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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. (1979) I d e n t i f y i n g e n v i r o n m e n t a l c h e m i c a l s m u t a t i o n s and c a n c e r , S c i e n c e , 204, 587-593.  causing  Ames, B.N. 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(1985) The r o l e o f 0 - m e t h y l g u a n i n e - D N A m e t h y l t r a n s f e r a s e i n c e l l s u r v i v a l , m u a t g e n e s i s and c a r c i n o g e n e s i s , M u t a t . R e s . , 145, 1-16. 6  Yeung, A.T., B.K. J o n e s , M. C a p r a r o a n d T. Chu (1987) T h e r e p a i r o f p s o r a l e n m o n o a d d u c t s by E s c h e r i c h i a c o l i UvrABC e n d o n u c l e a s e , N u c l . A c i d s R e s . , 15, 4957-4971. Yeung, A.T., W.B. M a t t e s , E.Y. Oh a n d L. Grossman (1983) Enzymatic p r o p e r t i e s o f the p u r i f i e d E s c h e r i c h i a c o l i u v r A B C c o m p l e x , i n : E.C. F r i e d b e r g a n d B.A. B r i d g e s (Eds.) C e l l u l a r R e s p o n s e s t o DNA Damage, A.R. L i s s , New Y o r k , pp. 77-86. 157  Z a r b l , H., S. S u k u m a r , A . L . A r t h u r , D. M a r t i n - Z a n c a a n d M. B a r b a c i d (1986) A c t i v a t i o n o f H - r a s - 1 oncogenes by chemical c a r c i n o g e n s , i n : M.G. S i m i c , L. G r o s s m a n a n d A.C. Upton ( E d s . ) M e c h a n i s m s o f DNA Damage a n d R e p a i r : I m p l i c a c t i o n s f o r C a r c i n o g e n e s i s a n d R i s k A s s e s s m e n t , P l e n u m , New York, pp. 385-397. Z i m m e r i n g , S. ( 1 9 8 2 ) A n o t e o n t h e u t i l i t y o f r e p a i r - d e f i c i e n t s t mus302 D r o s o p h i l a f e m a l e s i n d e t e c t i n g chromosome l o s s a n d s e x - l i n k e d r e c e s s i v e l e t h a l s i n d u c e d i n t h e m a l e genome b y m e t h y l m e t h a n e s u l f o n a t e (MMS) and dimethylnitrosamine (DMN), E n v i r o n . Mut., 4, 2 9 1 - 2 9 3 . Z i m m e r i n g , S. ( 1 9 8 3 ) T h e m e i - 9 t e s t f o r c h r o m o s o m e l o s s D r o s o p h i l a : A r e v i e w o f a s s a y s o f 21 c h e m i c a l s for chromosome b r e a k a g e , E n v i r o n . 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