that at least 10 of the 14 genotoxins are a l k y l a t i n g agents. Recall that the mutations mus205 B 1. mus208 B 1. mus210B1. and mus211 B 2 were o r i g i n a l l y i s o l a t e d for t h e i r MMS-sensitive properties (Henderson et a l . , 1987). (This i s not meant to imply that a l l a l k y l a t i n g agents react i d e n t i c a l l y with DNA (they do not) or are uniformly genotoxic (they are not).) These considerations aside, i t i s the fa l s e negative r e s u l t s (DMS, DES and SAF), more so than the p o s i t i v e r e s u l t s , which r a i s e a number of important issues concerning the use of mus s t r a i n s for mutagen te s t i n g . The reason(s) why DMS and DES tested as negative, even at comparatively high doses, i s not clear. (Although DMS appears to be weakly genotoxic at 30 mM, t h i s assessment has to be considered tentative u n t i l retests can be ca r r i e d out. Thus, for the sake of discussion the DMS re s u l t s are considered to be negative.) Both chemicals are re a d i l y active i n numerous t e s t systems, including apparently the sex-linked recessive l e t h a l t e s t i n Drosophila (see Hoffmann, 1980). The problem may be one of chemical delivery. Both DMS and DES are ra p i d l y hydrolyzed i n water (Hoffmann, 1980); the same may be true i n ethanol, the solvent employed here. This problem i l l u s t r a t e s the need to ensure both chemical-solvent and solvent-organism compatibility; however, t h i s i s not always easy to achieve. For example, whereas water would normally be the solvent of choice, not a l l 95 chemicals are s o l u b l e o r s t a b l e i n water. Moreover, whereas o t h e r s o l v e n t s such as dimethyl s u l f o x i d e (DMSO) can d i s s o l v e most chemicals, u n d i l u t e d DMSO i s t o x i c t o d e v e l o p i n g f l i e s ( unpublished o b s e r v a t i o n s ) , and a t lower c o n c e n t r a t i o n s i t may i n t e r f e r e w i t h the metabolism of x e n o b i o t i c s (Magnusson e t a l . , 1979). Although the problem o f s o l v e n t s u i t a b i l i t y i s not unique t o t h i s D r o s o p h i l a assay, e f f o r t s w i l l have t o be made t o f i n d a l t e r n a t i v e chemical d e l i v e r y v e h i c l e s a p p l i c a b l e t o t h i s t e s t . A second important p o i n t t o be drawn from the DMS/DES r e s u l t s i s the q u e s t i o n o f n e g a t i v i t y , i . e . , a t what dose does one f i n a l l y conclude t h a t a chemical i s not gen o t o x i c i n the mus t e s t ? In the case of SAF, f o r example, the upper dose l i m i t i s n e c e s s a r i l y somewhere between 5 and 10 mM. Doses h i g h e r than t h i s a re c y t o t o x i c . For DMS and DES i t may be necessary t o determine LD50 v a l u e s i n w i l d t y p e (b p_r cn) f l i e s and then t o a d j u s t the maximal t e s t c o n c e n t r a t i o n s a c c o r d i n g l y . While t h i s may not seem a v e r y s a t i s f a c t o r y method i n what i s supposed t o be a r a p i d t e s t system, i t w i l l , n e v e r t h e l e s s , have t o be employed f o r chemical unknowns. The mus t e s t j o i n s numerous o t h e r s h o r t - t e r m g e n o t o x i c i t y assays i n b e i n g unable t o d e t e c t as p o s i t i v e SAF (de S e r r e s and Ashby, 1981). There are a t l e a s t two p o s s i b l e reasons f o r t h i s n e g a t i v e response. For example, i f indeed SAF e x e r t s i t s c a r c i n o g e n i c e f f e c t by damaging DNA, the l e s i o n s i t generates may be r e p a i r e d by pathways d i f f e r e n t from those b l o c k e d by the 96 three mus mutations. An a l t e r n a t i v e explanation i s that SAF may be metabolized d i f f e r e n t l y i n Drosophila and mammalian c e l l s . Thus, f l i e s may convert SAF to nongenotoxic compounds, whereas mammals may produce genotoxic intermediates. To d i s t i n g u i s h between these two p o s s i b l i t i e s , known SAF metabolites (e.g., 1'-OH-safrole) could be tested i n the mus assay. The major t h e o r e t i c a l disadvantage of t h i s mus t e s t i s that the underlying mechanisms leading to c e l l k i l l i n g are unknown, i . e . , there i s no definable genetic endpoint. While t h i s i s a drawback i n terms of b i o l o g i c a l inquiry, i t does not necessarily lessen the value of the t e s t as a rapid genotoxicity screen. The working hypothesis i s that the mus homozygotes die following f a i l e d attempts to repair lesions i n DNA. The types of lesions w i l l of course vary with the chemical, the dose, the c e l l type etc., but DNA strand breaks, i n p a r t i c u l a r , are l i k e l y to be prominent c e l l k i l l i n g lesions (Gatti et a l . , 1974, 1975; P i m p i n e l l i et a l . , 1977; Levina and Sharygin, 1984). Such strand breaks could a r i s e either d i r e c t l y by the clastogenic action of the mutagen i t s e l f , or i n d i r e c t l y , e.g., following r e p l i c a t i o n arrest at bulky DNA adducts. (Transient r e p l i c a t i o n arrest leading to overreplication of DNA may i t s e l f be an important mechanism for generating a v a r i e t y of chromosomal aberrations (Schimke et a l . , 1986).) Regardless of t h e i r etiology, DNA strand d i s c o n t i n u i t i e s are highly recombinogenic, thus opening up the p o s s i b i l i t y for additional genomic a l t e r a t i o n s (e.g., deletions, inversions, translocations, 97 a n e u p l o i d y ) . I t must be emphasized t h a t v i r t u a l l y a l l of these events, not merely p o i n t mutations i n oncogenes (e.g., Z a r b l e t a l . , 1986; Guerrero and P e l l i c e r , 1987), may be f a c t o r s l e a d i n g t o cancer development (e.g., Radman e t a l . , 1982; Oshimura and B a r r e t t , 1986) . In view of our ignorance of the c a r c i n o g e n i c p r o c e s s , should we, f o r example, l i m i t our s h o r t - t e r m t e s t s only t o those t h a t monitor p o i n t mutations or aneuploidy as g e n e t i c endpoints? D r o s o p h i l a mus mutations w i l l c o ntinue t o have a r o l e i n the realm of s h o r t - t e r m t e s t s f o r environmental genotoxins. But whether they w i l l be employed d i r e c t l y o r simply as a d j u n c t s t o o t h e r t e s t s (e.g., Wurgler and Graf, 1985) w i l l have t o await f u r t h e r v a l i d a t i o n of the t e s t d e s c r i b e d here. 98 CHAPTER FOUR A GENETIC AND DEVELOPMENTAL ANALYSIS O F mus209 99 INTRODUCTION Mutations that render c e l l s hypersensitive to DNA-damaging agents often confer additional mutant phenotypes. For example, humans a f f l i c t e d with any one of a number of inherited diseases associated with defects i n DNA repair or r e p l i c a t i o n (e.g., xeroderma pigmentosum (XP), ataxia t e l a n g i e c t a s i a (AT), Fanconi's anemia (FA), Bloom's syndrome (BS) and Cockayne's syndrome(CS)) are predisposed to malignancy ( A r l e t t and Lehmann, 1978; Kraemer et a l . , 1984). Less easy to explain, but no less i n t r i g u i n g , are the observations that some of these syndromes also involve neurological complications (XP, AT), immunodeficiencies (AT), or growth retardation (FA, BS and CS) (Robbins, 1983; Hanawalt and Sarasin, 1986). In only one of these disorders, Bloom's syndrome, i s the molecular basis of the disease apparently understood. C e l l s from i n d i v i d u a l s with Bloom's syndrome appear to have an altered DNA l i g a s e I a c t i v i t y (Chan et a l . , 1987; W i l l i s and Lindahl, 1987). Since t h i s enzyme normally p a r t i c i p a t e s i n DNA r e p l i c a t i o n , that BS patients survive development at a l l implies that the mutant gene i s weakly hypomorphic and/or the dysfunction i s l i m i t e d to a r e l a t i v e l y small number of c e l l or tissue types. In Drosophila melanogaster many mutagen-sensitive (mus) and meiotic (mei) mutants adversely a f f e c t the s t a b i l i t y of chromosomes, even i n c e l l s not exposed to mutagens (Baker and Smith, 1979; G a t t i , 1979; Baker et a l . , 1980, 1982; Green, 1981; G a t t i et a l . , 1983). Many of these mutants also e x h i b i t 100 f e r t i l i t y problems, probably r e s u l t i n g from d e f e c t s i n chromosome recombination and/or s e g r e g a t i o n . Chromosome i n s t a b i l i t y i s a l s o a c h a r a c t e r i s t i c o f AT, FA and BS c e l l s (Hanawalt and S a r a s i n , 1986), and of numerous mutagen-sensitive f u n g a l mutants (see Baker and Smith (1979) f o r r e f e r e n c e s ) . In Saccharomyces c e r e v i s i a e , a t l e a s t t h r e e genes which i n f l u e n c e the s e n s i t i v i t y o f c e l l s t o mutagens encode v i t a l c e l l - d i v i s i o n - c y c l e (CDC) f u n c t i o n s (see Haynes and Kunz, 1981; K a s s i r e t a l . , 1985). For example, CDC9 encodes a DNA l i g a s e (Johnston and Nasmyth, 1978) and CDC8 encodes t h y m i d y l a t e kinase (Jong e t a l . , 1984). The ye a s t e x c i s i o n r e p a i r gene PAD3 i s a l s o e s s e n t i a l f o r v i a b l i t y (Higgins e t a l . , 1983; Naumovski and F r i e d b e r g , 1983), as are t h r e e X - l i n k e d mus l o c i i n D r o s o p h i l a (Baker e t a l . , 1982; G a t t i e t a l . , 1983). V i t a l m utagen-sensitive genes i n which the r e p a i r and e s s e n t i a l f u n c t i o n s are not sepa r a b l e by mutation ( i . e . , the gene p r o d u c t s do not possess separate domains f o r these f u n c t i o n s ) pose a problem f o r c o n v e n t i o n a l mutant scree n s . At be s t , o n l y v i a b l e and t h e r e f o r e presumably weak a l l e l e s can be i d e n t i f i e d . To r e c o v e r s t r o n g e r mutant a l l e l e s a t v i t a l mus l o c i , a l t e r n a t i v e s e l e c t i o n s t r a t e g i e s are r e q u i r e d . One such approach i s t o s e l e c t f o r t e m p e r a t u r e - s e n s i t i v e (ts) l e t h a l mutations t h a t e x h i b i t mutagen s e n s i t i v i t y a t the p e r m i s s i v e temperature (Henderson e t a l . , 1987). In o t h e r organisms t s mutations have proven t o be ex c e e d i n g l y u s e f u l as probes of complex c e l l u l a r p r ocesses t h a t otherwise are d i f f i c u l t t o access g e n e t i c a l l y (e.g., H a r t w e l l , 1978; P r i n g l e , 1 9 8 1 ) . The u t i l i t y o f t s mutations i n D r o s o p h i l a has been reviewed (Suzuki, 1970). To date, t h r e e t s l e t h a l mus mutations have been i d e n t i f i e d i n D r o s o p h i l a . The f i r s t of these, an a l l e l e o f muslOl. was a c t u a l l y r e covered i n a s c r e e n f o r mutations e x h i b i t i n g abnormal m i t o t i c chromosome beh a v i o r (Smith e t a l . , 1985). P r o p e r t i e s unique t o t h i s a l l e l e l e d t o the p r o p o s i t i o n t h a t muslOl"1" encodes a product necessary f o r proper condensation of h e t e r o c h r o m a t i c r e g i o n s of chromosomes d u r i n g m i t o s i s ( G a t t i e t a l . , 1983). T h i s o b s e r v a t i o n suggests t h a t the mutagen s e n s i t i v t y of muslOl mutants may be s e c o n d a r i l y r e l a t e d t o a d e f e c t i n chromosome s t r u c t u r e ( G a t t i e t a l . , 1983). The second t s l e t h a l mus mutation t o be i d e n t i f i e d i n D r o s o p h i l a i s rous209B1. T h i s r e c e s s i v e mutation was recovered from a c o l l e c t i o n of 63 t s l e t h a l s t r a i n s as the o n l y one e x h i b i t i n g s e n s i t i v i t y t o MMS a t the p e r m i s s i v e temperature (Henderson e t a l . , 1987). Subsequent analyses r e v e a l e d t h a t mus209 B 1 i s a l s o s e n s i t i v e t o gamma r a d i a t i o n , and t h a t homozygous females are s t e r i l e . Thus, t h i s novel mus gene encodes a product (or products) whose normal f u n c t i o n i s e s s e n t i a l f o r development, i s m o b i l i z e d i n response t o DNA damage, and i s necessary f o r female f e r t i l i t y . T h i s chapter p r o v i d e s a p r e l i m i n a r y g e n e t i c and developmental a n a l y s i s of mus209 B 1 as a f i r s t s t e p toward und e r s t a n d i n g the r o l e ( s ) of t h i s p l e i o t r o p i c gene i n chromosome metabolism. In the course of t h i s study a second t s l e t h a l 102 a l l e l e o f mus209. mus209" 2. was i d e n t i f i e d . 103 MATERIALS AND METHODS S t r a i n s For d e s c r i p t i o n s of most of the v i s i b l e mutations and s p e c i a l chromosomes used i n t h i s study, c o n s u l t L i n d s l e y and G r e l l (1968). The f o l l o w i n g mutants r e q u i r e s p e c i a l mention: M(2)173 (2 - 92.3) i s a spontaneous, c y t o l o g i c a l l y normal Minute mutation (see L i n d s l e y and G r e l l , 1968; S h e l l e n b a r g e r and Duttagupta, 1978). M(2)017. a l s o a Minute mutation, was r e c o v e r e d among the F^ progeny of EMS-treated Oregon-R males (Stone, 1974). The M(2)017 chromosome i s d e l e t e d f o r about f i v e bands i n the 56F r e g i o n of the s a l i v a r y g l a n d p o l y t e n e chromosome ( S h e l l e n b a r g e r and Duttagupta, 1978). I t f a i l s t o complement M(2)173. 36. 157. 775. 1991. 2735. D-292 and D-1368 are second chromosomal, EMS-induced r e c e s s i v e l e t h a l mutations t h a t f a i l t o complement K(2)017 and M(2)173 f o r l e t h a l i t y ( S h e l l e n b a r g e r and Duttagupta, 1978). The 2735-bearing chromosome a l s o c a r r i e s a s e c o n d - s i t e l e t h a l mutation (50.7 + 1.4 map u n i t s ) . b p_r cn mus209 B 1. h e r e a f t e r r e f e r r e d t o as mus209 B 1. i s a t e m p e r a t u r e - s e n s i t i v e (ts) r e c e s s i v e l e t h a l mus mutation t h a t was i s o l a t e d as d e s c r i b e d p r e v i o u s l y (Henderson e t a l . , 1987; Appendix A ) . fe ET. £Q mus209 B 2. h e r e a f t e r r e f e r r e d t o as mus209 B 2, was r e c o m b i n a t i o n a l l y d e r i v e d from the 2735-bearing chromosome. L i k e mus_209B1, mus209 B 2 i s a t s r e c e s s i v e l e t h a l mutation. B l a c k c e l l s (Be. 2 - 80.6) i s a dominant mutation which causes melanized c e l l s t o form beneath the integument i n Be / Be"1" l a r v a e , pupae, and a d u l t s (see R i z k i e t a l . , 1980). I3c i s employed here as a l a r v a l marker. C u l t u r e c o n d i t i o n s were as d e s c r i b e d i n Chapter 2. Unless noted otherwise, 22+l°C and 29+0.5°C are the p e r m i s s i v e and r e s t r i c t i v e temperatures, r e s p e c t i v e l y . 104 Genetic Mapping of MMS S e n s i t i v i t y and Temperature-Sensitive  L e t h a l i t y The t s l e t h a l and MMS-sensitive phenes associated with the mus209 B 1 s t r a i n were mapped separately using a multiply-marked chromosome that c a r r i e d the dominant mutations S (1.3), Sp_ (22.0), T f t (53.2), nwD (83), and P i n Y t (107.3). S Sp_ T f t nwD P i n v t / mus209 B 1 females were mated i n v i a l s to homozygous mus209 B 1 males. Parents were transferred to new v i a l s a f t e r 2 days of ovi p o s i t i o n . To map the MMS s e n s i t i v i t y , cultures were treated with 0.25 mL 0.08% v/v MMS and l e f t to develop at 22°C. To map the t s l e t h a l i t y , untreated cultures were s h i f t e d to 29°C and l e f t to develop at t h i s temperature. Recombinant progeny were counted and c l a s s i f i e d 2-3 weeks l a t e r . 95% confidence i n t e r v a l s were calculated as described i n O'Brien and Maclntyre (1978). Using genotypically appropriate parents, and following procedures s i m i l a r to those just described, two separate l e t h a l mutations on the o r i g i n a l 2735-bearing chromosome were mapped. One of these, designated mus209 B 2, i d e n t i f i e s a second t s l e t h a l a l l e l e of mus209. Cosegregation of MMS S e n s i t i v i t y and Female S t e r i l i t y with  Temperature-Sensitive L e t h a l i t y i n mus209 B 1 The procedures used to analyze the cosegregation of the mus, t s l e t h a l , and female s t e r i l e (fs) phenes of mus209 B 1 are outlined i n Figure 1. nwD Pu 2 / b mus209 B 1 females were mated i n v i a l s to homozygous b p_r cn mus209 B 1 males. Parents were 105 F i g u r e 1. Procedures used t o analyze the c o s e g r e g a t i o n of the mus. t s l e t h a l , and female s t e r i l e phenes o f mus209 B 1. mus209 B 1 = b p r cn mus209 B 1. 106 22 °C n o n w ° P y 2 + + b mus 209 nwD Pu 2 mus 209 9 nw p Pu 2 mus 209 or recomb mus 209 single £ per vial oviposition ? mus 209 mus 209 cTcT 29 °C nw Pu' mus 209 mus 209 die mus209 mus 209 recomb mus 209 b mus 209 mus 209 die mus209 CyO 22°C mus209 CyO recomb CvO 107 t r a n s f e r r e d t o new v i a l s w i t h i n 2 days, and the o r i g i n a l c u l t u r e s were s h i f t e d t o 29°C. At t h i s temperature, o n l y mus209 + recombinants or nw D Pu 2 heterozygotes s u r v i v e . To t e s t f o r the c o s e g r e g a t i o n o f MMS s e n s i t i v i t y and t s l e t h a l i t y (or, more c o r r e c t l y , the c o s e g r e g a t i o n of the c o r r e s p o n d i n g w i l d - t y p e f u n c t i o n s ) , nw D or P u 2 recombinant male s u r v i v o r s were mated i n d i v i d u a l l y t o 5-6 b p_r cn mus209 B 1 / CyO females. F o l l o w i n g 2 days of o v i p o s i t i o n , p a r e n t s were t r a n s f e r r e d t o new v i a l s ; the o r i g i n a l c u l t u r e s were t r e a t e d w i t h 0.09% MMS and l e f t t o develop a t 22°C. (MMS, a t t h i s c o n c e n t r a t i o n , k i l l s e s s e n t i a l l y a l l mus209 B 1 homozygotes.) A s e t o f r e p l i c a t e c u l t u r e s was t r e a t e d s i m i l a r l y . These experiments were p r e d i c a t e d on the e x p e c t a t i o n t h a t i f the mus and t s l e t h a l phenes cosegregate, then normal numbers of recombinant ( i . e . , mus209 +) / b p_r cn mus209 B 1 animals from each l i n e s h o u l d s u r v i v e MMS treatment. Homozygous mus209 B 1 females are completely s t e r i l e ; they do not o v i p o s i t . The c o s e g r e g a t i o n o f t h i s phenotype w i t h t s l e t h a l i t y ( o r , more c o r r e c t l y , the c o s e g r e g a t i o n of the c o r r e s p o n d i n g w i l d - t y p e f u n c t i o n s ) was a n a l y z e d as f o l l o w s (see F i g u r e 1) . S i n g l e recombinant females t h a t s u r v i v e d development a t 29°C were p l a c e d i n t o v i a l s w i t h s e v e r a l w i l d - t y p e males. A f t e r 1 week, each v i a l was examined f o r the presence of eggs. F e r t i l i t y assessments were made o n l y f o r those v i a l s i n which the females l i v e d f o r a t l e a s t 5 days. C y t o g e n e t i c Mapping and Complementation A n a l y s e s The chromosomal segment d e l e t e d i n M(2)017 corresponds t o a 108 g e n e t i c map p o s i t i o n of about 92 map u n i t s . S i n c e mus209 maps t o t h i s v i c i n i t y (see RESULTS), complementation experiments were c a r r i e d out t o determine whether M(2)017 uncovers the mus209 l o c u s . For t h i s a n a l y s i s , M(2)017 / SMI males were c r o s s e d i n v i a l s t o mus209 B 1 / CyO females. Progeny from these matings were r e a r e d a t 22°C or 29°C. H a l f the c u l t u r e s , a t each temperature, were t r e a t e d with MMS; the remainder were l e f t u n t r e a t e d . An absence of C y + f l i e s , among Cy s i b s , i d e n t i f i e s the c y t o g e n e t i c l o c a t i o n of mus209, as d e f i n e d by the d e f i c i e n c y ' s b r e a k p o i n t s . A l l e l i s m t e s t s between mus209 B 1 and a s e r i e s of EMS-induced l e t h a l mutations ( S h e l l e n b a r g e r and Duttagupta, 1978) were c a r r i e d out as f o l l o w s . R e p r e s e n t a t i v e males from extant l e t h a l l i n e s were mated i n v i a l s t o mus209 B 1 / CyO females. Progeny from these c r o s s e s were r e a r e d a t 29°C. In each case, complementarity was assessed by comparing the number of C y + t o Cy f l i e s . F o l l o w i n g s e v e r a l i n s t a n c e s o f non-complementation, these t e s t s were expanded t o i n c l u d e a l l p a i r w i s e combinations (m^/Cy X m^/Cy) of a v a i l a b l e mutants. R e t e s t s were c a r r i e d out a t both 22°C and 29°C. Temperature S h i f t A nalyses For a d e t a i l e d d i s c u s s i o n of the r a t i o n a l e and the methodology f o r determining t e m p e r a t u r e - s e n s i t i v e p e r i o d s (TSPs) of t s mutations, see Suzuki (1970). In t hese s t u d i e s , the u n c o n d i t i o n a l s t e r i l i t y o f mus209 females n e c e s s i t a t e d the use of the l a r v a l marker Be. Thus, from 109 matings between Be mus209 / SM5 females and b g r cn mus209 homozygous males, homozygous mus l a r v a e were i d e n t i f i e d by t h e i r Be phenotype. Standard egg-lay p r a c t i c e s (e.g., S i n c l a i r e t a l . , 1981) of 2 hr d u r a t i o n were employed t o o b t a i n synchronous c u l t u r e s . Immediately a f t e r egg d e p o s i t i o n , s e c t i o n s of food from the c o l l e c t i o n p l a t e s were t r a n s f e r r e d t o v i a l s c o n t a i n i n g f r e s h medium (approximately 70 eggs per v i a l ) . Some c u l t u r e s were l e f t t o develop a t 22°C (permissive c o n t r o l s ) ; o t h e r s were immediately s h i f t e d t o , and kept a t 29°C ( r e s t r i c t i v e c o n t r o l s ) . The remaining c u l t u r e s were s u b j e c t e d t o o v e r l a p p i n g 36-hr heat treatments (29°C) d u r i n g development, a t i n t e r v a l s staggered by 12 h r . ( P i l o t experiments, c o n s i s t i n g of contiguous heat p u l s e s o f e i t h e r 24, 3 6 or 48 hr d u r a t i o n , showed t h a t the 36-hr heat treatments p r o v i d e d the b e s t r e s o l u t i o n between l e t h a l i t y and v i a b i l i t y . P rolongated exposure of t s mutants t o the r e s t r i c t i v e temperature can obscure s u c c e s s i v e but s e p a r a t e TSPs (Poodry e t a l . , 1973; S i n c l a i r e t a l . , 1981).) The developmental stages were assessed a t both the b e g i n n i n g and the end of each heat treatment by examining the mouth p a r t s and the a n t e r i o r s p i r a c l e s o f l a r v a e (Bodenstein, 1950) from r e p r e s e n t a t i v e v i a l s . The s u r v i v a l of mus209 homozygotes r e l a t i v e t o mus209 / Cy h e t e r o z y g o t e s was measured by c o u n t i n g and c l a s s i f y i n g emergent a d u l t s . The s u r v i v a l v a l u e obtained f o r each developmental i n t e r v a l was normalized t o the homozygoterheterozygote r a t i o o b t a i n e d f o r the 22°C c o n t r o l s . 110 R a d i a t i o n Treatments During Development T r e a t e d animals were d e r i v e d from c r o s s e s between Be mus209 B 1 / SM5 females and b p_r cn mus209 B 1 homozygous males. Synchronous c u l t u r e s were ob t a i n e d as d e s c r i b e d above. A l l c u l t u r e s were maintained a t 22°C. F l i e s , a t f o u r d i f f e r e n t developmental stages (24 hr, embryonic; 96 hr, l a t e second i n s t a r ; 140 hr, m i d - t h i r d i n s t a r ; 216 hr, e a r l y p u p a l ) , were i r r a d i a t e d by exposing v i a l c u l t u r e s t o 6 0 C o i n a Gammacell 220 (Atomic Energy of Canada). Dose r a t e s v a r i e d between 10 and 12 rads per second. I l l RESULTS G e n e t i c Mapping and Cosegregation Analyses These i n i t i a l c h a r a c t e r i z a t i o n s of mus209 B 1 were undertaken t o determine whether i t s MMS-sensitive, t s l e t h a l , and f s phenes r e s u l t from a s i n g l e g e n e t i c l e s i o n . T h i s correspondence i s c r u c i a l t o any f u r t h e r e x perimentation w i t h d e s i g n s t o e x p l o i t the temperature s e n s i t i v i t y o f t h i s s t r a i n . The mutant s i t e ( s ) c o n f e r r i n g MMS s e n s i t i v i t y and t s l e t h a l i t y map(s) t o 92.8 + 2.6 and 94.0 + 2.9 map u n i t s , r e s p e c t i v e l y . (In subsequent experiments, the t s l e t h a l i t y a s s o c i a t e d w i t h the a l l e l i c mutation, mus209 B 2. was mapped t o t h i s same l o c a t i o n (92.6 + 1.5 map u n i t s ) . ) While these r e s u l t s are c o n s i s t e n t w i t h the n o t i o n t h a t mus209 B 1 i s a s i n g l e - s i t e mutation, they do not r u l e out the p o s s i b i l i t y t h a t the mus and t s l e t h a l phenes might r e s u l t from separate, a l b e i t t i g h t l y l i n k e d , mutations. To e l i m i n a t e t h i s l a t t e r p o s s i b i l i t y , an e x t e n s i v e c o s e g r e g a t i o n a n a l y s i s was c a r r i e d out (see F i g u r e 1). A t o t a l o f 852 recombinant males (99 nw D / mus209 B 1 and 753 P u 2 / mus209 B 1) t h a t s u r v i v e d development a t 29°C were i n d i v i d u a l l y mated t o mus209 B 1 / CyO females. Progeny from th e s e matings were t r e a t e d with MMS and allowed t o develop a t 22°C. In 64 nw D and 659 Pu 2 l i n e s (the remaining c u l t u r e s were s t e r i l e ) no s e p a r a t i o n of t s l e t h a l i t y from MMS s e n s i t i v i t y was found. Analogous experiments were conducted t o determine whether the t s l e t h a l and f s phenes of mus209 B 1 cosegregate. The 112 r e s u l t s of these s t u d i e s are presented i n T a b l e 1. A f t e r d i s c o u n t i n g the background s t e r i l i t y , these data i n d i c a t e t h a t the f s and t s l e t h a l phenes are due t o the same p l e i o t r o p i c mutation. In support of t h i s i d e a , i t was l a t e r observed t h a t mus209 B 2 homozygous females, l i k e t h e i r mus209 B 1 c o u n t e r p a r t s , f a i l t o o v i p o s i t . C y t o g e n e t i c Mapping and Complementation Analyses An approximate c y t o g e n e t i c l o c a l i z a t i o n of mus209 B 1 was i n f e r r e d from the r e s u l t s of the g e n e t i c mapping s t u d i e s . When M(2)017 d e f i c i e n c y - b e a r i n g f l i e s were c r o s s e d t o mus209 B 1 i n d i v i d u a l s , the Mf2)017 / mus209 B 1 heterozygous progeny f a i l e d t o s u r v i v e a t both 22°C and 29°C, even i n the absence of MMS. T h i s r e s u l t d e l i m i t s the c y t o g e n e t i c p o s i t i o n of mus209 t o a 5 band segment w i t h i n the 56F5-15 r e g i o n of the r i g h t arm of chromosome 2. In a d d i t i o n , the u n c o n d i t i o n a l l e t h a l i t y of the M(2)017 / mus209 B 1 heterozygotes strengthens the c o n t e n t i o n t h a t mus209 B 1 i d e n t i f i e s an e s s e n t i a l gene, and f u r t h e r , suggests t h a t mus209 B 1 i s a hypomorphic mutation. The c y t o g e n e t i c p o s i t i o n i n g of mus209 B 1 l e d t o a s e r e n d i p i t o u s f i n d . The chromosomal segment uncovered by the M(2)017 d e l e t i o n i s a r e g i o n p r e v i o u s l y s u b j e c t e d t o i n t e n s e g e n e t i c s c r u t i n y . Using EMS, S h e l l e n b a r g e r and Duttagupta (1978) s a t u r a t e d t h i s s m a l l p o r t i o n of 2R f o r l e t h a l mutations. To determine whether any of these i d e n t i f y l e t h a l a l l e l e s of mus209, i n t e r se complementation c r o s s e s were c a r r i e d out. At 22°C, mus209 B 1 f a i l e d t o complement 5 r e c e s s i v e l e t h a l 113 T a b l e 1. COSEGREGATION OF FEMALE STERILITY AND TEMPERATURE-SENSITIVE LETHALITY IN mus209 B 1 Genotype 3 Number of females t e s t e d Number o f s t e r i l e females % s t e r i l i t y 1. nw D P u 2 328 4 1.2 2. nw D 153 3 2.0 3. P u 2 639 8 1.3 4. T o t a l o f 2 and 3 792 11 1.4 a fe EE cn mus209 B 1 heterozygous females t h a t s u r v i v e d development a t 29°C (see F i g u r e 1). 114 mutations (36, 157. 775. 1991. and D-292) and a second Minute mutation, M(2)173• In c o n t r a s t , mus209 B 1 f u l l y complemented f o r l e t h a l i t y 2735. D-1368 and a s e r i e s of M i n u t e - l i k e mutations (M(2)U. 12, 29, 47, 51, 2362. and D-741) ( F i g u r e 2A). A l l other i n t e r a c t i o n s were as o r i g i n a l l y d e s c r i b e d ( S h e l l e n b a r g e r and Duttagupta, 1978). At 29°C, however, mus209 B 1 / 2735 h e t e r o z y g o t e s f a i l e d t o s u r v i v e . Furthermore, the v i a b i l i t i e s o f mus_209 B 1 / D-1368 and 2735 / D-1368 were reduced t o about 20% and 75%, r e s p e c t i v e l y , of the l e v e l s observed a t 22°C (data not shown). ( S i m i l a r l y , a t 22°C, exposure of mus209 B 1 / 2735. mus209 B 1 / D-1368. and 2735 / D-1368 t o MMS (0.09%) lowered t h e i r s u r v i v a l by as much as 70-90% (data not shown).) Based on l e t h a l i t y , the mus2 09 l o c u s i s r e p r e s e n t e d o n l y by the l e f t major group (subgroups I and II) from the complementation map of S h e l l e n b a r g e r and Duttagupta (1978) ( F i g u r e 2B). A l l , or a subset, of the mutations i n the r i g h t major group (subgroups IV and V) l i k e l y i d e n t i f y an adjacent Minute l o c u s . T h e i r argument t h a t mutant 12 e s t a b l i s h e s a f u n c t i o n a l r e l a t e d n e s s between the l e f t and r i g h t groups seems s p u r i o u s . That 12 s u r v i v e s over the M(2)017 d e f i c i e n c y but i s i t s e l f homozygous l e t h a l s t r o n g l y i m p l i e s t h a t i t maps o u t s i d e the chromosomal segment d e l e t e d i n M(2)017. The M i n u t e - l i k e phenotype o f mutant 12 when combined with c e r t a i n mutations from t h i s r e g i o n may be a s c r i b e d t o n o n s p e c i f i c t r a n s i n t e r a c t i o n s t y p i c a l o f Minute mutations i n g e n e r a l (e.g., see S i n c l a i r e t a l . , 1 9 8 4 ) . Figure 2. Complementation maps of l e t h a l mutations uncovered by the M(2)017 deficiency. A . Complementation diagram of a l l e l e s of the mus209 locus. This map was constructed from complementation data based on l e t h a l i t y at 29°C. [The shotgun (shg) locus (2-92) encodes a zygotic function that i s esse n t i a l for embryonic development (Nusslein-Volhard et a l . , 1984). The proximity of t h i s mutation to mus209 prompted a t e s t for a l l e l i s m . Both M(2)017 and mus209 B 1 f u l l y complemented s h g I H 8 1 at 29°C in d i c a t i n g that shg and mus209 are separate genes.] B. A reproduction of the complementation map of Shellenbarger and Duttagupta (1978). This map i s based on data for both l e t h a l i t y and short b r i s t l e (Minute) phenotype. 116 36 157 775 1991 D-292 mus209 B 1 shg D-1368 2735 M(2)173 M(2)017 D-91 D-292 D-932 157 2362 775 D-741 1991 29 36 47 D-1368 2735 12 51 M(2)U II III IV v~ M(2)173 M(2)017 117 Taken t o g e t h e r , the r e s u l t s of these mapping, c o s e g r e g a t i o n , and complementation s t u d i e s are c o m p e l l i n g evidence t h a t the mus. t s l e t h a l , and f s phenes of rous209B1 are due t o a s i n g l e mutation. Temperature S h i f t s During Development Given t h a t the MMS s e n s i t i v i t y and t s l e t h a l i t y o f mus209 B 1 are simply p l e i o t r o p i c e x p r e s s i o n s of a s i n g l e mutant gene, the temperature s e n s i t i v i t y may be e x p l o i t e d t o ask when and t o what ex t e n t d u r i n g development the e s s e n t i a l f u n c t i o n o f mus209 i s r e q u i r e d . These q u e s t i o n s were addressed by doing temperature s h i f t s , i n the form of heat p u l s e s between the p e r m i s s i v e and r e s t r i c t i v e temperatures, throughout development. The p a t t e r n of mus209 B 1 l e t h a l i t y , r e v e a l e d by 36-hour heat treatments, i s shown i n F i g u r e 3. These data i n d i c a t e t h a t the mus209 + gene product i s r e q u i r e d through most stages of development, and t o v a r y i n g degrees. In p a r t i c u l a r , those developmental i n t e r v a l s which begin a f t e r the f i r s t l a r v a l molt and extend i n t o the e a r l y stages of pupation are a c u t e l y s e n s i t i v e t o the g e n e t i c d e f e c t i n mus209 B 1. As no s i n g l e 36-hr heat p u l s e r e s u l t s i n 100% m o r t a l i t y , the f a i l u r e o f mus2Q9 B 1 f l i e s t o complete development when r e a r e d c o n t i n u o u s l y a t the r e s t r i c t i v e temperature i s a t t r i b u t e d t o the cumulative e f f e c t o f the mutation through d i f f e r e n t developmental stages, and perhaps i n d i f f e r e n t t i s s u e s . 118 F i g u r e 3. S e n s i t i v i t y of mus209 B 1 homozygotes t o heat or r a d i a t i o n treatments d u r i n g development. Progeny were d e r i v e d from matings between Be mus209 B 1 /SM5 females and mus209 B 1 homozygous males. S u r v i v a l v a l u e s have been normalized t o the homozygoterheterozygote r a t i o o b t a i n e d i n the p e r m i s s i v e c o n t r o l s (22°C). The l a t t e r v a l u e i s 0.76 f o r the temperature s h i f t experiments (2975 c o n t r o l f l i e s s c o r e d ) . The heat treatment data are i n d i c a t e d by c i r c l e s and s o l i d l i n e s . The h o r i z o n t a l bars i n d i c a t e the d u r a t i o n of the heat p u l s e . The r a d i a t i o n treatment data (see T a b l e 3) are i n d i c a t e d by t r i a n g l e s and dashed l i n e s ( i n v e r t e d t r i a n g l e s = 1.3 krad, r i g h t - s i d e - u p t r i a n g l e s = 2.5 k r a d ) . 119 To address the p o s s i b i l i t y t h a t the t e m p e r a t u r e - s e n s i t i v i t y p r o f i l e might r e s u l t from an a l l e l e - s p e c i f i c p e c u l i a r i t y o f mus209 B 1 r a t h e r than as a consequence of a d e f e c t i n the mus209 l o c u s per se, a s i m i l a r heat p u l s e regimen was a p p l i e d t o the mus2 0 9 B 2 a l l e l e . The r e s u l t s of t h i s a n a l y s i s are shown i n F i g u r e 4 . Except f o r some minor d i f f e r e n c e s i n v i a b i l i t y d u r i n g the t h i r d l a r v a l i n s t a r , the p a t t e r n o f t s l e t h a l i t y e l i c i t e d by mus209 B 2 p a r a l l e l s c l o s e l y t h a t of mus209 B 1. I f anything, the d e f e c t i n mus209 B 2 appears t o be more severe than t h a t i n mus209 B 1. Thus, the continuous TSPs are assumed t o r e f l e c t a fundamental developmental requirement f o r the mus209 + product. L e t h a l Phases The e f f e c t i v e l e t h a l phase of both mus209 B 1 and mus209 B 2 o c c u r s s h o r t l y a f t e r puparium formation (data not shown), a t a time when l a r v a l t i s s u e s are b e i n g h i s t o l y z e d and r e p l a c e d by i m a g i n a l s t r u c t u r e s . Although a s m a l l p r o p o r t i o n o f i n d i v i d u a l s d i e a t each l a r v a l stage, and some o t h e r s pupate but d i e as pharate a d u l t s p r i o r t o e c l o s i o n , most p u p a r i a (>80%) come t o encase an amorphous mass of d i s i n t e g r a t i n g l a r v a l t i s s u e , o f t e n w i t h gaping h o l e s , and without d i s c e r n i b l e a d u l t s t r u c t u r e s . By the c r i t e r i a o f Shearn (1977), these mutations are c l a s s i f i e d as prepupal-pupal l e t h a l s . M a t e r n a l E f f e c t s mus209 B 1 and mus209 B 2 are r e f r a c t o r y t o 36-hr heat 121 F i g u r e 4. S e n s i t i v i t y of mus209 B 2 homozygotes t o heat p u l s e s (29°C) d u r i n g development. Progeny were d e r i v e d from matings between Be mus209 B 2/SM5 females and mus209 B 2 homozygous males. S u r v i v a l v a l u e s have been normalized t o the homozygote:heterozygote r a t i o o b t a i n e d i n the p e r m i s s i v e c o n t r o l s (22°C). The l a t t e r v a l u e i s 0.38 (1710 c o n t r o l f l i e s s c o r e d ) . The h o r i z o n t a l bars i n d i c a t e the d u r a t i o n of the heat p u l s e . 122 treatments during embryogenesis and through most of the f i r s t l a r v a l i n s t a r (Figures 3 and 4). This suggests that the mus209 gene product may not be required i n these early developmental stages. A l t e r n a t i v e l y , the maternal genome might contribute to the egg s u f f i c i e n t mus209+ product to support the early development of the mus209 homozygote, even at the r e s t r i c t i v e temperature. (That the maternal genotype can profoundly influence the s e n s i t i v i t y of mus progeny to mutagens has been amply demonstrated - see Appendix B and the references therein.) To d i s t i n g u i s h between the p o s s i b i l i t i e s of non-requirement versus maternal expression, i t would be necessary to examine the temperature s e n s i t i v i t y of mus209 homozygous embryos obtained from s i m i l a r l y homozygous mothers. Obviously, the unconditional s t e r i l i t y of the mus209 homozygous female precludes t h i s d i r e c t approach. However, rous209B1 and mus209 B 2 p a r t i a l l y complement for s t e r i l i t y and so provide a means to t e s t for the maternal e f f e c t i n d i r e c t l y . To do t h i s , h e t e r o a l l e l i c mus209 B 1 / mus209 B 2 females were mated to Be mus209 B 2 / SM5 males (150-200 pa i r s per b o t t l e ) ; eggs were c o l l e c t e d on p e t r i plates (2 hr egg l a y s ) , counted, and subjected at once to a single 36-hr heat treatment (29°C) during embryogenesis. The r e s u l t s of t h i s experiment are given i n Table 2. I t can be seen that the mus209 B 1 / mus209 B 2 mothers produced very few v i a b l e offspring, i r r e s p e c t i v e of the temperature. However, following the embryonic heat treatment even fewer animals survived to adulthood, and a l l of these were 124 Table 2. THE MATERNAL-EFFECT LETHALITY OF mus209 B 1 / mus209 HETEROALLELIC FEMALES Temp.3 No.of Unfert. Lethal Phase c Adult Survivors (°C) Eggs and/or early E L P male female l e t h a l s b Be Bc + Be Bc + Be Bc + Be Bc + 22 862 750 21 20 3 31 4 4 11 6 12 29 877 807 45 2 0 3 4 0 3 0 13 3 Temperature: 22=progeny were developed at 22 UC. 29=progeny were subjected to a single 36-hr heat-pulse (29°C) immediately following o v i p o s i t i o n . F l i e s were then returned to 22°C and kept at t h i s temperature for the remainder of development. b Total number of u n f e r t i l i z e d eggs and/or early (pre-gastrula) l e t h a l embryos (white eggs). c Lethal stages: E=late embryonic (darkened embryos); L=larval; P=pupal. 125 heterozygous. These re s u l t s , taken together with the data i n Figures 3 and 4, show that the v i t a l function of mus209+ i s required not only throughout the second and t h i r d l a r v a l instars and i n the early pupal stages, but during embryogenesis (and presumably i n the f i r s t l a r v a l instar) as well. Moreover, while the maternal expression of mus209+ i s normally e s s e n t i a l for v i a b i l i t y , zygotic expression of a paternally-derived mus209+ gene may occasionally rescue the maternal-effect l e t h a l i t y observed at 29°C. Other factors may have contributed to the low fecundity as well. Scanning electron microscopy revealed that the vast majority of eggs from these h e t e r o a l l e l i c females were morphologically abnormal; most had defective egg s h e l l s , including rudimentary or malformed respiratory appendages (not shown). Thus, s t r u c t u r a l abnormalities i n the v i c i n i t y of the micropyle may have prevented many of the eggs from being f e r t i l i z e d . This observation i s p a r t i c u l a r l y i n t e r e s t i n g because i t suggests that mus209+ might have an i n d i r e c t role i n choriogenesis. A mutant a l l e l e at one other mus locus has been shown to disrupt the amplification of chorion genes that normally occurs l a t e i n oogenesis (Snyder et a l . , 1986) . F i n a l l y , meiotic problems i n mus209 B 1 and mus209 B 2 females have not been ruled out as factors leading to s t e r i l i t y . Radiation Treatments During Development As shown i n the previous section, the l e v e l of heat pulse-induced l e t h a l i t y i n mus209 B 1 varies according to the 126 developmental i n t e r v a l exposed ( F i g u r e 3 ) . Does the p a t t e r n of t s l e t h a l i t y m i r r o r t h a t f o r mutagen s e n s i t i v i t y ? A c l o s e correspondence between the t e m p e r a t u r e - s e n s i t i v e i n t e r v a l s f o r l e t h a l i t y and those f o r mutagen s e n s i t i v i t y might imply t h a t the proximate cause of death, r e s u l t i n g from exposure t o the r e s t r i c t i v e temperature, i s a f a i l u r e i n the mus209 + DNA r e p a i r f u n c t i o n . In o t h e r words, i s t h e r e a r e s i d u a l r e p a i r a c t i v i t y (e.g., f o r spontaneous l e s i o n s ) t h a t a l l o w s s u r v i v a l a t 22°C ( i n the absence of DNA-damaging agents) but which i s rendered n o n f u n c t i o n a l a t 29°C? On the other hand, a l a c k of temporal correspondence f o r the p h e n o c r i t i c a l i n t e r v a l s o f the two phenotypes ( l e t h a l i t y and mus) would n e c e s s i t a t e a more co m p l i c a t e d i n t e r p r e t a t i o n of the r o l e , o r r o l e s , o f the mus209 + product. To address t h i s i s s u e , the r e l a t i v e s e n s i t i v i t y o f mus209 B 1 homozygotes t o gamma-rays was determined a t f o u r s e l e c t times i n development: 24, 96, 140, or 216 hrs p o s t o v i p o s i t i o n . The r e s u l t s o f these experiments are shown i n F i g u r e 3 and i n Ta b l e 3. I t can be seen t h a t a t every stage the mus209 B 1 homozygotes are more s e n s i t i v e t o i o n i z i n g r a d i a t i o n than t h e i r heterozygous s i b s . T h i s i s e s p e c i a l l y t r u e e a r l y i n development; a t l a t e r stages the d i f f e r e n c e s are l e s s pronounced. T h i s p r o g r e s s i v e l o s s of r a d i o s e n s i t i v i t y p a r a l l e l s the response o f w i l d t y p e D r o s o p h i l a . and of holometabolous i n s e c t s g e n e r a l l y (Mavor, 1927; Grosch and Hopwood, 1979). Apparently, 127 T a b l e 3. THE EFFECTS OF IONIZING RADIATION ON THE RELATIVE SURVIVAL OF mus209 B 1 HOMOZYGOTES AT VARIOUS TIMES IN DEVELOPMENT Dose Normalized su r v i v a l r a t i o 3 (krads) Time of i r r a d i a t i o n postoviposition ( h r s ) b 24 96 140 216 1.3 0.00 0.27 0.38 0.73 (283) (432) (422) (448) 2.5 — 0. 00 0.01 0.38 (486) (377) (427) The normalized s u r v i v a l r a t i o i s the treated homozygote to heterozygote r a t i o divided by the untreated homozygote to heterozygote r a t i o . The l a t t e r value i s 0.84 (1189). The numbers i n parentheses indicate the t o t a l number of f l i e s scored per experiment. See Figure 3 for the corresponding developmental stage. 128 the l e v e l of r a d i o s e n s i t i v i t y i s a p r o p e r t y of the p a r t i c u l a r stage i n the animal's l i f e c y c l e , p o s s i b l y determined by both the m i t o t i c r a t e and the number of c o n s e q u e n t i a l t a r g e t c e l l s ( i . e . , the degree of d i f f e r e n t i a t i o n ) . The d e f e c t i n mus209 B 1 does not appear t o a l t e r t h i s i n t r i n s i c r a d i o s e n s i t i v i t y p r o f i l e . I t merely i n c r e a s e s the r e l a t i v e r a d i o s e n s i t i v i t y o f the homozygote, and only by an amount commensurate wi t h the animal's developmental stage. Consequently, the developmental p a t t e r n s of mutagen s e n s i t i v i t y and temperature s e n s i t i v i t y d i f f e r markedly ( F i g u r e 3). For example, the most s t r i k i n g c o n t r a s t i s seen i n embryogenesis. T h i s stage, although h i g h l y s e n s i t i v e t o r a d i a t i o n , i s u n a f f e c t e d by the heat treatments. These r e s u l t s imply t h a t the b i o c h e m i c a l b a s i s f o r the mutagen s e n s i t i v i t y may be d i f f e r e n t from t h a t f o r the temperature-induced l e t h a l i t y . In o t h e r words, the mus209 gene produ c t ( s ) may possess more than one f u n c t i o n . 129 DISCUSSION The mutation mus209 B 1 i d e n t i f i e s a gene whose normal f u n c t i o n (1) i n f l u e n c e s the s e n s i t i v i t y of f l i e s t o both i o n i z i n g r a d i a t i o n and MMS; (2) i s necessary f o r female f e r t i l i t y and (3) i s e s s e n t i a l f o r v i a b i l i t y (even i n the absence of DNA-damaging ag e n t s ) . I n c l u d i n g mus209 B 1, 8 l e t h a l mutations, 2 of them t s , denote t h i s p l e i o t r o p i c gene. While t h i s c o l l e c t i o n of l e t h a l a l l e l e s underscores the e s s e n t i a l nature of mus209. the f a c t t h a t u n c o n d i t i o n a l l y v i a b l e mutants are m i s s i n g may be an a r t i f a c t of the o r i g i n a l s c r e e n i n g p r o t o c o l . Except f o r mus209 B 1, each mutation was i d e n t i f i e d by i t s f a i l u r e t o s u r v i v e over the M(2)017 d e f i c i e n c y ( S h e l l e n b a r g e r and Duttagupta, 1978). Using t h i s approach, o n l y h a p l o - i n s u f f i c i e n t and s t r i c t l y l e t h a l mutations are s e l e c t a b l e . As S h e l l e n b a r g e r and Duttagupta had no p r i o r knowledge of the mus phenotype, a l l e l e s c o n f e r r i n g mutagen s e n s i t i v i t y but having no adverse e f f e c t s on v i a b i l i t y would have gone undetected. However, i n a s c r e e n t h a t should have allowed t h e i r d e t e c t i o n , Henderson e t a l . (1987) a l s o f a i l e d t o r e c o v e r noncomplementing ( f o r MMS s e n s i t i v i t y ) s t r i c t l y v i a b l e a l l e l e s o f mus209 B 1. While t h i s r e s u l t may not be s u r p r i s i n g g i v e n the c o m p a r a t i v e l y low number of chromosomes t e s t e d , the p o s s i b i l i t y t h a t the mus + and e s s e n t i a l f u n c t i o n s of mus209 are not m u t a t i o n a l l y s e p a r a b l e i n t o d i s c r e t e genie domains cannot be excluded. Thus, mutations which a f f e c t the r e p a i r - r e l a t e d f u n c t i o n o f mus209 may i n v a r i a b l y a f f e c t the e s s e n t i a l f u n c t i o n t o a g r e a t e r o r l e s s e r d e g r e e , a n d v i c e v e r s a . N a u m o v s k i a n d F r i e d b e r g (198 6) h a v e a t t e m p t e d t o a n s w e r a s i m i l a r q u e s t i o n a b o u t t h e g e n i e o r g a n i z a t i o n o f t h e e s s e n t i a l a n d e x c i s i o n r e p a i r f u c t i o n s o f t h e RAD3 ge n e o f S. c e r e v i s i a e . U s i n g i n v i t r o m u t a g e n e s i s , n e i t h e r o f t h e s e f u n c t i o n s c o u l d be l o c a l i z e d t o d i s c r e t e r e g i o n s o f t h e g e n e . H o w e v e r , u n l i k e t h e p r e s e n t s i t u a t i o n f o r mus209, m u t a t i o n s i n RAD3 a p p e a r t o i n a c t i v a t e more r e a d i l y t h e r e p a i r f u n c t i o n a s o p p o s e d t o t h e e s s e n t i a l f u n c t i o n , a n d p e r f e c t l y v i a b l e r a d 3 m u t a n t s t h a t a r e c o m p l e t e l y d e f e c t i v e i n DNA r e p a i r h a v e b e e n i s o l a t e d . Two i m p o r t a n t f i n d i n g s e merged f r o m t h e t e m p e r a t u r e s h i f t s t u d i e s c a r r i e d o u t on m u s 2 0 9 B 1 a n d m u s 2 0 9 B 2 . F i r s t , t h e e s s e n t i a l f u n c t i o n o f m u s 2 0 9 + i s r e q u i r e d t h r o u g h o u t m o s t o f d e v e l o p m e n t ( t h e l a t t e r h a l f o f t h e p u p a l p e r i o d a p p e a r s t o be an e x c e p t i o n ) , a n d s e c o n d , m a t e r n a l a s w e l l a s z y g o t i c e x p r e s s i o n o f t h i s g e n e i s a d e v e l o p m e n t a l s i n e qua n o n . D e s p i t e t h e more o r l e s s c o n t i n u o u s TSP, h e a t t r e a t m e n t s a p p l i e d d u r i n g t h e s e c o n d a n d t h i r d l a r v a l i n s t a r s h a v e no o b v i o u s e f f e c t o n l a r v a l v i a b i l i t y . N o t u n t i l p u p a r i a t i o n do t h e l e t h a l e f f e c t s become e v i d e n t . The b a s i s f o r t h i s d e l a y e d k i l l i n g i s u n d e r s t a n d a b l e i n t h e c o n t e x t o f D r o s o p h i l a o n t o g e n y . D u r i n g e a r l y e m b r y o g e n e s i s , t h e c e l l s d e s t i n e d t o g i v e r i s e t o a d u l t s t r u c t u r e s ( e . g . , i m a g i n a l d i s c c e l l s a n d h i s t o b l a s t s ) become s e p a r a t e d f r o m t h o s e t h a t p r o d u c e t h e l a r v a l t i s s u e s . A f t e r h a t c h i n g , t h e l a r v a l c e l l s g r o w i n s i z e a n d d e v e l o p l a r g e p o l y t e n e n u c l e i b u t t h e y do n o t d i v i d e . I n c o n t r a s t , t h e 131 i m a g i n a l d i s c c e l l s remain d i p l o i d and co n t i n u e t o p r o l i f e r a t e by m i t o t i c d i v i s i o n s . Although p r e s e n t i n the d e v e l o p i n g l a r v a , most of these m i t o t i c a l l y a c t i v e c e l l s are not e s s e n t i a l f o r l a r v a l f u n c t i o n s (some c e l l s i n the c e n t r a l nervous system and i n the r i n g glands of the f o r e g u t and s a l i v a r y glands may be e x c e p t i o n s ) . During metamorphosis (which begins a t puparium formation) most l a r v a l t i s s u e s degenerate. C e l l s i n the abdominal h i s t o b l a s t n e s t s begin t o d i v i d e f o r the f i r s t time, whereas those i n the imaginal d i s c s cease d i v i d i n g and d i f f e r e n t i a t e . At t h i s time the imaginal d i s c s evaginate t o g i v e r i s e t o the a d u l t s t r u c t u r e s and a f u l l y formed imago e v e n t u a l l y r e s u l t s . Thus, i t i s s i g n i f i c a n t t h a t the TSPs of mus209 B 1 and mus209 B 2 correspond t o developmental i n t e r v a l s d u r i n g which the im a g i n a l d i s c c e l l s and the h i s t o b l a s t s are m i t o t i c a l l y a c t i v e ( P o s t l e t h w a i t , 1978). (That a t 29°C the e a r l y embryonic n u c l e a r and c e l l d i v i s i o n s proceed normally i n mutants d e r i v e d from heterozygous mothers i s a t t r i b u t e d t o a maternal e f f e c t (see RESULTS).) T h i s correspondence suggests t h a t the i n d i s p e n s i b l e f u n c t i o n o f mus209 may be r e q u i r e d f o r c e l l p r o l i f e r a t i o n . S e v e r a l d i a g n o s t i c s c o u l d be used t o e s t a b l i s h such a r o l e . Most o b v i o u s l y , the imaginal d i s c s o f h e a t - t r e a t e d l a r v a e c o u l d be examined as t o t h e i r s i z e , t h e i r shape, and t h e i r c a p a c i t y t o d i f f e r e n t i a t e a f t e r t r a n s p l a n t a t i o n i n t o a w i l d t y p e host ( i . e . , i s t he mus209 d e f e c t d i s c autonomous?) (e.g., Shearn e t a l . , 1971; Shearn and Garen, 1974). V i t a l s t a i n i n g t e c h n i q u e s c o u l d 132 be used t o v i s u a l i z e n e c r o t i c c e l l s w i t h i n these d i s c s (Arking, 1974). A l s o , brooding experiments c o u l d be c a r r i e d out t o look f o r p r e - m e i o t i c t s m a l e - s t e r i l e e f f e c t s ( S h e l l e n b a r g e r and Cross, 1979). While p o s i t i v e f i n d i n g s i n these t e s t s would be c o n s i s t e n t w i t h a d e f e c t i n c e l l p r o l i f e r a t i o n , they would not c o n s t i t u t e i t s p r o o f . To c o n f i r m t h a t mus209 B 1 i s a c e l l -autonomous l e t h a l mutation, somatic recombination a n a l y s e s would have t o be c a r r i e d out (e.g., A r k i n g , 1974; R u s s e l l , 1974). In g e n e r a l , mutagen s e n s i t i v e mutations which i d e n t i f y v i t a l l o c i a re apt t o d i s r u p t one of two processes i n the c e l l c y c l e : DNA s y n t h e s i s (e.g., N j a g i and K i l b e y , 1982; Jong e t a l . , 1984; K a s s i r e t a l . , 1985; Peterson e t a l . , 1985; Kupiec and Simchen, 1986) or m i t o s i s (e.g., Baker e t a l . , 1982; G a t t i e t a l . , 1983). That DNA r e p l i c a t i o n proceeds normally i n mus209 mutants r e a r e d under r e s t r i c t i v e c o n d i t i o n s i s suggested by the o b s e r v a t i o n t h a t the l a r v a l t i s s u e s are p o l y t e n i z e d . However, the p o s s i b i l i t y t h a t mus209 encodes a DNA r e p l i c a t i o n f a c t o r t h a t i s a c t i v e i n d i p l o i d but not p o l y t e n e n u c l e i cannot be excluded. An a l t e r n a t e , perhaps more l i k e l y p o s s i b i l i t y i s t h a t mus209 has a r o l e p r e p a r a t o r y t o or d u r i n g m i t o s i s . Three o t h e r e s s e n t i a l mus l o c i i n D r o s o p h i l a appear t o encode such f u n c t i o n s . For example, a t the r e s t r i c t i v e temperature, the t s l e t h a l mutation m u ^ l 0 1 t s l d i s r u p t s p r e f e r e n t i a l l y the condensation of heterochromatic r e g i o n s of chromosomes d u r i n g the m i t o t i c c e l l c y c l e ( G a t t i e t a l . , 1983). T h i s suggests t h a t 133 t h e muslOl" 1" p r o d u c t may n o r m a l l y be i n v o l v e d i n c h r o m a t i n p a c k a g i n g . The o b s e r v a t i o n by D. S i n c l a i r ( p e r s o n a l c o m m u n i c a t i o n ) t h a t m u s l 0 1 t s l i s a n e n h a n c e r o f p o s i t i o n - e f f e c t v a r i e g a t i o n i s c o n s i s t e n t w i t h t h i s i d e a . Two o t h e r i n d i s p e n s i b l e mus l o c i , mus!05 a n d mus!09. e n c o d e p r o d u c t s t h a t a l s o a p p e a r t o h e l p m a i n t a i n t h e s t r u c t u r a l i n t e g r i t y o f chromosomes i n d i v i d i n g c e l l s . L a r v a l b r a i n g a n g l i a l c e l l s f r o m c e r t a i n m u t a n t a l l e l e s e x h i b i t h i g h f r e q u e n c i e s o f chromosome a b e r r a t i o n s (>0.5 a b e r r a t i o n s ( b r e a k s a n d e x c h a n g e s ) p e r c e l l p e r c y c l e ) ( B a k e r e t a l . , 1 9 8 2 ) . I n mus!05. m o s t o f t h e a b e r r a t i o n s o c c u r i n t h e e u c h r o m a t i c r e g i o n s o f t h e genome. By c o n t r a s t , i n mus!09. m o s t o f t h e chromosome b r e a k s a r e c o n c e n t r a t e d a t t h e e u c h r o m a t i c - h e t e r o c h r o m a t i c j u n c t i o n s ( G a t t i , 1 9 7 9 ; B a k e r e t a l . , 1 9 8 2 ) . B a k e r e t a l . ( 1982) o b s e r v e d t h a t l a r v a e c a r r y i n g l e t h a l a l l e l e s o f t h e s e two g e n e s h a v e s m a l l , d e g e n e r a t e i m a g i n a l d i s c s a n d d i e a t t h e l a r v a l - p u p a l b o u n d a r y . T h e s e r e s u l t s l e d them t o p o s t u l a t e t h a t l a r v a e h o m o z y g o u s o r h e m i z y g o u s f o r s e v e r e a l l e l e s o f muslOS o r rousl09 f a i l t o p u p a t e b e c a u s e e x t e n s i v e chromosome b r e a k a g e k i l l s a n i r r e t r i e v a b l e number o f c e l l s w i t h i n t h e i m a g i n a l d i s c s . A s i m i l a r a n a y s i s o f m i t o t i c f i g u r e s f r o m n e u r o b l a s t s o f m u s 2 0 9 B 1 ( a n d m u s 2 0 9 B 2 ) l a r v a e r a i s e d a t 22°C v e r s u s 29°C m i g h t r e v e a l a r o l e , i f a n y , f o r mus_209 + i n m a i n t a i n i n g chromosome s t a b i l i t y t h r o u g h o u t t h e m i t o t i c c e l l c y c l e . C u r i o u s l y , t h e t s a n d gamma r a y - s e n s i t i v e d e v e l o p m e t n a l i n t e r v a l s do n o t c o i n c i d e i n m u s 2 0 9 B 1 . T h i s may i m p l y t h a t t h e 134 e s s e n t i a l and r e p a i r - r e l a t e d a c t i v i t i e s o f mus209 are d i s t i n c t , t h a t i s , the t s l e t h a l i t y r e s u l t s not as a consequence of a d e f e c t i n DNA r e p a i r but from the absence of an e s s e n t i a l , second f u n c t i o n of mus209. To a t t a c h a d d i t i o n a l s i g n i f i c a n c e t o these f i n d i n g s may, however, be f a l l a c i o u s s i n c e these experiments measure the e f f e c t s of two q u a l i t a t i v e l y d i f f e r e n t p h y s i o l o g i c a l burdens - an acute dose of i o n i z i n g r a d i a t i o n a d m i n i s t e r e d over s e v e r a l minutes a t 22°C, v e r s u s a sub-acute, 36-hr, 29°C heat treatment. Notwithstanding these d i f f e r e n c e s , how i s i t t h a t the maternal genome can rescue the t s l e t h a l i t y but not the gamma ray s e n s i t i v i t y of the embryonic and f i r s t l a r v a l i n s t a r stages? The most s t r a i g h t f o r w a r d answer t o t h i s q u e s t i o n i s t h a t e a r l y i n development, the amount of mus2_09+ product needed t o perform the e s s e n t i a l r o l e i s c o n s i d e r a b l y l e s s t h a t t h a t r e q u i r e d f o r the r e p a i r - r e l a t e d a c t i v i t y . Thus, f o l l o w i n g i r r a d i a t i o n , the demand f o r the mus209 + product may simply o u t s t r i p the maternal s u p p l y . S p e c u l a t i o n s as t o the nature o f the mus209 + gene product In the absence of b i o c h e m i c a l and m o l e c u l a r b i o l o g i c a l c l u e s as t o the nature of the mus209 + gene p r o d u c t ( s ) , any hypotheses c o n c e r n i n g i t s p r e c i s e f u n c t i o n ( s ) are a t b e s t h i g h l y s p e c u l a t i v e , and s o l e l y r e l i a n t on g e n e t i c data. For example, the e x i s t e n c e of the complementary a l l e l i c mutations mus209 B 1, mus209 B 2 and D-1368. by convention, i m p l i e s t h a t i n i t s 135 e s s e n t i a l r o l e , the mus209 + gene product may be a m u l t i m e r i c complex, e.g., a homodimer. Because the same h e t e r o a l l e l i c f l i e s s t i l l e x h i b i t s e n s i t i v i t y t o mutagens, i t i s p o s s i b l e (although not o b l i g a t o r y ) t h a t the r e p a i r - r e l a t e d a c t i v i t y r e s i d e s i n s i n g l e p o l y p e p t i d e s and not i n multimers. Thus, s u b u n i t s t r u c t u r e may d i s t i n g u i s h between f u n c t i o n s . At t h i s time, perhaps the b e s t approach toward understanding the b i o c h e m i c a l nature of the mus209 + product i s t o l o o k f o r p h e n o t y p i c a l l y s i m i l a r mutants amongst the l a r g e c o l l e c t i o n o f w e l l c h a r a c t e r i z e d rad mutations i n S. c e r e v i s i a e . The r e l e v a n t mus209 B 1 phenes i n c l u d e s e n s i t i v i t y t o MMS and gamma r a y s , but not t o HN2 (Henderson e t a l . , 1987), and female s t e r i l i t y . In these r e s p e c t s mus209 B 1 most c l o s e l y resembles a number o f mutations b e l o n g i n g t o the RAD52 e p i s t a s i s group (Haynes and Kunz, 1981). For example, r a d 5 2 - l mutants are extremely s e n s i t i v e t o MMS and i o n i z i n g r a d i a t i o n , and d i p l o i d c e l l s f a i l t o produce v i a b l e spores (Game and Mortimer, 1974). The b i o c h e m i c a l b a s i s f o r these a b n o r m a l i t i e s appears t o be a m i s s i n g endo-exonuclease a c t i v i t y t h a t i s r e g u l a t e d (but not encoded) by PAD52 (Resnick e t a l . , 1984). More s p e c i f i c a l l y , r a d 5 2 - l c e l l s are d e f e c t i v e i n m i t o t i c and m e i o t i c recombination (Game e t a l . , 1980; Prakash e t a l . , 1980), and i n d o u b l e - s t r a n d break (DSB) r e p a i r (Ho, 1975). They a l s o e x h i b i t h i g h l e v e l s of spontaneous and r a d i a t i o n - i n d u c e d chromosome l o s s (Mortimer e t a l . , 1981). I t has been suggested t h a t t h i s l a t t e r phenotype c o u l d a r i s e because u n r e p a i r e d double 136 s t r a n d breaks i n DNA l e a d t o telomere l o s s , r e s u l t i n g i n chromosome i n s t a b i l i t y , or because u n r e s o l v e d chromatid exchanges l e a d t o a b e r r a n t chromosome s e g r e g a t i o n a t m i t o s i s (Mortimer e t a l . , 1981). E i t h e r mechanism c o u l d account f o r the mutagen s e n s i t i v i t y (and t s l e t h a l i t y ) of mus209 B 1. However, the mus209 gene cannot be s t r i c t l y analogous t o RAD52 or t o some o t h e r RAD52-like genes (e.g., RAD50 and RAD54) because u n l i k e mus209, thes e y e a s t genes do not encode e s s e n t i a l f u n c t i o n s ( S c h i l d e t a l . , 1983). 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(1982) A n o t e on t h e u t i l i t y o f r e p a i r - d e f i c i e n t s t mus302 D r o s o p h i l a f e m a l e s i n d e t e c t i n g chromosome l o s s a n d s e x - l i n k e d r e c e s s i v e l e t h a l s i n d u c e d i n t h e m a l e genome b y m e t h y l m e t h a n e s u l f o n a t e (MMS) a n d d i m e t h y l n i t r o s a m i n e (DMN), E n v i r o n . Mut., 4, 2 9 1 - 2 9 3 . Z i m m e r i n g , S. (1983) The m e i - 9 a t e s t f o r chromosome l o s s i n D r o s o p h i l a : A r e v i e w o f a s s a y s o f 21 c h e m i c a l s f o r chromosome b r e a k a g e , E n v i r o n . Mut., 5, 3 6 3 - 3 6 5 . 158 APPENDIX A I s o l a t i o n o f Mutaaen-Sensitive S t r a i n s A l l mus mutants b e a r i n g the alphanumeric s u p e r s c r i p t s B l or B2 were i s o l a t e d as o u t l i n e d ' i n F i g u r e 1. A d u l t males, c a r r y i n g i s o g e n i c second chromosomes marked wi t h the r e c e s s i v e mutations b, p_r and cn, were f e d e t h y l methanesulf onate (EMS, 0.24% v/v) a c c o r d i n g t o the method of Lewis and Bacher (1968) and mated en masse t o p_r T f t / CyO females. S i n g l e b pjr cn* / CyO F± males (where * i n d i c a t e s an EMS-treated chromosome) were c r o s s e d t o 3 or 4 p_r T f t / CyO females. F 2 b p r cn* / CyO females were c o l l e c t e d from each l i n e and mated t o t h e i r r e s p e c t i v e b p_r cn* / CyO male s i b s . Each c u l t u r e was examined f o r the presence of b p r cn homozygotes amongst the F 3 i n d i v i d u a l s . An absence of b p r cn f l i e s i n c u l t u r e s c o n t a i n i n g b p_r cn / CyO h e t e r o z y g o t e s i n d i c a t e d the presence of an induced r e c e s s i v e l e t h a l mutation, and such c u l t u r e s were d i s c a r d e d . A d u l t s from the remaining c u l t u r e s were t r a n s f e r r e d every 2 days t o v i a l s c o n t a i n i n g f r e s h medium t o o b t a i n 3 s u c c e s s i v e 2-day s u b c u l t u r e s . I n d i v i d u a l s i n the f i r s t s u b c u l t u r e (0-48 hr post o v i p o s i t i o n ) were t r e a t e d w i t h 0.25 mL of 0.08% v/v MMS ( i n water) and incubated a t 29°C u n t i l e c l o s i o n . The second s e t of r e p l i c a t e c u l t u r e s was t r e a t e d w i t h 0.25 mL of water and incubated a t 29°C u n t i l e c l o s i o n . The t h i r d s e t o f r e p l i c a t e c u l t u r e s was maintained a t 22°C t o e s t a b l i s h s t o c k s . A s t r a i n was c o n s i d e r e d t o be a p u t a t i v e MMS-sensitive mutant when the homozygoterheterozygote r a t i o o b t a i n e d i n the 159 F i g u r e 1. Mating and s e l e c t i o n p r o t o c o l used t o i s o l a t e second chromosome mus mutations. 160 EMS - b pr cn pr Tft P oo — x ^ ^ b pr cn . CyO b pr cn CyO y pr Tft CyO b pr cn CyO y b pr cn CyO b pr cn b pr cn b pr c n * ' CyO ' MMS © o 22,29 AAF HN2 gamma rays 161 MMS-treated c u l t u r e f e l l below 1/6 of t h a t o b t a i n e d i n the H 20-t r e a t e d c o n t r o l c u l t u r e . A l l p u t a t i v e MMS-sensitive mutants and those s t r a i n s d i s p l a y i n g a t e m p e r a t u r e - s e n s i t i v e l e t h a l phenotype a t 29°C were r e t e s t e d a t 22°C f o r s e n s i t i v i t y t o 0.08% MMS. A d d i t i o n a l e x t e n s i v e r e t e s t i n g was c a r r i e d out t o co n f i r m the mutant phenotypes. A t o t a l o f 4032 p a i r matings, each r e p r e s e n t i n g a s i n g l e EMS-treated second chromosome, were e s t a b l i s h e d . Approximately 83% o f these matings produced F 2 c u l t u r e s , and about 45% of the F 2 c u l t u r e s produced normal numbers of homozygous v i a b l e b pr cn o f f s p r i n g a t 22°C. In t o t a l , 18 MMS-sensitive s t r a i n s were re c o v e r e d . In 3 subsequent s t u d i e s , the EMS-treated chromosomes were t e s t e d f o r s e n s i t i v i t y t o the mutagens AAF, HN2 and gamma r a y s . F i v e A A F - s e n s i t i v e , 5 HN2-sensitive and 4 gamma r a y -s e n s i t i v e mutants ( i n a d d i t i o n t o those MMS-sensitive s t r a i n s t h a t show c r o s s - s e n s i t i v i t y t o these 3 mutagens) were recovered. 162 APPENDIX B An Examination o f the I n f l u e n c e of the Maternal Genotype on the  S e n s i t i v i t y o f mus O f f s p r i n g t o MMS Wurgler e t a l . (1972) p r o v i d e d some of the f i r s t evidence t h a t the maternal genotype c o u l d i n f l u e n c e the r e c o v e r y o f mutations ( s p e c i f i c a l l y , sex chromosome l o s s ) i n zygotes d e r i v e d from X - i r r a d i a t e d sperm. They concluded t h a t one or more m a t e r n a l l y - d e r i v e d f a c t o r s d e p o s i t e d i n t o the egg, c o u l d i n f l u e n c e the balance between f i x a t i o n and r e p a i r of p r e m u t a t i o n a l DNA l e s i o n s t h a t l e a d t o aneuploidy. Racine e t a l . (1979) o b t a i n e d s i m i l a r r e s u l t s , and f u r t h e r , found l i t t l e o r no evidence of a maternal i n f l u e n c e on the r e c o v e r y of dominant l e t h a l mutations or t r a n s l o c a t i o n s . The a v a i l a b i l i t y o f b i o c h e m i c a l l y c h a r a c t e r i z e d mutagen-s e n s i t i v e (mus) s t r a i n s made p o s s i b l e an examination of the e f f e c t s o f DNA r e p a i r d e f e c t s i n the oocyte on both spontaneous and induced mutagenesis i n sperm (Graf e t a l . , 1979). Pronounced maternal e f f e c t s , as measured by a s e x - l i n k e d r e c e s s i v e l e t h a l t e s t , were observed i n some cases. Depending on the p a r t i c u l a r r e p a i r - d e f i c i e n t s t r a i n used, and the type of mutagen employed, the frequency of l e t h a l mutations e i t h e r i n c r e a s e d , decreased, or remained unchanged. The maternal genotype can a l s o i n f l u e n c e the s e n s i t i v i t y of somatic c e l l s i n mus l a r v a e t o both spontaneous and MMS-effected l e t h a l i t y . G r af and Wurgler (1978) observed t h a t MMS-treated l a r v a e d e r i v e d from c e r t a i n r e p a i r - d e f i c i e n t females are much 163 more s e n s i t i v e t o k i l l i n g by MMS than are g e n o t y p i c a l l y i d e n t i c a l progeny d e r i v e d from r e p a i r - p r o f i c i e n t (heterozygous) mothers. Notably, these maternal e f f e c t s extend beyond the embryonic s t a g e s . T h i s maternal e f f e c t phenomenon i s p o t e n t i a l l y u s e f u l t o mutagen t e s t i n g regimens such as the mus t e s t d e s c r i b e d i n Chapter 3. In p r i n c i p a l , o f f s p r i n g d e r i v e d from matings between homozygous mus mothers and heterozygous f a t h e r s should be more s e n s i t i v e t o a suspect mutagen than g e n o t y p i c a l l y s i m i l a r o f f s p r i n g o b t a i n e d from the r e c i p r o c a l mating. Thus, a lower dose of mutagen c o u l d be used t o e f f e c t the same p o s i t i v e response. With these i s s u e s i n mind, the experiments r e p o r t e d here sought t o determine whether maternal e f f e c t s c o u l d i n f a c t be demonstrated f o r t h r e e s i n g l e mutants, mus205 B 1, mus2Q8 B 1. and mus210 B 1. MMS dose-response curves were generated as d e s c r i b e d i n Chapter 2, except homozygous mus females were c r o s s e d t o heterozygous males. The r e s u l t s of these experiments are shown i n F i g u r e 1. C l e a r l y , each mutant e x h i b i t s a s t r o n g maternal e f f e c t t h a t renders homozygous mus progeny some 2-3 times more s e n s i t i v e t o MMS (as measured by the LD50) than t h e i r g e n o t y p i c c o u n t e r p a r t s d e r i v e d from the r e c i p r o c a l c r o s s . These f i n d i n g s a r e i n agreement w i t h the e a r l i e r r e s u l t s o f Graf and Wurgler (1978) and suggest t h a t a t l e a s t some of the m a t e r n a l l y - d e r i v e d mutagenesis " f a c t o r s " h y p o t h e s i z e d by Wurgler e t a l . (1972) may i n f a c t be mus gene products (e.g., DNA r e p a i r enzymes). 164 F i g u r e 1. MMS s e n s i t i v i t y and maternal e f f e c t s i n mus205 B 1. mus208 B 1 and mus210 B 1. Progeny were d e r i v e d from matings between heterozygous females and homozygous mus males ( s o l i d symbols) or homozygous mus females and heterozygous males (open symbols). Data f o r the former c r o s s e s were taken from Chapter 2. C i r c l e s , mus205 B 1 (1455, 1337); t r i a n g l e s , mus208 B 1 (3098, 2175); squares, mus210 B 1 (3893, 3531). The data i n parentheses p e r t a i n t o matings between homozygous females and heterozygous males. The numbers i n d i c a t e the average, and the minimum number of f l i e s s c o r e d per non-zero dose p o i n t , r e s p e c t i v e l y . S u r v i v a l v a l u e s from the t r e a t e d c u l t u r e s have been normalized t o the homozygoterheterozygote r a t i o o b t a i n e d i n the u n t r e a t e d c o n t r o l s as d e s c r i b e d i n Chapter 2 (MATERIALS AND METHODS). The l a t t e r v a l u e s are : mus205 B 1. 0.94 (2042); mus208 B 1. 0.99 (3007) and mus210 B 1. 0.86 (3062). The numbers i n parentheses i n d i c a t e the t o t a l number of c o n t r o l f l i e s s c o r e d per genotype. The e r r o r b a r s i n d i c a t e 1 standard d e v i a t i o n o f the mean c a l c u l a t e d from 3-6 separate treatments. 165 0.01 0.02 0.03 0.04 % MMS 166