A GENETIC ANALYSIS OF MUTAGEN-SENSITIVE MUTATIONS ON THE SECOND CHROMOSOME OF DROSOPHILA MELANOGASTER By DARYL STEWART B.Sc, The U n i v e r s i t y HENDERSON of British C o l u m b i a , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department We a c c e p t t h i s to THE o f Zoology) thesis the required as conforming standard UNIVERSITY OF BRITISH COLUMBIA September 1987 © Daryl Stewart Henderson In presenting degree at this the thesis in University of partial fulfilment of British Columbia, I agree freely available for reference and study. I further copying of department this or publication of thesis for by his or her that the for an advanced Library shall make it It is granted by the understood that head of copying my or this thesis for financial gain shall not be allowed without my written Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1 Y 3 DE-6(3/81) representatives. requirements agree that permission for extensive scholarly purposes may be permission. Date the OCT" \ ABSTRACT Mutagen-sensitive (mus) mutations i n D r o s o p h i l a melanogaster render d e v e l o p i n g f l i e s h y p e r s e n s i t i v e t o the l e t h a l e f f e c t s o f DNA-damaging agents. mutations i d e n t i f y DNA new r e p a i r - r e l a t e d genes. second chromosome mus mus209 . mus210 B1 B1 In g e n e r a l , mus mutations (mus2_05 , mus208 . B1 B1 using a v a r i e t y of genetic t e s t s . s e n s i t i v i t y o f double mutant mus component s i n g l e mutants. of (MMS), were c h a r a c t e r i z e d One t e s t measured the Mutant i n t e r a c t i o n s were examined i n and i n 2 t r i p l e mus D1 B2 strains containing B1 B1 B1 combinations and mus211 (or B1 These a n a l y s e s have r e v e a l e d predominantly s y n e r g i s t i c and e p i s t a t i c responses t o MMS. the MMS- s t r a i n s compared t o t h e i r mus201 . mus205 . mus208 , mus210 mus211 ). B1 and m u s 2 1 1 ) , s e l e c t e d on the b a s i s o f s e n s i t i v i t y t o methyl methanesulfonate 8 double mus In t h i s study, 5 Taken t o g e t h e r with f i n d i n g s o f p r e v i o u s g e n e t i c and b i o c h e m i c a l s t u d i e s o f D r o s o p h i l a mus s t r a i n s , these r e s u l t s suggest t h a t 3 major r e p a i r pathways may operate i n f l i e s t o c o r r e c t 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 o f the i n t e r a c t i o n s t u d i e s suggest t h a t mus mutations might s e r v e as r a p i d and s e n s i t i v e b i o a s s a y s o f somatic g e n o t o x i c i t y caused by mutagens and c a r c i n o g e n s . To e x p l o r e t h i s p o s s i b i l i t y , mutagen t e s t system was d e v i s e d employing t r i p l e mutant strains. One strain (mus208 B1 mus210 s e n s i t i v i t y t o 14 mutagens/carcinogens ii B1 m u s 2 l l ) was B 2 a simple mus tested for and 2 non-carcinogens. E l e v e n o f the mutagens/carcinogens were r e a d i l y d e t e c t e d as g e n o t o x i c . Both non-carcinogens were non-genotoxic. p r e l i m i n a r y r e s u l t s demonstrate the f e a s i b i l i t y These (and some l i m i t a t i o n s ) o f the proposed somatic g e n o t o x i c i t y assay and emphasize t h e need f o r f u r t h e r t e s t v a l i d a t i o n u s i n g a l a r g e r chemical data base. The 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 mus209 B1 was s u b j e c t e d t o e x t e n s i v e g e n e t i c a n a l y s e s and t o temperature experiments d u r i n g development. shift T h i s l o c u s was found t o encode a p r o d u c t ( s ) t h a t (1) i s e s s e n t i a l f o r v i a b i l i t y a t v i r t u a l l y a l l p r e - i m a g i n a l developmental stages (the l a t t e r h a l f o f p u p a t i o n appears t o be an e x c e p t i o n ) , (2) i s necessary f o r w i l d t y p e l e v e l s o f r e s i s t a n c e t o the genotoxic e f f e c t s o f MMS and i o n i z i n g r a d i a t i o n , and (3) i s r e q u i r e d f o r female fertility. mutation C o n f i r m a t i o n o f the p l e i o t r o p i c nature o f t h i s was o b t a i n e d by m e i o t i c and c y t o g e n e t i c mapping s t u d i e s and by complementation t e s t s w i t h a s e r i e s o f a l l e l i c The mus209 mutations of for B1 mutations. phenotypes a r e s i m i l a r t o ones c o n f e r r e d by i n D r o s o p h i l a and y e a s t t h a t d i s r u p t v a r i o u s a s p e c t s chromosome metabolism. In t h i s context, some p o s s i b l e r o l e s mus209 B1 are discussed. iii TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES v i i ACKNOWLEDGEMENTS CHAPTER 1. ix GENERAL INTRODUCTION TO CELLULAR RESPONSES TO DNA DAMAGE I. Perspectives 2 I I . C e l l u l a r Responses t o DNA Damage A. Enzyme-catalyzed photoreversal of p y r i m i d i n e dimers B. DNA e x c i s i o n r e p a i r 5 8 C. P o s t r e p l i c a t i o n r e p a i r 20 D. I n d u c i b l e r e p a i r responses 23 I I I . M u t a g e n - s e n s i t i v e mutations i n D r o s o p h i l a melanogaster CHAPTER 2. 4 30 INTERACTIONS BETWEEN MMS-SENSITIVE MUTATIONS I. I n t r o d u c t i o n 36 I I . M a t e r i a l s and Methods I I I . Results 39 44 IV. D i s c u s s i o n 69 iv CHAPTER 3. MUTAGEN-SENSITIVE STRAINS AS GENOTOXICITY INDICATORS I. I n t r o d u c t i o n 79 I I . M a t e r i a l s and Methods I I I . Results 88 IV. D i s c u s s i o n CHAPTER 4. 82 94 A GENETIC AND DEVELOPMENTAL ANALYSIS OF mus209 B1 I. Introduction 100 I I . M a t e r i a l s and Methods I I I . Results 112 IV. D i s c u s s i o n 130 REFERENCES APPENDIX A. APPENDIX B. 104 138 ISOLATION OF MUTAGEN-SENSITIVE STRAINS 159 AN EXAMINATION OF THE INFLUENCE OF MATERNAL GENOTYPE ON THE SENSITIVITY OF mus OFFSPRING TO MMS 163 v LIST OF TABLES Table 1- 1 2- 1 2- 2 3- 1 3-2 3- 3 4- 1 4-2 Page Summary o f p r o p e r t i e s o f mus mutants o f the second chromosome 33 R e l a t i v e v i a b i l i t y o f mus homozygotes and heterozygotes i n untreated c u l t u r e s 45 A compilation of previously reported i n t e r a c t i o n s i n double mutant s t r a i n s 71 S e n s i t i v i t y o f mus208 mus210 t o simple a l k y l a t i n g agents B1 S e n s i t i v i t y o f mus205 mus208 t o simple a l k y l a t i n g agents B1 S e n s i t i v i t y o f mus208 mus210 t o m i s c e l l a n e o u s chemicals B1 B1 B1 B1 B2 89 mus210 B1 91 mus211 B2 Cosegregation o f female s t e r i l i t y and 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 i t y i n mus209 92 B1 114 The m a t e r n a l - e f f e c t l e t h a l i t y o f mus209 /mus209 h e t e r o a l l e l i c females 125 The e f f e c t s o f i o n i z i n g r a d i a t i o n on t h e r e l a t i v e s u r v i v a l o f mus209 homozygotes a t v a r i o u s times i n development 128 B1 4-3 mus211 B2 B1 vi LIST OF FIGURES Figure 1- 1 2- 1 2-2 Page A schematic diagram o f DNA r e p a i r pathways Procedure used t o c o n s t r u c t mutant mus s t r a i n s 2-4 2-5 2-6 2-7 2-8 2-9 2-10 multiply42 MMS s e n s i t i v i t y o f mus201 , mus205 , mus205 and mus201 mus205 D1 D1 2-3 7 A1 B1 MMS s e n s i t i v i t y o f mus205 mus208 t o i t s component s i n g l e mutants B1 MMS s e n s i t i v i t y o f mus205 mus210 t o i t s component s i n g l e mutants B1 MMS s e n s i t i v i t y o f mus208 mus211 t o i t s component s i n g l e mutants B1 MMS s e n s i t i v i t y o f mus210 mus211 t o i t s component s i n g l e mutants B1 MMS s e n s i t i v i t y o f mus_208 mus210 t o i t s component s i n g l e mutants B1 MMS s e n s i t i v i t y o f mus201 mus208 t o i t s component s i n g l e mutants B1 MMS s e n s i t i v i t y of rous201 mus210 t o i t s component s i n g l e mutants B1 MMS s e n s i t i v i t y o f mus205 mus208 and mus208 mus210 mus211 B1 B1 B1 B1 B1 B1 D1 D1 B1 B1 B1 b2 vii B1 48 compared 51 compared 53 compared 56 compared 58 compared 60 compared 63 compared 66 mus210 B1 68 2-11 4-1 A model o f 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 74 Procedures used t o a n a l y z e t h e c o s e g r e g a t i o n of t h e mus. t s l e t h a l , and female s t e r i l e phenes o f mus209 107 Complementation maps o f l e t h a l mutations uncovered by t h e M(2) 017 d e f i c i e n c y 117 S e n s i t i v i t y o f mus209 homozygotes t o heat o r r a d i a t i o n treatments d u r i n g development 120 S e n s i t i v i t y o f mus209 homozygotes t o heat p u l s e s (29 C) d u r i n g development 123 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 161 MMS s e n s i t i v i t y and maternal e f f e c t s i n mus205 . mus2Q8 and mus210 166 B1 4-2 4-3 4-4 B1 B1 D A-1 B-l B1 B1 B1 viii ACKNOWLEDGEMENTS I would l i k e t o thank Tom G r i g l i a t t i , my t h e s i s supervisor, f o r h i s u n f a i l i n g support, understanding and encouragement. his a d v i c e on matters s c i e n t i f i c and otherwise, I am most appreciative. and For Don S i n c l a i r and Bob D e v l i n o f f e r e d encouragement made many v a l u a b l e benefited suggestions about t h i s r e s e a r c h . I have immeasurably from t h e i r knowledge and e x p e r t i s e . Many o t h e r s i n t h e f l y l a b made t h e day-to-day drudgery o f f l y f l i c k i n g sufferable, i f not e n j o y a b l e — t o thank Murray R i c h t e r , Annette B a i l e y , Jo-Ann Brock and V i v i a n Ngan f o r t h e i r f r i e n d s h i p s . typing I would e s p e c i a l l y l i k e I am a l s o g r a t e f u l t o V i v i a n f o r some o f t h e f i r s t d r a f t s o f t h i s t h e s i s and f o r g e t t i n g me s t a r t e d on t h e wordprocessor. L a s t but not l e a s t I wish t o thank my p a r e n t s Anne and Ken Henderson f o r t h e i r support. ix CHAPTER ONE GENERAL INTRODUCTION TO C E L L U L A R R E S P O N S E S T O DNA DAMAGE 1 I. PERSPECTIVES In an open system, such as our b o d i e s r e p r e s e n t , compounded o f u n s t a b l e m a t e r i a l and s u b j e c t e d c o n t i n u a l l y t o d i s t u r b i n g c o n d i t i o n s , constancy i s i n i t s e l f evidence t h a t agencies a r e a c t i n g or ready t o a c t , t o m a i n t a i n t h i s constancy. Cannon, 1939 Deoxyribonucleic acid (DNA) i s not a s t a b l e molecule. For example, i n mammalian c e l l s t h e l o s s o f bases due t o spontaneous d e p u r i n a t i o n and d e p y r i m i d a t i o n may be as h i g h r e s i d u e s p e r genome p e r day ( L i n d a h l , 1982). as 10,000 At t h i s rate, Thielmann (1984) has c a l c u l a t e d t h a t over t h e l i f e t i m e o f a p o s t - m i t o t i c 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. the t o t a l number o f bases p r e s e n t . A d d i t i o n a l l y , each day about T h i s r e p r e s e n t s about 2% o f 100 c y t o s i n e r e s i d u e s and a s m a l l e r number o f adenine and guanine bases may undergo spontaneous deamination ( L i n d a h l , 1982). In p a r t i c u l a r , deamination o f 5-methylcytosine produces thymine, i t s e l f a normal base i n DNA. u n r e p a i r e d a r e p o t e n t i a l l y mutagenic. Thus, GT mismatchs left In E s c h e r i c h i a c o l i . 5- m e t h y l c y t o s i n e r e s i d u e s appear t o be " h o t s p o t s " f o r spontaneous GC t o 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 o f DNA i s but one p o t e n t i a l source o f i n j u r y t o the genome. Numerous o t h e r d e l e t e r i o u s f o r c e s a l s o impact upon t h e g e n e t i c m a t e r i a l . 2 For 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 r a d i a t i o n s , both n a t u r a l and man-made, c o n t i n u a l l y d i s r u p t 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 L i n d a h l , 1982; B a r t s c h and Montesano, 1984; Hutchinson, 1985). Yet d e s p i t e these genomic a s s a u l t s , the i n f o r m a t i o n a l content of the DNA i s p r e s e r v e d w i t h remarkable fidelity from one cell g e n e r a t i o n t o the next. T h i s p r e s e r v a t i o n i s achieved by an e x t r a o r d i n a r y c e l l u l a r a r s e n a l of DNA repair damage-surveillance and functions. The remainder of t h i s chapter p r o v i d e s a s e r i e s of b r i e f accounts of some of the major c e l l u l a r responses t o n u c l e a r damage. (The r e l a t i v e l y uncharted area of m i t o c h o n d r i a l r e p a i r i s not addressed (see Foury and Lahaye, 1987).) DNA DNA Many of these t o p i c s are f u r t h e r d i s c u s s e d i n r e l e v a n t s e c t i o n s of the thesis. Unavoidably, a s u b s t a n t i a l p o r t i o n of the m a t e r i a l i n t h i s i n t r o d u c t i o n has been gleaned from work c a r r i e d out i n E. coli• T h i s e n t e r i c bacterium i s by f a r the b e s t c h a r a c t e r i z e d organism i n terms of DNA repair. Discoveries of r e p a i r p r o c e s s e s i n E. c o l i spawned many s u c c e s s f u l searches f o r analogous r e p a i r responses i n o t h e r p r o k a r y o t e s and i n eukaryotes. Apparently, many of the major DNA are f u n c t i o n a l l y analogous v a s t p h y l o g e n e t i c range F r i e d b e r g , 1985). i f not m e c h a n i s t i c a l l y s i m i l a r over a (Hanawalt e t a l . , Indeed, r e p a i r processes 1979; c l o n e d E. c o l i DNA Lindahl, 1982; r e p a i r genes have r e c e n t l y been shown t o complement r e p a i r d e f e c t s i n both human 3 and hamster c e l l l i n e s (Samson e t a l . , 1986; Margison e t a l . , 1987) . D e s p i t e i t s u s e f u l n e s s , however, e x t r a p o l a t i n g from prokaryotes al., t o eukaryotes 1985). has c e r t a i n l i m i t a t i o n s There are aspects of e u k a r y o t i c DNA have no c o u n t e r p a r t s i n p r o k a r y o t i c r e p a i r (e.g., S i m i l a r l y , t h e r e are aspects of DNA (Walker e t r e p a i r which chromatin). p r o c e s s i n g and r e p a i r i n metazoans which have no c o u n t e r p a r t s i n u n i c e l l u l a r organisms (e.g., p o l y ( A D P - r i b o s y l a t i o n ) ) . r e p a i r processes For p r e c i s e l y these reasons are being s t u d i e d i n organisms other b a c t e r i a . The y e a s t Saccharomyces c e r e v i s i a e and the D r o s o p h i l a melanogaster are prominent examples. these two review. DNA than fruitfly Accordingly, e u k a r y o t i c organisms are emphasized i n the f o l l o w i n g At t h i s j u n c t u r e , those readers having a b a s i c knowledge o f DNA t h i s chapter. r e p a i r mechanisms can proceed t o s e c t i o n I I I of For an account of the mechanisms of DNA repair more d e t a i l e d than t h a t p r o v i d e d here, the reader i s r e f e r r e d t o the comprehensive monograph by F r i e d b e r g the primary II. source of t h i s as information. CELLULAR RESPONSES TO DNA In 1949 (1985) which served DAMAGE K e l n e r r e p o r t e d t h a t the s u r v i v a l of U V - i r r a d i a t e d Streptomyces g r i s e u s spores c o u l d be improved d r a m a t i c a l l y i f , f o l l o w i n g UV treatment, the c o n i d i a were exposed t o v i s i b l e 4 light. This observation r e p a i r phenomena. marks t h e Subsequently, phenomena were d i s c o v e r e d , to as and dark r e p a i r processess. light-mediated considered variety recovery Figure A. these The are alterations, pyrimidine UV), A of pyrimidine DNA dimers (TT, a schematic in 1986). G i v e n t h e CT, o r CC) wavelength biological (photolyases) dimers. range base cyclobutane 1985). This class the major c y t o t o x i c relevance i f not ( F r a n k l i n and of these DNA a l l , organisms which s p e c i f i c a l l y Photolyases the (Friedberg, i n U V - i r r a d i a t e d DNA s u r p r i s i n g t h a t most, i n the nm dimers s u f f e r s a c o n s t e l l a t i o n of l i k e l y represents mutagenic) l e s i o n pyrimidine 254 t h e most a b u n d a n t u s u a l l y b e i n g of photoproduct and, is 1. (ultraviolet, enzymes the r e p a i r pathways i s i l l u s t r a t e d Enzyme-catalyzed photoreversal not basis of damage. DNA referred B through D describe o f d a r k r e p a i r r e s p o n s e s t o DNA of repair (photoreactivation) When e x p o s e d t o s u n l i g h t i n t h e is study collectively biochemical Sections overview of c e r t a i n of these of the light-independent reaction i n s e c t i o n A. beginning (but Haseltine, lesions, i t possess act to repair bind to dimer-containing presence of v i s i b l e light, not monomerize t h e DNA joined b a s e s . T h i s UV-damage r e v e r s a l phenomenon i s t e r m e d photoreactivation (see F r i e d b e r g , taken advantage of the accompanies fact that UV. 5 1985). Evidently, cells i n nature, visible light have F i g u r e 1. A schematic diagram of DNA r e p a i r pathways ( m o d i f i e d from Hanawalt e t a l . (1979)). 6 SPONTANEOUS BASE LOSS BASE DEFECT HELIX DISTORTION ( e . g . , UV d i m e r ) glycosylase AP SITE damage-specific endonuclease (incision) AP endonuclease (incision) photolyase alkyltransferase insertase exonuclease / polymerase / ligase INTACT DNA 7 Recent f i n d i n g s in E. c o l i i m p l i c a t e the p h o t o r e p a i r phr. not o n l y i n p h o t o r e a c t i v a t i o n but i n e x c i s i o n r e p a i r well (Sancar e t a l . , stimulates dimers 1984; Hays e t a l . , 1985). the UvrABC e x c i s i o n endonuclease (but not o t h e r bulky l e s i o n s ) t h i s cooperative t o remove p y r i m i d i n e better or t h a t The nature of I t has been suggested t h a t photolyase might promote UvrABC turnover, as Photolyase from DNA. i n t e r a c t i o n i s not known. gene, endonuclease i t might make the d i m e r - c o n t a i n i n g DNA a s u b s t r a t e f o r the e x c i s i o n endonuclease (Sancar e t al., 1984). In D r o s o p h i l a . a mutation d e f e c t i v e in photoreactivation has been l o c a l i z e d t o the second chromosome at m e i o t i c map p o s i t i o n 56.8. The D r o s o p h i l a phr mutant a l s o appears t o influence excision repair B. (Boyd and H a r r i s , 1987). DNA E x c i s o n R e p a i r The e x c i s i o n removal of damaged or i n a p p r o p r i a t e bases from DNA c o n s t i t u t e s (Friedberg, refer the major mode of DNA r e p a i r i n any c e l l 1985). However, the term e x c i s i o n r e p a i r does not s p e c i f i c a l l y t o a s i n g l e enzymatic mechanism. number o f d i s t i n c t but i n t e r c o n n e c t e d routes and comprise the e x c i s i o n r e p a i r pathway l). 1. DNA q l y c o s y l a s e s : DNA g l y c o s y l a s e s (Figure Mediators o f base e x c i s i o n (reviewed by L i n d a h l , p r o t e i n s of r e l a t i v e l y low m o l e c u l a r weight, 8 Rather, activities repair 1982, 1986) are and g e n e r a l l y of a narrow s u b s t r a t e s p e c i f i c i t y . subunit s t r u c t u r e They appear t o have n e i t h e r nor c o f a c t o r requirements. 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 a c t i v i t i e s are u b i q u i t o u s . Although glycosylase These enzymes c l e a v e m o d i f i e d o r i n a p p r o p r i a t e bases a t the N - g l y c o s y l bond t o c r e a t e a p u r i n i c or apyrimidinic backbone. DNA (AP) s i t e s w h i l e l e a v i n g i n t a c t the p h o s p h o d i e s t e r For example, u r a c i l DNA g l c o s y l a s e and hypoxanthine g l y c o s y l a s e s p e c i f i c a l l y c a t a l y z e the removal from DNA of u r a c i l and hypoxanthine, the deamination products o f c y t o s i n e and adenine, r e s p e c t i v e l y . A major c y t o t o x i c l e s i o n i n t r o d u c e d i n t o DNA by simple m e t h y l a t i n g agents (e.g., methyl methanesulfonate, MMS) methyladenine. i s 3- Under normal circumstances t h i s m o d i f i e d base i s r a p i d l y r e l e a s e d from DNA by DNA b a c t e r i a l and mammalian c e l l s g l y c o s y l a s e s found i n both ( L i n d a h l , 1982) . In E_j_ c o l i . the enzyme l a r g e l y r e s p o n s i b l e f o r t h i s r e a c t i o n i s 3-methyladenine DNA g l y c o s y l a s e I (encoded by the tagA gene) (Karran e t a l . , 1982). T h i s 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 a weak s u b s t r a t e ) . by MMS (although 3-ethyladenine i s a l s o tagA mutants are h y p e r s e n s i t i v e t o (Evensen and Seeberg, killing 1982). The o b s e r v a t i o n t h a t tagA mutants are not c o m p l e t e l y d e v o i d of 3-methyladenine of the w i l d t y p e l e v e l ) l e d t o the i d e n t i f i c a t i o n o f a second methyladenine DNA 1982; DNA glycosylase a c t i v i t y g l y c o s l a s e i n E_j_ c o l i Karran e t a l . , 1982). 5-10% 3- (Evensen and Seeberg, 3-methyladenine 9 (they r e t a i n DNA glycosylase II, the p r o d u c t o f the alkA gene, i s an i n d u c i b l e enzyme t h a t has a much broader s u b s t r a t e s p e c i f i c i t y than i t s c o u n t e r p a r t . In a d d i t i o n t o removing 3-methyladenine, the alkA p r o d u c t e x c i s e s 3-methylguanine and two minor p y r i m i d i n e a l k y l a t i o n l e s i o n s , m e t h y l c y t o s i n e and 0 -methylthymine 2 ( L i n d a h l , 1986). 0 2 When induced as p a r t o f the a d a p t i v e response t o a l k y l a t i o n damage (see s e c t i o n II.D.2), 3-methyladenine DNA glycosylase II accounts f o r as much as 50-70% o f the t o t a l 3-methyladenine g l y c o s y l a s e a c t i v i t y i n E^. c o l i In (Karran e t a l . , c o n t r a s t t o E_j_ c o l i , mammalian c e l l s 1982). (from human p l a c e n t a and c a l f thymus) appear t o have o n l y a s i n g l e methyladenine DNA glycosylase (Lindahl,1986). DNA 3- The mammalian enzyme i s c o n s t i t u t i v e l y expressed and has a broad s u b s t r a t e s p e c i f i c i t y , but i t does not r e c o g n i z e p r e c i s e l y the same l e s i o n s as 3-methyladenine DNA g l y c o s y l a s e I I from E_j. c o l i . In a d d i t i o n t o r e l e a s i n g 3-methyladenine, the mammalian enzyme c a t a l y z e s the removal of 3-methylguanine and 7-methylguanine, but not the 0 - m e t h y l p y r i m i d i n e s . 2 I o n i z i n g r a d i a t i o n and c e r t a i n r a d i o m i m e t i c chemicals (e.g., d i m e t h y l s u l f a t e ) may cause base a l t e r a t i o n s which, under a p p r o p r i a t e c o n d i t i o n s , l e a d t o p u r i n e s w i t h opened rings. In the case o f 7-methylguanine i n methylated DNA imidazole (the most abundant lesion (Singer and Kusmierek, 1982)), c l e a v a g e o f the i m i d a z o l e r i n g produces a s u b s t i t u t e d 5-formamidopyrimidine. DNA A g l y c o s y l a s e which c a t a l y z e s the r e l e a s e of t h i s a l t e r e d base from DNA has been i d e n t i f i e d i n e x t r a c t s o f E. c o l i . rodent 10 l i v e r , c a l f thymus, Friedberg, 1985). and human f i b r o b l a s t s ( L i n d a h l , S e v e r a l other examples o f DNA can be found i n L i n d a h l (1982) and i n F r i e d b e r g 1982; glycosylases (1985). The extent t o which base e x c i s i o n r e p a i r i s u t i l i z e d i n Drosophila is difficult Drosophila DNA of u r a c i l , 3-methyladenine, or 7-methylguanine have been unsuccessful to assess. Attempts t o d e t e c t g l y c o s y l a s e s t h a t c a t a l y z e the removal from DNA ( F r i e d b e r g e t a l . , 1978; Deutsch and S p i e r i n g , 1982; Green and Deutsch, 1983). I t i s p o s s i b l e t h a t Drosophila r e l i e s on mechanisms other than base e x c i s i o n t o remove a l t e r e d or i n a p p r o p r i a t e bases from DNA (Green and Deutsch, 1983). However, f l i e s are not completely g l y c o s y l a s e s as a DNA l a c k i n g i n a l l DNA g l y c o s y l a s e which e x c i s e s o x i d a t i v e l y damaged thymine r e s i d u e s has r e c e n t l y been d e t e c t e d i n Drosophila 2. embryos (Breimer, 1986). AP endonucleases AP s i t e s , whether spontaneously o r e n z y m a t i c a l l y produced, are s u b s t r a t e s f o r a v a r i e t y o f d i f f e r e n t enzymes. For example, t h e r e i s some i n d i r e c t evidence t h a t enzymes termed DNA i n s e r t a s e s may purine r e i n s e r t the a p p r o p r i a t e m i s s i n g base d i r e c t l y i n t o a p u r i n i c s i t e s i n duplex DNA ( F r i e d b e r g , 1985). Activities of t h i s s o r t have been i d e n t i f i e d i n human c e l l e x t r a c t s , i n crude e x t r a c t s from E. c o l i . and more r e c e n t l y i n D r o s o p h i l a embryos (Deutsch and S p i e r i n g , 1985). However, i n s e r t a s e d e f e c t i v e mutants have y e t t o be i d e n t i f i e d i n any organism (Friedberg, 1985). 11 M o s t AP AP sites endonucleases a r e p r o b a b l y a c t e d upon by enzymes known a s (see F r i e d b e r g , p h o s p h o d i e s t e r bond e i t h e r 1985). These 3' o r 5' t o t h e AP DNases c l e a v e site rendering i t s u s c e p t i b l e t o t h e d e g r a d a t i v e a c t i o n o f an e x o n u c l e a s e . endonuclease activities a r e u b i q u i t o u s i n n a t u r e , and appear t o possess m u l t i p l e not appear endonuclease form, (Loeb and endonuclease glycosylase phage T4 f u n c t i o n may endonuclease AP endonuclease activities AP associated with a DNA i n E. c o l i This relatively endonuclease function, a 3' p h o s p h a t a s e glycosylases DNA from AP i s b u t one of several small protein activity, a 3' t o 5' activity and (28 kDa) has a 5'- exonuclease a ribonuclease H x t h A m u t a n t s a r e h y p e r s e n s i t i v e t o k i l l i n g by peroxide, but are only s l i g h t l y a g e n t s such as Most c e l l s , sensitive to direction alkylating MMS. prokaryotic and eukaryotic alike, numerous e x o n u c l e a s e 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 and/or to o f t h e x t h A gene p r o d u c t , e x o n u c l e a s e I I I a c t i n g AP hydrogen f r o m 20 gene) and M i c r o c o c c u s l u t e u s h a v e a s s o c i a t e d 1981). activity. be p h y s i c a l l y the does activities). The m a j o r (Weiss, Very r a r e l y (e.g., the p y r i m i d i n e dimer (denV catalytic P r e s t o n , 1986). There however. T h e y a r e 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 40 kDa AP cells s p e c i e s o f t h e s e enzymes. t o be a p r o t o t y p i c AP the of hydrolysis (Friedberg, possess specificity 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 events are not n e c e s s a r i l y uncoupled or uncoordinated) 12 (these and ligation (Figure 1). 3. N u c l e o t i d e e x c i s i o n r e p a i r The one removal of damaged bases by DNA of a t l e a s t two pathway (see F i g u r e glycosylases represents a l t e r n a t i v e routes i n the e x c i s i o n repair 1). However, i n view of the m u l t i t u d e of d i f f e r e n t base a l t e r a t i o n s which are known t o e x i s t , i t i s u n l i k e l y t h a t c e l l s possess a DNA every type of DNA glycosylase unique t o each 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 p y r i m i d i n e dimers) are removed by a g e n e r a l n u c l e o t i d e e x c i s i o n r e p a i r mechanism. UvrABC endonuclease found i n E. c o l i example (see F r i e d b e r g , The i s the best probably required excision repair. 1985). for nucleotide Mutations i n any hypersensitive t o k i l l i n g by UV mutagens), and severely (and defective (or oligonucleotide) a v a r i e t y of o t h e r i n dimer e x c i s i o n . event of the e x c i s i o n r e p a i r p r o c e s s . i n the uvrA i n c i s i o n r e a c t i o n , a l b e i t more s l o w l y do. A l l t h r e e uvr genes have been cloned and than w i l d t y p e nucleotide out cells t h e i r products p u r i f i e d , advances which have l e d t o the e l u c i d a t i o n of UvrA, B, and and i n i t i a l incision uvrC mutants c a r r y the e x c i s i o n r e p a i r mechanism i n E. uvrC. of these genes render c e l l s uvrB mutants are completely d e f e c t i v e The The characterized p r o d u c t s of t h r e e E. c o l i genes, uvrA, uvrB, and are a b s o l u t e l y and the coli. C proteins are b e l i e v e d e x c i s i o n r e p a i r as f o l l o w s . 13 to i n t e r a c t during F i r s t , the UvrA p r o t e i n binds weakly a t a n o n s p e c i f i c s i t e i n DNA c o n t a i n i n g lesions. bulky Next, t h e UvrB p r o t e i n a s s o c i a t e s with t h e bound UvrA p r o t e i n i n an i n t e r a c t i o n t h a t i n c r e a s e s the a f f i n i t y o f the l a t t e r f o r DNA. Then, by a mechanism t h a t i s p o o r l y understood, the UvrAB complex t r a n s l o c a t e s along the DNA duplex u n t i l t h e s i t e o f damage i s encountered. T h i s t r a n s l o c a t i o n i s dependent on t h e ATPase a c t i v i t y o f the UvrA s u b u n i t . The UvrAB complex forms a s t a b l e a s s o c i a t i o n with DNA i n t h e v i c i n i t y o f t h e l e s i o n , and i n t h e presence o f t h e UvrC p r o t e i n , t h e UvrABC endonuclease complex ("repairosome") i n c i s e s t h e same DNA s t r a n d at s i t e s which f l a n k the damage. Thus, the i n c i s i o n r e a c t i o n r e s u l t s i n two c l o s e l y spaced e n d o n u c l e o l y t i c s t r a n d breaks. the case o f p y r i m i d i n e In dimers and p s o r a l e n monoadducts, one o f the s t r a n d s c i s s i o n s occurs 7 n u c l e o t i d e s upstream o f the 5' member o f t h e dimer; the other n i c k occurs 3 or 4 nucleotides downstream o f t h e 3' member. Thus, a p o t e n t i a l l y e x c i s a b l e dimer-containing generated oligomer some 12 t o 13 n u c l e o t i d e s long i s (Sancar and Rupp, 1983; Yeung e t a l . , 1987). distance represents The about one t u r n o f t h e DNA h e l i x . i n c i s i o n events r e p r e s e n t only t h e f i r s t steps i n t h e o v e r a l l e x c i s i o n r e p a i r p r o c e s s . events such as fragment r e l e a s e and gap f i l l i n g synthesis) and catalytic Postincision (repair r e q u i r e the products o f a t l e a s t two a d d i t i o n a l genes, uvrD (DNA h e l i c a s e II) and polA et This al.,1985; Husain e t a l . , 1985). (DNA polymerase I) (Caron Beginning a t t h e 5' n i c k , i n t h e presence o f the unwinding p r o t e i n DNA h e l i c a s e I I , 14 the 5' t o 3' exonuclease f u n c t i o n o f DNA polymerase I e x c i s e s the o l i g o n u c l e o t i d e fragment. In a c o o r d i n a t e d s t r a n d being degraded i s r e s y n t h e s i z e d f a s h i o n , the DNA by t h e polymerase f u n c t i o n o f DNA polymerase I, which uses t h e undamaged opposite s t r a n d as a template. F i n a l l y , the r e s u l t i n g 3' n i c k i s s e a l e d by DNA l i g a s e . Most l e s i o n s y i e l d s h o r t r e p a i r patches i n s i z e from 13 t o 30 n u c l e o t i d e s ranging (Hanawalt e t a l . , 1979). E v i d e n t l y , many o f the gaps c r e a t e d by t h e e x c i s i o n removal o f the 12 t o 13 n u c l e o t i d e fragment a r e lengthened by e x o n u c l e o l y t i c degradation (Friedberg, 1985). While the molecular b a s i s o f n u c l e o t i d e excision repair i n E. c o l i i s understood i n some d e t a i l , comparatively little is known about the enzymology o f t h i s p r o c e s s i n eukaryotes. F o r example, genes encoding products analogous t o t h e UvrA, B, and C p r o t e i n s o f E. c o l i have not been found i n any eukaryote. I n f o r m a t i o n concerning eukaryotic e x c i s i o n r e p a i r has i s s u e d mostly from work c a r r i e d out i n humans, i n rodents, y e a s t Saccharomyces c e r e v i s i a e . complexity t o e u k a r y o t i c and i n the These s t u d i e s r e v e a l a g e n e t i c excision repair that i s unparalleled. For example, i n S. c e r e v i s i a e . a t l e a s t 10 genes, a l l o f them members o f t h e RAD3 e p i s t a s i s group, a r e r e q u i r e d f o r nucleotide e x c i s i o n r e p a i r (Haynes and Kunz, 1981). S i m i l a r l y , s t u d i e s o f r e p a i r d e f e c t s among i n d i v i d u a l s a f f l i c t e d w i t h xeroderma pigmentosum (XP) i n d i c a t e t h a t n u c l e o t i d e excision repair i n humans may i n v o l v e a t l e a s t 8 o r 9 d i f f e r e n t genes Hanawalt and S a r a s i n , 1986). (e.g., Other s t u d i e s suggest t h a t as many 15 as 15 genes may p a r t i c i p a t e i n t h i s p r o c e s s i n humans (see C l e a v e r and Karentz, 1987). Moreover, i n both man and y e a s t , no fewer than 5 genes p a r t i c i p a t e a t the i n c i s i o n s t e p alone (Friedberg, 1985). The b a s i s f o r t h i s complexity almost c e r t a i n l y r e s i d e s i n the f a c t t h a t e u k a r y o t i c DNA, u n l i k e b a c t e r i a l DNA, i s i n t i m a t e l y a s s o c i a t e d w i t h h i s t o n e s and non-histone chromosomal p r o t e i n s i n t h e form o f chromatin ( F r i e d b e r g e t a l . , 1986). Thus, a subset o f these e x c i s i o n r e p a i r genes might encode p r o d u c t s f o r r e c o g n i z i n g damaged DNA i n chromatin, and/or f o r making otherwise i n a c c e s s i b l e l e s i o n s a v a i l a b l e t o t h e a c t u a l r e p a i r enzymes. Support f o r t h i s idea comes from experiments conducted i n both humans and y e a s t . f r e e e x t r a c t s from human XP c e l l s To summarize b r i e f l y , cell- (complementation groups A, C and G) c a t a l y z e t h e removal o f p y r i m i d i n e dimers from d e p r o t e i n i z e d p u r i f i e d exogenous DNA, but not from endogenous chromatin. On the o t h e r hand, e x t r a c t s from normal human c e l l s c a r r y out both r e a c t i o n s . Furthermore, the l a t t e r extracts e x c i s e 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 n a t i v e chromatin (Mortelmans e t a l . , 1976; F u j i w a r a and Kano, 1983). These f i n d i n g s suggest t h a t none o f t h e aforementioned XP mutants a r e d e f e c t i v e i n t h e a c t u a l catalytic a c t i v i t i e s o f i n c i s i o n and e x c i s i o n , but r a t h e r i n events p r e p a r a t o r y t o t h e i n c i s i o n r e a c t i o n , e.g., a t s t e p s which render t h e chromatin-ensconced dimer a c c e s s i b l e t o a DNA damages p e c i f i c endonuclease. S i m i l a r approaches a p p l i e d t o 5 i n c i s i o n d e f e c t i v e y e a s t mutants ( r a d l , rad2, rad3 rad4 and r a d i o ) have y i e l d e d i d e n t i c a l r e s u l t s and c o n c l u s i o n s (Bekker e t a l . , 1980; f Reynolds e t a l . , 1981). supported These i n v i t r o f i n d i n g s are f u r t h e r by more r e c e n t i n v i v o experiments i n y e a s t . P y r i m i d i n e dimers i n U V - i r r a d i a t e d plasmid DNA transformed into a rad3 mutant were r e p a i r e d with w i l d t y p e p r o f i c i e n c y , whereas dimers i n i d e n t i c a l plasmids endonuclease-defective transformed coli uvrA mutant were not e x c i s e d a t a l l (Dominski and Jachymczyk, 1984). c r u c i a l determinant i n t o an E. Apparently, chromatin is a of the e x c i s i o n r e p a i r response i n eukaryotes. A f u l l a p p r e c i a t i o n of the mechanism of n u c l e o t i d e e x c i s i o n r e p a i r 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 c l o n e d and t h e i r products reconstituted in v i t r o . s i m i l a r s t r a t e g y i s being adopted i n a number of a l b e i t w i t h v a r y i n g degrees of success. to c l o n e human r e p a i r genes u s i n g DNA A eukaryotes, For example, attempts t r a n s f e c t i o n t o rescue the mutagen s e n s i t v i t y of XP c e l l s have been f r u s t r a t e d by t e c h n i c a l problems (Lehmann, 1985; S c h u l t z e t a l . , 1985). More s u c c e s s f u l approaches have been those which i n t r o d u c e human DNA r e p a i r - d e f e c t i v e hamster c e l l s C l e a v e r and Karentz, The (e.g., Westerveld into e t a l . , 1984; 1987). r e l a t i v e ease with which DNA can be manipulated c e r e v i s i a e has enabled most of the y e a s t RAD e x c i s i o n r e p a i r t o be cloned P e r o z z i and Prakash, 1986 i n S. genes i n v o l v e d i n (see F r i e d b e r g e t a l . , 1986; and r e f e r e n c e s t h e r e i n ) . 17 Nucleotide sequence data r e v e a l t h a t RAD1, RAD2 and RAD3 encode p r o t e i n s of comparable s i z e (estimated a t 110 kDa, 111 kDa and 90 kDa, r e s p e c t i v e l y ) , whereas t h e RADIO gene and i t s p r e d i c t e d product (22.6 kDa) a r e c o n s i d e r a b l y s m a l l e r The RAD1 and RAD2 p o l y p e p t i d e s ( F r i e d b e r g e t a l . , 1986). share only t h r e e s m a l l r e g i o n s o f amino a c i d sequence homology d e s p i t e t h e i r n e a r l y size. The RAD1, RAD3 and RADIO p o l y p e p t i d e s a s i n g l e l i m i t e d r e g i o n o f homology. identical have i n common only However, t h i s sequence i s a l s o homologous t o one found i n p r o k a r y o t i c and e u k a r y o t i c p r o t e i n s t h a t b i n d and/or h y d r o l y z e purine nucleotides. The RAD3 p u t a t i v e n u c l e o t i d e - b i n d i n g domain shows p a r t i c u l a r l y good amino a c i d sequence homology with t h e n u c l e o t i d e - b i n d i n g of t h e UvrA p r o t e i n . Somewhat region l e s s homology e x i s t s between t h i s RAD3 sequence and the ATP-binding r e g i o n o f t h e UvrD protein ( F r i e d b e r g e t a l . , 1986). The RAD3 p r o t e i n does not appear t o be f u n c t i o n a l l y analogous t o e i t h e r E. c o l i p r o t e i n , however, s i n c e i t f a i l s t o complement t h e UV s e n s i t i v i t y o f both uvrA and uvrD mutants (Naumovski and F r i e d b e r g , 1986). p r o t e i n a l s o c o n t a i n s a p u t a t i v e DNA-binding domain and Friedberg, The RAD3 (Naumovski 1986). Codon usage data suggest t h a t RAD1, RAD2. RAD3 and RADIO are weakly expressed genes ( F r i e d b e r g e t a l . , 1986). been confirmed e x p e r i m e n t a l l y et a l . (1985) estimate f o r both RAD2 and RAD3. Naumovski t h a t i n e x p o n e n t i a l l y growing c e l l s are l e s s than 5 c o p i e s o f RAD3 t r a n s c r i p t p e r c e l l . RAD1) gene e x p r e s s i o n T h i s has RAD3 (and i s n e i t h e r c e l l - c y c l e - r e g u l a t e d nor 18 there a l t e r e d by DNA damage (Nagpal e t a l . , 1985). Estimates of the amount o f RAD2 mRNA a r e even lower a t l e s s than 1 copy p e r c e l l (Naumovski and F r i e d b e r g , 1984). A c o m p l e t e l y unexpected p r o p e r t y o f RAD3 i s t h a t i t encodes a function essential f o r v i a b i l i t y Naumovski and F r i e d b e r g , 1983). (Higgins e t a l . , 1983; In t h i s r e s p e c t RAD3 i s unique among e x c i s i o n r e p a i r genes i n S. c e r e v i s i a e (Friedberg e t a l . , 1986). T h i s aspect o f RAD3 i s d i s c u s s e d b r i e f l y i n Chapter 4. In D r o s o p h i l a , 8 n o n - a l l e l i c mutations p a r t i a l l y o r completely d i s r u p t e x c i s i o n r e p a i r (see s e c t i o n I I I ) . At l e a s t 2 l o c i , mei-9 and mus201. a r e a b s o l u t e l y r e q u i r e d f o r t h i s p r o c e s s , and both genes encode products t h a t f u n c t i o n a t o r prior to incision mus201 D1 (Boyd e t a l . , 1987). C e l l s d e r i v e d from embryonic o r l a r v a l t i s s u e e x h i b i t reduced l e v e l s o f an AP endonuclease a c t i v i t y (Osgood and Boyd, 1982) and f a i l t o c a r r y out unscheduled DNA s y n t h e s i s f o l l o w i n g treatment w i t h UV or a l k y l a t i n g agents (Dusenbery e t a l . , 1983). S e v e r a l mei-9 a l l e l e s a r e a l s o d e f e c t i v e i n these parameters o f e x c i s i o n repair (Osgood and Boyd, 1982; Dusenbery et a l . , 1983). However, whereas t h e mus2 01 mutations p h e n o t y p i c a l l y resemble members o f t h e RAD3 e p i s t a s i s group i n y e a s t and t h e c l a s s i c a l forms o f XP i n humans, t h e mei-9 mutations a r e unique. Unlike e x c i s i o n d e f e c t i v e mutants i n any organism, mei-9 mutations are h i g h l y 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 and e x h i b i t meiotic effects (Boyd e t a l . , 1987). strong Based on p h e n o t y p i c s i m i l a r i t i e s between mei-9 mutants and uvrD mutations i n E. 19 coli. Smith e t a l . (1983) have s p e c u l a t e d t h a t m e i - 9 a DNA unwinding p r o t e i n . + may encode H a r r i s and Boyd (1987) have r e c e n t l y p r o v i d e d evidence a p r e i n c i s i o n chromatin remodeling process for i n D r o s o p h i l a which exposes p r e v i o u s l y i n a c c e s s i b l e p y r i m i d i n e dimers t o e x c i s i o n repair. C. P o s t r e p l i c a t i o n R e p a i r Damage t o the template s t r a n d s i n r e p l i c a t i n g DNA h i n d e r , or b l o c k completely, machinery. the progress of the may replicative Moreover, e x c i s i o n r e p a i r i s rendered ineffectual in s i n g l e - s t r a n d e d r e g i o n s of the chromosome (e.g., i n the vicinity o f the r e p l i c a t i o n f o r k ) . P o s t r e p l i c a t i o n r e p a i r mechanisms are those which allow replisomes templates t o resume DNA s y n t h e s i s on 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 q u a n t i f y i n g the molecular weight of p u l s e - l a b e l e d DNA a t v a r i o u s times f o l l o w i n g mutagen exposure (e.g., see Brown and Boyd, 1981a,b). Having encountered a r e p l i c a t i v e b l o c k dimer), (e.g., a p y r i m i d i n e any of s e v e r a l mechanisms c o u l d permit the polymerase t o continue s y n t h e s i z i n g DNA stalled on the damaged template. For example, one model p o s i t s t h a t the r e p l i c a s e , a l t e r e d i n some way as a r e s u l t of i t s f o r c e d stoppage (e.g., r e l a x e d i n its replicative fidelity), simply resumes DNA the non-coding l e s i o n and beyond. t r a n s l e s i o n DNA s y n t h e s i s , may synthesis across T h i s mechanism, termed be the b a s i s of the mutagenic component of the SOS response i n E. c o l i (see s e c t i o n I I . D . l . ) . A second mechanism used by c e l l s t o circumvent b l o c k s t o DNA et r e p l i c a t i o n i s termed daughter s t r a n d gap a l . , 1979). The biochemistry w e l l understood i n E. c o l i , discovered r e p a i r (Hanawalt of t h i s process is reasonably the organism i n which i t was (see Howard-Flanders, 1981). gaps are generated i n the nascent DNA first T h i s model p o s i t s t h a t s t r a n d as the polymerase stops s t r a n d e l o n g a t i o n o p p o s i t e the template damage, and r e i n i t i a t e s DNA s y n t h e s i s at p o i n t s downstream. daughter s t r a n d gap then As the name r e p a i r i n d i c a t e s , the gaps i n the nascent s t r a n d s are the a c t u a l s u b s t r a t e s f o r t h i s r e p a i r response. They range i n s i z e from 1 t o 40 k i l o b a s e s (Hanawalt e t a l . , 1979). These secondary DNA l e s i o n s are e l i m i n a t e d by a s e r i e s of mediated r e c o m b i n a t i o n a l recA- events as d e s c r i b e d below. (Other genes have a l s o been i m p l i c a t e d i n daughter s t r a n d gap r e p a i r , but the e x t e n t and manner of t h e i r involvement i s , f o r the most p a r t , unclear (see Hanawalt e t a l . , 1979; Friedberg, 1985).) RecA p r o t e i n s c o o p e r a t i v e l y b i n d t o gaps i n duplex DNA and a l i g n them w i t h the homologous p o r t i o n s of the undamaged s i s t e r duplexes. Once p a i r e d , an exchange event f i l l s each gap i n t a c t complementary DNA with from the i s o p o l a r p a r e n t a l s t r a n d . The p a r e n t a l s t r a n d d i s c o n t i n u i t i e s so produced are e l i m i n a t e d by r e p a i r s y n t h e s i s u s i n g the undamaged p o r t i o n s of the complementary s t r a n d s as templates. At t h i s stage, the primary l e s i o n can be removed by e x c i s i o n r e p a i r . 21 These events c o n s t i t u t e the 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 importance of t h i s pathway f o r c o n f e r r i n g E. c o l i UV resistance i s emphasized by the o b s e r v a t i o n t h a t uvr mutants are repair. recA double some 50 times more s e n s i t i v e t o k i l l i n g by r a d i a t i o n than e i t h e r s i n g l e mutant alone to UV (Howard-Flanders, 1981). P o s t r e p l i c a t i o n r e p a i r , as i t i s o p e r a t i o n a l l y defined, been demonstrated i n a v a r i e t y of eukaryotes, i n c l u d i n g (Veleminsky e t a l . , 1980), yeast Neurospora 1983) and (Calza and barley (Resnick et a l . , 1981), Schroeder, 1982), D r o s o p h i l a mammals (e.g., Hanawalt et a l . , 1979; (Boyd e t a l . , Lehmann and Karran, 1981). However, the mechanisms by which e u k a r y o t i c overcome b l o c k s t o DNA i s due mainly t o two r e p l i c a t i o n i s unclear. f a c t o r s : 1) the w i t h the g r e a t e r complexity of DNA p r e s e n t s t e c h n i c a l and e t a l . , 1979; suitable repair defective p a r t i a l defects Karran, 1981), and DNA synthesis i n the (see Hanawalt 2) a p a u c i t y of mutants. l a r g e s t c o l l e c t i o n of Boyd, 1981a; Boyd and n o n - a l l e l i c mutations e x h i b i t complete or i n t h i s r e p a i r pathway. have been grouped i n t o two Mutations i n one uncertainty l a r g e genome s i z e coupled p o s t r e p l i c a t i o n r e p a i r mutants (Brown and Ten cells r e p l i c a t i o n i n eukaryotes D r o s o p h i l a boasts probably the Shaw, 1982). This interpretative d i f f i c u l t i e s Lehmann and has classes group (mus302 D1 The 4 s t r o n g e s t mutants (Brown and and Boyd, 1981a). mus310 ) appear t o h a l t D1 a t s i t e s o p p o s i t e p y r i m i d i n e dimers, whereas those o t h e r group ( m e i - 4 1 D5 and 22 mus20_5 ) a l l o w s y n t h e s i s A1 to resume beyond t h e l e s i o n , c r e a t i n g gaps i n t h e p r o c e s s . contrast to bacteria, In a recombinational mode o f p o s t r e p l i c a t i o n r e p a i r does not appear t o operate t o any a p p r e c i a b l e e x t e n t i n Drosophila (Boyd e t a l . , 1983). In t h i s respect and mammalian mechanisms are s i m i l a r Boyd e t a l . , 1983). (Lehmann and Karran, 1981; However, e l u c i d a t i o n o f t h i s r e p a i r i n eukaryotes awaits f u r t h e r D. I n d u c i b l e the Drosophila pathway study. R e p a i r Responses The p r e c e e d i n g s e c t i o n s have d e a l t mainly with t h e m e c h a n i s t i c aspects o f c o n s t i t u t i v e DNA r e p a i r p r o c e s s e s . This s e c t i o n reviews how t h e e x p r e s s i o n o f some r e p a i r - r e l a t e d genes i s altered following 1. The S O S r e g u l a t o r y exposure t o DNA-damaging agents. network o f E. c o l i When exposed t o c o n d i t i o n s that i n t e r f e r e w i t h DNA replication (e.g., DNA damaging agents, n a l i d i x i c a c i d , thymine deprivation e t c . ) , E. c o l i undergo a s e r i e s o f 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 t h e SOS response (reviewed by W i t k i n , 1976; L i t t l e and Mount, 1982; Kenyon, 1984; Ossanna e t a l . , 1986). enhanced c a p a c i t y 1983; Walker, The SOS phenotype i n c l u d e s an f o r DNA r e p a i r (both e x c i s i o n r e p a i r and r e c o m b i n a t i o n a l r e p a i r ) , enhanced mutagenesis, delayed d i v i s i o n , prophage i n d u c t i o n , cell and r e s p i r a t o r y a r r e s t . Most o f t h e s e changes presumably serve t o enhance t h e s u r v i v a b i l i t y of the damaged c e l l . (Prophage i n d u c t i o n 23 i s likely an a d a p t a t i o n of the v i r u s f o r escaping the i m p e r i l e d c e l l . ) The products of two genes, recA and lexA, r e g u l a t e the response. The LexA p r o t e i n i s a r e p r e s s o r of a t l e a s t separate genes i n the SOS lexA i t s e l f ; regulon. SOS 17 These i n c l u d e : recA, and the e x c i s i o n r e p a i r genes uvrA. B, C and D; umuC,D. an operon necessary f o r UV mutagenesis; sulA, a gene i n v o l v e d 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 a t 4 f o u r "damage i n d u c i b l e " genes whose f u n c t i o n s are not known. (din) In e x p o n e n t i a l l y growing c e l l s the LexA p r o t e i n r e p r e s s e s i t s t a r g e t genes by b i n d i n g a t one o r two s i m i l a r upstream o p e r a t o r sequences (SOS the uninduced s t a t e , many of the SOS boxes). genes, i n c l u d i n g 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 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 . of Obviously, In at low i n the absence r e p l i c a t i o n a r r e s t , c e l l s must m a i n t a i n a steady s t a t e c o n c e n t r a t i o n of LexA r e p r e s s o r t o prevent g r a t u i t o u s i n d u c t i o n of SOS functions. S i m i l a r l y , c o n s t i t u t i v e e x p r e s s i o n of recA a p p a r e n t l y p r o v i d e s s u f f i c i e n t RecA p r o t e i n f o r i t s dual r o l e s i n SOS r e g u l a t i o n and g e n e t i c recombination. The need f o r c o n s t i t u t i v e e x p r e s s i o n of the uvr genes i s understandable in view of t h e i r r o l e i n e x c i s i o n r e p a i r . Most of the g e n e r a l f e a t u r e s of SOS f a i r l y w e l l understood r e p l i c a t i o n generates r e g u l a t i o n are a t the molecular l e v e l . Blocked DNA an as y e t undetermined i n d u c i n g s i g n a l which a c t i v a t e s the protease f u n c t i o n of the RecA p r o t e i n . The RecA p r o t e a s e c l e a v e s and thus i n a c t i v a t e s the LexA r e p r e s s o r s . T h i s r e s u l t s i n d e r e p r e s s i o n of the genes of the SOS 24 regulon and e x p r e s s i o n o f t h e SOS p h e n o t y p e . the inducing signal proteolytically the c e l l The search cells. inactive. f o r an a n a l o g o u s stress A number o f f i n d i n g s , differentiation, in As t h e l e v e l induction, o f LexA r e p r e s s o r r i s e s , state. d i s c o v e r y o f t h e SOS r e s p o n s e virus i n E. c o l i response including i t s replication, prompted a mechanism i n mammalian e n h a n c e d DNA and i n d u c e d a l l following treatments mammalian c e l l s i s repaired, d i m i n i s h e s , and t h e RecA p r o t e i n s become i s r e t u r n e d t o the uninduced mutagenesis, arrest As t h e DNA damage cellular t h a t damage DNA o r a r e s u g g e s t i v e o f an S O S - l i k e (Herrlich r e p a i r and e t a l . , 1984). response Furthermore, a R e c A - l i k e p r o t e i n t h a t p r o m o t e s homologous r e c o m b i n a t i o n 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 However, t h e r e i s a s y e t no e v i d e n c e cells respond t o these treatments (Cassuto e t a l . , 1987). t o suggest by e n g a g i n g that eukaryotic a previously r e p r e s s e d b a t t e r y o f g e n e s 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 s y s t e m 2. ( F r i e d b e r g , 1985). The a d a p t i v e response t o a l k y l a t i o n damage E. c o l i alkylating e x p o s e d t o low, n o n l e t h a l l e v e l s agents (e.g., N-methyl-N'-nitro-N-nitrosoguanidine, MNNG) s o o n become c o n s i d e r a b l y more r e s i s t a n t and the cytotoxic effects same c h e m i c a l s of simple o f subsequent h i g h e r doses (Samson and C a i r n s , 1 9 7 7 ) . termed t h e a d a p t i v e response t o t h e mutagenic T h i s phenomenon i s t o a l k y l a t i o n damage 25 of these ( r e v i e w e d by Walker, 1984; Demple, 1987). i n d u c i b l e DNA r e p a i r pathway t h a t operates independently of SOS response. r e p a i r e d by death i s not The The mutagenic and a d a p t i v e response i s an cell-killing lesions separate components of t h i s pathway. the are Thus, cell 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 p r i n c i p a l a n t a g o n i s t s of the a l k y l a t i o n damage. II, the alkA gene product DNA and 2 T h i s component of the a d a p t i v e response mutagenic l e s i o n s (0 -methylguanine and 6 m e t h y l p y r i m i d i n e s ) are r e p a i r e d l a t t e r reactions requires sites. the from The O4 i n s i t u by 0 -methylguanine 6 m e t h y l t r a n s f e r a s e , a product of the ada The the 0 -alkylpyrimidines polymerase I t o r e p a i r the r e s u l t i n g AP 1984). glycosylase (see s e c t i o n I I . B . l ) , e x c i s e s p o t e n t i a l l y l e t h a l 3-alkylpurines duplex DNA. DNA the gene (McCarthy e t a l . , are unusual i n t h a t the covalent t r a n s f e r of a s i n g l e methyl group from the a l k y l a t e d base i n to a s p e c i f i c cysteine residue methyltransferase protein, methyltransferase function. described Two as s u i c i d e r e p a i r ada. irreversibly inactivates (see Demple and I t s product may cytotoxic lesions The be been Karran, 1983). aidB, are a l s o induced as p a r t alkB gene forms a s m a l l involved (Kataoka and the the T h i s novel a c t i v i t y has o t h e r genes, alkB and the a d a p t i v e response. i n the carboxy terminus of DNA of the aidB gene i s unknown ( V o l k e r t and 26 operon w i t h i n the e x c i s i o n r e p a i r of Sekiguchi, 1985). of The function Ngyuen, 1984). C o n t r o l of the adaptive locus i t s e l f response r e s i d e s w i t h i n the (Teo e t a l . , 1984). The 39 kDa the RecA p r o t e i n , has m u l t i p l e f u n c t i o n s . r e s i d u e , t h i s one serves methyltransferase (MeP) of the DNA substrates. The receptor MeP cysteine of the Ada protein, phosphotriesters not a l k y l a t e d bases, are methyltransferase a c t i v i t y does not t o c o n f e r r e s i s t a n c e t o c y t o t o x i c i t y or m u t a g e n i c i t y and like in a different suicide r e a c t i o n i n which methyl backbone, and protein, A second a t the amino t e r m i n a l end as an a l k y l - g r o u p Ada ada L i n d a h l , 1985), but r a t h e r serves t r a n s c r i p t i o n i n a manner d e s c r i b e d the appear (McCarthy as a p o s i t i v e r e g u l a t o r below. Unadapted c e l l s each c o n t a i n about 20 molecules of the protein. agents are r e p a i r e d by the MeP of the Ada protein. m o d i f i c a t i o n converts and Ada methyltransferase This post-translational the Ada p r o t e i n i n t o an t r a n s c r i p t i o n a l a c t i v a t o r of at l e a s t ada modified Ada Some of the MePs generated f o l l o w i n g exposure t o methylating activity of and efficient alkA. p r o t e i n then b i n d s t o s i m i l a r sequences i n the alkA promoters a t s i t e s upstream of the RNA b i n d i n g domain (Teo et a l . , 1986). l e v e l s o f Ada The (150-fold) The ada polymerase r e s u l t i n g increased and AlkA p r o t e i n are r e s p o s i b l e f o r the a l k y l a t i o n r e s i s t a n c e phenotype. The a l k y l t r a n s f e r a s e may y i e l d an 18 kDa a 13 kDa polypeptide polypeptide undergo f u r t h e r p r o c e s s i n g that repairs O-alkylated t h a t r e p a i r s MePs. While the a c t i v i t i e s are r e t a i n e d upon p r o t e o l y t i c cleavage, to bases, and transferase the r e g u l a t o r y a c t i v i t y of the Ada 1986). Teo protein i s lost e t a l . (1986) s p e c u l a t e which the adaptive (Teo e t a l . , t h a t t h i s may be one way response i s switched o f f when the c e l l by i s no l o n g e r exposed t o a l k y l a t i n g agents. An detected adaptive response s i m i l a r t o t h a t i n E. c o l i has i n B a c i l l u s s u b t i l i s and i n M. been l u t e u s . but not i n Salmonella typhimurium or i n S. c e r e v i s i a e (see Yarosh, 1985). Mammalian c e l l s have 0 -methylguanine m e t h y l t r a n s f e r a s e s with 6 p h y s i c a l p r o p e r t i e s s t r i k i n g l y s i m i l a r t o those of the 18 kDa c o l i protein induced (Samson, 1986). Whether or not these are as p a r t of a mammalian adaptive issue. The much h i g h e r response remains a c o n s t i t u t i v e l e v e l s of molecules i n mammalian c e l l s (Friedberg, 1985) E. contentious methyltransferase may obviate the need f o r an i n d u c i b l e system. An adaptive response t o o x i d a t i o n damage t h a t may as many as 30 p r o t e i n s has and i n S. typhimurium. r e c e n t l y been d e s c r i b e d The involve i n E. coli k i n e t i c s of p r o t e i n a c t i v a t i o n and the f a c t t h a t some of these genes are under heat shock c o n t r o l i n d i c a t e t h a t the r e g u l a t i o n of t h i s response i s l i k e l y t o be complex (see Demple, 1987). 3 . M o l e c u l a r approaches t o the q u e s t i o n o f damagei n d u c i b l e f u n c t i o n s i n eukaryotes U s i n g random y e a s t gene : E. c o l i l a c Z c o n t r u c t s , Ruby Szostak (1985) i d e n t i f i e d 6 f u s i o n s r e p r e s e n t i n g d i f f e r e n t DNA and at l e a s t 4 damage-inducible (din) genes i n S. c e r e v i s i a e . F i v e of t h e s e are r e s p o n s i v e t o a v a r i e t y of agents i n c l u d i n g 28 UV, 4 - n i t r o c j u i n o l i n e - N - o x i d e (4NQ0) , gamma r a y s , MMS, EMS, MNNG, and methotrexate. A s i x t h d i n - l a c Z f u s i o n i s induced o n l y by UV and methotrexate. Depending on t h e f u s i o n c o n s t r u c t and t h e mutagen employed, b e t a - g a l a c t o s i d a s e a c t i v i t y was i n c r e a s e d over the b a s a l l e v e l by as much as 300 times. R e s t r i c t i o n enzyme p a t t e r n s o f 4 o f t h e c o r r e s p o n d i n g chromosomal DIN genes i n d i c a t e t h a t they a r e not RAD1. RAD2, RAD3, RAD6, RADIO. RAD50, RAD51. RAD52, RAD54. o r RAD55. U s i n g a d i f f e r e n t approach t o t h e same q u e s t i o n , and McEntee to RNA (1986) i s o l a t e d cDNA t h a t d i f f e r e n t i a l l y McClanahan hybridized from mutagen-treated and u n t r e a t e d y e a s t c e l l s . They i d e n t i f i e d two 4NQO-inducible genes, termed DDR (DNA damage r e g u l a t i o n ) , n e i t h e r o f which i s a l l e l i c t o any o f t h e DIN genes i s o l a t e d by Ruby and Szostak (1985). I n t e r e s t i n g l y , t r a n s c r i p t i o n o f both DDR genes i s a l s o induced by heat shock, and a t l e v e l s comparable t o those e f f e c t e d by 4NQO. Heat shock/DNA damage-responsive genes have a l s o been found i n E. coli (Krueger and Walker, 1984) and more r e c e n t l y i n D r o s o p h i l a (Vivino e t a l . , 1986). The D r o s o p h i l a gene encodes a 1 k i l o b a s e p o l y a d e n y l a t e d t r a n s c r i p t t h a t i s induced by UV o r heat shock. Its r e s t r i c t i o n enzyme p a t t e r n d i f f e r s from those o f t h e known D r o s o p h i l a heat shock genes, none o f which a r e induced by UV (Vivino e t a l . , 1986). Although presumed, i n none o f these s t u d i e s has i t been demonstrated t h a t DNA damage p e r se i s t h e actual inducing s i g n a l . din In p a r t i c u l a r , t h e o b s e r v a t i o n t h a t the genes a r e s e n s i t i v e t o t h e a n t i m e t a b o l i t e methotrexate would 29 more l i k e l y suggest t h a t they are responding t o s t r e s s caused by n u c l e o t i d e p o o l imbalances r a t h e r than DNA damage. I I I . MTJTAGEN-SENSITIVE MUTATIONS IN DROSOPHILA MELANOGASTER As evidenced by the many examples above, the r e c o v e r y and characterization of mutagen-sensitive m i c r o b i a l s t r a i n s p r o v i d e d c o n s i d e r a b l e i n s i g h t i n t o the mechanisms of metabolism i n these u n i c e l l u l a r organisms. has DNA These r e s u l t s p r o v i d e d the r a t i o n a l e and the impetus t o s e a r c h f o r analogous mutations i n the m u l t i c e l l u l a r eukaryote D r o s o p h i l a melanogaster. Mutations induced a t more than 30 d i s t i n c t mutagensensitive (mus) loci i n D. melanogaster render d e v e l o p i n g f l i e s abnormally s e n s i t i v e t o the g e n o t o x i c e f f e c t s of DNA-damaging agents 1987; (reviewed by Smith e t a l . , 1980; Wurgler e t a l . , 1986). Boyd e t a l . , 1980, The f i r s t mus 1983, mutations i n D r o s o p h i l a were i s o l a t e d i n s e v e r a l s y s t e m a t i c screens f o r and X r a y - s e n s i t i v e 1976; mus mutants of the X chromosome. (Smith, MMS- 1973, Boyd e t a l . , 1976a; Nguyen e t a l . , 1978). The n e a r l y mutants r e c o v e r e d i n those screens f a l l complementation groups 100 into 9 discrete (muslOl. mus!02. mus_105, musl06, musl08. musiog, m u s l l l . mei-9. and mei-41) (Boyd e t a l . , 1987). Some mutations were found t o be a l l e l e s of two p r e v i o u s l y d e s c r i b e d r e c o m b i n a t i o n - d e f e c t i v e m e i o t i c mutants, mei-9 and mei-41 and Carpenter, 1972). were r e t a i n e d . (Baker For these s t r a i n s the mei d e s i g n a t i o n s In view of the frequency of redundant 30 alleles, it i s d o u b t f u l t h a t any more MMS-sensitive l o c i w i l l be d i s c o v e r e d on the X chromosome. I f t h i s r e s u l t can be e x t r a p o l a t e d t o the r e s t of the genome, then as many as 50 t o 60 MMS-sensitive genes may e x i s t i n Drosophila. Analogous s c r e e n i n g procedures were subsequently to s e l e c t f o r mus employed mutations on both of the major autosomes. Boyd e t a l (1981) r e c o v e r e d 34 t h i r d chromosome mutations t h a t c o n f e r s e n s i t i v i t y t o MMS and/or t o the b i f u n c t i o n a l agent n i t r o g e n mustard (HN2). complementation (mus3_01, mus302. mus3 04-mus312) (Boyd e t al., groups 1987). On the second chromosome, 8 mus representing 7 separate l o c i two MMS by Khromykh and Zakharov 11 strains, (mus2 01-mus207), were r e c o v e r e d i n s c r e e n s (Boyd e t a l . , 1981; o t h e r second chromosome mus MMS These mutations d e f i n e alkylating Snyder and Smith, 1982). Two mutants had e a r l i e r been d e s c r i b e d (1978). One o f these i s s e n s i t i v e t o (mus201 ), the o t h e r i s s e n s i t i v e t o i o n i z i n g G1 radiation (rad20lGlj ^ More r e c e n t l y , 32 a d d i t i o n a l second chromosome mus were i s o l a t e d by Henderson e t a l . (1987). Each s t r a i n i s s e n s i t i v e t o one o r more of the f o l l o w i n g mutagens: MMS, the mutants HN2, b u l k y adduct-forming p r o c a r c i n o g e n N-acetyl-2-aminofluorene (AAF), and gamma r a d i a t i o n . Their screening protocol differed from e a r l i e r s e l e c t i o n schemes i n t h a t i t p e r m i t t e d the r e c o v e r y of t e m p e r a t u r e - c o n d i t i o n a l mus temperature-sensitive mutants. (ts) s t r a i n s i n a l l . mus209 , i s a t s l e t h a l mutation B1 They i d e n t i f i e d 5 One o f them, (see Chapter 4 ) . The 7 s t r o n g e s t MMS-sensitive s t r a i n s i n t h a t were c h a r a c t e r i z e d e x t e n s i v e l y Standard g e n e t i c mapping and (Henderson et a l . , 1987). complementation analyses t h a t these mutations i d e n t i f y 4 new by two showed second chromosome mus (mus208, mus209, mus210, and mus21l). mus211) are each represented collection Two mus loci a l l e l e s . One (mus208 and mutant (mus205 ) i s a l l e l i c t o a p r e v i o u s l y c h a r a c t e r i z e d mus locus B1 (Snyder and loci Smith, 1982). A summary of t h e i r p r o p e r t i e s i s p r e s e n t e d i n Table 1. Although these mutations were i n i t i a l l y s e l e c t e d on b a s i s of s e n s i t i v i t y t o MMS, a l l of them (or t h e i r e x h i b i t s e n s i t i v i t y t o a t l e a s t one some form of r a d i a t i o n . alleles) other mutagen, i n c l u d i n g Furthermore, i n most cases the of mutagen c r o s s - s e n s i t i v i t y vary s i m i l a r between a l l e l e s . the The patterns from l o c u s t o l o c u s , but former o b s e r v a t i o n are ( e s p e c i a l l y as i t a p p l i e s t o the r a d i a t i o n s e n s i t i v i t y ) suggests t h a t these mutations i d e n t i f y bona f i d e DNA t o genes i n v o l v e d simply exogenously a p p l i e d MMS. most of these mus at of DNA i n one n o t i o n t h a t mus r e p a i r has i n the uptake and/or metabolism The l a t t e r observation s t r a i n s represent d i f f e r e n t steps The r e p a i r - r e l a t e d genes as opposed suggests t h a t genes whose products operate of s e v e r a l d i f f e r e n t r e p a i r pathways. mutations are d e f e c t i v e i n some aspect i n f a c t been confirmed b i o c h e m i c a l l y of the o r i g i n a l mus of isolates. f o r many Thus, of the 18 d i f f e r e n t l o c i which have been examined f o r r e p a i r a b n o r m a l i t i e s , d e f e c t i v e i n the e x c i s i o n r e p a i r of UV-damaged DNA mus 4 are (Boyd et a l . , T a b l e 1. SUMMARY OF PROPERTIES OF mus MUTANTS OF THE SECOND CHROMOSOME Strain 201 D 1 Mutagen c r o s s - s e n s i t i v i t y MMS HN2 + + AAF - + + A 1 + 205 B 1 + 208 B 1 209 B 1 UV ionizing radiation + - 205 BP Map p o s i t i o n a 23 + - 54.9+1.6 + + - 89.8+3.3 - - - + 92.8+2.6 + 64 210 B 1 + + + + - 69.1±3.1 211 B 1 + + - - + 50.4+3.1 + = s e n s i t i v e , - = not s e n s i t i v e , blank = not t e s t e d . Although not i n d i c a t e d here, t h e degree o f mutagen s e n s i t i v i t y may v a r y c o n s i d e r a b l y between mutants (e.g., see Henderson e t a l . , 1987). Data f o r mus201 and mus2_05 ^ were compiled from Boyd e t a l . (1982, 1987) and Snyder and Smith (1982). a D1 A Map p o s i t i o n s and 95% c o n f i d e n c e i n t e r v a l s f o r t h e "B" s e r i e s mutants were determined as d e s c r i b e d i n Chapter 4 MATERIALS AND METHODS. Map p o s i t i o n s f o r mus201 and mus205 were o b t a i n e d from Boyd e t a l . (1987). b D1 33 A1 1976b, 1982; are d e f e c t i v e Brown and Boyd and Harris, 1981; Luchkina e t a l . , 1982), 6 in postreplication repair Boyd, 1981a; Boyd and (Boyd and Shaw, 1982), and p a r t i a l l y d e f i c i e n t i n both processes (Boyd and Brown and neither loci 1981; express abnormalities. However, some of these s t r a i n s do e x h i b i t minor d e f e c t s synthesis The the f o l l o w i n g mutagen treatment (Henderson e t a l . , 1987). D r o s o p h i l a mus functionally interrelated. Boyd, 1981b). using and genetic genes Chapter 3, u s i n g o b s e r v a t i o n s s t r a i n s may be u s e f u l as r a p i d and i n d i c a t o r s of chemical g e n o t o x i c i t y . further genetic further second chromosome mus made i n Chapter 2, e x p l o r e s the p r a c t i c a l p o s s i b i l i t y multiply-mutant mus DNA gene r e p e r t o i r e Chapter 2 d e s c r i b e s , t e s t s , the manner i n which the new are (Brown and in remaining chapters i n t h i s t h e s i s c h a r a c t e r i z e newest members of the 1976; 4 are a t l e a s t Harris, Boyd, 1981a). Mutations at 4 o t h e r mus e x c i s i o n nor p o s t r e p l i c a t i o n r e p a i r Setlow, 34 sensitive Chapter 4 s i n g l e s out developmental a n a l y s i s the mutagen-sensitive l o c u s mus209. that intriguing for CHAPTER INTERACTIONS TWO BETWEEN MMS-SENSITIVE 35 MUTATIONS INTRODUCTION The o v e r a l l c e l l u l a r response t o genome damage i s a complex m o l e c u l a r t o u r de f o r c e t h a t i s p o o r l y understood. i n D r o s o p h i l a melanogaster F o r example, n e a r l y 30 d i f f e r e n t genes s e n s i t i v i t y t o t h e DNA a l k y l a t i n g agent methyl control methanesulfonate (MMS), and t o add t o t h e complicacy, numerous o t h e r r e p a i r r e l a t e d genes have been i d e n t i f i e d (reviewed by Smith e t a l . , 1980; Boyd e t a l . , 1983, 1987; Wurgler e t a l . , 1986). yet powerful g e n e t i c approach t o the problem functional A simple of determining the 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 a n a l y s i s and Game, 1974). (Brendel and Haynes, 1973; Cox T h i s technique, pioneered i n t h e bakers' y e a s t Saccharomyces c e r e v i s i a e . has been i n s t r u m e n t a l i n shaping t h e c o n c e p t u a l framework which u n d e r l i e s t h e c u r r e n t view o f t h e e u k a r y o t i c response t o genome damage. Mutations i n double mutants may i n t e r a c t i n one o f t h r e e ways: e p i s t a t i c a l l y , a d d i t i v e l y , o r 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 , t h e double mutant s t r a i n i s no more s e n s i t i v e t o k i l l i n g by a mutagen than t h e most s e n s i t i v e s i n g l e mutant. E p i s t a s i s i m p l i e s t h a t t h e gene products ( c o n c e p t u a l i z e d as DNA r e p a i r enzymes) operate a t s e q u e n t i a l s t e p s i n a l i n e a r pathway. I n an a d d i t i v e repair interaction, the s e n s i t i v i t y of the double mutant i s simply t h e sum o f t h e s e n s i t i v i t i e s o f t h e s i n g l e mutants. A d d i t i v i t y i s thought t o a r i s e when d i s t i n c t gene p r o d u c t s use as s u b s t r a t e s d i f f e r e n t DNA l e s i o n s 36 produced by a s i n g l e mutagen. In a s y n e r g i s t i c i n t e r a c t i o n , t h e s e n s i t i v i t y o f the double mutant exceeds the l e v e l o f s e n s i t i v i t y expected f o r an a d d i t i v e i n t e r a c t i o n . Synergism i m p l i e s t h a t the gene products, as components o f d i f f e r e n t r e p a i r pathways, compete f o r the same DNA l e s i o n . The r e s u l t s o f numerous m u l t i p l e mutant a n a l y s e s i n y e a s t i n d i c a t e t h e e x i s t e n c e o f 3 e p i s t a s i s groups: RAD52 RAD3. RAD6, and (see Haynes and Kunz (1981) and r e f e r e n c e s t h e r e i n ) . Although some o v e r l a p e x i s t s 1987), (e.g., see Eckardt-Schupp e t a l . , i t i s w i d e l y h e l d t h a t these groups d e f i n e d i s c r e t e cellular (dark r e p a i r ) responses t o radiation-damaged DNA, and t h a t c o l l e c t i v e l y , they c o n s t i t u t e a complete s e t o f DNA r e p a i r pathways. These i d e a s have emerged from the f o l l o w i n g observations. Mutant members w i t h i n a group g e n e r a l l y g r o u p - s p e c i f i c patterns of mutagen-sensitivity see Haynes and Kunz, 1981; Hoekstra e t a l . , common, b i o c h e m i c a l l y - d e f i n e d r e p a i r d e f e c t s Kunz, 1981; Game, 1983). exhibit ( f o r exceptions 1986), and d i s p l a y (see Haynes and Furthermore, h a p l o i d t r i p l e mutant s t r a i n s , composed o f one mutant from each e p i s t a s i s group, e x h i b i t " s i n g l e - h i t " s u r v i v a l responses a t UV doses t h a t produce o n l y 1-2 p y r i m i d i n e dimers p e r genome (Cox and Game, 1974). F i n a l l y , mutants i s o l a t e d on the b a s i s o f s e n s i t i v i t y t o mutagens o t h e r than r a d i a t i o n fall ( i n c l u d i n g MMS) almost i n t o one o f the known e p i s t a s i s groups Prakash, 1977; invariably (e.g., Prakash and Henriques and Moustacchi, 1980; S i e d e and B r e n d e l , 1982). Taken t o g e t h e r , these o b s e r v a t i o n s suggest t h a t 37 no o t h e r major r e p a i r processes (apart from p h o t o r e a c t i v a t i o n ) 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 e u k a r y o t i c r e p a i r response remains t o be determined. of However, data from a number o t h e r e u k a r y o t i c sources, i n c l u d i n g the f i s s i o n y e a s t Schizosaccharomyces Neurospora crassa pombe (Phipps e t a l . , (Kafer, 1983), eleaans (Hartman, 1985) response t o DNA In 1985), the bread mold and the nematode C a e n o r h a b d i t i s are c o n s i s t e n t w i t h a t r i p a r t i t e damage. D r o s o p h i l a . the scope of double mutant a n a l y s e s has been r e s t r i c t e d l a r g e l y t o the study of i n t e r a c t i o n s among mutagens e n s i t i v e mutants of the X chromosome (Baker e t a l . , Smith, 1978; Nguyen e t a l . , 1979; Smith e t a l . , 1976, 1980). 1982; The t y p e s of i n t e r a c t i o n s seen p a r a l l e l those observed i n y e a s t . However, too few mutant combinations have been c h a r a c t e r i z e d t o p r o v i d e a view as comprehensive as t h a t i n S. c e r e v i s i a e . The p r e s e n t study i s an e x t e n s i o n of p r e v i o u s double mutant a n a l y s e s in Drosophila. I t r e p r e s e n t s the f i r s t a n a l y s i s o f i n t e r a c t i o n s among autosomal mus chromosome. loci, s p e c i f i c a l l y those on the second A t o t a l of 10 d i f f e r e n t m u l t i p l e mutant mus were t e s t e d f o r s e n s i t i v i t y t o 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 f i n d i n g s i n D r o s o p h i l a suggest t h a t DNA strains and r e p a i r pathways i n t h i s complex eukaryote be o r g a n i z e d s i m i l a r l y t o those i n y e a s t . 38 may MATERIALS AND METHODS Strains D e t a i l e d d e s c r i p t i o n s o f the v i s i b l e mutations and s p e c i a l chromosomes used can be found i n L i n d s l e y and G r e l l (1968). E i g h t double mus s t r a i n s and two t r i p l e mus s t r a i n s were analyzed i n t h i s study. They a r e as f o l l o w s : mus201 D1 mus205 . mus201 D1 mus208 . mus201 D1 mus210 . mus205 B1 mus208 . mus205 B1 mus210 , mus208 B1 mus210 . mus208 B1 mus211 . mus210 B1 B1 B1 mus2JL0 , and mus208 B1 B1 B1 B1 B1 mus211 . mus205 B1 mus210 B1 musll . B1 B1 mus208 B1 B1 The mus mutations 6 2 b e a r i n g t h e alphanumeric s u p e r s c r i p t BI o r B2 were i s o l a t e d as described (Henderson e t a l . , 1987; see a l s o Appendix A ) . A l l s i n g l e and m u l t i p l e mutant mus s t r a i n s c a r r y the r e c e s s i v e mutations b p_r cn except cn bw mus205 A1 J . B. Boyd, and cn mus201 D1 (Boyd e t a l . , 1982) and (Snyder and Smith, 1982), k i n d l y p r o v i d e d by Dr. mus201 through t h e g e n e r o s i t y G1 (Luchkina e t a l . , 1982), o b t a i n e d o f Dr. I . A. Zakharov. Mutant chromosomes were kept i n s t o c k over the m u l t i p l y - i n v e r t e d b a l a n c e r chromosomes In(2LR)CyO ( a l l s i n g l e mutants except mus201 ) o r In(2LR)SM5 (mus201 D1 D1 Both i n v e r s i o n s and a l l m u l t i p l e mutants). ( h e r e a f t e r r e f e r r e d t o as CyO and SM5. r e s p e c t i v e l y ) a r e i d e n t i f i e d by the dominant marker C u r l y wings (Cy.) . 1987). 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 Methyl methanesulfonate (MMS) o b t a i n e d from Sigma Chemical Company. 39 (Henderson e t a l . , (CAS No. 66-27-3) was Strain Construction Initially, combined double mutant s t r a i n s were c o n s t r u c t e d t h a t second and t h i r d chromosomal mus mutations. s u f f e r e d both v i a b i l i t y to These and f e c u n d i t y problems, p r o b a b l y owing the presence o f the two b a l a n c e r chromosomes. These early e f f o r t s were abandoned i n f a v o r o f c o n s t r u c t i n g double mutant s t r a i n s composed o n l y of mus l o c i from the second chromosome. Double mus s t r a i n s were c o n s t r u c t e d as diagrammed i n F i g u r e 1. Females, trans-heterozygous f o r the two mus l o c i of interest (e.g. A and B) were mated en masse t o G l a / SM5 males. P u t a t i v e 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 t o 3 or 4 G l a / SM5 females. cn Cy ( i . e . Gla" ") F2 females were c o l l e c t e d from each 1 l i n e and mated t o p h e n o t y p i c a l l y s i m i l a r male s i b s . Each p u t a t i v e double mus l i n e was t e s t e d f o r mutagen s e n s i t i v i t y and f a i l u r e t o complement a p p r o p r i a t e s i n g l e mus s t r a i n s . of A total 13 d i f f e r e n t double mus s t r a i n s were c o n s t r u c t e d (although o n l y 8 were a n a l y z e d i n t h i s s t u d y ) . Ten o f these r e p r e s e n t a l l p o s s i b l e p a i r w i s e combinations o f 5 d i s c r e t e second chromosomal mus l o c i (Henderson e t a l . , 1987). mus201 mutation. D1 synthesized The o t h e r 3 i n c l u d e the In a d d i t i o n , 2 t r i p l e mus s t r a i n s were (from a p p r o p r i a t e s i n g l e and double mus s t r a i n s ) by f o l l o w i n g procedures s i m i l a r t o those used t o make the double mutants. 40 Figure 1. Procedure used t o c o n s t r u c t m u l t i p l y - m u t a n t strains. 41 mus Gla mus A musB cfcT Gla mus A SM5 SM5 musB or Gla cr putative recombinants musA musB mus A musB SMS SM5 viable homozygotes ? 1. mutagen test 2. complementation test with single mus strains A and B 42 cfd 1 MMS Dose R e s p o n s e For these tests, mated i n v i a l s Initially, or controls 22°C. at t o about h a l f These f e d , mus / Cy. f e m a l e s were a s many homozygous mus c u l t u r e s were l e f t t o new v i a l s untreated (developmental temperature = 25°C). f e m a l e s were a l l o w e d t o o v i p o s i t hatching). following the start development. each day f o r 2 and s e r v e d a s In the test applying of oviposition A p p r o x i m a t e l y 12 h o u r s l a t e r , t o 2 5 ° C where t h e y r e m a i n e d males. f o r 12+1 h o u r s a t C u l t u r e s were t r e a t e d b y t o p i c a l l y 24+2 h o u r s shifted 5 t o 10 y e a s t p a r e n t s were t r a n s f e r r e d 3 days. series, Curves 0.25 mL (i.e., treated were f o r the duration of h o m o z y g o t e s t o mus / Cy_ h e t e r o z y g o t e s . o f mus T h e s e v a l u e s were normalized t o t h e homozygote:heterozygote 43 prior to cultures S u r v i v a l v a l u e s a r e presented as a r a t i o controls. MMS ratio obtained i n the RESULTS In t o t a l , 13 double mus i n t e r a c t i o n s were analyzed s t r a i n s were c o n s t r u c t e d in 8 strains only). Ten (although of these r e p r e s e n t a l l p o s s i b l e p a i r w i s e combinations o f 5 d i s c r e t e second chromosome mus 3 i n c l u d e mus201 D1 loci t o g e t h e r with mus2_05 , mus2_08 B1 In a d d i t i o n , 2 t r i p l e mus mus208 The B1 mus210 B1 (Henderson e t a l . , 1987). B1 s t r a i n s were s y n t h e s i z e d : and mus208 B1 mus210 i n t e r a c t i o n s may or mus210 . B1 mus205 B1 B2 v i a b l e s i n g l y , might i n t e r a c t t o cause l e t h a l i t y i n i t i a l l y a concern. other mus211 . B1 p o s s i b i l i t y t h a t the p r o g e n i t o r mutations, mutant was The although i n the double Such s y n t h e t i c l e t h a l be r i f e among combinations of mutants at i n v o l v e d i n chromosome metabolism loci ( f o r examples i n D r o s o p h i l a . Neurospora and Saccharomyces see Smith e t a l . ( 1 9 8 0 ) , 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 turned out t o be l e t h a l . s t r a i n s b e a r i n g mus2_09 B1 s t r a i n s d e s c r i b e d above Moreover, with the e x c e p t i o n (which are not i n c l u d e d i n t h i s a n a l y s i s - see below), none of the homozygous mus any individuals in s i n g l e or double mutant s t r a i n appears t o s u f f e r from v i a b i l i t y problems (Table 1). heterozygotes but one The r a t i o of homozygotes t o i s v e r y c l o s e t o the expected v a l u e of 1 f o r a l l strain. In the case of mus201 D1 mus210 , the B1 r a t i o i s skewed d r a m a t i c a l l y i n f a v o r of the mus The of reason f o r t h i s d e v i a t i o n i s unknown. The survival homozygotes. t r i p l e mutant s t r a i n s e x h i b i t only a s l i g h t reduction i n v i a b i l i t y . This T a b l e 1. RELATIVE VIABILITY OF mus HOMOZYGOTES AND HETEROZYGOTES IN UNTREATED CULTURES Strain R e l a t i v e v i a b i l i t y of homozygous mutants ' k a b p r cn 201 205 205 208 BI 210 BI 211 BI 0.96 0.94 0.93 1.00 0.98 0.97 0.98 [3869) 1668) [3990) [5073) 1710) 1534) ;i932) 201 DI 201 DI 201 DI 205 BI 205 208 208 210 BI 205 BI 208 BI 210 BI 208 BI 210 210 211 211 BI 1.05 0.93 1.61 0.99 1.03 1.07 1.02 0.96 [6115) [3576) [3998) ;3883) [5858) [4233) [3433) [3320) 205 BI 208 208 BI 210 BI 210 211 0.89 0.88 [2814) ;i3944) D 1 A 1 B 1 B 1 B 1 B 1 B 1 B 1 B 1 B 1 B 1 B 2 In a l l cases, progeny were d e r i v e d from matings between heterozygous mus / Cy females and homozygous mus males (see MATERIALS AND METHODS). C u l t u r e s were maintained a t 25°C. a R e l a t i v e v i a b i l i t y = no. o f mus/mus a d u l t s no. o f mus/Cy adults T o t a l number o f progeny a r e g i v e n i n parentheses. 45 situation contrasts with that viability o f d o u b l e mutant s t r a i n s p r e c l u d e s t h e s y n t h e s i s o f triple i n Neurospora mutant m u t a g e n - s e n s i t i v e l i n e s where t h e p o o r (Kafer, 1983). F o r t h o s e d o u b l e mutant s t r a i n s b e a r i n g m u s 2 0 9 . B 1 sensitivities will h a v e t o be e x a m i n e d a t 2 2 ° C . temperature-sensitive lethal, even a t 25°C S i n g l e Mutant V e r s u s Double Mutant al., and m u s 2 0 5 D 1 1982; Snyder mus209 is a i s nearly completely MMS Sensitivities r e c o v e r e d s e c o n d chromosomal mus A 1 a r e t h e most s e n s i t i v e t o MMS and S m i t h , 1982). For t h i s categorical r e a s o n , and both mutations e x h i b i t repair, they a r e i n c l u d e d here t o serve as y a r d s t i c k s o f t h e newest c o l l e c t i o n mutations, (Boyd e t because w h i c h members B 1 (see Chapter 4 ) . Among p r e v i o u s l y mus201 l e t h a l mutation that MMS defects i n DNA against o f second chromosomal mus m u t a n t s c a n be compared. Figure 2 shows t h e M M S - s u r v i v a l for the a l l e l i c that mus205 B 1 weaker m u s 2 0 5 mutations mus205 A 1 c u r v e s t h a t were g e n e r a t e d and m u s 2 0 5 . I t c a n be s e e n B 1 i s by f a r t h e more s e n s i t i v e allele. A 1 strain i s completely defective i n postreplication repair (Brown and Boyd, 1981a; 1982) and p a r t i a l l y Harris, that 1981), defective i t i s inferred i n excision that mus205 a r e s i m i l a r t o , and a t l e a s t mus205 . A 1 46 repair B 1 Since the Boyd a n d Shaw, (Boyd and has r e p a i r as severe as those i n defects F i g u r e 2. MMS s e n s i t i v i t y of mus201 mus201 2838); mus205 1591); D1 mus205 . B1 A1 f D1 open squares, mus201 number o f f l i e s respectively. deviation indicate circles, D1 mus205 mus205 . (2310, the average, and the minimum dose The e r r o r bars i n d i c a t e o f the mean c a l c u l a t e d were o b t a i n e d from 2 t r e a t m e n t s ) . point, 1 standard from a t l e a s t 3 (data f o r mus205 47 B1 The numbers B1 scored per non-zero separate treatments and (1718, 969); open c i r c l e s (1905, 989); s o l i d i n parentheses B1 S o l i d squares, b p_r cn (3575, t r i a n g l e s , mus201 A1 mus205 . mus205 D1 A1 a t 0.04% MMS 0.01 0.02 % MMS 48 0.03 0.04 Epistatic The Interactions biochemical pleiotropy encodes a product t h a t o f mus2 0 5 + suggests t h a t mus205 + i s common t o both t h e e x c i s i o n and p o s t r e p l i c a t i o n r e p a i r pathways. e f f e c t o f a mus205 A 1 I n order t o determine t h e d e f i c i t on o t h e r known o r suspected DNA r e p a i r d e f e c t i v e mutants, 3 d i f f e r e n t mus20J5 -containing double B1 mutant mus s t r a i n s were examined f o r s e n s i t i v i t y t o MMS. T e s t s o f double mutants i n v o l v i n g mus205 B1 mus201 . mus208 , o r mus210 , have r e v e a l e d D1 B1 interactions; that and any o f epistatic B1 i s , each double mutant s t r a i n i s no more s e n s i t i v e t o MMS than t h e most s e n s i t i v e s i n g l e mutant, mus205 (Figures 2, 3, and 4 ) . These r e s u l t s a r e s i m i l a r t o those o f Smith e t a l . (1980) who found t h a t mus205 epistatically. Based on these f i n d i n g s , A1 and m e i - 9 + i n a common l i n e a r r e p a i r pathway. repair defects assigned t o mei-9 (Boyd e t a l . , 1982), and mus210 a interact i t would appear t h a t mus_2j05 and t h e o t h e r MMS-sensitive l o c i d e s c r i b e d sequentially here a c t The e x c i s i o n (Boyd e t a l . , 1976b), mus201 a (see below) a r e c o n s i s t e n t B1 with t h i s notion (mus208 biochemically). However, t h i s i n t e r p r e t a t i o n i s complicated by the f a c t t h a t mus205 well. + B1 B1 has not been characterized has a p o s t r e p l i c a t i o n r e p a i r f u n c t i o n as Moreover, t h e i n t e r a c t i o n s observed i n t h e p a i r e d combinations o f mus201 . mus208 D1 B1 and mus210 suggest a complexity t h a t i s not r e v e a l e d interactions j u s t described. 49 B1 (see below) by t h e e p i s t a t i c D1 Figure 3. MMS s e n s i t i v i t y of mus205 component s i n g l e m u t a n t s . 1591); squares, mus205 B 1 mus208 information mus208 B 1 B 1 (2087, see legend 50 B 1 mus208 B 1 compared C i r c l e s , mus205 (2632, 1632). to Figure 1411); B 1 to i t s (2310, triangles For additional 2. 51 Figure 4. MMS sensitivity of mus205 component s i n g l e m u t a n t s . 1591); squares, mus210 the B 1 mus210 B 1 (2751, 2 2 6 1 ) . legend to Figure 2. 52 B 1 mus210 B 1 compared Triangles, mus205 (1675, 9 2 6 ) ; c i r c l e s to i t s B 1 (2310, mus205 B 1 F o r a d d i t i o n a l i n f o r m a t i o n see 0.01 0.02 % MMS 53 0.03 0.04 An A d d i t i v e Interaction The mutations mus208 additively (Figure 5). and mus211 B1 appear t o i n t e r a c t B1 T h i s r e s u l t suggests t h a t these mus loci operate i n d i s p a r a t e r e p a i r pathways, each capable o f removing a d i f f e r e n t type o f DNA l e s i o n caused by MMS. Consistent with t h i s h y p o t h e s i s i s the o b s e r v a t i o n t h a t the mutagen c r o s s s e n s i t i v i t i e s o f these two s i n g l e mutants d i f f e r (see Table 1 i n Chapter 1 ) . Synergistic Interactions S e v e r a l i n s t a n c e s o f synergism were observed. the combination o f mus210 s t r o n g synergism and mus211 B1 B1 u n l e s s extremely h i g h doses et a l . , and m u s 2 1 1 B1 exhibits a f a i r l y B1 (Figure 6 ) . T h i s i n t e r a c t i o n i s r a t h e r s t r i k i n g s i n c e mus211 . by i t s e l f , (Henderson 1987). i s not s e n s i t i v e t o MMS (0.12 t o 0.15 %) a r e a p p l i e d The synergism i m p l i e s t h a t mus210 B1 i d e n t i f y d e f e c t s i n d i f f e r e n t r e p a i r pathways t h a t normally compete f o r the same type o f MMS-induced Furthermore, F o r example, lesion. i t can be i n f e r r e d t h a t the pathway i d e n t i f i e d by mus211 . although u s u a l l y o f c o m p a r a t i v e l y minor importance f o r B1 the r e p a i r o f 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 t h e mus210 words, i n t h e absence o f normal + function. l e v e l s o f mus210 l e s i o n s a r e " c h a n n e l l e d " i n t o the mus211 B o r s t e l and H a s t i n g s , + In o t h e r + a c t i v i t y , DNA pathway (see von 1985). A second example o f synergism i s p r o v i d e d by the combination of mus208 B1 and mus210 B1 (Figure 7 ) . The heightened MMS- F i g u r e 5. MMS s e n s i t i v i t y o f mus208 component s i n g l e mutants. 1411); t r i a n g l e s , mus211 mus208 B1 mus211 B1 B1 mus211 B1 Squares, mus208 B1 B1 to i t s (2632, (2182, 1244); c i r c l e s , (3712, 3053). 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. 55 compared 20 H I 0.01 1 1 0.02 0.03 % MMS 56 1 • 0.04 Figure 6 . MMS sensitivity of mus210 component s i n g l e m u t a n t s . 926); t r i a n g l e s mus211 mus210 B 1 mus211 information B 1 B 1 B 1 mus211 B 1 Squares, mus210 (2182, (3520, 9 9 6 ) . 1244); B 1 toi t s (1675, circles, For additional see the legend t o Figure 57 compared 2. 0.01 0.02 % MMS 58 0.03 0.04 Figure 7. MMS s e n s i t i v i t y of mus208 component s i n g l e m u t a n t s . 1411); t r i a n g l e s , m u s 2 1 0 mus208 B 1 mus210 information B 1 (2735, B 1 mus210 compared Squares, mus208 B 1 B 1 to i t s (2632, (1675, 9 2 6 ) ; c i r c l e s , 1701). see legend t o Figure 59 B 1 For additional 2. 0.01 0.02 % MMS 60 0.03 0.04 s e n s i t i v i t y o f t h e mus208 e v i d e n t a t 0.02 % MMS. B1 inus210 repair and mus210 D1 B1 D1 and mus208 (Figure B1 f o r excision encodes an a c t i v i t y t h a t and mus208 B1 B1 i s consistent s i n g l e mutants mus201 indistinguishable a pattern D1 with t h i s i n t e r p r e t a t i o n . and mus210 are v i r t u a l l y B1 i n t h e i r responses t o mutagens; excision repair-defective mutations g e n e r a l l y . While the mus210 biochemically, Indeed, mus201 at or p r i o r B1 s t r a i n has not been i t f a i l s t o complement the MMS (data not shown) o f an independently i s o l a t e d mus mutation t h a t i s defective i n excision repair Thus, an e x c i s i o n r e p a i r d e f e c t the b a s i s displays i n c i s i o n step o f the e x c i s i o n r e p a i r response (Boyd e t a l . , 1982). sensitivity each i s c h a r a c t e r i s t i c of i d e n t i f i e s a gene whose product normally f u n c t i o n s characterized functions The e p i s t a s i s between o f mutagen c r o s s - s e n s i t i v i t y t h a t to the i n i t i a l (Luchkina e t a l . , almost c e r t a i n l y forms f o r the mutagen s e n s i t i v i t y o f mus210 A c c o r d i n g l y , mus201 D1 and mus210 B1 B1 as w e l l . both a r e assumed t o be members o f t h e same e p i s t a s i s group, a D r o s o p h i l a e q u i v a l e n t the RAD3 group ( e x c i s i o n r e p a i r ) The B1 r e p a i r pathway, f o r example, i n some aspect o f the p o s t r e p l i c a t i o n r e p a i r p r o c e s s . 1982) . 8 ) . As (see below), t h e i r s y n e r g i s t i c i n t e r a c t i o n s w i t h mus208 i n some a l t e r n a t e The of s e n s i t i v i t y i s both i d e n t i f y genes r e q u i r e d suggest t h a t the l a t t e r l o c u s mus205 homozygotes i s e s p e c i a l l y A very s i m i l a r p a t t e r n seen i n t h e combination o f mus201 mus201 B1 of of yeast. y e a s t model p r e d i c t s t h a t members o f the same e p i s t a s i s group should i n t e r a c t e p i s t a t i c a l l y . To t e s t t h i s i n regard t o 61 D1 F i g u r e 8. MMS sensitivity o f mus201 component s i n g l e mutants. 969); squares, mus201 D1 mus208 mus208 B1 B1 D1 mus208 B1 compared T r i a n g l e s , mus201 to i t s D1 (2632, 1411); c i r c l e s , (2839, 491). F o r 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. 62 (1718, 0.01 0.02 % MMS 63 0.03 0.04 mus201 L,x and mus210 -. an MMS dose-response curve was generated BJ f o r t h e mus201 mus210 D1 strain. B1 Instead o f t h e expected e p i s t a s i s , a v e r y s t r o n g synergism was observed (Figure 9). MMS S e n s i t i v i t i e s o f T r i p l e Mutant S t r a i n s Any p a i r w i s e combination o f mus208 . mus210 , and mus211 B1 e x h i b i t s a s e n s i t i v i t y t o MMS B1 B1 t h a t i s g r e a t e r than t h a t o f any component s i n g l e mutant (see above). In view o f p r e v i o u s examples from y e a s t (e.g., see Cox and Game, 1974), these f i n d i n g s suggest t h a t a t r i p l e mutant s t r a i n composed o f these mutations might e x h i b i t a s e n s i t i v i t y t o MMS of each double mutant combination. the s t r a i n mus208 B1 mus210 s u r p r i s i n g l y , mus208 MMS than mus208 B1 i s the mus21Q B1 B1 B1 mus211 mus210 most B1 To e x p l o r e t h i s B2 B2 B1 mus208 B1 Somewhat i s no more s e n s i t i v e t o double mutant strain, (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 mus205 possibility, was s y n t h e s i z e d . mus211 sensitive t h a t surpasses t h a t mus210 B1 tested. i s no more s e n s i t i v e t o MMS the h i g h l y s e n s i t i v e s i n g l e mutant, mus205 64 B1 than i s ( F i g u r e 10). Figure 9. MMS sensitivity component 969); single circles, mus201 D 1 mus208 B 1 of mus201 mutants. mus210 mus210 B 1 additional (1196, information 65 mus210 B 1 compared Triangles, mus201 (1675, B 1 were o b t a i n e d D 1 to i t s D 1 (1718, 926); squares, 9 4 6 ) . D a t a f o r 0.02% m u s 2 0 1 from 2 t r e a t m e n t s see legend only. to Figure 2. For D 1 0.01 0.02 % MMS 66 0.03 0.04 F i g u r e 10. MMS s e n s i t i v i t y o f mus205 mus208 B1 mus210 B1 B1 mus211 . 1701); open squares, 572). triangles, mus205 mus208 B1 mus210 - and B Circles, B2 (1905, 989); s o l i d squares, 497); inus208 mus208 B1 B1 mus208 mus210 B1 L mus205 mus210 B1 B1 B1 mus210 mus211 B2 (2735, B1 (3719, 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. 67 (698, 0.01 0.02 % MMS 68 0.03 0.04 DISCUSSION In p r i n c i p l e , f u n c t i o n a l i n t e r r e l a t i o n s h i p s among mutagen- s e n s i t i v e l o c i can be a s c e r t a i n e d interactions by a n a l y z i n g mus mutant i n double mutant s t r a i n s . should be p o s s i b l e Using t h i s approach, i t t o o r g a n i z e mus genes o f r e l a t e d i n t o common pathways o f DNA r e p a i r . In p r a c t i c e , function however, the v a l i d i t y o f a p a r t i c u l a r assignment i s l a r g e l y dependent upon the u n c e r t a i n nature o f both the mutant a l l e l e and t h e b i o c h e m i s t r y o f the gene product component o f a multimer). assumptions a r e made First, (e.g., l e a k i n e s s ) (e.g., monomer v e r s u s Thus, f o r s i m p l i c i t y , t h e f o l l o w i n g initially. i t i s assumed t h a t mus l o c i i d e n t i f y d i s c r e t e , repair-related DNA a c t i v i t i e s which operate a t s u c c e s s i v e steps i n l i n e a r pathways. Second, i n the absence o f a d d i t i o n a l mutagen c r o s s - s e n s i t i v i t y d i f f e r e n c e s fundamental f u n c t i o n a l differences data, a r e assumed t o r e f l e c t between mus gene p r o d u c t s . (However, t h e c o r o l l a r y , t h a t mutations having s i m i l a r p a t t e r n s of mutagen c r o s s - s e n s i t i v i t y a r e d e f i c i e n t i n s i m i l a r cannot always be assumed t o be t r u e functions, (cf mei-9 and mei-41).) F i n a l l y , mus mutations are assumed t o be amorphic (see M u l l e r , 1932), a t l e a s t i n terms o f t h e i r DNA r e p a i r f u n c t i o n s . v a l i d i t y o f these assumptions, and a d d i t i o n a l The l i m i t a t i o n s o f the methodology a r e c o n s i d e r e d below i n r e f e r e n c e t o s p e c i f i c mutant-mutant The interactions. most severe form o f mutant i n t e r a c t i o n i s t h a t results i n unconditional l e t h a l i t y . 69 which In D r o s o p h i l a . f o r example, 4 of 11 d i f f e r e n t double mus of 7 X - l i n k e d loci, s t r a i n s , constructed with exhibit synthetic lethality alleles (Table 2 ) . Perhaps s i g n i f i c a n t l y , these l e t h a l i n t e r a c t i o n s are r e s t r i c t e d t o combinations of mutants obtained from f o u r l o c i , l e a s t three specify e s s e n t i a l functions of which a t (Baker e t a l . , 1982; G a t t i e t a l . , 1983). In c o n t r a s t to e a r l i e r studies (Table 2), s y n t h e t i c i n t e r a c t i o n s are not r e p r e s e n t e d i n t h i s work. pattern i n t e r a c t i o n s seen p r e v i o u s l y of n o n l e t h a l lethal However, the closely p a r a l l e l s the d i s t r i b u t i o n of i n t e r a c t i o n s observed here. Thus, s y n e r g i s t i c i n t e r a c t i o n s are most numerous, f o l l o w e d i n t u r n e p i s t a t i c and additive surprising since, interactions. T h i s r e s u l t may not by be on the b a s i s 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 i n t e r a c t i o n s t o be more p r e v a l e n t than e p i s t a t i c ones i f indeed mus mutations are a l i g n e d into discrete r e p a i r pathways. The mus210 the s t r o n g s y n e r g i s t i c i n t e r a c t i o n between mus201 B1 was surprising. C o n v e n t i o n a l l y , synergism i m p l i e s that i n t e r a c t i n g genes encode products of d i f f e r e n t r e p a i r pathways ( e p i s t a s i s groups) which compete f o r the DNA and D1 lesion. However, t h i s i n t e r p r e t a t i o n i s d i f f i c u l t r e c o n c i l e w i t h the mutants. same k i n d i d e n t i c a l phenotypic p r o p e r t i e s of of to the Although the t r i v i a l e x p l a n a t i o n of hypomorphy cannot be d i s c o u n t e d , a p r o v o c a t i v e a l t e r n a t i v e i s t h a t mus201 mus210"*" encode components of a m u l t i m e r i c e x c i s i o n + and repair complex analogous t o the UvrABC e x c i s i o n complex i n E. coli T a b l e 2. A COMPILATION OF PREVIOUSLY REPORTED INTERACTIONS IN DOUBLE MUTANT STRAINS Double 101 101 105 105 mutant 105 mei-41 109 mei-41 A 1 A 1 A 1 A 1 102 mei-9 201 mei-9 205 mei-9 D 1 A 1 epistatic 5 3 6 additive 6 synergistic 4 5 1 4 6 4 1 A 3 A T 1 mei-41 101 mei-9 102 105 102 mei-41 103 mei-9 105 mei-9 mei-9 mei-41 mei-9 mei-41 D 1 A 3 a A 1 A 1 D 1 a A 1 a a D 5 a a k c Reference 6 6 2 6 a a 5 lethal A 3 D 1 A 1 mei-9 Interaction* a 0 F o r some m u t a t i o n s a l l e l i c d e s i g n a t i o n s were n o t p r o v i d e d , I n most c a s e s t h e i n t e r a c t i o n was a s s e s s e d b a s e d on s e n s i t i v i t y t o MMS. (1) B a k e r e t a l . , 1976; (2) B a k e r e t a l . , 1982; (3) D u s e n b e r y e t a l . , 1983; (4) Nguyen e t a l . , 1979; (5) S m i t h , 1978; (6) S m i t h e t a l . , 1980. 71 (Seeberg and Steinum, 1983; al., Yeung e t a l . , 1983; Grossman e t 1986). At p re sent, t h e r e i s no d i r e c t b i o c h e m i c a l evidence t o s u b s t a n t i a t e the e x i s t e n c e o f a m u l t i m e r i c complex f o r n u c l e o t i d e e x c i s i o n r e p a i r i n eukaryotes. I t s existence i s i n f e r r e d l a r g e l y through c i r c u m s t a n t i a l g e n e t i c evidence and from t h e paradigm p r o v i d e d by s t u d i e s i n E. c o l i . F o r example, i n S. c e r e v i s i a e a t l e a s t 5 genes o r c h e s t r a t e 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 o v e r a l l e x c i s i o n r e p a i r process itself. Likewise, the existence o f 9 d i s t i n c t complementation groups among i n d i v i d u a l s with xeroderma pigmentosum suggests t h a t e x c i s i o n r e p a i r i n humans may i n v o l v e a comparable number o f genes and thus perhaps a complex i n t e r a c t i o n o f gene products ( F r i e d b e r g , 1985). If a s i m i l a r l y complex e x c i s i o n r e p a i r mechanism e x i s t s i n D r o s o p h i l a . t h i s may e x p l a i n the apparent anomalous i n t e r a c t i o n between m u s 2 0 1 D1 and mus210 . B1 In support of t h i s notion, P e r o z z i and Prakash (1986) r e c e n t l y r e p o r t e d t h a t two amorphic members o f t h e RAD3 ( e x c i s i o n r e p a i r ) e p i s t a s i s group i n y e a s t interact synergistically. Based on t h e p r e c e d i n g been a s s i g n e d arguments, mus201 D1 and mus210 B1 have t o t h e same pathway, namely t h a t r e s p o n s i b l e f o r excision repair. I f the remaining double mutant i n t e r a c t i o n s are taken a t f a c e v a l u e , then one p o s s i b l e o r g a n i z a t i o n o f 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 11. s u b s t r a t e s S T and S 2 For s i m p l i c i t y , r e p r e s e n t two d i f f e r e n t types o f MMS72 Figure 11. A model o f DNA interactions r e p a i r pathways i n D r o s o p h i l a based between s e c o n d chromosome mus 73 on mutations. 74 induced DNA l e s i o n . substrates, mus210 The e x c i s i o n r e p a i r pathway a c t s upon both i n keeping with i t s major r o l e i n DNA r e p a i r . B1 and mus211 B1 have been assigned t o d i f f e r e n t r e p a i r 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 i n t e r a c t i o n i n mus210 e x c i s i o n r e p a i r pathway and t h a t f o r t h e same DNA l e s i o n , S The notion 2 B1 mus211 . Both t h e B1 i d e n t i f i e d by mus211 compete . t h a t p a r t i c u l a r types o f DNA l e s i o n can be " c h a n n e l l e d " through d i f f e r e n t r e p a i r pathways has important implications f o r mutagenesis. (e.g. RAD6 i n S . c e r e v i s i a e ) e r r o r - p r o n e o r mutagenic others are e r r o r - f r e e C e r t a i n r e p a i r pathways a r e (e.g. RAD3). an e r r o r - f r e e pathway c o u l d This f whereas implies that blocks i n force a greater l e s i o n s i n t o a pathway t h a t i s mutagenic. proportion o f DNA Conversely, b l o c k s i n an e r r o r - p r o n e pathway c o u l d r e s u l t i n more l e s i o n s being c h a n n e l l e d i n t o an e r r o r - f r e e pathway. explanations are tenable, Although o t h e r the c h a n n e l l i n g h y p o t h e s i s accounts f o r t h e o b s e r v a t i o n s t h a t a t l e a s t one rad3 mutant i s a mutator, and t h a t rev3 mutants (rev3 i s a member o f the RAD6 e p i s t a s i s group) a r e a n t i m u t a t o r s mus208 B1 (see von B o r s t e l and H a s t i n g s , 1985). has been assigned t o a unique pathway based on two l i n e s o f evidence. F i r s t , mus208 interact synergistically. mus208 B1 mus211 B1 B1 and mus210 B1 (or mus201 ) Furthermore, t h e double mutant e x h i b i t s an a d d i t i v e i n t e r a c t i o n . r e s u l t i m p l i e s t h a t mus208 D1 + and mus210 + The former c o n t r o l steps i n a l t e r n a t e pathways t h a t compete f o r the same l e s i o n ( S i ) ; the l a t t e r r e s u l t suggests t h a t the pathways i d e n t i f i e d by mus208 and m u s 2 1 1 of DNA B1 operate independently t o c o r r e c t d i f f e r e n t c l a s s e s lesion. mus205 B1 e x h i b i t s e p i s t a s i s with mus201 w e l l as w i t h mus208 . B1 D1 and rous210 . as B1 These r e s u l t s can be e x p l a i n e d i f d i s p a r a t e r e p a i r pathways share, a t some p o i n t , a common s t e p . In F i g u r e 11 t h i s i s r e p r e s e n t e d by the convergence o f the pathways near t h e i r t e r m i n i . the o b s e r v a t i o n t h a t mus205 This hypothesis i s c o n s i s t e n t with A1 i s d e f i c i e n t i n two r e p a i r , e x c i s i o n and p o s t r e p l i c a t i o n Boyd and Shaw, 1982). k i n d s of (Boyd and H a r r i s , 1981; However, t h i s key p o s i t i o n occupied by mus205 seems somewhat a t odds w i t h i t s l i m i t e d range of mutagen cross-sensitivity mus205 + (see Table 1 i n Chapter 1). Apparently, i s r e q u i r e d o n l y t o r e p a i r damage caused monofunctional radiation. by a l k y l a t i n g agents and l e s i o n s r e s u l t i n g from Damage a r i s i n g from bulky adduct-forming l i k e N-acetyl-2-aminofluorene, UV chemicals 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 n i t r o g e n mustard, must be r e p a i r e d by mechanisms t h a t bypass the requirement new f o r mus205 . + Obviously, pathways, i n a d d i t i o n t o those a l r e a d y d e p i c t e d i n F i g u r e 11, are necessary t o accommodate these o b s e r v a t i o n s . A f u r t h e r c o m p l i c a t i o n stems from the o b s e r v a t i o n t h a t the t r i p l e mutant, mus208 t o MMS B1 mus210 B1 mus211 . i s no more s e n s i t i v e B2 than the most s e n s i t i v e double mutant, mus208 However, s i n c e d i f f e r e n t mus21l a l l e l e s were used m u l t i p l e mutants, i n t e r s t r a i n comparisons may 76 B1 mus210 . B1 i n making the not be v a l i d even though mus211 sensitivities. B1 and mus211 B2 e x h i b i t v e r y s i m i l a r mutagen S t r a i n v a r i a b i l i t y and a l l e l i c d i f f e r e n c e s major sources o f anomalous i n t e r a c t i o n s i n S. c e r e v i s i a e and Cox, 1972; 1973). constructing were (Game T h i s p o s s i b i l i t y can be t e s t e d by the a p p r o p r i a t e m u s 2 1 1 - c o n t a i n i n g t r i p l e mutant B1 strain. In the absence o f b i o c h e m i c a l data concerning the r o l e o f these mus gene p r o d u c t s i n DNA r e p a i r , the c o n t r i b u t i o n s a d d i t i o n a l pathway a n a l y s e s are somewhat l i m i t e d . of Ultimately, model o f DNA r e p a i r i n D r o s o p h i l a t h a t more a c c u r a t e l y a reflects the t r u e r e p a i r response w i l l come from a combination o f genetic, b i o c h e m i c a l , and molecular b i o l o g i c a l of mus and o t h e r r e p a i r - r e l a t e d mutations. 77 characterizations CHAPTER MUTAGEN-SENSITIVE THREE STRAINS AS GENOTOXICITY A PILOT STUDY 78 INDICATORS: INTRODUCTION A r e c o g n i t i o n 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, e s p e c i a l l y i n r e l a t i o n t o human c a r c i n o g e n e s i s and h e r i t a b l e and somatic d i s e a s e (e.g., Ames, 1979, 1983; Hartman, 1983), t o g e t h e r w i t h the knowledge t h a t as many as 80% o f a l l cancers are caused by f a c t o r s i n our environment ( D o l l and Peto, 1981), has i n r e c e n t years made the t a s k o f i d e n t i f y i n g environmental priority. Because both l i f e s t y l e and o c c u p a t i o n appear t o be important determinants Peto, genotoxins a p u b l i c h e a l t h of the c a r c i n o g e n i c p r o c e s s 1981), r e c e n t e f f o r t s have c o n c e n t r a t e d on (Doll and identifying mutagens/carcinogens among the chemical c o n s t i t u e n t s found i n foods and i n d r i n k s (e.g., chemicals d e l i b e r a t e l y added, n a t u r a l l y o c c u r r i n g or r e s u l t i n g from p r e p a r a t i o n ) , i n non-food products (e.g., cosmetics, m e d i c i n a l s , p e s t i c i d e s ) , and work environment (Nagao e t a l . , i n the 1978). C u r r e n t e s t i m a t e s p l a c e the number of chemicals i n commercial use a t about 70,000, w i t h 700 t o 3000 new i n t r o d u c e d each year (Hoffmann, 1982). ones being However, e p i d e m i o l o g i c a l s t u d i e s have i d e n t i f i e d o n l y 22 chemicals, i n d u s t r i e s , or i n d u s t r i a l p r o c e s s e s f o r which t h e r e i s s u f f i c i e n t evidence t o support a c a u s a l a s s o c i a t i o n w i t h cancer occurrence i n (Bartsch et a l . , 1982). man Another 18 chemicals are h i g h l y suspect as human c a r c i n o g e n s based on data d e r i v e d from both e p i d e m i o l o g i c a l and animal s t u d i e s (Bartsch e t a l . , 1982). All t o l d , the human c a r c i n o g e n data base i s d i s t r e s s i n g l y s m a l l . For reasons o f p r a c t i c a l i t y , c a r c i n o g e n t e s t i n g i s n e c e s s a r i l y c a r r i e d out i n r a t s and mice (and v e r y r a r e l y i n o t h e r mammalian s p e c i e s ) . Yet even so, t o t e s t j u s t a s i n g l e chemical i n a rodent b i o a s s a y may take 2-4 years and c o s t as much as 1 m i l l i o n d o l l a r s (Lave and Omenn, 1986). p r e c l u d e t h e use o f animals These f a c t o r s i n any mass s c r e e n i n g programs f o r c a r c i n o g e n i c agents, and have spurred t h e development o f more than 100 s h o r t - t e r m b i o a s s a y s o f g e n o t o x i c a c t i v i t y (usually mutagenicity) u s i n g a v a r i e t y o f p r o k a r y o t i c and e u k a r y o t i c materials (e.g., H o l l s t e i n e t a l . , 1979; de S e r r e s and Ashby, 1981) . The Salmonella/microsome m u t a g e n i c i t y assay (i.e., t h e Ames t e s t ) i s t h e most w i d e l y used and t h e most e x t e n s i v e l y v a l i d a t e d short-term t e s t (e.g., Haroun and Ames, 1981). However, t h e Ames t e s t by i t s e l f does not appear t o be a r e l i a b l e enough p r e d i c t o r o f e i t h e r rodent o r human c a r c i n o g e n s 1983) o r o f mammalian germ c e l l mutagens (Bridges and Mendelsohn, 1986). environmental of (e.g., B r u s i c k , Thus, a p a r t i c u l a r t h r u s t o f c u r r e n t mutagenesis r e s e a r c h i s t o i d e n t i f y a s m a l l number e u k a r y o t i c t e s t s t h a t can complement t h e a l r e a d y Ames t e s t (Ashby e t a l . , 1985). In p r i n c p l e , entrenched these complementary assays would be capable o f d e t e c t i n g rodent mutagens and c a r c i n o g e n s t h a t a r e d i f f i c u l t or impossible t o d e t e c t as p o s i t i v e i n t h e Ames t e s t . D r o s o p h i l a has been f o r y e a r s , and c o n t i n u e s t o be, an important organism f o r use i n g e n e t i c t o x i c o l o g y . 80 I t s numerous advantages have been w e l l d e s c r i b e d Baars, 1984; 1980; Vogel, Graf W u r g l e r e t a l . , 1984, observation mus 1981; strains reported may possible to are extremely genotoxic two tester strains. of the proposed obtained assay identify Sobels, Valencia et a l . , W u r g l e r and Vogel, that certain may 1976; b a s i s of f o r 16 Nguyen e t a l . , 1 9 7 9 ) . triple mutant mus This chapter mutations are reported. 81 pursue a preliminary somatic g e n o t o x i c i t y assay. chemicals To l i n e s were c o n s t r u c t e d provides a In p r i n c i p l e , i t c o m b i n a t i o n s o f mus (e.g., The multiply-mutant form the i n Drosophila. 1986). s e n s i t i v e t o a wide spectrum o f d i f f e r e n t l y agents possibility, 1985; a r e h y p e r s e n s i t i v e t o MMS, somatic genotoxity and e t a l . , 1984; i n C h a p t e r 2, rapid, be (Vogel The that acting this as evaluation results MATERIALS AND METHODS Strains For a d e s c r i p t i o n o f t h e 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 t r i p l e mus mutant s t r a i n s , mus208 mus210 B1 and B1 b p_r cn mus208 b p_r cn mus205 B1 mus210 were c o n s t r u c t e d as d e s c r i b e d i n Chapter 2. mus208 . mus210 B1 B1 and mus211 B2 B1 mus211 . B1 B2 The mutations were s e l e c t e d as c o n s t i t u e n t s o f one t e s t e r s t r a i n because o f t h e i r unique mutagen c r o s s s e n s i t i v i t y and demonstrated p a i r w i s e i n t e r a c t i o n s . This combination o f mutations i s p o t e n t i a l l y extremely s e n s i t i v e t o a wide spectrum o f d i f f e r e n t l y a c t i n g mutagens. s t r a i n i n c o r p o r a t e d mus205 mus205 B1 B1 The second i n p l a c e o f mus211 . tester Although B2 d i d not i n t e r a c t o t h e r than e p i s t a t i c a l l y w i t h any o t h e r mus mutation (see Chapter 2 ) , p r e v i o u s u n p u b l i s h e d o b s e r v a t i o n s showed t h a t i t was h i g h l y s e n s i t i v e t o a number o f monofunctional a l k y l a t i n g agents. mus210 B1 Thus, mus205 B1 mus208 B1 may be a p a r t i c u l a r l y good i n d i c a t o r o f t h i s important mutagen c l a s s . C u l t u r e s were maintained 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). F l i e s were r a i s e d a t 22°C o r 25°C. Chemicals Benzo(a)pyrene (B(a)P, 50-32-8), 97-2), cyclophosphamide (DEB, (CP, 6055-19-2), 298-18-0), d i e t h y l n i t r o s a m i n e dimethylnitrosamine benzo(e)pyrene (DMN, 62-75-9), 82 (B(e)P, 192- 1,2,3,4-diepoxybutane (DEN, 55-18-5), hexamethylphosphoramide (HMPA, 680-31-9), methyl methanesulfonate methyl-N'-nitro-N-nitrosoguanidine methylnitrosourea 105-60-2), d i e t h y l s u l f a t e (DES, 77-78-1) were purchased E t h y l methanesulfonate Kodak. (MNNG, 70-25-7), (MNU, 684-93-5) and s a f r o l e were o b t a i n e d from Sigma Chemical (DMS, (MMS, 66-27-3), N- (EMS, Company. (SAF, 94-59-7) Caprolactam 64-67-5) and dimethyl from A l d r i c h Chemical (CAP, sulfate Company. 62-50-0) was o b t a i n e d from Eastman Formaldehyde (FA, 50-00-00), as f o r m a l i n , was obtained from F i s h e r S c i e n t i f i c . C r i t e r i a f o r Chemical selection 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 s e l e c t e d as a s e r i e s o f a l k y l a t i n g agents each having a d i f f e r e n t propensity t o enter into unimolecular bimolecular agents (S 2) N r e a c t i o n s (Hoffmann, 1980). (S^l) or S 1 alkylating N (e.g., MNU) are comparatively more e f f e c t i v e a t a l k y l a t i n g oxygens i n n u c l e i c a c i d s , and consequently are b e t t e r mutagens than S 2 a l k y l a t i n g agents N (e.g., MMS, DMS). On the o t h e r hand, Sjj2 a l k y l a t i n g agents r e a c t w i t h h i g h l y n u c l e o p h i l i c sites (e.g., r i n g n i t r o g e n s ) and are much more e f f e c t i v e a t p r o d u c i n g chromosomal a b e r r a t i o n s than t h e i r S 1 c o u n t e r p a r t s N (see Vogel and Natarajan, 1979). Chemicals such as EMS and DES r e a c t both u n i m o l e c u l a r l y and b i m o l e c u l a r l y (Hoffmannn, 1980) . MNU, MNNG, DMN and DEN belong t o the N - n i t r o s o chemical class. N - n i t r o s o compounds are w i d e l y o c c u r r i n g (e.g., i n tobacco smoke, n i t r i t e - p r e s e r v e d f i s h p r o d u c t s , and c e r t a i n 83 cosmetics) potent c a r c i n o g e n s (Scanian, 1984; B a r t s c h and Montesano, 1984; and DEN Preussmann, 1984). Both DMN are promutagens/procarcinogens. The s t r u c t u r a l isomers B(a)P and B(e)P were chosen f o r study as r e p r e s e n t a t i v e s of the u n s u b s t i t u t e d p o l y c y c l i c aromatic hydrocarbons. Both compounds occur u b i q u i t o u s l y i n p r o d u c t s o f incomplete combustion g a s o l i n e and d i e s e l exhaust, (e.g., c i g a r e t t e smoke, b r o i l e d or smoked foods, r o a s t e d c o f f e e e t c . ) , as w e l l as i n unburned f o s s i l f u e l s , i n vegetables and i n v e g e t a b l e o i l s e t c . (IARC, 1983); i . e . , they are major environmental p o l l u t a n t s t o which i t i s v i r t u a l l y i m p o s s i b l e t o avoid exposure. B(a)P (a bulky adduct-forming i s a c t i v e i n short-term t e s t s promutagen/procarcinogen) ( i t i s o f t e n used as a p o s i t i v e c o n t r o l ) and i s c a r c i n o g e n i c t o experimental animals 1983). B(a)P may (IARC, be a c a u s a t i v e f a c t o r i n s e v e r a l human cancers (e.g., s k i n , lung and c o l o n ) . reviewed by P h i l l i p s Many aspects of B(a)P have been (1983). There i s l i m i t e d evidence t h a t B(e)P i s a c t i v e i n s h o r t term t e s t s ( i t i s , however, mutagenic i n the Ames t e s t ) , and t h e r e i s i n s u f f i c i e n t data t o a l l o w an e v a l u a t i o n of the c a r c i n o g e n i c i t y of B(e)P — i n some s t u d i e s i t i s c o n s i d e r e d a n o n c a r c i n o g e n i c analog of B(a)P DEB (IARC, 1983). i s a potent genotoxin i n a v a r i e t y o f organisms (reviewed by Ehrenberg and Hussain, 1981). agent than can produce both mono- and b i f u n c t i o n a l 84 I t i s an alkylating alkylation products. links. The l a t t e r adducts a r e i n t e r - and i n t r a s t r a n d I n D r o s o p h i l a . g e n e t i c evidence suggests t h a t DEB may a c t by p r o d u c i n g d e l e t i o n s Green, cross- (Shukla and Auerbach, 1980; Olsen and 1982). CP, a human c a r c i n o g e n (Bartsch e t a l . , 1982), i s a w i d e l y used chemotherapeutic a l k y l a t i n g agent which, l i k e DEB, can e n t e r i n t o 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 reactions. However, f o r t h e l a t t e r r e a c t i o n s t o occur, CP must f i r s t be metabolically activated (Brendel and Ruhland, FA i s both mutagenic and c a r c i n o g e n i c Casanova-Schmitz, 1984). unclear, although (d'A. Heck and The b a s i s f o r FA g e n o t o x i c i t y i s FA i s known t o r e a c t r e v e r s i b l y w i t h amino groups o f n u c l e i c a c i d s and t o form c r o s s l i n k s Kusmierek, 1984). (Singer and 1982). HMPA and SAF were s e l e c t e d as c h e m i c a l s t h a t a r e i n a c t i v e o r d i f f i c u l t t o d e t e c t i n the Ames assay (de S e r r e s and Ashby, 1981). The mechanism by which HMPA e x e r t s i t s g e n o t o x i c e f f e c t s i s unknown. agent HMPA does n o t appear t o be a simple m e t h y l a t i n g (Vogel e t a l . , plant origin. 1985). SAF i s a n a t u r a l c a r c i n o g e n o f I t i s a major c o n s t i t u e n t (85%) o f o i l o f s a s s a f r a s , and i s found i n minor o r t r a c e q u a n t i t i e s i n cocoa, mace, nutmeg, b l a c k pepper and a number o f o t h e r p l a n t products ( H a l l , 1973). The u l t i m a t e r e a c t i v e p r o d u c t o f SAF appears t o be s a f r o l e - 1 ' - s u l f a t e , which r e a c t s w i t h 0 - g u a n i n e i n DNA 6 (Hathway, 1986). additional S i n g e r and Kusmierek reactions. 85 (1982) have r e p o r t e d CAP i s a chemical which shows no evidence o f c a r c i n o g e n icity i n long-term 1984). rodent b i o a s s a y s (Shelby and S t a s i e w i c z , I t i s used here as a n e g a t i v e c o n t r o l . Solvents A p p r o p r i a t e amounts o f B(a)P and B(e)P were f i r s t dissolved i n a s m a l l volume (< 1 mL) o f dimethyl s u l f o x i d e and d i l u t e d accordingly with ethanol. MNNG was s i m i l a r l y d i s s o l v e d i n DMSO but d i l u t e d w i t h 70% e t h a n o l . DMS, DES and MNU were d i s s o l v e d i n e t h a n o l . SAF was d i s s o l v e d i n 70% e t h a n o l . for a l l other chemicals. Water was used The v a r i o u s s o l v e n t s d i d not a l t e r t h e r e l a t i v e v i a b l i t y o f t h e genotypes. Mutagen S e n s i t i v i t y T e s t P r o t o c o l The h principal ET. c n mus208 strain, B1 tester mus210 b p_r cn mus205 B1 strain B1 mus211 . i n this A B2 mus208 o n l y a few s e l e c t a l k y l a t i n g used study was second mus210 . was used t o t e s t B1 B1 agents. The chemical t e s t i n g p r o t o c o l used here i s e s s e n t i a l l y t h e same as t h a t used i n Chapter 2 t o d e t e c t mus mutant i n t e r a c t i o n s . Yeast f e d mus / SM5 females (where "mus" i n d i c a t e s the b fjr c n - marked t r i p l e mus chromosome) were mated i n b o t t l e s t o homozygous mus males. S e v e r a l days l a t e r a d u l t s were p l a c e d i n t o f r e s h v i a l s to l a y eggs. these (5 p a i r s p e r v i a l ) and l e f t A f t e r 24 h r the parents were t r a n s f e r r e d t o new v i a l s and t h e o r i g i n a l v i a l c u l t u r e s (eggs) were t r e a t e d with 86 0.25 mL o f an general, l i t e r a t u r e v a l u e s were u s e d levels). f o r up adult appropriate concentration of a t e s t The same p a r e n t s were t r a n s f e r r e d t o 5 days t o e s t a b l i s h p r o g e n y were c o u n t e d treatment (FA d e l a y e d and / Cy_ h e t e r o z y g o t e s . survival in value obtained Chapter Principle new classified o f mus vials treatment. weeks o f 2 weeks). homozygotes t o T h e s e v a l u e s were n o r m a l i z e d using the f o r the u n t r e a t e d c o n t r o l s (see T a b l e 1 2). of the Method homozygotes r e l a t i v e rous/Cy genotoxic sensitivity of to phenotypically distinguishable, (mus"*") h e t e r o z y g o t e s . s e r v e as a r e l a t i v e l y t h i s way, dose to fresh w i t h i n 2-3 T h i s system measures the developmental proficient daily a s much a s as a r a t i o (In in selecting cultures for d e v e l o p m e n t by S u r v i v a l values are presented mus as g u i d e s chemical. mutagen-insensitive doses can are s y s t e m i c a l l y t o x i c , and be The repair- heterozygotes internal d i s t i n g u i s h e d from control. those t h e r e f o r e not n e c e s s a r i l y 87 mus In that DNA-damaging. RESULTS The r e s u l t s of t r e a t i n g varying concentrations DES) the genotoxic apparent. rous210 B1 of 8 d i f f e r e n t simple compounds are presented and rous208 i n Table 1. mus211 B1 alkylating In a l l but two cases e f f e c t s of these compounds are For example, MMS to B2 (DMS readily k i l l e d v i r t u a l l y a l l homozygous f l i e s a t a c o n c e n t r a t i o n of 2.5 mM. methylating produced very s i m i l a r l e v e l s of agents (except DMS) l e t h a l i t y at t h i s concentration. c o n c e n t r a t i o n of EMS was Indeed, a l l o f the By c o n t r a s t , a 30 mM r e q u i r e d t o e f f e c t n e a r l y complete developmental l e t h a l i t y of the mus homozygotes. In t h i s r e s p e c t , a q u a l i t a t i v e l y s i m i l a r t r e n d i s a l s o e v i d e n t f o r the d i a l k y l n i t r o s a m i n e s , DMN and DEN; i . e . , the mus homozygotes are s u b s t a n t i a l l y more s e n s i t i v e (by a t l e a s t a f a c t o r of 4) t o than t o DEN a t equimolar c o n c e n t r a t i o n s . explanations are t e n a b l e , these Although other l a t t e r f i n d i n g s may f a c t t h a t , on average, m e t h y l a t i n g DMN reflect the agents are about 20 times more r e a c t i v e than t h e i r e t h y l a t i n g homologs (Singer and Kusmierek, 1982). N e i t h e r of the d i a l k y l s u l f a t e s DMS genotoxic t o mus208 B1 mus210 B1 mus211 B2 and DES i s obviously d e s p i t e a wealth of i n f o r m a t i o n from other t e s t systems t o the c o n t r a r y Hoffmann, 1980). However, upon c l o s e r i n s p e c t i o n , DMS t o be weakly genotoxic of 30 mM. i s 0.66 The (reviewed a t the comparatively unadjusted high appears concentration r e l a t i v e s u r v i v a l v a l u e a t t h i s dose (432/659), a v a l u e too low t o be due 88 simply to by Table 1. S E N S I T I V I T Y OF mus2 0 8 ALKYLATING AGENTS Chemical B 1 Concentration (mM) mus210 B 1 mus211 B 2 TO SIMPLE Normalized r e l a t i v e survival 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 t h e homozygoterheterozygote r a t i o from t h e t r e a t e d c u l t u r e s d i v i d e d by t h e homozygoterheterozygote r a t i o from t h e u n t r e a t e d c o n t r o l cultures. T h e l a t t e r v a l u e i s 0.88. T h e numbers i n p a r e n t h e s e s i n d i c a t e t h e number o f f l i e s s c o r e d p e r concentration. 89 variability. (The r e l a t i v e s u r v i v a l v a l u e s from u n t r e a t e d c o n t r o l r e p e a t s range between 0.76 dose-response and 0.96, a v e r a g i n g 0.88). A r e l a t i o n s h i p may w e l l be demonstrable f o r DMS u s i n g c o n c e n t r a t i o n s above 3 0 mM. The response mus210 . t o MMS, B1 o f the second t e s t e r s t r a i n , mus205 rous208 B1 B1 EMS, DMN and DEN i s presented i n T a b l e 2. can be seen t h a t mus205 B1 mus208 B1 mus210 B1 i s extremely s e n s i t i v e t o a l l f o u r chemicals, even more so than mus208 mus210 B1 mus211 the presence to B2 is. o f mus205 It B1 This hypersensitivity i s attributed to B1 which, by i t s e l f , i s h i g h l y s e n s i t i v e a l l f o u r o f these monofunctional alkylating agents (unpublished o b s e r v a t i o n s ) . The remaining musJ210 B1 mus211 B2 g i v e n i n T a b l e 3, 8 chemicals were t e s t e d u s i n g mus208 only. B1 The r e s u l t s o f these experiments are and they a r e r e l a t i v e l y s t r a i g h t f o r w a r d . The r e s u l t s o b t a i n e d f o r FA i n d i c a t e t h a t i t i s weakly genotoxic ( i . e . , comparatively h i g h c o n c e n t r a t i o n s o f FA a r e r e q u i r e d t o e l i c i t a d e t e c t a b l e response). Similar conclusions about FA were reached by o t h e r s u s i n g t h e Ames assay al., 1983) al., 1983). (Connor e t and two m u t a g e n i c i t y t e s t s i n D r o s o p h i l a (Szabad e t DEB k i l l e d a l l mus208 B1 mus210 B1 mus211 B2 a p p l i e d a t c o n c e n t r a t i o n s o f 2.5 mM and above. f a i l e d t o e f f e c t a response. homozygotes when A 1.0 mM dose These r e s u l t s compare f a v o r a b l y w i t h t h e l e v e l s o f DEB-induced l a r v a l k i l l i n g observed number o f o t h e r D r o s o p h i l a mus s t r a i n s 90 for a (Olsen and Green, 1982). T a b l e 2. SENSITIVITY ALKYLATING AGENTS Chemical OF m u s 2 0 5 B 1 Concentration (mM) mus208 B 1 mus210 B 1 TO SIMPLE Normalized r e l a t i v e survival 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 t h e t r e a t e d c u l t u r e s d i v i d e d by t h e homozygoterheterozygote r a t i o from t h e u n t r e a t e d c o n t r o l cultures. T h e l a t t e r v a l u e i s 0.89. T h e numbers i n p a r e n t h e s e s i n d i c a t e t h e t o t a l number o f f l i e s s c o r e d p e r concentration. 91 Table 3. S E N S I T I V I T Y OF m u s 2 0 8 MISCELLANEOUS CHEMICALS Chemical B 1 Concentration (mM) 3 mus210 B 1 mus211 B 2 TO Normalized r e l a t i v e survival* 3 FA 2 4 6 0.73 0.31 0.05 (1541) (1243) (1180) CP 2.5 0.00 (1464) DEB 1.0 2.5 5.0 1.01 0.00 0.00 (765) (1419) (988) B(a)P 0.05 0. 10 0.42 0.02 (1933) (1568) B(e)P 0.10 1.01 (2548) 0. 35 <0.01 (981) (1056) HMPA 5.0 10.0 SAF 2.5 5.0 10.0 1.01 (937) 0.93 (663) toxic CAP 10.0 20.0 30.0 0.91 1.05 1.09 FA c o n c e n t r a t i o n i s (2185) (897) (1058) e x p r e s s e d a s % v / v f o r m a l i n :H 0 2 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 t h e t r e a t e d c u l t u r e s d i v i d e d by t h e homozygoterheterozygote r a t i o from t h e u n t r e a t e d c o n t r o l cultures. The l a t t e r v a l u e i s 0.88. The numbers i n p a r e n t h e s e s i n d i c a t e t h e t o t a l numbers o f f l i e s s c o r e d p e r concentration. 92 The p r o c a r c i n o g e n comparatively B(a)P i s decidedly genotoxic low doses o f 0.05 and 0.10 mM. a t the Previous i n v i t r o s t u d i e s have demonstrated t h a t D r o s o p h i l a microsomal p r e p a r a t i o n s a r e capable o f m e t a b o l i z i n g a v a r i e t y o f procarcinogens, i n c l u d i n g B(a)P (Baars e t a l . , 1977; Hallstrom and Grafstrom, 1981), t o t h e i r r e a c t i v e forms. r e p o r t e d here, and those o f Boyd e t a l . (1976a) and Nguyen e t al. (1979), suggest t h a t l a r v a l somatic s i m i l a r a c t i v a t i o n o f B(a)P genotoxic e f f e c t o f B(a)P, in vivo. The f i n d i n g s t i s s u e s can e f f e c t a In c o n t r a s t t o t h e s t r o n g B(e)P was n o t g e n o t o x i c a t t h e c o n c e n t r a t i o n used. HMPA and SAF a r e d i f f i c u l t o r i m p o s s i b l e t o d e t e c t as mutagens i n the Ames t e s t (see de S e r r e s and Ashby, 1981). these, o n l y HMPA i s genotoxic t o mus208 B1 mus210 B1 Of mus211 . B2 While SAF f a i l e d t o e f f e c t a response a t e i t h e r 2.5 o r 5.0 mM, the 10 mM c o n c e n t r a t i o n k i l l e d a l l d e v e l o p i n g o f t h e i r genotype. flies irrespective T h i s l a t t e r r e s u l t i s t h e o n l y example o f a t o x i c r e a c t i o n i n t h i s study. However, i t p o i n t s out t h e importance o f t h e i n t e r n a l c o n t r o l ( i . e . , t h e heterozygous flies) i n t h i s t e s t system. The non-carcinogen l e t h a l i t y i n mus208 B1 CAP d i d not e f f e c t any d e t e c t a b l e mus210 B1 mus211 . B2 CAP t e s t e d as m a r g i n a l l y p o s i t i v e i n the somatic mutation and recombination t e s t i n Drosophila (Wurgler e t a l . , 1985). 93 DISCUSSION To date, mus s t r a i n s have been used mainly t o e l u c i d a t e c e l l u l a r mechanisms of DNA mutagenesis. r e p a i r , recombination and T h e i r a p p l i c a t i o n t o chemical g e n o t o x i c i t y testing has p r i n c i p a l l y r e s i d e d i n e f f o r t s t o 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 r e c e n t l y - d e v e l o p e d m u t a g e n i c i t y assays i n Drosophila (e.g., Zimmering, 1982, 1983; Vogel e t a l . , Fujikawa e t a l . , t h a t mus 1985; Wurgler e t a l . , 1985). The 1983; possibility s t r a i n s themselves might serve d i r e c t l y as g e n o t o x i c i t y i n d i c a t o r s has remained v i r t u a l l y unexplored s i n c e Nguyen e t a l . (1979) f i r s t demonstrated X - l i n k e d mus t h i s idea. the p o t e n t i a l of t h i s approach u s i n g and mei mutations. The p r e s e n t study f o l l o w s up However, the t e s t p r o t o c o l employed here from t h a t o f Nguyen e t a l . (1979) i n t h a t mus on differs mutations o f the second chromosome are used. The mus inexpensive. efficiently. t e s t d e s c r i b e d here i s f a s t , easy t o do, and Thus, a l a r g e number of chemicals can be screened Moreover, a c o n s i d e r a b l e advantage of t h i s over comparable d i f f e r e n t i a l k i l l i n g assays t h a t u t i l i z e d e f i c i e n t mammalian c e l l s (e.g., Hoy p h e n o t y p i c a l l y d i f f e r e n t mus et a l . , test repair- 1984), i s t h a t mutations can be combined r e l a t i v e l y e a s i l y i n t o s i n g l e t e s t e r 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 d e t e c t i o n capability. A t o t a l of 16 chemicals were t e s t e d i n t h i s p i l o t Of the 14 c h e m i c a l s g e n e r a l l y c o n s i d e r e d g e n o t o x i c ( a l l B ( e ) P and CAP), 11 t e s t e d as p o s i t i v e . 94 study. but While t h i s l e v e l of concordance i s r e s p e c t a b l e , i t s s i g n i f i c a n c e i s somewhat tempered by t h e f a c t t h a t a t l e a s t 10 o f t h e 14 genotoxins a r e a l k y l a t i n g agents. R e c a l l t h a t the mutations mus208 . mus210 . and mus211 B1 B1 t h e i r MMS-sensitive B2 mus205 . B1 were o r i g i n a l l y i s o l a t e d f o r p r o p e r t i e s (Henderson e t a l . , 1987). (This i s not meant t o imply t h a t a l l a l k y l a t i n g agents r e a c t i d e n t i c a l l y w i t h DNA (they do not) o r a r e u n i f o r m l y g e n o t o x i c (they a r e not).) These c o n s i d e r a t i o n s a s i d e , i t i s the f a l s e n e g a t i v e results (DMS, DES and SAF), more so than t h e p o s i t i v e which r a i s e a number o f important results, i s s u e s concerning t h e use of mus s t r a i n s f o r mutagen t e s t i n g . The reason(s) why DMS and DES t e s t e d as n e g a t i v e , even a t c o m p a r a t i v e l y h i g h doses, i s not c l e a r . be weakly g e n o t o x i c a t 30 mM, (Although DMS appears t o t h i s assessment has t o be c o n s i d e r e d t e n t a t i v e u n t i l r e t e s t s can be c a r r i e d out. Thus, f o r t h e sake o f d i s c u s s i o n t h e DMS r e s u l t s a r e c o n s i d e r e d t o be negative.) Both chemicals are r e a d i l y a c t i v e i n numerous t e s t systems, i n c l u d i n g a p p a r e n t l y the 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 i n D r o s o p h i l a (see Hoffmann, 1980). o f chemical d e l i v e r y . The problem may be one Both DMS and DES a r e r a p i d l y h y d r o l y z e d i n water (Hoffmann, 1980); the same may be t r u e i n e t h a n o l , the s o l v e n t employed here. T h i s problem i l l u s t r a t e s t h e need t o ensure both c h e m i c a l - s o l v e n t and solvent-organism however, t h i s i s not always easy t o a c h i e v e . compatibility; F o r example, whereas water would normally be the s o l v e n t o f c h o i c e , not a l l 95 chemicals a r e s o l u b l e o r s t a b l e i n water. o t h e r s o l v e n t s such most c h e m i c a l s , (unpublished 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 flies c o n c e n t r a t i o n s i t may t h e m e t a b o l i s m o f x e n o b i o t i c s (Magnusson e t a l . , Although unique t o t h i s find whereas s u l f o x i d e (DMSO) c a n d i s s o l v e o b s e r v a t i o n s ) , and a t lower i n t e r f e r e with 1979). as dimethyl Moreover, t h e problem o f s o l v e n t s u i t a b i l i t y D r o s o p h i l a assay, alternative chemical efforts will i s not h a v e t o be made t o delivery vehicles applicable to this test. A second results one conclude I n t h e case necessarily this that a chemical may n o t seem a v e r y The test i n wildtype f o r chemical than (b p_r cn) f l i e s and t h e n t o concentrations accordingly. While this i t will, n e v e r t h e l e s s , h a v e t o be unknowns. 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 being unable Ashby, 1981). negative Doses h i g h e r s a t i s f a c t o r y method i n what i s s u p p o s e d t o system, mus t e s t i n t h e mus F o r DMS a n d DES i t may be n e c e s s a r y t o a d j u s t t h e maximal t e s t employed i s not genotoxic somewhere between 5 a n d 10 mM. are cytotoxic. a rapid i . e . , a t what d o s e d o e s o f SAF, f o r example, t h e u p p e r d o s e l i m i t i s determine LD50 v a l u e s be p o i n t t o be drawn f r o m t h e DMS/DES i s the question of negativity, finally test? important t o d e t e c t as p o s i t i v e There a r e a t l e a s t response. carcinogenic effect F o r example, SAF (de S e r r e s a n d two p o s s i b l e r e a s o n s i f indeed SAF e x e r t s i t s by damaging DNA, t h e l e s i o n s may b e r e p a i r e d b y p a t h w a y s d i f f e r e n t 96 for this from t h o s e i t generates blocked by t h e t h r e e mus mutations. An a l t e r n a t i v e e x p l a n a t i o n be m e t a b o l i z e d d i f f e r e n t l y i n D r o s o p h i l a and Thus, f l i e s may mammals may convert SAF i.e., i n the mus To metabolites genetic (e.g., 1'- assay. mechanisms l e a d i n g t o c e l l k i l l i n g t h e r e i s no d e f i n a b l e cells. distinguish major t h e o r e t i c a l disadvantage of t h i s mus the u n d e r l y i n g may t o nongenotoxic compounds, whereas p o s s i b l i t i e s , known SAF OH-safrole) c o u l d be t e s t e d The mammalian produce genotoxic i n t e r m e d i a t e s . between these two i s t h a t SAF endpoint. t e s t i s that are unknown, While t h i s i s a drawback i n terms of b i o l o g i c a l i n q u i r y , i t does not necessarily l e s s e n the v a l u e o f the t e s t as a r a p i d g e n o t o x i c i t y screen. The working h y p o t h e s i s i s t h a t the mus homozygotes d i e f a i l e d attempts t o r e p a i r l e s i o n s i n DNA. will The following types of lesions of course v a r y with the chemical, the dose, the c e l l e t c . , but DNA s t r a n d breaks, i n p a r t i c u l a r , are l i k e l y prominent c e l l k i l l i n g P i m p i n e l l i e t a l . , 1977; s t r a n d breaks c o u l d lesions ( G a t t i e t a l . , 1974, Levina and adducts. a r r e s t l e a d i n g t o o v e r r e p l i c a t i o n of DNA Such clastogenic a c t i o n of the mutagen i t s e l f , or i n d i r e c t l y , e.g., r e p l i c a t i o n a r r e s t a t bulky DNA be 1975; Sharygin, 1984). a r i s e e i t h e r d i r e c t l y by the to type following (Transient r e p l i c a t i o n may i t s e l f be an important mechanism f o r g e n e r a t i n g a v a r i e t y of chromosomal aberrations e t i o l o g y , DNA (Schimke e t a l . , 1986).) strand Regardless of t h e i r d i s c o n t i n u i t i e s are h i g h l y recombinogenic, thus opening up the p o s s i b i l i t y f o r a d d i t i o n a l genomic alterations (e.g., d e l e t i o n s , inversions, 97 translocations, aneuploidy). events, al., I t must be not merely 1986; point mutations G u e r r e r o and t o cancer development . emphasized Barrett, 1986) process, s h o u l d we, Pellicer, that v i r t u a l l y i n oncogenes 1987), may (e.g., Radman e t a l . , In view of our f o r example, be a l l of these (e.g., Z a r b l factors 1982; et leading Oshimura and ignorance of the carcinogenic limit t o those t h a t monitor point mutations our s h o r t - t e r m t e s t s only o r a n e u p l o i d y as g e n e t i c endpoints? D r o s o p h i l a mus mutations the realm of short-term t e s t s whether they w i l l other tests be c o n t i n u e t o have a r o l e i n f o r environmental employed d i r e c t l y (e.g., Wurgler further validation will and of the t e s t Graf, But o r s i m p l y as a d j u n c t s t o 1985) will described here. 98 genotoxins. have t o await CHAPTER FOUR A GENETIC AND DEVELOPMENTAL ANALYSIS O F mus209 99 INTRODUCTION Mutations t h a t render c e l l s h y p e r s e n s i t i v e t o DNA-damaging agents o f t e n c o n f e r a d d i t i o n a l mutant phenotypes. humans a f f l i c t e d w i t h any one of a number of i n h e r i t e d d i s e a s e s a s s o c i a t e d w i t h d e f e c t s i n DNA xeroderma pigmentosum (XP), For example, r e p a i r or r e p l i c a t i o n (e.g., a t a x i a 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 t o malignancy 1978; Less easy t o e x p l a i n , but no Kraemer e t a l . , 1984). i n t r i g u i n g , are the o b s e r v a t i o n s (Robbins, 1983; (XP, less AT), (AT), or growth r e t a r d a t i o n (FA, Hanawalt and Lehmann, t h a t some of these syndromes also involve neurological complications immunodeficiencies ( A r l e t t and Sarasin, 1986). BS and In o n l y one CS) of these d i s o r d e r s , Bloom's syndrome, i s the m o l e c u l a r b a s i s of disease apparently understood. C e l l s from i n d i v i d u a l s w i t h Bloom's syndrome appear t o have an a l t e r e d DNA (Chan e t a l . , 1987; the W i l l i s and ligase I activity L i n d a h l , 1987). enzyme n o r m a l l y p a r t i c i p a t e s i n DNA Since this r e p l i c a t i o n , that BS p a t i e n t s s u r v i v e development a t a l l i m p l i e s t h a t the mutant gene i s weakly hypomorphic and/or the d y s f u n c t i o n r e l a t i v e l y s m a l l number of c e l l or t i s s u e In D r o s o p h i l a meiotic (mei) i s l i m i t e d to a types. melanogaster many mutagen-sensitive mutants a d v e r s e l y a f f e c t the s t a b i l i t y (mus) of chromosomes, even i n c e l l s not exposed t o mutagens (Baker Smith, 1979; G a t t i , 1979; G a t t i e t a l . , 1983). Baker e t a l . , 1980, 1982; Green, Many of these mutants a l s o e x h i b i t 100 and and 1981; fertility problems, probably chromosome r e c o m b i n a t i o n instability resulting from d e f e c t s i n and/or s e g r e g a t i o n . i s also a characteristic Chromosome o f AT, FA a n d BS ( H a n a w a l t and S a r a s i n , 1986), and o f numerous f u n g a l mutants ( s e e B a k e r and S m i t h In Saccharomyces c e r e v i s i a e , influence the s e n s i t i v i t y cell-division-cycle (CDC) f u n c t i o n s mutagen-sensitive (1979) f o r r e f e r e n c e s ) . at least of cells cells t h r e e genes which t o mutagens e n c o d e vital ( s e e H a y n e s a n d Kunz, 1981; K a s s i r e t a l . , 1985). F o r example, (Johnston 1978) and CDC8 e n c o d e s t h y m i d y l a t e (Jong also a n d Nasmyth, e t a l . , 1984). essential Friedberg, (Baker e t a l . , 1982; G a t t i essential do n o t p o s s e s s pose a problem only viable identified. mutations separate (i.e., the domains f o r t h e s e f o r c o n v e n t i o n a l mutant s c r e e n s . and t h e r e f o r e p r e s u m a b l y weak a l l e l e s selection i s to select f o rtemperature-sensitive (ts) At c a n be at vital strategies are required. t h a t e x h i b i t mutagen s e n s i t i v i t y temperature mutations i n Drosophila g e n e s i n w h i c h t h e r e p a i r and T o r e c o v e r s t r o n g e r mutant a l l e l e s alternative approach ( H i g g i n s e t a l . , 1983; Naumovski and f u n c t i o n s a r e n o t s e p a r a b l e by m u t a t i o n functions) kinase e t a l . , 1983). mutagen-sensitive gene p r o d u c t s loci, for viablity ligase e x c i s i o n r e p a i r gene PAD3 i s 1 9 8 3 ) , a s a r e t h r e e X - l i n k e d mus l o c i Vital best, The y e a s t CDC9 e n c o d e s a DNA mus One such lethal a t the permissive (Henderson e t a l . , 1987). In other organisms t s h a v e p r o v e n t o be e x c e e d i n g l y u s e f u l as probes o f complex c e l l u l a r processes that otherwise are d i f f i c u l t to access genetically utility ( e . g . , H a r t w e l l , 1978; of t s mutations i n D r o s o p h i l a has Pringle,1981). The been reviewed (Suzuki, 1970). To in date, three t s l e t h a l Drosophila. actually mitotic The recovered encodes a product heterochromatic 1983). sensitivty defect ts i n a screen allele mutations of these, chromosome b e h a v i o r unique to t h i s al., first mus an allele f o r mutations (Smith f o r proper muslOl" " 1 condensation be that the of t o be secondarily related identified T h i s r e c e s s i v e mutation was collection o f 63 as t h e sensitivity t o MMS 1987). ts lethal strains at the permissive Subsequent a n a l y s e s s e n s i t i v e t o gamma r a d i a t i o n , sterile. Thus, t h i s products) whose n o r m a l mobilized i n response fertility. n o v e l mus recovered o n l y one temperature This chapter damage, and provides a n a l y s i s of mus209 understanding the metabolism. In the role(s) a B 1 i s also B 1 as a first study are (or f o r development, i s f o r female a preliminary genetic of t h i s 102 second (Henderson e t i s necessary of t h i s p l e i o t r o p i c course The a exhibiting gene e n c o d e s a p r o d u c t developmental to t h a t homozygous f e m a l e s function i s essential t o DNA from revealed that mus209 and et i n Drosophila i s rous209 . B1 (Gatti mutagen i n chromosome s t r u c t u r e ( G a t t i e t a l . , 1 9 8 3 ) . mutation abnormal Properties r e g i o n s o f chromosomes d u r i n g m i t o s i s o f m u s l O l m u t a n t s may was exhibiting l e d to the p r o p o s i t i o n t h a t necessary identified of muslOl. e t a l . , 1985). T h i s observation suggests l e t h a l mus al., have been step and toward gene i n chromosome a second t s lethal allele o f mus209. m u s 2 0 9 " . was 2 103 identified. MATERIALS AND METHODS Strains For d e s c r i p t i o n s special Grell o f most o f t h e v i s i b l e m u t a t i o n s chromosomes u s e d (1968). The in this and study, c o n s u l t L i n d s l e y f o l l o w i n g mutants r e q u i r e s p e c i a l and mention: M(2)173 (2 - 92.3) i s a s p o n t a n e o u s , c y t o l o g i c a l l y n o r m a l M i n u t e m u t a t i o n ( s e e 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 D u t t a g u p t a , 1 9 7 8 ) . M ( 2 ) 0 1 7 . a l s o a M i n u t e m u t a t i o n , was r e c o v e r e d among t h e F^ p r o g e n y o f E M S - t r e a t e d Oregon-R m a l e s ( S t o n e , 1974). The M(2)017 chromosome i s d e l e t e d f o r a b o u t f i v e b a n d s i n t h e 56F r e g i o n o f t h e 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 D u t t a g u p t a , 1978). I t f a i l s t o complement M ( 2 ) 1 7 3 . 36. 157. 775. 1991. 2735. D-292 and D-1368 a r e s e c o n d chromosomal, EMS-induced r e c e s s i v e l e t h a l m u t a t i o n s that 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 D u t t a g u p t a , 1 9 7 8 ) . The 2 7 3 5 - b e a r i n g 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 m u t a t i o n (50.7 + 1.4 map u n i t s ) . b p_r c n m u s 2 0 9 . h e r e a f t e r r e f e r r e d t o a s m u s 2 0 9 . i s a t e m p e r a t u r e - s e n s i t i v e ( t s ) r e c e s s i v e l e t h a l mus m u t a t i o n 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 ( H e n d e r s o n e t a l . , 1987; Appendix A ) . B 1 B 1 fe ET. £ Q m u s 2 0 9 . h e r e a f t e r r e f e r r e d t o a s m u s 2 0 9 , 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 f r o m t h e 2 7 3 5 - b e a r i n g chromosome. L i k e mus_209 , m u s 2 0 9 i s a t s recessive l e t h a l mutation. B 2 B 2 B1 B 2 B l a c k c e l l s (Be. 2 - 80.6) i s a d o m i n a n t m u t a t i o n w h i c h c a u s e s m e l a n i z e d c e l l s t o form beneath t h e integument i n Be / Be"" l a r v a e , pupae, and a d u l t s ( s e e R i z k i e t a l . , 1980). I3c i s employed h e r e a s a l a r v a l m a r k e r . 1 C u l t u r e c o n d i t i o n s were a s d e s c r i b e d n o t e d o t h e r w i s e , 22+l°C restrictive and temperatures, 29+0.5°C Unless a r e t h e p e r m i s s i v e and respectively. 104 i n C h a p t e r 2. G e n e t i c Mapping o f MMS Lethality S e n s i t i v i t y and The t s l e t h a l and MMS-sensitive mus209 B1 Temperature-Sensitive phenes a s s o c i a t e d w i t h the s t r a i n were mapped s e p a r a t e l y u s i n g a chromosome t h a t c a r r i e d the dominant mutations (22.0), Tft S Sp_ T f t nw D (53.2), Pin / mus209 v t homozygous mus209 nw B1 S (1.3), Sp_ and Pin Parents were t r a n s f e r r e d t o new a f t e r 2 days o f o v i p o s i t i o n . To map the MMS vials sensitivity, c u l t u r e s were t r e a t e d w i t h 0.25 mL 0.08% v/v MMS develop a t 22°C. (107.3). Y t females were mated i n v i a l s t o B1 males. (83), D multiply-marked and l e f t t o To map the t s l e t h a l i t y , u n 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 29°C and l e f t t o develop a t t h i s Recombinant progeny were counted temperature. and c l a s s i f i e d 2-3 weeks later. 95% c o n f i d e n c e i n t e r v a l s were c a l c u l a t e d as d e s c r i b e d i n O'Brien and M a c l n t y r e (1978). U s i n g g e n o t y p i c a l l y a p p r o p r i a t e p a r e n t s , and f o l l o w i n g procedures mutations s i m i l a r t o those j u s t d e s c r i b e d , two separate on t h e o r i g i n a l 2735-bearing One o f these, d e s i g n a t e d mus209 , B2 lethal chromosome were mapped. 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 o f mus209. C o s e g r e g a t i o n of MMS S e n s i t i v i t y and Female S t e r i l i t y 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 i t y i n mus209 with B1 The procedures used t o analyze the c o s e g r e g a t i o n o f the mus, t s l e t h a l , and female s t e r i l e o u t l i n e d i n F i g u r e 1. in v i a l s to homozygous nw D Pu ( f s ) phenes o f mus209 2 / b mus209 b p_r cn mus209 105 B1 B1 B1 are females were males. mated Parents were Figure 1. Procedures used t o analyze the cosegregation of the mus. t s l e t h a l , and f e m a l e s t e r i l e mus209 B 1 = b p r cn mus209 . B 1 106 phenes o f mus209 . B 1 n o n ° w P y mus 209 mus 209 2 + + b mus 209 cTcT 29 ° C nw P u mus 209 D 2 Pu' nw mus 209 mus 209 b mus 209 mus 209 die 9 nw P u mus 209 p 2 or mus209 CyO recomb mus 209 22 °C single £ per vial 22°C oviposition ? die 107 mus209 mus 209 mus209 CyO recomb mus 209 recomb CvO transferred t o new vials were s h i f t e d t o 29°C. recombinants or cosegregation correctly, nw individually of or D Pu t o 5-6 this i f the mus from each line experiments and ts at 1) . 29°C After 1 Fertility The (or, corresponding Figure were week, B 1 male / B 1 CyO each were females. Following vial assessments and kills vials; left was for at least 5 2 the t o develop essentially a l l cultures on the cosegregate, of correctly, this the was treated expectation then normal animals B 1 was with examined phenotype they with cosegregation a n a l y z e d as females t h a t vials mated treatment. were made o n l y C y t o g e n e t i c M a p p i n g and MMS t o new females are completely s t e r i l e ; into more wild-type + wild-type functions) placed f o r the (or, m u s 2 0 9 ) / b p_r c n m u s 2 0 9 recombinant + survivors replicate cosegregation more Single females l i v e d The corresponding phenes s h o u l d s u r v i v e MMS oviposit. lethality were p r e d i c a t e d lethal (i.e., Homozygous m u s 2 0 9 lethality set of test the concentration, A To cultures mus209 ts p a r e n t s were t r a n s f e r r e d numbers o f r e c o m b i n a n t not of b p_r c n m u s 2 0 9 at These only and recombinant 2 homozygotes.) B 1 the o r i g i n a l temperature, sensitivity cosegregation (MMS, similarly. that this and heterozygotes survive. 2 MMS 2 days, c u l t u r e s were t r e a t e d w i t h 0.09% 22°C. mus209 At Pu D oviposition, original at of the functions) , days nw within survived several f o r the ts of the follows (see development wild-type presence f o r those v i a l s do of males. eggs. i n which the days. Complementation c h r o m o s o m a l segment d e l e t e d 108 Analyses i n M(2)017 corresponds to a g e n e t i c map position this vicinity o f a b o u t 92 map ( s e e RESULTS), units. S i n c e mus209 maps t o c o m p l e m e n t a t i o n e x p e r i m e n t s were carried o u t t o d e t e r m i n e w h e t h e r M(2)017 u n c o v e r s t h e mus209 locus. For t h i s vials analysis, t o mus209 were r e a r e d / CyO B 1 M(2)017 / SMI females. a t 22°C o r 29°C. males Progeny were crossed from t h e s e matings Half the cultures, a t each t e m p e r a t u r e , were t r e a t e d w i t h MMS; t h e r e m a i n d e r were untreated. among Cy s i b s , the An a b s e n c e o f C y cytogenetic deficiency's identifies o f mus209, a s d e f i n e d by t h e t e s t s between mus209 and a s e r i e s o f B 1 EMS-induced m u t a t i o n s ( S h e l l e n b a r g e r and D u t t a g u p t a , 1978) carried out as f o l l o w s . l i n e s were mated R e p r e s e n t a t i v e males in vials to mus209 f r o m t h e s e c r o s s e s were r e a r e d c o m p l e m e n t a r i t y was flies. Following a t 29°C. females. In each lethal Progeny case, t o Cy i n s t a n c e s o f non-complementation, these t o i n c l u d e a l l p a i r w i s e c o m b i n a t i o n s (m^/Cy X R e t e s t s were c a r r i e d out a t both 29°C. Temperature For / CyO from e x t a n t + m^/Cy) o f a v a i l a b l e m u t a n t s . 2 2 ° C and B 1 were a s s e s s e d by c o m p a r i n g t h e number o f C y several t e s t s were e x p a n d e d S h i f t Analyses a detailed methodology of flies, left breakpoints. Allelism lethal location + in d i s c u s s i o n of the r a t i o n a l e and t h e f o r determining temperature-sensitive periods (TSPs) t s m u t a t i o n s , see S u z u k i (1970). In these studies, females n e c e s s i t a t e d the unconditional sterility the use o f the l a r v a l 109 marker o f mus209 Be. Thus, from m a t i n g s b e t w e e n Be mus209 / SM5 females homozygous m a l e s , homozygous mus Be and b g r cn l a r v a e were i d e n t i f i e d by their phenotype. Standard egg-lay practices (e.g., S i n c l a i r e t a l . , 1981) 2 h r d u r a t i o n were e m p l o y e d t o o b t a i n s y n c h r o n o u s I m m e d i a t e l y a f t e r egg collection medium (approximately a t 22°C immediately The treatments hr. t o , and kept treatments Some c u l t u r e s were a t 29°C experiments, 3 6 o r 48 consisting (restrictive of contiguous heat 36-hr p r o v i d e d t h e b e s t r e s o l u t i o n between l e t h a l i t y Prolongated exposure of t s mutants t o the temperature can s u c c e s s i v e but al., Sinclair 1973; were a s s e s s e d by e t a l . , 1981).) a t both the beginning examining survival heterozygotes was s e p a r a t e TSPs The and 1950) from adults. The survival interval was normalized obtained f o r the 22°C value obtained the a n t e r i o r and et stages heat spiracles t o mus209 / classifying f o r each Cy emergent developmental t o the homozygoterheterozygote 110 heat representative vials. c o u n t i n g and controls. by pulses (Poodry o f each o f mus209 h o m o z y g o t e s r e l a t i v e m e a s u r e d by heat restrictive developmental t h e end t h e mouth p a r t s and of l a r v a e (Bodenstein, The controls). staggered h r d u r a t i o n , showed t h a t t h e obscure left o t h e r s were viability. treatment the containing fresh (29°C) d u r i n g d e v e l o p m e n t , a t i n t e r v a l s 24, from 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 3 6 - h r (Pilot of e i t h e r eggs p e r v i a l ) . (permissive c o n t r o l s ) ; shifted remaining 70 to v i a l s of cultures. d e p o s i t i o n , s e c t i o n s of food p l a t e s were t r a n s f e r r e d to develop 12 mus209 ratio R a d i a t i o n Treatments Treated Be m u s 2 0 9 males. All animals / SM5 B 1 During were females Synchronous Development derived and from b p_r c n m u s 2 0 9 instar; 140 irradiated stages (Atomic Energy rads per Flies, (24 h r , e m b r y o n i c ; hr, mid-third by B 1 between homozygous c u l t u r e s were o b t a i n e d a s d e s c r i b e d c u l t u r e s were m a i n t a i n e d a t 2 2 ° C . developmental crosses exposing v i a l o f Canada). instar; 216 cultures to at four 96 h r , l a t e above. different second h r , e a r l y p u p a l ) , were 6 0 Co i n a Gammacell Dose r a t e s v a r i e d b e t w e e n 10 and second. Ill 220 12 RESULTS G e n e t i c M a p p i n g and These to initial Cosegregation Analyses characterizations of mus209 determine whether i t s MMS-sensitive, result from a s i n g l e g e n e t i c crucial the t o any sensitivity mutant s i t e ( s ) lethality of t h i s + 2.6 (In subsequent associated with the a l l e l i c are same l o c a t i o n and t h e y do n o t r u l e ts phenes might mutations. Pu 2 sensitivity 94.0 + 2.9 o f 852 individually result map units).) 22°C. sterile) no males B 1 / CyO were t r e a t e d w i t h MMS I n 64 nw D and 659 Pu While these B 1 is a results single-site from s e p a r a t e , a l b e i t carried out (99 nw / mus209 2 lines D and an (see F i g u r e 1 ) . a t 29°C females. B 1 and 753 were Progeny from allowed t o develop at (the remaining c u l t u r e s separation of t s l e t h a l i t y and tightly latter possibility, s u r v i v e d development mated t o m u s 2 0 9 these matings mapped t o o u t t h e p o s s i b i l i t y t h a t t h e mus recombinant B 1 ts units, B 2 To e l i m i n a t e t h i s / mus209 ) that map and m u t a t i o n , m u s 2 0 9 . was 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 A total exploit experiments, the t s l e t h a l i t y (92.6 + 1.5 mutation, linked, f s phenes strain. consistent with the notion that mus209 lethal and This correspondence i s c o n f e r r i n g MMS map(s) t o 92.8 respectively. this ts lethal, undertaken further experimentation with designs to temperature The lesion. were B 1 f r o m MMS sensitivity were was found. the Analogous e x p e r i m e n t s were c o n d u c t e d t o d e t e r m i n e whether ts lethal and The f s phenes o f m u s 2 0 9 112 B 1 cosegregate. results of these s t u d i e s are discounting the f s and mutation. mus209 fail B 2 the background s t e r i l i t y , ts lethal In phenes a r e support of t h i s homozygous f e m a l e s , An M a p p i n g and these data t o the same idea, i t was later like t h e i r mus209 Complementation from the After indicate that pleiotropic observed that counterparts, B 1 individuals, s u r v i v e at both result b a n d segment w i t h i n B 1 B 1 B 1 an serendipitous scrutiny. (1978) s a t u r a t e d The Using this At inter se B 1 mus209 B 1 failed absence of MMS. p o s i t i o n o f mus209 t o a of the unconditional strengthens right arm of lethality of the EMS, B 1 led to previously subjected Shellenbarger portion of of these failed the contention further, 5 that suggests mutation. complementation 22°C, m u s 2 0 9 to When a chromosomal segment u n c o v e r e d by small d e t e r m i n e w h e t h e r any mus209, even i n t h e e s s e n t i a l gene, and M(2)017 d e l e t i o n i s a r e g i o n genetic mapping s t u d i e s . p o s i t i o n i n g of mus209 find. was B 1 heterozygous progeny 56F5-15 r e g i o n i s a hypomorphic cytogenetic B 1 cytogenetic heterozygotes identifies that mus209 The the of mus209 were c r o s s e d 29°C, In a d d i t i o n , the M(2)017 / m u s 2 0 9 mus209 flies 2 2 ° C and d e l i m i t s the chromosome 2. genetic Mf2)017 / m u s 2 0 9 the Analyses localization r e s u l t s of the M(2)017 d e f i c i e n c y - b e a r i n g To 1. due approximate c y t o g e n e t i c inferred This i n Table to oviposit. Cytogenetic to presented 2R intense Duttagupta for lethal identify crosses and to lethal mutations. alleles were c a r r i e d t o complement 5 r e c e s s i v e 113 the of out. lethal T a b l e 1. COSEGREGATION OF FEMALE S T E R I L I T Y AND S E N S I T I V E LETHALITY IN m u s 2 0 9 TEMPERATURE- B 1 Genotype 1. nw % sterility 328 4 1.2 D 153 3 2.0 2 639 8 1.3 792 11 1.4 Pu D 2 2. nw 3. Pu 4. Total of 2 and 3 a Number o f s t e r i l e females Number o f females t e s t e d 3 fe E E c n m u s 2 0 9 heterozygous females t h a t d e v e l o p m e n t a t 29°C ( s e e F i g u r e 1 ) . B 1 114 survived mutations (36, 157. 775. m u t a t i o n , M(2)173• lethality (M(2)U. 1991. 29, 47, 51, 1978). 2362. and A t 29°C, heterozygotes f a i l e d o f mus_209 and 75%, (Similarly, mus209 / D-1368. B 1 survival Based the left group complementation ( F i g u r e 2B). major group Minute map spurious. That itself the 12 o f mutant r e g i o n may and 2735 the viabilities I and (0.09%) 2735. lowered i s represented and not shown).) I I ) from only by the Duttagupta of the mutations likely / B 1 (data 20% identify (1978) i n the an right adjacent 12 e s t a b l i s h e s a and right groups seems s u r v i v e s o v e r t h e M(2)017 d e f i c i e n c y b u t i s strongly implies that i n M(2)017. 12 when combined with i t maps The outside Minute-like c e r t a i n mutations from 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 of Minute mutations 1984). locus between t h e l e f t c h r o m o s o m a l segment d e l e t e d typical al., IV and V) homozygous l e t h a l phenotype this t h e mus2 09 T h e i r argument t h a t m u t a n t relatedness / of mus209 (data not of Shellenbarger functional B 1 o b s e r v e d a t 22°C exposure A l l , or a subset, locus. A l l other 2735 / D-1368 were r e d u c e d t o a b o u t (subgroups (subgroups mutations (Shellenbarger 2735 / D-1368 t o MMS on l e t h a l i t y , Minute complemented f o r Furthermore, by a s much as 70-90% major fully however, m u s 2 0 9 a t 22°C, and a second ( F i g u r e 2A). described r e s p e c t i v e l y , of the l e v e l s shown). their D-741) to survive. / D-1368 and B1 B 1 and a s e r i e s of Minute-like i n t e r a c t i o n s were a s o r i g i n a l l y Duttagupta, D-292) In c o n t r a s t , mus209 2735. D-1368 and 12, and i n general (e.g., see S i n c l a i r et F i g u r e 2. Complementation maps o f l e t h a l mutations uncovered by the M(2)017 d e f i c i e n c y . alleles A . Complementation diagram o f o f t h e mus209 l o c u s . T h i s map was c o n s t r u c t e d from complementation [The shotgun data based on l e t h a l i t y (shg) l o c u s (2-92) encodes a z y g o t i c function that i s essential (Nusslein-Volhard a t 29°C. f o r embryonic development e t a l . , 1984). The p r o x i m i t y o f t h i s mutation t o mus209 prompted M(2)017 and mus209 B1 a test for allelism. f u l l y complemented s h g I H 8 1 Both a t 29°C i n d i c a t i n g t h a t shg and mus209 a r e s e p a r a t e genes.] B. A reproduction Shellenbarger o f the complementation map o f and Duttagupta (1978). T h i s map i s based on data f o r both l e t h a l i t y and s h o r t b r i s t l e phenotype. 116 (Minute) 36 157 775 1991 D-292 shg mus209 2735 D-1368 B 1 M(2)173 M(2)017 D-91 D-292 D-932 157 775 1991 36 D-1368 2362 D-741 29 47 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 and complementation mus. ts lethal, o f t h e s e mapping, c o s e g r e g a t i o n , studies are compelling evidence that and f s phenes o f rous209 a r e due B1 to a the single mutation. Temperature S h i f t s During G i v e n t h a t t h e MMS sensitivity are simply p l e i o t r o p i c temperature Development sensitivity and ts lethality e x p r e s s i o n s o f a s i n g l e m u t a n t gene, may be exploited e x t e n t d u r i n g development the e s s e n t i a l required. shifts, The mus209 + temperatures, gene p r o d u c t developmental extend B 1 i s required heat pulse r e s u l t s t o complete restrictive perhaps i n 100% through in different and 36-hour h e a t indicate that the t h r o u g h most s t a g e s o f In p a r t i c u l a r , first stages of pupation are i n mus209 . B 1 mortality, the those larval i s attributed different As no failure single tissues. 118 36-hr of mus2Q9 to the cumulative developmental molt acutely d e v e l o p m e n t when r e a r e d c o n t i n u o u s l y a t temperature of the mutation temperature r e v e a l e d by i n t e r v a l s which b e g i n a f t e r the s e n s i t i v e t o the genetic defect flies f u n c t i o n o f mus209 i s These data t o v a r y i n g degrees. i n t o the e a r l y the development. lethality, i s shown i n F i g u r e 3. d e v e l o p m e n t , and and throughout B 1 t o what o f h e a t p u l s e s between t h e p e r m i s s i v e pattern of mus209 treatments, t o a s k when and T h e s e q u e s t i o n s were a d d r e s s e d by d o i n g i n t h e form restrictive of mus209 stages, B1 the effect and F i g u r e 3. Sensitivity radiation derived and of mus209 B 1 homozygotes t o h e a t treatments d u r i n g development. from matings mus209 B 1 between Be m u s 2 0 9 homozygous m a l e s . B 1 or P r o g e n y were /SM5 females S u r v i v a l v a l u e s have been n o r m a l i z e d t o the homozygoterheterozygote obtained i n the permissive c o n t r o l s l a t t e r v a l u e i s 0.76 experiments treatment lines. (2975 c o n t r o l horizontal the heat pulse. T a b l e 3) f o r the temperature flies d a t a a r e i n d i c a t e d by The The scored). circles r a d i a t i o n treatment ( i n v e r t e d t r i a n g l e s = 1.3 krad). 119 The shift The and heat solid bars i n d i c a t e the duration of a r e i n d i c a t e d by t r i a n g l e s t r i a n g l e s = 2.5 (22°C). ratio krad, and data dashed right-side-up (see lines To address the p o s s i b i l i t y p r o f i l e might mus209 B 1 result that the temperature-sensitivity f r o m an a l l e l e - s p e c i f i c r a t h e r than as a consequence of a defect l o c u s p e r se, a s i m i l a r heat p u l s e regimen mus2 0 9 Figure allele. B 2 4. the t h i r d mus209 defect Except larval i n mus209 instar, B 2 fundamental was a p p l i e d t o t h e a n a l y s i s a r e shown i n differences in viability the pattern of t s l e t h a l i t y of mus209 . elicited closely that appears t o be more s e v e r e t h a n t h a t i n B 1 during developmental requirement by I f anything, the f o r t h e mus209 + a product. Phases The effective occurs shortly t i m e when imaginal lethal phase o f both m u s 2 0 9 a f t e r puparium larval larval to eclosion, Maternal but d i e as most p u p a r i a (>80%) come t o and w i t h o u t d i s c e r n i b l e o f Shearn prepupal-pupal larval adult (1977), t h e s e m u t a t i o n s tissue, B 1 are c l a s s i f i e d lethals. and m u s 2 0 9 B 2 are refractory 121 often structures. Effects mus209 B 2 Although a small proportion o f i n d i v i d u a l s a n amorphous mass o f d i s i n t e g r a t i n g the c r i t e r i a and m u s 2 0 9 a n d r e p l a c e d by s t a g e , a n d some o t h e r s p u p a t e pharate adults p r i o r with gaping holes, B 1 f o r m a t i o n ( d a t a n o t shown), a t a tissues are being histolyzed structures. a t each encase i n t h e mus209 T h u s , t h e c o n t i n u o u s TSPs a r e assumed t o r e f l e c t B 1 die of this f o r some m i n o r parallels B 2 mus209 . Lethal The r e s u l t s peculiarity of t o 36-hr heat By as Figure 4. Sensitivity of mus209 (29°C) d u r i n g matings the between Be Progeny mus209 /SM5 Survival values controls control flies i n d i c a t e the ratio latter scored). The horizontal of the 122 heat pulse. B 2 normalized obtained The from mus209 have been (22°C). duration pulses were d e r i v e d f e m a l e s and B2 homozygote:heterozygote permissive (1710 homozygotes t o h e a t development. homozygous m a l e s . to B 2 value in is bars the 0.38 treatments d u r i n g embryogenesis larval instar ( F i g u r e s 3 and 4 ) . gene product may stages. and through most of the first T h i s suggests t h a t the mus209 not be r e q u i r e d i n these e a r l y developmental A l t e r n a t i v e l y , the maternal genome might c o n t r i b u t e t o the egg s u f f i c i e n t mus209 product t o support the e a r l y + development of the mus209 homozygote, even a t the temperature. (That the maternal genotype i n f l u e n c e the s e n s i t i v i t y of mus amply demonstrated progeny restrictive can p r o f o u n d l y t o mutagens has been - see Appendix B and the r e f e r e n c e s t h e r e i n . ) 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 v e r s u s maternal e x p r e s s i o n , i t would be necessary t o examine the temperature s e n s i t i v i t y of mus209 homozygous embryos o b t a i n e d from s i m i l a r l y homozygous mothers. sterility of the mus209 homozygous female p r e c l u d e s t h i s approach. for However, sterility B2 rous209 B1 B2 p a r t i a l l y complement females To do t h i s , h e t e r o a l l e l i c mus209 were 200 p a i r s per b o t t l e ) ; hr and mus209 direct and so p r o v i d e a means t o t e s t f o r the maternal effect indirectly. mus209 O b v i o u s l y , the u n c o n d i t i o n a l mated t o Be mus209 B2 / SM5 B1 / males (150- eggs were c o l l e c t e d on p e t r i p l a t e s (2 egg l a y s ) , counted, and s u b j e c t e d a t once t o a s i n g l e 36-hr heat treatment experiment (29°C) d u r i n g embryogenesis. The r e s u l t s of t h i s are g i v e n i n Table 2. I t can be seen t h a t the mus209 B1 / mus209 B2 v e r y few v i a b l e o f f s p r i n g , i r r e s p e c t i v e of the However, f o l l o w i n g the embryonic mothers produced temperature. heat treatment even animals s u r v i v e d t o adulthood, and a l l of these were 124 fewer T a b l e 2. THE MATERNAL-EFFECT LETHALITY OF mus209 HETEROALLELIC FEMALES Temp. (°C) 3 No.of Eggs Unfert. and/or early lethals Lethal Phase E b 22 862 750 21 29 877 807 45 L Be B c 20 2 0 3 + / mus209 B1 Adult Survivors c P Be B c 31 male Be B c + 4 3 4 + female Be B c + 4 11 6 12 0 3 0 13 Temperature: 22=progeny were developed a t 22 C. 29=progeny were s u b j e c t e d t o a s i n g l e 36-hr h e a t - p u l s e (29°C) immediately following oviposition. F l i e s were then r e t u r n e d t o 22°C and kept a t t h i s temperature f o r the remainder o f development. 3 U T o t a l number o f u n f e r t i l i z e d eggs and/or e a r l y l e t h a l embryos (white eggs). b L e t h a l s t a g e s : E=late embryonic P=pupal. c 125 (pre-gastrula) (darkened embryos); L = l a r v a l ; heterozygous. These r e s u l t s , taken t o g e t h e r with t h e data i n F i g u r e s 3 and 4, show t h a t t h e v i t a l f u n c t i o n o f mus209 i s + r e q u i r e d not only throughout t h e second and t h i r d l a r v a l and i n t h e e a r l y pupal stages, but d u r i n g embryogenesis (and presumably i n the f i r s t l a r v a l i n s t a r ) as w e l l . the maternal e x p r e s s i o n o f mus209 viability, instars + i s normally Moreover, while essential for z y g o t i c e x p r e s s i o n o f a p a t e r n a l l y - d e r i v e d mus209 gene may o c c a s i o n a l l y rescue the m a t e r n a l - e f f e c t + lethality observed a t 29°C. Other f a c t o r s may have c o n t r i b u t e d t o t h e low f e c u n d i t y as well. Scanning e l e c t r o n microscopy r e v e a l e d t h a t t h e v a s t m a j o r i t y o f eggs from these h e t e r o a l l e l i c females were morphologically abnormal; most had d e f e c t i v e egg s h e l l s , i n c l u d i n g rudimentary o r malformed r e s p i r a t o r y appendages (not shown). Thus, s t r u c t u r a l a b n o r m a l i t i e s i n the v i c i n i t y o f t h e micropyle may have prevented fertilized. many of t h e eggs from b e i n g This observation i s particularly because i t suggests t h a t mus209 choriogenesis. + interesting might have an i n d i r e c t r o l e i n A mutant a l l e l e a t one o t h e r mus l o c u s has been shown t o d i s r u p t t h e a m p l i f i c a t i o n o f c h o r i o n genes t h a t normally occurs l a t e i n oogenesis (Snyder e t a l . , 1 9 8 6 ) . F i n a l l y , m e i o t i c problems i n mus209 B1 and mus209 B2 females have not been r u l e d out as f a c t o r s l e a d i n g t o s t e r i l i t y . R a d i a t i o n Treatments During Development As shown i n t h e p r e v i o u s s e c t i o n , t h e l e v e l o f heat p u l s e induced l e t h a l i t y i n mus209 B1 v a r i e s according t o the 126 developmental ts interval exposed l e t h a l i t y mirror that correspondence (Figure 3). f o r mutagen s e n s i t i v i t y ? a n d t h o s e f o r mutagen s e n s i t i v i t y proximate cause function. (e.g., of death, temperature, resulting f o r spontaneous phenotypes lesions) a t 29°C? correspondence that to the but which DNA + repair allows survival repair activity a t 22°C ( i n i s rendered On t h e o t h e r hand, a l a c k o f t e m p o r a l f o r the phenocritical (lethality complicated close imply that the i n t h e mus209 i s there a residual o f DNA-damaging a g e n t s ) nonfunctional might from exposure i s a failure I n o t h e r words, the absence A between t h e 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 lethality restrictive Does t h e p a t t e r n o f intervals o f t h e two and mus) w o u l d n e c e s s i t a t e a more interpretation of the role, or roles, o f t h e mus209 the r e l a t i v e sensitivity + product. To address t h i s issue, h o m o z y g o t e s t o gamma-rays was d e t e r m i n e d development: results Table 3. of at four select these experiments are shown development; This at later The F i g u r e 3 and i n a t every stage the mus209 h o m o z y g o t e s a r e more s e n s i t i v e t o i o n i z i n g sibs. in i s especially B 1 times i n 24, 96, 140, o r 216 h r s p o s t o v i p o s i t i o n . I t c a n be s e e n t h a t heterozygous of mus209 B 1 r a d i a t i o n than their true early i n stages the differences are less pronounced. This progressive loss 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 r e s p o n s e o f w i l d t y p e D r o s o p h i l a . and o f h o l o m e t a b o l o u s generally (Mavor, 1927; Grosch and Hopwood, 127 1979). insects Apparently, T a b l e 3. THE EFFECTS OF IONIZING RADIATION ON THE RELATIVE SURVIVAL OF mus209 HOMOZYGOTES AT VARIOUS TIMES IN DEVELOPMENT B1 Dose (krads) Normalized survival ratio 3 Time of i r r a d i a t i o n p o s t o v i p o s i t i o n 24 96 140 (hrs) 216 1.3 0.00 (283) 0.27 (432) 0.38 (422) 0.73 (448) 2.5 — 0. 00 (486) 0.01 (377) 0.38 (427) b The n o r m a l i z e d s u r v i v a l r a t i o i s the t r e a t e d homozygote t o h e t e r o z y g o t e r a t i o d i v i d e d by the u n t r e a t e d homozygote t o heterozygote r a t i o . The l a t t e r v a l u e i s 0.84 (1189). The numbers i n parentheses i n d i c a t e t h e t o t a l number o f f l i e s s c o r e d p e r experiment. See F i g u r e 3 f o r t h e c o r r e s p o n d i n g developmental 128 stage. the level of r a d i o s e n s i t i v i t y stage i n the animal's l i f e the mitotic (i.e., does n o t appear the cycle, r a t e and t h e number the degree profile. i s a property of the p o s s i b l y determined I t merely The d e f e c t intrinsic h o m o z y g o t e , and o n l y by an amount stage. contrast i s seen sensitive lethality. sensitivity F o r example, t h e most striking This stage, although i s u n a f f e c t e d by t h e h e a t be d i f f e r e n t from t h a t of the developmental imply t h a t the b i o c h e m i c a l b a s i s s e n s i t i v i t y may may 3). B 1 commensurate w i t h t h e Consequently, i n embryogenesis. to radiation, These r e s u l t s induced (Figure i n mus209 radiosensitivity p a t t e r n s o f mutagen s e n s i t i v i t y and t e m p e r a t u r e d i f f e r markedly cells radiosensitivity increases the r e l a t i v e animal's developmental by b o t h of consequential target of d i f f e r e n t i a t i o n ) . to alter this particular highly treatments. f o r t h e mutagen f o r the temperature- I n o t h e r words, t h e mus209 gene p r o d u c t ( s ) p o s s e s s more t h a n one function. 129 DISCUSSION The mutation mus209 i d e n t i f i e s a gene whose B 1 function (1) i n f l u e n c e s t h e s e n s i t i v i t y o f f l i e s ionizing radiation fertility absence and and MMS; While (3) i s e s s e n t i a l essential collection protocol. Except haplo-insufficient and of 1978). strictly Duttagupta had Using t h i s no p r i o r mus209 . While B 1 s h o u l d have a l l o w e d this sensitivity) result may strictly n o t be t h a t the mus and essential separable into viable discrete invariably alleles given the the p o s s i b i l i t y f u n c t i o n s o f mus209 a r e Thus, m u t a t i o n s which f u n c t i o n o f mus209 may having their surprising number o f chromosomes t e s t e d , excluded. selectable. f a i l e d to recover low mutationally are only knowledge o f t h e comparatively + approach, w o u l d h a v e gone u n d e t e c t e d . H e n d e r s o n e t a l . (1987) a l s o ( f o r MMS by deficiency l e t h a l mutations on v i a b i l i t y However, i n a s c r e e n t h a t noncomplementing viable identified 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 b u t adverse e f f e c t s detection, the that unconditionally e a c h m u t a t i o n was B 1 Duttagupta, no gene. a l l e l e s underscores f a i l u r e t o s u r v i v e o v e r t h e M(2)017 phenotype, lethal an a r t i f a c t o f t h e o r i g i n a l s c r e e n i n g ( S h e l l e n b a r g e r and mus 8 B 1 of l e t h a l be (even i n t h e this pleiotropic f o r mus209 , A s S h e l l e n b a r g e r and both Including mus209 , n a t u r e o f mus209. t h e f a c t m u t a n t s a r e m i s s i n g may its for viability 2 o f them t s , d e n o t e this to (2) i s n e c e s s a r y f o r f e m a l e o f DNA-damaging a g e n t s ) . mutations, normal not g e n i e domains cannot affect affect the be repair-related the e s s e n t i a l function to a greater o r l e s s e r degree, Naumovski and Friedberg similar and question i nvitro localized present mutagenesis, function, studies neither of these o f t h e gene. f i n d i n g s emerged function development expression Despite o f mus209 (the latter exception), + B 1 could i n RAD3 a p p e a r t o as opposed t o t h e gene from t h e temperature i s required B 2 . shift First, the throughout most o f h a l f of t h e pupal period as w e l l i s a developmental as sine appears t o be zygotic qua non. 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 during t h e second effect on l a r v a l effects During adult larval viability. The b a s i s hatching, (e.g., for this the cells imaginal from those t h a t the larval pupariation of Drosophila e a r l y embryogenesis, structures i n s t a r s have no Not u n t i l i n t h e context become s e p a r a t e d polytene and t h i r d become e v i d e n t . understandable be However, u n l i k e t h e and mus209 and second, maternal of this functions 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 . c a r r i e d o u t on m u s 2 0 9 essential of the essential and p e r f e c t l y v i a b l e rad3 mutants t h a t a r e defective Two i m p o r t a n t After organization 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 completely to have attempted t o answer a s i t u a t i o n f o r mus209, m u t a t i o n s essential versa. f u c t i o n s o f t h e RAD3 g e n e o f S. c e r e v i s i a e . t o discrete regions inactivate an ( 1 9 8 6) about t h e genie excision repair Using and v i c e cells obvious do t h e l e t h a l killing i s ontogeny. destined disc cells t o give rise and h i s t o b l a s t s ) produce t h e l a r v a l tissues. grow i n s i z e and d e v e l o p n u c l e i b u t t h e y do n o t d i v i d e . 131 delayed applied In contrast, the large imaginal disc by mitotic cells remain d i p l o i d divisions. Although present most o f t h e s e m i t o t i c a l l y larval in functions the ring (some c e l l s mus209 B 2 imaginal At this cell Cells cease d i v i d i n g s t r u c t u r e s and a f u l l y that correspond t o developmental disc cells evaginate t o formed 1978). divisions intervals and t h e h i s t o b l a s t s proceed normally the imaginal discs examined as t o t h e i r differentiate after size, transplantation t h e mus209 d e f e c t d i s c 1971; their Shearn and Garen, i n mutants d e r i v e d 132 nuclear from t o a maternal e f f e c t (see forcell indispensible proliferation. such a of heat-treated s h a p e , and t h e i r into autonomous?) 1974). active embryonic T h i s correspondence suggests that the obviously, and B 1 d u r i n g which t h e S e v e r a l d i a g n o s t i c s c o u l d be u s e d t o e s t a b l i s h is imago are m i t o t i c a l l y ( T h a t a t 29°C t h e e a r l y f u n c t i o n o f mus209 may be r e q u i r e d be time, and t h e TSPs o f m u s 2 0 9 heterozygous mothers i s a t t r i b u t e d Most i n the f o r the f i r s t time the imaginal d i s c s i t i s significant RESULTS).) g l a n d s may be results. (Postlethwait, and degenerate. nests begin t o divide r i s e to the adult Thus, n e r v o u s s y s t e m and (which b e g i n s a t puparium i n the imaginal discs differentiate. larva, are not essential f o r i n the central most l a r v a l t i s s u e s whereas t h o s e eventually cells During metamorphosis abdominal h i s t o b l a s t give active i n the developing g l a n d s o f t h e f o r e g u t and s a l i v a r y exceptions). formation) and c o n t i n u e t o p r o l i f e r a t e Vital role. larvae could capacity to a wildtype host (i.e., (e.g., Shearn e t a l . , staining techniques could be used t o v i s u a l i z e 1974). for Also, brooding pre-meiotic Cross, n e c r o t i c c e l l s w i t h i n these 1979). ts male-sterile effects While p o s i t i v e consistent with constitute i t s proof. To carried out (Arking, out (Shellenbarger to t e s t s would they would that mus209 is a cell- somatic B 1 recombination (e.g., A r k i n g , 1974; look and proliferation, confirm mutation, carried f i n d i n g s i n these a defect in c e l l autonomous l e t h a l h a v e t o be e x p e r i m e n t s c o u l d be discs be not analyses would R u s s e l l , 1974). I n g e n e r a l , mutagen s e n s i t i v e m u t a t i o n s w h i c h i d e n t i f y loci DNA are apt t o d i s r u p t one synthesis ( e . g . , N j a g i and K a s s i r e t a l . , 1985; Simchen, al., 1986) 1983). o f two K i l b e y , 1982; Peterson or mitosis T h a t DNA e t a l . , 1985; (e.g., replication mutants r e a r e d under r e s t r i c t i v e observation t h a t the the that possibility larval processes cell Jong e t a l . , Kupiec Gatti proceeds normally i n mus209 c o n d i t i o n s i s suggested not 1984; and t i s s u e s are p o l y t e n i z e d . but cycle: B a k e r e t a l . , 1982; t h a t mus209 e n c o d e s a DNA i s active in diploid i n the vital nuclei the However, replication polytene by et factor cannot be excluded. An has a l t e r n a t e , p e r h a p s more l i k e l y a role preparatory e s s e n t i a l mus functions. loci For condensation to or during m i t o s i s . i n Drosophila example, a t t h e l e t h a l mutation m u ^ l 0 1 t s l cycle appear t o encode restrictive regions other such temperature, the ts the o f chromosomes ( G a t t i e t a l . , 1983). 133 i s t h a t mus209 Three disrupts preferentially of heterochromatic the m i t o t i c c e l l possibility during T h i s suggests that the m u s l O l " " p r o d u c t may 1 packaging. The o b s e r v a t i o n communication) variegation Two this cells t h e genome. junctions per cell per cycle) By c o n t r a s t , (Gatti, (1982) o b s e r v e d larval-pupal at degenerate mus209 reveal B 1 stability occur (breaks i n the euchromatic carrying lethal imaginal In regions Baker e t a l . alleles of these ) f o r severe alleles chromosome within the breakage imaginal r a i s e d a t 22°C v e r s u s i f a n y , f o r mus_209 + i n maintaining that o f muslOS o r o f m i t o t i c f i g u r e s from neuroblasts larvae two d i s c s and d i e a t t h e throughout the m i t o t i c c e l l Curiously, intervals B 2 high (Baker e t a l . , 1982). number o f c e l l s anaysis (and mus209 a role, exhibit (>0.5 a b e r r a t i o n s t o pupate because extensive A similar brain These r e s u l t s l e d them t o p o s t u l a t e an i r r e t r i e v a b l e discs. Larval i n mus!09. m o s t o f t h e chromosome l a r v a e homozygous o r hemizygous fail encode the euchromatic-heterochromatic larvae boundary. and mus!09. the structural 1979; Baker e t a l . , 1982). that genes have s m a l l , kills maintain from c e r t a i n mutant a l l e l e s are concentrated rousl09 mus!05 o f chromosome a b e r r a t i o n s exchanges) breaks idea. loci, a l s o appear t o help mus!05. most o f t h e a b e r r a t i o n s of (personal o f chromosomes i n d i v i d i n g c e l l s . frequencies and Sinclair i n chromatin i s an enhancer o f p o s i t i o n - e f f e c t t s l i n d i s p e n s i b l e mus products that ganglial that musl01 be i n v o l v e d b y D. i s consistent with other integrity normally 29°C of might chromosome cycle. 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 do n o t c o i n c i d e i n mus209 134 B 1 . This may imply that the essential that and r e p a i r - r e l a t e d i s , the t s lethality defect i n DNA second function these administered o f mus209. 36-hr, 29°C h e a t however, be f a l l a c i o u s since these o f two q u a l i t a t i v e l y these d i f f e r e n c e s , how radiation sub-acute, i s i t that the genome c a n r e s c u e t h e t s l e t h a l i t y sensitivity The most s t r a i g h t f o r w a r d answer t o t h i s o f t h e e m b r y o n i c and f i r s t d e v e l o p m e n t , t h e amount o f mus2_09 the e s s e n t i a l different treatment. ray in significance to s e v e r a l m i n u t e s a t 22°C, v e r s u s a Notwithstanding maternal To a t t a c h a d d i t i o n a l b u r d e n s - an a c u t e d o s e o f i o n i z i n g over are distinct, t h e absence o f an e s s e n t i a l , measure t h e e f f e c t s physiological o f mus209 r e s u l t s not as a consequence o f a r e p a i r b u t from f i n d i n g s may, experiments activities role the r e p a i r - r e l a t e d + b u t n o t t h e gamma larval question i s that product demand f o r t h e m u s 2 0 9 Thus, p r o d u c t may + following stages? early needed t o p e r f o r m i s considerably less that that activity. instar required f o r i r r a d i a t i o n , the simply o u t s t r i p the maternal supply. S p e c u l a t i o n s as t o t h e n a t u r e o f t h e mus209 In gene t h e absence o f b i o c h e m i c a l and m o l e c u l a r c l u e s as t o t h e n a t u r e o f t h e mus209 hypotheses concerning speculative, i t s precise and s o l e l y reliant + B 2 biological function(s) are at best on g e n e t i c d a t a . and D-1368. by c o n v e n t i o n , 135 product gene p r o d u c t ( s ) , any t h e e x i s t e n c e o f t h e complementary a l l e l i c mus209 + F o r example, mutations implies that highly mus209 , in i t s B 1 essential complex, flies role, e.g., still t h e mus209 a homodimer. exhibit Because sensitivity (although not obligatory) resides in subunit s t r u c t u r e may At gene p r o d u c t may + d i s t i n g u i s h between The i t i s possible activity of well relevant mus209 B 1 s i m i l a r mutants phenes toward n a t u r e o f t h e mus209 characterized product i s + amongst t h e r a d m u t a t i o n s i n S. include gamma r a y s , b u t n o t t o HN2 sterility. In t h e s e r e s p e c t s mus209 sensitivity (Henderson e t a l . , and Kunz, 1 9 8 1 ) . F o r example, e x t r e m e l y s e n s i t i v e t o MMS cells fail and t o produce v i a b l e The b i o c h e m i c a l b a s i s by PAD52 1980), and that (Resnick et a l . , chromosome l o s s latter and are diploid 1974). i s regulated levels break are defective (DSB) 1980; repair o f s p o n t a n e o u s and (Mortimer e t a l . , phenotype could 136 a (but not 1984). rad52-l c e l l s i n double-strand high group s p o r e s (Game and M o r t i m e r , r e c o m b i n a t i o n (Game e t a l . , also exhibit that this radiation, female f o r t h e s e a b n o r m a l i t i e s a p p e a r s t o be More s p e c i f i c a l l y , and m e i o t i c and resembles a r a d 5 2 - l mutants ionizing m i s s i n g endo-exonuclease a c t i v i t y encoded) cerevisiae. 1 9 8 7 ) , and number o f m u t a t i o n s b e l o n g i n g t o t h e RAD52 e p i s t a s i s (Haynes large t o MMS most c l o s e l y B 1 Thus, functions. time, perhaps the best approach for phenotypically collection t o mutagens, heteroallelic s i n g l e p o l y p e p t i d e s and n o t i n m u l t i m e r s . this look t h e same that the r e p a i r - r e l a t e d understanding the biochemical to be a m u l t i m e r i c 1981). in mitotic Prakash e ta l . , (Ho, 1975). They radiation-induced I t has been s u g g e s t e d a r i s e because u n r e p a i r e d double strand breaks i n DNA lead to telomere chromosome i n s t a b i l i t y , e t a l . , 1981). mutagen s e n s i t i v i t y (Schild o u t t o be mutation y e a s t g e n e s do deficient t o be strictly ) of mus209 . On i n DSB identified not B 1 functions t h e o t h e r hand, i f m u s 2 0 9 i t w o u l d be i n Drosophila 137 However, RAD54) b e c a u s e u n l i k e encode e s s e n t i a l repair, f o r the a n a l o g o u s t o RAD52 o r t o some ( e . g . , RAD50 and e t a l . , 1983). chromatid E i t h e r mechanism c o u l d a c c o u n t t h e mus209 gene c a n n o t be mus209, t h e s e in chromosome s e g r e g a t i o n a t m i t o s i s (and t s l e t h a l i t y o t h e r RAD52-like genes resulting or because unresolved exchanges l e a d t o a b e r r a n t (Mortimer loss, the (Dezzani B 1 first turns such e t a l . , 1982). References Ames, B.N. 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Mut., 5, 3 6 3 - 3 6 5 . a 158 in APPENDIX A Isolation All B2 of Mutaaen-Sensitive Strains mus mutants b e a r i n g the alphanumeric s u p e r s c r i p t s were i s o l a t e d isogenic b, p_r and as o u t l i n e d ' i n F i g u r e c n , were f e d e t h y l m e t h a n e s u l f o n a t e masse t o p_r T f t / CyO (where * i n d i c a t e s o r 4 p_r T f t / CyO / CyO an E M S - t r e a t e d females. from each l i n e male s i b s . flies F indicated and first with until 0.25 mL o f 0.08% eclosion. t r e a t e d w i t h 0.25 eclosion. 22°C The The mL third F± i n d i v i d u a l s . An second and set of r e p l i c a t e and absence lethal of b pr mutation, containing were stocks. 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 m u t a n t when t h e h o m o z y g o t e r h e t e r o z y g o t e ratio treated incubated at cultures c u l t u r e s was to establish fresh Individuals in i n c u b a t e d a t 29°C set of replicate 159 to 3 A d u l t s from t h e r e m a i n i n g ( i n water) o f water males heterozygotes hr post o v i p o s i t i o n ) v / v MMS en examined f o r t h e p r e s e n c e o f b every 2 days t o v i a l s (0-48 v/v) r e s p e c t i v e b p_r c 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 . subculture 0.24% f e m a l e s were induced r e c e s s i v e s u c h c u l t u r e s were d i s c a r d e d . mutations (1968) and mated c o n t a i n i n g b p_r c n / CyO c u l t u r e s were t r a n s f e r r e d the 3 carrying chromosome) were c r o s s e d and mated t o t h e i r t h e p r e s e n c e o f an medium t o o b t a i n Bacher b p r c n * / CyO 2 E a c h c u l t u r e was in cultures and (EMS, f e m a l e s . S i n g l e b pjr c n * / CyO p r c n h o m o z y g o t e s amongst t h e F cn A d u l t males, s e c o n d chromosomes marked w i t h t h e r e c e s s i v e a c c o r d i n g t o t h e method o f L e w i s collected 1. B l or 29°C was until maintained at MMS-sensitive obtained i n the F i g u r e 1. M a t i n g and selection protocol chromosome mus mutations. 160 used t o i s o l a t e second EMS P b pr cn b pr cn oo — b pr cn x . y pr Tft CyO CyO b pr cn pr Tft ^ ^ CyO y b pr cn CyO CyO b pr cn b pr cn b pr c n * ' CyO ' MMS © AAF HN2 o 22,29 161 gamma rays MMS-treated treated culture control fell culture. those s t r a i n s d i s p l a y i n g phenotype below 1/6 o f t h a t 2 A l l p u t a t i v e M M S - s e n s i t i v e mutants and a temperature-sensitive a t 2 9 ° C were r e t e s t e d MMS. A d d i t i o n a l obtained i n t h e H 0- a t 22°C extensive retesting lethal for sensitivity was c a r r i e d t o 0.08% out t o confirm t h e mutant phenotypes. A total o f 4032 p a i r matings, each r e p r e s e n t i n g E M S - t r e a t e d s e c o n d chromosome, were e s t a b l i s h e d . 83% F 2 o f these matings produced c u l t u r e s produced normal offspring a t 22°C. F 2 cultures, a single Approximately a n d a b o u t 45% o f t h e numbers o f homozygous v i a b l e b p r c n In total, 18 M M S - s e n s i t i v e s t r a i n s recovered. I n 3 subsequent studies, were t e s t e d for sensitivity t o t h e mutagens AAF, HN2 a n d gamma rays. Five AAF-sensitive, s e n s i t i v e mutants that chromosomes 5 H N 2 - s e n s i t i v e a n d 4 gamma r a y - (in addition show c r o s s - s e n s i t i v i t y t h e EMS-treated were t o those MMS-sensitive strains t o t h e s e 3 mutagens) were r e c o v e r e d . 162 APPENDIX B An E x a m i n a t i o n o f t h e I n f l u e n c e o f t h e M a t e r n a l G e n o t y p e on 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 that e t a l . (1972) p r o v i d e d some o f t h e f i r s t the maternal mutations from genotype could s e x chromosome l o s s ) X - i r r a d i a t e d sperm. They c o n c l u d e d t h a t al. DNA lesions that evidence of a maternal dominant l e t h a l mutations The availability sensitive effects and and (mus) o f DNA further, lethal maternal lethality. i n t h e o o c y t e on b o t h i n sperm (Graf t e s t , were o b s e r v e d the o r remained genotype i n mus from et little mutagenthe spontaneous a sex-linked i n some c a s e s . and Depending the type of either unchanged. can a l s o larvae t o both influence the s e n s i t i v i t y spontaneous (1978) o b s e r v e d and that of MMS-effected MMS-treated c e r t a i n r e p a i r - d e f i c i e n t females 163 of e t a l . , 1979). frequency of l e t h a l mutations G r a f and W u r g l e r larvae derived found characterized e f f e c t s , a s m e a s u r e d by decreased, somatic c e l l s Racine or t r a n s l o c a t i o n s . repair defects mutagen e m p l o y e d , The could r e p a i r of on t h e 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 u s e d , increased, o r more s t r a i n s made p o s s i b l e an e x a m i n a t i o n Pronounced maternal derived i n f l u e n c e on t h e r e c o v e r y o f of biochemically induced mutagenesis recessive one lead to aneuploidy. (1979) o b t a i n e d s i m i l a r r e s u l t s , and o r no i n zygotes f a c t o r s d e p o s i t e d i n t o t h e egg, i n f l u e n c e t h e b a l a n c e between f i x a t i o n premutational evidence influence the recovery of (specifically, maternally-derived the a r e much more s e n s i t i v e to k i l l i n g identical progeny mothers. Notably, embryonic stages. This maternal mutagen t e s t i n g Chapter 3. by MMS d e r i v e d from than are repair-proficient these maternal effects (heterozygous) extend beyond regimens s u c h a s t h e mus m o t h e r s and d e r i v e d from matings heterozygous offspring o b t a i n e d from t h e r e c i p r o c a l here With sought these issues t o determine be d e m o n s t r a t e d and dose-response 2, lower positive effects reported could i n fact mutants, m u s 2 0 5 , B 1 males. F i g u r e 1. effect that sensitive c u r v e s were g e n e r a t e d e x c e p t homozygous mus heterozygous The Clearly, mus2Q8 . B1 results mutagenesis the r e c i p r o c a l suggest that at l e a s t "factors" f a c t be mus progeny a r e shown a strong maternal some 2-3 t i m e s more ( a s m e a s u r e d by t h e LD50) t h a n t h e i r g e n o t y p i c i n agreement w i t h t h e e a r l i e r (1978) and of these experiments r e n d e r s homozygous mus t o MMS as d e s c r i b e d i n f e m a l e s were c r o s s e d t o each mutant e x h i b i t s c o u n t e r p a r t s d e r i v e d from in Thus, a B 1 Chapter are similar i n mind, t h e e x p e r i m e n t s whether maternal more mus210 . MMS in mating. t o e f f e c t t h e same for three single between f a t h e r s s h o u l d be t o a s u s p e c t mutagen t h a n g e n o t y p i c a l l y d o s e o f mutagen c o u l d be u s e d to test described i n sensitive response. the 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 In p r i n c i p a l , o f f s p r i n g homozygous mus genotypically cross. results o f G r a f and ( e . g . , DNA 164 findings Wurgler some o f t h e m a t e r n a l l y - d e r i v e d h y p o t h e s i z e d by W u r g l e r gene p r o d u c t s These repair e t a l . (1972) enzymes). may Figure 1. MMS sensitivity mus208 males and m u s 2 1 0 . B 1 matings and m a t e r n a l Progeny B 1 between h e t e r o z y g o u s ( s o l i d symbols) effects i n mus205 . B 1 were d e r i v e d f e m a l e s a n d homozygous mus o r homozygous mus f e m a l e s and heterozygous males (open s y m b o l s ) . Data c r o s s e s were t a k e n from C h a p t e r Circles, (1455, 1337); triangles, squares, mus210 B 1 (3893, parentheses p e r t a i n 2. mus208 3531). t o matings average, non-zero dose point, 2175); The numbers i n d i c a t e Survival ratio i n Chapter values 2 (MATERIALS AND B 1 (2042); m u s 2 0 8 . B 1 0.99 (3007) a n d m u s 2 1 0 . B 1 The numbers i n p a r e n t h e s e s number o f c o n t r o l flies 0.86 The e r r o r o f t h e mean 3-6 s e p a r a t e t r e a t m e n t s . 165 0.94 indicate the total scored p e r genotype. 1 standard deviation from scored per obtained i n the untreated The l a t t e r v a l u e s a r e : m u s 2 0 5 . calculated B 1 c u l t u r e s have been n o r m a l i z e d t o t h e c o n t r o l s as d e s c r i b e d bars indicate mus205 b e t w e e n homozygous respectively. homozygoterheterozygote (3062). (3098, B 1 and t h e minimum number o f f l i e s from t h e t r e a t e d METHODS). f o r t h e former The d a t a i n f e m a l e s and h e t e r o z y g o u s m a l e s . the from 0.01 0.02 % MMS 166 0.03 0.04
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A genetic analysis of mutagen-sensitive mutations on the second chromosome of Drosophila melanogaster Henderson, Daryl Stewart 1987
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Title | A genetic analysis of mutagen-sensitive mutations on the second chromosome of Drosophila melanogaster |
Creator |
Henderson, Daryl Stewart |
Publisher | University of British Columbia |
Date Issued | 1987 |
Description | Mutagen-sensitive (mus) mutations in Drosophila melanogaster render developing flies hypersensitive to the lethal effects of DNA-damaging agents. In general, mus mutations identify DNA repair-related genes. In this study, 5 new second chromosome mus mutations (mus205B¹, mus208B¹, mus209B¹, mus210B¹ and mus211B¹), selected on the basis of sensitivity to methyl methanesulfonate (MMS), were characterized using a variety of genetic tests. One test measured the MMS-sensitivity of double mutant mus strains compared to their component single mutants. Mutant interactions were examined in 8 double mus and in 2 triple mus strains containing combinations of mus201D¹, mus205B¹, mus208B¹, mus210B¹ and mus211B¹ (or mus211B²). These analyses have revealed predominantly synergistic and epistatic responses to MMS. Taken together with the findings of previous genetic and biochemical studies of Drosophila mus strains, these results suggest that 3 major repair pathways may operate in flies to correct damage caused by MMS. Mutagen cross-sensitivity data and the results of the interaction studies suggest that mus mutations might serve as rapid and sensitive bioassays of somatic genotoxicity caused by mutagens and carcinogens. To explore this possibility, a simple mutagen test system was devised employing triple mutant mus strains. One strain (mus208B¹ mus210B¹ mus211B²) was tested for sensitivity to 14 mutagens/carcinogens and 2 non-carcinogens. Eleven of the mutagens/carcinogens were readily detected as genotoxic. Both non-carcinogens were non-genotoxic. These preliminary results demonstrate the feasibility (and some limitations) of the proposed somatic genotoxicity assay and emphasize the need for further test validation using a larger chemical data base. The temperature-sensitive lethal mutation mus209B¹ was subjected to extensive genetic analyses and to temperature shift experiments during development. This locus was found to encode a product(s) that (1) is essential for viability at virtually all pre-imaginal developmental stages (the latter half of pupation appears to be an exception), (2) is necessary for wildtype levels of resistance to the genotoxic effects of MMS and ionizing radiation, and (3) is required for female fertility. Confirmation of the pleiotropic nature of this mutation was obtained by meiotic and cytogenetic mapping studies and by complementation tests with a series of allelic mutations. The mus209B¹ phenotypes are similar to ones conferred by mutations in Drosophila and yeast that disrupt various aspects of chromosome metabolism. In this context, some possible roles for mus209B¹ are discussed. |
Subject |
Genetic code Chromosome abnormalities Mutagens -- analysis Mutagenesis Drosophila melanogaster -- Genetics |
Genre |
Thesis/Dissertation |
Type |
Text |
Language | eng |
Date Available | 2010-07-14 |
Provider | Vancouver : University of British Columbia Library |
Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
DOI | 10.14288/1.0096960 |
URI | http://hdl.handle.net/2429/26418 |
Degree |
Master of Science - MSc |
Program |
Zoology |
Affiliation |
Science, Faculty of Zoology, Department of |
Degree Grantor | University of British Columbia |
Campus |
UBCV |
Scholarly Level | Graduate |
Aggregated Source Repository | DSpace |
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