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The effect of gamma radiation on recombination frequency in Caenorhabditis elegans Kim, Jong Sun 1985

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c / THE EFFECT OF GAMMA RADIATION ON RECOMBINATION FREQUENCY IN CAENORHABDITIS ELEGANS By JONG SUN KIM B.Sc, Yon-Sei University, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES GENETICS PROGRAMME We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d Dr. A. M. Ros-e A d v i s o r Dr> F. D L i l A d v i s o r y Committee Dr. D. H"ol{n, Advisioxy Committee Dr. D. B a i l l i e , A d v i s o r y Committee D r . D . S " u r i l b l f . Examiner Dr. S. Wood," Chairman THE UNIVERSITY OF BRITISH COLUMBIA December 1985 Jong Sun Kim, 1985 T h i s t h e s i s may not be r e p r o d u c e d i n whole o r p a r t , by photocopy o r o t h e r means, w i t h o u t p e r m i s s i o n o f the a u t h o r . +8 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a gree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Medical Genetics The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date January 15, 1986 ABSTRACT Treatment w i t h i o n i z i n g r a d i a t i o n i s known t o cause chromosome breakage i n many organisms i n c l u d i n g C a e n o r h a b d i t i s e l e g a n s . I n a d d i t i o n , gamma r a d i a t i o n i n c r e a s e s r e c o m b i n a t i o n f r e q u e n c y i n D r o s o p h i l a m e l a n o g a s t e r . I n o r d e r t o i n v e s t i g a t e t h e g e n e r a l i t y of r a d i a t i o n - i n d u c e d r e c o m b i n a t i o n , I have u n d e r t a k e n t o stu d y t h e e f f e c t o f gamma r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y i n C. e l e g a n s . T h i s i s the f i r s t s t udy t o d e s c r i b e r a d i a t i o n - i n d u c e d r e c o m b i n a t i o n i n C. e l e g a n s . R a d i a t i o n doses o f 2K rads i n c r e a s e d r e c o m b i n a t i o n f r e q u e n c y i n t h e dpy-5 unc-13 i n t e r v a l o f LGI_ a p p r o x i m a t e l y t w o - f o l d . The amount of the i n c r e a s e was a f f e c t e d by t h e de v e l o p m e n t a l s t a g e of gonads a t the time of r a d i a t i o n t r e a t m e n t and by r a d i a t i o n dose. X-chromosome n o n d i s j u n c t i o n was a l s o i n c r e a s e d by r a d i a t i o n t r e a t m e n t . A h i g h f r e q u e n c y of t h e recombinant progeny produced w i t h r a d i a t i o n t r e a t m e n t were s t e r i l e u n l i k e t h e i r nonrecombinant s i b l i n g s . When parameters a f f e c t i n g r e c o m b i n a t i o n f r e q u e n c y a r e h e l d c o n s t a n t , chromosomal r e g i o n s w i t h i n t h e gene c l u s t e r s of t he m e i o t i c map i n c r e a s e d most a f t e r r a d i a t i o n t r e a t m e n t . However, none of t h e i n c r e a s e s were of t h e magnitude d e s c r i b e d f o r c e n t r i c h e t e r o c h r o m a t i n i n D r o s o p h i l a . i i Acknowledgements I would l i k e t o s p e c i a l l y thank my s u p e r v i s o r , Dr. A Rose f o r her encouragement, p a t i e n c e , and i n v a l u a b l e g uidance d u r i n g my r e s e a r c h . I am a l s o t h a n k f u l t o my c o l l e a g u e s , Ann M a r i e H o w e l l and L i n d a H a r r i s ; t o Dr. F. D i l l f o r c y t o l o g i c a l e x p e r t i s e and d i s c u s s i o n ; t o my s u p e r v i s o r y committee f o r t h e i r generous t i m e : Dr. D.L. B a i l l i e , Dr. F. D i l l , Dr. D. Holm, and Dr. P. B a i r d ; and R a j a R o s e n b l u t h f o r comments on the t h e s i s . i i i TABLE OF CONTENTS A p p r o v a l i ABSTRACT i i ACKNOWLEDGEMENTS . - i i i L i s t of T a b l e s v i L i s t o f F i g u r e s * v i i i INTRODUCTION 1 MATERIALS AND METHODS 6 I . Nematode S t r a i n s and C u l t u r e C o n d i t i o n s 6 I I . M a t i n g P r o t o c o l and R a d i a t i o n Treatment 8 I I I . M e a s uring Recombination Frequency 10 a. I d e n t i f i c a t i o n of Recombinants 10 b. C a l c u l a t i o n of Reco m b i n a t i o n Frequency 10 IV. M e a s u r i n g R a d i a t i o n Dose Response.... 12 V. Measuring F1 S t e r i l i t y 12 V I . M e a s u r i n g Frequency of X-chromosome N o n d i s j u n c t i o n ..15 a. D e t e r m i n a t i o n of N o n d i s j u n c t i o n Frequency 15 b. Chromosome Loss o r N o n d i s j u n c t i o n 15 V I I . Developmental Stages 16 a. E f f e c t on r e c o m b i n a t i o n f r e q u e n c y 16 b. C y t o l o g y of gonads 16 c. E f f e c t on sper m a t o g e n e s i s o r o o g e n e s i s 17 V I I I . Chromosomal R e g i o n a l i t y 19 i v RESULTS 20 I. E f f e c t of Radiation on Recombination Frequency 20 a. Brood Analysis 20 b. Radiation Dose Response 23 c. Developmental Stages 26 i . Time of Treatment & Recombination Frequency ..26 i i . Cytology of Gonads 29 i i i . E f f e c t on Spermatogenesis or Oogenesis 33 d. S t e r i l i t y of Recombinants 33 II . E f f e c t of Radiation on X-chromosome Nondisjunction ...35 a. Rate of Non-disjunction 35 b. Stage E f f e c t on Nondisjunction 38 III. Comparison of Recombination and Nondisjunction 41 IV. Chromosomal Regionality 41 a. Recombination i n the Clustered Region 41 b. Recombination i n a Region Adjacent to the Cluster..46 c. Recombination at the Left-end of the Chromosome ..46 d. Summary Regarding Recombination at Different Intervals 53 e. Nondisjunction Curves at Different Intervals 53 DISCUSSION 56 PROPOSALS FOR FURTHER RESEARCH ...65 APPENDIX 1 The Genetic Map of C. elegans JO APPENDIX 2 The Gene Cluster on LG I of C. elegans <Y1 BIBLIOGRAPHY 66 v T a b l e 1. L i s t o f D o u b l e M u t a n t S t r a i n s U s e d i n t h i s S t u d y ... 7 L I S T OF TABLES  . T a b l e 2. D e s c r i p t i o n o f R e c o m b i n a n t s O b s e r v e d i n t h e E x p e r i m e n t s ...11 T a b l e 3. E f f e c t o f R a d i a t i o n on R e c o m b i n a t i o n F r e q u e n c y i n t h e d p y - 5 u n c - 1 3 I n t e r v a l 20 T a b l e 4. R a d i a t i o n Dose R e s p o n s e ....24 T a b l e 5. S t a g e E x p e r i m e n t ( I ) i n t h e d p y - 5 u n c - 1 3 I n t e r v a l ..26 T a b l e 6. S t a g e E x p e r i m e n t ( I I ) i n t h e dpy-5 dpy-14 I n t e r v a l ..29 T a b l e 7. D e v e l o p m e n t o f t h e R e p r o d u c t i v e S y s t e m o f C a e n o r h a b d i t i s e l e g a n s 31 T a b l e 8. E f f e c t on S p e r m a t o g n e n s i s o r O o g e n e s i s 33 T a b l e 9. E f f e c t o f R a d i a t i o n o n S t e r i l i t y o f R e c o m b i n a n t s ...35 v i T a b l e 10. E f f e c t of R a d i a t i o n on X-chromosome N o n d i s j u n c t i o n Frequency 38 T a b l e 11. E f f e c t of R a d i a t i o n on X-chromosome N o n d i s j u n c t i o n a t D i f f e r e n t Stages 41 T a b l e 12. Comparison of R e c o m b i n a t i o n and N o n d i s j u n c t i o n 43 Ta b l e 13. E f f e c t of R a d i a t i o n on R e c o m b i n a t i o n Frequency i n the C l u s t e r e d Regions 46 Ta b l e 14. E f f e c t of R a d i a t i o n on R e c o m b i n a t i o n Frequency i n t h e dpy-5 unc-11 I n t e r v a l 48 T a b l e 15. E f f e c t of R a d i a t i o n on R e c o m b i n a t i o n Frequency i n t h e b l i - 3 unc-35 I n t e r v a l 50 T a b l e 16. Summary R e g a r d i n g R e c o m b i n a t i o n a t D i f f e r e n t I n t e r v a l s 53 T a b l e 17. E f f e c t of R a d i a t i o n on N o n d i s j u n c t i o n Frequency a t D i f f e r e n t Regions 54 v i i * LIST OF FIGURES F i g u r e 1. M a t i n g P r o t o c o l f o r G e n e r a t i n g O f f s p r i n g Which C a r r y Recombinant Chromosomes 9 F i g u r e 2. Method f o r C a c u l a t i n g the R e c o m b i n a t i o n Frequency from Rose (1 980) 13 F i g u r e 3. Method f o r M e a s u r i n g R e c o m b i n a t i o n Frequency i n Hermaphrodite Oocytes 17 F i g u r e 4. E f f e c t of R a d i a t i o n on R e c o m b i n a t i o n Frequency f o r the dpy-5 unc-13 I n t e r v a l 21 F i g u r e 5. R a t i o of E x p e r i m e n t a l t o C o n t r o l f o r R e c o m b i n a t i o n Frequency f o r t h e dpy-5 unc-13 I n t e r v a l 23 F i g u r e 6. E f f e c t o f R a d i a t i o n on R e c o m b i n a t i o n Frequency f o r t h e dpy-5 unc-13 I n t e r v a l a t D i f f e r e n t D evelopmental Stages 27 F i g u r e 7. E f f e c t o f R a d i a t i o n on R e c o m b i n a t i o n Frequency f o r t h e dpy-5 dpy-14 I n t e r v a l a t D i f f e r e n t D evelopmental Stages 30 v i i i F i g u r e 8. E f f e c t o f R a d i a t i o n on S t e r i l i t y o f Recombinants a t 36-hr P r i o r t o E g g - l a y i n g Stage 36 F i g u r e 9. E f f e c t o f R a d i a t i o n on N o n d i s j u n c t i o n Frequency a t 12-hr P r i o r t o E g g - l a y i n g Stage 39 F i g u r e 10. E f f e c t o f R a d i a t i o n on N o n d i s j u n c t i o n Frequency f o r t h e dpy-5 unc-13 I n t e r v a l a t D i f f e r e n t D evelopmental Stages 42 F i g u r e 11. Comparison of t h e E f f e c t o f R a d i a t i o n on t h e Frequency of R e c o m b i n a t i o n and N o n d i s j u n c t i o n 44 F i g u r e 12. The Magnitude o f I n c r e a s e i n R e c o m b i n a t i o n Frequency w i t h R a d i a t i o n Treatment i n t h e Gene C l u s t e r 47 F i g u r e 1-3. E f f e c t o f R a d i a t i o n on R e c o m b i n a t i o n Frequency a c r o s s the dpy-5 unc-11 I n t r e v a l 49 F i g u r e 14. E f f e c t of R a d i a t i o n on R e c o m b i n a t i o n Frequency a c r o s s t h e b l i - 3 unc-35 I n t e r v a l 51 F i g u r e 15. E f f e c t o f R a d i a t o n on N o n d i s j u n c t i o n Frequency f o r D i f f e r e n t Regions 55 ix 1 INTRODUCTION G a m m a - r a d i a t i o n i s a n i o n i z i n g r a d i a t i o n known t o c a u s e c h r o m o s o m a l b r e a k s i n many o r g a n i s m s r e s u l t i n g i n c h r o m o s o m a l r e a r r a n g e m e n t s s u c h a s d e l e t i o n s and t r a n s l o c a t i o n s . I n a d d i t i o n t o c r e a t i n g c h r o m o s o m a l r e a r r a n g e m e n t s , i o n i z i n g r a d i a t i o n i s known t o i n c r e a s e c r o s s i n g - o v e r i n D r o s o p h i l a  m e l a n o g a s t e r ( M u l l e r , 1 9 2 5 ) . The e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y i n D r o s o p h i l a m e l a n o g a s t e r h a s b e e n e x t e n s i v e l y s t u d i e d ( M u l l e r , 1 9 2 5 ; P a t t e r s o n a nd S u c h e , 1 9 3 4 ; W h i t t i n g h i l l , 1 9 5 1 ; R o b e r t s , 1969; M g l i n e t s , 1 9 7 2 , 1 9 7 3 ; H a e n d l e , 1979; T a t t e r s a l l , 1 9 8 1 ) . I n o r d e r t o i n v e s t i g a t e t h e g e n e r a l i t y o f t h i s e f f e c t w i t h r e g a r d t o o t h e r o r g a n i s m s , I h a v e s t u d i e d t h e e f f e c t o f g a m m a - r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y i n C a e n o r h a b d i t i s e l e g a n s . I n C. e l e g a n s i t h a s b e e n d e m o n s t r a t e d t h a t i o n i z i n g r a d i a t i o n g e n e r a t e s c h r o m o s o m a l r e a r r a n g e m e n t s ( R o s e n b l u t h , C u d d e f o r d and B a i l l i e , 1 9 8 5 ) . However, p r i o r t o t h e w o r k p r e s e n t e d i n t h i s t h e s i s , t h e e f f e c t o f g a m m a - r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y i n C. e l e g a n s h a s n o t b e e n r e p o r t e d . G e n e t i c s t u d i e s i n D. m e l a n o g a s t e r h a v e d e m o n s t r a t e d a c o r r e l a t i o n b e t w e e n t h e c e n t r o m e r i c h e t e r o c h r o n y t i n a n d r a d i a t i o n - i n d u c e d map e x p a n s i o n ( i n c r e a s e d r e c o m b i n a t i o n f r e q u e n c y ) . V e r y l a r g e i n c r e a s e s i n r e c o m b i n a t i o n f r e q u e n c y h a v e b e e n o b s e r v e d p r i m a r i l y i n r e g i o n s composed o f c e n t r i c h e t e r o c h r o m a t i n ( T a t t e r s a l l , 1 9 8 1 ) . The m a g n i t u d e o f t h e i n c r e a s e was a t l e a s t 2 t o 6 - f o l d i n h e t e r o c h r o m a t i n a s c o m p a r e d t o i n c r e a s e s f r o m 0 t o 1 . 5 - f o l d i n e u c h r o m a t i c r e g i o n s . 2 S i n c e r a d i a t i o n has been shown t o a l t e r r e c o m b i n a t i o n p r i m a r i l y i n t h e c e n t r i c h e t e r c h o m a t i n r e g i o n of D. m e l a n o g a s t e r chromosomes, i t might be p o s s i b l e t o d e t e c t r e g i o n s of c e n t r i c h e t e r o c h r o m a t i n i n C. e l e g a n s chromosomes u s i n g r a d i a t i o n - i n d u c e d map e x p a n s i o n . For example, one c o u l d attempt t o s t u d y whether r a d i a t i o n d r a m a t i c a l l y i n c r e a s e s r e c o m b i n a t i o n i n r e g i o n s o f C. e l e g a n s chromosomes as i t does i n r e g i o n s o f h e t e r o c h r o m a t i n i n D. m e l a n o g a s t e r . The g e n e t i c map of C. e l e g a n s , which i s based on m e i o t i c r e c o m b i n a t i o n f r e q u e n c i e s , shows c l u s t e r s o f genes on the autosomes. That i s , most of t h e genes wh i c h were i d e n t i f i e d i n B r e n n e r ' s (1974) o r i g i n a l s c r e e n a r e c l u s t e r e d w i t h i n a r e g i o n of a few map u n i t s on each autosome. See Appendix 1. For example, on L i n k a g e Group (LG) I_ t h e r e a r e 30 genes l o c a t e d i n the unc-11 t o unc-13 i n t e r v a l , an i n t e r v a l o f 4 map u n i t s ; compared t o 3 genes l o c a t e d i n t h e a d j a c e n t unc-35 t o unc-11 i n t e r v a l , an i n t e r v a l o f 12 map u n i t s . The dpy-5 unc-13 r e g i o n d e s c r i b e d i n t h i s t h e s i s i s i n t h e LG I_ "gene c l u s t e r ' . Brenner (1 974) s u g g e s t e d t h a t t h e s e c l u s t e r s were th e r e s u l t o f s u p p r e s s e d r e c o m b i n a t i o n f r e q u e n c y i n d e f i n e d chromosomal r e g i o n s . I n D. m e l a n o g a s t e r , t h e a r e a o f c e n t r o m e r i c h e t e r o c h r o m a t i n i s known t o be a r e g i o n o f low r e c o m b i n a t i o n f r e q u e n c y r e l a t i v e t o t h e amount of DNA (Yeomans, 1979; G r e l l , 1978). S t u d i e s on C. e l e g a n s m i t o t i c chromosomes suggest t h a t t h e r e i s no s i n g l e s i t e f o r s p i n d l e attachment ( A l b e r t s o n and Thomson, 1982) s u g g e s t i n g t h e p o s s i b l e absence o f c e n t r i c h e t e r o c h r o m a t i n i n t h i s o rganism. The m e i o t i c chromosomes a r e 3 however t o o s m a l l t o s t u d y c y t o l o g i c a l l y . A l t h o u g h some a s p e c t s of the d e s c r i p t i v e c y t o l o g y of chromosomes i n C. e l e g a n s have been s t u d i e d (Nigon and Brun, 1 9 5 5 ; A l b e r t s o n and Thomson, 1 9 8 2 ; Abi-Rached and B r u n , 1 9 7 9 ; G o l d s t e i n , 1 9 8 2 ; G o l d s t e i n and S l a t o n , 1 9 8 2 ) , t h e o v e r a l l s t r u c t u r e o f the C. e l e g a n s chromosomes i s p o o r l y u n d e r s t o o d a t t h i s t i m e . S i n c e the amount of r e c o m b i n a t i o n i n d u c e d w i t h r a d i a t i o n seems t o be p r o p o r t i o n a l t o t h e amount of DNA p r e s e n t ( r a t h e r t h a n r e l a t e d t o t h e amount of m e i o t i c r e c o m b i n a t i o n ) , s t u d y i n g the e f f e c t s of r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y a c r o s s a C. e l e g a n s chromosome might p r o v i d e i n s i g h t i n t o t h e a r c h i t e c t u r e of t h e s e chromosomes. The work r e p o r t e d here i s an attempt t o c h a r a c t e r i z e a s p e c t s o f chromosomal o r g a n i z a t i o n u s i n g i o n i z i n g r a d i a t i o n . Having a method f o r d e t e r m i n i n g the r e l a t i v e amounts of DNA p r e s e n t i n d i f f e r e n t chromosomal r e g i o n s i s i m p o r t a n t w i t h r e g a r d t o u n d e r s t a n d i n g t h e o r g a n i z a t i o n of genes i n C. e l e g a n s chromosomes. On t h e o t h e r hand, t h e s t u d y o f m e i o t i c r e c o m b i n a t i o n i s i t s e l f i n t r i n s i c a l l y i n t e r e s t i n g . I n t h i s r e g a r d C. e l e g a n s p r o v i d e s a model system i n w h i c h t o s t u d y a s p e c t s of m e i o t i c r e c o m b i n a t i o n . T h i s organism i s w e l l s u i t e d f o r such s t u d i e s s i n c e g e n e r a l l y i t i s a good e x p e r i m e n t a l system and s i n c e s p e c i f i c a l l y t h e r e e x i s t s an enhancer o f m e i o t i c r e c o m b i n a t i o n . C a e n o r h a b d i t i s e l e g a n s i s an e x c e l l e n t e x p e r i m e n t a l g e n e t i c system f o r a number of reas o n s and has become th e model organi s m o f c h o i c e f o r many t y p e s o f s t u d i e s : i t has a s h o r t g e n e r a t i o n t i m e ( 3 . 5 days a t 2 0°C); i t has a l a r g e number 4 o f o f f s p r i n g ( e a c h a d u l t w i l d - t y p e h e r m a p h r o d i t e p r o d u c e s 250-300 o f f s p r i n g ) ; a nd i t h a s a s m a l l genome s i z e (300 map 7 u n i t s a nd 8x10 bp o f DNA). I n a d d i t i o n , C. e l e g a n s i s e a s y t o m a i n t a i n i n t h e l a b o r a t o r y and h a s t h e a d v a n t a g e t h a t g e n e t i c s t o c k s c a n be f r o z e n i n l i q u i d n i t r o g e n . S i n c e C. e l e g a n s i s a s e l f - f e r t i l i z i n g h e r m a p h r o d i t e , p o p u l a t i o n s t e n d t o become homozygous ( g e n e t i c a l l y i d e n t i c a l i n d i v i d u a l s ) . M a l e s a r e p r o d u c e d s p o n t a n e o u s l y by X-chromosome n o n d i s j u n c t i o n ( H o d g k i n , H o r v i t z a n d B r e n n e r , 1 9 7 9 ) . H e r m a p h r o d i t e s h a v e f i v e p a i r s o f a u t o s o m e s a n d one p a i r o f s e x chromosomes ( X X ) , w h e r e a s m a l e s h a v e f i v e p a i r s o f a u t o s o m e s and o n l y one X-chromosome ( X O ) . G e n e t i c a n a l y s i s r e l i e s on t h e e x i s t e n c e o f m a l e s , s i n c e t h e g e n e t i c m a r k e r s f r o m i n d e p e n d e n t l y i s o l a t e d m u t a n t h e r m a p h r o d i t e s c a n be t r a n s f e r r e d t o t h e f o l l o w i n g g e n e r a t i o n when m a l e s a r e c r o s s e d t o h e r m a p h r o d i t e s . A s t r a i n ( R e c - 1 ) h a s b e e n i s o l a t e d a n d c h a r a c t e r i z e d w h i c h h a s a t h r e e - f o l d i n c r e a s e i n m e i o t i c r e c o m b i n a t i o n ( R o s e a n d B a i l l i e , 1 979b; R o s e , 1980; R a t t r a y and R o s e , u n p u b l i s h e d r e s u l t s ) . I n c o n t r a s t t o t h e e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y i n D. m e l a n o g a s t e r , Rec-1 i n C. e l e g a n s shows i n c r e a s e d r e c o m b i n a t i o n f r e q u e n c i e s t h r o u g h o u t t h e genome ( R o s e a n d B a i l l i e , 1 9 79b; R o s e , u n p u b l i s h e d d a t a ) . T h e r e f o r e , r e c o m b i n a t i o n f r e q u e n c y may be i n c r e a s e d i n Rec-1 t h r o u g h d i f f e r e n t m e c h a n i s m s o r a l t e r n a t i v e l y , C. e l e g a n s chromosomes may h a v e a d i f f e r e n t o r g a n i z a t i o n t h a n D. m e l a n o g a s t e r c h romosomes. The e x i s t i n g map o f C. e l e g a n s g e n e s i s b a s e d on 5 r e c o m b i n a t i o n b e t w e e n g e n e s a l o n g e a c h l i n k a g e g r o u p . I n o r d e r t o s t a n d a r d i z e t h e c o n d i t i o n s f o r m e a s u r i n g r e c o m b i n a t i o n f r e q u e n c i e s , R o s e a n d B a i l l i e ( 1 9 7 9 a ) h a v e s t u d i e d some f a c t o r s a f f e c t i n g r e c o m b i n a t i o n . They f o u n d t h a t r e c o m b i n a t i o n f r e q u e n c y i n c r e a s e d w i t h t e m p e r a t u r e a n d d e c r e a s e d w i t h p a r e n t a l age a n d s u g g e s t e d t h a t i n o r d e r t o s t a n d a r d i z e m a p p i n g p r o c e d u r e s c o n t r o l l e d t e m p e r a t u r e s o f 20°C a n d s c o r i n g o f a l l t h e p r o g e n y be a d o p t e d . S i m i l a r l y i n o r d e r t o make c o m p a r i s o n s o f t h e e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n a c r o s s d i f f e r e n t c h r o m o s o m a l r e g i o n s , i t i s n e c e s s a r y t o s t a n d a r d i z e t h e c o n d i t i o n s u n d e r w h i c h r a d i a t i o n t r e a t m e n t i s a p p l i e d . The i n i t i a l o b j e c t i v e o f t h e r e s e a r c h r e p o r t e d i n t h i s t h e s i s was t o e x a m i n e t h e e f f e c t o f i o n i z i n g r a d i a t i o n o n r e c o m b i n a t i o n f r e q u e n c y w i t h i n t h e c l u s t e r o f L i n k a g e G r o u p I. S i n c e r e c o m b i n a t i o n w i t h r e g a r d t o t e m p e r a t u r e a n d p a r e n t a l age a c r o s s t h e d p y - 5 u n c - 1 3 i n t e r v a l h a s b e e n w e l l c h a r a c t e r i z e d ( R o s e a n d B a i l l i e , 1 9 7 9 a ) , t h i s r e g i o n was c h o s e n t o c h a r a c t e r i z e f a c t o r s a f f e c t i n g t h e i n c r e a s e . The g o a l s o f t h i s r e s e a r c h w e re t o d e t e r m i n e : 1) i f t h e d p y - 5 u n c - 1 3 r e g i o n o f t h e C. e l e g a n s g e n e t i c map w o u l d show i n c r e a s e d r e c o m b i n a t i o n f r e q u e n c y a f t e r r a d i a t i o n t r e a t m e n t , 2) t h e p a r a m e t e r s a f f e c t i n g t h e amount o f t h e i n c r e a s e ; 3) i f t h e amount o f t h e i n c r e a s e v a r i e d w i t h r e g i o n ; 4) i f t h e m a g n i t u d e o f t h e i n c r e a s e was c o m p a r a b l e t o t h a t s e e n i n t h e c e n t r i c h e t e r o c h r o m a t i n i n D. m e l a n o g a s t e r a f t e r r a d i a t i o n t r e a t m e n t 6 MATERIALS AND METHODS I . NEMATODE STRAINS AND CULTURE CONDITIONS. W i l d - t y p e C a e n o r h a b d i t i s e l e g a n s v a r . B r i s t o l , s t r a i n N2 a n d m u t a n t s t r a i n s (BC) w e r e o b t a i n e d f r o m t h e s t o c k c o l l e c t i o n a t Simon F r a s e r U n i v e r s i t y ( T a b l e 1 ) . KR s t r a i n s w e r e c o n s t r u c t e d h e r e f r o m s t r a i n s o b t a i n e d e i t h e r f r o m t h e M e d i c a l R e s e a r c h C o u n c i l , C a m b r i d g e , E n g l a n d o r t h e C a e n o r h a b d i t i s G e n e t i c s C e n t e r , U n i v e r s i t y o f M i s s o u r i , C o l u m b i a . The s t r a i n s w e r e m a i n t a i n e d a n d m a t e d on p e t r i p l a t e s c o n t a i n i n g n e m a tode g r o w t h medium (NGM) s t r e a k e d w i t h OP50 ( B r e n n e r , 1 9 7 4 ) . OP50 i s a u r a c i l - r e q u i r i n g m u t a n t o f E s c h e r i c h i a c o l i , u s e d t o p r e v e n t o v e r g r o w t h o f t h e b a c t e r i a l l a w n . The medium c o n t a i n s l i m i t e d u r a c i l so t h a t t h e b a c t e r i a c a n n o t o v e r g r o w a n d o b s c u r e t h e worms. C r o s s e s w e r e c a r r i e d o u t e i t h e r on 10x35 mm o r 10x60 mm p e t r i p l a t e s . S i n c e t e m p e r a t u r e a n d p a r e n t a l a g e a f f e c t r e c o m b i n a t i o n f r e q u e n c y i n C. e l e g a n s , a l l e x p e r i m e n t s w e r e p e r f o r m e d a t a s t a n d a r d t e m p e r a t u r e o f 20°C a n d a l l t h e p r o g e n y o f a n i n d i v i d u a l w e r e s c o r e d a s recommended b y R o s e a n d B a i l l i e ( 1 9 7 9 a ) . E a r l y e x p e r i m e n t s i n v o l v i n g t h e d p y - 5 u n c - 1 3 i n t e r v a l w e r e done u s i n g t h e BC 26 s t r a i n ( T a b l e 3 ) . S u b s e q u e n t e x p e r i m e n t s u s e d BC 41 5. 7 T a b l e 1 L i s t o f Double Mutant S t r a i n s Used i n t h i s Study Genotype LG A l l e l e s S t r a i n Name dpy-5 unc-13 I e61 e51 BC 26 dpy-5 unc-13 I e61 e450 BC 415 dpy-5 dpy-14 I e61 e1 88 BC 1 96 dpy-14 unc-13 I e1 88 e450 BC 69 dpy-5 unc-11 I e61 e47 BC 260 b l i - 3 unc-35 I e579 e259 KR 286 8 I I . MATING PROTOCOL AND RADIATION TREATMENT. C a e n o r h a b d i t i s e l e g a n s i s a s e l f - f e r t i l i z i n g h e r m a p h r o d i t e . I n d i v i d u a l d o u b l y m u t a n t h e r m a p h r o d i t e s ( T a b l e 1) w e r e c r o s s e d t o w i l d - t y p e m a l e s . The m a t e d h e r m a p h r o d i t e s p r o d u c e d two t y p e s o f p r o g e n y , t h o s e r e s u l t i n g f r o m s e l f - f e r t i l i z a t i o n a n d f r o m f e r t i l i z a t i o n w i t h m a l e s p e r m . I n o r d e r t o g e n e r a t e a p p r o p r i a t e h e t e r o z y g o u s h e r m a p h r o d i t e s , m a t i n g was c a r r i e d o u t by p l a c i n g 20-25 homozygous m u t a n t h e r m a p h r o d i t e s w i t h 30-40 w i l d - t y p e m a l e s on a 60mm p e t r i p l a t e ( F i g u r e 1 ) . 24 h o u r s l a t e r m a t e d d o u b l e m u t a n t h e r m a p h r o d i t e s w e re t r a n s f e r r e d t o f r e s h p l a t e s . A p p r o x i m a t e l y 3 d a y s l a t e r , t h e h e t e r o z y g o u s p r o g e n y (F1 g e n e r a t i o n ) o f t h e c r o s s b e g a n l a y i n g e g g s . S i n c e t h e r a d i a t i o n t r e a t m e n t t o a d u l t worms d i d n o t show an y e f f e c t on r e c o m b i n a t i o n , g a m m a - r a d i a t i o n t r e a t m e n t o f a p p r o x i m a t e l y 2K r a d s was a p p l i e d t o t h e e x p e r i m e n t a l p a r e n t s a t t h e 1 2 - h r p r i o r t o e g g - l a y i n g s t a g e . B o t h t h e e x p e r i m e n t a l ( t r e a t e d ) a n d c o n t r o l ( u n t r e a t e d ) p l a t e s w e r e c a r r i e d t o t h e c o b a l t - 6 0 s o u r c e ( G a m m a c e l l , A t o m i c E n e r g y o f C a n a d a ) , s o t h a t i d e n t i c a l t e m p e r a t u r e c o n d i t i o n s w e r e m a i n t a i n e d d u r i n g t h e i r a b s e n c e f r o m t h e i n c u b a t o r . F1 h e t e r o z y g o u s w i l d - t y p e h e r m a p h r o d i t e s w e r e p i c k e d f r o m e a c h s e t o f p l a t e s . T h e s e h e r m a p h r o d i t e i n d i v i d u a l s w e r e m a i n t a i n e d a t 20°C a n d t r a n s f e r r e d t o f r e s h p l a t e s e v e r y 12 h o u r s , t h u s g e n e r a t i n g °12-hr b r o o d s ' . The F1 h e r m a p h r o d i t e s w e r e t r a n s f e r r e d u n t i l t h e y w e r e e x h a u s t e d o f f e r t i l i z e d e g g s . F i g u r e 1. M a t i n g p r o t o c o l f o r g e n e r a t i n g o f f s p r i n g w h i c h c a r r y r e c o m b i n a n t chromosomes. *Po : dpy-5 u n c - 1 3 x __+ + dpy-5 u n c - 1 3 + + v **F1 : dpy-5 u n c - 1 3 W i l d Type + + h e r m a p h r o d i t e s i r r a d i a t i o n s e l f - c r o s s F2 Non-recombinants W i l d T ype dpy-5 u n c - 1 3 dpy-5 unc-13 , dpy-5 unc-13 W i l d Type Dumpy Unc R e c o m b i n a n t s dpy-5 + dpy-5 + + u n c - 1 3 + u n c - 1 3 + + , dpy-5 u n c - 1 3 , + + , dpy-5 u n c - 1 3 W i l d Type Dumpy W i l d T ype Unc P o : P a r e n t a l i n d i v i d u a l s . F 1 : F i r s t g e n e r a t i o n p r o g e n y . 10 I I I . MEASURING RECOMBINATION FREQUENCY. a. I d e n t i f i c a t i o n o f R e c o m b i n a n t s . I n t h i s s t u d y , t h e r e c o m b i n a n t p h e n o t y p e s a r e "Dumpy" ( s h o r t e r t h a n t h e w i l d t y p e s ) , " U n c o o r d i n a t e d " ( d e t e c t a b l e d e f e c t i n t h e n o r m a l p a t t e r n o f movement), a n d " B l i s t e r e d " ( b l i s t e r e d c u t i c l e ) ( T a b l e 2 ) . The number o f "Dumpy" p r o g e n y was u s e d t o d e t e r m i n e r e c o m b i n a t i o n f r e q u e n c y i n m o s t e x p e r i m e n t s . S i n c e t h e Dpy-5 r e c o m b i n a n t s f r o m t h e o u t c r o s s i n v o l v i n g Dpy-5 a r e more v i a b l e t h a n U n c - 1 3 s , o r U n c - 1 1 s ( R o s e , u n p u b l i s h e d d a t a ) , a more a c c u r a t e e s t i m a t e o f r e c o m b i n a n t e v e n t s was c a l c u l a t e d by d o u b l i n g t h e number o f D p y - 5 s . I n a n a n a l o g o u s way, B l i - 3 r e c o m b i n a n t s w e r e u s e d f o r c a l c u l a t i o n o f r e c o m b i n a t i o n f r e q u e n c i e s . A l l Dpy-5 o r B l i - 3 r e c o m b i n a n t s w e r e p r o g e n y t e s t e d t o c o n f i r m t h a t t h e y w e re t h e e x p e c t e d g e n o t y p e . b. C a l c u l a t i o n o f R e c o m b i n a t i o n F r e q u e n c y . R e c o m b i n a t i o n f r e q u e n c y was d e t e r m i n e d i n t h e f o l l o w i n g way. The r a t i o o f w i l d - t y p e h e r m a p h r o d i t e s t o d o u b l e m u t a n t h e r m a p r o d i t e s ( e . g . Dpy Unc, Dpy Dpy, B l i Unc) i s e x p e c t e d t o be 3 t o 1 , so t h e t o t a l number o f o f f s p r i n g was c a l c u l a t e d a s 4/3 t h e number o f w i l d t y p e s p l u s more v i a b l e r e c o m b i n a n t s ( R o s e , 1 9 7 9 a ) . S i n c e t h e d o u b l e m u t a n t s h a v e r e d u c e d v i a b i l i t y c o m p a r e d t o w i l d t y p e , t h i s i s a more a c c u r a t e e s t i m a t e o f t h e t o t a l number o f p r o g e n y t h a n s c o r i n g w i l d - t y p e s p l u s Dpy U n cs o r 11 T a b l e 2 D e s c r i p t i o n o f R e c o m b i n a n t s O b s e r v e d i n t h e E x p e r i m e n t s R e c o m b i n a n t s LG A l l e l e P h e n o t y p e Dpy-5 I e61 s h o r t , d e c r e a s e d l e n g t h t o w i d t h Dpy-14 I e1 88 s h o r t , s w o l l e n m e d i a l l y Unc-13 I e450 s e v e r e l y u n c o o r d i n a t e d B l i - 3 I e579 b l i s t e r e d c u t i c l e , a n d d a r k e r a n d f a t t e r t h a n W i l d - T y p e Unc-35 I e259 m o d e r a t e l y u n c o o r d i n a t e d 12 B l i Uncs. A l l t h e F2 phenotypes were s c o r e d and t h e r e c o m b i n a t i o n f r e q u e n c y was c a l c u l a t e d by a p p l y i n g t h e f o r m u l a p = 1 - / l - 2R, where R = the f r a c t i o n of o b s e r v e d r e c o m b i n a n t s over t o t a l progeny ( B r e n n e r , 1974; F i g u r e 2 ) . The 95% c o n f i d e n c e i n t e r v a l s ( C . I . ) were o b t a i n e d by u s i n g t h e f o r m u l a C.I. = 1.96ynpq, where n = t h e t o t a l number of progeny, p = r e c o m b i n a t i o n f r e q u e n c y , and q = 1 - p. I f t h e number of r e c o m b i n a n t s was l e s s than 15, P o i s s o n s t a t i s t i c s were used t o c a l c u l a t e the c o n f i d e n c e i n t e r v a l s ( S t e v e n s , 1942). Assuming t h a t t h e r e c o m b i n a t i o n events f o l l o w a b i n o m i a l d i s t r i b u t i o n , the P o i s s o n d i s t r i b u t i o n i s a good a p p r o x i m a t i o n of the b i n o m i a l when the e v e n t s a r e r a r e . IV. MEASURING RADIATION DOSE RESPONSE. I n o r d e r t o d e t e r m i n e the e f f e c t o f r a d i a t i o n dose on r e c o m b i n a t i o n f r e q u e n c y , t h r e e d i f f e r e n t dosages were a p p l i e d . The dpy-5 unc-13 and dpy-5 dpy-14 i n t e r v a l s were chosen f o r t h i s e x p e r i m e n t . Dpy-5 r e c o m b i n a n t s from dpy-5 unc-13 / + + and dpy-5 dpy-14/ + + h e r m a p h r o d i t e s were s c o r e d . Males were s c o r e d as an e s t i m a t e of the f r e q u e n c y of X-chromosome n o n d i s j u n c t i o n (Hodgkin, 1977; Rose and B a i l l i e , 1979a). V. MEASURING F l STERILITY. I n o r d e r t o i n v e s t i g a t e t h e e f f e c t of gamma-radiation on s t e r i l i t y o f t h e F2 o f f s p r i n g o f t r e a t e d i n d i v i d u a l s , progeny t e s t i n g of Dpy-5 r e c o m b i n a n t s and w i l d - t y p e non-recombinant s i b l i n g s , was performed. A l l Dpy-5 reco m b i n a n t s from u n t r e a t e d 13 F i g u r e 2. M e t h o d f o r c a l c u l a t i n g r e c o m b i n a t i o n f r e q u e n c i e s ( f r o m R o s e , 1980 ). The number o f r e c o m b i n a n t p r o g e n y (Dumpy a n d U n c o o r d i n a t e d ) 2 x (number o f more v i a b l e r e c o m b i n a n t s ) The t o t a l number o f p r o g e n y 4/3 x (number o f W i l d T y p e s + number o f more v i a b l e r e c o m b i n a n t s ) The f r a c t i o n o f o b s e r v e d r e c o m b i n a n t s , R 2 x number o f Dumpies t o t a l p r o g e n y The f r e q u e n c y o f r e c o m b i n a t i o n , p = 1 - ^ 1 - 2R , w h e r e 2R = t h e f r a c t i o n o f p r o g e n y c a r r y i n g r e c o m b i n a n t chromosomes 1 - 2R = t h e f r a c t i o n o f p r o g e n y n o t c a r r y i n g a r e c o m b i n a n t chromosome J\ - 2R = t h e f r e q u e n c y o f n o n - r e c o m b i n a n t g a m e t e s 1 - J\ - 2R = t h e f r e q u e n c y o f r e c o m b i n a n t g a m e t e s 1 4 ( c o n t r o l ) a n d t r e a t e d ( e x p e r i m e n t a l ) w e r e p l a c e d i n d i v i d u a l l y on f r e s h p l a t e s . I f t h e y p r o d u c e d no s e l f - f e r t i l i z e d p r o g e n y , t h e y w e r e s c o r e d as a s t e r i l e . F u r t h e r m o r e , a p p r o x i m a t e l y t h e same number o f w i l d - t y p e h e r m a p h r o d i t e s w e re a l s o i s o l a t e d a n d e x a m i n e d i n o r d e r t o i n v e s t i g a t e t h e s t e r i l i t y o f n o n - r e c o m b i n a n t p r o g e n y . V I . MEASURING X-CHROMOSOME NONDISJUNCTION. a. D e t e r m i n a t i o n o f N o n d i s j u n c t i o n F r e q u e n c y . The s e x o f t h e f r e e - l i v i n g s o i l n e m a t ode C a e n o r h a b d i t i s  e l e g a n s i s d e t e r m i n e d by t h e r a t i o o f X chromosomes t o a u t o s o m e s . N o r m a l h e r m a p h r o d i t e s a r e 5 AA XX (X:A = 1 . 0 ) , w h e r e a s m a l e s a r e 5AA XO (X:A = 0.5) ( H o d g k i n , H o r v i t z a n d B r e n n e r , 1 9 7 9 ) . H e r m a p h r o d i t e s n o r m a l l y p r o d u c e h e r m a p h r o d i t i c (XX) o f f s p r i n g by s e l f - f e r t i l i z a t i o n . M a l e s (XO) a r e g e n e r a l l y p r o d u c e d by s p o n t a n e o u s X-chromosome n o n d i s j u n c t i o n o r chromosome l o s s ( H o d g k i n , 1 977; R o s e a nd B a i l l i e , 1 9 7 9 a ) . The number o f m a l e s were s c o r e d as a m e a s u r e o f X-chromosome n o n d i s j u n c t i o n . b . Chromosome L o s s o r N o n d i s j u n c t i o n . I n a d d i t i o n t o m e a s u r i n g t h e f r e q u e n c y o f m a l e s , i t i s n e c e s s a r y t o c o n f i r m w h e t h e r t h e p r e s e n c e o f m a l e s i s a 15 c o n s e q u e n c e o f X-chromosome n o n d i s j u n c t i o n o r t h e l o s s o f X-chromosomes. I n C. e l e g a n s , 1X d i p l o i d s a r e m a l e s , 2X d i p l o i d s a r e h e r m a p h r o d i t e s , 3X d i p l o i d s a r e s h o r t h e r m a p h r o d i t e s , a n d 4X d i p l o i d s a r e i n v i a b l e ( H o d g k i n , H o r v i t z a n d B r e n n e r , 1 9 7 9 ) . As a n i n d i c a t i o n o f X-chromosome n o n d i s j u n c t i o n , t h e o c c u r r e n c e o f 3X h e r m a p h r o d i t e s h a s b e e n u s e d . T r i s o m i c - X i n d i v i d u a l s w e r e c o l l e c t e d a nd t h e s e g r e g a t i o n o f s e l f p r o g e n y was o b s e r v e d . V I I . DEVELOPMENTAL STAGES. a. E f f e c t on r e c o m b i n a t i o n f r e q u e n c y . I t i s o f i n t e r e s t t o know w h e t h e r t h e r a d i a t i o n - i n d u c e d i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y i s s t a g e - s p e c i f i c . To i n v e s t i g a t e t h i s , i t i s n e c e s s a r y t o c o n s i d e r t h e age o f t h e worms a t t h e t i m e o f i r r a d i a t i o n . The d py-5 u n c - 1 3 i n t e r v a l was u s e d f o r t h i s e x p e r i m e n t . The worms w e r e t r e a t e d w i t h 2000 r a d s o f g a m m a - r a d i a t i o n a t t h r e e d i f f e r e n t s t a g e s o f t h e l i f e c y c l e : 1) 3 6 - h r p r i o r t o e g g - l a y i n g , 2) 2 4 - h r p r i o r t o e g g - l a y i n g , a n d 3) 1 2 - h r p r i o r t o e g g - l a y i n g . b. C y t o l o g y o f g o n a d s . The s t r u c t u r e o f t h e a d u l t g o n a d o f w i l d t y p e worms i n b o t h t h e t r e a t e d a n d u n t r e a t e d c o n d i t i o n h a s b e e n s t u d i e d u s i n g 16 w h o l e worms. The t e c h n i q u e o f S u l s t o n a n d H o r v i t z ( 1 9 7 7 ) f o r m o u n t i n g l i v e worms f o r o b s e r v a t i o n was u s e d . A p p r o x i m a t e l y 3 t o 5 l i v e worms were p l a c e d on a t h i n a g a r p a d o n a m i c r o s c o p e s l i d e . A c o v e r s l i p was p l a c e d on t o p t o t r a p t h e worms b e t w e e n t h e a g a r s u r f a c e a n d t h e c o v e r s l i p i n a d r o p o f M9 b u f f e r . The m a t u r e worms were h e a t - k i l l e d , s i n c e a c c u r a t e m e a s u r e m e n t s c o u l d n o t be made b e c a u s e o f t h e i r movement. The p r i m o r d i a l g o n a d i a l c e l l s (p c e l l s ) o f t h e r e p r o d u c t i v e s y s t e m a t t h r e e d i f f e r e n t d e v e l o p m e n t a l s t a g e s w e r e o b s e r v e d u s i n g N o m a r s k i - i n t e r f e r e n c e m i c r o s c o p y . c. E f f e c t on s p e r m a t o g e n e s i s o r o o g e n e s i s S i n c e s p e r m a t o g e n e s i s a n d o o g e n e s i s t a k e p l a c e i n t h e same g o n a d i n C. e l e g a n s , i t was n e c e s s a r y t o i n v e s t i g a t e w h e t h e r r a d i a t i o n a f f e c t s r e c o m b i n a t i o n f r e q u e n c y t h r o u g h i t s e f f e c t o n t h e sperm o r t h e o o c y t e s i n t h e s e l f - f e r t i l i z i n g h e r m a p h r o d i t e s . I n o r d e r t o d e t e r m i n e i f t h e s p e r m were a f f e c t e d , i t was a l s o n e c e s s a r y t o e v a l u a t e t h e r e c o m b i n a t i o n e v e n t s i n h e r m a p h r o d i t e o o c y t e s . The d o u b l e m u t a n t h e r m a p h r o d i t e s w e r e mated t o w i l d t y p e m a l e s a n d t r a n s f e r r e d t o f r e s h p l a t e s o n t h e f o l l o w i n g d a y a s d e s c r i b e d i n s e c t i o n 2 o f M a t e r i a l s a n d M e t h o d s . A f t e r t r e a t m e n t w i t h g a m m a - r a d i a t i o n , F1 i n d i v i d u a l w i l d - t y p e h e r m a p h r o d i t e s w e r e p l a c e d o n 10x60 mm p e t r i p l a t e s w i t h 2-3 u n t r e a t e d s i b l i n g m a l e s (shown i n F i g u r e 3 ) . T r e a t e d h e r m a p h r o d i t e s w e r e t r a n s f e r r e d w i t h u n t r e a t e d s i b l i n g m a l e s a t 17 F i g u r e 3. Method f o r measuring r e c o m b i n a t i o n f r e q u e n c y i n h e r m a p h r o d i t e o o c y t e s . Po : Double mutant h e r m a p h r o d i t e s x N2 males v F1 : Heterozygous W i l d - t y p e h e r m a p h r o d i t e s a r e p i c k e d . v Gamma-radiation v Each i n d i v i d u a l h e r m a p h r o d i t e i s p l a c e d on a f r e s h p e t r i p l a t e w i t h 2-3 u n t r e a t e d s i b l i n g males ( h e t e r o z y g o u s , e.g. dpy-5 dpy-14/ + + male) and t r a n s f e r r e d e v e r y 12 hour. v F2 : By s c o r i n g the phenotypes o f males o n l y , r e c o m b i n a t i o n f r e q u e n c y i n h e r m a p h r o d i t e sperm i s i g n o r e d . 18 12-hour i n t e r v a l s u n t i l male sperm were e x h a u s t e d . I t has been r e p o r t e d t h a t male sperm a re used p r e f e r e n t i a l l y (Ward and C a r r e l , 1979). By s c o r i n g recombinant males, the r e c o m b i n a t i o n f r e q u e n c y i n herma p h r o d i t e sperm was e x c l u d e d . T h e r e f o r e , i t i s p o s s i b l e t o d e t e c t whether t h e e f f e c t of r a d i a t i o n on r e c o m b i n a t i o n i s o c c u r r i n g d u r i n g s p e r m a t o g e n e s i s o r o o g e n e s i s . V I I I . CHROMOSOMAL REGIONALITY. I n o r d e r t o e v a l u a t e i f the e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y i s s p e c i f i c f o r the gene c l u s t e r , i t s e f f e c t on a r e g i o n a t the t i p of the chromosome was a l s o measured. The b l i - 3 unc-35 i n t e r v a l on t h e l e f t end o f L i n k a g e Group I_ was chosen f o r t h i s purpose. The r e c o m b i n a t i o n f r e q u e n c y was measured i n the same manner as p r e v i o u s l y d e s c r i b e d . 1 9 RESULTS I . EFFECT OF RADIATION ON RECOMBINATION FREQUENCY. Heterozygous h e r m a p h r o d i t e s were g a m m a - i r r a d i a t e d and i n d i v i d u a l s of the same age i s o l a t e d i n o r d e r t o o b s e r v e and c h a r a c t e r i z e t h e e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y . The r e s u l t s f o r t h e dpy-5 unc-13 i n t e r v a l a r e p r e s e n t e d i n T a b l e 3. I n t h e absence of g a m m a - i r r a d i a t i o n , t h e f r e q u e n c y . o f r e c o m b i n a t i o n i n t h i s r e g i o n was 0.021. A f t e r t r e a t m e n t w i t h 2000 r a d s of gamma-radiation the r e c o m b i n a t i o n f r e q u e n c y i n c r e a s e d about t w o - f o l d (p = 0.039). a. Brood A n a l y s i s . S u c c e s s i v e 12-hr broods were r o u t i n e l y c o l l e c t e d . I n each c a s e , t h e r e c o m b i n a t i o n f r e q u e n c y f o r each brood was c a l c u l a t e d i n d i v i d u a l l y ( M a t e r i a l s and Methods, s e c t i o n 3 b ) . F i g u r e 4 summarizes th e changes i n r e c o m b i n a t i o n f r e q u e n c y f o r each b r o o d a f t e r r a d i a t i o n t r e a t m e n t . The f r e q u e n c y of r e c o m b i n a t i o n f o r u n t r e a t e d h e t e r o z y g o t e s d e c r e a s e d c h a r a c t e r i s t i c a l l y w i t h p a r e n t a l age. T r e a t e d i n d i v i d u a l s showed no i n c r e a s e i n t h e f i r s t 12-hr b r o o d , but d i d show i n c r e a s e s i n r e c o m b i n a t i o n f r e q u e n c y i n the l a t e r b roods. R a d i a t i o n t r e a t m e n t produced a peak a t t h e second brood ( B ) . However, t h e e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y d i d not appear t o be as TABLE 3 20 <Effect of radiation on recombination frequency in dpy-5 unc-13 > Stage: 12-hr prior to egg-laying. 12-hr BROOD PROGENY RECOMBINANTS [p]x100 95% C . I . 2000 rads, N = 52 c d 1333 ' 2347 2800 1275 40 1 42 84 24 3.1 6.5 3.1 1 .9 (2.0-4.2) (5.0-8.0) (2.0-4.2) (1.0-2.8) TOTAL 7755 290 3.9 (3.0-4.8) CONTROL, N = 26 a b c d 1051 1 656 1767 317 34 46 18 2 3.4 (2.0-4.8) 2.9 (2.0-3.8) 1.0 (0.4-1.6) 0.6 (0.01-1.32) TOTAL 4791 1 00 2.1 (1.6-2.6) Effect of rad iat ion on recombination frequency for dpy-5 unc-13 s t ra in at 12 hr pr ior to egg-laying stage. pronounced when the r e c o m b i n a t i o n f r e q u e n c i e s f o r a l l t h e broods were averaged. I n o r d e r t o d e t e r m i n e whether th e e f f e c t of r a d i a t i o n on r e c o m b i n a t i o n i s c o n t i n u o u s w i t h p a r e n t a l age, the e x p e r i m e n t a l t o c o n t r o l r a t i o o f r e c o m b i n a t i o n f r e q u e n c y was c a l c u l a t e d . F i g u r e 5 shows a c o n t i n u o u s e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n events w i t h p a r e n t a l age. b. R a d i a t i o n Dose Response. Experiments measuring dose response were done as d e s c r i b e d i n t h e M a t e r i a l s and Methods, s e c t i o n 4. T a b l e 4 shows the dose response f o r r e c o m b i n a t i o n f r e q u e n c y i n t h e dpy-5  unc-13 i n t e r v a l . The r e c o m b i n a t i o n f r e q u e n c i e s d i f f e r a t t h e t h r e e doses used, 1, 2 and 4K r a d s . A l i n e a r i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d f o r the doses examined. Above 1K r a d s t h e r e was a l s o a d e c r e a s e i n progeny number. When 4K r a d s was a p p l i e d , r e c o m b i n a t i o n f r e q u e n c y i n c r e a s e d more th a n t w o - f o l d compared t o u n t r e a t e d i n d i v i d u a l s . However, the h i g h dose r e s u l t e d i n a reduced number of progeny. I t might be p o s s i b l e t h a t t h e r a d i a t i o n can cause embryonic l e t h a l i t y . T h e r e f o r e , t h e o p t i m a l dose f o r t h e e x p e r i m e n t s appeared t o be 2K r a d s . 23 Ratio of Experimental to Control for recombination frequency in dpy-5 unc-13 s t r a i n . TABLE 4 24 < R a d i a t i o n d o s e r e s p o n s e ? . S t a g e : 1 2 - h r p r i o r t o e g g - l a y i n g . PROGENY/ DOSES N* PROGENY PARENT RECOMBINANTS NDJ** [ P i 9 5 % C . I . dp y - 5 u n c - 13: CONTROL 26 4791 184 100 1 .0 2.1 ( 1 . 7 - 2 . 5 ) 1 K r a d s 10 2120 212 64 2.0 3.1 ( 2 . 3 - 3 . 9 ) 2 K r a d s 52 7755 1 49 290 5.9 3.9 ( 3 . 2 - 4 . 6 ) 4 K r a d s 25 3491 139 1 68 5.7 4.9 ( 4 . 1 - 5 . 5 ) d p y - 5 dpy-•14: CONTROL 10 2578 257 46 0.5 1 .8 ( 1 . 3 - 2 . 3 ) 2 K r a d s 1 6 2650 1 65 58 3.6 2.2 (1 .6-2.8) 4 K r a d s 16 2398 149 78 3.5 3.3 ( 2 . 5 - 4 . 0 ) * N = Number o f p a r e n t . **NDJ =Number o f w i l d - t y p e m a l e s p e r 1000 p r o g e n y . 25 c. D e v e l o p m e n t a l S t a g e s . i . Time o f T r e a t m e n t and R e c o m b i n a t i o n F r e q u e n c y . S i n c e o o g e n e s i s b e g i n s d u r i n g t h e l a r v a l s t a g e s and c o n t i n u e s i n t o a d u l t h o o d p a s t t h e o n s e t o f egg l a y i n g ( A b i - R a c h e d a n d B r u n , 1 9 7 5 ; H i r s h , O ppenheim a n d K l a s s , 1 9 7 6 ) , t h e e f f e c t o f r a d i a t i o n t r e a t m e n t t h r o u g h t h e o o g e n i c s t a g e s c a n ,be e x a m i n e d t o d e t e r m i n e w h e t h e r t h e r a d i a t i o n s e n s i t i v i t y c h a n g e s w i t h t i m e o f t r e a t m e n t . T h e s e e x p e r i m e n t s w e r e done u s i n g d p y - 5 u n c - 1 3 / + + i n d i v i d u a l s a t t h r e e d i f f e r e n t s t a g e s : 1 2 - h r , 2 4 - h r , a n d 3 6 - h r p r i o r t o t h e b e g i n n i n g o f e g g - l a y i n g ( d e s c r i b e d i n M a t e r i a l s and M e t h o d , s e c t i o n 7 ) . When t h e h e r m a p h r o d i t e s w e re t r e a t e d 1 2 - h o u r s p r i o r t o e g g - l a y i n g , a t w o -t o t h r e e - f o l d i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d t h r o u g h t h e e g g - l a y i n g p e r i o d . H owever, when t h e h e r m a p h r o d i t e s w e r e t r e a t e d 3 6 - h o u r s p r i o r t o e g g - l a y i n g , a p p r o x i m a t e l y a f i v e - f o l d i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d (shown i n T a b l e 5 ) . The i n c r e a s e was o b s e r v e d i n t h e B b r o o d (shown i n F i g u r e 6 ) . T h e r e f o r e , i t seems t h a t a v e r y r a d i a t i o n - s e n s i t i v e s t a g e e x i s t s 3 6 - h o u r s b e f o r e t h e f i r s t e g g i s l a i d . I n o r d e r t o d e t e r m i n e i f t h i s s t a g e e f f e c t w o u l d o c c u r f o r d i f f e r e n t r e g i o n s , t h e d py-5 dpy-14 i n t e r v a l was s t u d i e d . The r e s u l t s a r e p r e s e n t e d i n T a b l e 6. As F i g u r e 7 s h ows, g a m m a - r a d i a t i o n t r e a t m e n t a t 3 6 - h r p r i o r t o egg l a y i n g a l s o c a u s e d a d r a m a t i c i n c r e a s e i n r e c o m b i n a t i o n . TABLE 5 26 <Stage experiment (I)> Region: dpy-5 unc-13 Dose : 2K rads. *STAGE±BROOD** a [P] per brood [ P i 95% C.I. -36hr 1.25 5.54 2.91 2.53 3.1 (2.39-3.98) (271) (641) (482) (542) (1936) -24hr 1.66 2.06 2.90 1.14 1.88 (1.33-2.48) (296) (599) (430) (686) (1624) -12hr 1.87 3.68 2.49 1.42 2.42 (1.77-2.91) (413) (684) (377) (649) (2123) NOT TREATED 1.76 1.14 0.92 (521) (897) (694) 0.86 1.00 (0.67-1.31) (755) (2867) * Hours b e f o r e e g g - l a y i n g . ** 12 hr broods. ( )Number of progeny. 27 RF(%) F i g u r e 6 a b e d Brood E f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y f o r dpy-5 unc-13 s t r a i n a t d i f f e r e n t d e v e l o p m e n t a l s t a g e s . i i . C y t o l o g y o f Gonads. 28 The number o f p r i m o r d i a l g o n a d a l c e l l s (p c e l l s ) was c o u n t e d f o r t h e t h r e e d i f f e r e n t d e v e l o p m e n t a l s t a g e s t h a t w e r e t r e a t e d w i t h g a m m a - r a d i a t i o n . F o r e a c h s t a g e u n t r e a t e d s i b l i n g s w e r e e x a m i n e d c y t o l o g i c a l l y . A t t h e 3 6 - h r p r i o r t o e g g - l a y i n g s t a g e , a p p r o x i m a t e l y t e n p r i m o r d i a l g o n a d a l c e l l s w e r e o b s e r v e d . A t 2 4 - h r p r i o r t o e g g - l a y i n g , 50-55 p r i m o r d i a l g o n a d a l c e l l s were o b s e r v e d . A t t h e 1 2 - h r p r i o r t o e g g - l a y i n g s t a g e , t h e number o f p c e l l s was t o o many t o c o u n t . My e x p e r i m e n t s w e re c a r r i e d o u t a t 20°C w h e r e a s t h o s e o f K i m b l e a nd H i r s h ( 1 9 7 9 ) w e r e done a t 25°C, s o t h a t t h e d e v e l o p m e n t a l t i m e i n h o u r s d i d n o t c o r r e s p o n d e x a c t l y w i t h t h e i r p u b l i s h e d w o r k ( T a b l e 7 ) . No d e t e c t a b l e m o r p h o l o g i c a l d i f f e r e n c e was f o u n d b e t w e e n t r e a t e d a n d u n t r e a t e d i n d i v i d u a l s when e x a m i n e d i m m e d i a t e l y a f t e r t r e a t m e n t . TABLE 6 29 <Stage e x p e r i m e n t ( I I ) > . R e g i o n : d p y - 5 dpy-14 Dose : 2K r a d s . [ p ] p e r b r o o d *STAGE±BROOD** a t p ] 95% C . I . -36 h r 0.66 7.54 3.69 2.56 3.47 ( 2 . 4 2 - 4 . 5 1 ) (196) ( 2 5 9 ) ( 2 4 3 ) ( 3 4 5 ) ( 1 0 4 3 ) -24 h r 1.37 1.14 1.20 1.42 1.28 ( 0 . 7 2 - 1 . 8 3 ) (48 0 ) ( 861) ( 4 0 5 ) ( 5 0 7 ) ( 2 2 5 3 ) -12 h r 2.50 3.90 1.40 1.30 2.10 ( 1 . 2 5 - 2 . 5 4 ) (30 0 ) ( 456) ( 3 9 8 ) ( 7 4 6 ) ( 1 9 0 0 ) NOT TREATED 0 1.05 2.37 2.03 1.92 ( 1 . 3 9 - 2 . 4 6 ) ( 1 2 0 ) ( 4 4 5 ) ( 3 6 5 ) ( 5 9 0 ) ( 1 0 7 5 ) * H o u r s b e f o r e e g g - l a y i n g . * * 1 2 - h r b r o o d s . ( )Number o f p r o g e n y . 3 0 F i g u r e 7 a b c d Brood Effect of rad iat ion on recombination frequency for dpy-5 dpy-14 at d i f fe rent developmental stages. 31 TABLE 7 d e v e l o p m e n t o f the r e p r o d u c t i v e system o f C. elegans>. Hours a t 25 degrees Celsius 0 Hatch 0-6 4 p r i m o r d i a l gonadal c e l l s (p c e l l s ) L1 6-11 6 c e l l s < 36-hr p r i o r t o e g g - l a y i n g L2 11.5 F i r s t l a r v a l m olt 12-13 10 p c e l l s 1 4-18 30 p c e l l s L3 18.5 Second l a r v a l m o l t 20-22 60 p c e l l s . Hypodermal c e l l s e n l a r g e . < 24-hr p r i o r t o e g g - l a y i n g 24-26 120 p c e l l s L3 26 Gonad b e g i n s 1 80 2 t u r n L4 26 T h i r d l a r v a l m olt 28-32 Hypodermal c e l l s form v u l v a and v a g i n a 32-35 Sperm f o r m a t i o n i n p r o x i m a l arm; < 12-hr p r i o r Spermathecae f o r m a t i o n . t o e g g - l a y i n g L4 35.5 F o u r t h l a r v a l m o l t A d u l t 35-36 Oocytes appear 37 F e r t i l i z e d eggs p r e s e n t 45-46 Egg l a y i n g b e g i n s Retyped from t h e r e s u l t s r e p o r t e d by Kimble and H i r s h (1979). 32 i i i . E f f e c t on Spermatogenesis o r Oogenesis. To i n v e s t i g a t e whether the i n c r e a s e d r e c o m b i n a t i o n f r e q u e n c i e s o c c u r r e d d u r i n g s permatogenesis o r o o g e n e s i s , sperm i n t r e a t e d h e r m a p h r o d i t e s were r e p l a c e d by i n t r o d u c i n g t h e sperm from u n t r e a t e d s i b l i n g males (the M a t e r i a l s and Methods, s e c t i o n 8 ) . The male o u t c r o s s progeny r e s u l t e d from f e r t i l i z a t i o n o f t r e a t e d o o c y t e s by u n t r e a t e d sperm. The number of s e l f - c r o s s males would be n e g l i g i b l e . The r e c o m b i n a t i o n e v e n t s i n t h e h e r m a p r o d i t e sperm a r e not s c o r e d , i f o n l y male progeny were s c o r e d . When e v e n t s i n h e r m a p h r o d i t e sperm a r e e x c l u d e d , t h e r e i s l e s s i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y a f t e r r a d i a t i o n t r e a t m e n t (shown i n T a b l e 8 ) . I f a l l of t h e o b s e r v e d i n c r e a s e s i n t h e s e l f - c r o s s e x p e r i m e n t s were th e r e s u l t o f r a d i a t i o n on s p e r m a t o g e n e s i s , one would expect no i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y w i t h u n t r e a t e d male sperm. S i n c e some i n c r e a s e o c c u r r e d , t h i s i n c r e a s e must be due t o an a f f e c t o f r a d i a t i o n on o o g e n e s i s . S i n c e the amount of the i n c r e a s e i s reduced some a f f e c t on h e r m a p h r o d i t e spermatogenesis may a l s o be o c c u r r i n g . d. S t e r i l i t y o f Recombinants. Dumpy, U n c o o r d i n a t e d , and B l i s t e r e d r e c o m b i n a n t s from the e x p e r i m e n t s were t e s t e d t o c o n f i r m t h a t t h e y were t r u e r e c o m b i n a n t s as w e l l as t o i n v e s t i g a t e t h e i r s t e r i l i t y . The TABLE 8 33 <Effect on spermatogenesis or oogenesis). Region: dov-5 unc-13 Stage : 36-hr p r i o r to egg - l a y i n g . Dose : 2K rads. [o] 95% C, OUTCROSS: CONTROL, N = 17: WILD-TYPE MALES 909 RECOMBINANT MALES 35 2.85 (2.05-3.56) 2000 rads, N = 18: WILD-TYPE MALES 1167 RECOMBINANT MALES 54 3.41 (2.47-4.24) SELF-CROSS: CONTROL, N = 13: TOTAL PROGENY 3721 .RECOMBINANTS 37 1 .00 (0.67-1 .31 ) 2000 rads, N = 10: TOTAL PROGENY 1820 RECOMBINANTS 58 3.1 (2.39-3.98) d a t a i s r e c o r d e d i n T a b l e 9 . I n u n t r e a t e d i n d i v i d u a l s , a l l the reco m b i n a n t s were f e r t i l e . However, some f r a c t i o n o f reco m b i n a n t s r e c o v e r e d from the t r e a t e d i n d i v i d u a l s were found t o be s t e r i l e . From t h e d a t a which were c o l l e c t e d a t 1 2-hr i n t e r v a l s , t h e h i g h e s t s t e r i l i t y was o b s e r v e d i n t h e B brood (shown i n F i g u r e 8 ) . T h i s c o i n c i d e s w i t h the h i g h e s t peak o f r e c o m b i n a t i o n f r e q u e n c y caused by gamma-radiation. T h e r e f o r e , r a d i a t i o n appears t o cause g e n e t i c damage r e s u l t i n g s t e r i l i t y accompanied by an i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y . I n o r d e r t o de t e r m i n e whether the e f f e c t of r a d i a t i o n on s t e r i l i t y i s a s s o c i a t e d w i t h t h e r e c o m b i n a t i o n e v e n t , t h e non-recombinant w i l d - t y p e progeny from t r e a t e d i n d i v i d u a l s were a l s o c o l l e c t e d and examined. A l l of t h e w i l d - t y p e progeny h e r m a p h r o d i t e s t e s t e d were found t o be f e r t i l e . T h e r e f o r e i t appears t h a t t h e r a d i a t i o n - i n d u c e d s t e r i l i t y i s a s s o c i a t e d w i t h t h e r e c o m b i n a t i o n e v e n t . I I . EFFECT OF RADIATION ON X-CHROMOSOME NONDISJUNCTION. a. Rate o f N o n d i s j u n c t i o n . The e f f e c t of r a d i a t i o n on X-chromosome n o n d i s j u n c t i o n was o b s e r v e d ( T a b l e 1 0 ) . The number o f w i l d - t y p e males was s c o r e d as a measure o f X-chromosome n o n d i s j u n c t i o n . TABLE 9 < E f f e c t o f r a d i a t i o n on s t e r i l i t y o f r e c o m b i n a n t s > . 35 GENOTYPE *DOSE(STAGE) - a b C d QDV- 5 u n c - 13 2 R ( 1 2 h r ) C/36 = : 0 2 6 / 1 3 2 = : 0. ,20 1 1 / 7 9 = 0. 14 0 /43 = 0 dTDV- 5 u n c - 13 4 R ( 1 2 h r ) 1/28 = : 0 . 04 2 8 / 76 = : 0. ,37 13/41 = • 0. 32 0 /23 = 0 C o n t r o l 1/29 = : 0 . 03 0 / 34 = = 0 0/26 = : 0 0/11 = : 0 d o v - •5 •1 4 2 R ( 1 2 h r ) 0 / 3 = : 0 3 / 22 = = 0. .14 0/16 = < 0 0 /17 = : o d o v - •5 d o v - •1 4 4 R ( 1 2 h r ) 0/ 9 = : 0 6/ 34 = = 0. .18 1/20 = : 0 . 05 1/15 = : 0 . 07 C o n t r o l 0/10 = : 0 0/ 19 = = 0 0/ 9 = : 0 0 / 8 = : 0 d n v --5 ur . c - •1 3 2R(3 6 h r ) 0 / 4 = = 0 8/ 26 : = 0 .31 3 /20 = * 0 . ,15 1/18 = = 0 . 05 d o v - -5 d o v - -1 4 2 R ( 3 6 h r ) 0/ 3 = = 0 5 / 18 : = 0 .28 0 / 8 -• 0 0 / 5 = = 0 CDV- -1 4 u n c - -1 3 2R(3 6 h r ) 0/ 2 = = 0 4 / 11 : = 0 .36 0 / 5 : = 0 0 / 6 = = 0 C o n t r o l 0/ 2 = = 0 0/ 3 = o 0/ 2 : = 0 0 / 0 : = 0 cov- -5 unc- -1 1 2R(3 6 h r ) 1 / 6 : = 0. .17 • - 3 / 14 = 0 .21 1/14 : = 0 . 0 7 0 / 6 : = 0 C o n t r o l 0 / 7 : = 0 0/ 8. = 0 0 / 8 : = 0 0 / 1 , = 0 b l i - -3 unC' -35 2 R ( 3 6 h r ) 0 / 9 : = 0 1/10 = 0 . 1 2 /10 = 0 . 2 0 / 8 = o. C o n t r o l 0 /22 = 0 0/22 = 0 1/22 = 0 . 05 0 /27 = 0 * DOSE : 2R = 2K r a d s , 4R = 4K r a d s . 36 E f f e c t of r a d i a t i o n on s t e r i l i t y of recombinants a t 36 h r p r i o r to egg-laying stage. 37 A f t e r t r e a t m e n t w i t h 2K r a d s o f g a m m a - r a d i a t i o n , t h e f r a c t i o n o f m a l e s o b s e r v e d i n c r e a s e d d r a m a t i c a l l y . The r e s u l t i s s u m m a r i z e d i n F i g u r e 9 w h i c h shows t h e number o f m a l e s p e r t h o u s a n d w i l d - t y p e h e r m a p h r o d i t e s a f t e r i r r a d i a t i o n . The f r e q u e n c y o f X-chromosome n o n d i s j u n c t i o n c a n be u s e d a s a means o f c o n f i r m i n g t h e d e v e l o p m e n t a l s y n c h r o n y o f t r e a t e d i n d i v i d u a l s . T h a t i s , o n l y d a t a f r o m e x p e r i m e n t s i n w h i c h t h e n o n d i s j u n c t i o n p e a k s c o i n c i d e d w e r e c o m p a r e d t o s t u d y r e g i o n a l e f f e c t s . I n o r d e r t o d e t e r m i n e w h e t h e r t h e m a l e s were a c o n s e q u e n c e o f X-chromosome n o n d i s j u n c t i o n , t r i s o m i c h e r m a p h r o d i t e s f r o m F1 h e t e r o z y g o u s i n d i v i d u a l s w e re c o l l e c t e d a nd p r o g e n y t e s t e d . I n d i v i d u a l t r i s o m i c h e r m a p h r o d i t e s s e g r e g a t e d o n l y 3X and 2X h e r m a p h r o d i t e s (3X : 2X = 2 : 1 ) . The number o f 3X h e r m a p h r o d i t e s was a l m o s t e q u i v a l e n t t o t h a t o f m a l e s o b s e r v e d . The d o s e r e s p o n s e f o r X-chromosome n o n d i s j u n c t i o n was a l s o s t u d i e d ( T a b l e 4 ) . I t a p p e a r e d t o i n c r e a s e w i t h i n c r e a s i n g d o s e s e x a m i n e d . b. S t a g e E f f e c t on N o n d i s j u n c t i o n . The e f f e c t o f r a d i a t i o n o n n o n d i s j u n c t i o n f r e q u e n c y a t d i f f e r e n t d e v e l o p m e n t a l s t a g e s was s t u d i e d ( T a b l e 1 1 ) . The p e a k o f n o n d i s j u n c t i o n o c c u r r e d i n C b r o o d when i n d i v i d u a l s w e r e t r e a t e d 3 6 - h r p r i o r t o e g g - l a y i n g ( F i g u r e 10) c o m p a r e d t o aB b r o o d p e a k f o l l o w i n g a m i n u s 1 2 - h r t r e a t m e n t . 38 TABLE 10 < E f f e c t o f r a d i a t i o n on n o n - d i s j u n c t i o n f r e q u e n c y ^ . Stage: 12-hr p r i o r t o e g g - l a y i n g . Dose : 2K r a d s . Number of males per 1000  GENOTYPE±BROOD a b C d dpy-5 unc-13 CONTROL: 1/763 = 1.3 1/1208 = 0.8 2/1312 = 1.5 1/236 = 4.2 2000 r a d s : 2/967 = 2.1 14/1654 = 8.5 10/2037 = 4.9 3/938 = 3.2 dpy-5 dpy-1 4 CONTROL: 0/381 = 0 1/ 673 =1.5 1/ 540 = 1.9 1/306 = 3.4 2000 r a d s : 1/278 = 3.6 3/ 497 = 6.0 1/ 352 = 2.8 0/292 = 0 i g u r e 9 Brood E f f e c t o f r a d i a t i o n on n o n - d i s j u n c t i o n f r e q u e n c y a t 12 h r p r i o r t o e g g - l a y i n g s t a g e s . 40 I I I . COMPARISON OF RECOMBINATION AND NON-PISJUNCTION I n o r d e r t o i n v e s t i g a t e t h e g a m m a - r a d i a t i o n s e n s i t i v e b r o o d f o r r e c o m b i n a t i o n a n d X-chromosome n o n d i s j u n c t i o n , t h e r a t i o o f e x p e r i m e n t a l ( t r e a t e d ) t o c o n t r o l ( u n t r e a t e d ) d a t a w e r e p l o t t e d ( T a b l e 12 a n d F i g u r e 1 1 ) . The r a t i o s o f e x p e r i m e n t a l t o c o n t r o l showed a c o n t i n u o u s i n c r e a s e w i t h p a r e n t a l a g e i n r e c o m b i n a t i o n f r e q u e n c y , w h e r e a s a p e a k i n X-chromosome n o n d i s j u n c t i o n was o b s e r v e d i n t h e B b r o o d . I V . CHROMOSOMAL REGIONALITY. I n o r d e r t o d e t e r m i n e i f r e c o m b i n a t i o n a n d X-chromosome n o n d i s j u n c t i o n i n c r e a s e d i n o t h e r r e g i o n s o f t h e chromosome w i t h r a d i a t i o n t r e a t m e n t , s e v e r a l i n t e r v a l s w e r e c h o s e n a n d c o m p a r e d : 1) c l u s t e r e d i n t e r v a l s ( d p y - 5 d p y - 1 4 , dpy-14 u n c - 1 3 , dpy-5  u n c - 1 3 ) , 2) a n i n t e r v a l a d j a c e n t t o a c l u s t e r ( d p y - 5 u n c - 1 1 ) , and 3) an i n t e r v a l a t t h e l e f t e n d o f t h e chromosome ( b l i - 3  u n c - 3 5 ) . See map i n A p p e n d i x 2 a. R e c o m b i n a t i o n i n t h e C l u s t e r e d R e g i o n . The w e l l c h a r a c t e r i z e d c l u s t e r e d r e g i o n b e t w e e n d p y - 5 a n d u n c - 1 3 was c h o s e n f o r my r e s e a r c h . . T h i s c l u s t e r e d r e g i o n b e t w e e n d p y - 5 a n d u n c - 1 3 i s s u b d i v i d e d i n t o two i n t e r v a l s , d p y - 5  dpy-14 a n d dpy-14 u n c - 1 3 . R e s u l t s f o r t h e i n t e r v a l s a r e 41 TABLE 11 <Effect of r a d i a t i o n on n o n - d i s j u n c t i o n frequency at d i f f e r e n t stages>. Region: dpy-5 unc-13 Dose : 2K r a d s . Number of males per 1 000 *STAGS±3R00D** a b c d -36hr 1/239 = 4.18 2/342 = 5.84 6/507 = 11.83 2/234 = 8.55 -24hr 2/445 = 4.49 2/504 = 3.96 0/306 = 0 2/327 = 6.12 -12hr 1/318 = 3.14 3/401 = 7.48 3/715 = 4.19 2/631 = 3.17 * Hours bef o r e e g g - l a y i n g . ** 12-hr broods. 42 Effect of rad ia t ion on nondisjunction'frequency for dpy-5 unc-13 s t ra in at d i f fe rent developmental stages. 43 TABLE 12 <Comparison o f r e c o m b i n a t i o n and n o n - d i s j u n c t i o n > . R e g i o n : dpy-5 unc-13 Stage : 12-hr p r i o r t o e g g - l a y i n g . Dose : 2K r a d s . E x p e r i m e n t a l / C o n t r o l  BROOD a b e d R e c o m b i n a t i o n Frequency 0.91 2.24 3.10 3.17 N o n - d i s j u n c t i o n Frequency 2.73 12.80 6.18 1.22 44 F i a u r e 11 Brood Comparison of the effect of rad ia t ion on the frequency of recombination and nondisjunction for dpy-5 unc-13 s t ra in at 12 hr pr ior to egg-laying stage. 45 r e c o r d e d i n T a b l e 13. As F i g u r e 12 summarizes, t h e maximum magnitude of i n c r e a s e i s obse r v e d i n t h e dpy-14 unc-13 i n t e r v a l . I n c r e a s e s i n r e c o m b i n a t i o n f r e q u e n c y by r a d i a t i o n t r e a t m e n t seems t o be a p p r o x i m a t e l y t h e same i n b o t h t h e dpy-5 dpy-14 i n t e r v a l and t h e dpy-5 unc-13 i n t e r v a l . b. R e c o m b i n a t i o n i n a Region A d j a c e n t t o t h e C l u s t e r . These e x p e r i m e n t s i n t h e c l u s t e r e d r e g i o n between dpy-5 and unc-13 prompted a f u r t h e r study on a c o n t i g u o u s r e g i o n . The dpy-5 unc-11 i n t e r v a l was chosen f o r t h i s purpose and showed a l i t t l e l e s s o f an i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y t h a n i n the c l u s t e r e d r e g i o n ( T a b l e 1 4 ) . A s i m i l a r b rood e f f e c t i s observed ( F i g u r e 1 3 ) . c. R e c o m b i n a t i o n a t the L e f t - e n d o f the Chromosome. I t i s c l e a r t h a t t h e above o b s e r v a t i o n s have s i g n i f i c a n c e f o r an u n d e r s t a n d i n g o f t h e e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y : gamma-radiation has an i n c r e a s e d e f f e c t on r e c o m b i n a t i o n f r e q u e n c y i n t h e c l u s t e r e d r e g i o n o f LG I_. I t i s , t h e n , i m p o r t a n t t o t e s t a r e g i o n f a r t h e r a p a r t from t h i s c l u s t e r . The b l i - 3 unc-35 i n t e r v a l from t h e l e f t end o f LG I was s t u d i e d . The r e g i o n a l r e s p o n s e s d e s c r i b e d a r e r e c o r d e d i n T a b l e 15. The b l i - 3  unc-35 i n t e r v a l seems t o show a l m o s t no r e s p o n s e t o a r a d i a t i o n t r e a t m e n t . 46 TABLE 13 < E f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n i n t h e c l u s t e r e d r e g i o n s > . S t a g e : 3 6 - h r p r i o r t o e g g - l a y i n g . Dose : 2K r a d s . GENOTYPE *N WTS DPYS UNCS [ p ] 95% C . I . dpy-5 u n c - 1 3 CONTROL 26 4691 2K r a d s 18 1447 dpy-5 dpy-14 CONTROL 10 2532 2K r a d s 9 861 dpy-14 u n c - 1 3 CONTROL 17 2340 2K r a d s 13 1092 61 39 2.11 (1 .61 -2.62) 29 39 3.46 ( 2 . 6 0 - 5 . 8 1 ) 23 19 1.64 ( 1 . 2 3 - 1 . 6 5 ) 20 14 2.92 ( 1 . 9 2 - 3 . 8 4 ) 4 3 0.26 ( 0 . 0 7 - 0 . 6 6 ) 9 14 1.23 ( 0 . 6 7 - 1 . 7 9 ) * N = Number o f p a r e n t s . 47 £ 3 a — 3 p 1 CJ ( 1 . 9 / 1 . 3 ) 1.4 unc-11 ( 2 . 9 / 1 . 6 ) 1.8 1.6 ( 3 . 5 / 2 . 1 ) • c - v - 5 4.7 ( 1 . 2 / 0 . 3 ) * G^V—14 unc—13 Chromo social Resion (Experimental/Control) value. TABLE 14 48 < E f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n i n dpy-5 unc-11>. S t a g e : 3 6 - h r p r i o r t o e g g - l a y i n g . 1 2 - h r BROOD PROGENY RECOMBINANTS [ p ] 9 5 % C . I . 2000 r a d s , N = 10 a 450 6 1.34 ( 0 . 2 7 - 2 . 4 0 ) b 440 14 3.23 ( 1 . 5 3 - 4 . 8 3 ) c 906 14 1.56 ( 0 . 7 4 - 2 . 3 5 ) d 469 6 1.29 ( 0 . 2 6 - 2 . 3 0 ) TOTAL 2265 40 1 .85 (1 .48-2.59) CONTROL, N = 15 a 325 7 2.17 ( 0 . 5 7 - 3 . 7 4 ) b 502 8 1.60 ( 0 . 5 0 - 2 . 6 9 ) c 953 8 0.84 ( 0 . 2 6 - 1 . 4 2 ) d 127 1 0.78 ( - -2.32) TOTAL 1907 24 1.27 ( 0 . 7 6 - 1 . 7 6 ) 49 F i g u r e 13 RF (%) Effect of rad iat ion on recombination frequency for dpy-5 unc-11 s t r a i n . TABLE 15 50 < E f f e c t of r a d i a t i o n on r e c o m b i n a t i o n i n b l i - 3 unc-35>. S t a g e : 36-hr p r i o r t o e g g - l a y i n g . 1 2 - h r BROOD PROGENY RECOMBINANTS [p] 9 5 % C . I . 2000 r a d s , N = 28 a 1 1 1 7 9 0.82 ( 0 . 2 8 - 1 . 3 3 ) b 7 74 10 1.30 ( 0 . 4 9 - 2 . 0 9 ) c 611 10 1.65 ( 0 . 6 3 - 2 . 6 5 ) d 680 8 1.18 ( 0 . 3 6 - 2 . 3 8 ) TOTAL 3174 3 J 1 .17 ( 0 . 7 9 - 1 .54) CONTROL, N = 43 a 1462 22 1.52 ( 0 . 8 8 - 2 . 1 3 ) b 1 6 5 2 22 1.34 ( 0 . 7 8 - 1 . 8 9 ) c 2 1 6 0 22 1.02 ( 0 . 5 9 - 1 . 4 4 ) d 2 3 9 6 27 1.13 ( 0 . 7 0 - 1 . 5 5 ) TOTAL 7671 93 1.22 ( 0 . 9 7 - 1.46) RF (%) 2.0 51 F i g u r e 14 1.5 - — e x p . c o n t . 1.0 0.5 Effect of radiation on recombination frequency for bl i-3 unc-35 s t r a i n . 52 d. Summary R e g a r d i n g R e c o m b i n a t i o n a t D i f f e r e n t I n t e r v a l s I n summary, d a t a on t h e e f f e c t o f g a m m a - r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y o v e r t h e l e f t p o r t i o n o f LG J_ w e re c o l l e c t e d , a n d t h e m a g n i t u d e o f i n c r e a s e was c o m p a r e d i n e a c h i n t e r v a l ( T a b l e 1 6 ) . R e g i o n a l d i f f e r e n c e s i n t h e r e l a t i v e i n c r e a s e s o f r e c o m b i n a t i o n f r e q u e n c y seemed t o be a p p a r e n t i n t h e m a g n i t u d e o f t h e 2K r a d s / C o n t r o l v a l u e s . The dpy-14 u n c - 1 3 i n t e r v a l a p p e a r e d t o i n c r e a s e t h e most a f t e r r a d i a t i o n t r e a t m e n t . The b l i - 3 u n c - 3 5 i n t e r v a l i n a n o n - c l u s t e r e d r e g i o n d i d n o t i n c r e a s e a f t e r t r e a t m e n t . e. N o n - d i s j u n c t i o n c u r v e s f o r d i f f e r e n t s t r a i n s . The f r e q u e n c i e s o f X-chromosome n o n - d i s j u n c t i o n w e r e a l s o c o m p a r e d f o r d i f f e r e n t s t r a i n s . T a b l e 17 a n d F i g u r e 15 s u m m a r i z e t h e r e s u l t s . I t a p p e a r s t h a t t h e e f f e c t o f r a d i a t i o n on X-chromosome n o n d i s j u n c t i o n o c c u r s a t t h e same d e v e l o p m e n t a l s t a g e f o r d i f f e r e n t m u t a n t s t r a i n s . T h i s c o n f i r m s t h a t t h e p a r e n t a l h e r m a p h r o d i t e s w e re t r e a t e d a t t h e same d e v e l o p m e n t a l s t a g e . 53 TABLE 16 <Summary>. BROOD WITH RECOMBINATION RATIO OF REGION HIGHEST INCREASE CONTROL 2 000 r a d s 2K r a d s / C o n t r o l OF RECOMBINATION dpy-5 unc-13 b .2.11 3.46 1 .64 dpy-5 dpy-14 b 1 .64 2.92 1 .78 dpy-14 unc-13 b 0.26 1 .23 4.73 dpy-5 unc-11 b 1 .27 1 .85 1 .46 b l i - 3 unc-35 c 1 .22 1 .17 0.96 54 TABLE 17 <Effect of r a d i a t i o n on n o n - d i s j u n c t i o n frequency at d i f f e r e n t regions>. Stage: 36-hr p r i o r t o e g g - l a y i n g . GENOTYPE±RROOD a b c d d-ov-5 unc-13 1/158 = 6* 3/349 = 9 10/432 = = 23 1/281 = 4 doy-5 dr>v-1 4 3/226 = 13 3/1 77 = 17 8/283 = = 28 3/175 = 17 dDv-14 unc-13 0/ 78 = 0 0/362 = 0 4/302 = = 13 2/350 = 5 dov-5 unc-11 0/333 = 0 1 /320 = 3 8/671 : = 12 2/498 = 4 b l i - 3 unc-35 3/831 = 4 3/573 = 5 9/451 : = 20 2/498 = 4 * F r a c t i o n of w i l d type progeny t h a t were male converted to number of males per 1000. F i g u r e 15 a b c d Brood E f f e c t o f r a d i a t i o n on n o n - d i s j u n c t i o n f r e q u e n c y f o r d i f f e r e n t r e g i o n s . 56 DISCUSSION The e f f e c t o f gamma r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y f o r i n t e r v a l s o f L i n k a g e G r o u p 1 i n C. e l e g a n s h a s b e e n s t u d i e d . R e c o m b i n a t i o n f r e q u e n c y i n c r e a s e d a p p r o x i m a t e l y t w o - f o l d a c r o s s t h e dp y - 5 u n c - 1 3 i n t e r v a l a f t e r t r e a t m e n t w i t h 2K r a d s o f gamma r a d i a t i o n . T h i s t h e s i s i s t h e f i r s t d e s c r i p t i o n o f t h e e f f e c t o f gamma r a d i a t i o n on r e c o m b i n a t i o n f r e q u e n c y i n t h i s o r g a n i s m . S e v e r a l f a c t o r s a f f e c t i n g t h e m a g n i t u d e o f t h e i n c r e a s e h a v e b e e n c h a r a c t e r i z e d . R e c o m b i n a t i o n f r e q u e n c y i n c r e a s e d more w i t h h i g h e r d o s e s o f r a d i a t i o n . H o wever, t h e i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y w i t h i n c r e a s i n g d o s e was a c c o m p a n i e d by a r e d u c e d a v e r a g e number o f p r o g e n y i n r a d i a t i o n - t r e a t e d i n d i v i d u a l s . T h u s , b e c a u s e gamma r a d i a t i o n a l s o a f f e c t s f e c u n d i t y , a d o s e o f 2K r a d s was c h o s e n a s t h e o p t i m a l d o s e f o r my e x p e r i m e n t s . I n o r d e r t o d e t e r m i n e w h e t h e r t h e e f f e c t o f r a d i a t i o n on r e c o m b i n a t i o n i s s p e c i f i c f o r a c e r t a i n s t a g e o f o o g e n e s i s , a b r o o d a n a l y s i s was p e r f o r m e d f o r e v e r y 1 2 - h r i n t e r v a l . I n t h i s s e l f - f e r t i l i z i n g h e r m a p h r o d i t e , s p e r m a t o g e n e s i s i s c o m p l e t e d p r i o r t o t h e o n s e t o f o o g e n e s i s w h i c h c o n t i n u e s t h r o u g h o u t t h e a d u l t l i f e o f t h e h e r m a p h r o d i t e . T h u s , t h e p r o g e n y f r o m e a c h 1 2 - h r b r o o d a r e t h e r e s u l t o f f e m a l e g a m e t e s w h i c h were a t d i f f e r e n t d e v e l o p m e n t a l s t a g e s a t t h e t i m e o f t r e a t m e n t . The e a r l i e s t (A) b r o o d was most m a t u r e when t h e y w e r e t r e a t e d . The r e s u l t s p r e s e n t e d i n T a b l e 3 a n d F i g u r e 4 show t h e g r e a t e s t i n c r e a s e i n t h e s e c o n d (B) b r o o d . T h e r e was no i n c r e a s e i n the f i r s t (A) brood, and as i n the u n t r e a t e d i n d i v i d u a l s , d e c r e a s e s i n r e c o m b i n a t i o n f r e q u e n c y were seen i n l a t e r b r o o d s . The A brood progeny r e s u l t e d from eggs t h a t were p o s t - m e i o t i c a t t h e time of t r e a t m e n t . Thus, t h e l a c k of i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y i n t h e f i r s t brood i s most l i k e l y because the o o c y t e s sampled i n the f i r s t brood have a l r e a d y completed r e c o m b i n a t i o n e v e n t s p r i o r t o t r e a t m e n t w i t h r a d i a t i o n . Because spermatogenesis o c c u r s p r i o r t o o o g e n e s i s and t h e mature sperm a r e used t h r o u g h o u t a l l t h e b r o o d s , g e n e r a l i n c r e a s e s t h r o u g h o u t a l l t h e broods would have been e x p e c t e d i f i n c r e a s e s i n r e c o m b i n a t i o n f r e q u e n c y a f t e r r a d i a t i o n t r e a t m e n t were the r e s u l t o f an e f f e c t on s p e r m a t o g e n e s i s . These e x p e r i m e n t s demonstrate t h e r e f o r e t h a t t r e a t m e n t w i t h r a d i a t i o n caused an i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y i n o o c y t e s t h a t are i n a m e i o t i c s t a g e which o c c u r s between 12 and 24 hours p r i o r t o e g g - l a y i n g . The s e n s i t i v e s t a g e i s p o s s i b l y (but not n e c e s s a r i l y ) prophase of m e i o s i s I ( t h e a c t u a l event o f r e c o m b i n a t i o n ) . Treatment of h e r m a p h r o d i t e s a t d i f f e r e n t d e v e l o p m e n t a l t i m e s has shown t h a t r a d i a t i o n a p p l i e d p r i o r t o m e i o s i s can have an a f f e c t on r e c o m b i n a t i o n f r e q u e n c y i n t h e s e n s i t i v e B b r o o d . However not a l l d e v e l o p m e n t a l s t a g e s a r e s e n s i t i v e t o r a d i a t i o n t r e a t m e n t ( w i t h r e g a r d t o r e c o m b i n a t i o n f r e q u e n c y ) . The e x p e r i m e n t s were performed a t t h r e e d i f f e r e n t d e v e l o p m e n t a l s t a g e s . When he r m a p h r o d i t e s were t r e a t e d w i t h r a d i a t i o n 24-hr p r i o r t o e g g - l a y i n g no s i g n i f i c a n t i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d . I t may be t h a t a t t h i s t i m e i n t h e 58 d e v e l o p m e n t o f t h e g o n a d t h e DNA i s i n s e n s i t i v e t o r a d i a t i o n damage. The maximum a f f e c t o f r a d i a t i o n was p r o d u c e d by-t r e a t i n g t h e h e r m a p h r o d i t e s 36 h o u r s p r i o r t o e g g - l a y i n g ( s e e t h e R e s u l t s s e c t i o n I - c - i ; T a b l e s 5 a n d 6 ) . The d i f f e r e n c e i n c o n t r o l v a l u e s b e t w e e n T a b l e 3 a n d T a b l e 5 m i g h t be due t o a s t r a i n e f f e c t s i n c e t h e f i r s t e x p e r i m e n t ( T a b l e 3) was done u s i n g t h e e51 a l l e l e (BC 26 s t r a i n ) a nd t h e l a t t e r e x p e r i m e n t s u s i n g e450 (BC 4 1 5 ) . The amount o f i n c r e a s e r e l a t i v e t o t h e c o n t r o l i s t h e same i n b o t h c a s e s e v e n t h o u g h a l l t h e r e c o m b i n a t i o n f r e q u e n c i e s a r e r e d u c e d when t h e BC 415 s t r a i n was u s e d . T h i r t y - s i x h o u r s p r i o r t o e g g - l a y i n g i s a n e a r l y p r e - m e i o t i c s t a g e p r i o r t o a t i m e o f e x t e n s i v e c e l l d i v i s i o n a n d DNA r e p l i c a t i o n i n t h e g o n a d s ( N e l s o n , Lew and Ward, 1978; K i m b l e a n d H i r s h , 1 9 7 9 ) . P r e s u m a b l y , c h r o m o s o m a l b r e a k s c a u s e d a t t h i s s t a g e a r e n o t d i r e c t l y r e s p o n s i b l e f o r t h e i n c r e a s e d r e c o m b i n a t i o n b e c a u s e t h e s e c e l l s w i l l h a v e t o go t h r o u g h many more c e l l d i v i s i o n s b e f o r e e n t e r i n g m e i o s i s . B a s e - p a i r m o d i f i c a t i o n s w h i c h a r e known t o be c a u s e d by r a d i a t i o n c o u l d o c c u r and s i n c e many o f t h e s e a l t e r a t i o n s a r e u n s t a b l e , c h e m i c a l c h a n g e s i n t h e DNA a t l a t e r s t a g e s m i g h t c a u s e n i c k s o r b r e a k s w h i c h c o u l d become s u b s t r a t e f o r r e c o m b i n a t i o n e v e n t s . O t h e r t y p e s o f e x p l a n a t i o n s a r e f e a s i b l e . F o r e x a m p l e r a d i a t i o n may have, a s t i m u l a t o r y a f f e c t on r e c o m b i n a t i o n e n z y m e s . C u r r e n t l y h o w e v e r t h e r e i s no e v i d e n c e t h a t t h i s o c c u r s . When h e r m a p h r o d i t e s a r e t r e a t e d 3 6 - h r s p r i o r t o e g g - l a y i n g , a n i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d i n t h e B b r o o d j u s t a s when r a d i a t i o n was a p p l i e d 1 2 - h r p r i o r t o e g g - l a y i n g . T h e s e r e s u l t s s u g g e s t t h a t i n d u c e d l e s i o n s p r o d u c e d by r a d i a t i o n a t e a r l i e r ( p r e m e i o t i c ) s t a g e s a r e c a r r i e d t h r o u g h t o l a t e r ( m e i o t i c ) s t a g e s . A l t h o u g h t h e n a t u r e o f t h e s e l e s i o n s i s n o t known, t h i s p r o v i d e s a r e a s o n a b l e e x p l a n a t i o n f o r t h e g r e a t e r i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y a t t h e -36 h o u r s t a g e . I n D. m e l a n o g a s t e r , s t u d i e s on t h e c o r r e l a t i o n b e t w e e n t h e r a d i a t i o n - s e n s i t i v e p e r i o d and t h e d e v e l o p m e n t a l s t a g e s o f t h e m a l e h a v e b e e n r e p o r t e d ( B o n n e r and L u n i n g , 1950; L u n i n g , 1 9 5 2 ) . My o b s e r v a t i o n s s u p p o r t t h o s e made i n D r o s o p h i l a t h a t more chromosome damage c a n be i n d u c e d i n c e l l s i n e a r l i e r m e i o t i c s t a g e s t h a n i n m a t u r e s t a g e s . R a d i a t i o n t r e a t m e n t a t -36 h o u r s a p p e a r s t o a f f e c t s p e r m a t o g e n e s i s as w e l l a s o o g e n e s i s . R o se and B a i l l i e ( 1 9 7 9 a ) h a v e d e m o n s t r a t e d t h a t i n e l e g a n s , a s i n D. m e l a n o g a s t e r ( S t e r n , 1926; S c h u l t z a n d R e d f i e l d , 1 9 5 1 ) , r e c o m b i n a t i o n f r e q u e n c y d e c r e a s e s w i t h m a t e r n a l a g e . I n C. e l e g a n s t h e d e c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y w i t h s u c c e s s i v e b r o o d s must be a r e f l e c t i o n o f c h a n g i n g r e c o m b i n a t i o n f r e q u e n c y i n o o g e n e s i s s i n c e s p e r m a t o g e n e s i s i s c o m p l e t e d p r i o r t o o o g e n e s i s . A f t e r t r e a t m e n t w i t h r a d i a t i o n , a l t h o u g h r e c o m b i n a t i o n f r e q u e n c y d e c r e a s e s w i t h age i t r e m a i n s e l e v a t e d a b o v e t h e c o n t r o l v a l u e s . I n o r d e r t o e v a l u a t e w h e t h e r t h i s e l e v a t i o n i n t h e l a t e r b r o o d s was t h e r e s u l t o f a n a f f e c t o f r a d i a t i o n on s p e r m a t o g e n e s i s , o u t c r o s s i n g o f t r e a t e d h e r m a p h r o d i t e s t o u n t r e a t e d m a l e s was d o n e . I n t h e s e e x p e r i m e n t s t h e r a d i a t i o n t r e a t e d s p e r m i n h e r m a p h r o d i t e s w e re r e p l a c e d w i t h u n t r e a t e d m a l e s p e r m ( s e e t h e R e s u l t s , s e c t i o n I - c - i i i ) w h i c h a r e u s e d 60 p r e f e r e n t i a l l y by t h e h e r m a p h r o d i t e . F e r t i l i z a t i o n w i t h m a l e sperm (XO) r e s u l t s i n 50% m a l e p r o g e n y w h i c h were c o u n t e d and u s e d t o c a l c u l a t e r e c o m b i n a t i o n f r e q u e n c y (a c o m b i n a t i o n o f r e c o m b i n a t i o n e v e n t s i n t h e h e r m a p h r o d i t e o o c y t e and t h e m a l e sperm b u t e x c l u d i n g r e c o m b i n a t i o n e v e n t s i n h e r m a p h r o d i t e s p e r m ) . The d a t a i s r e p o r t e d i n T a b l e 8. An i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d w i t h m a l e s p e r m i n b o t h t h e t r e a t e d and u n t r e a t e d i n d i v i d u a l s . T h i s i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was s u s p e c t e d t o be a r e s u l t o f t h e h i g h e r r e c o m b i n a t i o n f r e q u e n c y w h i c h h a s b e e n o b s e r v e d i n m a l e s ( R o s e , u n p u b l i s h e d d a t a ) . A f t e r t r e a t m e n t w i t h r a d i a t i o n , r e c o m b i n a t i o n f r e q u e n c y i n c r e a s e d i n b o t h s e l f - c r o s s e d a n d o u t - c r o s s e d h e r m a p h r o d i t e s . The m a g n i t u d e o f i n c r e a s e i s g r e a t e r i n t r e a t e d s e l f - c r o s s e d h e r m a p h r o d i t e s t h a n i n t h e t r e a t e d h e r m a p h r o d i t e s c r o s s e d t o u n t r e a t e d m a l e s . T h u s , r a d i a t i o n i s a f f e c t i n g o o g e n e s i s and may be h a v i n g some a f f e c t o n h e r m a p h r o d i t e s p e r m a t o g e n e s i s . The e f f e c t o f r a d i a t i o n on X-chromosome n o n d i s j u n c t i o n was i n v e s t i g a t e d . A t 20°C, a p p r o x i m a t e l y 1 m a l e p e r 700 w i l d - t y p e p r o g e n y was o b s e r v e d f o r t h e c o n t r o l s , a s p r e v i o u s l y r e p o r t e d ( H o d g k i n , 1 9 7 7 ; R o s e and B a i l l i e , 1 9 7 9 a ) . As w e l l , X-n o n d i s j u n c t i o n i n c r e a s e d w i t h m a t e r n a l age a n d t e m p e r a t u r e i n C. e l e g a n s ( R o s e a n d B a i l l i e , 1 9 7 9 a ) . I n D. m e l a n o g a s t e r , t e m p e r a t u r e ( G r e l l , 1 9 7 1 , 1973) and m a t e r n a l age ( T o k u n a g a , 1970) e f f e c t s on n o n d i s j u n c t i o n h a v e h a v e b e e n r e p o r t e d . I o b s e r v e d a n i n c r e a s e i n t h e r a t e o f X-chromosome n o n d i s j u n c t i o n w i t h r a d i a t i o n t r e a t m e n t ( T a b l e 4 ) . The d a t a p r e s e n t e d shows a 61 l i n e a r i n c r e a s e w i t h i n c r e a s i n g dose. I n o r d e r t o compare r a d i a t i o n - i n d u c e d n o n d i s j u n c t i o n and r e c o m b i n a t i o n e v e n t s , E x p e r i m e n t a l / C o n t r o l v a l u e s were p l o t t e d f o r each brood ( F i g u r e 1 1 ) . As p r e v i o u s l y d i s c u s s e d r e c o m b i n a t i o n f r e q u e n c y i s i n c r e a s e d o v e r more t h a n one b rood. N o n d i s j u n c t i o n , on the o t h e r hand, has a d e f i n i t e peak i n one of t h e b r o o d s . T h i s peak i n n o n d i s j u n c t i o n d i f f e r e d , when d i f f e r e n t d e v e l o p m e n t a l s t a g e s were t r e a t e d . The g r e a t e s t i n c r e a s e appeared i n C b rood f o r t h e 36-hr p r i o r t o e g g - l a y i n g s t a g e and i n B brood f o r t h e 12-hr p r i o r t o e g g - l a y i n g s t a g e . These r e s u l t s were somewhat unexpected ( T a b l e 1 1 ) . One might e x p e c t t h a t t h o s e i n d i v i d u a l s t h a t were t r e a t e d e a r l i e r would have an e a r l i e r peak i n n o n d i s j u n c t i o n or t h a t the peak would o c c u r i n the same br o o d r e g a r d l e s s of the time of t r e a t m e n t as was seen f o r t h e g r e a t e s t i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y . The r e a s o n f o r t h e o b s e r v e d r e s u l t i s not known, p o s s i b l e a d i f f e r e n t mechanism e x i s t s f o r the p r e - m e i o t i c r a d i a t i o n - i n d u c e d n o n d i s j u n c t i o n t h a n f o r the m e i o t i c . S i n c e the brood i n which the peak n o n d i s j u n c t i o n appeared v a r i e d w i t h t h e time of t r e a t m e n t , I have used the brood peak as means of c o n f i r m i n g t h a t s y n c h r o n i z e d p o p u l a t i o n s were t r e a t e d w i t h r a d i a t i o n i n s e p a r a t e e x p e r i m e n t s . I n o r d e r t o do so, t h e n o n - d i s j u n c t i o n c u r v e s were p l o t t e d f o r t h e d i f f e r e n t mutant s t r a i n s used i n s e p a r a t e e x p e r i m e n t s ( T a b l e 1 ? ) . I n o r d e r t o d e t e r m i n e whether r a d i a t i o n a f f e c t e d t h e f e c u n d i t y o f t h e p r o g e n y o f t r e a t e d i n d i v i d u a l s , a l l t h e r e c o m b i n a n t i n d i v i d u a l s r e c o v e r e d f r o m t h e e x p e r i m e n t s were p r o g e n y t e s t e d . As many a s 30% o f t h e s e r e c o m b i n a n t s w e r e f o u n d t o be s t e r i l e i n t h e a f f e c t e d b r o o d s a f t e r r a d i a t i o n t r e a t m e n t ( T a b l e 9 ) . H o w e v e r , none o f t h e n o n - r e c o m b i n a n t s t e s t e d w e r e s t e r i l e . Thus g a m m a - r a d i a t i o n a p p e a r e d t o c a u s e s t e r i l i t y p r e d o m i n a n t l y i n r e c o m b i n a n t i n d i v i d u a l s . F u r t h e r m o r e , t h e s t a g e o f maximum i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y by r a d i a t i o n t r e a t m e n t c o i n c i d e s w i t h t h a t o f maximum s t e r i l i t y o f r e c o m b i n a n t s : a l l t h e i n t e r v a l s w i t h t h e g r e a t e s t i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y a t B b r o o d show t h e maximum s t e r i l i t y a t t h e same d e v e l o p m e n t a l s t a g e ( T a b l e s 9 and 1 6 ) . C o m p a r a b l e r e s u l t s w e r e p r e v i o u s l y o b s e r v e d i n D r o s o p h i l a ( I v e s , 1 9 6 0 ) : t h e r e c o m b i n a t i o n f r e q u e n c y r e a c h e d i t s h i g h e s t l e v e l i n t h e same m e i o t i c s t a g e a t w h i c h t h e g r e a t e s t n e g a t i v e r a d i a t i o n e f f e c t on f e c u n d i t y o c c u r r e d . One o f t h e g o a l s i n t h i s t h e s i s was t o i n v e s t i g a t e t h e r e g i o n a l r e s p o n s e t o r a d i a t i o n t r e a t m e n t i n C. e l e g a n s . I n D. m e l a n o g a s t e r , a r e g i o n a l r e s p o n s e i n r e c o m b i n a t i o n f r e q u e n c y h a d b e e n d e m o n s t r a t e d p r e v i o u s l y ( T a t t e r s a l l , 1 9 8 1 ) : a d r a m a t i c maximum i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d i n a r e g i o n o f c e n t r i c h e t e r o c h r o m a t i n . B r e n n e r ( 1 9 7 4 ) s u g g e s t e d t h a t t h e a u t o s o m a l gene c l u s t e r s i n C. e l e g a n s w e r e a c o n s e q u e n c e o f r e c o m b i n a t i o n s u p p r e s s i o n i n a d e f i n e d r e g i o n o f a chromosome. S i n c e c l u s t e r e d r e g i o n s i n C. e l e g a n s may be c o m p a r a b l e t o r e g i o n s o f c e n t r i c h e t e r o c h r o m a t i n i n D. m e l a n o g a s t e r w i t h r e g a r d t o t h e r e d u c e d r e c o m b i n a t i o n f r e q u e n c y , 63 one might e x p e c t a s i m i l a r r e g i o n a l response t o r a d i a t i o n t r e a t m e n t i n C. e l e g a n s . I have s t u d i e d the c l u s t e r e d r e g i o n between dpy-5 and unc-13. I n o r d e r t o e v a l u a t e t h e e f f e c t of chromosomal r e g i o n a l i t y w i t h r e g a r d t o r a d i a t i o n - i n d u c e d r e c o m b i n a t i o n , an i n t e r v a l from th e n o n - c l u s t e r e d r e g i o n ( b l i - 3  unc-35) on L i n k a g e Group (LG) I was a l s o s t u d i e d . As can be seen i n T a b l e 16, the l a r g e s t i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d i n the dpy-14 unc-13 r e g i o n , which happens t o c o i n c i d e w i t h the r e g i o n of h i g h e s t gene d e n s i t y on LG I_. I n c o n t r a s t , no e f f e c t on r e c o m b i n a t i o n f r e q u e n c y was o b s e r v e d w i t h r a d i a t i o n t r e a t m e n t i n t h e b l i - 3 unc-35 i n t e r v a l , w h i c h c o i n c i d e s w i t h a gene poor r e g i o n . A l t h o u g h a l t e r n a t e e x p l a n a t i o n s a r e p o s s i b l e , t h i s f i n d i n g s u p p o r t s the p r o p o s a l o f B r enner (1974) t h a t the gene c l u s t e r s a r e a consequence of r e c o m b i n a t i o n s u p p r e s s i o n . Rec-1 i s a m e i o t i c r e c o m b i n a t i o n enhancer known t o cause an i n c r e a s e d r e c o m b i n a t i o n f r e q u e n c y t h r o u g h o u t th e whole genome. I t expands the map u n i f o r m l y . I n c o n t r a s t , "map e x p a n s i o n 1 by r a d i a t i o n t r e a t m e n t i s not p r o p o r t i o n a l t o the m e i o t i c map, t h a t i s , gamma-radiation produces r e g i o n a l d i f f e r e n c e s . T h e r e f o r e , gamma-radiation and rec-1 a r e p r o b a b l y a c t i n g t h r o u g h d i f f e r e n t mechanisms t o cause i n c r e a s e s i n r e c o m b i n a t i o n f r e q u e n c i e s . The r e g i o n a l response t o r a d i a t i o n t r e a t m e n t i n C. e l e g a n s i s comparable t o t h a t of D. m e l a n o g a s t e r . However, the magnitude of t h e i n c r e a s e i n the c l u s t e r e d r e g i o n i n C. e l e g a n s was not comparable t o the magnitude o f i n c r e a s e d e s c r i b e d f o r the c e n t r i c h e t e r o c h r o m a t i n i n D. m e l a n o g a s t e r . A more e x t e n s i v e s t u d y encompassing the e n t i r e chromosome seems t o be n e c e s s a r y t o s e a r c h f o r a r e g i o n w i t h p r o p e r t i e s o f c e n t r i c h e t e r o c h r o m a t i n . T h i s work has demonstrated f o r t h e f i r s t t i me t h a t gamma-radiation i n c r e a s e s r e c o m b i n a t i o n f r e q u e n c y i n C. e l e g a n s . F a c t o r s a f f e c t i n g the magnitude o f t h e i n c r e a s e have been i n v e s t i g a t e d . The amount of i n c r e a s e v a r i e d w i t h d i f f e r e n t chromosomal r e g i o n s presumably r e f l e c t i n g more a c c u r a t e l y the amount o f DNA th a n does t h e m e i o t i c map. I n c o m p a r i s o n , t h e r e c o m b i n a t i o n enhancer, Rec-1 a p p a r a n t l y i n c r e a s e s r e c o m b i n a t i o n a c c o r d i n g t o m e i o t i c r u l e s . The o b s e r v e d s t e r i l i t y a s s o c i a t e d w i t h t h e r a d i a t i o n - i n d u c e d r e c o m b i n a t i o n i s w o r t h f u r t h e r i n v e s t i g a t i o n and may be a r e f l e c t i o n of e x t e n s i v e DNA damage i n the r e g i o n o f t h e r e c o m b i n a t i o n e v e n t . 65 PROPOSALS FOR FURTHER RESEARCH A number o f e x p e r i m e n t s , which a r e proposed f o r f u r t h e r s t u d y from the r e s u l t s of my r e s e a r c h , a r e l i s t e d below. 1. I n v e s t i g a t i o n of t h e n a t u r e of F1 s t e r i l i t y w i t h r e g a r d t o l i n k e d l e t h a l e v e n t s . 2. E x t e n s i o n o f the i n v e s t i g a t i o n t o t h e r e s t o f t h e chromosomes t o s e a r c h f o r a r e g i o n w i t h the p r o p e r t i e s o f c e n t r i c h e t e r o c h r o m a t i n . 3. I n v e s t i g a t i o n of d i f f e r e n t methods f o r s y n c h r o n i z i n g the t ime o f r a d i a t i o n t r e a t m e n t (e.g. dauer l a r v a e , e t c . ) . 4. A n a l y s i s o f s h o r t e r brood i n t e r v a l ( e . g . 3-hr o r 4 - h r ) , t o d e t e r m i n e t h e e f f e c t o f r a d i a t i o n on s p e r m a t o g e n e s i s . 5. A n a l y s i s o f t h e i n t e r a c t i o n between rec-1 and gamma-radiation. BIBLIOGRAPHY Ab i - R a c h e d , M. and J . L . Brun. 1975. Changes i n t h e synaptonemal complex i n t h e o o c y t e n u c l e u s i n m e i o t i c prophase o f C. e l e g a n s . Rev Nematol. 1_: 63-72. A l b e r t s o n , D.G. and J.N. Thomson. 1982. The k i n e t o c h o r e s o f C a e n o r h a b d i t i s e l e g a n s . Chromosoma 8_6: 409-428. B o n n i e r , G. and K.G. L u n i n g . 1950. X-ray i n d u c e d dominant l e t h a l s i n D r o s o p h i l a m e l a n g a s t e r . H e r e d i t a s 36: 445-456 B r e n n e r , S. 1974. The g e n e t i c s of C a e n o r h a b d i t i s e l e g a n s . G e n e t i c s 77: 71-94. B u c k t o n , K.E. 1976. I d e n t i f i c a t i o n w i t h G and R b a n d i n g o f t h e p o s i t i o n o f breakage p o i n t s i n d u c e d i n human chromosomes by i n v i t r o x - i r r a d i a t i o n . I n t . J . R a d i a t . B i o l . 29: 475-488. Dougherty, E.S., E.L. Hansen, W.L. N i c h o l a s , J.H. M o l l e t t , and E.A. Yarwood. 1959. 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Pos t e m b r y o n i c c e l l l i n e a g e s of t h e h e r m a p h r o d i t e and male gonads i n C a e n o r h a b d i t i s  e l e g a n s . Develop B i o l . 70: 396-417. L i n d s l e y , D.L. and L. S a n d l e r . 1977. Segmental a n e u p l o i d y and t h e g e n e t i c s t r u c t u r e of the D r o s o p h i l a genome. G e n e t i c s 71: 157-184. L u n i n g , K.G. 1952. X-ray i n d u c e d chromosome b r e a k s i n D r o s o p h i l a m e l a n o g a s t e r . H e r e d i t a s 38_: 321 -338 M g l i n e t s , V.A. 1972. I r r a d i a t i o n - i n d u c e d c r o s s i n g o v e r i n D r o s o p h i l a males. C y t o l o g i c a l i n v e s t i g a t i o n o f c r o s s o v e r s . G e n e t i k a 8_: No 2: 82-92. M g l i n e t s , V.A. 1973. C y t o l o g i c a l a n a l y s i s o f c r o s s o v e r s i n d u c e d by i r r a d i a t i o n i n females of D r o s o p h i l a  m e l a n o g a s t e r . G e n e t i k a j ) : No 3: 69-75. Moerman, D.G., and D.L. B a i l l i e . 1978. G e n e t i c o r g a n i z a t i o n i n C a e n o r h a b d i t i s e l e g a n s : F i n e - s t r u c t u r e a n a l y s i s o f t h e unc-22 gene. G e n e t i c s 1_9: 95-103. M u l l e r , H.J. 1925. The r e g i o n a l l y d i f f e r e n t i a l e f f e c t o f x - r a y s on c r o s s i n g over i n autosomes o f D r o s o p h i l a . G e n e t i c s 10: 470-507. 68 N e l s o n , G.A., K.K. Lew, a n d S. Wards. 1982. I n t e r s e x , a t e m p e r a t u r e - s e n s i t i v e m u t a n t s o f t h e nematode C a e n o r h a b d i t i s e l e g a n s . D e v e l o p B i o l . 66: 3 8 6 - 4 0 9 . N i g o n , V. a n d J . B r u n . 1 9 5 5 . L ' e v o l u t i o n d e s s t r u c t u r e s n u c l e a i r e s d a n s l ' o v o g e n e s e de C. e l e g a n s Maupas 1900. Chromosoma 2^ 129-169. P a t t e r s o n , J . T . , and M.L. S u c h e . 1934. C r o s s i n g o v e r i n d u c e d by x - r a y s i n D r o s o p h i l a m a l e s . 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G e n e t i c s 7J7: 95-1 04. Tokunaga, C. 1970. The e f f e c t s of low te m p e r a t u r e and a g i n g on n o n - d i s j u n c t i o n i n D r o s o p h i l a . G e n e t i c s 65: 75-94. T a t t e r s a l l , P . J . 1981. Induced r e c o m b i n a t i o n i n t h e p r o x i m a l r e g i o n s f o r chromosome 2_ i n fe m a l e s o f D r o s o p h i l a m e l a n o g a s t e r . M a s t e r ' s T h e s i s , U.B.C. Ward, S., and J.S. C a r r e l . 1979. F e r t i l i z a t i o n and sperm c o m p e t i t i o n i n t h e nematode C. e l e g a n s . Develop. B i o l . 73: 304-321 W h i t t i n g h i l l , M. 1951. Some e f f e c t s o f gamma r a y s on r e c o m b i n a t i o n and c r o s s i n g o v e r i n D r o s o p h i l a  m e l a n o g a s t e r . G e n e t i c s 3_6: 332-355. Yeomans, T.C. 1972. Induced exchange and compound autosome f o r m a t i o n i n females o f D r o s o p n i l a m e l a n o g a s t e r . B. Sc. T h e s i s , U.B.C. A P P E N D I X 1: o •mblO mabl m,tfA mab2 u n c M \ unc-55 •mb6 unc 6 3 - l i r -7 CAENORHABDITIS ELEGANS GENETIC M A P A P R I L 1979 n ni l u M unc-45 1 dp»1 — I — dal 7 — I cal 3 da(2 1— nub 0 ~" n C- 7 9 —Tr 3 « . - 2 - 3 6 lon-1 d p y l 9 , u n c 3 2 s m a 4 \ d a f 4 / / u n c 00 unc 50 unc 49 • unc 47 0 I 2 3 4 5 % R E C O M B I N A T I O N him 10 1— t—mab-4 —t vah-7 ^dpy-IB , u p . , unc 81 unc-25 b l i - 5 —I unc-64 I TL dal 1 dal 10 1 l in l —+— : -»ab-2-unc 3 3 unc-17 i oim-4 t-unc-77-< —dpy 16 1 f ~ X ± = la , „ r •>•> ^-unc-4 3 i him 6 unc-/-; , himO. unc 82 ^ o n c a 0 . unc?8 .unci ° p y ' ^ iwc-5 ^ / u n c 3 unc44 himfl unc24 m t c - 3 corz •1555 • Op 6 unc-67 1 0 / / ' " 2 Alt'* 3 ' p y 4 - c i t - 5 -unc 60 I flu 1 unc-34 1 hrm-7 —I unc-72 1 unc 0 3 -siip 8 dpy-11 unc 42 unc 46 — osm-6 — dpy 15 — unc 70—• „ i - r - t>-vab8-t ' n < ' : 9 iot-3 i—ca l4 unc 23 unc€0 .jnac-1 .ol 4 »ql-3 unc 6 5 unc 61 ' unc-39 dpy-21 ojm-1 d»l 3 unc-1 I dp»3 1— unc-2 \ dal 9 i—osm-5-unc-20 llu-2 ton 2 mnDp33 mn cal-1 4 -Iev9 unc6 leva . m . c - 7 — > * M him-4 • U P 2 H unc27 „ a 4 , unc58 . 5 unc-18 „ lel-4 Ipy V y«ncf une-84 If <r V V unc 9 m«c 5 » mec 4 leM4 lettB «--del 6--> hl-35 111 3 3 •mb-4 unc 51 1 mn0p32 mnDp30 mnDpl ^mnDp2.mnDp8. mnDp26,mnDp27 ^mnnp3.nuiDp4, mnOpIO ' mnDp9 ' mnOp2S 'mnOll.mnDIQ ,mnDf2.(wiDII3 , mnDIS ,mnDI4 mnr)l5,mnOII8 mnDI7.mnOIIO,mnDI21 mnDI8,mnOI17 v ^ _ ~ m n M 9 . m n O l t 5 NS>- innDII I X"mnDI 19 x mnOI20 APPENDIX 2: RADIATION-INDUCED MAP EXPANSION dpy-5 unc-13 b l i - 3 unc-35 unc-11 MEIOTIC MAP RADIATION MAP • • • • dpy-14 • • • 0.9 1.4 1.8 4.7 

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