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Position effect variegation in Drosophila melanogaster : chemical modification and mutational analysis Mottus, Randall C. 1983

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POSITION EFFECT VARIEGATION IN DROSOPHILA MELANOGASTER; CHEMICAL MODIFICATION AND MUTATIONAL ANALYSIS by RANDALL C. MOTTUS B.Sc..U.B.C.,Vancouver,1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Z o o l o g y ) We a c c e p t t h i s t h e s i s a s 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 THE UNIVERSITY OF BRITISH COLUMBIA September 1983 © R a n d a l l C. M o t t u s , 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department o The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date (QcVr. D E - 6 ( 3 / 8 1 ) i i ABSTRACT P o s i t i o n e f f e c t v a r i e g a t i o n i s an event i n which the t r a n s c r i p t i o n a l f a t e of a gene i s de termined long b e f o r e the p r o d u c t of t h a t gene i s r e q u i r e d . T h u s , i t p a r a l l e l s the normal p r o c e s s e s of d e t e r m i n a t i o n and d i f f e r e n t i a t i o n a s s o c i a t e d w i t h development in h i g h e r organ i sms . P o s i t i o n e f f e c t v a r i e g a t i o n i s thought to r e s u l t from a l t e r a t i o n s i n chromat in s t r u c t u r e a t the new h e t e r o c h r o m a t i c - e u c h r o m a t i c boundary . I have found tha t when f r u i t f l i e s ( D r o s o p h i l a m e l a n o q a s t e r ) , b e a r i n g a v a r i e g a t i n g gene, a r e r e a r e d on meduim c o n t a i n i n g low c o n c e n t r a t i o n s of e i t h e r n - b u t y r a t e or n - p r o p i o n a t e , the gene i n a c t i v a t i o n a s s o c i a t e d w i t h p o s i t i o n e f f e c t v a r i e g a t i o n i s s u p p r e s s e d . T h i s i s t r u e of both X - l i n k e d and autosomal v a r i e g a t i n g genes r e g a r d l e s s of the type of h e t e r o c h r o m a t i n wi th which they are a s s o c i a t e d . These c h e m i c a l s are on ly e f f e c t i v e as s u p p r e s s o r s of v a r i e g a t i o n d u r i n g the embryonic and l a r v a l s tages s u g g e s t i n g they may o n l y a c t to modify v a r i e g a t i o n i n d i v i d i n g c e l l s . Removal of the Y chromosome i n males c o m p l e t e l y a b o l i s h e s the e f f e c t s of n - b u t y r a t e and n - p r o p i o n a t e . I examined the a b i l i t y of these c h e m i c a l s to. suppress v a r i e g a t i o n i n f l i e s h a p l o i d f o r the h i s t o n e gene c l u s t e r and found tha t the combined e f f e c t was a d d i t i v e . These r e s u l t s are d i s c u s s e d wi th re spec t to c u r r e n t knowledge of chromat in s t r u c t u r e and the known e f f e c t s of n - b u t y r a t e and n - p r o p i o n a t e . In a d d i t i o n a l a r g e s c a l e screen was undertaken to i s o l a t e and r e c o v e r dominant mutat ions which e i t h e r suppress or enhance the v a r i e g a t e d phenotype . In t o t a l , 54 mutat ions were r e c o v e r e d : 51 s u p p r e s s o r s , and 3 e n h a n c e r s . P r e l i m i n a r y c h a r a c t e r i z a t i o n has r e v e a l e d t h a t t h e m a j o r i t y of t h e s u p p r e s s o r m u t a t i o n s a r e c l u s t e r e d a t s p e c i f i c s i t e s i n t h e D. M e l a n o g a s t e r genome. A m a j o r i t y of t h e s u p p r e s s o r s a r e s e n s i t i v e t o t h e h e t e r o c h r o m a t i c c o n s t i t u t i o n o f t h e o r g a n i s m but most a r e i n s e n s i t i v e t o a l t e r a t i o n s i n d e v e l o p m e n t a l t e m p e r a t u r e . The p o s s i b i l i t y that' some of t h e s e m u t a t i o n s r e p r e s e n t l e s i o n s i n genes c o d i n g f o r s t r u c t u r a l components of c h r o m a t i n s u g g e s t s f u r t h e r c h a r a c t e r i z a t i o n , b o t h a t t h e g e n e t i c and m o l e c u l a r l e v e l , may p r o v i d e i n s i g h t s n o t o n l y i n t o p o s i t i o n e f f e c t v a r i e g a t i o n b u t a l s o p r o v i d e i n f o r m a t i o n r e g a r d i n g t h e b a s i c s t r u c t u r e o f c h r o m a t i n . i v TABLE OF CONTENTS ABSTRACT i i L I S T OF TABLES v L I S T OF FIGURES v i i ACKNOWLEDGEMENTS v i i i GENERAL INTRODUCTION 1 CHAPTER ONE 11 INTRODUCTION 11 MATERIAL and METHODS 14 RESULTS 22 DISCUSSION 51 CHAPTER TWO 62 INTRODUCTION 62 MATERIALS and METHODS 64 RESULTS 69 DISCUSSION 97' SUMMARY 104 REFERENCES CITED 106 A p p e n d i x A 115 A p p e n d i x B 118 V LIST OF TABLES T a b l e I . The e f f e c t of exposure t o - b u t y r a t e on v a r i e g a t i o n of # v 29 T a b l e I I . The e f f e c t of exposure to n - b u t y r a t e or n -v32e p r o p i o n a t e on v a r i e g a t i o n of bw 31 T a b l e I I I . The e f f e c t of exposure to n - b u t y r a t e or n -. v p r o p i o n a t e on v a r i e g a t i o n of Sb 32 T a b l e I V . The e f f e c t of exposure of pupae to n - b u t y r a t e or n - p r o p i o n a t e on v a r i e g a t i o n of wm^ 45 T a b l e V . The e f f e c t of exposure to n - b u t y r a t e on v a r i e g a t i o n of w m 4 i n X / 0 males 47 T a b l e V I . The e f f e c t of exposure to n - b u t y r a t e on ni4 v a r i e g a t i o n of w in s t r a i n s b e a r i n g a h i s t o n e d e f i c i e n c y 50 T a b l e V I I . Complementat ion a n a l y s i s of s e l e c t e d s u p p r e s s o r s of p o s i t i o n e f f e c t v a r i e g a t i o n l o c a t e d on chromosome 2 . 77 T a b l e V I I I . Complementation a n a l y s i s of s u p p r e s s o r s of p o s i t i o n e f f e c t v a r i e g a t i o n l o c a t e d on chromosomes 2 and 3 •-. 82 T a b l e I X . The i n t e r a c t i o n between s u p p r e s s o r s l o c a t e d on chromosome 2 and v a r i o u s genes s u b j e c t to p o s i t i o n . e f f e c t v a r i e g a t i o n 84 T a b l e X . The i n t e r a c t i o n between s u p p r e s s o r s l o c a t e d on chromosome 3 and v a r i o u s genes s u b j e c t to p o s i t i o n e f f e c t v a r i e g a t i o n 87 v i T a b l e X I . The i n t e r a c t i o n between enhancers of v a r i e g a t i o n and v a r i o u s genes s u b j e c t to p o s i t i o n e f f e c t v a r i e g a t i o n 89 T a b l e X I I . The e f f e c t of s u p p r e s s o r s on v a r i e g a t i o n of w m 4 i n X / 0 males 91 T a b l e X I I I . The e f f e c t of deve lopmenta l temperature on s u p p r e s s o r s of v a r i e g a t i o n i n s t r a i n s b e a r i n g I n ( 1 ) w m 4 . 94 v i i L I S T OF FIGURES F i g u r e I . M a l e I n ( l ) w m 4 f l i e s e x p o s e d t o v a r i o u s c o n c e n t r a t i o n s of n - b u t y r a t e , n - p r o p i o n a t e , o r t e g o s e p t f r o m egg d e p o s i t i o n t o p u p a r i u m f o r m a t i o n 24 F i g u r e I I . Female I_n(Owra4 f l i e s e x p o s e d t o v a r i o u s c o n c e n t r a t i o n s o f n - b u t y r a t e , n - p r o p i o n a t e , o r t e g o s e p t f r o m egg d e p o s i t i o n t o p u p a r i u m f r o m a t i o n 26 F i g u r e I I I . I n ( l ) w m 4 f l i e s e x p o s e d t o 0.15 M n - b u t y r a t e f o r 48 h o u r p e r i o d s 35 F i g u r e I V . I_n(j_)w m /* f l i e s e x p o s e d t o 0.15 M n - p r o p i o n a t e f o r 48 hou r p e r i o d s 37 F i g u r e V. l n ( l ) w m 4 f l i e s e x p o s e d t o 0.15 M n - b u t y r a t e f o r 24 h o u r p e r i o d s 40 F i g u r e V I . I n . ( l ) w m 4 f l i e s e x p o s e d t o 0.15 M n - p r o p i o n a t e f o r 24 hou r p e r i o d s 42 F i g u r e V I I . The s c r e e n i n g p r o t o c o l f o r i s o l a t i o n o f dom i n a n t m u t a t i o n s w h i c h m o d i f y p o s i t i o n e f f e c t v a r i e g a t i o n 70 F i g u r e V I I I . The map p o s i t i o n s o f e n h a n c e r s and s e l e c t e d s u p p r e s s o r s o f p o s i t i o n e f f e c t v a r i e g a t i o n 74 F i g u r e IX. A c o m p l e m e n t a t i o n map o f s u p p r e s s o r s o f p o s i t i o n e f f e c t v a r i e g a t i o n l o c a t e d on chromosome 2 79 v i i i ACKNOWLEDGEMENTS I would l i k e t o thank D r . T.A. G r i g l i a t t i f o r h i s e n t h u s i a s m , g u i d a n c e , and h e l p f u l c r i t i c i s m t h r o u g h o u t t h e c o u r s e o f t h i s work. In a d d i t i o n , I thank D r . D.A. S i n c l a i r f o r h i s c o l l a b o r a t i o n and c r i t i c a l r e a d i n g o f t h i s m a n u s c r i p t . L a s t , b u t n o t l e a s t , I thank t h e members o f t h e F l y L a b f o r t h e good t i m e s and good t a l k s . 1 GENERAL INTRODUCTION I t has become c l e a r i n r e c e n t y e a r s t h a t t h e r e i s an i n t i m a t e r e l a t i o n s h i p between gene e x p r e s s i o n and c h r o m a t i n s t r u c t u r e . One s y s t e m t h a t may p r o v i d e u n i q u e o p p o r t u n i t i e s t o s t u d y t h i s r e l a t i o n s h i p i s p o s i t i o n e f f e c t v a r i e g a t i o n . P o s i t i o n e f f e c t v a r i e g a t i o n u s u a l l y o c c u r s when an e u c h r o m a t i c r e g i o n of a chromosome i s j u x t a p o s e d t o an h e t e r o c h r o m a t i c b r e a k p o i n t by a chromosomal r e a r r a n g e m e n t . I n some c e l l s e u c h r o m a t i c g e n e s l y i n g a d j a c e n t t o t h e h e t e r o c h r o m a t i n a r e c o n d e n s e d and t h e r e b y i n a c t i v a t e d w h i l e i n o t h e r c e l l s i n t h e same t i s s u e t h e p a c k a g i n g o f t h e e u c h r o m a t i c g e n e s i s n o r m a l and t h e y a r e e x p r e s s e d . T h i s g i v e s r i s e t o t h e m o s a i c o r v a r i e g a t e d p h e n o t y p e a s s o c i a t e d w i t h p o s i t i o n e f f e c t v a r i e g a t i o n . Thus, i n p o s i t i o n e f f e c t v a r i e g a t i o n t h e r e i s a c l e a r a s s o c i a t i o n between t h e s t a t e o f c h r o m a t i n p a c k a g i n g and t h e t r a n s c r i p t i o n a l c a p a c i t y o f a gene. A c c o r d i n g l y , an u n d e r s t a n d i n g o f t h i s phenomenon p r o m i s e s t o p r o v i d e i n s i g h t i n t o t h e r e l a t i o n s h i p between c h r o m a t i n s t r u c t u r e and gene e x p r e s s i o n . In a d d i t i o n , much o f t h e a n a l y s i s o f t h e m o s a i c p h e n o t y p e i n d i c a t e s t h a t t h e d e c i s i o n a s t o whether a v a r i e g a t i n g gene w i l l be a c t i v e o r not i s made e a r l y i n d e v e l o p m e n t . Once made, t h e d e c i s i o n i s o f t e n i r r e v o c a b l e and p r o p o g a t e d c l o n a l l y . T h u s , t h e r e a r e c l o s e p a r a l l e l s between p o s i t i o n e f f e c t v a r i e g a t i o n a n d t h e no r m a l p r o c e s s e s of d e t e r m i n a t i o n and d i f f e r e n t i a t i o n w h i c h o c c u r d u r i n g d e v e l o p m e n t i n a l l h i g h e r o r g a n i s m s as w e l l a s X chromosome i n a c t i v a t i o n i n mammals. An u n d e r s t a n d i n g o f t h e m o l e c u l a r b a s i s o f p o s i t i o n e f f e c t 2 v a r i e g a t i o n may shed l i g h t on t h e s e phenomena. P o s i t i o n e f f e c t v a r i e g a t i o n has been o b s e r v e d i n a wide v a r i e t y o f o r g a n i s m s , from f u n g i t o man and i s t h e r e f o r e c o n s i d e r e d t o be a u b i q u i t o u s phenomenon i n e u c a r y o t e s . A l t h o u g h u n i v e r s a l i n e x p r e s s i o n and r e l a t e d t o gene r e g u l a t i o n , u n t i l r e c e n t l y , l i t t l e p r o g r e s s has been made i n u n d e r s t a n d i n g t h i s phenomenon a t t h e m o l e c u l a r l e v e l . The b u l k of i n f o r m a t i o n a b o u t p o s i t i o n e f f e c t v a r i e g a t i o n comes from s t u d i e s i n v o l v i n g D r o s o p h i l a m e l a n o g a s t e r ( f o r r e v i e w s see LEWIS,1950; BAKER, 1968; SPOFFORD, 1976). A l t h o u g h i t was f i r s t d e s c r i b e d by BRIDGES and MORGAN (1923) f o r t h e p l e x u s (p_x) gene, i t was STURTEVANT ( 1925) who r e p o r t e d t h a t m o s a i c e x p r e s s i o n o f t h e p_x l o c u s was a s s o c i a t e d w i t h a c hromosomal r e a r r a n g e m e n t . MULLER's (1930) work w i t h X - r a y i n d u c e d chromosomal r e a r r a n g e m e n t s marked t h e b e g i n n i n g o f a s y s t e m a t i c a n a l y s i s o f p o s i t i o n e f f e c t v a r i e g a t i o n . He o b s e r v e d m o s a i c e x p r e s s i o n o f s e v e r a l genes a s s o c i a t e d w i t h chromosomal r e a r r a n g e m e n t s . S i n c e t h a t t i m e s t u d i e s have r e v e a l e d t h a t v i r t u a l l y e v e r y gene e x a m i n e d i n D r o s o p h i l a c a n be i n d u c e d t o u n d e r g o p o s i t i o n e f f e c t v a r i e g a t i o n (SPOFFORD, 1976; HENIKOFF, 1979). T h u s , t h i s phenomenon i s n o t l i m i t e d t o a c e r t a i n c l a s s o f g e n e s . A l a r g e body o f e v i d e n c e now e x i s t s w h i c h c l e a r l y i n d i c a t e s t h a t t h e m o s a i c p h e n o t y p e o b s e r v e d i n p o s i t i o n e f f e c t v a r i e g a t i o n i s a r e s u l t o f v a r i a b l e gene e x p r e s s i o n r a t h e r t h a n m u t a t i o n . R e c o m b i n a t i o n o r s t r u c t u r a l r e a r r a n g e m e n t s w h i c h r e t u r n v a r i e g a t i n g genes t o a e u c h r o m a t i c l o c a t i o n r e s u l t i n t h e r e s t o r a t i o n o f f u l l gene a c t i v i t y (HINTON, 1950; GRUNEBERG, 3 1937). F o r example, t h e w h i t e m o t t l i n g a s s o c i a t e d w i t h T ( J _ ;4)w m 1 1 was r e d u c e d o r e l i m i n a t e d when t h e t i p o f t h e X chromosome c o n t a i n i n g t h e w h i t e gene (wj_) was r e l o c a t e d t o e u c h r o m a t i n (PANSHIN, 1938). R e c o m b i n a n t s have been r e c o v e r e d between v a r i e g a t i o n i n d u c i n g t r a n s l o c a t i o n and s t r u c t u r a l l y n o r m a l homologous chromosomes w h i c h e x c h a n g e d a v a r i e g a t i n g a l l e l e w i t h i t s mutant. The r e c o m b i n a n t s showed n o r m a l gene a c t i v i t y i n d i c a t i n g t h a t t h e h e t e r o c h r o m a t i c e n v i r o n m e n t , r a t h e r t h a n m u t a t i o n o f t h e l o c u s , was r e s p o n s i b l e f o r t h e m o s a i c gene e x p r e s s i o n (PANSHIN, 1938; JUDD, 1955). W h i l e many t h e o r i e s have been a d v a n c e d t o e x p l a i n p o s i t i o n e f f e c t v a r i e g a t i o n , o n l y two have g e n e r a t e d s i g n i f i c a n t a t t e n t i o n . PATERSON (1932b) s u g g e s t e d t h a t t h e p r o x i m i t y o f t h e h e t e r o c h r o m a t i c b r e a k p o i n t r e s u l t e d i n s o m a t i c i n s t a b i l i t y of t h e g e n e . T h u s , m o s a i c e x p r e s s i o n o f a v a r i e g a t i n g gene was t h o u g h t t o be t h e r e s u l t o f s o m a t i c gene l o s s . T h i s now seems u n l i k e l y s i n c e i n s i t u h y b r i d i z a t i o n o f a h e a t shock gene p r o b e t o t h e p o l y t e n e chromosomes o f a s t r a i n w h i c h v a r i e g a t e d f o r t h e s e g e n e s showed h y b r i d i z a t i o n i n n u c l e i i n w h i c h t h e bands c o n t a i n i n g t h e s e g e n e s were a b s e n t . R a t h e r t h a n b e i n g l o s t s o m a t i c a l l y i t a p p e a r s t h e e u c h r o m a t i n c o n t a i n i n g t h e s e genes h a d t a k e n on an h e t e r o c h r o m a t i c a p p e a r a n c e (HENIKOFF, 1979). I n a d d i t i o n , a n a l y s i s o f t h e m o s a i c p h e n o t y p e p r o d u c e d by v a r i e g a t i n g a l l e l e s o f t h e w_; gene i n d i c a t e s t h a t t h e d e c i s i o n a s t o whether o r n o t t h e v a r i e g a t i n g gene w i l l be a c t i v e i n a c e l l a p p e a r s t o be made e a r l y i n d e v e l o p m e n t , p r o d u c i n g l a r g e c l o n e s o f p i g m e n t e d and u n p i g m e n t e d o m m a t i d i a (BECKER, 1957). 4 However, i n some c a s e s , s u c h as Iri(J_)w , t h e e y e s c o n t a i n s m a l l c l o n e s o f p i g m e n t e d and u n p i g m e n t e d o m m a t i d i a s u p e r i m p o s e d on t h e o r i g i n a l p a t t e r n i n d i c a t i n g t h a t t h e o r i g i n a l d e t e r m i n a t i v e e v e n t i s not i r r e v o c a b l e . C l e a r l y , t h e w_^  was not l o s t o r e l i m i n a t e d i n t h o s e c e l l s w h i c h r e g a i n wj_ gene a c t i v i t y l a t e i n d e v e l o p m e n t . The n o t i o n t h a t v a r i e g a t i o n i s a r e s u l t of s o m a t i c gene l o s s i s i n c o m p a t i b l e w i t h t h e s e o b s e r v a t i o n s . An h y p o t h e s i s more c o m p a t i b l e w i t h t h e e x i s t i n g e v i d e n c e was o r i g i n a l l y p r o p o s e d by DEMEREC and SLIZYNSKA ( 1 9 3 7 ) . They s u g g e s t e d t h a t t h e m o s a i c e x p r e s s i o n o f genes c o n t i g u o u s w i t h an h e t e r o c h r o m a t i c b r e a k p o i n t was t h e r e s u l t o f a c e l l autonomous s p r e a d o f h e t e r o c h r o m a t i c m a t e r i a l i n t o t h e a d j a c e n t e u c h r o m a t i n . P r e s u m a b l y , i n some c e l l s t h e h e t e r o c h r o m a t i n would s p r e a d f a r enough t o c o n d e n s e and t r a n s c r i p t i o n a l l y i n a c t i v a t e n e a r b y g e n e s w h i l e i n o t h e r s t h e s e l o c i would r e m a i n a c t i v e . T h i s a c c o u n t s f o r t h e o b s e r v a t i o n t h a t t h e l e v e l o f v a r i e g a t i o n a p p e a r s t o e x t e n d a l o n g t h e e u c h r o m a t i c r e g i o n o f t h e chromosome i n a l i n e a r f a s h i o n . Genes c l o s e s t t o t h e b r e a k p o i n t a r e e x p r e s s e d l e s s o f t e n t h a n t h o s e f u r t h e r away. The d i s t a n c e o v e r w h i c h v a r i e g a t i o n c a n be i n d u c e d v a r i e s w i t h e a c h r e a r r a n g e m e n t and has been r e p o r t e d t o e x t e n d as f a r a s 80 p o l y t e n e bands away f r o m t h e h e t e r o c h r o m a t i c b r e a k p o i n t (SPOFFORD, 1976). In a d d i t i o n , t h e above h y p o t h e s i s i s a l s o c o n s i s t e n t w i t h t h e o b s e r v e d c o r r e l a t i o n between t h e l e v e l o f v a r i e g a t i o n and t h e e x t e n t o f h e t e r o c h r o m a t i n i z a t i o n i n t h e p o l y t e n e chromosomes o f D. m e l a n o g a s t e r (HARTMANN-GOLDSTEIN, 1967) s u g g e s t i n g a d i r e c t l i n k between c h r o m a t i n p a c k a g i n g as 5 h e t e r o c h r o m a t i n and gene i n a c t i v a t i o n . A l s o i n l i n e w i t h t h i s h y p o t h e s i s a r e r e c e n t e x p e r i m e n t s i n w h i c h TARTOF ( p e r s o n n a l c o m m u n i c a t i o n ) f o u n d t h a t p o s i t i o n e f f e c t v a r i e g a t i o n o f t h e wj_ gene i n D r o s o p h i l a may be a s s o c i a t e d w i t h Type I i n s e r t s . He f o u n d t h a t i n two o f t h r e e v a r i e g a t i n g s t r a i n s ( w m 4 and w m M c ) p a r t o f a Type I i n s e r t was a s s o c i a t e d w i t h t h e wj_ gene. S i n c e i t i s t h o u g h t t h a t Type I i n s e r t s p r e v e n t t r a n s c r i p t i o n o f r i b o s o m a l g e n e s w i t h w h i c h t h e y a r e a s s o c i a t e d (LONG e t . a l . , 1981), TARTOF b e l i e v e s t h a t Type I i n s e r t s may a l s o d i s r u p t t r a n s c r i p t i o n o f genes s u b j e c t t o p o s i t i o n e f f e c t v a r i e g a t i o n on t h e X chromosome. Type I i n s e r t s c a n n o t a c c o u n t f o r a l l c a s e s o f p o s i t i o n e f f e c t v a r i e g a t i o n s i n c e t h e y a r e n o t f o u n d on t h e Y chromosome (TARTOF and DAWID, 1976) and one o f t h e wj_ v a r i e g a t o r s e x a m i n e d by TARTOF ( w m 5 1 b ) i s n o t a s s o c i a t e d w i t h a Type I i n s e r t . However, he may have i d e n t i f i e d s p e c i f i c DNA s e q u e n c e s from w h i c h h e t e r o c h r o m a t i n i z a t i o n may be i n i t i a t e d ( f o r d i s c u s s i o n see SPOFFORD, 1976) on t h e X chromosome. A s u b s t a n t i a l p r o p o r t i o n of t h e e a r l i e r r e s e a r c h on p o s i t i o n e f f e c t v a r i e g a t i o n i n v o l v e d t h e s t u d y o f e x t r i n s i c and i n t r i n s i c f a c t o r s w h i c h m o d i f y t h i s phenomenon. The most e x t e n s i v e l y a n a l y s e d e x t r i n s i c f a c t o r i s t e m p e r a t u r e . The e f f e c t o f t e m p e r a t u r e on v a r i e g a t i o n was f i r s t i d e n t i f i e d by GOWAN and GAY ( 1 9 3 4 ) . In g e n e r a l , i t has been shown t h a t t e m p e r a t u r e and v a r i e g a t i o n a r e i n v e r s e l y r e l a t e d . The l o w e r t h e t e m p e r a t u r e a t w h i c h a v a r i e g a t i n g s t r a i n d e v e l o p s , t h e g r e a t e r t h e p r o p o r t i o n o f c e l l s i n a p a r t i c u l a r t i s s u e i n w h i c h 6 t h e v a r i e g a t i n g gene i s i n a c t i v e ( f o r a r e v i e w see SPOFFORD, 1976). T h i s e f f e c t has been e x p l o i t e d t o d e t e r m i n e t h e t e m p e r a t u r e s e n s i t i v e p e r i o d s ( t s p s ) f o r s e v e r a l v a r i e g a t i n g a l l e l e s . The t s p c a n be e i t h e r c o n t i n u o u s o r b i p h a s i c . I n c a s e s of t h e l a t t e r , one o f t h e t s p u s u a l l y o c c u r s v e r y e a r l y i n e m b r y o g e n e s i s a r o u n d t h e t i m e o f b l a s t o d e r m f o r m a t i o n and t h e o t h e r l a t e r , d u r i n g t h e l a r v a l o r p u p a l p e r i o d . I t has been s u g g e s t e d t h a t t h e e a r l y t s p c o i n c i d e s w i t h t h e t i m e a t w h i c h t h e t r a n s c r i p t i o n a l f a t e o f t h e v a r i e g a t i n g a l l e l e i s d e t e r m i n e d w h i l e t h e l a t e r t s p c o i n c i d e s w i t h t h e t i m e t h e gene i s a c t i v e ( r e v i e w SPOFFORD, 1976; SCHULTZ, 1956; HARTMANN-GOLDSTEIN, 1967). However, t h e r e a r e many e x c e p t i o n s t o t h i s g e n e r a l p a t t e r n and s i n c e t h e e f f e c t s o f t e m p e r a t u r e a r e complex and n o t w e l l u n d e r s t o o d l i t t l e i n s i g h t i n t o t h e m o l e c u l a r n a t u r e o f p o s i t i o n e f f e c t v a r i e g a t i o n has been g a i n e d f r o m t h e s e s t u d i e s . G e n e t i c m o d i f i e r s have a l s o been i d e n t i f i e d w h i c h c a n p r o f o u n d l y a f f e c t p o s i t i o n e f f e c t v a r i e g a t i o n . SCHULTZ ( c i t e d i n GSELL, 1971) i s o l a t e d a m u t a t i o n , E ( v a r ) 7 , w h i c h a p p e a r s t o be a s t r o n g e n h a n c e r o f v a r i e g a t i o n (BROSSEAU, 1960; GSELL, 1971; LINDSLEY e t a l . , 1960). SPOFFORD (1967) r e c o v e r e d a s p o n t a n e o u s s u p p r e s s o r o f p o s i t i o n e f f e c t v a r i e g a t i o n , S u ( v a r ) , w h i c h mapped t o t h e l e f t arm o f t h e t h i r d chromosome. T h e r e i s e v i d e n c e t h a t g e n e t i c m o d i f i e r s o f p o s i t i o n e f f e c t v a r i e g a t i o n may be q u i t e numerous. HENIKOFF (1979) o b s e r v e d two s u p p r e s s o r s o f v a r i e g a t i o n i n a r e g i o n on 3R u n c o v e r e d by a s m a l l d e l e t i o n . B a s e d on t h i s o b s e r v a t i o n and a s s u m i n g a random d i s t r i b u t i o n o f t h e s e genes he e s t i m a t e d t h e r e were s e v e r a l h u n d r e d s u p p r e s s o r s 7 of p o s i t i o n e f f e c t v a r i e g a t i o n i n t h e D. m e l a n o g a s t e r genome. However, u n t i l r e c e n t l y no s y s t e m a t i c a t t e m p t has been made t o i d e n t i f y t h e g e n e t i c components of t h e v a r i e g a t i n g p r o c e s s . The b e s t c h a r a c t e r i z e d i n t r i n s i c m o d i f i e r o f p o s i t i o n e f f e c t v a r i e g a t i o n i s t h e e f f e c t of v a r i a t i o n s of Y chromosome h e t e r o c h r o m a t i n . In t h e v a s t m a j o r i t y o f c a s e s t h e a d d i t i o n o f Y h e t e r o c h r o m a t i n t o t h e genome r e s u l t s i n an i n c r e a s e i n t h e e x p r e s s i o n o f a v a r i e g a t i n g gene, t h a t i s s u p p r e s s i o n o f t h e v a r i e g a t e d p h e n o t y p e . The r e m o v a l o f Y h e t e r o c h r o m a t i n from t h e genome has t h e o p p o s i t e e f f e c t , r e d u c i n g e x p r e s s i o n of t h e v a r i e g a t i n g gene r e s u l t i n g i n enhancement o f v a r i e g a t i o n (GOWAN and GAY, 1934; SPOFFORD, 1976). The s u p p r e s s o r e f f e c t o f t h e Y chromosome has been l o c a l i z e d t o s p e c i f i c r e g i o n s (BAKER and SPOFFORD, 1959; BAKER and REIN, 1962; BROSSEAU, 1964) however m o d i f i c a t i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n i s n o t l i m i t e d t o Y chromosome h e t e r o c h r o m a t i n . The b a s a l h e t e r o c h r o m a t i n o f t h e X i s a l s o a p o t e n t s u p p r e s s o r o f v a r i e g a t i o n (NOUJDIN, 1944) w h i l e d e f i c i e n c e s o f t h e 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 o f t h e s e c o n d chromosome enh a n c e t h e v a r i e g a t i n g p h e n o t y p e (SCHULTZ, 1936; BROSSEAU, 1960; LINDSLEY e t a l . , 1960). Taken t o g e t h e r t h e s e s t u d i e s i n d i c a t e t h a t t h e l e v e l o f v a r i e g a t i o n o b s e r v e d i n an i n d i v i d u a l i s a r e f l e c t i o n o f t h e amount and p o s s i b l y t h e t y p e o f c o n s t i t u t i v e h e t e r o c h r o m a t i n p r e s e n t i n t h e genome. C o n s t i t u t i v e h e t e r o c h r o m a t i n i s d e n s e l y s t a i n i n g c h r o m a t i n w h i c h r e m a i n s i n a c o n d e n s e d c o n f o r m a t i o n t h r o u g h o u t t h e c e l l c y c l e . I t i s p r i m a r i l y composed of s h o r t r e p e t i t i v e s a t e l l i t e DNA s e q u e n c e s w i t h v e r y few c o d i n g r e g i o n s (YASMENEH and YUNIS, 8 1969; BLUMENFELD and FORREST, 1971; PEACOCK e t a l . , 1974; PEACOCK e t a l . , 1977),. I f , a s has been p r o p o s e d , p o s i t i o n e f f e c t v a r i e g a t i o n i s a r e s u l t of h e t e r o c h r o m a t i c e l e m e n t s s p r e a d i n g i n t o t h e a d j a c e n t e u c h r o m a t i n t h e n f a c t o r s i n v o l v e d i n c h r o m a t i n p a c k a g i n g and s p e c i f i c a l l y t h e f o r m a t i o n of h e t e r o c h r o m a t i n might be d i r e c t l y i n v o l v e d i n t h e v a r i e g a t i o n p r o c e s s . S e v e r a l a u t h o r s have n o t e d t h a t t h e p r o t e i n component of h e t e r o c h r o m a t i n i s d i f f e r e n t from t h a t i n e u c h r o m a t i n . BERLOWITZ (1965) has shown t h e h e t e r o c h r o m a t i c chromosome s e t i n t h e mealy bug i s e n r i c h e d i n h i s t o n e c o n t e n t and i t has been d e m o n s t r a t e d t h a t c e r t a i n s u b s p e c i e s o f p h o s p h o r y l a t e d h i s t o n e H1 a r e a s s o c i a t e d w i t h D r o s o p h i l a h e t e r o c h r o m a t i n (BLUMENFELD, 1978). R e c e n t l y , e x p e r i m e n t s on D. m e l a n o g a s t e r i n our l a b o r a t o r y have d e m o n s t r a t e d t h a t t h e h i s t o n e p r o t e i n s a r e i n d e e d i n v o l v e d i n p o s i t i o n e f f e c t v a r i e g a t i o n . MOORE ejt. a l . (1979) and MOORE (1980) f o u n d t h a t f l i e s h a p l o i d f o r h i s t o n e gene c l u s t e r e x h i b i t marked i n c r e a s e s i n v a r i e g a t i n g gene e x p r e s s i o n . The s i m p l e s t i n t e r p r e t a t i o n of t h i s f i n d i n g i s t h a t a d e c r e a s e i n t h e number o f h i s t o n e genes r e s u l t s i n a r e d u c t i o n i n t h e amount of h i s t o n e p r o t e i n . a v a i l a b l e a t t h e t i m e o f DNA p a c k a g i n g l e a d i n g t o a d e c r e a s e i n t h e s p r e a d o f h e t e r o c h r o m a t i n i n t o t h e a d j a c e n t e u c h r o m a t i n . A p r e d i c t i o n o f t h i s h y p o t h e s i s i s t h a t f a c t o r s w h i c h b i o c h e m i c a l l y a l t e r t h e h i s t o n e p r o t e i n s m i g h t be e x p e c t e d t o m o d i f y p o s i t i o n e f f e c t v a r i e g a t i o n . We have t e s t e d t h i s h y p o t h e s i s by e x a m i n i n g t h e e f f e c t s o f n-b u t y r a t e ( b u t y r a t e ) and n - p r o p i o n a t e ( p r o p i o n a t e ) , c h e m i c a l s known t o c a u s e h y p e r a c e t y l a t i o n o f h i s t o n e s (CANDIDO e t . a l . , 9 1978; SEALY and CHALKLEY, 1978), on v a r i e g a t i o n o f t h e wj_ gene i n t h e s t r a i n I_n(_1^)wm4 . We o b s e r v e d t h a t f l i e s r e a r e d on medium c o n t a i n i n g low c o n c e n t r a t i o n s of e i t h e r o f t h e s e c h e m i c a l s e x h i b i t e d s t r o n g s u p p r e s s i o n o f w m 4 v a r i e g a t i o n (MOTTUS e t a l . , 1980). However, b e f o r e t h e p r e c i s e n a t u r e of t h e e f f e c t s o f b u t y r a t e and p r o p i o n a t e on v a r i e g a t i o n c a n be d e t e r m i n e d , many q u e s t i o n s r e m a i n unanswered. T h e s e i n c l u d e : 1) a r e most o r a l l v a r i e g a t i n g genes s e n s i t i v e t o - c h e m i c a l i n d u c e d s u p p r e s s i o n o f v a r i e g a t i o n ; 2) a t what s t a g e s o f d e v e l o p m e n t d o e s e x p o s u r e t o t h e s e c h e m i c a l s c a u s e s u p p r e s s i o n of t h e v a r i e g a t e d p h e n o t y p e ; 3) i s t h e r e any r e l a t i o n s h i p between t h e e f f e c t s o f Y h e t e r o c h r o m a t i n and t h e s e c h e m i c a l s ; and 4) what, i f any, i s t h e r e l a t i o n s h i p between t h e s e c h e m i c a l s and h i s t o n e d e f i c i e n c i e s w i t h r e g a r d t o s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n ? H i s t o n e s a r e t h e b a s i c b u i l d i n g b l o c k s o f c h r o m a t i n and t h e r e f o r e m i g h t be e x p e c t e d t o p l a y a r o l e i n t h e f o r m a t i o n o f h e t e r o c h r o m a t i n and t h u s i n f l u e n c e p o s i t i o n e f f e c t v a r i e g a t i o n . However, i n a d d i t i o n t o h i s t o n e s , s e v e r a l h u n d r e d o t h e r p r o t e i n s a r e i n t i m a t e l y i n v o l v e d i n c h r o m a t i n s t r u c t u r e and f u n c t i o n (PETERSON and McCONKEY, 1976). I t i s l i k e l y t h a t a s u b s e t o f t h e s e n o n - h i s t o n e chromosomal p r o t e i n s may a l s o p l a y a p a r t i n t h e f o r m a t i o n o f h e t e r o c h r o m a t i n and t h u s i n t h e phenomenon o f p o s i t i o n e f f e c t v a r i e g a t i o n . T h e o r e t i c a l l y , l o c i e n c o d i n g e i t h e r chromosomal p r o t e i n s , o r p r o d u c t s w h i c h f u n c t i o n t o m o d i f y chromosomal p r o t e i n s , s h o u l d be amenable t o g e n e t i c d i s s e c t i o n . I t seems l i k e l y t h a t many o f t h e l e s i o n s o b t a i n e d 10 i n s u c h a s t u d y m i g h t a f f e c t chromosome s t r u c t u r e and p h e n o t y p i c a l l y would m a n i f e s t t h e m s e l v e s by m o d i f y i n g p o s i t i o n e f f e c t v a r i e g a t i o n . A c c o r d i n g l y we have u n d e r t a k e n a l a r g e s c r e e n t o r e c o v e r m u t a t i o n s w h i c h e i t h e r s u p p r e s s o r enhance p o s i t i o n e f f e c t v a r i e g a t i o n i n t h e hopes t h a t a n a l y s i s o f t h e m u t a n t s m i g h t l e n d i n s i g h t n ot o n l y i n t o t h e phenomenon of p o s i t i o n e f f e c t v a r i e g a t i o n but s h e d l i g h t on f a c t o r s i n v o l v e d i n t h e f o r m a t i o n o f h e t e r o c h r o m a t i n . T h i s t h e s i s w i l l be d i v i d e d i n t o two p a r t s . The f i r s t w i l l e xamine t h e e f f e c t s o f b u t y r a t e and p r o p i o n a t e on t h e e x p r e s s i o n o f v a r i e g a t i n g g e n e s . The s e c o n d w i l l f o c u s on t h e i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f genes whose p r o d u c t s have a s t r o n g i n f l u e n c e on p o s i t i o n e f f e c t v a r i e g a t i o n . A summary w i l l a t t e m p t t o r e l a t e t h e f i n d i n g s p r e s e n t e d i n t h i s t h e s i s w i t h t h o s e o f r e s e a r c h e r s c u r r e n t l y w o r k i n g i n t h e f i e l d a s w e l l a s t h o s e who have worked on p o s i t i o n e f f e c t v a r i e g a t i o n o v e r t h e l a s t 50 y e a r s . 11 CHAPTER ONE INTRODUCTION The m o l e c u l a r mechanism u n d e r l y i n g p o s i t i o n e f f e c t v a r i e g a t i o n has r e m a i n e d u n c l e a r . The f a c t t h a t o n l y a few a c t i v e genes r e s i d e i n h e t e r o c h r o m a t i n may i n d i c a t e t h a t t h i s h i g h l y c o n d e n s e d form o f c h r o m a t i n i s i n c o m p a t i b l e w i t h t h e t r a n s c r i p t i o n o f most genes (BALDWIN and SUZUKI, 1971; HILLIKER and HOLM, 1975; HILLIKER, 1976). T h i s n o t i o n i s c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t h e s u p p r e s s i o n o f t h e a c t i v i t y of v a r i e g a t i n g genes l y i n g p r o x i m a l t o an h e t e r o c h r o m a t i c b r e a k p o i n t r e s u l t s from t h e " s p r e a d i n g " o f h e t e r o c h r o m a t i c e l e m e n t s w h i c h c o n d e n s e and t r a n s c r i p t i o n a l l y i n a c t i v a t e t h e e u c h r o m a t i n i n a c e r t a i n p r o p o r t i o n o f c e l l s (DEMERIC and SLIZYNSKA, 1937). H i s t o n e s a r e t h e p r i m a r y a g e n t s i n DNA p a c k a g i n g and as s u c h i t h a s been s u g g e s t e d t h e y may p l a y a r o l e i n t h e r e g u l a t i o n o f n o r m a l gene a c t i v i t y (STEDMAN and STEDMAN, 1950; r e v i e w MCGHEE and FELSENFELD, 1980). U n t i l r e c e n t l y h i s t o n e s have n o t been i m p l i c a t e d i n t h e r e g u l a t i o n o f v a r i e g a t i n g g e n e s ; however, t h e h i s t o n e c o n t e n t o f - h e t e r o c h r o m a t i n d i f f e r s i n b o t h amount and t y p e from t h a t f o u n d i n e u c h r o m a t i n . The p a t e r n a l chromosome s e t i n t h e mealy bug i s e n t i r e l y h e t e r o c h r o m a t i c and BERLOWITZ (1965) has shown i t i s e n r i c h e d i n i t s h i s t o n e c o n t e n t . In D r o s o p h i l a , s p e c i f i c s u b s p e c i e s o f p h o s p h o r y l a t e d H1 a r e a s s o c i a t e d w i t h h e t e r o c h r o m a t i n (BLUMENFELD, 1978). I f h i s t o n e s a r e i n v o l v e d i n t h e f o r m a t i o n o f h e t e r o c h r o m a t i n t h e n 1 2 one m i g h t e x p e c t p e r t u r b a t i o n s o f t h e h i s t o n e s t o m o d i f y p o s i t i o n e f f e c t v a r i e g a t i o n . R e c e n t e x p e r i m e n t s w h i c h examined t h e e f f e c t o f h e t e r o z y g o u s d e f i c i e n c e s of t h e h i s t o n e gene c l u s t e r on p o s i t i o n e f f e c t v a r i e g a t i o n i n D r o s o p h i l a a p p e a r t o s u p p o r t t h i s h y p o t h e s i s . P o s i t i o n e f f e c t v a r i e g a t i o n o f t h e wj_ gene i n J_n.(l)w m 4 was m a r k e d l y s u p p r e s s e d i n s t r a i n s of f l i e s h a p l o i d f o r t h e h i s t o n e genes (KHESIN and LEIBOVITCH, 1978; MOORE e t a l . , 1979). One i n t e r p r e t a t i o n o f t h e s e e x p e r i m e n t s i s t h a t h a p l o i d y f o r t h e h i s t o n e gene complex r e s u l t s i n r e d u c e d l e v e l s o f c e l l u l a r h i s t o n e s a v a i l a b l e f o r t h e p a c k a g i n g o f h e t e r o c h r o m a t i n . T h e o r e t i c a l l y , t h i s r e d u c t i o n i n t h e amount of h i s t o n e s f o r DNA p a c k a g i n g would be e x p e c t e d t o r e d u c e t h e " s p r e a d " o f h e t e r o c h r o m a t i n t h o u g h t t o o c c u r i n p o s i t i o n e f f e c t v a r i e g a t i o n and r e s u l t i n an i n c r e a s e i n t h e number of c e l l s e x p r e s s i n g t h e v a r i e g a t i n g gene. A l t h o u g h t h i s i s an a t t r a c t i v e h y p o t h e s i s i t has y e t t o be d e m o n s t r a t e d t h a t h a p l o i d y f o r t h e h i s t o n e genes r e s u l t s i n r e d u c e d l e v e l s o f h i s t o n e p r o t e i n . However, i f h i s t o n e s a r e i n v o l v e d i n p o s i t i o n e f f e c t v a r i e g a t i o n t h e n a f u r t h e r p r e d i c t i o n o f t h i s model i s t h a t f a c t o r s w h i c h b i o c h e m i c a l l y m o d i f y t h e h i s t o n e s m i g h t a l s o be e x p e c t e d t o a f f e c t t h e v a r i e g a t i n g p h e n o t y p e . Two a g e n t s known t o c a u s e m o d i f i c a t i o n o f h i s t o n e s a r e n-b u t y r a t e ( b u t y r a t e ) and n - p r o p i o n a t e ( p r o p i o n a t e ) . The e f f e c t s o f b u t y r a t e were f i r s t n o t e d i n mammalian t i s s u e c u l t u r e (PACE e t a l . , 1967). S u b s e q u e n t i n v e s t i g a t i o n s i n t o t h e e f f e c t s o f t h i s c h e m i c a l , p r i m a r i l y i n t i s s u e c u l t u r e , have shown t h a t i t c a u s e s a wide v a r i e t y o f m o r p h o l o g i c a l and p h y s i o l o g i c a l c h a n g e s 13 (see review KRUH, 1982). For example, RIGGS et a l . (1977) r e p o r t e d tha t treatment of He la c e l l s wi th m i l l i m o l a r c o n c e n t r a t i o n s of b u t y r a t e r e s u l t e d i n a r a p i d and e x t e n s i v e i n c r e a s e in h i s t o n e a c e t y l a t i o n , p a r t i c u l a r l y that of H3 and H4. I t was l a t e r found tha t p r o p i o n a t e had a s i m i l a r though l e s s d r a m a t i c e f f e c t (SEALY and CHALKLEY, 1978). F u r t h e r i n v e s t i g a t i o n s have demonstrated tha t the h y p e r a c e t y l a t i o n of h i s t o n e s i s due to i n h i b i t i o n of the h i s t o n e d e a c e t y l a s e enzymes by these c h e m i c a l s r a t h e r than to i n c r e a s e s i n the r a t e of h i s t o n e a c e t y l a t i o n (REEVES and CANDIDO, 1978). S i n c e i n c r e a s e d a c e t y l a t i o n of h i s t o n e s has been c o r r e l a t e d wi th t r a n s c r i p t i o n a l l y a c t i v e chromat in (DAVIE and CANDIDO, 1978; LEVY-WILSON et a l . , 1979) the e f f e c t s of these c h e m i c a l s were t e s t e d on p o s i t i o n e f f e c t v a r i e g a t i o n . I t was found tha t both b u t y r a t e and p r o p i o n a t e can s i g n i f i c a n t l y suppress p o s i t i o n e f f e c t v a r i e g a t i o n of the X - l i n k e d wj_ gene i n the D. melanogaster s t r a i n I_n(l)wm < !* when f l i e s are r e a r e d on medium c o n t a i n i n g low c o n c e n t r a t i o n s of e i t h e r of these c h e m i c a l s (MOTTUS et a l . , 1980) The presen t s tudy i s an e x t e n s i o n of the a n a l y s i s of these compounds i n v e s t i g a t i n g : 1) the g e n e r a l i t y of t h e i r e f f e c t s w i th r e s p e c t to p o s i t i o n e f f e c t v a r i e g a t i o n ; 2) the deve lopmenta l p r o f i l e of b u t y r a t e and p r o p i o n a t e s e n s i t i v i t y ; 3) t h e i r i n t e r a c t i o n w i t h h i s t o n e d e f i c i e n c e s ; and 4) the dependence of t h e i r e f f e c t on the h e t e r o c h r o m a t i c c o n s t i t u t i o n of the o r g a n i s m . 1 4 MATERIAL and METHODS 1) S t o c k s and C u l t u r e C o n d i t i o n s F l i e s were r e a r e d on s t a n d a r d s u c r o s e - c o r n m e a l - a g a r medium t o w h i c h t e g o s e p t ( m e t h y l - p - h y d r o x y b e n z o a t e ) was added as a mould i n h i b i t o r . S t o c k s o l u t i o n s o f 8M n - b u t y r i c and n-p r o p i o n i c a c i d were p r e p a r e d and a d j u s t e d t o pH. 5.0 w i t h s o d i u m h y d r o x i d e . A l i q u o t s o f t h e s t o c k s o l u t i o n s were added t o t h e medium t o b r i n g t h e f i n a l c o n c e n t r a t i o n t o t h a t s p e c i f i e d i n t h e e x p e r i m e n t a l d e s i g n s . The medium c o n t a i n s s o d i um p o t a s s u i m t a r t r a t e w h i c h a c t s as a b u f f e r . In a l l c a s e s , r e g a r d l e s s o f w hether c h e m i c a l s were added or n o t , t h e f i n a l pH. was 5.75. Most o f t h e s t r a i n s and m u t a t i o n s employed i n t h i s s t u d y a r e d e s c r i b e d i n LINDSLEY and GRELL ( 1 9 6 8 ) . The e x c e p t i o n s a r e : S_V A) Bar o f S t o n e - V a r i e g a t e d (B ) : a Y chromosome d e r i v e d from an i n s e r t i o n of t h e B gene i n t o t h e Y chromosome w h i c h e x h i b i t s p o s i t i o n e f f e c t v a r i e g a t i o n f o r B d e s c r i b e d by BROSSEAU ( i 9 6 0 ) . B) D f ( 2 L ) D S 6 / C y O (DS6) and D f ( 2 L ) D S 9 / C y O (DS9) : s t r a i n s g e n e r a t e d by SINCLAIR e t a l . (1980) b e a r i n g d e l e t i o n s o f m a t e r i a l i n t h e p r o x i m a l p o r t i o n o f t h e l e f t arm o f t h e s e c o n d chromosome. PS9 d e l e t e s bands 39A1-39B1-2 w h i c h a r e d i s t a l t o t h e h i s t o n e gene c l u s t e r . DS6 i s somewhat l a r g e r r e m o v i n g bands 38F5-39E7 w h i c h i n c l u d e s t h e h i s t o n e g e n e s . C) l n ( l ) w m 4 ( w m 4 ) : 15 R e c e n t s t u d i e s by APPELS and HILLIKER (1982) s u g g e s t t h e p r o x i m a l b r e a k p o i n t o f t h i s i n v e r s i o n l i e s w i t h i n t h e d i s t a l p o r t i o n of t h e n u c l e o l u s o r g a n i z e r r a t h e r t h a n i n 20A a s p r e v i o u s l y r e p o r t e d by COOPER ( 1 9 5 0 ) . 2) G e n e r a t i o n o f X/0 m a l e s . To s t u d y t h e i n t e r a c t i o n between b u t y r a t e and t h e Y chromosome i t was n e c e s s a r y t o g e n e r a t e m a l e s w h i c h do n o t have a Y chromosome (X/0 m a l e s ) . T h i s was a c c o m p l i s h e d by c r o s s i n g C(1)RMpn/0 v i r g i n f e m a l e s t o w m 4 m a l e s . A l l males r e s u l t i n g f r o m t h i s c r o s s a r e g e n o t y p i c a l l y I_n(]_)w m^/0 . 3) B u t y r a t e and H i s t o n e D e f i c i e n c e s . R e c e n t S t u d i e s ( CHERNYSHEV e t . a l . ,1980; CHERNYSHEV ,1982; and MOORE, p e r s o n n a l c o m m u n i c a t i o n ) i n d i c a t e t h a t i n b r e d s t r a i n s of f l i e s w h i c h a r e h a p l o i d f o r t h e h i s t o n e gene c l u s t e r a m p l i f y t h e i r h i s t o n e gene c o n t e n t o v e r t i m e . T h i s would o b v i o u s l y c o n f o u n d s t u d i e s o f t h e i n t e r a c t i o n s between h i s t o n e d e f i c i e n c e s a n d b u t y r a t e . A c c o r d i n g l y DS6 a n d DS9 s t r a i n s were o u t c r o s s e d t o w m 4 v i r g i n f e m a l e s t o p r o d u c e w m 4/Y; DS6/+ o r DS9/+ m a l e s . T h e s e m a l e s were employed i n s t u d i e s o f h i s t o n e d e f i c i e n c e s and b u t y r a t e . 4) Method o f A d m i n i s t e r i n g C h e m i c a l s . A) C o n t i n u o u s E x p o s u r e T h r o u g h o u t D e v e l o p m e n t ; S y n c h r o n o u s l y d e v e l o p i n g c u l t u r e s of f l i e s were p r o d u c e d by a l l o w i n g young, w e l l f e d f e m a l e s t o d e p o s i t 16 eggs on a g a r c o v e r e d w i t h a t h i n l a y e r of y e a s t p a s t e . The eggs were c o l l e c t e d f o r two h o u r i n t e r v a l s . The f i r s t major egg l a y was d i s c a r d e d t o e x c l u d e embryos r e t a i n e d by t h e f e m a l e s a f t e r f e r t i l i z a t i o n . S u b s e q u e n t 2 hour egg c o l l e c t i o n s were u s e d f o r t h e e x p e r i m e n t s . The eggs were c o u n t e d and p l a c e d on f i l t e r p a p e r w e t t e d w i t h d i s t i l l e d w a t er o r t h e a p p r o p r i a t e c o n c e n t r a t i o n of t h e c h e m i c a l b e i n g t e s t e d . The number of eggs p e r v i a l v a r i e d d e p e n d i n g on t h e e x p e c t e d v i a b i l i t y o f t h e s t r a i n under c o n s i d e r a t i o n . S i n c e t h e l e v e l o f v a r i e g a t i o n i n an o r g a n i s m c a n be a f f e c t e d by c r o w d i n g i t i s i m p o r t a n t t o c o n t r o l t h i s p a r a m e t e r . A c c o r d i n g l y , an a p p r o p r i a t e number of eggs were p l a c e d on t h e f i l t e r p a p e r t o g i v e an e x p e c t e d l a r v a l d e n s i t y o f 50 p e r v i a l . The f i l t e r p a p e r was imbedded i n c o n t r o l medium o r i n medium c o n t a i n i n g b u t y r a t e o r p r o p i o n a t e a t t h e same c o n c e n t r a t i o n as t h e f i l t e r p a p e r and t h e f l i e s a l l o w e d t o d e v e l o p n o r m a l l y . A l l e x p e r i m e n t s were done a t 2 2 ° C . The a d u l t s were c o l l e c t e d w i t h i n 24 h o u r s o f e c l o s i o n , a g e d 7-10 d a y s , and t h e l e v e l of v a r i e g a t i o n d e t e r m i n e d a s o u t l i n e d below. 24 and 48 Hour P u l s e s T h r o u g h o u t E a r l y D e v e l o p m e n t : In t h e d e v e l o p m e n t a l s t u d i e s f l i e s were e x p o s e d t o 150 mM b u t y r a t e o r p r o p i o n a t e f o r 24 o r 48 h o u r p e r i o d s . S y n c h r o n o u s b a t c h e s o f w m 4 eggs were p r o d u c e d by t h e method d e s c r i b e d a b o v e . D u r i n g t h e f i r s t 48 h o u r s 17 embryos were e x p o s e d by p l a c i n g them on f i l t e r p a p e r w e t t e d w i t h 150 mM b u t y r a t e o r p r o p i o n a t e and i m b e d d i n g t h e f i l t e r p a p e r i n medium o f t h e same c o n c e n t r a t i o n . A f t e r t h e e x p o s u r e p e r i o d t h e embryos were washed e x t e n s i v e l y w i t h d i s t i l l e d w a t e r , t r a n s f e r r e d t o f r e s h f i l t e r p a p e r w e t t e d w i t h d i s t i l l e d w a t e r , and p l a c e d on c o n t r o l medium. L a t e r i n d e v e l o p m e n t , l a r v a e were c o l l e c t e d f r o m s y n c h r o n o u s c u l t u r e s by f l o a t i n g them f r o m t h e medium i n a 20 % s u c r o s e s o l u t i o n . They were washed e x t e n s i v e l y and p l a c e d on e x p e r i m e n t a l medium f o r e i t h e r 24 o r 48 hour p e r i o d s . They were r e c o l l e c t e d by t h e same method, washed, and t r a n s f e r r e d t o c o n t r o l medium. The d e v e l o p m e n t a l s t a g e o f t h e l a r v a e was d e t e r m i n e d by e x a m i n i n g t h e m o r p h o l o g y o f t h e m o u t h p a r t s and a n t e r i o r s p i r a c l e s i m m e d i a t e l y b e f o r e and a f t e r t r e a t m e n t . C) E x p o s u r e o f P u p a l S t a g e s t o B u t y r a t e ; Pupae were e x p o s e d t o v a r i o u s c o n c e n t r a t i o n s o f b u t y r a t e r a n g i n g f r o m 0.2 t o 5.0 M. S y n c h r o n o u s c u l t u r e s were e s t a b l i s h e d by c o l l e c t i n g w m 4 p r e p u p a e f r o m uncrowded b o t t l e s e v e r y h o u r . The p r e p u p a e were p l a c e d on f i l t e r p a p e r w e t t e d w i t h t h e a p p r o p r i a t e c o n c e n t r a t i o n o f b u t y r a t e . E a c h g r o u p was p l a c e d i n a v i a l c o n t a i n i n g f i l t e r p a p e r i n t h e b o t t o m s o a k e d i n t h e same c o n c e n t r a t i o n o f t h e c h e m i c a l . The v i a l s were c a p p e d w i t h p a r a f i l m and t h e f l i e s a l l o w e d t o d e v e l o p t o 18 e c l o s i o n . A d u l t s were c o l l e c t e d w i t h i n 24 h o u r s of e c l o s i o n and t r a n s f e r r e d t o v i a l s c o n t a i n i n g c o n t r o l medium. They were aged 7-10 days b e f o r e t h e l e v e l o f v a r i e g a t i o n was m e a s u r e d . 5) Measurement o f v a r i e g a t i o n A) Ey_e P i g m e n t s ; I n ( l ) w m 4 ( w m 4 ) and I n ( 2 R ) b w v 3 2 s , ( b w v 3 2 8 ) Two methods were employed t o measure t h e eye p i g m e n t s . In t h e f i r s t , a d u l t s were a g e d 7-10 d a y s and t h e n q u i c k f r o z e n i n e t h a n o l c o o l e d by d r y i c e . The heads removed by a g i t a t i o n and s i n g l e heads were s q u a s h e d on t h i n l a y e r c h r o m a t o g r a p h y p l a t e s (Kodak Eastman Chromogram s h e e t s 13255 c e l l u l o s e No. 6 0 6 4 ) . The p l a t e s were d e v e l o p e d i n t h e d a r k f o r t h r e e h o u r s i n a p r o p a n o l : 1% aqueous ammonia (2:1) s o l v e n t . The amount o f t h e eye p i g m e n t , d r o s o p t e r i n ( EPHRUSSI and HEROLD ,1944), e l u t e d f r o m t h e eye was d e t e r m i n e d by i l l u m i n a t i n g t h e s p o t w i t h U.V. o f 500 nm and l o w e r and q u a n t i t a t i n g t h e r e s u l t i n g f l u o r e s c e n c e above 560 nm u s i n g an MPS-1 Z e i s s m i c r o s c o p e p h o t o m e t e r . The d r o s o p t e r i n l e v e l s o b t a i n e d f r o m t h e e x p e r i m e n t a l s t r a i n s ( e i t h e r w m 4 o r b w v 3 2 g ) were s t a n d a r d i z e d a g a i n s t t h a t f o u n d i n t h e c o n t r o l s t r a i n Oreqon-R (OR-R) • As t h e c o u r s e o f t h i s s t u d y p r o g r e s s e d an a l t e r n a t e , and more s e n s i t i v e t e c h n i q u e was d e v e l o p e d . The heads o f t h e f l i e s were removed by t h e above method, 19 p o o l e d , and s o n i c a t e d i n a 25 m i c r o l i t e r s o l u t i o n o f 0.25 M B - m e r c a p t o e t h a n o l and 1% aqueous ammonium h y d r o x i d e . F i v e m i c r o l i t e r a l i q u o t s were s p o t t e d on c h r o m a t o g r a p h i c p a p e r and t h e amount o f d r o s o p t e r i n p r e s e n t d e t e r m i n e d by i l l u m i n a t i n g t h e s p o t w i t h U.V. o f 500 nm a n d l o w e r a n d m e a s u r i n g t h e r e s u l t i n g f l u o r e s c e n c e above 560 nm. I t was e s t a b l i s h e d t h a t no o t h e r compounds i n t h e head c o n t r i b u t e t o f l u o r e s c e n c e above t h i s w a v e l e n g t h by m e a s u r i n g f l u o r e s c e n c e i n s t r a i n s o f f l i e s w h i c h l a c k a l l eye p i g m e n t s , w/w . In a d d i t i o n a s t r a i n of f l i e s ( bw/bw ) was measured w h i c h d o e s n ' t p r o d u c e any p t e r i d i n e s , t h e r e d p i g m e n t s o f w h i c h d r o s o p t e r i n i s one, a n d no d e t e c t a b l e f l u o r e s c e n c e was o b s e r v e d . T h i s t e c h n i q u e was employed i n t h e s t u d i e s w h i c h a s s e s s e d t h e i n t e r a c t i o n between b u t y r a t e t r e a t m e n t a n d : (1) a r e d u c t i o n i n h i s t o n e gene m u l t i p l i c i t y ; and (2) t h e a b s e n c e o f a Y chromosome i n m a l e s . The amount o f d r o s o p t e r i n m easured i n t h e e x p e r i m e n t a l s t r a i n s was s t a n d a r d i z e d a g a i n s t t h a t f o u n d i n OR-R B) B r i s t l e V a r i e g a t i o n : T ( 2 ; 3 ) S b v , ( S b v ) Seven m a j o r b r i s t l e s on e a c h s i d e o f t h e t h o r a x were i n d i v i d u a l l y examined and a s s i g n e d e i t h e r a mutant, "Sb," o r w i l d - t y p e , "+," p h e n o t y p e . T h e s e i n c l u d e d : a n t e r i o r and p o s t e r i o r s t e r n o p l e u r a l s , p o s t e r i o r s u p e r a l a r , a n t e r i o r p o s t a l a r , p o s t e r i o r d o r s o c e n t r a l , 20 and t h e a n t e r i o r and p o s t e r i o r s c u t e l l a r s . A l l o f t h e s e b r i s t l e s e x h i b i t a s t u b b l e p h e n o t y p e i n s t r a i n s w h i c h do not v a r i e g a t e f o r e x p r e s s i o n o f t h e Sb m u t a t i o n . A c c o r d i n g l y t h e amount o f v a r i e g a t i o n o b s e r v e d i s g i v e n a s a p e r c e n t a g e o f f u l l Sb e x p r e s s i o n . C) Eye S i z e V a r i e g a t i o n : B s _ v The amount o f B a r - S t o n e ( B s ) e x p r e s s i o n i n t h e v a r i e g a t i n g s t r a i n , B s _ v , was m o n i t o r e d by s k e t c h i n g t h e eye s h a p e s t o s c a l e on g r i d d e d p a p e r u s i n g a d i s s e c t i n g m i c r o s c o p e e q u i p p e d w i t h an o c u l a r m i c r o m e t e r . The o u t l i n e s were c u t o u t and w e i g h e d t o e s t i m a t e eye s i z e r e l a t i v e t o OR-R c o n t r o l s . I n t h i s c a s e i t i s n e c e s s a r y t o use eye s i z e r a t h e r t h a n p e r c e n t a g e o f B s - V e x p r e s s i o n s i n c e i t ' i s n o t known how s m a l l an eye i s p r o d u c e d by f u l l e x p r e s s i o n o f t h e B s - V gene. F a c t o r s w h i c h s u p p r e s s v a r i e g a t i o n i n c r e a s e t h e number o f eye c e l l s i n w h i c h t h e B s - V i s a c t i v e p r o d u c i n g a s m a l l e r e y e . 6) S t a t i s t i c s A n a l y s i s o f t h e d a t a showed t h a t t h e v a r i a n c e was o f t e n p r o p o r t i o n a l t o t h e mean. S t u d e n t ' s " t - t e s t " i s a s t a t i s t i c a l t o o l f o r e s t i m a t i n g t h e s i g n i f i c a n c e o f d i f f e r e n c e between means when t h e i r v a r i a n c e s a r e s i m i l a r . A l t h o u g h t h e o b s e r v e d v a r i a n c e s were s i g n i f i c a n t l y d i f f e r e n t t h i s t e s t i s s u f f i c i e n t l y r o b u s t t o be u s e d when t h e m o d i f i c a t i o n s o f WELCH (1953) a r e 21 e m p l o y e d . T h i s m o d i f i c a t i o n a l l o w s one t o u s e t h e same method t o g e n e r a t e a " t " v a l u e but a l t e r s t h e d e g r e e s o f f r e e d o m a c c o r d i n g t o t h e f o l l o w i n g e q u a t i o n : f< 22 RESULTS 1) Cont inuous Exposure to B u t y r a t e or P r o p i o n a t e The f o r m a t i o n of pigment i n the normal red eye of D. melanogaster r e q u i r e s the presence of f u n c t i o n a l pigment g r a n u l e s . S t r a i n s which are mutant for the white (w) gene produce eye pigment p r e c u r s o r s (BAKER, 1967) but the g r a n u l e s on which the pigments mature appear deformed under the e l e c t r o n microscope and the eye i s c o l o u r l e s s (SHOUP, 1966). The s t r a i n I_n(j_)wm 4, ( w m 4 ) , c a r r i e s a normal w_^  gene which v a r i e g a t e s due to i t s j u x t a p o s i t i o n to the 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 of the X chromosome. Thus , the eye of wm^ i s a mosaic of normal red eye c e l l s , i n which the wj_ gene i s f u l l y a c t i v e , and c o l o u r l e s s eye c e l l s i n which the wj_ gene i s c o m p l e t e l y i n a c t i v e . S i n c e the a c t i v i t y of the wj_ gene i s c e l l autonomous, the amount of pigment i n the eye i s a d i r e c t measure of the percentage of c e l l s i n which the wj_ gene i s a c t i v e . The r e s u l t s of e a r l i e r work i n which synchronous c u l t u r e s of w m 4 or OR-R were exposed c o n t i n u o u s l y to b u t y r a t e or p r o p i o n a t e from p r e - b l a s t o d e r m to p u p a r i a t i o n are summarized i n F i g u r e s 1 and 2 ( taken from MOTTUS e t . a l . f 1980).. In the w i l d - t y p e s t r a i n , OR-R, no s i g n i f i c a n t d i f f e r e n c e i n the amount of pigment c o u l d be d e t e c t e d between u n t r e a t e d f l i e s and those exposed to c o n c e n t r a t i o n s of b u t y r a t e or p r o p i o n a t e as h i g h as 200 mM. On the o ther hand, the amount of d r o s o p t e r i n i n f l i e s of the v a r i e g a t i n g s t r a i n , w m 4 , i s s i g n i f i c a n t l y i n c r e a s e d by both of these c h e m i c a l s . Medium c o n t a i n i n g b u t y r a t e at 23 . c o n c e n t r a t i o n s as low as 70 mM c a u s e s s i g n i f i c a n t s u p p r e s s i o n of w* v a r i e g a t i o n b o t h i n m a l e s ( F i g u r e 1) and i n f e m a l e s ( F i g u r e 2 ) . P r o p i o n a t e i s a l m o s t as e f f e c t i v e as b u t y r a t e i n i n c r e a s i n g t h e number of c e l l s i n w h i c h t h e w_^  gene i s a c t i v e . M a l e s and f e m a l e s showed s i g n i f i c a n t s u p p r e s s i o n o f v*_ gene v a r i e g a t i o n when r a i s e d on medium a t 70 mM ( F i g u r e 1) and 100 mM ( F i g u r e 2) r e s p e c t i v e l y . The d e g r e e of s u p p r e s s i o n o f v a r i e g a t i o n i s p o s i t i v e l y and l i n e a r l y c o r r e l a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f e i t h e r b u t y r a t e o r p r o p i o n a t e . A l t h o u g h b o t h c h e m i c a l s c a u s e d a d e v e l o p m e n t a l d e l a y a t h i g h e r c o n c e n t r a t i o n s , t h i s i n I t s e l f i s n o t s u f f i c i e n t t o c a u s e s u p p r e s s i o n o f v a r i e g a t i o n . O t h e r c h e m i c a l s , s u c h a s t e g o s e p t , c a u s e a s i m i l a r o r more p r o n o u n c e d d e l a y i n d e v e l o p m e n t but do n o t s u p p r e s s v a r i e g a t i o n ( F i g u r e 1 and F i g u r e 2 ) . One p o s s i b l e e x p l a n a t i o n o f t h e s e r e s u l t s i s t h a t b u t y r a t e and p r o p i o n a t e a c t s p e c i f i c a l l y t o a l t e r e x p r e s s i o n o f t h e w_^  gene and t h a t t h e s e r e s u l t s have l i t t l e b e a r i n g on t h e g e n e r a l phenomenon o f p o s i t i o n e f f e c t v a r i e g a t i o n . In o r d e r t o t e s t t h i s p o s s i b i l i t y we examined t h e e f f e c t s o f b u t y r a t e on t h e e x p r e s s i o n o f a v a r i e g a t i n g a l l e l e o f Bar ( B ) , B S ~ V » Y . B s - V i s a d u p l i c a t i o n o f t h e X - l i n k e d gene, B, w h i c h i s i n s e r t e d i n t o t h e h e t e r o c h r o m a t i n o f t h e Y chromosome. F u l l e x p r e s s i o n o f t h i s gene r e s u l t s i n a narrow e y e . D e c r e a s e d e x p r e s s i o n o f B, due t o p o s i t i o n e f f e c t v a r i e g a t i o n , r e d u c e s t h i s n a r r o w i n g e f f e c t and t h e r e f o r e any f a c t o r w h i c h s u p p r e s s e s v a r i e g a t i o n , r e s u l t i n g i n i n c r e a s e d e x p r e s s i o n o f t h e B a l l e l e , w i l l r e s u l t i n a n a r r o w e r , more mutant eye phene. Thus, one c a n e s t i m a t e B 24 FIGURE I. HALE inCDw" 1 * EXPOSED TO VARIOUS CONCEN-TRATIONS OF n-BUTYRATE, n-PROPIONATE, OR TEGOSEPT FROM EGG DEPOSITION TO PUPARIUM FORMATION CONTROL n-BUTYRATE • ••••••• • n-PROPIONATE O — O TEGOSEPT O O 26 FIGURE I I . FEMALE i n ( i ) w ™ A FLIES EXPOSED TO VARIOUS CONCENTRATIONS OF n-BUTYRATE, n-PROPIOANTE, OR TEGOSEPT FROM EGG DEPOSITION TO PUPARIUM FORMATION CONTROL n-BUTYRATE + n-pROPIONATE Q Q TEGOSEPT O ---O 28 e x p r e s s i o n by m e a s u r i n g t h e s i z e o f t h e a d u l t eye (MOORE et. a l . , 1980). M a l e s b e a r i n g t h e B S ~ V » Y were r e a r e d on medium a t b u t y r a t e c o n c e n t r a t i o n s of 0.0, 150-or 200 mM medium from p r e b l a s t o d e r m t o p u p a r i a t i o n . The r e s u l t s , shown i n T a b l e 1, d e m o n s t r a t e t h a t v a r i e g a t i o n of t h e B s _ v gene was s u p p r e s s e d i n t h o s e c u l t u r e s e x p o s e d t o b u t y r a t e , t h a t i s , t h e B s - V gene was e x p r e s s e d i n a g r e a t e r p r o p o r t i o n o f c e l l s r e s u l t i n g i n a n a r r o w e r e y e . Note t h a t , a s was t h e c a s e w i t h w , a p o s i t i v e , l i n e a r c o r r e l a t i o n was f o u n d between i n c r e a s i n g b u t y r a t e c o n c e n t r a t i o n s and i n c r e a s e d e x p r e s s i o n o f t h e v a r i e g a t i n g gene. B o t h wj_ and B a r e X - l i n k e d genes and t h e r e f o r e s u b j e c t t o X - l i n k e d d o s a g e c o m p e n s a t i o n . I t c o u l d t h e r e f o r e be a r g u e d t h a t r a t h e r t h a n a c t i n g d i r e c t l y on p o s i t i o n e f f e c t v a r i e g a t i o n , b u t y r a t e i n c r e a s e s e x p r e s s i o n of t h e s e genes by a c t i n g p r i m a r i l y on t h e d o s a g e c o m p e n s a t i o n mechanisms. T h i s p o s s i b i l i t y was e l i m i n a t e d by t e s t i n g t h e e f f e c t s o f b u t y r a t e and p r o p i o n a t e on a u t o s o m a l v a r i e g a t i n g l o c i . The bw* gene, l o c a t e d on chromosome 2, i s i n v o l v e d i n t h e p r o d u c t i o n o f t h e r e d p i g m e n t s , p t e r i d i n e s , w h i c h c o n t r i b u t e t o t h e n o r m a l d a r k r e d eye c o l o u r o f D r o s o p h i l a . M u t a t i o n s a t t h i s l o c u s r e s u l t i n t h e r e d u c t i o n o r a b s e n c e o f t h e p t e r i d i n e s p r o d u c i n g a brown eye phene. I n ( 2 L R ) b w v 3 2 g j u x t a p o s e s t h e bw+ gene t o t h e 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 of chromosome 2 r e s u l t i n g i n t h e m o s a i c e x p r e s s i o n o f t h i s l o c u s . T h e r e f o r e t h e a c t i v i t y o f t h e bw+ gene c a n be m o n i t o r e d by m e a s u r i n g the- r e d p i g m e n t , d r o s o p t e r i n , i n a manner s i m i l a r t o t h a t employed f o r 2 9 TABLE 1 THE EFFECT OF EXPOSURE TO BUTYRATE ON VARIEGATION OF B BUTYRATE CONCENTRATION ( M ) 0,00 (CONTROL) 0.15 0.20 EYE SIZE 64 59 52** s x 4.2 4.2 4.9 *EXPRESSED AS PERCENTAGE OF THE WILD-TYPE OR-R EYE **SIGNIFICANTLY DIFFERENT FROM CONTROL So = STANDARD ERROR 30 t h e wj_ gene. The e f f e c t o f c o n t i n u o u s e x p o s u r e of b w v 3 2 g i n d i v i d u a l s t o v a r i o u s c o n c e n t r a t i o n s of b u t y r a t e o r p r o p i o n a t e ( f r o m 50 t o 200 mM) d u r i n g t h e d e v e l o p m e n t a l i n t e r v a l f r o m o v i p o s i t i o n t o p u p a r i u m f o r m a t i o n a r e shown i n T a b l e 2. The r e s u l t s show t h a t b o t h b u t y r a t e and p r o p i o n a t e c a n e l i c i t s i g n i f i c a n t s u p p r e s s i o n o f b w v 3 2 g v a r i e g a t i o n . F u r t h e r m o r e , t h e r e i s a l i n e a r and p o s i t i v e c o r r e l a t i o n between s u p p r e s s i o n o f v a r i e g a t i o n and i n c r e a s i n g c o n c e n t r a t i o n s o f e i t h e r o f t h e s e c h e m i c a l s . A f u r t h e r t e s t o f t h e g e n e r a l i t y o f s u p p r e s s i o n of p o s i t i o n e f f e c t v a r i e g a t i o n by b u t y r a t e and p r o p i o n a t e was c o n d u c t e d by m o n i t o r i n g t h e e f f e c t o f t h e s e c h e m i c a l s on v a r i e g a t i o n o f t h e dominant t h i r d chromosome m u t a t i o n S t u b b l e , (Sb) • B r i s t l e f o r m i n g c e l l s i n w h i c h t h i s gene i s a c t i v e p r o d u c e a s h o r t e n e d t h i c k b r i s t l e . T ( 2 ; 2 ) S b v ( S b V ) i s a t r a n s l o c a t i o n w h i c h a b u t s t h e Sb a l l e l e t o t h e 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 o f chromosome 2 r e s u l t i n g i n m o s a i c e x p r e s s i o n o f t h i s gene. The l e v e l o f Sb e x p r e s s i o n i n e a c h i n d i v i d u a l c a n be d e t e r m i n e d by e x a m i n i n g t h e major b r i s t l e s on t h e f l y and a s s i g n i n g e a c h b r i s t l e a "Sb" o r w i l d - t y p e "+" p h e n o t y p e . F u l l e x p r e s s i o n o f t h i s d ominant m u t a t i o n r e s u l t s i n a l l b r i s t l e s b e i n g s t u b b l e . The r e s u l t s o f t h e e x p e r i m e n t s i n w h i c h S b v i n d i v i d u a l s were e x p o s e d t o e i t h e r b u t y r a t e o r p r o p i o n a t e t h r o u g h o u t e a r l y d e v e l o p m e n t a r e shown i n T a b l e 3. C l e a r l y , e x p o s u r e t o b u t y r a t e o r p r o p i o n a t e s i g n i f i c a n t l y s u p p r e s s v a r i e g a t i o n o f Sb_v r e s u l t i n g i n t h e Sb gene b e i n g a c t i v e i n a g r e a t e r p r o p o r t i o n o f t h e b r i s t l e - f o r m i n g c e l l s o f e a c h i n d i v i d u a l . As was t h e c a s e TABLE 2 T H E E F F E C T O F E X P O S U R E T O B U T Y R A T E A N D P R O P I O N A T E V A R I E G A T I O N O F b w V 3 ? 9 B U T Y R A T E C O N C E N T R A T I O N ( M ) 0.00 ( C O N T R O L ) 0.075 0.10 0.20 ? a ? a 9 a 9 P I G M E N T L E V E L 30 30 34 36* 33 36* 39* 35* ** P R O P I O N A T E C O N C E N T R A T I O N ( M ) 0.00 ( C O N T R O L ) 0.075 0.10 0.20 9 a 9 ? 9 a P I G M E N T L E V E L 30 30 29 35* 36* 38* 44* 43* S I G N I F I C A N T L Y D I F F E R E N T F R O M C O N T R O L E X P R E S S E D A S P E R C E N T A G E O F W I L D - T Y P E P I G M E N T L E V E L S S ^ = S T A N D A R D E R R O R 32 TABLE 3 T H E E F F E C T O F E X P O S U R E T O B U T Y R A T E A N D P R O P I O N A T E V A R I E G A T I O N O F Sb V B U T Y R A T E C O N C E N T R A T I O N ( M ) Sb E X P R E S S I O N * * 0.00 ( C O N T R O L ) ? 44 2,4 64 3,1 0.10 9 61* 2.0 76* 2.1 0.15 9 59* 1.6 74* 2.4 P R O P I O N A T E C O N C E N T R A T I O N (M ) Sb E X P R E S S I O N 0.00 ( C O N T R O L ) ? 44 2.4 64 3.1 0.10 * 60* 2.5 78* 2.1 0.15 • 61* 2.3 79* 1.8 * S I G N I F I C A N T L Y D I F F E R E N T F R O M C O N T R O L * * E X P R E S S E D A S P E R C E N T A G E O F F U L L Sb E X P R E S S I O N S ^ = S T A N D A R D E R R O R 33 w i t h b w v ; 3 Z 8 , b u t y r a t e and p r o p i o n a t e a r e e q u a l l y e f f e c t i v e as s u p p r e s s o r s of p o s i t i o n e f f e c t v a r i e g a t i o n . Taken t o g e t h e r t h e s e r e s u l t s demonstrate t h a t exposure t o b u t y r a t e or p r o p i o n a t e can e l i c i t s i g n i f i c a n t s u p p r e s s i o n of p o s i t i o n e f f e c t v a r i e g a t i o n . T h i s o c c u r s r e g a r d l e s s of the type of v a r i e g a t i n g gene, X - l i n k e d ( w m 4, B s _ v ) or autosomal ( S b v , b w v 3 2 g ), w i l d - t y p e ( wj_ i n w m 4, bw+ i n b w v 3 2 g ) or mutant ( B s v , S b v ) , and r e g a r d l e s s of the type of h e t e r o c h r o m a t i n , whether X, Y, or autosomal w i t h which the v a r i e g a t i n g a l l e l e s a r e a s s o c i a t e d . However, the a b i l i t y of the s e c h e m i c a l s t o suppr e s s v a r i e g a t i o n d i f f e r e d m a r kedly. The e x p r e s s i o n of the in A-w* gene i n c r e a s e d f o u r - f o l d i n the w males, whereas i n c r e a s e s • . s — v v3 2 c v • i n e x p r e s s i o n of B , bw , and Sb were l i m i t e d t o a p p r o x i m a t e l y 20-25%. Whether the s e d i f f e r e n c e s r e p r e s e n t p e c u l i a r i t i e s of the s p e c i f i c gene under o b s e r v a t i o n or r e f l e c t d i f f e r e n c e s i n the h e t e r o c h r o m a t i n w i t h which each v a r i e g a t i n g gene i s a s s o c i a t e d has y e t t o be de t e r m i n e d . T h i s q u e s t i o n c o u l d p o s s i b l y be r e s o l v e d by examining the e f f e c t s of t h e s e c h e m i c a l s on a s i n g l e gene a s s o c i a t e d w i t h s e v e r a l d i f f e r e n t h e t e r o c h r o m a t i c b r e a k p o i n t s . 2) Developmental A n a l y s i s Other m o d i f i e r s of p o s i t i o n e f f e c t v a r i e g a t i o n , such as temperature or Y h e t e r o c h r o m a t i n , a r e known t o a c t a t s p e c i f i c t i m e s d u r i n g development. A c c o r d i n g l y , i t was of some i n t e r e s t t o d e t e r m i n e i f the s u p p r e s s i o n evoked by b u t y r a t e or p r o p i o n a t e was a l s o l i m i t e d t o a s p e c i f i c s t a g e or s t a g e s i n development. 34 S i n c e c o n t i n u o u s e x p o s u r e t o b u t y r a t e o r p r o p i o n a t e a t a c o n c e n t r a t i o n o f 150 mM e l i c i t e d s t r o n g s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n but d i d n o t c a u s e h i g h l e t h a l i t y nor u n d u l y p r o l o n g d e v e l o p m e n t I e x p o s e d s y n c h r o n o u s l y d e v e l o p i n g w c u l t u r e s t o t h i s c o n c e n t r a t i o n f o r b r i e f d e f i n e d p e r i o d s . Summaries o f t h e r e s u l t s of t h e d e v e l o p m e n t a l s t u d i e s i n v o l v i n g b u t y r a t e and p r o p i o n a t e a r e p r e s e n t e d i n F i g u r e s 3 and 4 r e s p e c t i v e l y . F o r t y - e i g h t h o u r e x p o s u r e s t o b u t y r a t e a t any t i m e d u r i n g d e v e l o p m e n t p r i o r t o p u p a r i a t i o n r e s u l t e d i n s i g n i f i c a n t s u p p r e s s i o n of v a r i e g a t i o n i n b o t h males ( F i g u r e 3a) and f e m a l e s ( F i g u r e 3 b ) . L a t e s e c o n d and e a r l y t h i r d i n s t a r l a r v a e a p p e a r e d t o be s l i g h t l y more s e n s i t i v e t o b u t y r a t e - i n d u c e d s u p p r e s s i o n o f v a r i e g a t i o n , w h i l e t h e l a t e e m b r y o - f i r s t i n s t a r s t a g e was l e a s t s e n s i t i v e . E x p o s u r e t o 150 mM p r o p i o n a t e d u r i n g s i m i l a r 48 hour p e r i o d s gave r e s u l t s c o m p a r a b l e t o t h o s e o b s e r v e d w i t h b u t y r a t e , b u t t h e a m p l i t u d e o f r e s p o n s e was somewhat l o w e r . In b o t h males ( F i g u r e 4a) and f e m a l e s ( F i g u r e 4 b ) , l a t e s e c o n d t o e a r l y t h i r d i n s t a r was t h e s t a g e most s e n s i t i v e t o p r o p i o n a t e , w h i l e t h e l a t e e m b r y o - f i r s t i n s t a r s t a g e was t h e l e a s t s e n s i t i v e . S i n c e 48 h o u r e x p o s u r e s t o b u t y r a t e o r p r o p i o n a t e r e s u l t e d i n s i g n i f i c a n t s u p p r e s s i o n o f w_ gene v a r i e g a t i o n d u r i n g a l l e m b r y o n i c and l a r v a l s t a g e s , e s p e c i a l l y l a t e s e c o n d a n d e a r l y t h i r d i n s t a r , I d e c i d e d t o t e s t t h e s e n s i t i v i t y o f f l i e s t o v e r y s h o r t e x p o s u r e s o f t h e s e c h e m i c a l s . A c c o r d i n g l y , c u l t u r e s were e x p o s e d t o 150 mM b u t y r a t e o r p r o p i o n a t e f o r s e q u e n t i a l 24 hour p e r i o d s f r o m egg d e p o s i t i o n t o p u p a r i u m f o r m a t i o n . B o t h 35 FIGURE I I I . In (1 )w" A F L I E S E X P O S E D T O 0.15 M N - B U T Y R A T E F O R 48 H O U R P E R I O D S I I I ( A ) . M A L E S I I I ( B ) . F E M A L E S C O N T R O L n - B U T Y R A T E 36 A) 50 UJ S 40 J a. 30 H UJ 20 1 24 r 48 72 96 T " 120 TIME OF EXPOSURE 144 — r 168 192 B) embryo j 1 s t instar j 2nd . , r d . i n s t a r j 3 i n s t a r 25 -g 20 1 0. 15 H Ui 10 J 1 24 DEVELOPMENTAL STAGE 48 72 96 " ~ T 120 ~T— 144 168 192 TIME OF EXPOSURE 3 7 FIGURE VI. I n C D w " 4 F L I E S E X P O S E D T O 0.15 M n - P R O P I O N A T E F O R 48 H O U R P E R I O D S I I I ( A ) . M A L E S I I I ( B ) . F E M A L E S C O N T R O L n - P R O P I O N A T E 38 A) 50 -. 5 40 -| Q . U J o. 30 H * 20 H —r 24 r 48 72 120 96 TIME OF EXPOSURE 144 — r 168 192 6) embryo 25 -2 20 3 15 H I - I 10 J I | 1 s t instar | 2n t* instar j 3 r c* instar DEVELOPMENTAL STAGE T 24 — r 48 —r 72 T 96 — r -120 144 i 168 192 TIME OF EXPOSURE 39 c h e m i c a l s p r o d u c e d s i m i l a r r e s u l t s i n m a l e s ( F i g u r e 5a and 6a) and f e m a l e s ( F i g u r e 5b and 6 b ) . A 24 hour e x p o s u r e t o e i t h e r b u t y r a t e o r p r o p i o n a t e c a u s e d s u p p r e s s i o n o f v a r i e g a t i o n a t a l l s t a g e s . However, not a l l s t a g e s were e q u a l l y s e n s i t i v e . The most s e n s i t i v e i n t e r v a l s c o r r e s p o n d e d t o t h e p e r i o d f r o m l a t e f i r s t t o e a r l y s e c o n d i n s t a r and t h e 24 hour p e r i o d i m m e d i a t e l y f o l l o w i n g t h e m o l t t o t h i r d i n s t a r . T h e s e two p e r i o d s a p p e a r e d t o be e q u a l l y s e n s i t i v e t o s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n by b u t y r a t e o r p r o p i o n a t e . The c e l l s w h i c h g i v e r i s e t o t h e a d u l t eye a r e d e t e r m i n e d e a r l y d u r i n g e m b r y o g e n e s i s and s e t a s i d e i n a p a c k e t known a s t h e e y e - a n t e n n a l i m a g i n a l d i s k . Once s e t a s i d e t h e p r e s u m p t i v e eye c e l l s a p p e a r t o u n d e r g o l i t t l e o r no c e l l d i v i s i o n u n t i l a p p r o x i m a t e l y m i d - f i r s t i n s t a r . The e y e - a n t e n n a l d i s k c e l l s t h e n b e g i n r a p i d p r o l i f e r a t i o n w h i c h a p p e a r s t o be c o n t i n u o u s , w i t h s l i g h t r e d u c t i o n s i n r a t e a t e a c h m o l t , u n t i l t h e end o f t h i r d i n s t a r . A t t h a t p o i n t c e l l d i v i s i o n shows and no d e t e c t a b l e d i v i s i o n s o c c u r i n t h e eye t i s s u e a f t e r p u p a r i u m f o r m a t i o n (BECKER, 1957). The d e v e l o p m e n t a l p e r i o d s w h i c h a r e most s e n s i t i v e t o c h e m i c a l - i n d u c e d s u p p r e s s i o n of v a r i e g a t i o n c o r r e s p o n d t o t h e p e r i o d s o f r a p i d c e l l d i v i s i o n i n t h e eye a n l a g e n . Thus i t may be t h a t c e l l s u n d e r g o i n g r a p i d c e l l d i v i s i o n a r e p a r t i c u l a r l y s e n s i t i v e t o b u t y r a t e i n d u c e d s u p p r e s s i o n o f v a r i e g a t i o n w h i l e n o n - d i v i d i n g c e l l s a r e l e s s s e n s i t i v e o r i n s e n s i t i v e . A s i m p l e t e s t o f t h i s h y p o t h e s i s i s t o e x p o s e pupae t o b u t y r a t e o r p r o p i o n a t e . I f t h e s e c h e m i c a l s a c t i n n o n - d i v i d i n g c e l l s t o a l t e r p o s i t i o n e f f e c t v a r i e g a t i o n , 40 FIGURE V. In (1 )w" 4 FLIES E X P O S E D T O 0.15 M n - B U T Y R A T E F O R 24 H O U R P E R I O D S V ( A ) . M A L E S V ( B ) . F E M A L E S C O N T R O L n-BUTYRATE 40 1 30 1 20 1 i — I 1 1 1 1 1 I 1 24 48 72 96 120 144 168 192 TIME OF EXPOSURE e m b r y o I 1 instar l 2 instar | 3 instar I DEVELOPMENTAL STA6E 20 -15 10 I "T 1 1 1 1 1 1 1 24 48 72 96 120 144 168 192 TIME OF EXPOSURE 42 FIGURE VI. In (1 )w" 4 FLIES EXPOSED TO 0.15 M n-PROPIONATE FOR 24 HOUR PERIODS V I (A ) . MALES V I ( B ) . FEMALES CONTROL n-BUTYRATE 43 A) 40 n UJ E 19 D. UJ 30 £ 2 0 1 1 24 48 — r 72 — r 96 — I — 120 TIME OF EXPOSURE — I -144 —T" 168 192 B) embryo | 1 s t instar | 2 n d instar | 3 r d instar DEVELOPMENTAL STA6E 20 is t-i a. >-15 10 cc w a. r 24 48 72 " T " 96 — 120 144 168 192 TIME OF EXPOSURE 44 or i f t h e y a c t d u r i n g t r a n s c r i p t i o n o f a v a r i e g a t i n g a l l e l e , t h e n e x p o s u r e of pupae t o e i t h e r c h e m i c a l s h o u l d i n c r e a s e p i g m e n t a t i o n . A l t e r n a t i v e l y , i f t h e y a c t o n l y on d i v i d i n g c e l l s w i t h i n a t i s s u e t o r e a c t i v a t e a v a r i e g a t i n g a l l e l e , t h e n t h e r e s h o u l d be no e f f e c t on t h e v a r i e g a t e d p h e n o t y p e when t h e s e c h e m i c a l s a r e a d m i n i s t e r e d t o pupae. W h i t e p r e p u p a e were c o l l e c t e d i n s y n c h r o n o u s g r o u p s , p l a c e d i n v i a l s on f i l t e r p a p e r s o a k e d i n v a r i o u s c o n c e n t r a t i o n s o f b u t y r a t e o r p r o p i o n a t e . The v i a l s were s e a l e d and t h e c u l t u r e s a l l o w e d t o d e v e l o p t o e c l o s i o n . T h o s e f l i e s w h i c h e c l o s e d were' c o l l e c t e d w i t h i n 24 h o u r s , aged 7-10 d a y s , and t h e amount o f d r o s o p t e r i n i n t h e i r e y e s was mea s u r e d . C o n c e n t r a t i o n s o f b u t y r a t e and p r o p i o n a t e w h i c h a r e a b l e t o a l t e r t h e d e t e r m i n e d s t a t e o f a v a r i e g a t i n g gene when a d m i n i s t e r e d t o embryos o r l a r v a e had a b s o l u t e l y no e f f e c t when a d m i n i s t e r e d t o pupae. T h e r e f o r e , i n an a t t e m p t t o a l t e r t h e programmed s t a t e o f a v a r i e g a t i n g gene, v e r y h i g h c o n c e n t r a t i o n s o f b u t y r a t e and p r o p i o n a t e were u s e d , up t o 4.5M. O n l y d a t a f r o m t h e h i g h e s t c o n c e n t r a t i o n s a r e shown i n T a b l e 4. C l e a r l y , a d m i n i s t r a t i o n o f b u t y r a t e o r p r o p i o n a t e d u r i n g t h e p u p a l p e r i o d has no e f f e c t on p o s i t i o n e f f e c t v a r i e g a t i o n . I t c o u l d be a r g u e d t h a t a l t h o u g h v e r y h i g h c o n c e n t r a t i o n s o f t h e s e c h e m i c a l s were u s e d , l i t t l e o r none e n t e r e d t h e pupae. However, t h e p a t t e r n o f l e t h a l i t y t h a t r e s u l t e d f r o m e x p o s u r e t o h i g h c o n c e n t r a t i o n s o f b u t y r a t e o r p r o p i o n a t e v e r y much r e s e m b l e d t h e e f f e c t s o f f e e d i n g l o w e r c o n c e n t r a t i o n s o f t h e s e c h e m i c a l s t o l a r v a e . I n a d d i t i o n , b u t y r a t e had a s i g n i f i c a n t e f f e c t when TABLE 4 T H E E F F E C T O F E X P O S U R E O F P U P A E T O B U T Y R A T E P R O P I O N A T E O N V A R I E G A T I O N O F w m 4 B U T Y R A T E C O N C E N T R A T I O N ( M ) P I G M E N T L E V E L * 0.00 ( C O N T R O L ) ? 12 24 3.5 ? 8 18 4.0 * 7 21 4.5 ? 7 23 P R O P I O N A T E C O N C E N T R A T I O N ( M ) P I G M E N T L E V E L * 0.00 ( C O N T R O L ) 9 12 24 3.5 ? 10 31 4.0 ? 10 20 4.5 ? 9 26 * E X P R E S S E D A S P E R C E N T A G E S ^ = S T A N D A R D E R R O R O F W I L D - T Y P E P I G M E N T L E V E L S 46 a d m i n i s t e r e d t o embryos a t c o n c e n t r a t i o n s a s low a s 150 mM, and t h e p r o b l e m s o f a d m i n i s t e r i n g b u t y r a t e t o pupae a n d embryos a r e s i m i l a r . T h u s , we a r e r e a s o n a b l y c o n f i d e n t t h a t i n d i v i d u a l s u n d e r g o i n g m e t a m o r p h o s i s were e x p o s e d t o c e l l u l a r c o n c e n t r a t i o n s a t l e a s t c o m p a r a b l e t o , i f n o t e x c e e d i n g , t h e i r e m b r y o n i c o r l a r v a l c o u n t e r p a r t s . However, none o f t h e f l i e s t h a t e s c a p e d t h e l e t h a l e f f e c t s of t h e s e c h e m i c a l s d u r i n g p u p a t i o n e x h i b i t e d any s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n . 3) The I n t e r a c t i o n of B u t y r a t e and Y Chromosome H e t e r o c h r o m a t i n The Y chromosome i s a known s u p p r e s s o r o f p o s i t i o n e f f e c t v a r i e g a t i o n (GOWAN and GAY, 1934; r e v i e w e d SPOFFORD, 1976). I n most c a s e s r e m o v a l o f Y h e t e r o c h r o m a t i n d e c r e a s e s e x p r e s s i o n o f a v a r i e g a t i n g gene w h i l e e x t r a Y m a t e r i a l s u p p r e s s e s v a r i e g a t i o n r e s u l t i n g i n i n c r e a s e d gene e x p r e s s i o n . T h u s , i t was o f some i n t e r e s t t o examine t h e co m b i n e d e f f e c t s o f e x p o s u r e t o b u t y r a t e and r e m o v a l o f t h e Y chromosome. A c c o r d i n g l y male f l i e s l a c k i n g a Y chromosome were g e n e r a t e d by c r o s s i n g C(1)RMpn/0 f e m a l e s t o w m 4/Y m a l e s . S y n c h r o n o u s e g g s were c o l l e c t e d , p l a c e d on medium c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f b u t y r a t e and a l l o w e d t o d e v e l o p t o e c l o s i o n . The r e s u l t s i n T a b l e 5 show t h a t i n w m 4/Y m a l e s e x p r e s s i o n o f t h e w*_ gene was i n c r e a s e d 3 - f o l d by e x p o s u r e t o low c o n c e n t r a t i o n s o f b u t y r a t e . I n c o n t r a s t , r e m o v a l o f t h e Y chromosome c o m p l e t e l y a b o l i s h e d t h e s u p p r e s s o r e f f e c t s o f b u t y r a t e . E v e n a t t h e h i g h e s t c o n c e n t r a t i o n s , no d e t e c t a b l e i n c r e a s e i n wj_ gene e x p r e s s i o n c o u l d be o b s e r v e d . I t has y e t t o TABLE 5 THE EFFECT OF EXPOSURE TO BUTYRATE ON VARIEGATION OF w™4 IN X/0 MALES BUTYRATE CONCENTRATION ( M ) X/Y MALE** (CONTROL) 0.00 (CONTROL) 5 0.075 13* 0.10 14* 0.15 23* 0.20 21* B U T Y R A T E C O N C E N T R A T I O N ( M ) X/0 M A L E 0.00 ( C O N T R O L ) 1.0 0.075 0.8 0.10 2.1 0.15 1.7 0.20 1.7 *SIGNIFICANTLY DIFFERENT FROM CONTROL **EXPRESSED AS PERCENTAGE OF WILD~TYPE PIGMENT LEVELS F-, MALES FROM AN OUTCROSS So = STANDARD ERROR 48 be d e t e r m i n e d whether a b o l i t i o n of b u t y r a t e ' s e f f e c t i s a c o n s e q u e n c e o f t h e r e d u c t i o n h e t e r o c h r o m a t i n i n g e n e r a l o r whether i t i s p e c u l i a r t o l o s s o f Y chromosome h e t e r o c h r o m a t i n . In a d d i t i o n , s i n c e Y chromosome h e t e r o c h r o m a t i n i s not u n i f o r m i n i t s a b i l i t y t o s u p p r e s s v a r i e g a t i o n ( r e v i e w SPOFFORD, 1976), i t may be t h a t c e r t a i n r e g i o n s of t h e Y a r e r e s p o n s i b l e f o r b l o c k i n g t h e e f f e c t o f b u t y r a t e . 4) The I n t e r a c t i o n of B u t y r a t e and H i s t o n e D e f i c i e n c e s One o f t h e known e f f e c t s o f b u t y r a t e i s i n h i b i t i o n o f t h e h i s t o n e d e a c e t y l a s e enzymes ( C a n d i d o e t . a l . , 1978; MOTTUS e t . a l . , 1980). S i n c e a c e t y l a t i o n o f h i s t o n e s has been a s s o c i a t e d w i t h t r a n s c r i p t i o n (LEVY-WILSON e t . a l . , 1979) and p o s i t i o n e f f e c t v a r i e g a t i o n i s s u p p r e s s e d i n s t r a i n s o f f l i e s b e a r i n g o n e - h a l f t h e n o r m a l h i s t o n e gene complement (KHESIN and LEIBOVITCH, 1978; MOORE e t . a l . r 1979) i t was o f c o n s i d e r a b l e i n t e r e s t t o examine t h e e f f e c t s o f b u t y r a t e on s t r a i n s o f f l i e s h a p l o i d f o r t h e h i s t o n e g e n e s . M a l e s b e a r i n g t h e w m 4 chromosome and e i t h e r a n o r m a l s e c o n d chromosome, a d e f i c i e n c y w h i c h does n o t i n c l u d e t h e h i s t o n e complex ( D S 9 ) , o r a d e f i c i e n c y f o r t h e e n t i r e h i s t o n e gene c l u s t e r (DS6) were e x p o s e d t o v a r i o u s c o n c e n t r a t i o n s o f b u t y r a t e c o n t i n u o u s l y f r o m egg l a y t o p u p a r i a t i o n . I t i s known t h a t s t r a i n s w h i c h a r e h a p l o i d f o r t h e h i s t o n e complex a m p l i f y t h e i r h i s t o n e gene number o v e r t i m e ( CHERNYSHEV e t . a _ l . , 1980; CHERNYSHEV e t . a l . , 1982 MOORE e t . a l . , p e r s o n n a l c o m m u n i c a t i o n ) . To a v o i d t h i s p r o b l e m t h e e x p e r i m e n t s were 49 conducted on f i r s t g e n e r a t i o n f l i e s from c r o s s e s of e i t h e r c o n t r o l or d e f i c i e n c y s t r a i n s to w m 4 f emales . Only males produced from these c r o s s e s c a r r i e d the w m ^ chromosome and the v a r i o u s second chromosomes under c o n s i d e r a t i o n . T a b l e 6 shows the r e s u l t s of c o n t i n u o u s exposure to s e v e r a l c o n c e n t r a t i o n s of b u t y r a t e . As was expected u n t r e a t e d f l i e s b e a r i n g the h i s t o n e d e f i c i e n c y (DS6) had a much h i g h e r base l e v e l of wj_ gene a c t i v i t y than e i t h e r the c o n t r o l chromosome ( b pjr cn - - chosen because both DS6 and DS9 were imposed on the chromosome) or the n o n - h i s t o n e d e f i c i e n c y s t r a i n , DS9 . In both the c o n t r o l s t r a i n s and the h i s t o n e d e f i c i e n t s t r a i n s , exposure to b u t y r a t e suppressed v a r i e g a t i o n . Low c o n c e n t r a t i o n s (75 mM) i n c r e a s e d wj_ gene e x p r e s s i o n 2-3 f o l d i n a l l s t r a i n s , and exposure to i n c r e a s e d b u t y r a t e c o n c e n t r a t i o n s r e s u l t e d i n a s i m i l a r l i n e a r i n c r e a s e i n pigment l e v e l s i n a l l 3 s t r a i n s . T h u s , i t would appear tha t the e f f e c t s of a d e f i c i e n c y f o r the h i s t o n e gene c l u s t e r and b u t y r a t e on p o s i t i o n e f f e c t v a r i e g a t i o n are a d d i t i v e . TABLE 6 T H E E F F E C T O F E X P O S U R E T O B U T Y R A T E O N V A R I E G A T I O N O F w m 4 I N S T R A I N S B E A R I N G A H I S T O N E D E F I C I E N C Y B U T Y R A T E C O N C E N T R A T I O N (M) 0.00 ( C O N T R O L ) 0.075 0.10 0.15 0.20 CONTROL STRAINS ' pr cn s x DS9 5 0.4 5 13 1.3 14 14 1.3 22 23 4.0 19 21 3.6 B U T Y R A T E C O N C E N T R A T I O N (M ) DS6 0.00 ( C O N T R O L ) 23 0.075 59 0.10 61 0.15 67 0.20 67 VALUES ARE PERCENTAGE OF WILD-TYPE PIGMENT LEVELS Sn = STANDARD ERROR 51 DISCUSSION The p r e s e n t s t u d y c l e a r l y d e m o n s t r a t e s t h a t b u t y r a t e and p r o p i o n a t e n o t o n l y s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n o f t h e w* gene, but a l s o s u p p r e s s v a r i e g a t i o n of b w v 3 2 g , S b v , and B s _ v . T h e s e v a r i e g a t i n g a l l e l e s r e p r e s e n t genes s c a t t e r e d t h r o u g h o u t t h e genome j u x t a p o s e d t o a u t o s o m a l , X - l i n k e d , and Y - l i n k e d h e t e r o c h r o m a t i n . Thus, b u t y r a t e a n d p r o p i o n a t e a p p e a r t o a c t on t h e phenomenon of p o s i t i o n e f f e c t v a r i e g a t i o n i n g e n e r a l . However, t h e l e v e l of s u p p r e s s i o n o b s e r v e d v a r i e d d e p e n d i n g on t h e . s t r a i n e x amined. I f , as i s now w i d e l y b e l i e v e d , p o s i t i o n e f f e c t v a r i e g a t i o n r e s u l t s f r o m a s p r e a d o f h e t e r o c h r o m a t i n w h i c h c o n d e n s e s and t r a n s c r i p t i o n a l l y i n a c t i v a t e s t h e v a r i e g a t i n g genes i n a c e r t a i n p r o p o r t i o n o f c e l l s one m i ght e x p e c t a more u n i f o r m s e n s i t i v i t y t o b u t y r a t e o r p r o p i o n a t e . However, h e t e r o c h r o m a t i n i s n o t a u n i f o r m e n t i t y . E i g h t y p e r c e n t o f t h e 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 i s composed o f s h o r t r e p e t i t i v e s a t e l l i t e DNA s e q u e n c e s w h i c h c a n be r e s o l v e d i n t o f a m i l i e s b a s e d on b u o y a n t d e n s i t i e s and s e q u e n c e a n a l y s i s . They o c c u r i n s p e c i f i c r e g i o n s o f s e x - l i n k e d and a u t o s o m a l h e t e r o c h r o m a t i n (PEACOCK e t a l . , 1977). In a d d i t i o n , i t i s p o s s i b l e t h a t e a c h o f t h e e u c h r o m a t i c segments c o n t a i n i n g a g i v e n v a r i e g a t i n g gene p o s s e s s u n i q u e s t r u c t u r a l and b i o c h e m i c a l p r o p e r t i e s . Hence, i t i s l i k e l y t h a t t h e l e v e l o f v a r i e g a t i o n o f a p a r t i c u l a r a l l e l e maybe a p r o d u c t o f a t l e a s t t h e s e two v a r i a b l e s . The o b s e r v a t i o n t h a t e a c h v a r i e g a t i n g a l l e l e r e s p o n d s t o b u t y r a t e o r p r o p i o n a t e w i t h a u n i q u e l e v e l o f e x p r e s s i o n i s c o n s i s t e n t w i t h s u c h a n o t i o n . One p o s s i b l e t e s t 52 of t h i s h y p o t h e s i s would be to examine the e f f e c t s of these c h e m i c a l s on v a r i e g a t i o n of a s i n g l e gene jux taposed to s e v e r a l d i f f e r e n t h e t e r o c h r o m a t i c b r e a k p o i n t s . However, wh i l e there i s v a r i a b i l i t y in the response of d i f f e r e n t v a r i e g a t i n g systems to b u t y r a t e or p r o p i o n a t e , i t i s important to emphasize that in a l l c a s e s , exposure to r e l a t i v e l y low c o n c e n t r a t i o n s of these c h e m i c a l s s i g n i f i c a n t l y i n c r e a s e d e x p r e s s i o n of v a r i e g a t i n g genes . In p o s i t i o n e f f e c t v a r i e g a t i o n , as i n d e t e r m i n a t i o n , the u l t i m a t e t r a n s c r i p t i o n a l f a t e of a gene i s d e c i d e d upon e a r l y i n development , l ong be fore that gene i s t r a n s c r i b e d . In some cases t h i s d e c i s i o n appears to be i r r e v o c a b l e (HADORN et a l . , 1970; G S E L L , 1971) whi l e i n o t h e r s the d e c i s i o n may be l e s s s t a b l e ( f o r d i s c u s s i o n see SPOFFORD, 1976). In the former i n s t a n c e s , once the fa t e of a v a r i e g a t i n g a l l e l e i s d e t e r m i n e d , i t i s passed on f a i t h f u l l y to daughter c e l l s p r o d u c i n g c l o n e s of c e l l s w i t h i n a t i s s u e in which the v a r i e g a t i n g gene w i l l be a c t i v e or i n a c t i v e . D u r i n g d i f f e r e n t i a t i o n , t h i s e a r l y d e c i s i o n becomes man i f e s t p r o d u c i n g a mosaic phenotype . S i n c e c o n t i n u o u s exposure to b u t y r a t e or p r o p i o n a t e r e s u l t s i n a s i g n i f i c a n t i n c r e a s e i n the number of c e l l s i n which a v a r i e g a t i n g gene i s a c t i v e , i t was of c o n s i d e r a b l e i n t e r e s t to determine whether these agents suppress p o s i t i o n e f f e c t v a r i e g a t i o n by a c t i n g a t the t ime of the o r i g i n a l " d e t e r m i n a t i v e " event or whether they d i s r u p t e d maintenance or t r a n s m i s s i o n of the e a r l y d e c i s i o n . Exposures to b u t y r a t e or p r o p i o n a t e for 48 hours d u r i n g embryonic or l a r v a l l i f e demonstrated that these c h e m i c a l s can 53 s i g n i f i c a n t l y s u p p r e s s v a r i e g a t i o n a t a l l s t a g e s of e a r l y d e v e l o p m e n t . However, n o t a l l s t a g e s o f e m b r y o g e n e s i s and l a r v a l d e v e l o p m e n t a p p e a r t o be e q u a l l y s e n s i t i v e t o s u p p r e s s i o n of p o s i t i o n e f f e c t v a r i e g a t i o n by t h e s e c h e m i c a l s . The t i m e d u r i n g d e v e l o p m e n t when t h e f l i e s a r e most s e n s i t i v e t o c h e m i c a l - i n d u c e d s u p p r e s s i o n o f v a r i e g a t i o n we d e f i n e as t h e e f f e c t o r s e n s i t i v e p e r i o d , ESP. In t h e 48 hou r p u l s e s , l a t e s e c o n d and e a r l y t h i r d i n s t a r s t a g e s a r e most s e n s i t i v e t o b u t y r a t e ( F i g u r e 3) o r p r o p i o n a t e ( F i g u r e 4) i n d u c e d s u p p r e s s i o n , w h i l e f i r s t i n s t a r and l a t e t h i r d i n s t a r s t a g e s a r e l e a s t s e n s i t i v e . E x p o s u r e t o b u t y r a t e ( F i g u r e 5) o r p r o p i o n a t e ( F i g u r e 6) f o r 24 hour p e r i o d s show a s i m i l a r d i f f e r e n t i a l s t a g e s e n s i t i v i t y . Two p r i n c i p l e ESPs e x i s t , one b e g i n n i n g a t a b o u t t h e m i d d l e o f f i r s t i n s t a r and t h e s e c o n d b e g i n n i n g j u s t a f t e r t h e m o l t t o t h i r d i n s t a r . B o t h s t a g e s a p p e a r t o be e q u a l l y s e n s i t i v e t o s h o r t e x p o s u r e s o f t h e s e c h e m i c a l s . T h e r e f o r e , i t a p p e a r s t h a t b u t y r a t e and p r o p i o n a t e may .acts on t h e c l o n a l p r o p a g a t i o n o r m a i n t e n a n c e o f t h e e a r l y d e c i s i o n . However, t h e s e e x p e r i m e n t s do not r u l e o u t t h e p o s s i b i l i t y t h a t t h e s e c h e m i c a l s a l s o a f f e c t t h e e a r l y d e t e r m i n a t i v e e v e n t . T h i s q u e s t i o n c o u l d be r e s o l v e d by f u r t h e r e x p e r i m e n t s i n v o l v i n g c a r e f u l l y s y n c h r o n i z e d embryos and v e r y s h o r t e x p o s u r e s , l e s s t h a n 24 h o u r s , t o b u t y r a t e o r p r o p i o n a t e . C o n t r a r y t o our f i n d i n g d u r i n g e a r l y d e v e l o p m e n t , c o n t i n u o u s e x p o s u r e t o b u t y r a t e or p r o p i o n a t e d u r i n g p u p a t i o n had a b s o l u t e l y no e f f e c t on p o s i t i o n e f f e c t v a r i e g a t i o n a t any c o n c e n t r a t i o n up t o 4.5 M. A t h i g h e r c o n c e n t r a t i o n s many o f t h e 54 pupae d i e d p r i o r t o e c l o s i o n i n d i c a t i n g t h a t t h e s e c a r b o x y l i c a c i d s were e n t e r i n g t h e p u p a l c a s e s . However, e v e n t h o s e f l i e s w h i c h s u r v i v e d t h e s e h i g h e x p o s u r e s had p i g m e n t l e v e l s s i m i l a r t o t h e i r U n t r e a t e d s i b l i n g s . A l t h o u g h s u f f i c i e n t t i m e e x i s t s d u r i n g p u p a t i o n , a t l e a s t 48 h o u r s , f o r a l t e r i n g t h e d e t e r m i n e d s t a t e o f t h e v a r i e g a t i n g gene, b u t y r a t e and p r o p i o n a t e c a n n o t r e v e r s e t h e t r a n s c r i p t i o n a l commitment made e a r l i e r i n d e v e l o p m e n t . One p l a u s i b l e i n t e r p r e t a t i o n o f t h e s e r e s u l t s i s t h a t b u t y r a t e and p r o p i o n a t e c a n a l t e r t h e d e c i s i o n a s t o w h ether a v a r i e g a t i n g a l l e l e w i l l be a c t i v e o r r e p r e s s e d i n d i v i d i n g c e l l s o n l y . In n o n - d i v i d i n g t i s s u e t h e i n i t i a l d e c i s i o n r e m a i n s f i x e d . T h i s c o n c l u s i o n i s s u p p o r t e d by t h e o b s e r v a t i o n t h a t t h e ESPs f o r c h e m i c a l - i n d u c e d s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n a r e p r e c i s e l y c o n g r u e n t w i t h v a r i a t i o n s i n c e l l d i v i s i o n r a t e s w i t h i n t h e eye d i s c a n l a g e n . H i s t o l o g i c a l s t u d i e s (MADHAVEN and SCHNEIDERMAN, 1977) o f t h e eye i m a g i n a l d i s c a n d r e c e n t s t u d i e s e m p l o y i n g c l o n a l a n a l y s i s o f c a r e f u l l y s t a g e d young embryos (WIESCHAUS, 1975; WIESCHAUS and GEHRING, 1976) e s t a b l i s h e d t h a t between b l a s t o d e r m f o r m a t i o m , 3 h o u r s a f t e r f e r t i l i z a t i o n , and 10 h o u r s , t h e p r e s u m p t i v e eye c e l l s u n d e r g o an a v e r a g e o f one c e l l d i v i s i o n a n d t h e n m i t o t i c a c t i v i t y c e a s e s . M i t o t i c a c t i v i t y among t h e eye i m a g i n a l d i s c c e l l s resumes midway t h r o u g h f i r s t i n s t a r , a b o u t 12-14 h o u r s a f t e r h a t c h i n g . D u r i n g l a r v a l d e v e l o p m e n t t h e eye d i s c c e l l s c o n t i n u e t o d i v i d e however r e d u c t i o n s i n t h e r a t e o f c e l l d i v i s i o n o c c u r j u s t p r i o r t o e a c h l a r v a l m o l t (BECKER, 1957). 55 M o l t i n g i s c l o s e l y f o l l o w e d by an i n c r e a s e i n c e l l d i v i s i o n r a t e . D u r i n g l a t e t h i r d i n s t a r , c e l l d i v i s i o n i n t h e p r e s u m p t i v e e y e c e l l s c e a s e s . E a r l y f i r s t i n s t a r , l a t e t h i r d i n s t a r , and p u p a r i a t i o n a r e l e a s t s e n s i t i v e o r c o m p l e t e l y i n s e n s i t i v e t o t h e e f f e c t s o f e i t h e r b u t y r a t e o r p r o p i o n a t e and t h e s e p e r i o d s c o r r e s p o n d t o s t a g e s of d e v e l o p m e n t when c e l l d i v i s i o n i n t h e eye i m a g i n a l d i s c i s r e d u c e d or a b s e n t . S i m i l a r l y , l a t e f i r s t and e a r l y t h i r d i n s t a r a r e t h e two p e r i o d s most s e n s i t i v e t o s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n . T h e s e c o r r e s p o n d t o t h e p e r i o d s when t h e eye i m a g i n a l d i s c i s u n d e r g o i n g r a p i d p r o l i f e r a t i o n . T h u s , a s t r o n g c o r r e l a t i o n e x i s t s between t h e s t a g e s e n s i t i v i t y t o b u t y r a t e a n d p r o p i o n a t e and c e l l d i v i s i o n . O t h e r known m o d i f i e r s o f p o s i t i o n e f f e c t v a r i e g a t i o n h a v e been t e s t e d t o d e t e r m i n e i f t h e y a c t c o n t i n u o u s l y o r a t s p e c i f i c t i m e s d u r i n g d e v e l o p m e n t . T e m p e r a t u r e has been t h e most e x t e n s i v e l y s t u d i e d m o d i f i e r i n t h i s r e g a r d . Two p r i n c i p a l t e m p e r a t u r e - s e n s i t i v e p e r i o d s (TSP) f o r wj_ m o t t l i n g have been i d e n t i f i e d i n D. m e l a n o g a s t e r . One o c c u r s e a r l y d u r i n g e m b r y o g e n e s i s , and t h e o t h e r two d a y s a f t e r p u p a r i u m f o r m a t i o n (CHEN, 1948; SCHUTZ, 1956; HARTMANN-GOLDSTEIN, 1967; SPOFFORD, 19 7 6 ) . T h e s e c o r r e s p o n d t o t h e a p p r o x i m a t e t i m e s a t w h i c h i n i t i a l d e t e r m i n a t i o n i s made and t h e t i m e a t w h i c h t h e w/_ i s t r a n s c r i b e d . The major TSPs d i f f e r m a r k e d l y from t h e p r i n c i p a l E SPs we o b s e r v e d a f t e r s h o r t e x p o s u r e t o b u t y r a t e o r p r o p i o n a t e . T h i s s u g g e s t t h a t t e m p e r a t u r e a n d t h e s e c a r b o x y l i c a c i d s p r o b a b l y a f f e c t d i f f e r e n t a s p e c t s o f t h e p o s i t i o n e f f e c t 56 v a r i e g a t i o n phenomenon. The Y chromosome i s a potent suppressor of p o s i t i o n e f f e c t v a r i e g a t i o n (GOWAN and GAY, 1934). U s i n g somatic r e c o m b i n a t i o n , induced at s p e c i f i c s tages of development , BECKER and JANNING (1977) produced c l o n e s of t i s s u e which v a r i e d i n t h e i r Y h e t e r o c h r o m a t i n c o n t e n t . T h e i r exper iments demonstrated tha t a d d i t i o n of Y h e t e r o c h r o m a t i n a t anyt ime d u r i n g l a r v a l development suppressed p o s i t i o n e f f e c t v a r i e g a t i o n . However, i t e x e r t e d i t s s t r o n g e s t suppres sor e f f e c t when added near the end of f i r s t i n s t a r . To t h e i r s u p r i s e , removal of Y h e t e r o c h r o m a t i n a t any t ime had no s i g n i f i c a n t e f f e c t on v a r i e g a t i o n of the wj_ gene. One of the major ESPs for b u t y r a t e or p r o p i o n a t e , l a t e f i r s t to e a r l y second i n s t a r , c o r r e s p o n d s to the major Y -s e n s i t i v e p e r i o d . In a d d i t i o n , exposure to these c h e m i c a l s at t imes o ther than the major ESPs r e s u l t s i n s i g n i f i c a n t s u p p r e s s i o n of p o s i t i o n e f f e c t v a r i e g a t i o n as does the a d d i t i o n , but not the s u b t r a c t i o n , of Y h e t e r o c h r o m a t i n . T h u s , i t appears t h a t the a d d i t i o n of Y h e t e r o c h r o m a t i n a c t s i n a manner analagous to exposure to b u t y r a t e or p r o p i o n a t e . I t has been suggested t h a t the Y h e t e r o c h r o m a t i n suppresses p o s i t i o n e f f e c t v a r i e g a t i o n by competing f o r the agents r e s p o n s i b l e f o r " h e t e r o c h r o m a t i n i z a t i o n " of a v a r i e g a t i n g l o c u s (ZUCKERKANDL, 1974). In o r d e r to f u r t h e r i n v e s t i g a t e the s i m i l a r i t y between the e f f e c t of the Y chromosome and exposure to b u t y r a t e or p r o p i o n a t e , w m 4 males l a c k i n g a Y chromosome were exposed to these c h e m i c a l s c o n t i n u o u s l y from egg l a y to puparium f o r m a t i o n . 57 N e i t h e r b u t y r a t e nor p r o p i o n a t e s u p p r e s s e d v a r i e g a t i o n i n t h e s e f l i e s ( T a b l e 5 ) . T h i s c a n be i n t e r p r e t e d i n a manner c o n s i s t e n t w i t h ZUCKERKANDL's h y p o t h e s i s . Removal o f t h e Y chromosome mi g h t e l i m i n a t e c o m p e t i t i o n f o r t h e a g e n t s r e s p o n s i b l e f o r " h e t e r o c h r o m a t i n i z a t i o n " o f t h e v a r i e g a t i n g gene, t h e r e b y a b o l i s h i n g t h e e f f e c t s of b u t y r a t e o r p r o p i o n a t e . However, ZUCKERKANDL d i d n o t o f f e r any s u g g e s t i o n s a s t o what t h e m o l e c u l a r n a t u r e o f t h e s e " a g e n t s " m i g h t be. One p o s s i b l e c a n d i d a t e f o r t h e s e h e t e r o c h r o m a t i c a g e n t s i s t h e h i s t o n e p r o t e i n s . S i n c e t h e y a r e m a j o r s t r u c t u r a l components o f c h r o m a t i n , h i s t o n e s may p l a y a c r i t i c a l r o l e i n t h e f o r m a t i o n o f h e t e r o c h r o m a t i n and t h e r e f o r e i n p o s i t i o n e f f e c t v a r i e g a t i o n . T h a t t h i s may i n d e e d be t h e c a s e i s shown by r e c e n t e x p e r i m e n t s i n v o l v i n g s t r a i n s o f D. m e l a n o g a s t e r h a p l o i d f o r t h e h i s t o n e gene c l u s t e r . T h e s e s t r a i n s show s i g n i f i c a n t s u p p r e s s i o n o f v a r i e g a t i n g g e n e s (KHESIN and LEIBOVITCH, 1978; MOORE e t a l . , 1979). The a u t h o r s s u g g e s t t h a t h a p l o i d y f o r t h e h i s t o n e gene complex r e s u l t s i n a r e d u c t i o n o f t o t a l c e l l u l a r h i s t o n e w h i c h , i n t u r n , s u p p r e s s e s p o s i t i o n e f f e c t v a r i e g a t i o n . Thus, h i s t o n e s o r a s u b s e t o f t h e h i s t o n e s , a p p e a r t o be good c a n d i d a t e s f o r t h e m o l e c u l e s r e s p o n s i b l e f o r " h e t e r o c h r o m a t i n i z a t i o n " of v a r i e g a t i n g l o c i . E x p o s u r e t o b u t y r a t e o r p r o p i o n a t e c a u s e s s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n w h i c h i s as s t r o n g a s t h a t e v o k e d by e i t h e r h i s t o n e d e f i c i e n c i e s o r t h e a d d i t i o n o f a Y chromosome. T h i s l e a v e s t h r e e major q u e s t i o n s u n r e s o l v e d . How do t h e s e c h e m i c a l s a c t t o s u p p r e s s v a r i e g a t i o n , what r e l a t i o n s h i p , i f 58 any, e x i s t s between h i s t o n e gene m u l t i p l i c i t y a nd t h e s e c h e m i c a l s , and why a r e d i v i d i n g c e l l s more s e n s i t i v e t o t h e i r e f f e c t s ? S e v e r a l e x p e r i m e n t s have shown t h a t e x p o s u r e t o b u t y r a t e (RIGGS e t a l . , 1977) or p r o p i o n a t e (SEALY and CHALKLEY, 1978) l e a d s t o an a c c u m u l a t i o n o f h y p e r a c e t y l a t e d h i s t o n e s due t o i n h i b i t i o n o f t h e h i s t o n e d e a c e t y l a s e enzymes (CANDIDO e t a l . , 1978; MOTTUS e t a l . , 1980). A c e t y l a t e d h i s t o n e s have been a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e genes (LEVY-WILSON e t . a l . , 1 979). I n a d d i t i o n , i t has been w e l l documented t h a t a d d i t i o n o f s m a l l amounts o f b u t y r a t e (5mM) t o t h e medium c a u s e d marked a l t e r a t i o n s i n t h e t r a n s c r i p t i o n p a t t e r n s o f c e l l s g r o w i n g i n c u l t u r e ( f o r a r e v i e w see KRUH, 1982, REEVES and CSERJESI, 1979). T h e r e f o r e , t h e s u p p r e s s i o n o f p o s i t i o n e f f e c t v a r i e g a t i o n o b s e r v e d a f t e r e x p o s u r e t o b u t y r a t e or p r o p i o n a t e can be i n t e r p r e t e d t o r e s u l t , d i r e c t l y o r i n d i r e c t l y , f r o m i n  v i v o i n h i b i t i o n o f t h e h i s t o n e d e a c e t y l a s e enzymes l e a d i n g t o an a c c u m u l a t i o n o f h y p e r a c e t y l a t e d h i s t o n e s p e c i e s and r e d u c i n g t h e s p r e a d o f h e t e r o c h r o m a t i n i n t o t h e a d j a c e n t e u c h r o m a t i n . Thus, i t i s e a s y t o v i s u a l i z e how b u t y r a t e t r e a t m e n t m ight e f f e c t t h e d e t e r m i n a t i v e e v e n t i n p o s i t i o n e f f e c t v a r i e g a t i o n . However, t h e d e v e l o p m e n t a l s t u d i e s c l e a r l y d e m o n s t r a t e t h a t b u t y r a t e a l s o a f f e c t s m a i n t e n a n c e o f t h e e a r l y d e c i s i o n . T r e a t m e n t w i t h b u t y r a t e d u r i n g e a r l y d e v e l o p m e n t c an r e a c t i v a t e v a r i e g a t i n g genes w h i c h had p r e v i o u s l y been r e p r e s s e d . F u r t h e r m o r e , t h i s new d e c i s i o n i s t h e n c l o n a l l y p r o p o g a t e d i n th e a b s e n c e o f b u t y r a t e . How c a n b u t y r a t e a l t e r t h e d e t e r m i n e d 59 s t a t e o f a v a r i e g a t i n g gene? P r i o r t o i n c o r p o r a t i o n , newly s y n t h e s i z e d h i s t o n e s H3 and H4 a r e a c e t y l a t e d , p e r h a p s t o f a c i l i t a t e b i n d i n g t o t h e n a s c e n t DNA (RUIZ-CARRILLO e t . a l . , 1975) F u r t h e r m o r e , r e c e n t e v i d e n c e i n d i c a t e s t h a t i n t a c t n u c l e o s o m e s , or a t l e a s t h i s t o n e s H3 and H4, s e g r e g a t e a s y m m e t r i c a l l y d u r i n g DNA r e p l i c a t i o n (TSANEV and RUSSEV, 1974; LEFFAK e t a l . , 1977; SEIDMAN e t a l . , 1979; JACKSON and CHALKLEY, 1981). The c o n t i n u o u s l y r e p l i c a t i n g DNA s t r a n d r e c e i v e s t h e p a r e n t a l n u c l e o s o m e s w h i l e t h e d i s c o n t i n u o u s l y r e p l i c a t i n g DNA s t r a n d r e c e i v e s n ewly s y n t h e s i z e d h i s t o n e s . The a u t h o r s have s u g g e s t e d t h a t a s y m m e t r i c s e g r e g a t i o n o f n u c l e o s o m e s p r o v i d e s a s y s t e m whereby chromosomal i n f o r m a t i o n c a n be t r a n s m i t t e d t o d a u g h t e r c e l l s . T h e s e o b s e r v a t i o n s s u g g e s t a s i m p l e model t o a c c o u n t f o r t h e s u p p r e s s i o n of p o s i t i o n e f f e c t v a r i e g a t i o n c a u s e d by b u t y r a t e and p r o p i o n a t e . I f p a r e n t a l n u c l e o s o m e s a c t as t e m p l a t e s f o r d e t e r m i n i n g t h e b i o c h e m i c a l s t a t e o f t h e n ewly s y n t h e s i z e d c h r o m a t i n and t e m p l a t e a c t i v i t y o c c u r s d u r i n g o r v e r y s h o r t l y a f t e r DNA r e p l i c a t i o n , t h e n any f a c t o r , s u c h as b u t y r a t e , w h i c h i n h i b i t s t h e h i s t o n e d e a c e t y l a s e enzymes m i g h t r e s u l t i n h y p e r a c e t y l a t i o n o f t h e newly formed h i s t o n e s on t h e n o n - t e m p l a t e d a u g h t e r d u p l e x . S i n c e t h e r e i s a c o r r e l a t i o n between h i s t o n e a c e t y l a t i o n and t r a n s c r i p t i o n a l a c t i v i t y (LEVY-WILSON e_t a l . , 1979), i t i s r e a s o n a b l e t o s u g g e s t t h a t i n c r e a s e d a c e t y l a t i o n m i g h t l e a d t o i n c r e a s e d p o t e n t i a l f o r t r a n s c r i p t i o n , t h a t i s , a l t e r e d c h r o m a t i n p a c k a g i n g a n d t h u s , r e a c t i v a t i o n of a v a r i e g a t i n g a l l e l e . T h i s assumes t h a t t h e d e t e r m i n a t i o n o f gene 6 0 a c t i v i t y i s c o n t r o l l e d by c h r o m a t i n s t r u c t u r e . A l t e r n a t i v e l y , chromat in s t r u c t u r e may o n l y i n d i r e c t l y i n f l u e n c e , or be r e f l e c t i v e o f , d e t e r m i n a t i o n . In tha t c a s e , i n c r e a s e d a c e t y l a t i o n of H3 and H4 d u r i n g nucleosome assembly and m a t u r a t i o n of chromat in may a l l o w the DNA to be marked i n a manner analogous to the i n i t i a l d e t e r m i n a t i v e event which occurs at the t ime of a l l o c a t i o n of the presumpt ive eye c e l l s . T h i s model i s n e c e s s a r i l y n o n - s p e c i f i c and c o n t a i n s many s u p p o s i t i o n s s i n c e d e t a i l s of chromat in r e p l i c a t i o n and d e t e r m i n a t i o n are s t i l l very much at i s s u e . However, one assumption of t h i s model i s t h a t h i s t o n e d e a c e t y l a t i o n i s important i n the f o r m a t i o n of h e t e r o c h r o m a t i n . Thus , one might p r e d i c t t h a t the removal of l a r g e b l o c k s of h e t e r o c h r o m a t i n c o u l d i n c r e a s e the a v a i l a b i l i t y of h i s t o n e d e a c t y l a s e enzymes a t the v a r i e g a t i n g s i t e thereby r e d u c i n g or a b o l i s h i n g the e f f e c t of b u t y r a t e or p r o p i o n a t e . T h i s h y p o t h e s i s i s c o n s i s t e n t w i t h exper iments i n which males l a c k i n g a Y chromosome, the l a r g e s t s i n g l e b l o c k of h e t e r o c h r o m a t i n i n the D. melanogaster genome, demonstrated complete i n s e n s i t i v i t y t o these c h e m i c a l s (Tab le 5 ) . I t i s p o s s i b l e to determine whether t h i s e f f e c t i s r e s t r i c t e d to the Y chromosome or whether i s i s a g e n e r a l p r o p e r t y of h e t e r o c h r o m a t i n by expos ing v a r i e g a t i n g s t r a i n s b e a r i n g l a r g e d e l e t i o n s of X h e t e r o c h r o m a t i n , f o r example s c 4 L s c 8 R , or autosomal h e t e r o c h r o m a t i n , f o r example MS210, which bears a l a r g e d e l e t i o n of 2L h e t e r o c h r o m a t i n , to these c h e m i c a l s . A l s o c o n s i s t e n t w i th t h i s model are the r e s u l t s of 61 e x p e r i m e n t s i n w h i c h f l i e s b e a r i n g d e f i c i e n c i e s o f t h e h i s t o n e gene c l u s t e r were e x p o s e d t o b u t y r a t e ( T a b l e 6 ) . I f t h i s c h e m i c a l a c t s v i a i n h i b i t i o n of t h e h i s t o n e d e a c e t y l a s e enzymes t h e n one wo u l d not n e c e s s a r i l y e x p e c t t o see i n c r e a s e d s e n s i t i v i t y t o b u t y r a t e i n f l i e s w i t h r e d u c e d h i s t o n e gene m u l t i p l i c i t y . One s i m p l y m i g h t e x p e c t t o see an a d d i t i v e e f f e c t w i t h r e d u c t i o n i n h i s t o n e t e m p l a t e s and b u t y r a t e t r e a t m e n t . As was e x p e c t e d u n t r e a t e d f l i e s b e a r i n g o n e - h a l f t h e n o r m a l h i s t o n e gene complement had an i n c r e a s e d l e v e l o f v a r i e g a t i n g gene e x p r e s s i o n r e l a t i v e t h e t h e c o n t r o l s . E x p o s u r e t o low c o n c e n t r a t i o n s o f b u t y r a t e (75mM) r e s u l t e d i n a 2-3 f o l d i n c r e a s e i n e x p r e s s i o n o f t h e wj_ gene i n b o t h t h e c o n t r o l s t r a i n s (b p_r cn and DS9) and h a p l o i d s t r a i n (DS6). In a d d i t i o n , when e x p o s e d t o i n c r e a s i n g c o n c e n t r a t i o n s o f b u t y r a t e , b o t h t h e c o n t r o l s t r a i n s and DS6 showed i d e n t i c a l l i n e a r i n c r e a s e s i n wj_ gene e x p r e s s i o n . T h u s , t h e r e s u l t s o f t h e s e e x p e r i m e n t s a r e c o n s i s t e n t w i t h t h e n o t i o n t h a t b u t y r a t e and p r o p i o n a t e s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n a s a r e s u l t o f t h e i r a b i l i t y t o i n h i b i t t h e h i s t o n e d e a c e t y l a s e enzymes. However, t h i s h y p o t h e s i s i s o n l y t e n t a t i v e , s i n c e b o t h b u t y r a t e and p r o p i o n a t e a r e known t o e f f e c t a wide v a r i e t y o f c e l l u l a r f u n c t i o n s i n c l u d i n g m o d i f i c a t i o n o f some n o n - h i s t o n e chromosomal p r o t e i n s . 62 CHAPTER TWO INTRODUCTION T h e r e i s abundant e v i d e n c e t h a t t h e m o s a i c e x p r e s s i o n o f a r e l o c a t e d gene s u b j e c t t o p o s i t i o n e f f e c t v a r i e g a t i o n i s not due t o m u t a t i o n o f t h e gene ( r e v i e w SPOFFORD, 1976) n o r t o s o m a t i c gene l o s s (HENIKOFF, 1979). I t now a p p e a r s a l m o s t c e r t a i n t h a t p o s i t i o n e f f e c t v a r i e g a t i o n i s a r e s u l t o f a l t e r a t i o n s i n c h r o m a t i n s t r u c t u r e c a u s e d by t h e new a s s o c i a t i o n o f e u c h r o m a t i n and h e t e r o c h r o m a t i n i n t h e r e a r r a n g e d chromosome. S i n c e c h r o m a t i n i s a complex a s s o c i a t i o n o f DNA and p r o t e i n , i t seems r e a s o n a b l e t o assume t h a t p r o t e i n s p l a y a f u n d a m e n t a l r o l e i n p o s i t i o n e f f e c t v a r i e g a t i o n . F o r example, t h e r e i s good e v i d e n c e t h a t t h e h i s t o n e p r o t e i n s a r e i n v o l v e d i n t h i s phenomenon (KHESIN and LEIBOVITCH, 1978; MOORE e t a l . , 1979; MOORE e t . a l . , 1980; MOTTUS e t a l . , 1980). T h i s f i n d i n g i s n o t s u p r i s i n g s i n c e h i s t o n e s f o r m t h e b a s i c b u i l d i n g b l o c k o f e u c a r y o t i c c h r o m a t i n . However, i n a d d i t i o n t o h i s t o n e s , s e v e r a l h u n d r e d o t h e r p r o t e i n s a r e a l s o a s s o c i a t e d w i t h c h r o m a t i n (PETERSON and McCONKEY, 1975) and i t seems l i k e l y t h a t a s u b s e t o f t h e s e m i g h t be d i r e c t l y o r i n d i r e c t l y i n v o l v e d i n v a r i e g a t i o n . I f s o , t h e n t h e s e p r o t e i n s s h o u l d be amenable t o a g e n e t i c a n a l y s i s . P o s s i b l e examples o f s u c h m u t a t i o n s m i g h t be S u ( v a r ) (SPOFFORD, 1967) a n d E ( v a r ) (SCHULTZ, c i t e d i n GSELL, 1971), s p o n t a n e o u s mutants w h i c h s u p p r e s s o r enhance p o s i t i o n e f f e c t v a r i e g a t i o n r e s p e c t i v e l y . S e v e r a l a u t h o r s h a v e s u g g e s t e d t h a t g e n e t i c m o d i f i e r s o f p o s i t i o n e f f e c t v a r i e g a t i o n may be 63 q u i t e numerous i n the genome (SCHULTZ, 1950; SPOFFORD, 1976; HENIKOFF, 1979). However, u n t i l r e c e n t l y (REUTER and WOLFE, 1981) no s y s t e m a t i c attempt has been made to recover and a n a l y s e mutat ions m o d i f y i n g p o s i t i o n e f f e c t v a r i e g a t i o n . A c c o r d i n g l y , we undertook a l a r g e s c a l e s creen to i s o l a t e muta t ions which e i t h e r enhance or suppress p o s i t i o n e f f e c t v a r i e g a t i o n . F i f t y -seven mutants which modify the v a r i e g a t i n g phenotype a s s o c i a t e d w i t h p o s i t i o n e f f e c t v a r i e g a t i o n were r e c o v e r e d and we have begun to map and c h a r a c t e r i z e these m u t a t i o n s . I t i s hoped tha t a d e t a i l e d a n a l y s i s of these mutat ions w i l l l e a d to a b e t t e r u n d e r s t a n d i n g of p o s i t i o n e f f e c t v a r i e g a t i o n at the m o l e c u l a r l e v e l . In t u r n , t h i s may shed l i g h t on problems r e l a t i n g to the r e l a t i o n s h i p between chromat in s t r u c t u r e and g e n e t i c a c t i v i t y i n e u c a r y o t e s . 64 MATERIALS and METHODS 1) Stock and C u l t u r e C o n d i t i o n s The f l i e s were r e a r e d on s t a n d a r d s u c r o s e - c o r n m e a l - agar medium to which tegosept (methy l -p -hydroxybenzoate ) was added as a mould i n h i b i t o r . B a c t e r i a l growth was i n h i b i t e d by the a d d i t i o n of s t r e p t o m y c i n and t e t r a c y c l i n e or a m p i c i l l a n (10 m g / l i t e r e a c h ) . A l l of the s t r a i n s and mutat ions employed in t h i s s tudy are d e s c r i b e d in LINDSLEY and GRELL (1968) or i n Chapter One w i t h the e x c e p t i o n of b I t r l , a second chromosome l i n e d e r i v e d from a s i n g l e spontaneous m e i t o t i c r e c o m b i n a t i o n event between b l and r l (TATTERSALL, 1981). 2) I s o l a t i o n P r o t o c o l Dominant mutat ions which e i t h e r s t r o n g l y suppress or enhance w m 4 v a r i e g a t i o n were i s o l a t e d a c c o r d i n g t o the p r o t o c o l i n F i g u r e 7. Young males , homozygous f o r the b It: r l chromosome, were fed 0.025 M EMS ( e t h y l m e t h a n e s u l f o n a t e ) i n a 1% sucrose s o l u t i o n u s i n g the method of LEWIS and BACKER (1968) and mated to w m 4 / w m 4 ; +/CyO v i r g i n f emales . The eyes of F1 males were examined under d i s s e c t i n g microscopes for s t r o n g enhancement (reduced p i g m e n t a t i o n ) or s u p p r e s s i o n ( i n c r e a s e d p i g m e n t a t i o n ) of w m 4 v a r i e g a t i o n . 23,734 F1 males were examined and 548 p u t a t i v e s u p p r e s s o r s and 145 p u t a t i v e enhancers were r e c o v e r e d . P u t a t i v e mutat ions were p a i r - m a t e d to w m 4 / w m 4 ; DS6/CyO females and the r e s u l t i n g progeny examined. R e t e s t i n g 65 these s t r a i n s y i e l d e d 51 dominant s u p p r e s s o r s and 3 dominant enhancers of w m 4 v a r i e g a t i o n . 3) C h a r a c t e r i z a t i o n The above s c r e e n i n g p r o t o c o l p r e c l u d e s the r e c o v e r y of X -l i n k e d s u p p r e s s o r s or enhancers and s i n c e the mutat ions were e q u a l l y e f f e c t i v e in both sexes i t i s u n l i k e l y t h a t . a n y are Y -l i n k e d . A c c o r d i n g l y , the m u t a t i o n s were i n i t i a l l y a s s i g n e d to e i t h e r of the 2 major autosomes by c r o s s i n g each to e i t h e r I n ( 2 L R ) C y O / T f t or In(3LR)TM3 Sb S e r / L y and m o n i t o r i n g t h e i r s e g r e g a t i o n from the b a l a n c e r chromosomes in the F1 males . Once the mutat ions were a s s i g n e d to a p a r t i c u l a r chromosome, 5 randomly chosen s u p p r e s s o r s from each chromosome ( e i t h e r chromosome 2 or 3) and the 3 enhancers , a l l of which map to chromosome 3, were s u b j e c t e d to f u r t h e r a n a l y s i s as d e s c r i b e d below. A) Mapping and Complementat ion S t a n d a r d mapping p r o c e d u r e s were employed to l o c a t e the suppressor and enhancer mutat ions i n a w m 4 background . Suppressors on chromosome 2 were mapped by u s i n g the dominant ly marked second chromosome: S t a r (S, 2 - 1 . 3 ) , S t e r n o p l e u r a l (Sp_, 2 - 2 2 . 0 ) , T u f t ( T f t , 2 - 5 3 . 2 ) , narrow-wing dominant (nw D , 2 -83) , and P i n y e l l o w - t i p ( P Y T 2 - 1 0 7 . 3 ) . These markers span the e n t i r e second 66 chromosome and thus the mutants c o u l d be mapped r e l a t i v e l y a c c u r a t e l y . The enhancers and those s u p p r e s s o r s a s s i g n e d to the t h i r d chromosome were mapped r e l a t i v e to Glued ( G l , 3 - 4 1 . 4 ) , S t u b b l e (Sb, 3 - 5 8 . 2 ) , and H a i r l e s s (H, 3 - 6 9 . 5 ) . S ince these markers span o n l y about the middle 30% of the chromosome, muta t ions mapping more than 10 map u n i t s to the l e f t of G l or to the r i g h t of H can o n l y be l o c a t e d a p p r o x i m a t e l y . A l l r e c o m b i n a t i o n exper iments were conducted a t 2 2 ° C . In most cases no mutant was mapped u s i n g l e s s than 1000 progeny per experiment and none was mapped u s i n g l e s s than 400 progeny . I n i t i a l l y , a l l of the second chromosome s u p p r e s s o r s and a p p r o x i m a t e l y 60% of the t h i r d chromosome s u p p r e s s o r s were r e c e s s i v e l e t h a l s . In mapping exper iments in which a suppressor was a s s o c i a t e d w i t h r e c e s s i v e l e t h a l i t y , an attempt was made to s e p a r a t e the s u p p r e s s o r mutat ion from the r e c e s s i v e l e t h a l i t y . In cases where r e c o m b i n a t i o n events had o c c u r r e d i n the r e g i o n of the s u p p r e s s o r , the r e s u l t a n t organisms (minimum of 20) were b a c k c r o s s e d to the o r i g i n a l s u p p r e s s o r s t r a i n and the progeny examined for s u r v i v a l of the homozygous s u p p r e s s o r . Complementation a n a l y s i s was c a r r i e d out by c r o s s i n g the s u p p r e s s o r s to each o ther i n a l l p o s s i b l e p a i r - w i s e c o m b i n a t i o n s . In a d d i t i o n , the 5 second chromosome mutants under study were c r o s s e d to 7 o ther 67 chromosome 2 suppressor s t o c k s . In t h i s a n a l y s i s the a p p r o p r i a t e h e t e r o z y g o t e s were examined f o r v i a b i l i t y or a l t e r e d phenotype . The E f f e c t s of Suppres sors and Enhancers on Other  V a r i e g a t i n g Genes In order to determine whether the m o d i f y i n g e f f e c t of these m u t a t i o n s was r e s t r i c t e d to w m 4 or more g e n e r a l i n na ture the mutants were c r o s s e d to s e v e r a l o ther v a r i e g a t i n g a l l e l e s . In a l l cases except B s - V , 5 v i r g i n females of the v a r i e g a t i n g s t r a i n were c r o s s e d to 5 males of the suppressor or enhancer s tock i n v i a l s and the a d u l t s t u r n e d over i n t o new v i a l s a t t h r e e day i n t e r v a l s to prevent o v e r c r o w d i n g . The a p p r o p r i a t e progeny were c o l l e c t e d and assayed by the t e c h n i q u e s d e s c r i b e d i n Chapter One. In each experiment the c o n t r o l l e v e l of v a r i e g a t i o n was de termined by c r o s s i n g w m 4 / Y ; b I t r l males to females of the v a r i e g a t i n g s t r a i n . The mutat ions were imposed on the b I t r l chromosome and thus i t p r o v i d e s a r e l a t i v e l y homogeneous background . In t e s t s for the e f f e c t s of the s u p p r e s s o r s and enhancers B s _ v e x p r e s s i o n , B s - V males were c r o s s e d t o v i r g i n females from a s p e c i f i c mutant s tock or the c o n t r o l s t o c k . The amount of B s - V v a r i e g a t i o n was assayed as d e s c r i b e d i n Chapter One. 68 C) The A c t i v i t y of S u p p r e s s o r s i n X / 0 males The e f f e c t of the s u p p r e s s o r s on w v a r i e g a t i o n i n males l a c k i n g a Y chromosome was examined as f o l l o w s . Males wi thout a Y chromosome and he terozygous for the s u p p r e s s o r s were produced by c r o s s i n g males from each of the suppressor s t o c k s to v i r g i n C(1)RMpn/0 f e m a l e s . The e f f e c t of absence of the Y chromosome a lone was determined by c r o s s i n g w m 4 / Y ; b I t r l males to the compound b e a r i n g f emale s . In a l l cases the amount of pigment presen t i n the F1 males was q u a n t i t a t e d by the second method d e s c r i b e d in Chapter One. D) The E f f e c t of Temperature on w m 4 V a r i e g a t i o n i n V a r i o u s Suppressor S t r a i n s The e f f e c t of t h r e e d i f f e r e n t t emperatures on w v a r i e g a t i o n i n the s u p p r e s s o r was examined. Synchronous ly d e v e l o p i n g eggs were c o l l e c t e d at 2 2 ° C (Chapter One) from the suppressor s t o c k s - and p r e b l a s t o d e r m embryos were s h i f t e d to 1 7 ° C , 2 9 ° C , or r e t a i n e d at 2 2 ° C f o r the d u r a t i o n of development . A f t e r e c l o s i o n the a d u l t s were c o l l e c t e d , s h i f t e d back to 2 2 ° C , aged 7-10 d a y s , and the pigment l e v e l s of the males and females assayed s e p a r a t e l y (method 2, Chapter One) . 69 RESULTS 1) I s o l a t i o n o f M u t a t i o n s Dominant m u t a t i o n s w h i c h e i t h e r s u p p r e s s o r enhance p o s i t i o n e f f e c t v a r i e g a t i o n o f w m 4 were i n d u c e d by f e e d i n g EMS t o +/Y; b I t r l / b I t r l m a l e s . T h e s e were mated en mass t o w m 4/w m 4; +/CyO v i r g i n f e m a l e s and t h e F1 p r o g e n y were examined v i s u a l l y f o r s u p p r e s s i o n o r enhancement o f w m 4 v a r i e g a t i o n ( F i g u r e 7 ) . , O n l y t h e ma l e s w h i c h a p p e a r e d t o have w i l d - t y p e l e v e l s o f eye p i g m e n t a t i o n were k e p t a s p u t a t i v e s u p p r e s s o r s , w h i l e t h o s e w h i c h r e s e m b l e d t h e mutant w were r e t a i n e d a s p u t a t i v e e n h a n c e r s . U s i n g t h i s c r i t e r i o n 548 p u t a t i v e s u p p r e s s o r s and 145 p u t a t i v e e n h a n c e r s were r e c o v e r e d from amoung t h e 23,734 F l ma l e s examined ( w m 4 /Y; b I t r l * / + ; +*/+ ). From t h e s e 51 c o n f i r m e d s u p p r e s s o r s a n d 3 c o n f i r m e d e n h a n c e r s were r e c o v e r e d a f t e r e x t e n s i v e r e t e s t i n g . The f r e q u e n c y o f r e c o v e r y o f EMS i n d u c e d d o m i n a n t m u t a t i o n s w h i c h s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n (2.1 x 1 0 " 3 ) i s much h i g h e r t h a n t h e f r e q u e n c y o f i n d u c e d dominant e n h a n c e r s o f v a r i e g a t i o n (1.3 x 1 0 ~ 4 ) . Whether t h i s r e f l e c t s a l o w e r p r o p o r t i o n o f l o c i i n t h e genome c a p a b l e o f e n h a n c i n g v a r i e g a t i o n o r i s an a r t i f a c t o f o u r s c r e e n i n g p r o t o c o l i s u n c l e a r . I t i s i n t e r e s t i n g t o n o t e t h a t t h e f r e q u e n c y o f r e c o v e r y o f s u p p r e s s o r s i n t h i s s t u d y compares f a v o u r a b l y t o t h a t f o u n d by REUTER and WOLFE i n a s t u d y done s i m u l t a n e o u s l y ( 1 9 8 1 ) . The m u t a t i o n s were a s s i g n e d t o p a r t i c u l a r chromosomes b a s e d on t h e i r s e g r e g a t i o n from I n ( 2 L R ) C y O o r In(3LR)TM3 Sb S e r . The 70 FIGURE VII. THE SCREENING PROTOCOL FOR ISOLATION OF DOMINANT MUTATIONS WHICH MODIFY POSITION EFFECT VARIEGATION. 71 EMS X/Y; b It r l / b It r l w m 4 /Y ; b It r l * /+ AND w m 4 /Y ; b It r l */CyO w /w ; +/CyO TRANSFER EVERY 3~5 DAYS FOR 14 DAYS EXAMINE PROGENY FOR MODIFICATION OF w™ 4 VARIEGATION, MATE PUTATIVES, SINGLY, TO m4. m4 n « . , _ _ w /u ;DS6/CyO RESULTS: EXAMINE FOR ENHANCEMENT OR SUPPRESSION OF w PROGENY EXAMINED 23,734 PUTATIVES 548 SUPPRESSORS 145 ENHANCERS RETESTS 51 SUPPRESSORS 17 CHROMOSOME 2 30 CHROMOSOME 3 4 NOT LOCATED 3 ENHANCERS ALL CHROMOSOME 3 m4 72 r e s u l t s o f t h e s e g r e g a t i o n t e s t s r e v e a l e d t h a t a l l 3 e n h a n c e r s map t o chromosome 3 and a l l a r e v i a b l e when homozygous. Thus f a r 17 of t h e s u p p r e s s o r s map t o chromosome 2 and o f t h e r e m a i n i n g 34 s u p p r e s s o r s , 30 map t o t h e t h i r d chromosome and 4 have n o t , a s y e t , been l o c a l i z e d . I n i t i a l l y , a l l t h e s e c o n d chromosome s u p p r e s s o r s and a m a j o r i t y o f t h e t h i r d chromosome s u p p r e s s o r s (22 o u t of 30) were r e c e s s i v e l e t h a l s . However, s u b s e q u e n t r e c o m b i n a t i o n a l a n a l y s i s ( s e e below) has shown n e a r l y a l l t h e t h i r d chromosome s u p p r e s s o r s a r e homozygous v i a b l e ( 2 e x c e p t i o n s — 326 and 328) whereas i n t h e c a s e o f t h e s e c o n d chromosome m u t a t i o n s , w i t h t h e e x c e p t i o n o f 209, t h e l e t h a l i t y and t h e s u p p r e s s o r p h e n o t y p e c o - s e g r e g a t e . I n summary, 35% o f t h e m u t a n t s r e c o v e r e d e x h i b i t e d l e t h a l i t y when homozygous. T h i s i s somewhat l o w e r t h a n t h e f r e q u e n c y o f l e t h a l i t y (94%) a s s o c i a t e d w i t h s u p p r e s s o r s f o u n d by REUTER and WOLFF ( 1 9 8 1 ) . However, t h e p r o t o c o l f o r i s o l a t i o n of s u p p r e s s o r s u s e d by t h e s e w o r k e r s employed an e n h a n c e r o f p o s i t i o n e f f e c t v a r i e g a t i o n and t h u s i t i s p o s s i b l e t h e y s e l e c t e d f o r v e r y s t r o n g s u p p r e s s o r s w h i c h may have e n r i c h e d f o r r e c e s s i v e l e t h a l i t y . However, t h e d i s t r i b u t i o n o f s u p p r e s s o r m u t a t i o n s on t h e s e c o n d and t h i r d chromosomes we o b s e r v e d , 1:2, i s v e r y s i m i l a r t o t h a t f o u n d by REUTER and WOLFF ( 1 9 8 1 ) . S i n c e i t was n o t f e a s i b l e t o u n d e r t a k e an e x t e n s i v e a n a l y s i s o f a l l 54 m u t a t i o n s r e c o v e r e d i n t h e p r e s e n t s t u d y , 5 s e c o n d chromosome s u p p r e s s o r s and 5 t h i r d chromosome s u p p r e s s o r s were s e l e c t e d a t random, and, a l o n g w i t h t h e 3 e n h a n c e r s , s u b j e c t e d t o f u r t h e r a n a l y s i s a s o u t l i n e d i n t h e M a t e r i a l s and 73 Methods . The r e s u l t s of these v a r i o u s a n a l y s e s are p r e s e n t e d below. A l i s t of a l l the muta t ions i s o l a t e d i s g i v e n i n the Appendix A . 2) Mapping and Complementation A) Chromosome 2 Suppressors The r e s u l t s of the mapping experiments i n v o l v i n g the f i v e second chromosome mutat ions are p r e s e n t e d in F i g u r e 8a . I n i t i a l l y the mutants 214, 213, 215, 216, and 209 were chosen f o r t h i s a n a l y s i s . However, 209 proved too v a r i a b l e in phenotype to be r e l i a b l y mapped when o u t c r o s s e d to the m u l t i p l y marked chromosome. Subsequent a n a l y s i s demonstrated that i t was not induced on the b I t r_l chromosome s u g g e s t i n g i t may be a spontaneous m u t a t i o n . A l l f o u r remain ing mutants map to a 3 map u n i t c l u s t e r , g e n e t i c map p o s i t i o n 31 to 34, on the l e f t arm of chromosome 2. In cases where a r e c o m b i n a t i o n • event o c c u r e d in the r e g i o n of a suppres sor m u t a t i o n , the r e s u l t a n t organisms were b a c k c r o s s e d to the o r i g i n a l suppressor s t r a i n to determine whether i t was p o s s i b l e to s epara te the r e c e s s i v e l e t h a l i t y from the suppressor phenotype . At l e a s t 20 recombinant chromosomes were t e s t e d for each s t r a i n . In o n l y one i n s t a n c e , 209, was i t p o s s i b l e to s e p a r a t e the dominant s u p p r e s s o r from the r e c e s s i v e FIGURE VIII. THE MAP POSITIONS OF ENHANCERS AND SELECTED SUPPRESSORS OF POSITION EFFECT VARIEGATION 8 ( A ) 8 ( B ) 3 L 207(32.0) ' 214(32.6) 215(32.9) 213(32.9) 216(34.2) 209(35.4) 206(51.3) b — i Tf t GL Sb I- nw [- P in 2R Yt 326 328 125(53.3) 330(54.7) 327(55.5) 3R 8(c) 3L A106 A131 GL - J © _ Sb A102(49.3) H 76 l e t h a l i t y phenotype . I t i s i n t r i g u i n g tha t a l l four of these randomly chosen s u p p r e s s o r s map to c l u s t e r which g e n e t i c a l l y r e p r e s e n t s l e s s tha t 3% of the t o t a l second chromosome. Whi l e t h i s r e p r e s e n t s a l i m i t e d subset of the t o t a l number of s u p p r e s s o r s induced on the second chromosome, i t s t r o n g l y suggests tha t these l o c i may not be d i s t r i b u t e d randomly. For t h i s reason two a d d i t i o n a l second chromosome m u t a t i o n s , 207 and 206, a l s o chosen at random, were mapped. 207 mapped w i t h i n the c l u s t e r w h i l e 206 mapped to 52 .4 . Thus , 5 of the 6 mutat ions are c l u s t e r e d w i t h i n a 3 map u n i t r e g i o n . Of course the q u e s t i o n a r i s e s , do these s imply r e p r e s e n t a l l e l e s of a h i g h l y mutable gene or are they d i f f e r e n t genes which are c l u s t e r e d ? S i n c e a l l but one of the s u p p r e s s o r s induced on the second chromosome are a l s o r e c e s s i v e l e t h a l s , complementat ion a n a l y s i s c o u l d be performed u s i n g the r e c e s s i v e l e t h a l phenotype as a t e s t for a l l e l i s m . Complementat ion a n a l y s i s was performed.between a l l 5 mutants which map to the c l u s t e r to determine the number of genes . In a d d i t i o n , complementat ion a n a l y s i s was performed between each of these 5 mutat ions and 7 o ther second chromosome s u p p r e s s o r s . The r e s u l t s of t h i s a n a l y s i s are shown in T a b l e 7. The 5 m u t a t i o n s , mapped i n the c l u s t e r , r e p r e s e n t four complementat ion groups based on r e c e s s i v e l e t h a l i t y . The complementat ion p a t t e r n i s a s imple one TABLE 7 COMPLEMENTATION ANALYSIS OF SUPPRESSORS OF VARIEGATION LOCATED ON CHROMOSOME 2 214 213 216 215 209 206 207 204 208 212 210 205 214 + + + + + - + - + 213 + + + - + + - + 216 + + + + + + + - + 215 - + + + + + + + 209 n II + A VIABLE A LETHAL + CROSS CROSS + + + + + 78 w i t h four m u t a t i o n s , 215, 214, 216, and 207 behaving as p o i n t mutants and 213 behaving as a s m a l l d e l e t i o n . The r e s u l t s from the complementat ion a n a l y s i s w i t h the 7 a d d i t i o n a l m u t a t i o n s , which have not been mapped, i n d i c a t e tha t 208 i s a l l e l i c to 214 o n l y , wh i l e 210 i s a l l e l i c to 216, 213, and 214. The complementat ion map ( F i g u r e 9) suggests t h a t there are 4 genes which i n f l u e n c e p o s i t i o n e f f e c t v a r i e g a t i o n w i t h i n the g e n e t i c i n t e r v a l 31-34. Chromosome 3 S u p p r e s s o r s F i g u r e 8b shows the r e s u l t s of mapping the t h i r d chromosome s u p p r e s s o r s . F i v e randomly chosen mutants were mapped a g a i n s t the G l Sb H chromosome. As mentioned p r e v i o u s l y t h i s method p r e c l u d e s the a c c u r a t e l o c a l i z a t i o n of mutat ions s i t u a t e d s u b s t a n t i a l l y to the l e f t of G l or the r i g h t of H . For example , both 326 and 328 map more than 20 map u n i t s to the l e f t of G l . The remain ing 3 m u t a t i o n s , 325, 327, and 330, map to a 2 map u n i t c l u s t e r on 3 L . The l a t t e r f i n d i n g i s i n d i c a t i v e of a c l u s t e r i n g comparable to that observed on the second chromosome. Exper iments conducted subsequent to t h i s s tudy have shown tha t the suppressor mutat ions do indeed f a l l i n t o s e v e r a l d e s c r e t e c l u s t e r s on the t h i r d chromosome. Appendix B i s an updated map of the FIGURE IX. A COMPLEMENTATION MAP OF SUPPRESSORS OF POSITION EFFECT VARIEGATION LOCATED ON CHROMOSOME 2 . 80 213 215 207 210 214 216 209 81 s u p p r e s s o r s on t h e s e c o n d and t h i r d chromosomes ( t a k e n from SINCLAIR e t . a l . , i n p r e s s ) . Twenty of t h e 25 t h u s f a r mapped o c c u p y one o f f o u r c l u s t e r e d g r o u p s ( f o r t h i s s t u d y a c l u s t e r i s d e f i n e d as any g r o u p of m u t a t i o n s w h i c h o c c u p y t h e same 3 map u n i t i n t e r v a l and whose most w i d e l y s e p a r a t e d members have map p o s i t i o n s w i t h o v e r l a p p i n g 95% c o n f i d e n c e i n t e r v a l s ) . The m u t a n t s were c r o s s e d i n a l l p a i r - w i s e c o m b i n a t i o n s ( T a b l e 8) t o t e s t f o r l e t h a l i n t e r a c t i o n s . 326 and 328 a r e r e c e s s i v e l e t h a l s w h i c h f a i l t o complement one a n o t h e r . W h i l e no a t t e m p t was made t o s e p a r a t e t h e l e t h a l from t h e s u p p r e s s o r p h e n o t y p e , i t i s v e r y u n l i k e l y t h a t two i n d e p e n d e n t l y i n d u c e d dominant s u p p r e s s o r s would have s e c o n d s i t e i n d u c e d r e c e s s i v e l e t h a l s w h i c h a r e a l l e l i c . T h e r e f o r e , i t i s r e a s o n a b l e t o assume t h e s e two s u p p r e s s o r s a r e a l l e l e s , however, any c o n c l u s i o n s must a w a i t more e x a c t m a p p i n g . The r e m a i n i n g c o m b i n a t i o n s were v i a b l e a s m i g h t be e x p e c t e d s i n c e 325, 327, and 330 a r e v i a b l e as h o m o z y g o t e s . E n h a n c e r s o f P o s i t i o n E f f e c t V a r i e g a t i o n The r e s u l t s o f mapping t h e e n h a n c e r m u t a t i o n s , f o l l o w i n g t h e same p r o t o c o l as a p p l i e d t o t h e t h i r d chromosome s u p p r e s s o r s , a r e shown i n F i g u r e 8 c . As was TABLE 8 COMPLEMENTATION ANALYSIS OF SUPPRESSORS LOCATED ON CHROMOSOME 3 328 327 325 326 330 328 . + + . + 327 + + + + 325 + + + 326 - + 330 + COMPLEMENTATION ANALYSIS OF SUPPRESSORS LOCATED ON CHROMOSOMES 2 AND 3 214 213 216 215 209 328 + + + + + 327 + + + + + 325 + + + + + 326 + + + + + 330 + + + + + II II = A VIABLE CROSS = A LETHAL CROSS 83 the case wi th two of the s u p p r e s s o r s , the enhancers , A106 and A131, mapped f a r t o the l e f t of G l and t h e r e f o r e c o u l d not be a c c u r a t e l y p o s i t i o n e d . A102 maps to 49.3 on 3R. A l l mutat ions are homozygous v i a b l e . As a homozygote, Al02 i s female s t e r i l e however, as y e t , no attempt has been made to s epara te the female s t e r i l i t y from the enhancer phene. 3) The Response of Other V a r i e g a t i n g Genes The mutat ions were r e c o v e r e d because they e i t h e r suppress or enhance w m 4 v a r i e g a t i o n . O b v i o u s l y , i t i s neces sary to determine whether these mutat ions modify p o s i t i o n e f f e c t v a r i e g a t i o n i n g e n e r a l or a r e s p e c i f i c to the w_^  gene or perhaps in ^ \ the 121(1)" chromosome. S e v e r a l v a r i e g a t i n g genes were chosen which r e p r e s e n t a c r o s s s e c t i o n of the types of p o s i t i o n e f f e c t v a r i e g a t i o n observed i n D . melanogaster (see Chapter One) . These s t r a i n s r e p r e s e n t genes from a l l the major chromosomes jux taposed to h e t e r o c h r o m a t i n from the X and Y chromosomes as w e l l as the autosomes. T h e r e f o r e , a n a l y s i s of them s h ou ld c o n s t i t u t e an adequate t e s t of whether the s u p p r e s s o r s and enhancers a l t e r the e x p r e s s i o n of v a r i e g a t i n g genes i n g e n e r a l . A) Chromosome 2 S u p p r e s s o r s The e f f e c t s of the second chromosome s u p p r e s s o r s on the v a r i e g a t i n g s t r a i n s are shown i n T a b l e 9. Each va lue r e p r e s e n t s the percentage of f u l l e x p r e s s i o n of TABLE 9 THE INTERACTION BETWEEN SUPPRESSORS LOCATED ON CHROMOSOME 2 AND VARIOUS GENES SUBJECT TO POSITION EFFECT VARIEGATION CONTROL 2 1 4 2 1 3 2 1 6 2 1 5 2 0 9 w ? s x s x s x s x s x s x 4 0 . 4 83 5 . 4 8 0 6 . 0 1 2 5 3 . 5 1 0 7 6 . 5 1 0 0 2 . 5 11 1 . 3 1 1 3 2 . 3 1 1 0 3 . 5 1 5 9 5 . 5 1 3 1 7 . 5 1 1 2 5 . 4 . v D e 2 bw ? 15 1 . 8 1 0 7 6 . 5 1 0 3 1 1 . 5 1 4 6 6 . 0 1 0 5 7 . 0 5 7 6 . 5 d 1 9 2 . 2 1 0 6 7 . 5 1 3 1 8 . 0 1 3 2 9 . 5 1 1 1 6 . 5 5 6 9 . 0 S b V * 5 9 3 . 6 88 3 . 1 8 3 3 . 0 8 5 2 . 0 8 0 2 . 5 6 2 4 . 0 a 6 6 3 . 8 91 1 . 6 8 9 2 . 1 93 1 . 9 9 0 1 . 9 7 2 3 . 2 B S _ V « 35 1 . 7 3 0 1 . 4 2 9 1 .1 22 0 . 8 3 2 1 . 6 31 1 . 9 Sr; = STANDARD ERROR 00 85 t h e v a r i e g a t i n g gene, whether i t be f u l l y mutant ( S b v ) or f u l l y w i l d - t y p e ( b w v D e 2 o r w1"4 ) w i t h t h e e x c e p t i o n of B s - V , where t h e v a l u e s r e p r e s e n t t h e s i z e o f t h e a d u l t e y e . The s m a l l e r t h e eye t h e g r e a t e r t h e number of c e l l s i n wh i c h t h e B gene i s e x p r e s s e d ( s e e M a t e r i a l s and M e t h o d s ) . S u p p r e s s o r s s h o u l d d e c r e a s e t h e i n a c t i v a t i o n c a u s e d by p o s i t i o n e f f e c t v a r i e g a t i o n a nd t h u s i n c r e a s e e x p r e s s i o n o f a v a r i e g a t i n g a l l e l e . A g l a n c e a t t h e d a t e i n T a b l e 9 c o n f i r m s t h i s e x p e c t a t i o n . A l l o f t h e m u t a t i o n s s i g n i f i c a n t l y s u p p r e s s e d p o s i t i o n e f f e c t v a r i e g a t i o n . I n most i n s t a n c e s , w*,bw+, and Sb, t h e e x p r e s s i o n o f t h e v a r i e g a t i n g gene was i n c r e a s e d t o , or v e r y n e a r t o , 100%. In a d d i t i o n , v a r i e g a t i o n o f B was s i g n i f i c a n t l y s u p p r e s s e d by a l l t h e m u t a t i o n s . Thus, i n t h e p r e s e n c e o f t h e s u p p r e s s o r s t h e v a r i e g a t i n g a l l e l e i s e x p r e s s e d i n a l l , o r a l m o s t a l l , t h e c e l l s i n w h i c h t h e n o n - v a r i e g a t i n g a l l e l e i s n o r m a l l y e x p r e s s e d . The o n l y e x c e p t i o n i s 209 whose e f f e c t was weak o r in A-v a r i a b l e i n a l l c a s e s e x c e p t w i n b r e d l i n e s . O u t c r o s s i n g t h i s s t r a i n , f o r example, t o map t h e m u t a t i o n , l e d t o s u c h v a r i a b l e e x p r e s s i o n t h a t i t was i m p o s s i b l e t o map by r e c o m b i n a t i o n . I n t e r e s t i n g l y , t h i s m u t a t i o n i s s p o n t a n e o u s r a t h e r t h a n EMS i n d u c e d . B) Chromosome 3 S u p p r e s s o r s S u p p r e s s o r s mapping t o chromosome 3 were a l s o 86 t e s t e d f o r t h e i r e f f e c t on v a r i e g a t i o n of wj_, Sb, bw +, and B . The r e s u l t s of these exper iments are p r e s e n t e d i n T a b l e 10. I t appears from these r e s u l t s t h a t the t h i r d chromosome s u p p r e s s o r s e x h i b i t a h i g h e r degree of h e t e r o g e n e i t y i n t h e i r a b i l i t y to suppress p o s i t i o n e f f e c t v a r i e g a t i o n than d i d those mapping to the second m L\ v chromosome. V a r i e g a t i o n of w and Sb was markedly suppressed by a l l 5 mutants . With the e x c e p t i o n of females b e a r i n g 328 or 326 t h i s s u p p r e s s i o n r e s u l t e d i n almost f u l l e x p r e s s i o n of w*_ and Sb . P o s i t i o n e f f e c t v a r i e g a t i o n of b w v D e 2 was s i g n i f i c a n t l y suppressed i n a l l cases but the l e v e l s of gene e x p r e s s i o n v a r i e d s u b s t a n t i a l l y from m u t a t i o n to m u t a t i o n . 325 s t r o n g l y suppressed b w v D e 2 i n both males (93%) and females (74%) however the o ther m u t a t i o n s caused i n t e r m e d i a t e l e v e l s of gene e x p r e s s i o n i n both sexes . The e f f e c t of the s u p p r e s s o r s on males b e a r i n g the B S ~ V » Y was ex tremely v a r i a b l e . 327 and 330 caused s i g n i f i c a n t s u p p r e s s i o n of B s v w h i l e 325 and 328 had l i t t l e or no e f f e c t . In c o n t r a s t , 326 s i g n i f i c a n t l y reduced the e x p r e s s i o n of B s - V i n o p p o s i t i o n to i t s e f f e c t s on a l l o ther v a r i e g a t i n g a l l e l e s t e s t e d . Chromosome 3 Enhancers T A B L E 1 0 THE INTERACTION BETWEEN SUPPRESSORS LOCATED ON CHROMOSOME 3 AND VARIOUS GENES SUBJECT TO POSITION EFFECT VARIEGATION CONTROL 3 2 6 3 2 8 3 2 5 3 2 7 3 3 0 s x s x s x s x s x s x w ? 4 0 . 4 1 2 6 1 . 8 7 4 6 . 7 1 2 8 1 . 9 1 2 5 3 . 6 1 2 5 3 . 1 a 11 1 . 3 1 1 6 3 . 6 8 7 5 . 4 131 6 . 7 1 1 3 7 . 2 1 2 0 3 . 1 bw v D e 2 ? 15 1 . 8 55 2 . 0 4 8 5 . 9 74 7 . 5 31 6 . 0 3 9 2 . 0 <f 1 9 2 . 2 37 5 . 5 5 0 4 . 0 93 8 . 3 2 9 4 5 5 . 0 SbV ? 5 9 3 . 6 72 5 . 2 7 2 3 . 0 96 1 . 0 8 9 2 . 0 91 2 . 0 <f 6 6 3 . 8 91 2 . 1 9 3 2 . 0 9 9 0 . 4 9 4 1 . 4 9 4 2 . 5 Bs-v a 35 1 . 7 51 3 . 8 3 8 2 . 4 33 0 . 6 2 3 1 .1 2 5 1 . 3 S^  = STANDARD ERROR 00 88 M u t a t i o n s which enhance p o s i t i o n e f f e c t v a r i e g a t i o n r e s u l t i n a r e d u c t i o n i n the number of c e l l s w i t h i n a t i s s u e i n which a v a r i e g a t i n g gene i s e x p r e s s e d . Three mutat ions which s i g n i f i c a n t l y decreased e x p r e s s i o n of the wj_ gene i n I n ( l ) w m 4 were r e c o v e r e d and these were t e s t e d for t h e i r a b i l i t y to enhance p o s i t i o n e f f e c t v a r i e g a t i o n g e n e r a l l y . Only v a r i e g a t i o n of w m 4 and B s - V , both X - l i n k e d genes , was u n i f o r m l y enhanced by these mutat ions (Tab le 11) . The autosomal v a r i e g a t i n g genes v a r i e d w i d e l y in t h e i r response to the enhancer m u t a t i o n s . A102 enhanced S_bv i n both females and males but had no e f f e c t on b w v D e 2 i n e i t h e r sex . The o p p o s i t e r e s u l t was observed wi th A131; the e x p r e s s i o n of S b v was u n a f f e c t e d whereas tha t of b w v D e 2 was enhanced weakly i n females and o n l y m a r g i n a l l y i n males . A106 enhanced the e x p r e s s i o n of b w v D e 2 i n both males and females and t h a t V . v of Sb i n males but had no e f f e c t on Sb i n f emales . No c l e a r p a t t e r n emerges from these r e s u l t s . However i t i s of some i n t e r e s t to note t h a t the m u t a t i o n s appear to be most e f f e c t i v e a t enhancing p o s i t i o n e f f e c t v a r i e g a t i o n of genes jux taposed to s e x - l i n k e d h e t e r o c h r o m a t i n . 4) S u p p r e s s o r s Of P o s i t i o n E f f e c t V a r i e g a t i o n And X / 0 Males P o s i t i o n e f f e c t v a r i e g a t i o n appears to be v e r y s e n s i t i v e to the t o t a l amount of h e t e r o c h r o m a t i n present i n the genome. The a d d i t i o n of supernumery y chromosomes r e s u l t s i n s t r o n g s u p p r e s s i o n of p o s i t i o n e f f e c t v a r i e g a t i o n (GOWAN and GAY, 1934) TABLE 11 THE INTERACTION BETWEEN ENHANCERS LOCATED ON CHROMOSOME 3 AND VARIOUS GENES SUBJECT TO POSITION EFFECT VARIEGATION CONTROL s x m A w ? 4 0 . 4 tf 11 1 . 3 . vDe2 bw 9 1 5 1 . 8 a 1 9 2 . 2 S b V 9 5 9 3 . 6 a 6 6 3 . 8 B S _ V , 4 5 2 . 2 A 1 0 6 A 1 3 1 s x 1 0 . 2 1 2 0 . 4 2 12 0 . 4 11 1 2 0 . 9 1 5 61 4 . 6 6 3 4 8 5 . 4 6 4 8 0 4 . 7 81 A 1 0 2 s x s x 0 . 2 1 0 . 2 0 . 4 2 0 . 4 1 . 3 14 1 . 3 1 . 3 2 0 0 . 9 4 . 3 3 9 2 . 4 4 . 3 3 2 2 . 2 3 . 6 8 0 3 . 3 S£ = STANDARD ERROR 90 w h i l e r e m o v a l o f t h i s chromosome o r i n d e e d , d e l e t i o n o f e i t h e r X - l i n k e d (NOUJDIN, 1938) o r a u t o s o m a l h e t e r o c h r o m a t i n (SCHULTZ, 1938; BROSSEAU, 1960; LINDSLEY e t a l . , 1960) r e s u l t s i n s t r o n g enhancement o f p o s i t i o n e f f e c t v a r i e g a t i o n . T h u s , i t was o f c o n s i d e r a b l e i n t e r e s t t o d e t e r m i n e whether t h e m u t a t i o n s we have i s o l a t e d w h i c h s t r o n g l y s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n were a l s o s e n s i t i v e t o v a r i a t i o n s i n t h e h e t e r o c h r o m a t i c • • • • ni A-c o n s t i t u t i o n o f t h e f l i e s . A c c o r d i n g l y , w m a l e s h e t e r o z y g o u s f o r one o f t h e m u t a t i o n s w h i c h s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n were c r o s s e d i n d i v i d u a l l y t o C(1)RMpn/0 v i r g i n f e m a l e s and t h e w m 4/0; s u p p r e s s o r / + F1 m a l e s r e c o v e r e d and a n a l y z e d . E x a m i n a t i o n o f t h e r e s u l t s of t h e s e c r o s s e s ( T a b l e 12) r e v e a l s t h a t t h e d e g r e e o f s u p p r e s s i o n o f v a r i e g a t i o n e l i c i t e d by t h e s e m u t a t i o n s i s e x t r e m e l y s e n s i t i v e t o Y chromosome a n e u p l o i d y . In many c a s e s t h e l e v e l o f gene e x p r e s s i o n o b s e r v e d i n f l i e s c a r r y i n g a s u p p r e s s o r m u t a t i o n was r e d u c e d t o o r v e r y n e a r t o t h e l e v e l s o b s e r v e d i n c o n t r o l f l i e s . W i t h t h e e x c e p t i o n o f 214 t h e e f f e c t s of t h e s e c o n d chromosome s u p p r e s s o r s a r e a l m o s t c o m p l e t e l y a b o l i s h e d i n m a l e s w h i c h l a c k a Y chromosome. On t h e o t h e r hand, t h e r e s u l t s o b s e r v e d f o r t h e t h i r d chromosome s u p p r e s s o r s were somewhat more h e t e r o g e n e o u s . I n X/0 m a l e s t h e e f f e c t s of 326 and 328 were a l m o s t c o m p l e t e l y a b o l i s h e d w h i l e 330 and 327 o n l y weakly s u p p r e s s e d w m 4 v a r i e g a t i o n . 325 m a r k e d l y s u p p r e s s e d p o s i t i o n e f f e c t v a r i e g a t i o n o f wj_ and t h u s a p p e a r s t o be t h e o n l y m u t a t i o n r e l a t i v e l y i n s e n s i t i v e t o t h e l o s s o f Y chromosome TABLE 12 THE EFFECT OF SUPPRESSORS ON VARIEGATION ( A ) CHROMOSOME 2 O F w m 4 I N X/0 M A L E S GENOTYPE w m 4/Y w m 4/0 S X +/+ CONTROL 11 1.3 5 214/+ 113* 2.3 10 213/+ 110* 3.5 4 216/+ 159* 5.5 8 215/+ 131* 7.5 5 209/+ 112* 5.4 4 ( B ) C H R O M O S O M E 3 GENOTYPE w m V Y w m V 0 s x +/+ CONTROL 11 1.3 5 328/+ 87* 5.4 4 326/+ 116* 3.6 5 325/+ 131* 6.7 41* 327/+ 113* 7.2 12* 330/+ 120* 3.1 27* 'SIGNIFICANTLY DIFFERENT FROM CONTROL Sr, = STANDARD ERROR VALUES ARE PERCENTAGE OF WILD-TYPE PIGMENT LEVELS 92 h e t e r o c h r o m a t i n . T h e s e r e s u l t s a r e i n c o n t r a s t t o t h o s e r e p o r t e d by REUTER and WOLFF ( 1 9 8 1 ) . They r e p o r t t h a t t h e m u t a t i o n s t h e y have i s o l a t e d w h i c h s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n a r e dominant t o t h e e f f e c t s o f Y h e t e r o c h r o m a t i n . T h i s may be a c o n s e q u e n c e of t h e i r s c r e e n i n g p r o t o c o l w h i c h employed a s t r o n g e n h a n c e r o f w m 4 and t h u s may have s e l e c t e d f o r a s p e c i f i c s u b s e t of s u p p r e s s o r m u t a t i o n s p r e s e n t i n t h e genome. 5) T e m p e r a t u r e Numerous s t u d i e s have shown t h a t t h e e x t e n t o f v a r i e g a t i o n o b s e r v e d i n p o s i t i o n e f f e c t v a r i e g a t i o n c a n be m o d i f i e d by t h e t e m p e r a t u r e a t w h i c h d e v e l o p m e n t o c c u r s (GOWAN and GAY, 1934; HARTMANN-GOLDSTEIN, 1967; r e v i e w SPOFFORD, 1976). G e n e r a l l y t h e h i g h e r t h e t e m p e r a t u r e a t w h i c h a v a r i e g a t i n g s t r a i n i s r e a r e d t h e g r e a t e r t h e p r o p o r t i o n o f c e l l s w h i c h e x p r e s s a v a r i e g a t i n g gene, t h a t i s , h i g h t e m p e r a t u r e s u p p r e s s e s v a r i e g a t i o n . I t was o f i n t e r e s t t o d e t e r m i n e what, i f any, i n t e r a c t i o n o c c u r s between t e m p e r a t u r e and t h e domi n a n t s u p p r e s s o r s o f v a r i e g a t i o n . I f t e m p e r a t u r e a l s o m o d u l a t e s t h e e f f e c t o f some o r a l l o f t h e s u p p r e s s o r s t h e n i t may be p o s s i b l e t o use t e m p e r a t u r e s h i f t s t u d i e s t o d e t e r m i n e t h e t i m e d u r i n g d e v e l o p m e n t when t h e s e m u t a t i o n s a c t . S i n c e t h e e f f e c t s o f t e m p e r a t u r e on w_^  gene v a r i e g a t i o n a r e w e l l c h a r a c t e r i z e d i t was d e c i d e d t h a t t h e combined e f f e c t s o f t e m p e r a t u r e and t h e s u p p r e s s o r m u t a t i o n s on w m 4 v a r i e g a t i o n would be examined a s f o l l o w s . 93 P r e b l a s t o d e r m embryos o f t h e t e n mutant s t r a i n s w h i c h s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n and b e a r i n g t h e w chromosome ( e i t h e r w m 4 ; s u p p r e s s o r / b a l a n c e r o r s u p p r e s s o r / s u p p r e s s o r d e p e n d i n g on whether t h e s u p p r e s s o r i s a r e c e s s i v e l e t h a l o r n o t ) were c o l l e c t e d by methods d e s c r i b e i n C h a p t e r One. Two h o u r s a f t e r d e p o s i t i o n t h e eggs were s h i f t e d t o e i t h e r 1 7 ° , 2 2 ° , o r 29°C. and a l l o w e d t o d e v e l o p u n t i l e c l o s i o n . The a d u l t s were c o l l e c t e d , s h i f t e d t o 2 2 ° C , a g e d 7-10 d a y s , and t h e p i g m e n t l e v e l s a s s a y e d . The r e s u l t s a r e shown i n T a b l e 13. In most c a s e s , t e m p e r a t u r e d i d n o t have a marked e f f e c t on l e v e l s o f wj_ gene e x p r e s s i o n e v e n a t low d e v e l o p m e n t a l t e m p e r a t u r e s . E x p r e s s i o n of t h e wj_ was somewhat r e d u c e d i n 214/CyO and 213/CyO f e m a l e s a t 17°C. ( T a b l e 13a). 328/TM3 ( T a b l e 13b) showed s i g n i f i c a n t l y r e d u c e d l e v e l o f gene e x p r e s s i o n a t 2 9 ° C . i n b o t h m a l e s and f e m a l e s . At 2 9 ° C . f l i e s b e a r i n g t h e 325/325 m u t a t i o n d i d n o t s u r v i v e . I t has y e t t o be d e t e r m i n e d whether t h i s t e m p e r a t u r e - s e n s i t i v e l e t h a l i t y i s a s s o c i a t e d w i t h t h e s u p p r e s s o r m u t a t i o n o r n o t . The f l i e s a l l d i e d v e r y e a r l y d u r i n g e m b r y o g e n e s i s w h i c h may be s i g n i f i c a n t s i n c e one o f t h e m a j o r TSPs f o r w_^  v a r i e g a t i o n i s a l s o e a r l y , a r o u n d t h e t i m e o f b l a s t o d e r m f o r m a t i o n (SPOFFORD, 1976). E x a m i n a t i o n o f T a b l e 13 r e v e a l s t h a t i n some c a s e s , n o t a b l y 216/CyO m a l e s a t 2 2 ° C . and 214/CyO, 213/CyO, 215/CyO, 216/CyO, and 326/TM3 males a t 1 8 ° C , t h e l e v e l s o f p i g m e n t f o u n d i n t h e e y e s i s much h i g h e r t h a n t h e c o n t r o l , w i l d - t y p e s t r a i n , OR-R. T h i s i s n o t an i s o l a t e d i n c i d e n t , nor i s i t an e f f e c t o f t e m p e r a t u r e . The amount o f pigment i n b w v D e 2 s t r a i n s TABLE 13 THE EFFECT OF DEVELOPMENTAL TEMPERATURE ON SUPPRESSORS OF VARIEGATION IN STRAINS BEARING I N O ) w m 4 (A) CHROMOSOME 2 CONTROL 214/CyO 213/c yo 216/CyO 215/CyO 209/CyO TEMP . s x s x s x s x s x s x 17° 0 4 0.4 69 11.2 50 4.5 117 7.2 84 5.8 102 7.6 a 8 0.9 158 4.0 143 7.2 181 7.6 166 8.5 126 2.7 22° ? 12 G.8 83 5.4 80 5.4 125 2.7 107 5.8 100 2.2 <f 24 2.5 113 1.8 110 5.1 159 4.9 131 6.7 112 5.4 29° 9 20 2.7 106 12.7 119 2.8 133 14.1 119 3.5 120 5.7 cf 16 2.2 107 4.9 96 6.4 111 14.1 99 4.2 108 2.1 S^  = STANDARD ERROR VALUES ARE PERCENTAGE OF WILD-TYPE PIGMENT LEVELS TABLE 13 C O N T I N U E D ( B ) C H R O M O S O M E 3 C O N T R O L 3 2 8 / T M 3 3 2 6 / T M 3 325/325 327/327 330/330 s x s x s x s x s x s x 17° 4 0,4 72 3.6 117 11.0 115 3.1 120 5.4 106 1.8 <f 8 0.9 116 5.8 142 4.0 123 2.7 127 4.9 118 3.1 22 9 12 0.8 74 6.7 12b 1.8 128 1.8 125 3.6 125 3.1 a 2 4 2.5 87 5.4 116 3.6 131 6.7 113 7.2 120 3.1 29 9 20 2.7 56 1.8 103 1.8 L E T H A L 109 3.1 114 2.2 d 16 2.2 40 3.6 82 4.0 L E T H A L 111 3.6 122 3.1 S ^ = S T A N D A R D E R R O R V A L U E S A R E P E R C E N T A G E O F W I L D - T Y P E P I G M E N T L E V E L S 96 h e t e r o z y g o u s f o r the second chromosome s u p p r e s s o r , 216, i s i n c r e a s e d t o l e v e l s w e l l above t h a t o b s e r v e d i n t h e OR-R c o n t r o l s (Table 9 ) . T h i s s u g gests t h a t not o n l y a r e the v a r i e g a t i n g genes a c t i v e l y t r a n s c r i b e d i n most c e l l s but a l s o t h a t the r a t e or d u r a t i o n of t r a n s c r i p t i o n i s a l s o i n c r e a s e d . I t has been r e p o r t e d t h a t a n o t h e r s u p p r e s s o r of p o s i t i o n e f f e c t v a r i e g a t i o n , Y b b s u - v a r 5 (CLARK e t . a l . , 1977) i n c r e a s e s the o v e r a l l t r a n s c r i p t i o n r a t e i n some s t r a i n s of f l i e s . E l e v a t e d t r a n s c r i p t i o n r a t e s might be e x p e c t e d t o r e s u l t i n i n c r e a s e d l e v e l s of pigment d e p o s i t i o n and t h u s p r o v i d e s one p o s s i b l e e x p l a n a t i o n f o r the a b n o r m a l l y h i g h l e v e l s of pigment o b s e r v e d . 97 DISCUSSION T h i s s tudy was undertaken to s y s t e m a t i c a l l y i s o l a t e and c h a r a c t e r i z e dominant mutat ions which a l t e r the mosaic phenotype a s s o c i a t e d wi th p o s i t i o n e f f e c t v a r i e g a t i o n . In t o t a l , 54 mutat ions have been i s o l a t e d ; 51 of which suppress and 3 of which enhance v a r i e g a t i o n of the wj_ gene i n I_n(_1.) w m 4 . S i n c e a d e t a i l e d study of a l l the muta t ions was i m p r a c t i c a l an e x t e n s i v e c h a r a c t e r i z a t i o n was performed on a subset of the t o t a l . These i n c l u d e 5 randomly chosen s u p p r e s s o r s from each of the second and t h i r d chromosomes and a l l 3 e n h a n c e r s . The g e n e t i c map p o s i t i o n of each was de termined and the e f f e c t s of the s u p p r e s s o r s and enhancers on a v a r i e t y of l o c i s u b j e c t to p o s i t i o n e f f e c t v a r i e g a t i o n was t e s t e d . F i n a l l y , the i n t e r a c t i o n of the s u p p r e s s o r s w i t h o ther w e l l c h a r a c t e r i z e d m o d i f i e r s of p o s i t i o n e f f e c t v a r i e g a t i o n was examined. Mapping of a random sample of suppres sor muta t ions r e v e a l e d t h a t these genes are not randomly d i s t r i b u t e d throughout the genome but appear to be c l u s t e r e d i n s p e c i f i c r e g i o n s on 2L (31-34, F i g u r e 8a) and 3R (53-55, F i g u r e 8 b ) . T h i s c l u s t e r i n g of muta t ions c o u l d r e p r e s e n t s e v e r a l d i f f e r e n t genes or one h i g h l y mutable l o c u s a t each s i t e which has s e v e r a l a l l e l e s . Complementat ion a n a l y s i s based on the r e c e s s i v e l e t h a l i t y of the second chromosome s u p p r e s s o r s i n d i c a t e s tha t t h e r e i s a t l e a s t 4 genes i n the c l u s t e r on 2 L . HENIKOFF (1979) r e p o r t e d tha t at l e a s t 2 l o c i which suppress p o s i t i o n e f f e c t v a r i e g a t i o n are l o c a t e d very c l o s e to the c l u s t e r we have i d e n t i f i e d on 3R. The 98 t h i r d chromosome s u p p r e s s o r s we have i s o l a t e d may be a l l e l e s o f t h e s e p r e v i o u s l y i d e n t i f i e d l o c i o r t h e y may r e p r e s e n t newly i d e n t i f i e d l o c i w h i c h a r e p a r t o f a l a r g e r c l u s t e r of genes w h i c h i n f l u e n c e v a r i e g a t i o n . The r e s u l t s o f t h e p r e s e n t s t u d y i n c o n j u n c t i o n w i t h f u r t h e r mapping e x p e r i m e n t s ( A p p e n d i x B) p r o v i d e c o m p e l l i n g e v i d e n c e t h a t genes w h i c h s u p p r e s s p o s i t i o n e f f e c t v a r i e g a t i o n a r e c l u s t e r e d i n a few s p e c i f i c r e g i o n s . T h i s c l u s t e r i n g o f genes may i n d i c a t e t h e y have r e l a t e d f u n c t i o n s . I t h a s been r e p o r t e d t h a t t h e r e i s a p o s i t i v e c o r r e l a t i o n between t h e e f f e c t s o f s u p p r e s s o r a n d e n h a n c e r m u t a t i o n s and a l t e r a t i o n s i n c h r o m a t i n s t r u c t u r e (REUTER e t . a _ l . , 1982). M u t a t i o n s w h i c h s u p p r e s s v a r i e g a t i o n t e n d t o r e d u c e t h e s p r e a d o f h e t e r o c h r o m a t i n a t t h e v a r i e g a t i n g s i t e w h i l e e n h a n c e r s do t h e o p p o s i t e . T h i s s u g g e s t s t h a t s u c h m u t a t i o n s , e i t h e r d i r e c t l y o r i n d i r e c t l y , a f f e c t chromosome s t r u c t u r e . One i n t r i g u i n g p o s s i b i l i t y i s t h e s e m u t a t i o n s r e p r e s e n t l e s i o n s i n g e n e s w h i c h encode s t r u c t u r a l components o f c h r o m a t i n w h i c h a r e c l u s t e r e d , p e r h a p s t o f a c i l i t a t e t h e i r r e g u l a t i o n . O n l y 3 m u t a t i o n s were r e c o v e r e d w h i c h e n h a n c e d v a r i e g a t i o n o f w m 4 . T h e i r low f r e q u e n c y (1.3 x 10~ 4) r e l a t i v e t o t h e s u p p r e s s o r s (2.1 x 10 ) may be a r e f l e c t i o n of t h e i r r a r i t y i n t h e genome, t h e i r r e l a t i v e r e f r a c t o r i n e s s t o EMS i n d u c e d m u t a g e n e s i s , o r an a r t i f a c t o f o u r s c r e e n i n g p r o t o c o l . I f , however, p o s i t i o n e f f e c t v a r i e g a t i o n r e s u l t s f r o m t h e p a c k a g i n g o f n o r m a l l y e u c h r o m a t i c r e g i o n s o f t h e chromosome i n t o h e t e r o c h r o m a t i n t h e n one m i g h t e x p e c t t o f i n d v e r y few m u t a t i o n s 99 which would promote th i s process. By the same l o g i c , mutations which disrupt the formation of a complex organization such as heterochromatin might be more readily isolated. Since the mutations were isola t e d as suppressors or enhancers of w ™ 4 , i t was necessary to determine whether they were general modifiers of position effect variegation or sp e c i f i c to the w_^  gene or perhaps the I_n(l)w m 4 chromosome. We monitored t h e i r effects on 3 other variegating l o c i which represent a cross-section of the types of position effect variegation found in D. melanogaster. Sb v and bw v D e 2 are autosomal genes abutted to the centromeric heterochromatin of chromosome 2 and B s ~ v i s an X-linked gene juxtaposed to the heterochromatin of the Y chromosome. The suppressors mapping to chromosome 2 uniformly suppressed position effect variegation of a l l the variegating genes. In most cases the l e v e l of variegating gene expression caused by these mutations was increased to almost 100%. Sim i l a r l y , the th i r d chromosome suppressors a l l strongly suppressed w m 4 and Sb v and moderately suppressed bw v D e 2 . Position e f f e c t variegation of B s - V was only suppressed by 327 and 330 while 326 s i g n i f i c a n t l y reduced expression of the B s _ v a l l e l e . Thus, with only a few exceptions, a l l the suppressor mutations appear to act on position effect variegation in general rather than on the expression of s p e c i f i c genes. The main exception was B s - V which d i f f e r e d markedly in i t s response to the thi r d chromosome suppressors. Since t h i s gene i s the only one associated with the Y heterochromatin i t may be 100 s — v tha t B J_s anomalous response i n d i c a t e s some fundamental d i f f e r e n c e between Y h e t e r o c h r o m a t i n and the remainder of the genomic h e t e r o c h r o m a t i n . A l t e r n a t i v e l y , s i n c e the B mutat ion i s a tandem d u p l i c a t i o n of a gene s u b j e c t of c i s - t r a n s p o s i t i o n -e f f e c t s , i s non-autonomous over s h o r t d i s t a n c e s , and i s s —v s e n s i t i v e to e n v i r o n m e n t a l m o d i f i c a t i o n s perhaps B 's anomalous response i s due to v a g a r i e s of the B mutat ions i t s e l f . S i m i l a r l y the m u t a t i o n s which enhanced p o s i t i o n e f f e c t v a r i e g a t i o n were t e s t e d f o r t h e i r a b i l i t y to enhance p o s i t i o n e f f e c t v a r i e g a t i o n in g e n e r a l . Only p o s i t i o n e f f e c t v a r i e g a t i o n m4 s —v of w and B were u n i f o r m l y enhanced by a l l the m u t a t i o n s . vD e 2 v Enhancement of bw and Sb was v a r i a b l e and i n some cases sex s p e c i f i c . T h u s , the o n l y v a r i e g a t o r s u n i f o r m l y a f f e c t e d were X -l i n k e d genes a s s o c i a t e d w i t h s e x - l i n k e d h e t e r o c h r o m a t i n ( w m 4 with X h e t e r o c h r o m a t i n and B w i t h Y h e t e r o c h r o m a t i n ) . No c l e a r p a t t e r n of enhancement of autosomal genes i s e v i d e n t . However, b e f o r e any c o n c l u s i o n s can be drawn the e f f e c t of the enhancers must be t e s t e d on a l a r g e r sample of v a r i e g a t i n g genes. The l e v e l of v a r i e g a t i o n observed i n an organism appears to be very dependent on i t s h e t e r o c h r o m a t i c c o n s t i t u t i o n . For example, i n c r e a s i n g the amount of h e t e r o c h r o m a t i n w i t h supernumery Y chromosomes r e s u l t s i n s t r o n g s u p p r e s s i o n of v a r i e g a t i o n (GOWAN and GAY, 1934) w h i l e r e d u c i n g the amount of h e t e r o c h r o m a t i n enhances i t (NOUJDIN, 1938; SCHULTZ, 1936; BROSSEAU, 1960; LINDSLEY e t . a l . , 1960). I t was t h e r e f o r e of i n t e r e s t to determine whether the s u p p r e s s o r s are a l s o dependent 101 on the h e t e r o c h r o m a t i c content of an organism or whether v a r i e g a t i o n would be suppressed r e g a r d l e s s of the h e t e r o c h r o m a t i c c o n s t i t u t i o n . T h i s was t e s t e d by g e n e r a t i n g w m 4 / 0 males he terozygous for the 10 suppressor m u t a t i o n s . In almost a l l cases the l e v e l s of wj_ gene e x p r e s s i o n was reduced to or very near to c o n t r o l w m 4 / 0 l e v e l s (Table 12) . Only 327 and 325 c o n t i n u e d to evoke s t r o n g s u p p r e s s i o n of w m 4 v a r i e g a t i o n in X / 0 m a l e s . Thus , i n almost a l l c a s e s , the e f f e c t of the s u p p r e s s o r mutants i s dependent on the h e t e r o c h r o m a t i c c o n s t i t u t i o n of the f l y . One p o s s i b l e e x p l a n a t i o n of t h i s dependence i s tha t the s u p p r e s s o r s r e p r e s e n t d o s e - s e n s i t i v e l o c i i n v o l v e d i n the f o r m a t i o n of h e t e r o c h r o m a t i n . However, when one removes the Y chromosome, r e p r e s e n t i n g a p p r o x i m a t e l y 25% of the c o n s t i t u t i v e h e t e r o c h r o m a t i n , the l o s s of f u n c t i o n of the s u p p r e s s o r s i s a m e l i o r a t e d and they no l onger have a d e t e c t a b l e e f f e c t on v a r i e g a t i o n . A knowledge of the p e r i o d d u r i n g development when mutat ions which modify p o s i t i o n e f f e c t v a r i e g a t i o n ac t might p r o v i d e c l u e s to the m o l e c u l a r na ture of these mutat ions and thus p r o v i d e i n s i g h t i n t o the m o l e c u l a r b a s i s u n d e r l y i n g p o s i t i o n e f f e c t v a r i e g a t i o n . One method employed to determine the deve lopmenta l p e r i o d of gene a c t i v i t y i s to s creen for temperature s e n s i t i v e ( t s ) m u t a t i o n s . By s e q u e n t i a l l y s h i f t i n g the t s mutants between r e s t r i c t i v e and p e r m i s s i v e temperatures the deve lopmenta l p e r i o d ( s ) d u r i n g which a gene, or i t s p r o d u c t , a r e a c t i v e can be de termined (reviewed by SUZUKI e t . al., 1976). In the hope t h a t some of the mutat ions we have r e c o v e r e d are t s and thus 1 02 amenable to the above a n a l y s i s , we t e s t e d the e f f e c t s of temperature on the 10 l i n e s of suppres sor muta t ions we have been c h a r a c t e r i z i n g . Most of the mutat ions are r e l a t i v e l y i n s e n s i t i v e to the e f f e c t s of temperature (Table 13) . Two mutants , 328 and 325 (Tab le 13b), do, show marked s e n s i t i v i t y to t empera ture . Pigment l e v e l s in 328 are very much reduced at 2 9 ° C . and f l i e s b e a r i n g the 325 chromosome d i e very e a r l y i n development , p r o b a b l y p r i o r to g a s t r u l a t i o n , a t 2 9 ° C . I t has ye t to be demonstrated t h a t the t s l e t h a l i t y a s s o c i a t e d w i t h 325 i s at the same s i t e as the suppressor m u t a t i o n , however i f t h i s i s the case then both 328 and 325 would be good c a n d i d a t e s f o r temperature s h i f t a n a l y s i s . The present s tudy concerns c h a r a c t e r i z a t i o n of a r e l a t i v e l y s m a l l number of the the t o t a l mutat ions i s o l a t e d which modify p o s i t i o n e f f e c t v a r i e g a t i o n and t h e r e f o r e i t would be premature to form any c o n c l u s i o n s . However, the i d e n t i f i c a t i o n of major chromosomal c l u s t e r s of suppressor genes a f f o r d s an e x c e l l e n t o p p o r t u n i t y for a m u l t i f a c e t e d approach to the f u n c t i o n a l d i s s e c t i o n of these l o c i . I n t e n s i v e g e n e t i c a n a l y s i s s h o u l d e l u c i d a t e both the o r g a n i z a t i o n and the i n t e g r i t y of the c l u s t e r s , as w e l l as y i e l d a wide spectrum of mutants , i n c l u d i n g c o n d i t i o n a l a l l e l e s . U s i n g somatic r e c o m b i n a t i o n and c o n d i t i o n a l m u t a t i o n s , we s h o u l d be a b l e to d e l i n e a t e both the time of s u p p r e s s o r gene t r a n s c r i p t i o n and the t ime at which the p r o d u c t f u n c t i o n s . In a d d i t i o n , r e c e n t advances i n the s tudy of D r o s o p h i l a chromosomal p r o t e i n s (HSIEH and BRUTLAG, 1979; RODRIGUEZ-ALFAGEME e t . a l . , 1980; LEVINGER and VARSHAVSKY, 1 03 1982a, b) o f f e r the p o t e n t i a l f or the b i o c h e m i c a l c h a r a c t e r i z a t i o n of s p e c i f i c enhancer and suppressor gene p r o d u c t s . 104 SUMMARY An u n d e r s t a n d i n g o f p o s i t i o n e f f e c t v a r i e g a t i o n a t t h e m o l e c u l a r l e v e l w i l l u n d o u b t e d l y p r o v i d e i n f o r m a t i o n r e g a r d i n g t h e r e l a t i o n s h i p between c h r o m a t i n s t r u c t u r e and gene e x p r e s s i o n . I t i s l i k e l y t h a t v a r i e g a t i o n r e s u l t s f r o m t h e s p r e a d o f h e t e r o c h r o m a t i n , t h e r e f o r e an u n d e r s t a n d i n g o f f a c t o r s w h i c h e n h a n c e o r s u p p r e s s t h i s s p r e a d s h o u l d g i v e us c l u e s r e g a r d i n g t h e s t r u c t u r e and f u n c t i o n o f h e t e r o c h r o m a t i n . T h i s s t u d y came a t t h i s p r o b l e m f r o m two d i r e c t i o n s . The f i r s t was t o t e s t t h e e f f e c t s o f e x p o s u r e t o c h e m i c a l s known t o c a u s e m o d i f i c a t i o n o f t h e h i s t o n e p r o t e i n s , b u t y r a t e and p r o p i o n a t e (RIGGS e t . a l . , 1977; SEALY and CHALKLEY, 1978), on p o s i t i o n e f f e c t v a r i e g a t i o n . S i n c e h i s t o n e s a r e t h e f u n d a m e n t a l b u i l d i n g b l o c k s o f c h r o m a t i n and h i s t o n e d e f i c i e n c e s were known t o m a r k e d l y a f f e c t v a r i e g a t i o n (MOORE e_t. a l . , 1979; MOORE, 1980) i t seems l i k e l y t h e y a r e i n v o l v e d i n t h e v a r i e g a t i n g p r o c e s s . We f o u n d t h a t e x p o s u r e o f d e v e l o p i n g f l i e s t o low c o n c e n t r a t i o n s o f e i t h e r c h e m i c a l r e s u l t e d i n s u p p r e s s i o n o f v a r i e g a t i o n (MOTTUS e t . a l . , 1980) s u g g e s t i n g e x p o s u r e t o t h e s e c h e m i c a l s r e s u l t s i n a d e c r e a s e i n t h e s p r e a d o f h e t e r o c h r o m a t i n . S i n c e t h e s e c h e m i c a l s a r e known t o i n h i b i t t h e h i s t o n e d e a c e t y l a s e enzymes (C AND I DO e t . a _ l . , 1978; MOTTUS e t . a l . , 1980) a s i m p l e e x p l a n a t i o n o f t h e o b s e r v e d r e s u l t s i s t h a t e x p o s u r e t o e i t h e r c h e m i c a l r e s u l t s i n h y p e r a c e t y l a t i o n o f h i s t o n e s . S i n c e h y p e r a c e t y l a t e d h i s t o n e s have an a l t e r e d a f f i n i t y f o r DNA t h i s c o u l d r e d u c e t h e s p r e a d o f h e t e r o c h r o m a t r i n r e s u l t i n g i n s u p p r e s s i o n o f v a r i e g a t i o n . 105 An a n a l y s i s of the time d u r i n g development when exposure to b u t y r a t e or p r o p i o n a t e i s most e f f e c t i v e suggests tha t these c h e m i c a l s o n l y a c t in d i v i d i n g c e l l s to suppress v a r i e g a t i o n . An h y p o t h e s i s was proposed to account for the f a c t tha t these c h e m i c a l s may o n l y be e f f e c t i v e at the t ime of DNA r e p l i c a t i o n to reduce the spread of h e t e r o c h r o m a t i n and thus i n c r e a s e e x p r e s s i o n of a v a r i e g a t i n g a l l e l e . The second stage i n t h i s a n a l y s i s was to screen f o r dominant mutat ions which e i t h e r suppress or enhance v a r i e g a t i o n . F i f t y - o n e m u t a t i o n s which suppress and 3 mutat ions which enhance the v a r i e g a t e d phenotype were r e c o v e r e d . Mapping of the suppres sor m u t a t i o n s has y i e l d e d the s u p r i s i n g f i n d i n g tha t these genes are c l u s t e r e d a t s p e c i f i c s i t e s i n the genome of D . m e l a n o g a s t e r . S ince i t i s known tha t suppressor mutat ions tend to reduce , and enhancers i n c r e a s e , the spread of h e t e r o c h r o m a t i n (REUTER e t . a l . , 1982) i t may be tha t these mutat ions r e p r e s e n t l e s i o n s i n genes which code for n o n - h i s t o n e chromosomal p r o t e i n s i n v o l v e d i n the s t r u c t u r a l o r g a n i z a t i o n of h e t e r o c h r o m a t i n . T h i s p o s s i b i l i t y , c o u p l e d w i t h the i n g t r i g u i n g c l u s t e r i n g of the s u p p r e s s o r s p r o v i d e s an e x c e l l e n t o p p o r t u n i t y f o r f u r t h e r r e s e a r c h . 1 06 REFERENCES CITED B a k e r , W.K. (1968) P o s i t i o n e f f e c t v a r i e g a t i o n . Adv. Genet . 14:641 . B a k e r , W.K. and A . Rein (1962) The dichotomous a c t i o n of Y -chromosomes on the e x p r e s s i o n of p o s i t i o n e f f e c t v a r i e g a t i o n . G e n e t i c s 47:1399. B a k e r , W.K. and J . B . 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Yasmineh (1971) H e t e r o c h r o m a t i n s a t e l l i t e DNA and c e l l f u n c t i o n S c i e n c e 174:1200. Z u c k e r k a n d l , E . (1974) r e c h e r c h e s sur l e s p r o p r i e t e s et l ' a c t i v i t e b i o l o g i q u e de l a chromat ine B i o c h i m i e 56:937. 115 APPENDIX A CHROMOSOME 2 SUPPRESSORS OF POSITION EFFECT VARIEGATION MUTATION MAP POSITION HOMOZYGOUS VIABILITY 201 40.1* 202 — 203 — 204 -205 28.4* 206 51.3 207 32.0 208 — 209 35.4* + 210 32.6* 211 — 212 — 213 32.9 214 32.6 215 32.9 216 34.2 217 _ *MAP POSITIONS COURTESY OF D. SINCLAIR "+" = VIABLE "-" = LETHAL 116 APPENDIX A CONTINUED (B) CHROMOSOME 3 SUPPRESSORS OF POSITION EFFECT VARIEGATION MUTATION MAP POSITION HOMOZYGOUS VIABILITY 301 + 302 62.4* + 303 + 304 ' + 305 + 306 61.6* + 307 + 308 + 309 60.3* ' + 310 + 311 + 312 + 313 + 314 60.8* + 315 62.5* + 316 47.4* + 317 + 318 + 319 48.6* + 320 59.9* + *MAP POSITIONS COURTESY OF D. SINCLAIR + "+" = VIABLE " - " = LETHAL 117 APPENDIX A CONTINUED (B) CHROMOSOME 3 SUPPRESSORS CONTINUED M U T A T I O N H A P P O S I T I O N H O M O Z Y G O U S 321 + 322 + 323 47.3* + 324 60.2* + 325 53.3 + 326 3 L T I P -327 55.5 + 328 3 L T I P -329 + 330 54.7 + (C) CHROMOSOME 3 ENHANCERS OF POSITION EFFECT VARIEGATION M U T A T I O N M A P P O S I T I O N H O M O Z Y G O U S V I A B I L I T Y A102 49.3 + A 1 0 6 3 L . T I P + A 1 3 1 3L T I P + * M A P P O S I T I O N S C O U R T E S Y O F D . S I N C L A I R " + " = V I A B L E " - " = L E T H A L 118 APPENDIX B 2L 3L 205(28.9+2.0) 207(32, 210(32. 214(32, 215(32, 213(32, 216(34, 209(35, 201(40.1+3.4) 206(51.3+0.9)-GU -Tft Sb-—rtw -326,328 -Pin Yt 23(47.3+1.2) 316(47.4+0.8) 319(48.6+1.4) 325(53.3+1.4) 330(54.7+1.4) 327(55.5+1.0) 320(59.9+1.0) 324(60.2+1.1) 309(60.3+0.9) 314(60.8+0.7) 306(61.1+1.0) 302(62.4+1.5) 315(62.5+0.9) 2R 3R 

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