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Histone gene multiplicity and position-effect variegation in Drosophila melanogaster Moore, Gerald Douglas 1980

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HISTONE GENE MULTIPLICITY AND POSITION - EFFECT VARIEGATION IN DROSOPHILA MELANOGASTER by GERALD DOUGLAS MOORE B.Sc., The U n i v e r i s t y o f Western O n t a r i o , 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACUx/El. 08- GRADUATE STUDIES PROGRAMME'IN GENETICS We acc e p t t h i s t h e s i s as con f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA October 1980 © G e r a l d Douglas Moore, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date i i ABSTRACT The e f f e c t of a l t e r e d h i s t o n e gene m u l t i p l i c i t y on c h r o m a t i n s t r u c t u r e was assayed by measuring changes i n gene a c t i v i t y 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 . Heterozygous d e f i c i e n c i e s o f the h i s t o n e gene c l u s t e r o f D r o s o p h i l a melanogaster i n c r e a s e d the p r o p o r t i o n o f c e l l s w i t h i n a t i s s u e i n wh i c h a v a r i e g a t i n g gene was a c t i v e . T h i s e f f e c t was not dependant on the Y chromosome and a p p l i e d t o b o t h X-l i n k e d and autosomal v a r i e g a t i n g genes. D e l e t i o n s o f the h i s t o n e gene complex imposed on d i f f e r e n t s ource chromosomes e l i c i t e d t he same re s p o n s e . P a r t i a l d e f i c i e n c i e s , w hich d e l e t e d i f f e r e n t r e g i o n s o f the c l u s t e r , v a r i e d i n t h e i r e f f e c t on v a r i e g a t i o n . D u p l i c a t i o n s o f the h i s t o n e gene c l u s t e r d i d not i n c r e a s e the p r o p o r t i o n o f c e l l s i n which a v a r i e g a t i n g gene was i n a c t i v e . The presence of d e f i c i e n c i e s or a d u p l i c a t -i o n o f the c l u s t e r i n the m a t e r n a l genome d i d not m o d i f y the e x t e n t 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 n t h e i r progeny.. These;' r e s u l t s are d i s c u s s e d w i t h r e s p e c t t o c u r r e n t knowledge o f the o r g a n i z a t i o n of the h i s t o n e gene c l u s t e r and c o n t r o l o f i t s e x p r e s s i o n . - i i i -TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 6 RESULTS 16 DISCUSSION 40 SUMMARY 46 BIBLIOGRAPHY 48 - i v -LIST OF FIGURES FIGURE PAGE 1. C y t o l o g i c a l Map o f the p r o x i m a l (2L) d e f i c i e n c i e s 7 o f WRIGHT et a l . (55) . 2. C y t o l o g i c a l Map o f the p r o x i m a l (2L) d e f i c i e n c i e s 9 of SINCLAIR et a l . ( 4 6 ) . 3. The p r o d u c t i o n of segmental a n e u p l o i d s from T(Y;A) 10 t r a n s l o c a t i o n s . 4. Scheme f o r the g e n e r a t i o n of the Dp(2,-l)C239 11 s t o c k . 5. P h o t o m i c r o g r a p h of the p o l y t e n e chromosome spre a d 12 from the Dp (2,• 1) C239 s t o c k showing i n - s i t u h y b r i d i z a t i o n of cRNA :from'plasmid cDm500\ 6. F l u o r o m e t r i c response v e r s u s amount o f d r o s o p t e r i n 14 pigment. m 4 7. Procedure f o r the r e c o v e r y o f i n ( l ) w f l i e s w i t h 18 segmental a n e u p l o i d y of the h i s t o n e gene r e g i o n . 8. Procedure f o r the r e c o v e r y o f i n ( l ) w m 4 f l i e s w i t h 18 s t a b l e ; d e f i c i e n c i e s o f the h i s t o n e gene r e g i o n . 277 4 9. Cross f o r the r e c o v e r y o f i n ( l ) w females w i t h 25 d e f i c i e n c i e s o f the h i s t o n e gene r e g i o n . s v 10. Scheme f o r the r e c o v e r y o f s Y -progeny which have 27 p r o x i m a l (2L) d e f i c i e n c i e s . 11. Cross to generate T(2;3)SbV progeny w i t h p r o x i m a l 29 (2L) d e f i c i e n c i e s . 12. Scheme t o t e s t the m a t e r n a l e f f e c t o f p r o x i m a l 33 (2L) d e f i c i e n c i e s o f wm4 v a r i e g a t i o n . 13. C r o s s e s to t e s t the d i r e c t and m a t e r n a l e f f e c t o f 35 Dp (2,• l) C2.39 on v a r i e g a t i o n a s s o c i a t e d w i t h T (2 ; 3) Sbv . - v-LIST OF TABLES TABLE I . I I . I I I . IV. V. VI . V I I . V I I I . IX. H i s t o n e gene r e i t e r a t i o n i n v a r i o u s s p e c i e s . Mean p e r c e n t a g e of the w i l d - t y p e amount o f d r o s o p t e r i n i n eyes o f in (1) wm4/0 / T (Y,• 2)/ + f l i e s . Mean p e r c e n t a g e o f the w i l d - t y p e amount o f d r o s o p t e r i n i n eyes o f i n ( l ) w m 4 / y F i f l i e s . E f f e c t o f the p r o x i m a l (2L) d e f i c i e n c i e s o f SINCLAIR et a l . (46) on wm4 v a r i e g a t i o n . Mean p e r c e n t a g e o f the w i l d - t y p e amount o f d r o s o p t e r i n i n wm4/wm4 +/Df (2') females and t h e i r female +/CyO s i b l i n g s . Mean p e r c e n t a g e of w i l d - t y p e (B~*~) eye s u r f a c e i n +/BSVY F i f l i e s . E f f e c t o f p r o x i m a l (2L) d e f i c i e n c i e s on b r i s t l e l e n g t h v a r i e g a t i o n a s s o c i a t e d w i t h T(2;3)sbv. Mean p e r c e n t a g e of the w i l d - t y p e amount o f d r o s o p t e r i n i n the eyes o f wm4/Y;CyO/+ and w m4 /t.7m4 /w'" ;CyO/+ progeny from CyO/Df(2) mothers Mean p e r c e n t a g e of d o r s o - c e n t r a l and s c u t e l l a r b r i s t l e s w i t h a sb phenotype i n T(2;3)Sbv progeny from p r o x i m a l (2L) d u p l i c a t i o n and n o n - d u p l i c a t i o n mothers. PAGE •'•2 19 21 23 26 32 30 36 39 - v i -ACKNOWLEDGEMENTS I o f f e r my s i n c e r e s t thanks t o : Dr. D a v i d S u z u k i f o r h i s h e l p f u l c r i t i c s m ; Drs. Doug P r o c u n i e r and D a v i d Cross f o r c o n c e i v i n g o f t h i s p r o j e c t ; Dr. James Berger f o r the use o f h i s q u a n t i t a t i v e m i c r o s c o p y f a c i l i t y ; Dr. S h i z u H a y a s h i f o r her a i d i n p e r f o r m i n g i n - s i t u h y b r i d i z a t i o n ; Dr. D a v i d Hogness f o r h i s g i f t o f the cDm500 p l a s m i d ; Dr. Donald S i n c l a i r f o r h i s a c t i v e c o l l a b o r a t i o n i n v a r i o u s f a c e t s o f t h i s r e s e a r c h ; Mr s t u d e n t c o l l e a g u e s f o r s t i m u l a t i n g r a p p o r t ; and'Dr. Thomas G r i g l i a t t i f o r h i s u n s t i n t i n g •' a i d and encouragement. 1 INTRODUCTION The f i v e h i s t o n e p r o t e i n s are s m a l l , b a s i c p o l y p e p t i d e s whose r e s p o n s i b i l i t y f o r the p a c k a g i n g o f DNA i n t o the p r i m a r y u n i t o f c h r o m a t i n , the nucleosome, has been e x t e n s i v e l y documented (27) . 146 base p a i r s o f DNA are c o i l e d around the e x t e r i o r o f a core p a r t i c l e composed of two m o l e c u l e s each of h i s t o n e s H2A, H2B, H3 and H4 ( 1 8 ) . A s i n g l e m o l e c u l e o f h i s t o n e HI i s complexed w i t h a more v a r i a b l e l e n g t h o f DNA ( u s u a l l y about 60 base p a i r s ) which l i n k s a d j a c e n t nucleosomes ( 4 3 ) . The a c t i o n o f HI i s thought to c o n f e r h i g h e r o r d e r s t r u c t u r e on c h r o m a t i n ( 1 3 ) . The amino a c i d sequence of each of the f i v e h i s t o n e p r o t e i n s i s h i g h l y c o n s e r v e d among the m a j o r i t y o f e u k a r y o t e s . T h i s suggests t h a t each r e g i o n o f t h e s e m o l e c u l e s has an i n v a r i a n t i n t e r a c t i v e f u n c t i o n . Ready a v a i l a b i l i t y o f n e a r l y pure p r e p a r a t i o n s o f h i s t o n e messenger RNA from sea u r c h i n embryos, which c o u l d be d i r e c t e d as a h y b r i d i z a t i o n probe a g a i n s t the h i s t o n e genes:of " d i v e r s e s p e c i e s , a l l o w e d these genes to be among the f i r s t a c c e s s i b l e to b i o r - ' -. c h e m i c a l a n a l y s i s (31,32). In a l l s p e c i e s which have been examined (!with the e x c e p t i o n o f the y e a s t Saccharomyces cerevisae) t h e r e ' i s e v i d e n c e t h a t the c o d i n g sequences of the f i v e p r i n c i p l e h i s t o n e p r o t e i n s are grouped -, together., s e p a r a t e d by r e l a t i v e l y s h o r t l e n g t h s o f non-coding DNA (33) . T h i s u n i t i s tandemly r e i t e r a t e d t o a moderate r e p e t i t i v i t y w hich v a r i e s between s p e c i e s (Table I ) . In D r o s o p h l l a and* humans, 2 TABLE I . H i s t o n e Gene R e i t e r a t i o n i n V a r i o u s S p e c i e s Number of Repeat S p e c i e s U n i t s p e r H a p l o i d Genome Refer e n c e •• Drosophlla 110 (36) Man 40 (54) C h i c k e n 10 (14) X&nopiid 2 0 - 5 0 (28) Y e a s t 1* (33) Sea u r c h i n 200 - 500 (31) * H2A and H2B o n l y . 3 the r e p e a t u n i t s are c l u s t e r e d i n a s i n g l e chromosomal segment (12,44). The r e a s o n f o r t h i s redundancy has not been e l u c i d a t e d . I t has been suggested t h a t i t i s n e c e s s a r y to meet the demand e x e r t e d on the 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 thes e genes d u r i n g p e r i o d s o f r a p i d chromosome r e p l i c a t i o n ( 2 8 ) . S e v e r a l s t u d i e s have been attempted to c o r r e l a t e changes i n the a c t i v i t y of h i s t o n e genes t o events i n the c e l l and d e v e l o p m e n t a l c y c l e s ( f o r a r e v i e w see BORUN, (6)'). M u t a t i o n a l a n a l y s e s , which a s s e s s a l t e r a t i o n s i n h i s t o n e gene p r o d u c t s or c h r o m a t i n s t r u c t u r e r e s u l t i n g from s p e c i f i c changes i n h i s t o n e gene s t r u c t u r e or m u l t i p l i c i t y , have been r a r e . Few organisms posses s s u f f i c i e n t l y w e l l e s t a b l i s h e d g e n e t i c s to r e c o v e r such m u t a t i o n s ( a l t h o u g h the new " p s u e d o - g e n e t i c s " which uses c l o n e d and s p e c i f i c a l l y r e s t r i c t e d DNA i n oocyte or c e l l - f r e e systems o f t r a n s c r i p t i o n and t r a n s l a t i o n , i s r a p i d l y s i d e s t e p p i n g t h i s p r o b l e m ) . The h i s t o n e gene u n i t o f D r o s o p h i l a melanogaster i s r e i t e r a t e d about 110 times p e r h a p l o i d genome ( 3 6 ) . These r e p e a t e d u n i t s are c l u s t e r e d i n the p r o x i m a l l e f t arm o f the second chromosome i n s a l i v a r y g l a n d chromosome bands 39D2-3 to E l - 2 ( 4 4 ) . Hence, they are a c c e s s i b l e to g e n e t i c m a n i p u l a t i o n . D u p l i c a t i o n o f , or d e f i c i e n c y f o r , one e n t i r e c l u s t e r does not r e s u l t i n l e t h a l i t y , nor does i t have an o v e r t p h e n o t y p i c e f f e c t . The aim o f t h i s i n v e s t i g a t i o n i s to examine the e f f e c t of a l t e r e d h i s t o n e gene m u l t i p l i c i t y on c h r o m a t i n morphology and gene e x p r e s s i o n by m o n i t o r i n g a g e n e t i c phenomenon, 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 , which i s s e n s i t i v e t o changes i n c h r o m a t i n s t r u c t u r e . 4 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 o r i g i n a l l y o b s e r v e d i n D r o s o p h i l a by MULLER (40) and has s i n c e been demonstrated i n p l a n t s and mammals (11,2). When c e l l s bear a chromosomal rearrangement which j u x t a p o s e s a e u c h r o m a t i c gene t o h e t e r o -c h r o m a t i n , a f r a c t i o n o f the c e l l s e x h i b i t no e x p r e s s i o n of t h a t gene. The r e s u l t a n t organism i s a mosaic f o r the a c t i v i t y of the r e a r r a n g e d gene. The degree o f m o s a i c i s m can be m o d i f i e d ; f a c t o r s such as e l e v a t e d d e v e l o p m e n t a l t e m p e r a t u r e , a d d i t i o n a l Y chromosome h e t e r o c h r o m a t i n i n the genome, and v a r i o u s m o d i f i e r genes enhance the p r o p o r t i o n o f c e l l s i n which a v a r i e g a t e d gene i s a c t i v e . A l t h o u g h the phenomenon has been e x t e n s i v e l y c a t a l o g u e d ( r e v i e w s by BAKER, ( 2 ) ; and SPOFFORD, ( 4 9 ) ) t h e u n d e r l y i n g m o l e c u l a r mechanism 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 u n c l e a r . Two l i n e s o f e v i d e n c e suggest the v a r i a b l e g e n e t i c a c t i v i t y r e s u l t i n g from the p o s i t i o n - e f f e c t i s r e l a t e d t o a l t e r e d c h r o m a t i n morphology a t the v a r i e g a t i n g l o c u s : i>) I f two genes are i n v o l v e d i n a rearrangement, the gene n e a r e s t to the h e t e r o c h r o m a t i c break p o i n t w i l l be i n a c t i v a t e d i n a g r e a t e r ' p r o p o r t i o n "of • c e l l s '(,16]i, i i ) For gene l o c i which can be a s s i g n e d to s p e c i f i c s a l i v a r y g l a n d chromomeres ( b a n d s ) , the i n t e n s i t y o f gene i n a c t i v a t i o n f o l l o w i n g rearrangement i s p r o p o r t i o n a l to the f r a c t i o n on s a l i v a r y c e l l s i n which the l o c u s has assumed a h e t e r o -c h r o m a t i c morphology ( 2 5 ) . I t has been proposed t h a t the v a r i e g a t i n g gene l o c u s i s condensed 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 d by a " s p r e a d i n g 5 e f f e c t " or l i m i t e d s p a t i a l d i f f u s i o n o f m o l e c u l e s from the a d j a c e n t h e t e r o c h r o m i n ( 5 9 ) . The n a t u r e o f h e t e r o c h r o m a t i c elements w h i c h c o u l d e f f e c t t h i s c o n d e n s a t i o n i s open t o " : s p e c u l a t i o n . The c y t o l o g i c a l and g e n e t i c p r o p e r t i e s which d e f i n e h e t e r o c h r o m a t i n such as condensed s t a i n i n g throughout the c e l l c y c l e , a^paucity,of genes , and reduced r e c o m b i n a t i o n do not i l l u m i n a t e i t s m o l e c u l a r c o n s t i t u t i o n (56)'. As opposed to i t s n u c l e i c a c i d component, which c o n s i s t s p r i m a r i l y o f h i g h l y r e i t e r a t e d , v e r y s h o r t sequences, t h e r e i s l i t t l e i n f o r m a t i o n about the d i s t i n c t i v e f e a t u r e s o f h e t e r o c h r o m a t i c p r o t e i n s ( 1 0 ) . BERLOWITZ (3) r e p o r t e d t h a t the h e t e r o c h r o m a t i c chromosome s e t of the mealybug 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 . I n the genus D r o s o p h i l a , the t y p e s o f p h o s p h o r y l a t e d s u b - s p e c i e s o f the h i s t o n e HI v a r y between s p e c i e s d i f f e r i n g i n h e t e r o c h r o m a t i c c o m p o s i t i o n ( 5 ) . One can h y p o t h e s i z e t h a t h e t e r o c h r o m a t i c h i s t o n e s are agents o f the s p r e a d i n g e f f e c t . A p r e d i c t i o n o f t h i s model i s t h a t v a r i a t i o n i n the amount of c e l l u l a r h i s t o n e p r o t e i n s h o u l d a l t e r the e x t e n t o f any m o r p h o l o g i c a l change i n c h r o m a t i n a s s o c i a t e d w i t h the p o s i t i o n e f f e c t ' . To t e s t t h i s " p r e d i c t i o n , the e f f e c t o f ' . a l t e r e d "'" h i s t o n e gene m u l t i p l i c i t y on the degree of m o s a i c i s m a s s o c i a t e d w i t h v a r i e g a t i n g genes has been examined. 6 MATERIALS AND METHODS 1. C u l t u r e C o n d i t i o n s and Mutant S t o c k s F l i e s - w e r e r e a r e d i n % p i n t m i l k b o t t l e s or s h e l l v i a l s on a s u c r o s e - cornmeal - agar medium, seeded w i t h b a k e r s ' y e a s t . Tegosept (methyl - p - hydroxybenzoate) was i n c l u d e d i n the food as a mould i n h i b i t o r . To suppress b a c t e r i a l growth, a c o m b i n a 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 i n and t e t r a c y c l i n e was i n c l u d e d ( l O m g / l i t r e , e a c h ) . D e s c r i p t i o n s o f the m u t a t i o n s and chromosomes used can be found i n LINDSLEY and GRELL ( 3 7 ) , w i t h the f o l l o w i n g e x c e p t i o n s : •" ' ( i ) Bar of S t o n e - v a r i e g a t e d ( B ) . T h i s chromosome i s an s i r r a d i a t i o n - i n d u c e d d e r i v a t i v e o f B Y which e x h i b i t s a v a r i e g a t e d p o s i t i o n - e f f e c t of the m u t a t i o n Bar. I t i s d e s c r i b e d by BROUSSEAU ( 8 ) . ( i i ) P r o x i m a l (2L) d e f i c i e n c i e s . D f ( 2 ) l , 12,65,84 and 161 were g e n e r a t e d i n scr e e n s f o r d e f i c i e n c i e s o f the dopa decarboxy-l a s e l o c u s perfomed by WRIGHT e t a l . ( 5 5 ) . 1,12,65 and 84 are X - i r r a d i a t i o n i n d u c e d d e r i v a t i v e s o f - a Tuft l e t h a l ( 2 ) 7 4 i chromosome, w h i l e 161' was ind u c e d by X-rays on a cinnabar (cn) brown(b'w)'' chromosome. The o r i g i n a l c y t o l o g i c a l d e s c r i p t i o n was r e c o n f i r m e d and i s p r e s e n t e d i n F i g u r e 1. T h e i r g e n e t i c c o n s t i t u t i o n i s i n d i c a t e d below: Df (2) 1 ,12 ,64 ,84 are deleted for purple(pr), B r i s t l e ( B l ) and l e t h a l (2) cryptocephal (crc) . Df (2)161 i s d e l e t e d "for pr , B l , l ' ( 2 ) c r c , Minute(2)H(M(2)H). 7 IVV i AAI < n ( j < < 1 0 CD < < — < L U < 1 1IV < * U * m < — r\j V t() (O V* 00 CO * CM CM CM CM CM W * ' W v W M — ' | M — M — M -O O Q O Q \k ^ Y v y 5 < * W CO m LL CD CO n *¥ I ' l l N f ; m m ® LU < LL. LL < oo s r- oo CO CO CO CO CO 7a F i g u r e 1. C y t o l o g i c a l map o f the p r o x i m a l (2L) d e f i c i e n c e s o f WRIGHT et a l . ( 5 5 ) . 8 Nine m u t a t i o n s of p r o x i m a l (2L) were imposed w i t h the mutagen, t r i e t h y l m e l a m i n e , on a W a c i / i j p i en::, chromosome by SINCLAIR'et a l . (46):. A c y t o l o g i c a l e x a m i n a t i o n o f these mutants (D'si-9) r e v e a l e d t h a t at l e a s t f i v e o f the mutants are v i s i b l e d e f i c i e n c i e s , as i n d i c a t e d i n F i g u r e 2. ( i i i ) Translocation(Y;Autosome) a n e u p l o i d s . The g e n e r a t i o n o f t h i s s e r i e s o f chromosomes, and t h e i r use i n c r e a t i n g segmental a n e u p l o i d s o f autosomal l o c i , i s d e s c r i b e d i n LINDSLEY and SANDLER e t a l . ( 3 8 ) . An example o f segmental a n e u p l o i d s y n t h e s i s by t h i s t e c h n i q u e i s i l l u s t r a t e d i n F i g u r e 3. ( i v ) D u p l i c a t i o n (21) C239 . T h i s X - l i n k e d d u p l i c a t i o n o f p r o x i m a l (2L) i s a s e g r e g a n t from a T r a n s l o c a t i o n (1; 2) C2 J9':stobk . The scheme f o r the c r e a t i o n o f a d u p l i c a t i o n s t o c k i s i n d i c a t e d i n f i g u r e 4. I n - s i t u h y b r i z a t i o n o f H l a b e l l e d RNA t r a n s c r i b e d from recombinant p l a s m i d cDM500, which c a r r i e s the D r o s o p h i l a h i s t o n e gene u n i t , r e v e a l e d t h a t the d u p l i c a t e d segment c o n t a i n s a l l , or a p o r t i o n o f , the h i s t o n e gene c l u s t e r ( F i g u r e 5 ) ( 3 6 ) . 2. Eye Pigment Measurement In o r d e r t o l i m i t the s c a l e o f the experiments i t was d e s i r a b l e t o measure the eye pigment c o n t e n t o f i n d i v i d u a l heads. A f l u o r o m e t r i c t e c h n i q u e was d e v e l o p e d to p e r m i t measurements h a v i n g the n e c e s s a r y p r e c i s i o n . F l i e s were a l l o w e d t o age 1-7 days p o s t - e c l o s i o n , then d e c a p i t a t e d by f r e e z i n g and a g i t a t i o n . S i n g l e heads were c r u s h e d on c e l l u l o s e chromatography p l a t e s (Eastman 13255) and pigments were s e p a r a t e d w i t h a 2:1 p r o p a n o l r l l aqueous NH^ s o l u t i o n . The d r o s o p t e r i n spot was l o c a t e d and the r e l a t i v e l e v e l o f f l u o r e s c e n c e was measured u s i n g a Z e i s s m i c r o -Histone l(2)DS2 l(2)DSS l(2)DS6 i(2)DS8 l(2)DS9 9a F i g u r e 2. C y t o l o g i c a l map o f the p r o x i m a l (2L) d e f i c i e n c i e s of SINCLAIR et a l . ( 4 6 ) . XX ? X r—i 1 rv c? + B OVA DERIVED FROM ADJACENT I DISJUNCTION SPERM DERIVED FROM ADJACENT I DISJUNCTION TRIPLO X ( L E T H A L ) ^^^^ . , r— D F I J B * B DPI ? NULLOX ( L E T H A L ) 10a F i g u r e 3. The p r o d u c t i o n of segmental a n e u p l o i d s from T(Y,-A) t r a n s l o c a t i o n s . From LINDSLEY and SANDLER (.38). 11 T (1 ; 2 ) C239/FM4 ;+ (X)dl49/Y; Cy/bw VI T (1 ; 2 ) C239/149 ; Cy (2)dl49/Y; Cy/bw VI Stock: Dp (2 ; 1) C239/dl49 ;Cy/bwV1 (g) d 1 4 9 / Y ; C y / bwVl dl49=Inversion(1)scute + delta 49, sc v f car FM4 = In(l)FM4, y 3 1 d sc8 w dm B Cy = In(2LR)SM5, al Cy l t V cn2 sp2 11a F i g u r e 4. Scheme f o r the g e n e r a t i o n o f the Dp(2;1)C239 S t o c k . 12 12a F i g u r e 5. P h o t o m i c r o g r a p h o f a p o l y t e n e chromosome sp r e a d from the Dp (2;1)C239 s t o c k . s h o w i n g i n - s i t u h y b r i d i z a t i o n o f cRNA from p l a s m i d cDm 500. 13 scope equipped w i t h a UV l i g h t s ource and a p h o t o m u l t i p l i e r . The f l u o r o m e t e r was s t a n d a r d i z e d a g a i n s t the amount o f d r o s o p t e r i n f l u o r e s c e n c e from whi-'te+ (Oregon-R s t r a i n ) heads. To ensure t h a t the f l u o r e s c e n t response was l i n e a r over the range of p i g m e n t a t i o n measured,, a d r o s o p t e r i n e x t r a c t ( i n e t h a n o l , pH2) d i l u t i o n s e r i e s was p r e p a r e d , chromatographed and measured (17) . The response curve i s i l l u s t r a t e d by F i g u r e 6. 3. Eye S i z e Measurement Eye shapes were s k e t c h e d t o s c a l e on g r i d d e d bond paper w i t h the a i d o f a d i s s e c t i n g m i c r o s c o p e equipped w i t h an o c 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 out and weighed to e s t i m a t e s u r f a c e a r e a r e l a t i v e to w i l d - t y p e eyes. 4. B r i s t l e Phenotype Measurement B r i s t l e phenotype was s c o r e d by e i t h e r o f two methods, dependant on the genotypes i n v o l v e d i n the e x p e r i m e n t s : ( i ) The d o r s o - c e n t r a l and s c u t e l l a r . b r i s t l e s were observed by d i s s e c t i n g , m i c r o s c o p e and a s s i g n e d a Sb (stubble) or sb+ phenotype i n d i v i d u a l l y . ( i i ) Genotypes b e a r i n g Tft or BI were d i f f i c u l t to s c o r e unambiguously by the p r e v i o u s method s i n c e b oth of these m u t a t i o n s p e r t u r b b r i s t l e morphology. In these cases the l e n g t h of" the p o s t e r i o r s t e r n o - p l e u r a l and p o s t e r i o r dorso-c e n t r a l b r i s t l e s were measured 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 equipped w i t h an o c c u l a r micrometer. The v a l u e s o b t a i n e d from each f l y were summed to a s i n g l e v a l u e from which was s u b s t r a c t e d the l e n g t h o f the f o u r c o r r e s p o n d i n g b r i s t l e s i n a sb f l y . T h i s new v a l u e was d i v i d e d by the d i f f e r e n c e i n 14 14a F i g u r e 6. F l u o r o m e t r i c response v e r s u s amount of d r o s o p t e r i n pigment. 15 l e n g t h between the c o r r e s p o n d i n g sb+ p a r e n t a l b r i s t l e s and sb b r i s t l e s . The f o r m u l a i s i l l u s t r a t e d below: (sbV s.-p. and d.-c. b r i s t l e l e n g t h — sb b r i s t l e l e n g t h ) ^ i Q o (sb+ b r i s t l e l e n g t h — sb b r i s t l e l e n g t h ) A v a r i e g a t e d b r i s t l e phenotype which i s more w i l d - t y p e (sb+) w i l l have a v a l u e a p p r o a c h i n g 1001. Those phenotypes which are more sb i n appearance w i l l have v a l u e s a p p r o a c h i n g 0% . 5. S t a t i s t i c s A n a l y s i s o f d a t a o b t a i n e d from q u a n t i f i c a t i o n o f v a r i e g a t e d phenotypes r e v e a l e d t h a t the v a r i a n c e o f a parameter i s o f t e n p r o p o r t i o n a l t o i t s mean. St u d e n t ' s t - t e s t , which e s t i m a t e s the s i g n i f i c a n c e o f d i f f e r e n c e i n means between groups can o n l y be used to compare groups whose v a r i a n c e s are not s i g n i f i c a n t l y d i f f e r e n t . I t was n e c e s s a r y t o employ a m o d i f i c a t i o n o f the t - t e s t w h i c h accounts f o r d i f f e r e n c e i n v a r i a n c e s between groups i n o r d e r t o a n a l y s e the d a t a . The m o d i f i c a t i o n of SUTTERTHWAITE (51) , and WELCH (53);/,' r e t a i n s the method of e s t i m a t i n g the t v a l u e , but s u b s t i t u t e s an a l t e r e d e s t i m a t e of v ( t o t a l degrees o f freedom) which i s f . f= i< ( f x + f 2 ) 16 RESULTS 1. The E f f e c t o f T(Y,-2) Segmental A n e u p l o d i e s o f the H i s t o n e 777 4 Gene C l u s t e r on V a r i e g a t i o n of w S t o c k s b e a r i n g segmental d e f i c i e n c i e s or d u p l i c a t i o n s can be c r e a t e d from c r o s s e s o f a p p r o p r i a t e l y marked T(Y;A) s t o c k s ( F i g u r e 3 ) . The b o u n d a r i e s o f the segmental a n e u p l o i d y are d e f i n e d by the t r a n s l o c a t i o n break p o i n t s . KHESIN and LIEBVOTICH (34) e x p l o i t e d the ease and f l e x i b i l i t y o f t h i s t e c h n i q u e t o t e s t the e f f e c t of a d e f i c i e n c y o f the h i s t o n e gene c l u s t e r on the 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 the white gene a s s o c i a t e d w i t h T(1,-3)WVCO, and X-chromosome t r a n s c r i p t i o n i n i n t e r s e x e s . There i s , however, a s e r i o u s t h e o r e t i c a l r e s e r v a t i o n t o the use o f Y chromosome rearrangements i n the study 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 Y chromosome c o n t a i n s r e g i o n s which are p o t e 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 , and t h e i r i n t e g r i t y i n T(Y,-A) chromosomes i s u n c e r t a i n ( 9 ) . Moreover, the T(Y;A) a n e u p l o i d g e n e r a t i o n t e c h n i q u e produces p o o r l y d e f i n e d d e f i c i e n c i e s and d u p l i c a t i o n s of Y chromosome m a t e r i a l . I n h e r e n t i n i t s use are d i f f i c u l t i e s i n p r o p e r l y c o n t r o l l i n g f o r the e f f e c t of the Y chromosome on v a r i e g a t i o n . In o r d e r t o determine whether the segmental a n e u p l o i d y t e c h n i q u e might be o f f u r t h e r use i n the s t u d y o f h i s t o n e gene m u l t i p l i c i t y e f f e c t s on v a r i e g a t i o n , a s e r i e s o f T(Y;2) d e f i c i e n c y , non-d e f i c i e n c y and d u p l i c a t i o n chromosomes were t e s t e d f o r m o d i f i c a t i o n of the v a r i e g a t e d phenotype a s s o c i a t e d w i t h 17 i n v e r s i o n ( 1 ) w h i t e - m o t t l e d . A g e n e r a l i z e d scheme f o r these t e s t s i s d i s p l a y e d i n F i g u r e 7. The rearrangement, in(1)wm4, r e l o c a t e s the white* gene from i t s normal p o s i t i o n near the t i p o f the X chromosome t o 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 . In i t s normal chromosomal p o s i t i o n , the white+ gene f u n c t i o n s i n the f o r m a t i o n o f pigment g r a n u l e s i n secondary pigment c e l l s of the eye ( 5 8 ) . I n d i v i d u a l c e l l s b e a r i n g the i n v e r s i o n genotype e x h i b i t e i t h e r a f u l l complement of pigment g r a n u l e s or none (45 ) . T h i s r e s u l t s i n eyes which have a mosaic p i g m e n t a t i o n p a t t e r n i n i n ( l ) w m 4 f l i e s . I n these t e s t s , the e x t e n t o f the white-v a r i e g a t e d phenotype was q u a n t i f i e d by measuring the r e l a t i v e amount o f the eye pigment, d r o s o p t e r i n , i n i n d i v i d u a l heads. The e f f e c t o f the T(Y:2) a n e u p l o i d i e s 777 4 on thew p o s i t i o n e f f e c t i s d e t a i l e d i n T a b l e I I . The v a r i o u s segmental a n e u p l o i d i e s o f the h i s t o n e gene c l u s t e r and a d j a c e n t r e g i o n s a f f e c t the mosaic phenotype m a r k e d l y , but t h e r e i s no, c o n s i s t e n t p a t t e r n o f s u p p r e s s i o n or enhancement of v a r i e g a t i o n w i t h r e s p e c t to d e l e t i o n or d u p l i c a t i o n o f the h i s t o n e gene c l u s t e r . T(Y;2) t r a n s l o c a t i o n s which were not a n e u p l o i d had a s i m i l a r pronounced e f f e c t on the v a r i e g a t e d phenotype. These r e s u l t s suggest t h a t a l t e r a t i o n s o f Y chromosome l o c i , r a t h e r than h i s t o n e gene m u l t i p l i c i t y , " i s r e s p o n s i b l e f o r the observed m o d i f i c a t i o n o f the v a r i e g a t e d 18 17° XY/0;Cy/T(Y;2)dv(g) W™4 /wm4 ; +/+ i wm4/o; +/T(Y;2) OW 17° + /Y; CyO/Df (2)<&$ W™4/w™4; +/+ W i W m4 /Y ; Df(2)/+ C f t f " Wm4/y; CyO/+ cTrf 18a F i g u r e 7. Pro c e d u r e f o r the r e c o v e r y o f in(i)wm f l i e s w i t h segmental a n e u p l o i d y o f the h i s t o n e gene r e g i o n . F i g u r e 8. Procedure f o r the r e c o v e r y o f in(i)wm f l i e s w i t h s t a b l e d e f i c i e n c i e s o f the h i s t o n e gene r e g i o n . 19 TABLE I I . Mean Per c e n t a g e o f the w i l d - t y p e amount o f d r o s o p t e r i n i n eyes o f in (1) wm4/o; T(Y;2)/'+•••£lies , No. of. histone Second chromosome genotype of F 1 "gene * wm /o <f<f and breakpoints. clusters % w+ drosopterin s y +/T(Y;2)J59* (43A) 2 2 0 .1 +/T(Y;2)L138* (39C) 2 42 3 .8 +/T(Y;2)B190* (40) 2 25 3 .0 +/T(Y;2)B209* (40) 2 35 5 . 7 +/T(Y;2)H54* (40) 2 28 4 .2 +/T(Y;2)B26* (43E) 2 7 1 .1 +/T(Y;2)A107* (40) 2 47 5 .6 +/T(Y;2)B110* (38C) 2 5 1 .0 +/T(Y;2)DfBllO-Ll38 (38C-39C) 2 7 1 . 7 + /T (Y;2)DfB2 6-J59 (43A-43E) 2 63 2 .8 +/T(Y;2)DfBllO-Bl90 (38C-40) 1 43 4 .1 +/T(Y;2)DpLl38-B209 (39C-40) 3 11 1 .3 +/T(Y;2)DpBllO-Al07* (38C-40) 3 1 0 .1 +/T(Y;2)DpBll0-H54* (38C-40) 3 30 3 .'3 n 'v 3 0 f o r each genotype * Genotypes c a r r y i n g BS, i n +/BSY heads. e x p r e s s e d as % w d r o s o p t e r i n 20 phenotype by T(Y,-2) segmental a n e u p l o i d i e s . m 4 2. The E f f e c t of P r o x i m a l (2L) D e f i c i e n c i e s on w  V a r i e g a t i o n The p r e c e d i n g r e s u l t s mandate the use of s t a b l e d e f i c i e n c i e s , not i n v o l v i n g a l t e r e d Y chromosomes, t o stu d y the e f f e c t ; o f h i s t o n e gene m u l t i p l i c i t y on v a r i e g a t i o n . F i v e p r o x i m a l (2L) d e f i c i e n c y s t o c k s were o b t a i n e d from WRIGHT et a l . :(5:5)". A l l have s i m i l a r d i s t a l break p o i n t s ( F i g u r e 1 ) . Two o f the d e f i c i e n c i e s , D f ( 2 ) l and 12 extend p r o x i m a l l y towards the h i s t o n e gene c l u s t e r , but do not i n c l u d e i t . They s e r v e as c o n t r o l s f o r any e f f e c t of d e l e t i n g e u c h r o m a t i c segments d i s t a l t o the c l u s t e r . One d e f i c i e n c y , Df(2)84, has i t s p r o x i m a l break p o i n t w i t h i n the h i s t o n e gene c l u s t e r . The p r o p o r t i o n o f the c l u s t e r which i t d e l e t e s i s u n c e r t a i n . The r e m a i n i n g d e f i c i e n c i e s , Df(2)65 and 161, d e l e t e the e n t i r e c l u s t e r and ext e n d p r o x i m a l l y . The d e f i c i e n c i e s were t e s t e d f o r m o d i f i c a t i o n o f the v a r i e g a t e d 7734 phenotype a s s o c i a t e d w i t h i n ( l ) w ; the t e s t scheme i s d i s p l a y e d i n F i g u r e 8. In o r d e r t o p r e c l u d e any m a t e r n a l e f f e c t s o f the d e f i c i e n c y , the d e f i c i e n c y chromosome was c o n t r i b u t e d by the male p a r e n t . A wm4/y.: +/+ eye c o n t a i n s about 4% o f the w i l d - t y p e amount o f the r e d pigment d r o s o p t e r i n , when f l i e s are r a i s e d at 17°C. H e t e r o z y g o s i t y f o r the c o n t r o l d e f i c i e n c i e s , D f ( 2 ) l and 12, and the Cyo i n v e r s i o n chromosome does not s i g n i f i c a n t l y a l t e r t h i s l e v e l ( T a ble I I I ) . i n ( l ) w m 4 f l i e s h e t e r o z y g o u s f o r the t h r e e d e f i c i e n c i e s which d e l e t e , a l l , or a p a r t , of the h i s t o n e gene c l u s t e r e x h i b i t s i g n i f i c a n t l y 21 TABLE I I I . Mean p e r c e n t a g e o f the w i l d - t y p e o f d r o s o p t e r i n m4 i n eyes o f i n ( i ) w /Y F-.d'flies. cf Genotype No. o f H i s t o n e Gene C l u s t e r s D r o s o p t e r i n m4 w /Y;Df(2L)1/ + 2 4 1.2 m4 , /Y;CyO/+ 2 3 0.7 m4 W Y;Df(2 L)12/ + 2 4 0.9 m4 , ' Y;CyO/+ 2 3 0.5 m4 W /Y;Df(2L)84/+ 1-2 19 2.4 m4 , /Y;CyO/+ 2 6 0.7 m4 W 'Y;Df(2L)65/+ 1 9 2.1 m4 , /Y;CyO/+ 2 3 0.4 m4 W Y ; Df ( 2 L) 161 / + 1 24 3.2 m4 , /Y;CyO/+ 2 8 1. 7 m4 , W /Y;+/+ 2 4 0.9 >0.05 >0 . 05 « 0.05 < 0.05 « 0.05 22 e l e v a t e d l e v e l s o f d r o s o p t e r i n i n the eyes. A c t i v e e x p r e s s i o n o f the white*- gene occurs i n a l a r g e r p r o p o r t i o n o f pigment c e l l s i n wm4 i n d i v i d u a l s w i t h a s i n g l e h i s t o n e gene c l u s t e r , t han i n those w i t h a normal d i p l o i d complement. I t i s p o s s i b l e t h a t the observed s u p p r e s s i o n o f v a r i e g a t i o n was due to p e c u l i a r i t i e s a s s o c i a t e d w i t h the source o f chromosomes o f the d e f i c i e n c i e s g e n e r a t e d by WRIGHT et a l . ( 5 5 ) . T h i s seems u n l i k e l y g i v e n the s i m i l a r e f f e c t of d e f i c i e n c i e s 84 and 1 6 1 , which were i n d u c e d on d i f f e r e n t chromosomes. To ensure t h a t a d e l e t i o n o f the h i s t o n e gene c l u s t e r was " r e s p o n s i b l e f o r the obse r v e d e f f e c t , p r o x i m a l (2L) d e f i c i e n c i e s were i n d u c e d on a. • d i f f e r e n t chromosome , and t e s t e d u s i n g the same scheme, shown p r e v i o u s l y i n F i g u r e 8. 777 4 The e f f e c t on w v a r i e g a t i o n o f the d e f i c i e n c i e s o f SINCLAIR et a l . (46) was s c o r e d v i s u a l l y . The two d e f i c i e n c i e s d i s t a l t o , but not i n c l u d i n g , the h i s t o n e gene c l u s t e r (DS8, DS9) d i d not enhance the p r o p o r t i o n o f c e l l s i n which the white gene was a c t i v e (Table I V ) . DS6, which e n t i r e l y d e l e t e s the c l u s t e r , and DS5, a p a r t i a l d e f i c i e n c y which d e l e t e s the d i s t a l r e g i o n o f the c l u s t e r , caused a marked i n c r e a s e i n p i g m e n t a t i o n . DS2, a p a r t i a l d e f i c i e n c y f o r the p r o x i m a l p o r t i o n of the c l u s t e r d i d not cause an i n c r e a s e i n v a r i e g a t i n g gene a c t i v i t y . The obscure c y t o l o g i c a l c o n f i g u r a t i o n o f the h i s t o n e gene r e g i o n makes e s t i m a t i o n o f the s i z e o f the p a r t i a l d e f i c i e n c i e s d i f f i c u l t . These r e s u l t s c o n t r a d i c t the h y p o t h e s i s t h a t p e c u l a r i t i e s o f the source chromosome are r e s p o n s i b l e f o r the observed 2 3 T A B L E I V . E f f e c t of the p r o x i m a l ( 2 L ) d e f i c i e n c i e s o f m4 S I N C L A I R e t , a l . , . (45j)LC ; . on w v a r i e g a t i o n . NO.OF H I S T O N E V I S I B L E G E N O T P YE J'GENE C L U S T E R S S U P P R E S S I O N Df(2)DS8/+ 2 no of(2)DS9/+ 2 no Df(2)DS2/+ 1-2 no Df(2)DS5/+ 1-2 yes Df(2)DS6/+ 1 yes 24 m o d i f i c a t i o n o f v a r i e g a t i o n . However, they do suggest t h a t a d e l e t i o n of the d i s t a l p o r t i o n of the h i s t o n e gene c l u s t e r i s n e c e s s a r y t o e l i c i t s u p p r e s s i o n o f the p o s i t i o n - e f f e c t . In o r d e r t o determine i f the observed s u p p r e s s i o n o f the p o s i t i o n e f f e c t was r e s t r i c t e d t o males, a d i f f e r e n t c r o s s was used t o o b t a i n w m 4 females b e a r i n g the d e f i c i e n c i e s ( F i g u r e 9 ) . The r e s u l t s i l l u s t r a t e d i n T a b l e "V r e v e a l a p a t t e r n on pheno-type m o d i f i c a t i o n s i m i l a r t o males. ( C o n t r o l d e f i c i e n c y 12, which e x h i b i t s a m a r g i n a l l y s i g n i f i c a n t d i f f e r e n c e t o i t s cyO s i b l i n g s , i s not s i g n i f i c a n t l y d i f f e r e n t from the o v e r a l l cyo v a l u e ) . The presence of the Y chromosome i s not n e c e s s a r y to e l i c i t the 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 s s o c i a t e d w i t h h e t e r o z y g o u s d e f i c i e n c i e s of the h i s t o n e gene c l u s t e r . 3. The E f f e c t of P r o x i m a l (2L) D e f i c i e n c i e s on V a r i e g a t i o n A s s o c i a t e d w i t h B S V Y • The r e s u l t s p r e v i o u s l y t a b l e d do not e x c l u d e the p o s s i b i l i t y t h a t the h i s t o n e gene d e f i c i e n c i e s e x e r t a s p e c i f i c i n f l u e n c e on the white l o c u s , r a t h e r than 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 . I t was n e c e s s a r y t o t e s t a n other v a r i e g a t i n g rearrangement to demonstrate a g e n e r a l i z e d e f f e c t sv ( F i g u r e 10). Bar of Stone - V a r i e g a t e d (B ) was used f o r t h i s purpose. The narrow eye phenotype a s s o c i a t e d w i t h the m u t a t i o n Bar i s the r e s u l t of a tandem d u p l i c a t i o n i n the X chromosome sv r e g i o n 16A ( 7 ) . The rearrangement B Y a t t a c h e s t h i s d u p l i c a t e d r e g i o n t o the h e t e r o c h r o m a t i n of the Y chromosome(8). 25 Wm4/Y; CyO/Df (2)<Sf(E) W™4/W™4 ; +/+ <ft 4 Wm4/Wm4; + /Df(2) $? 25a F i g u r e 9. Cross f o r the r e c o v e r y o f i n ( l ) w females w i t h d e f i c i e n c i e s of the h i s t o n e gene r e g i o n . 26 TABLE V. Mean p e r c e n t a g e of the w i l d - t y p e amount o f I t l 4 Ttl4 d r o s o p t e r i n i n w /w ;+/Df(2) females and t h e i r female+/CyO s i b l i n g s . Second chromosome genotype o f wm4/ wm4 f c m a i c s No. o f h i s t o n e Gene c l u s t e r s D r o s o p t e r i n p +/Df(2)1 2 3 0.8 >0. 05 + /CyO 2 4 0.6 +/Df(2)12 2 5 1.0 <0.05 + /CyO 2 2 0.4 +/Df(2)84 1-2 24 4.4 «0. 0 5 + /CyO 2 5 0.8 +/Df(2)65 1 28 3.5 «0.05 + /CyO 2 4 0.9 + Df(2) 161 , 1 12 5.3 <0 . 05 + /CyO 2 2 0.4 20 f o r each genotype + /Y; CyO/Df (2)<fcf ® XX/B Y; +/+ I SV + /B Y; Df(2)/+ d V + /BSVY; CyO/+ d V 2 7a F i g u r e 10. Scheme f o r the r e c o v e r y o f B y progeny which have p r o x i m a l (2L) d e f i c i e n c i e s . 28: As the t r a n s p o s e d r e g i o n i s i n a c t i v a t e d i n a p r o p o r t i o n o f the sv p r e s u m p t i v e o m m a t i d i a l (eye f a c e t ) c e l l s , B f l i e s e x h i b i t a phenotype i n t e r m e d i a t e between the narrow eye of Bar, and the o v a l eye of Bar*. i n t h i s c a s e , s u p p r e s s i o n o f the s p r e a d i n g sv e f f e c t s h o u l d r e s u l t i n a narrower eye i n s f l i e s , s i n c e the t r a n s p o s e d d u p l i c a t i o n segment would be a c t i v e l y 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 p r e s u m p t i v e o m m a t i d i a l . c e l l s T h i s sv was observed i n B Y f l i e s b e a r i n g d e f i c i e n c i e s 65,84 and 161, c o n f i r m i n g the g e n e r a l i z a t i o n t h a t h e t e r o z y g o u s d e f i c i e n c i e s o f the h i s t o n e gene c l u s t e r i n c r e a s e the p r o p o r t i o n of c e l l s i n w h i c h a v a r i e g a t i n g gene i s a c t i v e (Table V I ) . The p a r t i a l d e f i c i e n c y ,84,had an i n t e r m e d i a t e s u p p r e s s i v e e f f e c t . on t h i s v a r i e g a t i n g a l l e l e . 4. The E f f e c t of P r o x i m a l (2L) D e f i c i e n c i e s on the V a r i e g a t i o n o f Autosomal Rearrangement T(2,-3) stubble-variegated. S i n c e b o t h white and Bar are X l i n k e d genes, the f o r e g o i n g d a t a would be c o n s i s t e n t w i t h an i n t e r p r e t a t i o n t h a t the e f f e c t of h i s t o n e gene d e f i c i e n c i e s i s r e s t r i c t e d t o the e x p r e s s i o n of v a r i e g a t i n g genes on the X chromosome, perhaps by a m o d i f i c a t i o n of the dosage compensation mechanism. T h i s h y p o t h e s i s was t e s t e d by o b s e r v i n g the e f f e c t o f the d e f i c i e n c i e s " on a v a r i e g a t i n g autosomal gene ( F i g u r e 11). The rearrangement v T(2,-3)Sb j u x t a p o s e s the mutant a l l e l e sb (which has a s h o r t b r i s t l e phene) t o the h e t e r o c h r o m a t i n of the second chromosome. The r e s u l t a n t v a r i e g a t i o n produces a mosaic p a t t e r n o f s h o r t b r i s t l e s (sb a c t i v e i n the b r i s t l e - f o r m i n g c e l l s ) and n o r m a l , l o n g e r b r i s t l e s {Sb i n a c t i v a t e d ) . H e t e r o z y g o s i t y f o r 29, T 12 ; 3) SvV; + i$ ® CyO/Df(2); + / + d'cf T (2 ; 3) SbV/Df (2•)• ;+ o*o» T(2;3)SbV/Df(2);+ <ft 29a F i g u r e 11. Cross t o generate T(2-3)sb progeny w i t h p r o x i m a l (2L) d e f i c i e n c i e s . 30 TABLE V I . Mean Per c e n t a g e o f Wild-Type O ) Eye S u r f a c e , sv i n +/B Y F . c f f i i e s . F^d* Genotype No. o f H i s t o n e Gene C l u s t e r s % B + Eye S i z e Sy P , SV +/B y;Df(2L)1/ + 2 53 1.9 , sv +/B Y;Df;CyO/+ .2 47 < 0.5 1.9 SV +/B Y;Df(2L)12/+ 2 43 1.1 SV +/B Y;Df;CyO/+ 2 48 >0.05 1.2 , sv +/B Y;Df(2L)84/+ 1-2 38 1.3 SV +/B Y;CyO/+ 2 44 <0.05 1.6 SV +/B Y;Df(2L)65/+ 1 26 0.9 SV +/B Y;CyO/+ 2 46 «0.05 1.1 SV +/B Y;Df(2L)161/+ 1 18 0.7 SV +/B Y;CyO/+ 2 43 « 0.05 1.9 n^ 100 f o r each genotype 31 d e f i c i e n c i e s o f the h i s t o n e gene c l u s t e r {.65,84, 161) r e s u l t s , i n a s h o r t e r mean b r i s t l e l e n g t h i n . sbV f l i e s t han does h e t e r o z y g o s i t y f o r the c o n t r o l d e f i c i e n c i e s {1,12)(Table V I I ) . The v a r i e g a t i n g autosomal gene i s 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 b r i s t l e - f o r m i n g c e l l s i n those f l i e s w h ich have a reduced gene c o n t e n t than i n those which have a normal complement. While t h i s r e s u l t does not p r e c l u d e d e f i c i e n c y e f f e c t s on dosage compensation, i t does c o n f i r m the g e n e r a l i z e d e f f e c t of h i s t o n e gene d e f i c i e n c 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 . 5. M a t e r n a l E f f e c t on V a r i e g a t i o n o f P r o x i m a l (2L) D e f i c i e n c i e s Oocytes o f s p e c i e s which undergo r a p i d c e l l u l a r p r o l i f e r -a t i o n a f t e r f e r t i l i z a t i o n c o n t a i n p o o l s o f h i s t o n e p r o t e i n and mRNA' t o f u l f i l l the needs o f chromosome r e p l i c a t i o n (1) . While i t i s not known whether D r o s o p h i l a o o c y t e s have such m-RNA p o o l s , abundant endogenous h i s t o n e p r o t e i n can be e x t r a c t e d from e a r l y embryos (42) . A h i s t o n e gene d e f i c i e n c y i n the m a t e r n a l p a r e n t would r e s u l t i n oocy t e s w i t h reduced h i s t o n e mRNA or p r o t e i n p o o l s , i f t r a n s c r i p t i o n a l c a p a c i t y was a l i m i t i n g f a c t o r i n t h e i r p r o d u c t i o n . S i n c e such a r e d u c t i o n would have i t s e f f e c t d u r i n g e a r l y development (t h e p e r i o d when some rearrangements are s e n s i t i v e t o v a r i e g a t i o n m o d i f i e r s ) , the n o n - d e f i c i e n c y progeny of h i s t o n e gene d e f i c i e n t mothers might be exp e c t e d t o e x h i b i t m o d i f i c a t i o n o f the v a r i e g a t e d phenotype (29) . The scheme o f F i g u r e 12 was d e s i g n e d t o t e s t t h i s h y p o t h e s i s , by the response o f m(i)w progeny t o m a t e r n a l h i s t o n e gene d e f i c i e n c i e s . No s i g n i f i c a n t d i f f e r e n c e can be d i s c e r n e d between the Cyo/+ progeny o f d e f i c i e n c y or 32 TABLE V I I , E f f e c t of p r o x i m a l C2L) d e f i c i e n c i e s on b r i s t l e l e n g t h v a r i e g a t i o n a s s o c i a t e d w i t h T(2;3)sbV. Genotype No. o f H i s t o n e Gene C l u s t e r s A.o£ p a r e n t a l b r i s t l e l e n g t h cf Sb + and 9 T(2;3)SbV/Df(2)1 2 77 ' (3) 97 :(4) T(2;3)SbV/Df(2)12 2 85 (4) 90 (5) T(2;3)SbV/Df(2)84 1-2 64 (3) 62 (4) T(2;3)SbV/Df(2)65 1 71 (4) 74 (3) T(2;3) SbV/Df(2 ) 1 6X". 1 49 (3) 53 (5) n^ 30 f o r each genotype V a l u e s were t e s t e d f o r t h e i r s i g n i f i c a n c e o f d i f f e r e n c e from the average c o n t r o l v a l u e f o r each sex. p< 0.05 f o r each e x p e r i m e n t a l v a l u e . 33 W™*/Wm<l; CyO/Df(2)$$ (2) W""i/Y; +/+ oV m4 , W /Y; CyO/+ d'dT rm4 , m4 nc, W /W ; CyO/+ 33a F i g u r e 12. Scheme to t e s t the m a t e r n a l e f f e c t o f p r o x i m a l (2L) d e f i c i e n c i e s on wm4 v a r i e g a t i o n , 34. c o n t r o l mothers ( T a b l e V I I I ) , H i s t o n e gene d e f i c i e n c i e s e x e r t no apparent m a t e r n a l e f f e c t on v a r i e g a t i o n . V a r i o u s hypotheses can be d e v i s e d t o e x p l a i n t h i s r e s u l t : (i) ' There are no p o o l s o f h i s t o n e m-RNA or p r o t e i n i n Drdsdphila o o c y t e s . T h i s i s i m p r o b a b l e , s i n c e h i s t o n e p r o t e i n i s abundant i n the embryo, p r i o r to the onset of z y g o t i c t r a n s c r i p t i o n (42 ,5 7). ( i i ) R e d u c t i o n o f gene m u l t i p l i c i t y i s not l i m i t i n g i n the p r o d u c t i o n o f such p o o l s . ( i i i ) The t r a n s c r i p t i o n a l f a t e o f the v a r i e g a t i n g gene i s d e t e r m i n e d a f t e r the time at which z y g o t i c h i s t o n e s supercede m a t e r n a l l y coded h i s t o n e s . A l l of the above hypotheses c o u l d be t e s t e d by a c o m b i n a t i o n o f b i o c h e m i c a l and g e n e t i c t e c h n i q u e s . 6. 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 by D u p l i c a t i o n  o f P r o x i m a l ( 2 L ) . I f v a r i e g a t i n g a l l e l e s are a c t i v e l y 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 whose genotypes bear h i s t o n e gene d e f i c i e n c i e s , i s the i n v e r s e o b s e r v e d i n genotypes which bear a d u p l i c a t i o n o f p r o x i m a l (2L)? A procedure, was d e v i s e d to answer t h i s q u e s t i o n and to a s s e s s any m a t e r n a l d u p l i c a t i o n e f f e c t s ( F i g u r e 1 3 ) . Dp(2,• 1 )c2.3.9. i n s e r t s a l l , or p a r t , o f the h i s t o n e gene c l u s t e r i n t o the e u c hromatin o f the X chromosome ( F i g u r e 5 ) . The i n t e n s i t y o f i n - s i t u h i s t o n e cRNA b i n d i n g t o the i n s e r t e d segment suggests t h a t a m a j o r i t y o f the h i s t o n e gene c l u s t e r has been d u p l i c a t e d by t h i s rearrangement. The d u p l i c a t i o n 35 CROSS 1 CROSS 2 Df (2 ; 1)C239/dl49;Cy/bwV ® v/Y;Cy/T(2;3)SbV 1 V dor /dl49;Cy/bw DpC2 39/v;Sb /Cy DpC239/v;SbV/bwV dl49/v;SbV/Cy dl49/v;SbV/bwV dl49/Y;SbV/Cy dl49/Y;SbV/bwV 1 , , V / dor /v;Sb /Cy 1 V V dor /v;Sb /bw dl49/v;SbV/Cy dl49/v;SbV/bwV dl49/Y;SbV/Cy dl49/Y;SbV/bwV 3 5a C r o s s e s t o t e s t the d i r e c t and m a t e r n a l e f f e c t o f Dp(2;1)C239 on v a r i e g a t i o n a s s o c i a t e d w i t h T(2;3)sbV. 36 TABLE V I I I . Mean p e r c e n t a g e the w i l d - t y p e amount of 777 4 d r o s o p t e r i n i n the eyes o f w /y; cyO/+ and w- /w ;CyO/+ progeny from Cyo/Df(2) mothers. No. o f m a t e r n a l M a t e r n a l genotype h i s t o n e gene c l u s t e r s % w+ d r o s o p t e r i n and CyO/Df(2)1 2 6 CO. .6) 2 CO. •2) CyO/Df(2)12 2 3 CO. •4) 1 Co. .2) CyO/Df(2)84 1-2 4 CO. •7) 2 Co. ,3) CyO/Df(2)65 1 3 CO. •4) 1 Co. .2) CyO/Df(2)161 1 3 CO. .5) 1 Co. •1) n-v 20 f o r each genotype. V a l u e s were t e s t e d f o r t h e i r s i g n i f i c a n c e o f d i f f e r e n c e from the average c o n t r o l v a l u e f o r each sex. p> 0.05 f o r each o f the e x p e r i m e n t a l v a l u e s . 37 chromosome i s l e t h a l i n the homozygous and hemizygous s t a t e s . The v a r i e g a t e d b r i s t l e phenotype a s s o c i a t e d w i t h the r e a r r a n g e -ment T(2,-3)SbV was measured i n females h e t e r o z y g o u s f o r the d u p l i c a t i o n chromosome. I t was a l s o measured i n two c o n t o l s ; female s i b l i n g s c a r r y i n g the m u l t i p l e i n v e r s i o n X chromosome, si i n ( l ) s c +dl49, and females from another c r o s s b e a r i n g the X 1 7 A chromosome p o i n t mutant, deep-orange . The l a t t e r two X chromosomes d i d not v a r y i n t h e i r a b i l i t y to m o d i f v v a r i e g a t i o n . The r e s u l t s enumerated i n T a b l e IX r e v e a l no e f f e c t on the v a r i e g a t e d phenotype when h i s t o n e gene m u l t i p l i c i t y i s i n c r e a s e d by a p p r o x i m a t e l y 50%. The p r o p o r t i o n o f b r i s t l e -f o r m i n g c e l l s which e x h i b i t e d i n a c t i v a t i o n o f the v a r i e g a t i n g gene was not s i g n i f i c a n t l y d i f f e r e n t than i n the c o n t r o l s . T h i s r e s u l t i s c o n s i s t e n t w i t h models of h i s t o n e gene a c t i v i t y w hich i n c o r p o r a t e a maximal l i m i t to the p r o d u c t i o n o f h i s t o n e p r o t e i n , independent o f h i s t o n e gene m u l t i p l i c i t y . A l t e r n a t e l y , a s u r f e i t o f c e l l u l a r h i s t o n e s may have no e f f e c t on c h r o m a t i n morphology, as r e v e a l e d by the a c t i v i t y o f v a r i e g a t i n g genes. The m a t e r n a l e f f e c t o f the h i s t o n e gene d u p l i c a t i o n c o u l d a l s o be a s s e s s e d , s i n c e i d e n t i c a l dl49/v and dl49/y progeny were o b t a i n e d from b o t h d u p l i c a t i o n or n o n - d u p l i c a t i o n mothers. ( F i g u r e 13). The v a r i e g a t e d phenotype of the progeny was not s i g n i f i c a n t l y a f f e c t e d by d i f f e r e n c e s i n m a t e r n a l h i s t o n e gene m u l t i p l i c i t y ( T a ble I X ) . T h i s r e s u l t i m p l i e s t h a t the s i z e o f the oocyte p o o l o f histone-mRNA or p r o t e i n i s not expanded by a 50% i n c r e a s e i n m a t e r n a l h i s t o n e gene m u l t i p l i c i t y . The r e s u l t c o u l d a l s o be e x p l a i n e d by p r o p o s i n g t h a t s u r p l u s 38 m a t e r n a l l y - c o d e d h i s t o n e s are not assembled i n t o embryonic c h r o m a t i n , or t h a t such m a t e r n a l l y - c o d e d h i s t o n e s are e f f e c t i v e p r i o r to the d e t e r m i n a t i o n o f the t r a n s c r i p t i o n a l f a t e o f the v a r i e g a t i n g gene. 39 TABLE IX. Mean p e r c e n t a g e o f d o r s o - c e n t r a l and s c u t e l l a r b r i s t l e s w i t h a sb phenotype i n T(2;3)Sbv progeny from p r o x i m a l (2L) d u p l i c a t i o n and n o n - d u p l i c a t i o n mothers. No. o f No. o f h i s t o n e m a t e r n a l gene h i s t o n e gene 1 Sb Cross 1 progeny genotype c l u s t e r s c l u s t e r s b r i s t l e s Sy DpC239/v;SbV/Cy 3 3 47 14 DpC2 3 9/v;SbV/bwV 3 3 52 9 dl49/v;SbV/cy 2 3 55 9 dl49/v;sbV/bwV 2 3 44 6 dl49/Y;SbV/Cy * 2 3 49 11 dl49/Y;sbV/bwV * 2 3 67 9 Cross 2 progeny genotype dor1/v;SbV/Cy 2 2 45 10 dor1/v;SbV/bwV 2 2 46 6 dl49/v;SbV/Cy 2 2 48 5 dl49/v;SbV/bwV 2 2 46 7 dl49/Y;SbV/Cy * 2 2 45 10 dl49/Y;SbV/bwV * 2 2 69 6 * Due t o the SC phenotype, o n l y the d o r s o - c e n t r a l b r i s t l e s c o u l d be s c o r e d , n rj 20. f o r each genotype. 40 DISCUSSION A l t e r a t i o n o f h i s t o n e gene m u l t i p l i c i t y has not been d i r e c t l y c o r r e l a t e d to changes i n the amount of c e l l u l a r h i s t o n e p r o t e i n . I t seems improbable t h a t any r e d u c t i o n i n h i s t o n e amount i s e q u i v a l e n t t o the 50% 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 a s s o c i a t e d w i t h the h e t e r o z y g o u s d e f i c i e n c i e s . However, the r e s u l t s can be i n t e r p r e t e d t o suggest t h a t compensation f o r the r e d u c t i o n i n h i s t o n e c o d i n g c a p a c i t y i s i n c o m p l e t e , and t h a t the l e v e l o f c e l l u l a r h i s t o n e i s p a r t i a l l y r e duced d u r i n g the p e r i o d when the t r a n s c r i p t i o n a l f a t e o f v a r i e g a t i n g g-enes i s d e t e r m i n e d . T h i s i n t e r p r e t a t i o n i s s u p p o r t e d by the o b s e r v a t i o n o f MOTTUS et a l . (40) t h a t • n - b u t y r a t e , an agent which a p p a r e n t l y reduces h i s t o n e - DNA i n t e r a c t i o n , a l s o enhanced the a c t i v i t y o f v a r i e g a t i n g genes. The l a c k o f e f f e c t o f i n c r e a s e d h i s t o n e gene numbers on v a r i e g a t i o n suggests t h a t h i s t o n e p r o t e i n p r o d u c t i o n has an upper l i m i t , not dependent on gene m u l t i p l i c i t y ; or t h a t c h r o m a t i n s t r u c t u r e i s u n a f f e c t e d by a super - abundance of h i s t o n e s . The l a t t e r e x p l a n a t i o n i s not f a v o u r e d , s i n c e SPERLING and WEISS (48) have demonstrated t h a t c h r o m a t i n •-w i t h a c h a r a c t e r i s t i c i n t e r n u c l e o s o m a l s p a c i n g w i l l a l t e r i t s s p a c i n g i n response t o c e l l f u s i o n w i t h a c e l l type h a v i n g a d i f f e r e n t s p a c i n g l e n g t h . NELSON et a l . (42) have i s o l a t e d a f a c t o r from D r o s o p h i l a embryos which mediates the assembly of nucleosomes on DNA. I t i s p o s s i b l e t h a t the abundance of t h i s f a c t o r l i m i t s the r a t e at which h i s t o n e i s i n c o r p o r a t e d i n t o c h r o m a t i n . 41 The r e s u l t s do not e n t i r e l y e l i m i n a t e the p o s s i b i l i t y t h a t the observed 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 i s due to the d e l e t i o n o f a gene t i g h t l y l i n k e d t o the h i s t o n e gene c l u s t e r , r a t h e r than d e l e t i o n o f the c l u s t e r i t s e l f . I t s h o u l d be n o t e d , however, t h a t h e t e r o z y g o t e s f o r any c o n t r o l d e f i c i e n c y d i s t a l t o the c l u s t e r (Df(2) 1,12, DS8 , DS9 ) do not e x h i b i t more a c t i v e e x p r e s s i o n o f v a r i e g a t i n g genes t h a n do n o n - d e f i c i e n c y c o n t r o l s . F l i e s h e t e r o z y g o u s f o r d e f i c i e n c i e s t h a t do not extend p r o x i m a l l y beyond the c l u s t e r (of(2)84,DS5) do e x h i b i t s u p p r e s s i o n o f v a r i e g a t i o n . Hence, i f d e l e t i o n o f a l o c u s o u t s i d e the h i s t o n e gene c l u s t e r i s r e s p o n s i b l e f o r the r e s u l t s o b t a i n e d , i t must be c l o s e l y l i n k e d d i s t a l t o the c l u s t e r . A r e c e n t s t u d y by HENIKOFF (26) suggests t h a t v a r i e g a t i o n m o d i f i e r l o c i may be as f r e q u e n t as one per 25 chromomeres, and p o s s i b l y are s i t e s o f n o n - h i s t o n e chromosomal p r o t e i n genes. The p a r t i a l d e f i c i e n c i e s o f the d i s t a l r e g i o n o f the h i s t o n e gene c l u s t e r (.Df (2) 84, DS5 ) do not cause an i n t e r -mediate enhancement of the v a r i e g a t i n g gene a c t i v i t y a s s o c i a t e d w i t h wm4 or sbv, a l t h o u g h an i n t e r m e d i a t e e f f e c t sv on B was ob s e r v e d . The p o r t i o n o f the c l u s t e r w h i c h t h e s e d e f i c i e n c i e s do not d e l e t e may be so s m a l l as to be i n c o n s e q u e n t i a l . A l t e r n a t e l y , the p o r t i o n s o f the c l u s t e r w h ich they do d e l e t e may c o n t a i n a subset o f h i s t o n e genes r e s p o n s i b l e f o r c h r o m a t i n c o n d e n s a t i o n d u r i n g the p e r i o d when the t r a n s c r i p t i o n a l f a t e o f p a r t i c u l a r v a r i e g a t i n g genes i s de t e r m i n e d . The genes removed by DS2, which d e l e t e s the 42 p r o x i m a l segment o f the c l u s t e r do not have an apparent e f f e c t on the v a r i e g a t i o n of wm4. These f i n d i n g s are c o n s i s t e n t w i t h a model i n which the h i s t o n e gene c l u s t e r i s f u n c t i o n a l l y d i f f e r e n t i a t e d . Is t h e r e e v i d e n c e f o r d i f f e r e n t i a t i o n among the genes f o r a h i s t o n e p r o t e i n ? Is t h i s d i f f e r e n t i a t i o n r e f l e c t e d i n the o r g a n i z a t i o n o f the h i s t o n e genes? E x t r a c t i o n of the HI, H2A and H2B mRNAs from the b l a s t u l a , mesenchyme b l a s t u l a and g a s t r u l a s t a g e s o f s t o n g y l o c e n t r o t u s purpuratus sea u r c h i n embryos, and t h e i r t r a n s l a t i o n i n - v i t r o , y e i l d s s t a g e - s p e c i f i c sequence v a r i a n t s o f these p r o t e i n s ( 5 2 ) . Sequence v a r i a t i o n i s obser v e d i n the H4 mRNA from e a r l y and l a t e d e v e l o p m e n t a l s t a g e s o f another sea u r c h i n , L y t e c h i n u s p i c t u s ( 2 3 ) . H i s t o n e mRNAs a l s o v a r y i n l e n g t h d u r i n g echinoderm development ( 2 3 ) . The de v e l o p m e n t a l s i g n i f i c a n c e of thes e s h i f t s i n histone' gene e x p r e s s i o n has not been e l u c i d a t e d . LIFTON e t a l . (36) r e p o r t e d two p r i n c i p a l v a r i a n t s of the D r o s o p h i l a h i s t o n e gene r e p e a t u n i t , d i f f e r i n g by a 250 base p a i r i n s e r t i n a non-coding s p a c e r segment. Rare v a r i a n t s , c o n t a i n i n g l a r g e r i n s e r t s i n spa c e r r e g i o n s have a l s o been observed (30) . STRASBAUGH and WEINBERG (50) i d e n t i f i e d more e x t e n s i v e v a r i a t i o n i n r e p e a t u n i t l e n g t h among D r o s o p h i l a s t r a i n s and i n d i v i d u a l s . A g a i n , the f u n c t i o n a l s i g n i f i c a n c e o f these v a r i a n t s i s u n c l e a r . The o r g a n i z a t i o n o f the r e p e a t u n i t v a r i a n t s w i t h i n the h i s t o n e gene c l u s t e r i s unknown. I t i s apparant from the occurence and te m p o r a l s p e c i f i c i t y o f h i s t o n e v a r i a n t s i n sea u r c h i n development, t h a t s u b s e t s of the h i s t o n e genes have p a r t i c u l a r d e v e l o p m e n t a l f u n c t i o n s . 43 The n a t u r e o f these f u n c t i o n s w i l l be e l u c i d a t e d o n l y by the development o f i n v i t r o t r a n s c r i p t i o n systems which can a s s e s s the s p e c i f i c e f f e c t s of h i s t o n e s a r i s i n g from these v a r i a n t sequences. The q u e s t i o n of f u n c t i o n a l s e g r e g a t i o n o f v a r i a n t sequences w i t h i n the h i s t o n e gene c l u s t e r may y i e l d to a g e n e t i c approach. The stage - s p e c i f i c e f f e c t s o f s m a l l , p a r t i a l d e l e t i o n s o f the c l u s t e r on phenomena i n f l u e n c e d by c h r o m a t i n morphology c o u l d be assayed. In a d d i t i o 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 , t h e s e phenomena i n c l u d e m i t o t i c r e c o m b i n a t i o n and mutagen s e n s i t i v i t y ( 4 7 ) . B i o c h e m i c a l parameters such as n u c l e a s e s e n s i t i v i t y and i n t e r n u c l e o s o m a l s p a c i n g c o u l d a l s o be t e s t e d . C e r t a i n f e a t u r e s o f the s t r u c t u r e and o r g a n i z a t i o n of the h i s t o n e genes have been i m p l i c a t e d i n t h e c o n t r o l o f t h e i r e x p r e s s i o n . I t i s assumed t h a t the arrangement of c o d i n g sequences i n t o a u n i t a r y sequence s e r v e s t o ensure c o o r d i n a t e t r a n s c r i p t i o n , s i n c e , w i t h the e x c e p t i o n of H I , the h i s t o n e s are p r e s e n t i n e q u i m o l a r amounts i n the nucleosome ( 3 6 ) . However, no p o l y c i s t r o n i c t r a n s c r i p t c o r r e s p o n d i n g t o the r e p e a t u n i t has been fou n d , a l t h o u g h h i g h m o l e c u l a r weight p r e c u r s o r s are observed i n some systems (39,35). IL\CKETT et a l . ""(.24) .concluded t h a t p o l y c i s t r o n i c h i s t o n e messengers do not e x i s t i n HeLa c e l l s , s i n c e the i n d u c t i o n o f t h y m i d i n e dimers (which a c t as t r a n s c r i p t i o n t e r m i n a t o r s ) does not s e l e c t i v e l y reduce the message f o r any subset o f the h i s t o n e s . In D r o s o p h i l a , the "sense" sequences f o r H2B and H4 are a r r a y e d on the complementary s t r a n d to those o f the o t h e r h i s t o n e s , e l i m i n a t i n g 44 the p o s s i b i l i t y o f a s i n g l e t r a n s c r i p t f o r a l l f i v e p r o t e i n s ( 3 6 ) . E x t e n s i v e s t u d y of the te m p o r a l a c t i v i t y o f h i s t o n e genes i n s o m a t i c c e l l s has g e n e r a l l y r e v e a l e d a c l o s e l i n k a g e t o DNA r e p l i c a t i o n . H i s t o n e mRNA can o n l y be r e c o v e r e d from S phase somati c c e l l c h r o m a t i n , and agents which a b o l i s h DNA r e p l i c a t i o n a b r u p t l y reduce h i s t o n e mRNA te m p l a t e a c t i v i t y ( 6 , 4 2 ) . MELLI et. a l . (39) r e p o r t e d the presence o f n u c l e a r h i s t o n e RNA t r a n s c r i p t s made throughout the HeLa c e l l c y c l e , but DETKE e t a l , (15) u s i n g a more e f f e c t i v e c e l l s y n c h r o n i z a t i o n p r o c e d u r e , were unable t o d u p l i c a t e t h i s f i n d i n g . D u r i n g gametogenesis, h i s t o n e p r o d u c t i o n i s independ-ant o f DNA r e p l i c a t i o n . H i s t o n e s are made some weeks subsequent t o the c e s s a t i o n o f DNA r e p l i c a t i o n i n gras s h o p p e r s p e r m a t i d s and o t h e r systems o f spermatogenesis ( 4 ) . H i s t o n e p r o t e i n and message are s t o c k p i l e d d u r i n g echinoderm and amphibian oogenesis f o r use a f t e r f e r t i l i z a t i o n ( 1 , 2 1 ) . The mechanism r e s p o n s i b l e f o r the te m p o r a l l i n k a g e o f h i s t o n e gene a c t i v i t y and DNA r e p l i c a t i o n i n som a t i c c e l l s i s unknown; however, i t i s i n t e r e s t i n g t o note t h a t the h i s t o n e gene r e g i o n i s the l a s t e u c h r o m a t i c segment r e p l i c a t e d d u r i n g the D r o s o p h i l a c e l l c y c l e ( 2 0 ) . At the l e v e l o f the i n d i v i d u a l h i s t o n e gene, a "pseudo-g e n e t i c " approach has probed the f u n c t i o n o f the a d j a c e n t 5' non-coding sequences (22) . A c l o n e d fragment c o n t a i n i n g a sea u r c h i n H2A gene was s u b j e c t e d t o r e s t r i c t i o n n u c l e a s e d i g e s t i o n s so as t o c r e a t e a s e r i e s o f d e l e t i o n s o f the 5' l e a d e r sequence. The d e l e t i o n "mutants" were t e s t e d f o r 45 t r a n s c r i p t i o n a l e f f i c i e n c y i n the Xenopus oocyte system. The l e a d e r sequence was r e v e a l e d t o have b o t h p o s i t i v e and n e g a t i v e c o n t r o l f u n c t i o n s . In c o n c l u s i o n , w h i l e t h e r e i s a c o m p a r a t i v e w e a l t h o f i n f o r m a t i o n about the sequence, o r g a n i z a t i o n and a c t i o n o f the h i s t o n e genes, those f e a t u r e s o f t h e i r arrangement r e s p o n s i b l e f o r d i f f e r e n t i a l e x p r e s s i o n a r e , as y e t , p o o r l y c h a r a c t e r i z e d . I t i s hoped t h a t f u r t h e r m u t a t i o n a l a n a l y s i s o f the D r o s o p h i l a h i s t o n e gene c l u s t e r w i l l i d e n t i f y those components which c o n t r o l i t s e x p r e s s i o n d u r i n g the c e l l and d e v e l o p m e n t a l c y c l e s . 46 SUMMARY H i s t o n e p r o t e i n s are r e s p o n s i b l e f o r the compaction of DNA i n t o the p r i m a r y s t r u c t u r a l u n i t o f c h r o m a t i n , the nucleosome, and a c t as non - s p e c i f i c r e p r e s s o r s o f t r a n s c r i p t i o n . The arrangement o f h i s t o n e genes i s s i m i l a r i n most s p e c i e s , but l i t t l e i s known about the s t r u c t u r a l f e a t u r e s which c o n t r o l t h e i r e x p r e s s i o n . The e f f e c t o f a l t e r e d h i s t o n e gene m u l t i p l i c i t y has been assayed by m o n i t o r i n g m o d i f i c a t i o n o f a phenomenon which i s s e n s i t i v e t o change i n c h r o m a t i n m o r p h o l o g y : p o s i t i o n - e f f e e t v a r i e g a t i o n . In D r o s o p h i l a , h e t e r o z y g o s i t y f o r a d e f i c i e n c y which removes the h i s t o n e gene c l u s t e r r e s u l t s i n an i n c r e a s e i n the p r o p o r t i o n of c e l l s i n which a v a r i e g a t i n g l o c u s i s t r a n s c r i p t i o n a l l y a c t i v e . T h i s e f f e c t i s o b s e r v e d i n males and f e m a l e s , and a p p l i e s t o v a r i e g a t i n g a l l e l e s t h r o u g h o u t the genome. The r e s u l t s are c o n s i s t e n t w i t h a h y p o t h e s i s which p r e d i c t s t h a t a r e d u c t i o n i n the amount of c e l l u l a r h i s t o n e p r o t e i n would cause a s t r u c t u r a l m o d i f i c a t i o n o f the c h r o m a t i n a t the v a r i e g a t i n g l o c u s . D u p l i c a t i o n of the h i s t o n e gene c l u s t e r does not a f f e c t v a r i e g a t i o n , s u g g e s t i n g an upper l i m i t to the p r o d u c t i o n o f h i s t o n e p r o t e i n , independent of gene m u l t i p l i c i t y . N e i t h e r i n c r e a s e , nor r e d u c t i o n o f the h i s t o n e gene number i n the m a t e r n a l genome a l t e r e d the v a r i e g a t e d phenotype of the progeny. T h e r e f o r e , the e f f e c t i s not oocyte t r a n s m i s s i b l e . P a r t i a l d e f i c i e n c i e s o f the h i s t o n e gene c l u s t e r do not have c o n s i s t e n t e f f e c t s on the m o d i f i c a t i o n of v a r i e g a t i o n , 47 i m p l y i n g a f u n c t i o n a l h e t e r o g e n e i t y w i t h i n the c l u s t e r , as has been proposed f o r o t h e r systems. 48 BIBLIOGRAPHY Adamson, E.D. and Woodland, H.R. (1977) Changes i n the r a t e o f h i s t o n e s y n t h e s i s d u r i n g oocyte m a t u r a t i o n and v e r y e a r l y development o f xenopus l a e v i s . Develop. B i o l . 5J7: 136-149. Baker, 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 . In "Advances i n G e n e t i c s " (E.W. C a s p a r i , Ed.) 14_: 133-169. B e r l o w i t z , L. (1965) A n a l y s i s o f h i s t o n e i n - s i t u i n d e v e l o p m e n t a l l y i n a c t i v a t e d c h r o m a t i n . P r o c . Nat. Acad. S c i . (U.S.A.) 54: 476-480. B l o c h . D.P. and B r o c k , S.D. (1964) E v i d e n c e f o r the c y t o p l a s m i c s y n t h e s i s of n u c l e a r h i s t o n e d u r i n g s p e r m i o g e n e s i s i n the grasshopper chortophaga v i v i d i f a s c i a t a . (DE GEER). J . C e l l B i o l . 2_2: 327-340. B l u m e n f e l d , M., O r f , J.W., S i n a , B . J . , K r e b e r , R.A. C a l l a h a n , M.A., M u l l i n s , J . I . and Snyder, L.A. (1978) C o r r e l a t i o n between p h o s p h o r y l a t e d HI h i s t o n e s and s a t e l l i t e DNAs i n D r o s o p h i l a v i r i l i s . P r o c . Nat. Acad. S c i . (U.S.A.) 75_: 866-870. Borun. T.W. (1975) H i s t o n e s , d i f f e r e n t i a t i o n , and the c e l l c y c l e . In. " R e s u l t s and Problems' i n C e l l D i f f e r e n t i a t i o n " , V o l 7. ( J . R e i n e r t and H. H o l t z e r , e d s . ) , p. 249-290. B r i d g e s , C. (1936) The Bar "gene" a d u p l i c a t i o n . S c i e n c e 83: 210-211. B r o s s e a u , G.E. J r . (1960) V-type p o s i t i o n e f f e c t s i n f l u e n c i n g the a c t i o n o f the Bar l o c u s i n D r o s o p h i l a . G e n e t i c s 45_: 979. B r o s s e a u , G.E. J r . (1964) E v i d e n c e t h a t h e t e r c h r o m a t i n does not suppress V-type p o s i t i o n - e f f e c t . G e n e t i c s 50_: 237. B r u t l a g , D., C a r l s o n , M., F r y , K. and H s i e h , T.S. (1977) DNA sequence o r g a n i z a t i o n i n 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 . C o l d S p r i n g Harb. Symp. Quant. B i o l . 4_2: 1137-1146. C a t t a n a c h , B.M. (1974) 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 the mouse. Genet. Res. Camb. 23: 921-306. 49 C h a n d l e r , M. , Kedes, L.H., Cohn, R.H. and Y u n i s , J . J . (1979) Genes c o d i n g f o r h i s t o n e p r o t e i n s i n man are " l o c a t e d on the d i s t a l end o f the l o n g arm o f chromosome 7. S c i e n c e 205: 908-910. C o l e , R.D. ,'Lawson, G.M. and H s i a n g , M.W. (1977) HI h i s t o n e and the c o n d e n s a t i o n o f c h r o m a t i n and DNA. C o l d S p r i n g Harb. Symp. Quant. B i o l . 42_: 253-263. C r a w f o r d , R.J., K r i e g , P., Harvey, R.P., Hewish, D.A. and W e l l s , J.R.E. (1979) H i s t o n e genes are c l u s t e r e d w i t h a 1 5 - k i l o l o a s e r e p e a t i n the c h i c k e n genome. Nature 279:132-136. Detke, S., L i c h t l e r , A., P h i l l i p s , I . , S t e i n , J . and S t e i n , G. (1979) Reassessment o f h i s t o n e gene e x p r e s s i o n d u r i n g the c e l l c y c l e i n human c e l l s by u s i n g homologous H4 h i s t o n e cDNA. P r o c . Nat. Acad. S c i . (U.S.A.) 76: 4995-4999. Demerec, M. and S l i z y n s k a , H. (1937) M o t t l e d w h i t e 258-18 o f D r o s o p h i l a melanogaster G e n e t i c s 22: 641-649. E p h r u s s i , B. and H e r o l d , J.L. (1944) S t u d i e s o f eye pigments o f D r o s o p h i l a . " I . 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G r o s s c h e d l , R. and B i r n s t i e l , M.L. (1980) I d e n t i f i c a t i o n o f r e g u l a t o r y sequences i n the p r e l u d e sequences of an H2A h i s t o n e gene by the stu d y o f s p e c i f i c d e l e t i o n mutants in_ v i v o . P r o c . Nat. Acad. S c i . (U.S.A.) 77: 1432-1436. 50 G r u n s t e i n , M. (1978) H a t c h i n g i n the sea u r c h i n L y t e c h i n u s p i c t u s i s accompanied by a s h i f t i n H4 gene a c t i v i t y . P r o c . Nat. Acad. S c i . (U.S.A.) 75: 4135-4139. H a c k e t t , P.B., Traub, P. and G a l l w i t z , D. (1978) The h i s t o n e gene i n HeLa c e l l s are on i n d i v i d u a l t r a n s c r i p t i o n a l u n i t s . J . M o l . B i o l . 126: 619-649. H a r t m a n n - G o l d s t e i n , I . J . 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(1970) Bestimmung des H e t e r o c h r o m a t i n i s i e r u n g s s t a d i u m beim white- P o s i t i o n e f f e c k t m i t t l e s r o n t g e n -i n d u z i e r t e r m i t o t i s c h e r Rekombination i n der Augenanlage von D r o s o p h i l a melanogaster. Molec. Genet. 107: 128-149. Karp, R. (1979) Ph.D. T h e s i s . S t a n f o r d U n i v . C a l i f o r n i a . Kedes, L.H. and B i r n s t i e l , M.L. (1971) R e i t e r a t i o n and c l u s t e r i n g o f DNA sequences complementary to h i s t o n e messenger RNA. Nat. New B i o l . 230: 165-169. Kedes, L.H., Cohn, R.H., Lowry, J.C., Chang, A.C.Y. and Cohen, S.N. (1975) The o r g a n i z a t i o n o f the sea u r c h i n h i s t o n e genes. C e l l . 6_: 359-369. Kedes, L.H. (1979) H i s t o n e genes and h i s t o n e messengers, In "Annual Revue o f B i o c h e m i s t r y " (E.E. S n e l l e t a l . , eTJs.) 48: 837-870. 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(1976) The g e n e t i c s o f d o p a - d e c a r b o x y l a s e i n D r o s o p h i l a melanogaster. I . I s o l a t i o n and c h a r a c t e r i z a t i o n of d e f i c i e n c i e s t h a t d e l e t e the do p a - d e c a r b o x y l a s e -d o s a g e - s e n s i t i v e r e g i o n and the -methyl-dopa-h y p e r s e n s i t i v e l o c u s . G e n e t i c s 84^: 267-285. Y u n i s , J . J . and Yasmineh, W.G. (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 174:1200-1209 Z a l o k a r , M. (1976) A u t o r a d i o g r a p h i c s t u d y o f p r o t e i n and RNA f o r m a t i o n d u r i n g e a r l y development o f D r o s o p h i l a eggs. Develop. B i o l . 49: 425-437. 53 58. Z i e g l e r , I . (1961) G e n e t i c a s p e c t s o f oomochrome and p t e r i n pigments. Ln "Advances i n G e n e t i c s . " (E.W. C a s p a r ! and J.M. Thodav, eds.) 10: 349-403. 59. Z u c k e r k a n d l , E. (1974) A p o s s i b l e r o l e o f " i n e r t " h e t e r o c h r o m a t i n i n c e l l d i f f e r e n t i a t i o n . A c t i o n o f and c o m p e t i t i o n f o r " l o c k i n g " m o l e c u l e s . B i o c h i m i e 56: 937-954. 

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