Science, Faculty of
Zoology, Department of
DSpace
UBCV
Fitzpatrick, Kathleen Anne
Master of Science - MSc
University of British Columbia
Dysgenic male recombination (MR) induced by the P strains T-007 and OKI rarely, if ever, occurs in the heterochromatin of chromosome two. One possible explanation is that the lack of heterochromatic exchange is due to the highly condensed chromatin in this region. Butyrate (a suspected modifier of chromatin structure) induced significant levels of heterochromatic MR in dysgenic hybrids derived from crosses involving two different P strains. This finding is consistent with the hypothesis that chromatin structure can influence the insertion and excision of P elements and hence MR. Analogous experiments were performed using third chromosome suppressor of variegation (Su(var)) mutations. Neither suppressor mutation induced any heterochromatic MR, suggesting that the mode of action of these Su(var) genes is different from, and more specific than, that of butyrate. One of the mutations (325) which is thought to influence meiotic recombination frequencies, causes some alterations in euchromatic MR in crosses involving the OKI strain. The other mutation, 318, affects neither meiotic nor dysgenic recombination. Su(var) 325 is the first known "factor" to influence meiotic and dysgenic recombination similarly.
THE EFFECT OF CHROMATIN STRUCTURE ON P ELEMENT-INDUCED MALE RECOMBINATION IN DROSOPHILA MELANOGASTER BY KATHLEEN ANNE FITZPATRICK B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA © September, 1985 Kathleen F i t z p a t r i c k , 1985 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 or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoo^O^Y The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT Dysgenic male recombination (MR) induced by the P s t r a i n s T-007 and OKI r a r e l y , i f ever, o c c u r s i n the h e t e r o c h r o m a t i n of chromosome two. One p o s s i b l e e x p l a n a t i o n i s that the lack of h e t e r o c h r o m a t i c exchange i s due to the h i g h l y condensed chromatin i n t h i s region. Butyrate (a suspected m o d i f i e r of c h r o m a t i n s t r u c t u r e ) i n d u c e d s i g n i f i c a n t l e v e l s o f h e t e r o c h r o m a t i c MR i n d y s g e n i c h y b r i d s d e r i v e d from c r o s s e s i n v o l v i n g two d i f f e r e n t P s t r a i n s . T h i s f i n d i n g i s c o n s i s t e n with the hypothesis that chromatin s t r u c t u r e can i n f l u e n c e the i n s e r t i o n and e x c i s i o n of P elements and hence MR. Analogous experiments were performed using t h i r d chromosome suppressor of v a r i e g a t i o n ( S u ( v a r ) ) m u t a t i o n s . N e i t h e r s u p p r e s s o r mutation induced any heterochromatic MR, suggesting that the mode of a c t i o n of t h e s e Su(var) genes i s d i f f e r e n t from, and more s p e c i f i c than, t h a t of b u t y r a t e . One of the m u t a t i o n s (325) which i s thought to i n f l u e n c e m e i o t i c r e c o m b i n a t i o n f r e q u e n c i e s , causes some a l t e r a t i o n s i n e u c h r o m a t i c MR i n c r o s s e s i n v o l v i n g the OKI s t r a i n . The o t h e r m u t a t i o n , 3_18, a f f e c t s n e i t h e r m e i o t i c nor dysgenic recombination. Su(var) 325 i s the f i r s t known " f a c t o r " to i n f l u e n c e m e i o t i c and dysgenic recombination s i m i l a r l y . i i i TABLE OF CONTENTS ABSTRACT i i L I S T OF TABLES i i i L I S T OF FIGURES i v ACKNOWLEDGEMENTS v INTRODUCTION 1 MATERIALS AND METHODS 16 RESULTS 24 DISCUSSION 39 CONCLUSIONS 53 L I T E R A T U R E C I T E D 55 APPENDIX I 64 iv LIST OF TABLES TABLE 1. TABLE 2. Recombination data, in each genetic i n t e r v a l , for control dysgenic crosses involving P strains T-007 and OKI .... Page 25 Recombination data, in each genetic i n t e r v a l , for butyrate treated dysgenic crosses involving P strains T-007 and OKI 29 TABLE 3. TABLE 4. Recombination data, in each genetic i n t e r v a l , for dysgenic crosses involving P strains T-007 and OKI, to which the mutant Su(var)325 has been introduced 33 Recombination data, in each genetic i n t e r v a l , for dysgenic crosses involving P strains T-007 and OKI, to which the mutant Su(var)318 has been introduced 35 V LIST OF FIGURES Page FIGURE 1. M u l t i p l y marked second chromosome used i n a l l experiments 18 FIGURE 2. Mating scheme used i n untreated and butyrate t r e a t e d (a) and Su(var) (b) experiments 19 FIGURE 3. Second chromosome maps de r i v e d from recombination data of dysgenic c r o s s e s i n v o l v i n g the P s t r a i n T-007 37 FIGURE 4. Second chromosome maps d e r v i e d from recombination data of dysgenic crosses .involving the P s t r a i n OKI 38 v i ACKNOWLEDGEMENTS I w i s h t o t h a n k my t h e s i s s u p e r v i s o r , Dr. T.A. G r i g l i a t t i , f o r h i s support of, and i n t e r e s t i n , my research. Dr. D.A.R. S i n c l a i r was an u n f a i l i n g s o u r c e of encouragement and suggestions. For i l l u m i n a t i n g d i s c u s s i o n s (some of them about s c i e n c e ) , I am g r a t e f u l to J. Leung, Dr. R.H. D e v l i n , B. Hansen, and Dr. A. R u d d e l l . In moments of c r i s i s ( r e a l or i m a g i n e d ) , my f r i e n d s : Y v e t t e L l o y d , A n n e t t e B a i l e y , and Kathy K a f e r , made the p r o s p e c t of f a c i n g the f l i e s a g a i n a bearable one. 1 INTRODUCTION While l o o k i n g f o r s e g r e g a t i o n d i s t o r t i o n e f f e c t s i n w i l d caught s t r a i n s of D r o s o p h i l a m e l a n o g a s t e r , H i r a i z u m i (1971) noted low but s i g n i f i c a n t l e v e l s of male recombination. This f i n d i n g c o n t r a d i c t e d the long e s t a b l i s h e d b e l i e f that m e i o t i c r e c o m b i n a t i o n d i d n o t o c c u r i n male D^ m e^a. ii££ a^ js _t e_ r_. H i r a i z u m i ' s o b s e r v a t i o n i m m e d i a t e l y prompted the e x t e n s i v e i n v e s t i g a t i o n of the s p e c i a l circumstances under which MR can a r i s e . Since the i n i t i a l r e p o r t s ( H i r a i z u m i , 1971; H i r a i z u m i e t a 1., 1973), male r e c o m b i n a t i o n among the progeny of w i l d caught D r o s o p h i l a melanogaster i n d i v i d u a l s has been observed and s t u d i e d by numerous i n v e s t i g a t o r s : Voelker, 1974; Waddle and Oster, 1984; Sved, 1974; 1976; C a r d i l l i n o and Mukai, 1975; K i d w e l l and K i d w e l l , 1975; Yamaguchi, 1976;Yannopou1os and P e l e c a n o s , 1977; Woodruff and Thompson, 1977; Green, 1977; G o l u b o v s k y ^_t a l . , 1977. In a l l c a s e s the r e c o m b i n a t i o n was o b s e r v e d i n male h y b r i d s d e r i v e d from c r o s s e s between male parents from r e c e n t l y w i l d caught s t r a i n s and female parents f r o m e s t a b l i s h e d l a b o r a t o r y s t r a i n s . However, male r e c o m b i n a t i o n i s o n l y one of s e v e r a l t r a i t s d i a g n o s t i c of a syndrome termed hyb r i d dysgenesis ( K i d w e l l e_t a l . , 1977). H y b r i d d y s g e n e s i s ( r e v i e w e d i n B r e g l i a n o e_t a l . , 1980; Green, 1980; B r e g l i a n o and K i d w e l l , 1983; and E n g e l s , 1983) 2 c o n s i s t s of a set of genetic and p h y s i o l o g i c a l a b n o r m a l i t i e s which i n c l u d e the i n d u c t i o n of s i n g l e s i t e v i s i b l e and l e t h a l m u t a t i o n s , chromosome r e a r r a n g e m e n t s (Berg e_t a l . , 1980; Yannopoulos et a l . , 1983), m i t o t i c r e c o m b i n a t i o n i n f e m a l e s ( S l a t k o , 1978; S i n c l a i r and Green, 1979) as w e l l as males, chromosomal non-dysjuction, unequal t r a n s m i s s i o n of homologous chromosomes, and g o n a d a l d y s g e n e s i s ( G. D.) s t e r i l i t y . These a b n o r m a l i t i e s seem to be r e s t r i c t e d to the germ l i n e and occur before and/or during m e i o s i s . H y b r i d d y s g e n e s i s o c c u r s o n l y i n t h e F l o f f s p r i n g r e s u l t i n g from matings between males from w i l d p o p u l a t i o n s and females from l a b o r a t o r y s t r a i n s . Progeny from the r e c i p r o c a l c r o s s ( l a b o r a t o r y males c r o s s e d to w i l d caught f e m a l e s ) d i s p l a y the t r a i t s at a frequency at l e a s t t e n - f o l d lower than do those from the dysgenic c r o s s . I n t r a - s t r a i n c r o s s e s r a r e l y i f ever produce dysgenic o f f s p r i n g . Thus, w i l d caught s t r a i n s were named P ( f o r p a t e r n a l ) , and l a b o r a t o r y s t r a i n s M ( f o r maternal) . E a r l y s p e c u l a t i o n s i n t o the e t i o l o g y of the d y s g e n i c anomalies i n c l u d e d : i n f e c t i o u s agents such as v i r u s e s causing random chromosome breakage (Sved, 1978), s i n g l e l o c u s mutator e f f e c t s , and c h r o m o s o m a l - c y t o p l a s m i c i n t e r a c t i o n s (Sved, 1976). The l a t t e r i s c o n s i s t e n t w i t h the n o n - r e c i p r o c a l n a t u r e of the syndrome. To produce the d y s g e n i c symptoms, P chromosomes must be placed i n an M-type cytoplasm. In theory 3 some agent (or the l a c k t h e r e o f ) i n the M cytoplasm i n t e r a c t s w i t h some component or p r o d u c t of the P chromosomes. The n a t u r e of the " a g e n t s " or "components" was vague, a l t h o u g h there was some suggestion (Engels, 1979) of an analogy between h y b r i d d y s g e n e s i s and z y g o t i c i n d u c t i o n of b a c t e r i o p h a g e lambda. S i n g l e l o c u s e f f e c t s were r u l e d out when major "mutator" e l e m e n t s c o u l d not be a c c u r a t e l y mapped to any s i n g l e chromosomal p o s i t i o n ( S l a t k o and Green, 1980). A p i v o t a l o b s e r v a t i o n w h i c h h e l p e d to p r o m o t e t h e i n f e c t i o u s agent or v i r a l hypothesis was the f i n d i n g that i n hybrids P chromosomes could "contaminate" M chromosomes i n a matter of s e v e r a l generations, i.e. the M chromosomes took on P - l i k e c h a r a c t e r i s t i c s and c o u l d produce d y s g e n e s i s when introduced back i n t o the o r i g i n a l M s t r a i n (see B r e g l i a n o and K i d w e l l , 1983). However, v i r u s e s were n o t o b s e r v e d c o n s i s t e n t l y i n P s t r a i n s and f u r t h e r m o r e , the v i r u s t h e o r y < a l o n e c o u l d not e x p l a i n the n o n - r e c i p r o c a l a s p e c t of the i n t e r a c t i o n . D e s p i t e the f a c t t h a t male r e c o m b i n a t i o n (MR) was the f i r s t known i n d i c a t o r of dysgenesis, the emphasis soon s h i f t e d to the i n v e s t i g a t i o n of m u t a t i o n i n d u c t i o n , a somewhat more e a s i l y observed aspect of dysgenesis. E a r l y i n s i g h t s i n t o the n a t u r e of d y s g e n i c a l l y i n d u c e d m u t a t i o n s came from Green (1977). He had o b s e r v e d t h a t v i s i b l e d y s g e n i c m u t a t i o n s 4 behaved l i k e mutable white (w) a l l e l e s i n that they d i s p l a y e d a high degree of i n s t a b i l i t y . Mutations could r e v e r t to w i l d type or, a l t e r n a t i v e l y , to a s t a b l e mutant s t a t e . Based p u r e l y on t h e s e g e n e t i c d a t a , Green s u g g e s t e d t h a t the mutations observed i n dysgenic hybrids were i n s e r t i o n mutation s i m i l a r to t h o s e found i n p r o k a r y o t e s (Green, 1976). He p o s t u l a t e d t h a t the abnormal t r a i t s a s s o c i a t e d w i t h h y b r i d dysgenesis r e s u l t e d from the t r a n s p o s i t i o n of a mobile element a s s o c i a t e d with P chromosomes. More s p e c i f i c a l l y , he proposed that s i n g l e s i t e mutations were the consequence of the element i n s e r t i n g i n t o s p e c i f i c g e n e t i c l o c i , and t h a t r e v e r s i o n s c o u l d o c c u r upon r e m o v a l of the e l e m e n t s by subsequent t r a n s p o s i t i o n or e x c i s i o n events. Green's hypothesis has been confirmed through molecular a n a l y s i s ( C o l l i n s and R u b i n , 1982; R u b i n , K i d w e l l , and Bingham, 1982; Bingham, K i d w e l l , and Rubin, 1982; O'Hare and Rubin, 1983; reviewed i n Engels, 1983). D y s g e n i c a l l y induced w mutations were shown to be a s s o c i a t e d with the presence of i n s e r t e d f o r e i g n DNA i n t o the l o c u s . R e v e r s i o n of t h o s e mutations which were unstable was i n v a r i a b l y accompanied by the e x c i s i o n of most or a l l of the i n s e r t . The i n s e r t e d elements were named P elements. A l l P s t r a i n s have been found to c a r r y P e l e m e n t s : about 30-50 per genome, s c a t t e r e d over a l l major chromosomes. Most M s t r a i n s l a c k P elements; a l l M s t r a i n s l a c k f u n c t i o n a l P elements. Because the elements are 5 a p p a r e n t l y n o n - m o b i l e i n the P s t r a i n , i t i s b e l i e v e d t h a t P s t r a i n s produce a r e p r e s s o r that accumulates c y t o p l a s m i c a l l y . M s t r a i n s l a c k b o t h t h e t r a n s p o s a b l e e l e m e n t and t h e r e p r e s s o r . The absence of the r e p r e s s o r i n hybrids between P males and M f e m a l e s a l l o w s m o b i l i t y of the element. T h i s system i s c o n s i s t e n t w i t h the v a r i o u s p r o p e r t i e s of h y b r i d dysgenesis, although a r e p r e s s o r has not been i s o l a t e d . The P element has been i s o l a t e d and c h a r a c t e r i z e d (O'Hare and Rubin, 1983)... The c o m p l e t e element i s 2.9 kb i n l e n g t h , w i t h 31 bp t e r m i n a l i n v e r t e d r e p e a t s and c o n t a i n s 4 open reading frames. I n c o m p l e t e or d e f e c t i v e P e l e m e n t s e x i s t . A l l of t h e s e c o n t a i n i n t e r n a l d e l e t i o n s . F o r e x a m p l e , d e f e c t i v e e l e m e n t s a s s o c i a t e d w i t h m u t a t i o n s at the w h i t e l o c u s (O'Hare and Rubin, 1983), ranged i n s i z e from 0.5 to 1.6 kb i n l e n g t h . I n c o m p l e t e P e l e m e n t s are m o b i l e o n l y so l o n g as they are accompanied by c o m p l e t e e l e m e n t s . Thus, i t appears that the open reading frames i n the complete element encode a product r e q u i r e d f o r t r a n s p o s i t i o n . T h i s product has been termed "transposase". F r a m e s h i f t mutations i n any of the f o u r r e a d i n g frames of the c o m p l e t e e l e m e n t a b o l i s h the transposase f u n c t i o n . In a d d i t i o n , p a i r w i s e combinations of t h e s e m u t a t i o n s f a i l t o r e s t o r e t h i s f u n c t i o n . I t i s t h e r e f o r e b e l i e v e d that a l l four reading frames are i n v o l v e d i n the production of a "transposase" (Karess and Rubin, 1984). 6 As n o ted p r e v i o u s l y , a s i m i l a r a n a l y s i s of the proposed " r e p r e s s o r " f u n c t i o n has not yet been p o s s i b l e . The s i t e s of P-induced rearrangements (thought to r e f l e c t s i t e s of P element r e s i d e n c e ) are non-random and s p e c i f i c " h o t - s p o t s " e x i s t ( E n g e l s and P r e s t o n , 1981). A r e c o g n i t i o n sequence i n t o which the P e lement i n s e r t s i s 8 bp l o n g , and t h i s sequence i s d u p l i c a t e d upon i n s e r t i o n of the P element i n t o a new c h r o m o s o m a l l o c a t i o n . The e x i s t e n c e of a p a r t i c u l a r r e c o g n i t i o n sequence might c o n t r i b u t e to the a p p a r e n t s i t e - s p e c i f i c i t y e x h i b i t e d by t h e t r a i t s of d y s g e n e s i s , such as chromosome r e a r r a n g e m e n t s (Simmons and Lim, 1980). The consensus sequence f o r P element i n s e r t i o n i s -GGCCAGAC- (O'Hare and Rubin, 1983). A l t h o u g h a p r e f e r r e d i n s e r t i o n s i t e e x i s t s , i t s sequence i s not h i g h l y c o n s e r v e d . Thus, i t i s l i k e l y t h a t c o n t r o l s o t h e r than a r e c o g n i t i o n sequence can i n f l u e n c e the p o s i t i o n at which i n s e r t i o n occurs. Although the nature of d y s g e n i c a l l y induced mutation i s now w e l l u n d e r s t o o d , the mechanism by which t r a n s p o s a b l e e l e m e n t s c a u s e r e c o m b i n a t i o n r e m a i n s u n c l e a r . M a l e r e c o m b i n a t i o n i s not i n f l u e n c e d by t h o s e f a c t o r s known to a l t e r m e i o t i c r e c o m b i n a t i o n ( E n g e l s , 1983). Recombinant e v e n t s o b s e r v e d are c l u s t e r e d and f r e q u e n t l y o n l y one of the two p o s s i b l e r e c i p r o c a l p r o d u c t s i s r e c o v e r e d (due to the p r e m e i o t i c t i m i n g of the exchanges). S e v e r a l s t u d i e s provide e v i d e n c e t h a t P-mediated c r o s s o v e r e v e n t s are s y m m e t r i c a l 7 exchanges ( V o e l k e r , 1974; Sved, 1978; I s a a k s o n , Johnson and D e n e l l , 1981; S i n c l a i r and G r i g l i a t t i , 1985) and thus not the products of random breakage which would be expected to produce s m a l l d e l e t i o n s and d u p l i c a t i o n s . The p a t t e r n o f recombination i s non-random, and does not r e f l e c t the m e i o t i c p a t t e r n n o r m a l l y o b s e r v e d ( E n g e l s , 1983). The p a t t e r n of recombination d i f f e r s from s t r a i n to s t r a i n as do the s i t e s of r e s i d e n c e and thus, recombination may be r e l a t e d to the s i t e s of r e s i d e n c e of t h e e l e m e n t s . In an e x t e n s i o n of t h e rearrangement hypothesis of Engels and Preston (1984), i t has been s u g g e s t e d t h a t DNA s t r a n d b r e a k s at the t e r m i n i of P e l e m e n t s may r e s u l t i n h o s t s t r a n d i n f i l t r a t i o n of the DNA duplex of the c l o s e l y p a i r e d homologous chromosome, l e a d i n g to p r e c i s e recombination ( S i n c l a i r and G r i g l i a t t i , 1985). A number of s t u d i e s have shown ( H i r a i z u m i et a1. , 1973; Woodruff and Thompson, 1977) t h a t r e c o m b i n a n t e v e n t s o c c u r p r e f e r e n t i a l l y i n the i n t e r v a l s nearest the centromere. T h i s l e d to the s u g g e s t i o n t h a t the m a j o r i t y of the r e c o m b i n a n t breaks took place i n c e n t r i c heterochromatin. However, t h i s could not be v e r i f i e d because a p p r o p r i a t e l y marked chromosomes d i d not e x i s t . In each case examined, the i n t e r v a l s w i t h d i s p o r p o r t i o n a t e 1 y h i g h l e v e l s of MR c o n t a i n e d b o t h euchromatin and heterochromatin. Therefore i t could not be e s t a b l i s h e d w h e t h e r t h e r e c o m b i n a t i o n t o o k p l a c e 8 p r e f e r e n t i a l l y i n one or the other chromatin type. S i n c l a i r and G r i g l i a t t i (1985) u t i l i z e d a s p e c i a l l y c o n s t r u c t e d second chromosome to examine the p a t t e r n of dysgenic recombination induced by four P s t r a i n s : h i 2, T-007, OKI, 1978. They used the h e t e r o c h r o m a t i c markers l i g h t ( l t ) and r o l l e d ( r l ) to d e l i n e a t e a region of the chromosome which 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 . M a l e r e c o m b i n a t i o n i n euchromatic segments was observed i n a l l s t r a i n s . However, i n experiments i n v o l v i n g 3 of the 4 s t r a i n s , no recombination was observed between the heterochromatic markers. T-007, OKI, and 19 7 8 each showed h i g h f r e q u e n c i e s of MR i n the r e g i o n s f l a n k i n g the c e n t r i c h e t e r o c h r o m a t i n but none a c t u a l l y o c c u r r e d i n the h e t e r o c h r o m a t i n between JL_t and j _ l . In c o n t r a s t , i n the e x p e r i m e n t i n v o l v i n g h-12, h e t e r o c h r o m a t i c c r o s s i n g over was observed, but at a very low frequency. The p a t t e r n s of r e c o m b i n a t i o n o b s e r v e d were s t r a i n s p e c i f i c and d i f f e r e n t from t h a t o b t a i n e d by i r r a d i a t i o n . S i n c l a i r and G r i g l i a t t i a t t r i b u t e the s t r a i n s p e c i f i c i t i e s to d i f f e r e n c e s between the s t r a i n s i n the s i t e s of r e s i d e n c e of P e l e m e n t s . They argue that the extremely low i n c i d e n c e of heterochromatic r e c o m b i n a t i o n might r e s u l t from the absence, or from a low frequency of the P r e c o g n i t i o n sequence, or from the i n a b i l i t y of the P element to i n s e r t i n t o heterochromatic regions due to s t e r i c c o n s t r a i n t s . C h r o m a t i n s t r u c t u r e can i n f l u e n c e gene a c t i v i t y . For 9 example, t r a n s c r i p t i o n a l l y a c t i v e genes are a s s o c i a t e d w i t h chromatin i n which the core h i s t o n e s - e s p e c i a l l y H3 and H4 -are h y p e r a c e t y l a t e d (Davie and Candido, 1978; Levy-Wilson et a l . , 1979). The a c e t y l groups on the h i s t o n e s may i n t e r f e r e w i t h and thus weaken the histone-DNA i n t e r a c t i o n i n the t y p i c a l nucleosome core s t r u c t u r e and t h i s may lead to a more open, l o o s e c h r o m a t i n s t r u c t u r e . P o s s i b l y t h i s "open" conformation f a c i l i t a t e s the b i n d i n g of RNA polymerases to the DNA, thereby promoting t r a n s c r i p t i o n . By analogy, one could argue that chromatin s t r u c t u r e a l s o i n f l u e n c e s P element i n s e r t i o n . Thus, a l t h o u g h r e c o g n i t i o n sequences may e x i s t i n both heterochromatin and euchromatin, r e g i o n s of the DNA i n the f o r m e r may be so h i g h l y condensed t h a t they are e s s e n t i a l l y i n a c c e s s i b l e to the P e l e m e n t s . T h e r e f o r e , one m i g h t e x p e c t l i t t l e i f any d y s g e n i c r e c o m b i n a t i o n i n h e t e r o c h r o m a t i n b e c a u s e i t i s h i g h l y c o n d e n s e d . One p r e d i c t i o n of t h i s h y p o t h e s i s i s t h a t a l t e r a t i o n s i n chromatin s t r u c t u r e might allow P i n s e r t i o n i n d i f f e r e n t areas of the genome and so may a l t e r the p a t t e r n of d y s g e n i c a l l y i n d u c e d r e c o m b i n a t i o n . M o st n o t a b l y , recombination would be expected i n heterochromatin where none had been p r e v i o u s l y o b s e r v e d , a l t h o u g h changes might a l s o occur i n euchromatic i n t e r v a l s . One p o s s i b l e means of a l t e r i n g c h r o m a t i n s t r u c t u r e i s 10 through treatment with n-butyrate. In HeLa c e l l s , n-butyrate i n d u c e s a r a p i d and e x t e n s i v e i n c r e a s e i n the amount of a c e t y l a t e d h i s t o n e s ( R i g g s e_t a l . , 1977; V i d a l i e_t a l . , 19 7 8; Simpson, 1978; Candido et a l . , 1978; Sealy and Chalkley, 1978; Cousens e_t a l . , 1979; D'anna e_t a l . , 1980). T h i s e f f e c t a p p e a r s to be due to n o n - c o m p e t i t i v e i n h i b i t i o n of the d e a c e t y l a s e enzyme (Reeves and Candido, 1978; B o f f a e_t a l . , 1978; V i d a l i et a l . , 1978), r a t h e r than to i n c r e a s e d r a t e s of a c e t y l a t i o n . In a d d i t i o n to t h e s e o b s e r v a t i o n s , i t has been e s t a b l i s h e d t h a t b u t y r a t e t r e a t m e n t i n d u c e s new p r o t e i n and mRNA s y n t h e s i s i n F r i e n d c e l l s (Reeves and C s e r j e s i , 1979). Changes i n the p a t t e r n s of t r a n s c r i p t i o n due to b u t y r a t e t r e a t m e n t have been noted ( T r a l k a e_t a l . , 1979; G r i f f e n et a l . , 1974; Leder and L e d e r , 1975; P r a s a d and S i n h a , 1976; P r a s a d , 1980; Chou e_t a l . , 1977; T a l l m a n e_t a l . , 1977; Ghosh and Cox, 1977; L e a v i t t and Moyzis, 1978; F a l l o n and Cox, 1979; R u b e n s t e i n et a l . , 1979; Hayman et a l . , 1980). B u t y r a t e a l s o r e v e r s e s malignant t r a n s f o r m a t i o n , y i e l d i n g c e l l s with normal morphological and b i o c h e m i c a l c h a r a c t e r i s t i c s . Moreover, i t renders chromatin more a c c e s s i b l e to nucleases such as DNAase I and I I , and m i c r o c o c c a l n u c l e a s e ( r e v i e w e d by Kruh, 1982). The e f f e c t s of b u t y r a t e on c e l l s i n c u l t u r e are r e v e r s i b l e . In a d d i t i o n to the e f f e c t s on c e l l c u l t u r e , In v i v o s t u d i e s have demonstrated that butyrate can i n h i b i t p h o s p h o r y l a t i o n of h i s t o n e s HI and H2A and i m p a i r m e t h y l a t i o n of a r g i n i n e and 11 l y s i n e r e s i d u e s i n h i s t o n e s and other nuclear p r o t e i n s (Boffa et a l . , 1981). An important phenomenon r e l e v a n t to the d i s c u s s i o n of i n v i v o e f f e c t s of b u t y r a t e on gene e x p r e s s i o n i s t h a t 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 D r o s o p h i l a m e l a n o g a s 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 i s the mosaic expr e s s i o n of a gene r e s u l t i n g f r o m i t s p r o x i m i t y t o a b r o k e n p i e c e o f heterochromatin. The gene may be a c t i v e i n some c e l l s and not i n o t h e r s . O f t e n the t r a n s c r i p t i o n a l f a t e of a v a r i e g a t i n g gene i s d e t e r m i n e d e a r l y i n development and i s c l o n a l l y propagated. Most l o c i examined have been found to v a r i e g a t e , and i n the m a j o r i t y of cases a v a r i e g a t i n g system i n v o l v e s a e u c h r o m a t i c gene or g e n e s j u x t a p o s e d t o a c e n t r i c h e t e r o c h r o m a t i c segment. In c a s e s where the a c t i v i t i e s of s e v e r a l l o c i are d e t e c t a b l e , l o c i c l o s e s t to the rearrangement b r e a k p o i n t are most l i k e l y to be i n a c t i v a t e d (Hartmann-G o l d s t e i n , 1967). In a homozygote, the i n a c t i v a t i o n of each l o c u s i s an i n d e p e n d e n t event. That v a r i e g a t i o n i s n o t . t h e r e s u l t of any permanent a l t e r a t i o n or mutation i n the gene can be shown by r e c o m b i n a t i o n e x p e r i m e n t s ( f o r r e v i e w , see S p o f f o r d , 1976). In many r e s p e c t s , v a r i e g a t i o n r e s e m b l e s X-chromosome i n a c t i v a t i o n i n mammals. I t i s thought to r e s u l t from " h e t e r o c h r o m a t i c s p r e a d i n g " ( f i r s t o b s e r v e d by Demerec and 12 S l i z y n s k a , 1937): t h e p r o g r e s s i v e c o n d e n s a t i o n or h e t e r o c h r o m a t i n i z a t i o n of the c h r o m a t i n , extending d i s t a l l y from the r e a r r a n g e m e n t b r e a k p o i n t to a g r e a t e r or l e s s e r e x t e n t i n i n d i v i d u a l c e l l s . There i s a good c o r r e l a t i o n between c y t o l o g i c a l o b s e r v a t i o n s on the number of c e l l s i n which a p a r t i c u l a r l o c u s i s h e t e r o c h r o m a t i n i z e d and the degree of mosaicism i n an i n d i v i d u a l ' s phenotype (Hartmann-Goldstein, 1967). I t has been shown (M o t t u s (it a l . , 1980, 1982), t h a t sodium butyrate e f f e c t i v e l y suppresses the i n a c t i v a t i o n of w+ i n w m 4 i n d i v i d u a l s and ( M o o r e ^ t a l . , 1979, 1983) t h a t r e d u c t i o n s i n h i s t o n e gene m u l t i p l i c i t y a l s o s u p p r e s s v a r i e g a t i o n . Thus r e d u c t i o n s i n the number of h i s t o n e s present or i n the a f f i n i t y of h i s t o n e s f o r DNA a l t e r chromatin s t r u c t u r e to the e x t e n t t h a t v a r i e g a t i o n ( h e t e r o c h r o m a t i -z a t i o n ) i s a f f e c t e d . In summary, butyrate i s a s s o c i a t e d with c e r t a i n chemical changes which are thought to r e s u l t i n a l t e r e d c h r o m a t i n s t r u c t u r e . In a d d i t i o n , i t i s known t o s u p p r e s s t h e i n a c t i v a t i o n of e u c h r o m a t i c genes near a h e t e r o c h r o m a t i c breakpoint. I t i s b e l i e v e d that t h i s e f f e c t r e s u l t s from the i n a b i l i t y of heterochromatin to "spread" over the euchromatic r e g i o n (due to the l o o s e n i n g of i t s c h r o m a t i n s t r u c t u r e ) . B e c a u s e o f t h i s s u s p e c t e d a c t i o n o f b u t y r a t e u p o n heterochromatin, i t was used i n t h i s study to t r e a t dysgenic 13 h y b r i d s of two P s t r a i n s . I f b u t y r a t e r e d u c e s t h e h y p o t h e s i z e d s t e r i c c o n s t r a i n t s upon P element i n s e r t i o n i n heterochromatin one would expect to observe heterochromatic male recombination as a r e s u l t of the butyrate treatment. Chemicals added to the c u l t u r e medium of any organism may produce m u l t i p l e e f f e c t s . In the case of b u t y r i c a c i d , a l t e r e d c h r o m a t i n s t r u c t u r e may be o n l y one of them. V a r i e g a t i o n s u p p r e s s o r genes, thought to be i n v o l v e d i n the assembly or maintenance of c h r o m a t i n s t r u c t u r e , o f f e r the o p p o r t u n i t y to d i s r u p t chromatin s t r u c t u r e i n a more p r e c i s e way, w i t h o u t the s i d e e f f e c t s which may be produced by exogenous chemical treatments. Using wm^ v a r i e g a t i o n as, an assay, S i n c l a i r e_t a l . (1983) i s o l a t e d 51 dominant v a r i e g a t i o n s u p p r e s s o r ( S u ( v a r ) ) and 3 v a r i e g a t i o n e n h a n c e r ( E n ( v a r ) ) m u t a t i o n s i n D £ £.s £ j) h. i^ 1 a. melanogaster. The m a j o r i t y of the Su(var) genes are c l u s t e r e d on chromosomes 2 and 3. Su(var) mutants s u p p r e s s s e v e r a l d i f f e r e n t v a r i e g a t i n g systems. Because of t h i s a p p a r e n t g e n e r a l e f f e c t on v a r i e g a t i n g systems, i t i s argued t h a t the products of Su(var) genes are i n v o l v e d i n chromatin s t r u c t u r e ; perhaps as s t r u c t u r a l , n o n - h i s t o n e chromosomal p r o t e i n s or enzymes f o r a s s e m b l y , m o d i f i c a t i o n or m a i n t e n a n c e of chromatin. One of t h e c l u s t e r s of S u ( v a r ) genes on t h e t h i r d 14 chromosome i s l o c a t e d near an u n u s u a l p a i r of s i t e s at 87B-E ( H e n i k o f f , 1979). D e l e t i o n of t h e s e s i t e s r e s u l t s i n suppression of v a r i e g a t i o n . S i n c l a i r e_t a l . t h e r e f o r e suggest t h a t many of the S u ( v a r ) m u t a t i o n s may be hypomorphs or amorphs. There i s some i n d i c a t i o n that these genes normally code f o r p r o d u c t s 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 . For i n s t a n c e i n males i n which the Y chromosome i s a b s e n t , the l e v e l of suppression of the Su(var) mutations i s s u b s t a n t i a l l y reduced. The Y chromosome i s almost e n t i r e l y heterochromatic; the i m p l i c a t i o n i s that i t s absence f r e e s up an otherwise very l i m i t e d l e v e l of p r o d u c t which can then p a r t i c i p a t e i n the heterochromatic spreading, i n a c t i v a t i n g the v a r i e g a t i n g locus. A l s o , c y t o l o g i c a l e v i d e n c e ( H a y a s h i , u n p u b l i s h e d ) i n d i c a t e s that the v a r i e g a t i n g white gene i s more of t e n euchromatic i n the presence than i n the absence of the suppressor mutations. B i o c h e m i c a l evidence (Ruddell, i n p r e p a r a t i o n ) i n d i c a t e s that the v a r i e g a t i n g w gene of w m^ a l t e r s c h r o m a t i n s t r u c t u r e (DNAase s e n s i t i v e s i t e s and nucleosome s p a c i n g ) and t h a t at l e a s t some suppressor genes r e t u r n the chromatin s t r u c t u r e to that observed f o r w i l d type f l i e s . In p r i n c i p l e , a s u p p r e s s o r m u t a t i o n s u c h as t h o s e d e s c r i b e d above c o u l d be used to a l t e r c h r o m a t i n s t r u c t u r e (perhaps s p e c i f i c a l l y that of heterochromatin, i n c o n t r a s t to the general e f f e c t of bu t y r a t e ) , without the c o m p l i c a t i o n of t h e p o s s i b l e s i d e e f f e c t s of c h e m i c a l t r e a t m e n t s . 15 A c c o r d i n g l y , dysgenic and non-dysgenic c r o s s e s were performed as i n the b u t y r a t e experiments with the a d d i t i o n of one of two t h i r d chromosome S u ( v a r ) genes chosen f o r the s t r e n g t h of t h e i r s u ppression of v a r i e g a t i o n . 16 MATERIALS AND METHODS Cu 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 i n h a l f p i n t m i l k b o t t l e s or 8 dram s h e l l v i a l s on a 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. 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 . A combination of a m p i c i l l i n with e i t h e r s t r e p t o m y c i n or t e t r a c y c l i n e was used (10 mg/1 each) to s u p p r e s s b a c t e r i a l growth. Where noted, some c r o s s e s were r a i s e d on food to which b u t y r i c a c i d was added to a f i n a l c o n c e n t r a t i o n of 150 mM. T h i s c o n c e n t r a t i o n was used to maximize the e f f e c t on chromatin s t r u c t u r e while m i n i m i z i n g the d e l a y i n development which i s o b s e r v e d as a r e s u l t of butyrate treatment (Mottus, 1979). In a l l cases, the f i n a l pH of the food was a d j u s t e d to 5.5. A l l c r o s s e s were p e r f o r m e d at 22°C. P - S t r a i n s and marked chromosomes: The P - s t r a i n s used were: T-007/CyO (T-007 i s o l a t e d i n Texas, H i r a i z u m i , 1971; H i r a i z u m i et a l . , 1973) and OKI/OKI (OKI i s o l a t e d i n Oklahoma, Woodruff and Thompson, 1977). The h i g h l y inbred l a b o r a t o r y s t r a i n Oregon R was used i n place of t h e P s t r a i n s i n t h e c o n t r o l c r o s s e s ( O r e g o n R i s an M s t r a i n ) . The s t r a i n used to monitor recombination was an M s t r a i n 17 b e a r i n g the second chromosome: S t a r (JS, g e n e t i c p o s i t i o n 1.3), b l a c k (b, 48.5), l i g h t ( l _ t , 55.0), r o l l e d ( r l , 55.1), TJ y*. narrow wing-dominant (n.w , 83.0) and P i n - y e l l o w t i p ( P i n , 107.3). F o r a d e s c r i p t i o n of t h e s e m u t a t i o n s and t h e i r p henotypes, see L i n d s l e y and G r e l l (1968; see a l s o S i n c l a i r and G r i g l i a t t i , 1985). The m u l t i p l y marked chromosome can be d i v i d e d i n t o f i v e i n t e r v a l s (see F i g u r e 1). Note t h a t the i n t e r v a l between Jl_t and r\L ( i n t e r v a l number 3) 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 , w h e r e a s i n t e r v a l s 2 and 4 span b o t h h e t e r o c h r o m a t i n and e u c h r o m a t i n , and i n t e r v a l s 1 and 5 are e n t i r e l y e u c h r o m a t i c . T h i s chromosome was m a i n t a i n e d i n c u l t u r e as a h e t e r o z y g o t e w i t h t h e m u l t i p l y i n v e r t e d chromosome: In: (2L, 2R) CyO ( h e r e a f t e r r e f e r r e d to as CyO). The M s t r a i n used i n the s u p p r e s s o r c r o s s e s was the same, with the a d d i t i o n of a t h i r d chromosome homozygous f o r e i t h e r the gene Su(var)325, or Su(var)318; h e r e a f t e r r e f e r r e d to as 325 and 318 ( d e s c r i b e d i n S i n c l a i r et a l . , 1983). Genetic c r o s s e s : The p r o c e d u r e f o r the u n t r e a t e d and b u t y r a t e t r e a t e d c rosses i s o u t l i n e d i n Figure 2 ( a ) . (a) Standard recombination experiments: T-007 or OKI males were c r o s s e d to m u l t i p l y marked M s t r a i n f e m a l e s . A p p r o x i m a t e l y 25 f l i e s of each sex were p l a c e d i n h a l f - p i n t milk b o t t l e s on normal food and allowed to lay eggs f o r three 18 1.3 K 3 C H 2 b nw Pin1 .Yt 4b 83.0 5a 107.3 F i g u r e 1. M u l t i p l y marked second chromosome used i n a l l experiments. a i n t e r v a l e n t i r e l y euchromatic b i n t e r v a l spans both euchromatin and heterochromatin c i n t e r v a l e n t i r e l y heterochromatic 19 Untreated and butyrate treated crosses P S b It rl nwD PinYt ^ ^ ^ T-007 CyO o r m or 0 r e R II S b It rl nwD Pin* T-007 or OK1 or OreR CyO OK1 OreR Half the parents lay eggs on standard medium,the other half on butyrate treated medium oV F2 Progeny scored for recombination on chromosome 2 B Suppressor crosses P S b It rl nwD PinYt Su(var) Su(var) CyO FI S b It rl nwD PinYt _ Su(var) * * b It rl T-007 or OK1 or OreR ' + ^ O H V CyO OK1 OreR a O JUL $8 Itrl T T F2 Progeny scored for recombination on chromosome 2 F i g u r e 2. Mating scheme used i n untreated and buyrate-t r e a t e d (A) and Su(var) (B) experiments. Except where noted, a l l crosses were r a i s e d on standard medium. 20 days. The p a r e n t s were t r a n s f e r r e d to f r e s h b o t t l e s and a l l o w e d to l a y eggs f o r t h r e e more days, and were then d i s c a r d e d . At l e a s t 5 b o t t l e s were s e t up f o r each of the P s t r a i n s . F l hybrid male progeny bearing the m u l t i p l y marked second chromosome and a second chromosome from the P s t r a i n s were c o l l e c t e d and then back c r o s s e d to v i r g i n _b l_t _r_l f e m a l e s . In each case 3 males were mated w i t h 10 v i r g i n f e m a l e s i n 8 dram s h e l l v i a l s . For each c r o s s at l e a s t 50 v i a l s were s e t up. The p a r e n t s were a l l o w e d to l a y eggs f o r t h r e e days and the males were then t r a n s f e r r e d to new v i a l s with 10 new females. The o r i g i n a l females were t r a n s f e r r e d to a h a l f - p i n t m i l k b o t t l e and a l l o w e d to c o n t i n u e to l a y eggs f o r 5 more days. T h i s p r o c e s s was r e p e a t e d t w i c e to p r o v i d e t h r e e r e p l i c a t e s f o r a t o t a l of at l e a s t 150 v i a l s f o r each c r o s s . P r o g e n y f r o m t h e s e c r o s s e s were e x a m i n e d f o r recombination on the second chromosome f o r 21 days a f t e r the p a r e n t s were i n t r o d u c e d i n t o the b o t t l e s or v i a l s . As a c o n t r o l f o r the s e c r o s s e s , the same p r o c e d u r e was f o l l o w e d except t h a t i n s t e a d of a P s t r a i n , males from the Oregon R s t r a i n were used. (b) B u t y r a t e r e c o m b i n a t i o n e x p e r i m e n t s : The p a r e n t s f o r t h e s e ^experiments were o b t a i n e d from the same c u l t u r e s as those used i n the aforementioned study. The butyrate crosses were performed e s s e n t i a l l y as d e s c r i b e d above, except that the 21 FI i n d i v i d u a l s were grown on medium c o n t a i n i n g b u t y r a t e (150mM). (c) Suppressor experiments: The p r o t o c o l f o r the suppressor c r o s s e s i s g i v e n i n F i g u r e 2(b). Two S u ( v a r ) m u t a t i o n s were t e s t e d f o r t h e i r e f f e c t on MR by procedures analogous to those d e s c r i b e d above. In t h i s case T-007, OKI or ( f o r a c o n t r o l ) Oregon R males were c r o s s e d to f e m a l e s b e a r i n g the m u l t i p l y marked second chromosome S_ b_ lt_ r_l jiw^ P i n ^ t / C y O and a t h i r d chromosome c a r r y i n g one of two S u ( v a r ) m u t a t i o n s : e i t h e r Su(var):325 or Su(var):318. Each p o p u l a t i o n of e i t h e r the P s t r a i n or the Oregon R males was d i v i d e d i n h a l f so t h a t one h a l f of the group was used i n the 3 2 5 c r o s s and the o t h e r i n the 318 c r o s s . Thus, the parents i n the 325 c r o s s e s were from the same c u l t u r e s as those used f o r the 318 c r o s s e s . A n a l y s i s of the data: Because the exchange events occur p r e m e i o t i c a l l y , i t i s p o s s i b l e f o r t h e p r o d u c t s of a s i n g l e e x c h a n g e t o be p r o p a g a t e d through subsequent m i t o t i c d i v i s i o n s p r i o r to meiosis. Both products of exchange may be represented i n the progeny, or l a r g e numbers of progeny bearing one or the other p r o d u c t may be o b s e r v e d . Most o f t e n , a s i n g l e p r o d u c t of exchange i s r e p r e s e n t e d i n one of the o f f s p r i n g . When a s i n g l e F2 i n d i v i d u a l bears a s i n g l e product of exchange, i t i s 22 c l e a r that one exchange event ( i n the regions of i n t e r e s t ) has taken p l a c e . When both r e c i p r o c a l p r o d u c t s a r e o b s e r v e d , e i t h e r s i n g l y or i n groups, or l a r g e groups of a s i n g l e p r o d u c t a r e ob s e r v e d ( e i t h e r of t h e s e p o s s i b i l i t i e s f a l l s under the t e r m . " c l u s t e r " ) , i t i s c l e a r that at l e a s t one event has taken p l a c e , but t h e r e i s a s m a l l p o s s i b i l i t y t h a t t h e r e has been more than one exchange. In a n a l y s i n g the data g e n e r a t e d i n t h i s s tudy, i t was of l e s s i n t e r e s t to know the t o t a l number of r e c o m b i n a n t i n d i v i d u a l s than the number of recombinant events which took place; each of which, d i r e c t l y or otherwise, represents the a c t i o n of one P element. Because the F l males were mated i n groups of t h r e e ( h i g h l e v e l s of s t e r i l i t y o b s e r v e d p r i o r to t h e s e e x p e r i m e n t s made mating males i n d i v i d u a l l y i m p r a c t i c a l ) , i t i s i m p o s s i b l e to t e l l whether a l l members of a c l u s t e r of r e c o m b i n a n t progeny o r i g i n a t e d from the same parent. I t was t h e r e f o r e necessary to estimate the number of s i n g l e events which occurred. T h i s was done by c o n s i d e r i n g each c l u s t e r which occurred to be the product of a s i n g l e event. In T a b l e s 1-4, the number of e s t i m a t e d s i n g l e e v e n t s i s shown f o r each i n t e r v a l on chromosome two as w e l l as the t o t a l e s t i m a t e d s i n g l e e v e n t s . On the l i n e below, i n p a r e n t h e s e s , i s given the t o t a l number of recombinant i n d i v i d u a l s observed. T h i s a l l o w s some e s t i m a t i o n of the degree of c l u s t e r i n g f o r each i n t e r v a l . In the r e s u l t s s e c t i o n , the number and percent 23 of c l u s t e r s are given f o r each experiment. T h i s i n f o r m a t i o n was i n c l u d e d because i t may i n d i c a t e whether the b u t y r a t e treatments or suppressors had any e f f e c t on the t i m i n g of the exchange e v e n t s , which would be e x p e c t e d to show up i n the p r o p o r t i o n of r e c o m b i n a n t e v e n t s which were c l u s t e r e d . The e s t i m a t e d number of s i n g l e e v e n t s i s n e c e s s a r i l y an underestimate, since i n a s m a l l p r o p o r t i o n of cases, a c l u s t e r may c o n t a i n the i d e n t i c a l p r o d u c t s of two s e p a r a t e exchange e v e n t s ( t h a t i s , from two s e p a r a t e p a r e n t s ) , but t h i s i s a very minor source of e r r o r . The data were analysed f o r s t a t i s t i c a l s i g n i f i c a n c e using the G t e s t (Sokal and Rohlf, 1969) at the standard p=.05 l e v e l of s i g n i f i c a n c e . 24 RESULTS (a) Standard recombination experiments M e i o t i c r e c o m b i n a t i o n does not o c c u r i n D r o s o p h i l a melanogaster males that l a c k P elements. To demonstrate t h i s g e n e r a l absence of m e i o t i c r e c o m b i n a t i o n , Oregon R males (M s t r a i n ) were mated w i t h f e m a l e s b e a r i n g the m u l t i p l y marked second chromosome and t h e i r male FI o f f s p r i n g , h e t e r o z y g o u s f o r the m u l t i p l y marked second chromosome were backcrossed to _b l._t r_ 1 v i r g i n f e m a l e s . As e x p e c t e d , t h e r e were no recombinants among the 7,919 F2 o f f s p r i n g examined (data not shown). The male r e c o m b i n a t i o n d a t a f o r t h e T-007_ and 0K_l e x p e r i m e n t s a r e p r e s e n t e d i n T a b l e 1. In b o t h c a s e s , s u b s t a n t i a l r e c o m b i n a t i o n was o b s e r v e d among male h y b r i d s r e s u l t i n g from P male and M female matings. The T-007 and OKI untreated crosses each produced a c h a r a c t e r i s t i c p a t t e r n and f r e q u e n c y of r e c o m b i n a t i o n . The f r e q u e n c y of r e c o m b i n a t i o n events among the T-007 males was 1.15%, whereas the frequency among the OKI s t r a i n males was only 0.74 percent. The number and frequency of recombinants that occurred i n each i n t e r v a l of the chromosome f o r the T-007 and OKI experiments are a l s o g i v e n i n T a b l e 1. 25 S t r a i n s Genetic I n t e r v a l 1 2 3 F l i e s T o t a l Scored T-007 number 3 25 33 (27) (67) 34 24 116 10,087 (82) (34) (220) per .248 .327 c e n t 3 (.367) (.664) 0 .337 .238 1.15 (.813) (.337) (2.18) d i s t r i b -t i o n b .216 .285 .293 .207 1.000 OKI number 3 14 13 (23) (49) 0 20 (63) 11 (18) 58 7,885 (152) per .178 .165 c e n t 3 (.292) (.621) 0 .254 .140 .736 (.786) (.228) (1.93) d i s t r i b u t i o n b 241 224 .345 . 190 1.000 Table 1. R e c o m b i n a t i o n d a t a , i n each g e n e t i c i n t e r v a l , f o r c o n t r o l d y s g e n i c c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI. a d a t a f o r r e c o m b i n a t i o n e v e n t s g i v e n f i r s t w i t h t o t a l r e c o m b i n a n t s g i v e n i n p a r e n t h e s e s (see " M a t e r i a l s and Methods"). b data based on recombination events only. 26 S i n c l a i r and G r i g l i a t t i (1985) found t h a t of the f o u r P s t r a i n s they examined, T-007 was the most p o t e n t i n d u c e r of MR. T h i s study c o n f i r m s t h i s o b s e r v a t i o n . The d a t a f o r the OKI s t r a i n i n both s t u d i e s are n e a r l y i d e n t i c a l i n both p a t t e r n and f r e q u e n c y of r e c o m b i n a n t e v e n t s . The f r e q u e n c y and p a t t e r n of e v e n t s f o r the T-007 s t r a i n o b s e r v e d i n t h i s s t u d y showed one major d i f f e r e n c e from t h a t of S i n c l a i r and G r i g l i a t t i . In e v e r y r e c o m b i n a n t i n t e r v a l as w e l l as i n t o t a l , the frequency of events observed was c o n s i s t e n t l y two-t h i r d s that observed i n the previous study. Perhaps t h i s can be e x p l a i n e d by the f a c t t h a t the T-007 c u l t u r e used i n t h i s study was d e r i v e d from a s i n g l e b o t t l e of the c u l t u r e used by S i n c l a i r and G r i g l i a t t i . There may be some v a r i a t i o n i n the s t o c k such t h a t the p r o p o r t i o n used to found my c u l t u r e was not r e p r e s e n t a t i v e of the e n t i r e p o p u l a t i o n . A l t e r n a t i v e l y , s m a l l v a r i a t i o n s i n l a b o r a t o r y c o n d i t i o n s , s u c h as temperature, may have i n f l u e n c e d the l e v e l s of recombination i n d u c e d by T-007, a l t h o u g h i t i s not c l e a r why one P s t r a i n and n o t t h e o t h e r w o u l d be s e n s i t i v e t o t h e s e s l i g h t v a r i a t i o n s . In any case, the general trends observed f o r T-007 were s i m i l a r f o r both s t u d i e s . The highest l e v e l s of MR were a l w a y s o b s e r v e d i n i n t e r v a l s 2 and 4; the i n t e r v a l s c o n t a i n i n g both 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 . Most i m p o r t a n t l y , f o r both s t u d i e s , t h e r e was no h e t e r o c h r o m a t i c 27 r e c o m b i n a t i o n o b s e r v e d f o r e i t h e r of t h e s t r a i n s . In a d d i t i o n , a c o m p a r i s o n of r e c o m b i n a n t f r e q u e n c i e s based on events (data c o r r e c t e d to account f o r c l u s t e r i n g ) showed much gre a t e r s i m i l a r i t y between the two s t u d i e s than that based on the number of r e c o m b i n a n t i n d i v i d u a l s o b s e r v e d . S i n c e the s t u d i e s were p r a c t i c a l l y i d e n t i c a l i n procedure, t h i s may be taken as an i n d i c a t i o n t h a t the c o r r e c t e d d a t a r e p r e s e n t the more accurate estimate of recombination frequency. D e s p i t e the f a c t t h a t the two P s t r a i n s each i n d u c e a c h a r a c t e r i s t i c p a t t e r n and frequency of recombination, there were some i n t e r e s t i n g s i m i l a r i t i e s between the two. The most i m p o r t a n t o b s e r v a t i o n ( a s n o t e d a b o v e ) i s t h a t no recombination occurred i n i n t e r v a l number 3; the only wholly h e t e r o c h r o m a t i c i n t e r v a l , i n e i t h e r the T-007 or the OKI s t r a i n s . In both s t r a i n s , a s u b s t a n t i a l p r o p o r t i o n of the t o t a l recombination (approximately 60%) occurred i n i n t e r v a l s 2 and 4. These i n t e r v a l s are both adjacent to the centromere and are c o m p r i s e d of both 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 . While the m a j o r i t y of the recombination was observed i n these two i n t e r v a l s , the p a t t e r n was somewhat d i f f e r e n t between the two s t r a i n s . (b) Butyrate recombination experiments To t e s t the e f f e c t of butyrate on male recombination the 28 same crosses as i n the untreated set were performed and the FI h y b r i d males were r a i s e d on mediume to which b u t y r i c a c i d had been added to a c o n c e n t r a t i o n of 150mM. Tr e a t m e n t w i t h butyrate i n the absence of P elements ( i n the Oregon R cross) had no e f f e c t on male r e c o m b i n a t i o n . No r e c o m b i n a n t s were d i s c o v e r e d among the 10,462 F2 progeny of the Oregon R c r o s s e s . T h e r e f o r e , i t can be c o n c l u d e d t h a t b u t y r a t e a l o n e does not induce p e r c e p t i b l e l e v e l s of male recombination. In c o n t r a s t , b u t y r a t e t r e a t m e n t a l t e r e d t h e male r e c o m b i n a t i o n e v e n t s which o c c u r r e d i n the T-007 and OKI c r o s s e s . In t h e T - CJ CJ _7 c r o s s ( T a b l e 2), t h e o v e r a l l recombination (event) frequency i n c r e a s e d s i g n i f i c a n t l y from 1.15% to 1.49% (G t e s t , p=.05). Moreover, the p a t t e r n of r e c o m b i n a t i o n was a l t e r e d by b u t y r a t e t r e a t m e n t . Most i m p o r t a n t l y , a low, but p e r c e p t i b l e l e v e l of r e c o m b i n a t i o n (.048%) was o b s e r v e d f o r the h e t e r o c h r o m a t i c i n t e r v a l . T h i s e f f e c t i s s t a t i s t i c a l l y s i g n i f i c a n t (using the G-test, Sokal and Rohlf, 1969). R e c a l l that recombination i s never observed i n t h i s i n t e r v a l i n the absence of butyrate. In a d d i t i o n , T - 0 0 7 - i n d u c e d exchange on the r i g h t arm of chromosome two ( i n t e r v a l s 3, 4, and 5) showed a s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e r e l a t i v e to comparable c o n t r o l l e v e l s . On the other hand, i n t e r v a l s 1 and 2 (on the l e f t arm) were u n a f f e c t e d by the b u t y r a t e t r e a t m e n t . F i n a l l y , a s m a l l i n c r e a s e i n the number of p r e m e i o t i c exchange e v e n t s as shown by a s l i g h t 29 S t r a i n s Genetic I n t e r v a l 2 3 4 F l i e s T o t a l Scored T-007 number b 30 48 (45) (77) 7 74 56 (7) (221) (87) 215 14,453 (437) per .208 .332 .048 .512 .388 1.49 c e n t b (.311) (.533) (.048) (1.53) (.602) (3.02) d i s t r i b - .140 t i o n 223 033 .344 261 1.000 OKI number 0 13 34 4 50 17 (22) (78) (4) (106) (44) 118 16,803 (254) .202 .024 ,298 ,101 per .077 c e n t b (.131) (.464) (.024) (.631) (.262) .702 (1.51) d i s t r i -b u t i o n 0 110 .288 .034 .424 .133 1 .000 Table 2. R e c o m b i n a t i o n d a t a , i n each g e n e t i c i n t e r v a l , f o r v b u t y r a t e 3 t r e a t e d dysgenic c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI. a . 15M b data f o r recombination events given f i r s t w ith t o t a l recombinants given i n parentheses (see " M a t e r i a l s and Methods"). b data based on recombination events only. 30 i n c r e a s e i n the p e r c e n t of c l u s t e r s was o b s e r v e d . In the u n t r e a t e d ( o r s t a n d a r d ) e x p e r i m e n t , 29 o f t h e 116 r e c o m b i n a t i o n e v e n t s o b s e r v e d appeared as c l u s t e r s . T h i s r e p r e s e n t s 25% of the e v e n t s o b s e r v e d . In the b u t y r a t e e x p e r i m e n t 78 of 215 e v e n t s o b s e r v e d were c l u s t e r e d , which c o r r e s p o n d s to 36 p e r c e n t of the e v e n t s o b s e r v e d . T h i s i n c r e a s e i s not s t a t i s t i c a l l y s i g n i f i c a n t . In c o n t r a s t with the T-007 s t r a i n , the o v e r a l l frequency of r e c o m b i n a t i o n e v e n t s i n d u c e d by the OKI s t r a i n r e mained u n a l t e r e d (about 0.8% f o r both t r e a t e d and untreated crosses) by butyrate treatment. However, as with the T-007 s t r a i n , the p a t t e r n of r e c o m b i n a t i o n e v e n t s was a l t e r e d by exposure to b u t y r a t e . Most n o t a b l y , a s m a l l (0.24%), but s i g n i f i c a n t amount of r e c o m b i n a t i o n o c c u r r e d w i t h i n 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 r e g i o n 3. In c o n t r a s t , t h e r e was a s i g n i f i c a n t d e c r e a s e i n exchange f r e q u e n c y f o r r e g i o n 1, whereas f o r i n t e r v a l s 2, 4 and 5, t h e f r e q u e n c y o f r e c o m b i n a t i o n e v e n t s r e m a i n e d at about the same l e v e l as i n the untreated crosses. There was no a p p r e c i a b l e change i n the percent of c l u s t e r s produced i n response to butyrate. In the u n t r e a t e d , or s t a n d a r d , e x p e r i m e n t , 20 of 58 o b s e r v e d r e c o m b i n a t i o n e v e n t s were c l u s t e r e d (34%). In the b u t y r a t e e x p e r i m e n t , e x a c t l y the same p e r c e n t of the e v e n t s was c l u s t e r e d (40 of 118 e v e n t s ) . One of the most i m p o r t a n t e f f e c t s of b u t y r a t e i s the 31 occurrence of recombination i n the heterochromatin, where i t has not p r e v i o u s l y been o b s e r v e d . In the case of both T-007 and OKI, a l l of the heterochromatic exchange events observed were s i n g l e , u n c l u s t e r e d events. Based on the data f o r a l l of the c r o s s e s , an ave r a g e degree of c l u s t e r i n g of 32% can be o b t a i n e d . G i v e n t h i s , and i f i t i s assumed t h a t the t i m i n g and nature of the heterochromatic events i s the same as those of the e u c h r o m a t i n , 4 of the 11 h e t e r o c h r o m a t i c c r o s s o v e r s c o u l d have been e x p e c t e d to be p r e m e i o t i c and as such would have appeared as a c l u s t e r of r e c o m b i n a n t s . The absence of c l u s t e r s may r e f l e c t the s m a l l sample s i z e , or i t may be an i n d i c a t i o n of an important d i f f e r e n c e i n heterochromatic vs. euchromatic c r o s s i n g over. (c) Su(var)325 P r e l i m i n a r y mapping experiments suggest that the Su(var) 3 2 5 mutant may i n f l u e n c e m e i o t i c c r o s s i n g over ( T e j a n i and K r o i t s c h , 1982, p e r s o n a l c o m m u n i c a t i o n ) . S i n c e many of the b a s i c p r o c e s s e s which o c c u r i n m e i o t i c c r o s s i n g over are probably important f o r male recombination, i t was of i n t e r e s t to d e t e r m i n e whether the s u p p r e s s o r 325 c o u l d i n f l u e n c e d y s g e n i c c r o s s i n g o v e r . A l s o , s i n c e s u p p r e s s o r s of v a r i e g a t i o n and butyrate have the same e f f e c t on v a r i e g a t i o n , there may be some s i m i l a r i t i e s i n t h e i r e f f e c t s on chromatin 32 s t r u c t u r e . Of key i n t e r e s t here i s whether suppressor genes can cause heterochromatic recombination as does butyrate. If the mutant has a g e n e r a l e f f e c t on r e c o m b i n a t i o n , male r e c o m b i n a t i o n might be o b s e r v e d even i n the absence of P e l e m e n t s . On t h e o t h e r hand, i f 3^ _2_5 a c t s i n a manner analogous to butyrate, one might expect s i m i l a r a l t e r a t i o n s i n recombination p a t t e r n or frequency only i n dysgenic c r o s s e s . When Oregon R males are crossed to M females bearing the m u l t i p l y marked second chromosome and Su(var)325 on the t h i r d chromosome (see F i g u r e 2(b) f o r a d i a g r a m of t h i s c r o s s ) , no r e c o m b i n a n t s were o b s e r v e d i n the 7,576 F2 progeny examined ( d a t a n o t shown). T h u s , w h a t e v e r e f f e c t _3 2__5 has on recombination, i t does not promote e i t h e r m e i o t i c or somatic exchange i n males. The e f f e c t s of i n t r o d u c i n g .3 2J5 i n t o t h e d y s g e n i c backgrounds of T-007 and OKI are shown i n T a b l e 3. The r e s u l t i n g l e v e l s and d i s t r i b u t i o n p atterns of recombination i n T-007 d y s g e n i c males b e a r i n g 325 are not 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 t h o s e of t h e Xz.9_ _0 _7 c o n t r o l ( s t a n d a r d ) experiment. Furthermore, the percent of c l u s t e r s observed i s not s i g n i f i c a n t l y a l t e r e d (37 c l u s t e r e d events of 125 observed i n t o t a l , or 30%). S i m i l a r l y , when 325 i s i n t r o d u c e d i n t o the OKI d y s g e n i c c r o s s , the e f f e c t s on MR are o n l y s l i g h t . For example, the o n l y s i g n i f i c a n t d i f f e r e n c e i n r e g i o n a l r e c o m b i n a t i o n 33 P S t r a i n s Genetic I n t e r v a l 1 2 3 4 F l i e s T o t a l Scored T-007 number 3 31 38 (40) (52) 0 38 18 (58) (31) 125 9,921 (181) per .312 .383 c e n t 3 (.403) (.524) 0 .383 .181 1.26 (.585) (.313) (1.82) d i s t r i b -t i o n b 248 304 0 .304 144 1.000 OKI number 3 35 68 0 85 24 (58) (106) 0 (141) (33) 212 22,448 (338) per .156 cent® (.258) .303 0 .379 .107 .944 (.472) (.628) (.147) (1.51) d i s t r i -b u t i o n ^ . 165 321 .401 .113 1.000 Table 3. R e c o m b i n a t i o n d a t a , i n each g e n e t i c i n t e r v a l , f o r dysgenic c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI, to which the mutant Su(var)325 had been i n t r o d u c e d . a data f o r recombination events given f i r s t , with t o t a l recombinants given i n parentheses (see " M a t e r i a l s and Methods"). b data based on recombination events only. 34 f r e q u e n c y t h a t was o b s e r v e d f o r e x p e r i m e n t a l v_s. c o n t r o l males, was that f o r i n t e r v a l 2. Here again, c l u s t e r i n g i s not a f f e c t e d by the p r e s e n c e of 3 2 5. Seventy-one of the 212 e v e n t s o b s e r v e d were c l u s t e r e d , which r e p r e s e n t s 34% of the events. Taken together, these r e s u l t s c l e a r l y i n d i c a t e that t h e _3 2_ 5_ m u t a t i o n h s a v i r t u a l l y no e f f e c t on m a l e r e c o m b i n a t i o n . M o s t s i g n i f i c a n t l y , i t i n d u c e s no heterochromatic recombination. (d) Su(var)318 P r e l i m i n a r y mapping s t u d i e s gave no i n d i c a t i o n that the mutant Su(var)318 i n f l u e n c e s m e i o t i c c r o s s i n g over. I t t h u s p r o v i d e s a c o n t r a s t w i t h the 3 2 5 m u t a t i o n . As was t r u e f o r 325, Su(var)318 f a i l e d to produce any male rec o m b i n a t i o n among the 7,358 F2 progeny of the Oregon R c o n t r o l c r o s s ( d a t a not shown). The r e s u l t s of the experiments to t e s t the e f f e c t s of 318 on male recombination i n the dysgenic backgrounds of T-007 and OKI are p r e s e n t e d i n T a b l e 4. I t can be seen from the data that 318 d i d not s i g n i f i c a n t l y a l t e r recombination i n the T-007 (13,662 f l i e s s c o r e d ) or OKI (18,676 f l i e s s c o r e d ) experiments. For T-007 48 of the 155 observed recombination e v e n t s were c l u s t e r e d (31%). For OKI, 50 of 151 e v e n t s o b s e r v e d were c l u s t e r e d (33%). N e i t h e r of t h e s e v a l u e s re p r e s e n t s any s i g n i f i c a n t d i f f e r e n c e from c o n t r o l l e v e l s . 35 P S t r a i n s Genetic I n t e r v a l 2 3 F l i e s T o t a l Scored T-007 number 3 35 43 (49) (86) 49 29 155 13,662 (86) (40) (261) per .256 .315 c e n t 3 (.359) (.630) 0 .359 .212 1.14 (.630) (.293) (1.91) d i s t r i b -t i o n b .226 .277 .316 .187 1.000 OKI number 3 23 42 0 63 23 (48) (58) 0 (137) (27) 151 18,676 (270) per c e n t 8 (.257) 123 .225 0 .335 .123 .809 (.311) (.734) (.734) (1.45) d i s t r i -b u t i o n * 152 278 .417 152 1 .000 Table 4. R e c o m b i n a t i o n d a t a , i n each g e n e t i c i n t e r v a l , f o r dysgenic c r o s s e s i n v o l v i n g P s t r a i n s T-007 and OKI, to which the mutant Su(var)325 had been i n t r o d u c e d . a data f o r recombination events given f i r s t , with t o t a l recombinants given i n parentheses (see " M a t e r i a l s and Methods"). b data based on recombination events only 36 F i g u r e s 3 and 4 (T-007 and OKI, r e s p e c t i v e l y ) show the second chromosome maps de r i v e d from data on the p r o p o r t i o n a l d i s t r i b u t i o n of r e c o m b i n a t i o n e v e n t s f o r c o n t r o l d y s g e n i c c r o s s e s and the butyrate and suppressor treatments. A g e n e t i c map d e r i v e d from female m e i o t i c recombination d a t a i s provided i n each f i g u r e , f o r pur p o s e s of c o m p a r i s o n . An a s t e r i s k i n d i c a t e s any region which shows a s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l d y s g e n i c c r o s s . In each f i g u r e , i t can be seen that there i s very l i t t l e d i f f e r e n c e between the c o n t r o l c ross and e i t h e r suppressor c r o s s . Thus, these experiments have re v e a l e d no Su(var) e f f e c t s on heterochromatic MR that are analogous to those of b u t y r a t e . 37 FIGURE 3. Second chromosome maps derived from recombination data of dysgenic c r o s s e s i n v o l v i n g the P s t r a i n T- 007. The upper l i n e i s d e r i v e d from female m e i o t i c recombination data, and i s i n c l u d e d f o r comparison. An a s t e r i s k i n d i c a t e s i n t e r v a l s which e x p e r i e n c e l e v e l s of r e c o m b i n a t i o n s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s (second l i n e ) . 38 F I G U R E 4 . S e c o n d c h r o m o s o m e maps d e r i v e d f r o m r e c o m b i n a t i o n d a t a o f d y s g e n i c c r o s s e s i n v o l v i n g t h e P s t r a i n , OKI. The u p p e r l i n e i s d e r i v e d f r o m f e m a l e m e i o t i c r e c o m b i n a t i o n d a t a , a n d i s i n c l u d e d f o r c o m p a r i s o n . A n a s t e r i s k i n d i c a t e s i n t e r v a l s w h i c h e x p e r i e n c e l e v e l s o f r e c o m b i n a t i o n 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 s ( s e c o n d l i n e ) . 39 DISCUSSION S i n c l a i r and G r i g l i a t t i (1985) r e p o r t e d t h a t the T-007 and OKI second chromosomes induce a c h a r a c t e r i s t i c frequency and p a t t e r n of recombination. They found a non-random, s t r a i n s p e c i f i c d i s t r i b u t i o n of exchange along the second chromosome f o r t h e s e P s t r a i n s . The^ d a t a p r e s e n t e d i n t h i s r e p o r t c o n f i r m t h e i r r e s u l t s . The pat t e r n s generated by the c o n t r o l c r o s s e s are very s i m i l a r to those of S i n c l a i r and G r i g l i a t t i , e s p e c i a l l y that of OKI which i s n e a r l y i d e n t i c a l . I t has been shown p r e v i o u s l y ( H i r a i z u m i e_t a l . , 1973; Woodruff and Thompson, 1977), t h a t most of the exchange i n d u c e d by the P s t r a i n s T-007 and OKI o c c u r r e d w i t h i n the proximal i n t e r v a l s of the second chromosome. I t was assumed t h a t t h i s exchange took p l a c e i n and around the c e n t r i c h e t e r o c h r o m a t i n . S i n c l a r i and G r i g l i a t t i (1985) and t h i s r e p o r t show t h a t t h i s i s not the case. Most, i f not a l l , of the exchange i n these i n t e r v l a s i s euchromatic. Combining the da t a from both s t u d i e s : 15,639 chromosomes f o r T-007 and 23,399 f o r OKI have been examined to date with no evidence of heterochromatic exchange w i t h i n the e n t i r e l y heterochromatic c e n t r o m e r i c segment d e l i n e a t e d by l i g h t and r o l l e d . S i n c e both l i g h t and r o l l e d are l o c a t e d w i t h i n the heterochromatin of the l e f t and r i g h t arms of chromosome 2, i t i s p o s s i b l e 40 t h a t r e c o m b i n a t i o n c o u l d have o c c u r r e d i n the segment of heterochromatin d i s t a l to each of these markers. While i t i s f o r m a l l y p o s s i b l e , i t i s not c l e a r why r e c o m b i n a t i o n would occur i n t h i s p o r t i o n of the c e n t r i c heterochromatin but not w i t h i n the r e g i o n of heterochromatin f l a n k e d by l_t and r_l. In summary then, i n both of t h e s e s t r a i n s , h e t e r o c h r o m a t i c c r o s s i n g over i f i t occurs at a l l i s extremely rare. The s i m p l e s t e x p l a n a t i o n f o r the lac k of heterochromatic exchange events i s that P elements do not normally i n s e r t i n t o and/or subsquently e x c i s e from, the heterochromatic segments of a chromosome. This i s supported by a number of s t u d i e s i n w h i c h t h e P e l e m e n t c a r r y i n g a f u n c t i o n i n g gene was transformed i n t o the D r o s o p h i l a embryo (Goldberg e_t a l . , 1983; R i c h a r d et^ a l . , 1983; S c h o l n i c k et a l . , 1983; S p r a d l i n g and Rubin, 1983; H a z e l r i g g ejt a l . , 1984). These P e l e m e n t s r e a d i l y i n s e r t i n t o the genome but r a r e l y , i f ever, do they i n s e r t i n t o h e t e r o c h r o m a t i n . In o v e r 200 i n s e r t i o n s i d e n t i f i e d to date o n l y 2 e l e m e n t s e x h i b i t e d a v a r i e g a t i o n e f f e c t , s u g g e s t i n g t h a t they may have i n s e r t e d i n t o or near heterochromatin ( S p r a d l i n g and Rubin, 1983; and H a z e l r i g g et a l . , 1984). However, one of the e l e m e n t s i n s e r t e d at the t i p of the chromosome, p o s s i b l y near the t e l o m e r e . The o t h e r i n s e r t s at the euchromatin/heterochromatin j u n c t i o n and i t i s not p o s s i b l e to t e l l i f i t i n s e r t e d i n t o or s i m p l y near the heterochromatin. 41 Why would P e l e m e n t s be abse n t from h e t e r o c h r o m a t i n ? There are at l e a s t two p o s s i b l e e x p l a n a t i o n s . Heterochromatin may l a c k the 8 bp r e c o g n i t i o n sequence i n t o which the P elements i n s e r t . T h i s r e c o g n i t i o n sequence i s composed mostly of the bases G and C whereas h e t e r o c h r o m a t i n i s known to be very A-T r i c h . On the other hand, the r e c o g n i t i o n sequence i s not h i g h l y c o n s e r v e d i n c o m p o s i t i o n , and c o n s i d e r i n g t h a t heterochromatin makes up approximately one f i f t h of the t o t a l DNA i n a c e l l , i t i s d i f f i c u l t to i m a g i n e t h a t some form of the r e c o g n i t i o n sequence i s not p r e s e n t i n h e t e r o c h r o m a t i n . Another p o s s i b i l i t y i s that an accep t a b l e r e c o g n i t i o n sequence does e x i s t i n the h e t e r o c h r o m a t i c r e g i o n s of the D r o s o p h i l a genome, but i s n o r m a l l y i n a c c e s s i b l e to P element i n s e r t i o n due to s t e r i c c o n s t r a i n t s imposed by the h i g h l y condensed s t a t e of the DNA. Transposable elements much l a r g e r than the r e l a t i v e l y s m a l l P el e m e n t are found i n h e t e r o c h r o m a t i n ( F o l d b a c k and c o p i a e l e m e n t s , f o r example; T r u e t t e_t a l . , 1981, Ruddell, personal communication; f o r review see Rubin, 1983). I f one h y p o t h e s i z e s t h a t P e l e m e n t s are b a r r e d from the h e t e r o c h r o m a t i c segments f o r s t e r i c r e a s o n s , then i t i s n e c e s s a r y to p o s t u l a t e f u r t h e r t h a t the c o n d i t i o n s and/or l i m i t a t i o n s on i n s e r t i o n of P elements must d i f f e r from those of t h o s e of the l a r g e r e l e m e n t s which are not s i m i l a r l y hampered. There i s no a p r i o r i r e a s o n why a l l e u c h a r y o t i c 42 e l e m e n t s s h o u l d o p e r a t e by the same " r u l e s " ; the v a r i o u s c l a s s e s of p r o k a r y o t i c t r a n s p o s a b l e e l e m e n t s o c c a s i o n a l l y d i f f e r q u i t e r a d i c a l l y i n t h e i r behaviour. B u t y r a t e was used to d i s r u p t c h r o m a t i n s t r u c t u r e i n an a t t e m p t to l e a r n whether or not i t i s p o s s i b l e to reduce the " s t e r i c r e s t r a i n t s " of h i g h l y condensed h e t e r o c h r o m a t i n and allow P-induced recombination to take place. The o b s e r v a t i o n that b u t y r a t e induced no recombination i n the Oregon R cross i s important because some chemicals are known to induce male r e c o m b i n a t i o n i n the absence of P e l e m e n t s ( F e r r e s e_t a l . , 1984). F o r example, two i n t e r c a l a t i n g a g e n t s , e t h i d i u m bromide and a c r i d i n e orange, cause s i g n i f i c a n t l e v e l s of r e c o m b i n a t i o n on t h e t h i r d c h r o m o s o m e s of JJ r_ <) j3 £P_ h_ J L 1^ a_ m e l a n o g a s t e r males. P r e s u m a b l y t h e s e i n t e r c a l a t i n g a g e n t s d i s r u p t the DNA h e l i x to the extent that chromosome breakage o c c u r s which i s o c c a s i o n a l l y r e s o l v e d v i a a homologous r e c o m b i n a t i o n event. However, ethoxy c a f f e i n e , a n o t h e r i n t e r c a l a t i n g agent, f a i l s to produce any male recombination. F e r r e s e_t a l . s u g g e s t t h a t e t h i d i u m bromide and a c r i d i n e orange somehow s u p p r e s s normal r e p a i r mechanisms so t h a t broken s t r a n d s are o c c a s i o n a l l y r e a n n e a l e d i n c o r r e c t l y to y i e l d recombinant chromosomes. The a l t e r a t i o n i n chromatin s t r u c t u r e i n d u c e d by b u t y r i c a c i d i s c l e a r l y a l e s s d i r e c t e f f e c t than t h a t of the above a g e n t s , or perhaps b u t y r a t e i s unable to suppress the normal r e p a i r mechanisms. In any case, A3 the i m p o r t a n t p o i n t i s t h a t b u t y r a t e f a i l s to i n d u c e male recombination i n a non-dysgenic c r o s s . In c o n t r a s t to the above l a c k of e f f e c t of b u t y r a t e on t h e p r e v i o u s c r o s s e s , b u t y r a t e does i n f l u e n c e male r e c o m b i n a t i o n i n the d y s g e n i c c r o s s e s . I t appears t h a t b u t y r a t e can o n l y a f f e c t male r e c o m b i n a t i o n i n c o n j u n c t i o n with the r e g u l a r dysgenic machinery. The s i m p l e s t c o n c l u s i o n t h a t can be made i s t h a t b u t y r a t e f a c i l i t a t e s i n s e r t i o n or e x c i s i o n of the elements by reducing the c o n s t r a i n t s imposed upon them by chromatin condensation. The most i m p o r t a n t o b s e r v a t i o n r e g a r d i n g the b u t y r a t e -t r e a t e d d y s g e n i c c r o s s e s i s t h a t f o r b o t h P s t r a i n s , a s i g n i f i c a n t l e v e l of h e t e r o c h r o m a t i c exchange was produced. T h i s s t r o n g l y f a v o u r s t h e h y p o t h e s i s t h a t r e c o g n i t i o n sequences may be found i n h e t e r o c h r o m a t i n but are n o r m a l l y i n a c c e s s i b l e to the e l e m e n t s . A l l of the h e t e r o c h r o m a t i c e x c h a n g e s o b s e r v e d were s i n g l e ( n o n - c 1 u s t e r e d ) e v e n t s . Whether t h i s r e f l e c t s a d i f f e r e n c e i n t i m i n g of the exchange event i n h e t e r o c h r o m a t i n r e l a t i v e to e u c h r o m a t i n or i n the n a t u r e of the exchange i t s e l f ( i f i t were an i m p r e c i s e exchange, f o r example, one would e x p e c t the " r e c i p r o c a l " product to be l e t h a l ) , or merely the r e s u l t of a s m a l l sample s i z e i s not c l e a r . Of the 11 e v e n t s which were o b s e r v e d , about 32% of them, or four events, could have been expected to 44 be c l u s t e r s (as noted i n the r e s u l t s ) . F u r t h e r c r o s s e s to i n c r e a s e the sample s i z e may i n d i c a t e whether or not the lac k of c l u s t e r i n g of the heterochromatic exchanges i s s i g n i f i c a n t . S i n c l a i r and G r i g l i a t t i (1985) observed some heterochromatic exchange i n a cross i n v o l v i n g the P s t r a i n hi2. Seven events were o b s e r v e d , among a p p r o x i m a t e l y 21,000 c h r o m o s o m e s examined, and each of t h e s e was a l s o a s i n g l e , u n c l u s t e r e d event. I t i s q u i t e i n t e r e s t i n g to note t h a t i n the above study the s t r a i n h12 i n d u c e d some h e t e r o c h r o m a t i c exchange. why would t h i s s t r a i n and none of the o t h e r s t e s t e d d i s p l a y h e t e r o c h r o m a t i c MR? S i n c l a i r and G r i g l i a t t i s u g g e s t t h a t P e l e m e n t s are p r e s e n t i n the h e t e r o c h r o m a t i n of t h i s s t r a i n . There i s another p o s s i b l e e x p l a n a t i o n . h y b r i d dysgenesis i s known to have m o b i l i z e d c o p i a e l e m e n t s (Rubin e_t a l . , 1982). Copia elements and other members of the c o p i a - l i k e f a m i l y show d e c i d e d p r e f e r e n c e s f o r l o c a t i o n s i n h e t e r o c h r o m a t i n ( F i n n e g a n , 1984; a l s o R u d d e l l , p e r s o n a l c o m m u n i c a t i o n ) . I suggest the p o s s i b i l i t y that hi2 normally c a r r i e s a quiescent c o p i a - l i k e element (perhaps gypsy) i n the h e t e r o c h r o m a t i n , near the lt_ l o c u s , and that t h i s element was m o b i l i z e d during the d y s g e n i c c r o s s e s to produce some r e c o m b i n a t i o n and chromosome rearrangements (which might be the b a s i s f o r the JLt m u t a t i o n s o b s e r v e d f o r t h i s s t r a i n ) . T h i s s u g g e s t i o n c o u l d e a s i l y be t e s t e d by jLn s i t u h y b r i d i z a t i o n . 45 I t i s i n t e r e s t i n g t h a t the two P s t r a i n s responded d i f f e r e n t l y to butyrate treatment. The T-007 s t r a i n shows an i n c r e a s e d l e v e l of r e c o m b i n a t i o n i n a l l t h r e e chromosomal i n t e r v a l s e x a m i n e d on t h e r i g h t arm of chromosome two ( i n c l u d i n g the c e n t r o m e r i c r e g i o n ) . I t may be t h a t some regions of euchromatin are more h i g h l y condensed than others; i n f a c t a c t i v e c h r o m a t i n i s known to have a more "open" c o n f o r m a t i o n than i n a c t i v e (as d e t e r m i n e d , f o r example, by n u c l e a s e s e n s i t i v i t y s t u d i e s ) , and perhaps b u t y r a t e r e l a x e s t h e s e r e g i o n s and p e r m i t s i n s e r t i o n and/or e x c i s i o n of P elements with concommittant recombination i n these regions. S t e r i c c o n s t r a i n t s , i n other words, may be imposed by c e r t a i n r e g i o n s of e u c h r o m a t i n as w e l l as h e t e r o c h r o m a t i n , and d e f i n i n g such regions might prove q u i t e i n t e r e s t i n g . The OKI s t r a i n showed very l i t t l e change i n p a t t e r n of r e c o m b i n a t i o n ( a s i d e from i n t e r v a l number 3) as a r e s u l t of butyrate treatment and the o v e r a l l frequency of recombination was s i m i l a r to t h a t of the c o n t r o l . Why s h o u l d the two s t r a i n s r e a c t so d i f f e r e n t l y to butyrate treatment? P o s s i b l y there e x i s t s some sequence polymorphism between them such that when the more condensed euchromatic regions of the OKI s t r a i n are opened up by butyrate treatment there are no r e c o g n i t i o n sequences newly exposed to permit element i n s e r t i o n i n t o these r e g i o n s . A l t h o u g h i t i s l i k e l y t h a t the two s t r a i n s p o s s e s s 46 some sequence d i f f e r e n c e s (as they are g e o g r a p h i c a l l y and temporally d i s t i n c t ) , i n v o king sequence polymorphism may be unnecessary. A s i m p l e r e x p l a n a t i o n e x i s t s . The d i f f e r e n c e i n response may simply r e f l e c t d i f f e r i n g s i t e s of reside n c e of P elements i n the two s t r a i n s ; the elements are known to r e s i d e i n d i f f e r e n t l o c a t i o n s among the v a r i o u s P s t r a i n s examined (see B r e g l i a n o and K i d w e l l , 1983). Another p o s s i b i l i t y i s that male recombination r e s u l t s from m o b i l i z a t i o n of elements o t h e r than P, such as c o p i a e l e m e n t s , which a r e a l s o p r e s e n t at d i f f e r e n t s i t e s i n the two P s t r a i n s . I t i s known t h a t h y b r i d dysgenesis causes m o b i l i z a t i o n of other elements ( f o r example, the element m d g 4 ; Ge r a s i m o v a e_t a 1., 1984); whether t h e m o b i l i z a t i o n of t h e s e e l e m e n t s i s a s s o c i a t e d w i t h c r o s s o v e r i s not known. In any case i t i s i m p o s s i b l e to d i s t i n g u i s h whether e v e n t s o b s e r v e d here are the r e s u l t of m o b i l i z a t i o n of P elements or other transposons. Su(var)325 r e s i d e s at p o s i t i o n 53.3 (j^l.4 map u n i t s ) on the l e f t arm of chromosome 3. I t l i e s i n the i n t e r v a l between the markers Gl_ ( g l u e d ) and S_b ( s t u b b l e ) which i n c l u d e s the c e n t r i c heterochromatin. In the process of mapping the gene, T e j a n i and K r o i t s c h (1982, personal communication) found that _3 2 5_ c a u s e s a s i g n i f i c a n t ( 5 0 % ) i n c r e a s e i n t h e m e i o t i c r e c o m b i n a t i o n f r e q u e n c y i n t h i s i n t e r v a l . I chose to study the e f f e c t of Su(var)325 on male recombination because of t h i s p o s s i b l e e f f e c t on m e i o t i c recombination. In c o n t r a s t to 325, 47 the o t h e r S u ( v a r ) m u t a t i o n used i n t h i s s t u d y , 318, had no such e f f e c t on m e i o t i c r e c o m b i n a t i o n . 318 l i e s i n the same g e n e t i c i n t e r v a l as 325 at p o s i t i o n 56.1. Both 3 2 5 and 318 are part of a c l u s t e r of Su(var) genes and may be s t r u c t u r a l l y and/or f u n c t i o n a l l y r e l a t e d ( S i n c l a i r e_t a l . , 1983). When a cross i s made between M s t r a i n females bearing the m u l t i p l y marked second chromosome and a S u ( v a r ) 3 2 5 - b e a r i n g chromosome and M s t r a i n O r e g o n R m a l e s , t h e r e i s no r e c o m b i n a t i o n o b s e r v e d i n the r e s u l t i n g FI progeny. T h i s e l i m i n a t e s t h e p o s s i b i l i t y t h a t .3 2_ 5_ c a u s e s l o c a l i z e d chromosome breakage and male r e c o m b i n a t i o n . I f Su(var)325 a l t e r s male recombination f r e q u e n c i e s at a l l , i t appears to do so by augmenting the normal mechanisms of r e c o m b i n a t i o n — e i t h e r m e i o t i c or d y s g e n i c — a n d not v i a some other independent process. The p r e s e n c e of 325 i n the d y s g e n i c c r o s s i n which the o r i g i n a l male p a r e n t i s of the OKI s t r a i n i n c r e a s e d the o v e r a l l amount of male r e c o m b i n a t i o n . The d i s t r i b u t i o n of recombinant events was s i m i l a r to that found i n the dysgenic c r o s s l a c k i n g 325, with the exception of the _b to l_t i n t e r v a l . In t h i s i n t e r v a l (number 2), the p e r c e n t r e c o m b i n a t i o n was n e a r l y t w i c e t h a t of the s t a n d a r d d y s g e n i c c r o s s . T h i s i s comparable to the 1.5-fold i n c r e a s e i n m e i o t i c recombination f r e q u e n c y o b s e r v e d d u r i n g t h e m a p p i n g of t h e gene on 48 chromosome t h r e e . 3 2 5_ d i d not c a u s e any c h a n g e s i n recombination frequency i n the T-007 c r o s s . Su(var)318 had no e f f e c t on e i t h e r P s t r a i n , which s u g g e s t s t h a t i t may a c t q u i t e d i f f e r e n t l y from 325. I f 318 and 3 2 5 are r e a l l y p a r t of a f u n c t i o n a l l y r e l a t e d c l u s t e r of genes, t h e i r f u n c t i o n s are s u f f i c i e n t l y d i s t i n c t to cause d i f f e r e n t responses i n the dysgenic c r o s s e s . 318 may act upon some aspect of the chromatin s t r u c t u r e r i g h t i n the t r a n s i t i o n r e gion, so s p e c i f i c a l l y that recombination, both dysgenic and m e i o t i c a r e u n a f f e c t e d . I t a p p a r e n t l y does not a c t upon heterochromatin, or does so i n a manner which does not allow dysgenic recombination to take place i n the i n t e r v a l between 11 and r _ l . T h i s i s as e x p e c t e d g i v e n the e f f e c t of 3 2 5 and o n m e i o t i c r e c o m b i n a t i o n . .3.2_5 a l t e r s m e i o t i c recombination as w e l l as dysgenic, while 318 a l t e r s n e i t h e r . P r e v i o u s s t u d i e s have shown (see E n g e l s , 1983) t h a t the f a c t o r s which are known to a l t e r m e i o t i c recombination do not seem to a f f e c t male r e c o m b i n a t i o n . The m u t a n t _3 2_ 5_ i s a p p a r e n t l y an e x c e p t i o n to t h i s o b s e r v a t i o n as i t seems to a f f e c t both types of recombination i n an analogous manner. 3 2 5, then, a c t s on a n a l a g o u s a r e a s : the l e f t hand e u c h r o m a t i n / h e t e r o c h r o m a t i n boundary of chromosome two (MR) and the r i g h t of l e f t hand boundary of chromosome t h r e e ( m e i o t i c recombination i n females), to i n c r e a s e recombination f r e q u e n c y . I t a l s o a c t s on the X chromosome ,to s u p p r e s s the 49 i n a c t i v a t i o n of w + i n w , and i t i s l i k e l y t h a t i t a c t s on the e u c h r o m a t i n / h e t e r o c h r o m a t i n boundary of t h i s chromosome a l s o . The euchromatin/heterochromatin boundary i s a r e g i o n of g r a d u a l r a t h e r than a b r u p t change ( L i f s c h y t z , 1978). I t a p p e a r s c y t o l o g i c a l l y as a r a t h e r d i f f u s e s t r e t c h of "B" h e t e r o c h r o m a t i n , g r a d u a l l y c h a n g i n g to the more h i g h l y condensed "A" ( c e n t r i c ) h e t e r o c h r o m a t i n (Lakhotia and Jacob, 1974). In the t r a n s i t i o n zone there are euchromatic sequences i n t e r s p e r s e d between b l o c k s of i n t e r c a l a r y heterochromatin. This apparently makes up the s t r u c t u r e of the l e s s d i f f u s e B-' type heterochromatin. I f , as seems l i k e l y , s u p p r e s s o r genes a r e i n v o l v e d i n e s t a b l i s h i n g , m a i n t a i n i n g or r e g u l a t i n g chromatin s t r u c t u r e , then i t i s l o g i c a l to l o o k f o r s i m i l a r i t i e s i n the makeup of the chromatin i n those regions which appear to be a f f e c t e d by the 325 mutant: the l e f t euchromatin/heterochromatin boundary of chromosome two, the l e f t and/or r i g h t (because both are i n c l u d e d i n the i n t e r v a l between GJL and ^_b) boundary of chromosome three, and the t r a n s i t i o n zone of the X chromosome. I t may be p o s s i b l e i n t h i s way to p i n p o i n t the a s p e c t of chromatin s t r u c t u r e upon which 325 has i t s e f f e c t . About 80% of h e t e r o c h r o m a t i n i s composed of s a t e l l i t e sequences: short h i g h l y repeated sequences occupying d i s t i n c t 50 r e g i o n s on i n d i v i d u a l chromosomes. There are s i x major s a t e l l i t e s e q u e n c e s i n JJ j_ o_ js o_j> ti i_ 1^ a_ m 3. a n o_£ a.^ t_^ J_» e a c h d i s t i n g u i s h a b l e on the b a s i s of i t s s e d i m e n t a t i o n i n c e s i u m c h l o r i d e d e n s i t y g r a d i e n t s . A s a t e l l i t e sequence i s an o b v i o u s a s p e c t of chromosome makeup which can be e a s i l y examined f o r p r e s e n c e or absence i n the a r e a s of i n t e r e s t . I n t e r a c t i o n s between e u c h r o m a t i c genes or gene p r o d u c t s and heterochromatic sequencs are not unknown. For example, there are c e r t a i n p r o t e i n s known to be p r e f e r e n t i a l l y a s s o c i a t e d w i t h c e r t a i n s a t e l l i t e sequences ( H s i e h and B r u t l a g , 1979; L e v i n g e r and V a r s h a v s k y , 1982). In a d d i t i o n , S a n d l e r (1977) observed that two maternal e f f e c t mutants of IX/ melanogaster, a_bo ( a b n o r m a l o o c y t e ) and d_ a ( d a u g h t e r l e s s ) , c o u l d be p a r t i a l l y rescued i n the presence of e x t r a copies of c e r t a i n X h e t e r o c h r o m a t i c sequences. Each gene i n t e r a c t e d w i t h one s p e c i f i c sequence. I e n v i s i o n the p o s s i b i l i t y of an analogous r e l a t i o n s h i p between the 325 mutant and a s a t e l l i t e sequence. The 325 p r o d u c t may i n t e r a c t w i t h t h i s sequence to cause l o o s e n i n g of the DNA s t r u c t u r e such t h a t r e c o m b i n a t i o n i s enhanced or suppression of v a r i e g a t i o n i s p o s s i b l e . There i s one sequence -1.697g/cc- which i s found on the X heterochro-m a t i n and l e f t t r a n s i t i o n b o u n d a r i e s of chromosomes two and t h r e e (Peacock ejt a 1., 1973). However, i t i s a l s o found on the r i g h t boundary of chromosome two, where 3 2 5 a p p e a r s to have no e f f e c t . T h i s may be e x p l a i n e d i n one of two ways: 51 e i t h e r 3 2 5 i s not i n any way i n v o l v e d w i t h t h i s p a r t i c u l a r s a t e l l i t e sequence, and i t s p r e s e n c e i n the r e g i o n s of i n t e r e s t i s c o i n c i d e n t a l ; or i t does i n t e r a c t w i t h the 1.697 sequence, but no d y s g e n i c r e c o m b i n a t i o n i s o b s e r v e d on the r i g h t boundary of chromosome two due to the p a u c i t y of ap p r o p r i a t e r e c o g n i t i o n s i t e s — o r P e l e m e n t s - - i n that region. Given the h i g h l y conserved sequence s t r u c t u r e of s a t e l l i t e s , i t seems u n l i k e l y t h a t r e c o g n i t i o n sequences p r e s e n t i n the l e f t hand b l o c k of 1.697 would not a l s o be p r e s e n t i n the r i g h t hand b l o c k . There i s some e v i d e n c e , a l s o , t h a t the s u p p r e s s o r gene a c t s on e u c h r o m a t i c r a t h e r than h e t e r o c h r o -matic sequences (see l a t e r d i s c u s s i o n ) . The e f f e c t o f t h e 3_ 2 _5 m u t a t i o n may be v e r y s t r a i g h t f o r w a r d . I t may act upon any heterochromatin/euchro-matin boundary with the net e f f e c t of r e l e a s i n g some sequences from the t i g h t l y compressed regions of the t r a n s i t i o n zone to a l e s s c o n d e n s e d f o r m . The e f f e c t , or l a c k of one, on d y s g e n i c r e c o m b i n a t i o n may then stem d i r e c t l y from the p r e s e n c e or absence of P-element r e c o g n i t i o n s i t e s i n the a r e a s which have been newly exposed. These newly exposed regions need not be s a t e l l i t e sequences, as unique sequences, as w e l l as b l o c k s of e u c h r o m a t i n , are known to r e s i d e i n the t r a n s i t i o n zone. These sequences w i l l not be the same f o r any two t r a n s i t i o n r e g i o n s , and can thus e x p l a i n the d i f f e r e n c e s 52 between the r e s p o n s e s of v a r i o u s t r a n s i t i o n r e g i o n s i n the presence of 325. I f the s u p p r e s s i o n a c t i v i t y of 3 2 5 o p e r a t e s by the same mechanism as does the e f f e c t on r e c o m b i n a t i o n — w h i c h i s the s i m p l e s t p o s s i b i l i t y - - t h e n the suppressor gene may not a f f e c t heterochromatin at a l l , c o n t r a r y to i n i t i a l e x p e c t a t i o n s (see I n t r o d u c t i o n ) . Rather, the mutant may confer upon euchromatin t h e a b i l i t y t o r e s i s t h e t e r o c h r o m a t i c s p r e a d i n g . Two o b s e r v a t i o n s s u g g e s t t h a t t h e m u t a n t J3 2 5_ may a f f e c t euchromatin r a t h e r than heterochromatin. No heterochromatic c r o s s i n g over was o b s e r v e d i n e i t h e r d y s g e n i c c r o s s i n the presence of 325. 325 i s a stronger suppressor of v a r i e g a t i o n than i s butyrate, yet i t d i d not a f f e c t the heterochromatin as d i d b u t y r a t e . Thus, the mutant must a c t on a d i f f e r e n t component of chromosome s t r u c t u r e than does b u t y r a t e , and probably more s p e c i f i c a l l y . Therefore, 325 probably a c t s upon the e u c h r o m a t i n i n and around the t r a n s i t i o n zone (the o n l y r e g i o n where i t ap p e a r s to have any e f f e c t at a l l ) . In a d d i t i o n , the f a c t t h a t m e i o t i c r e c o m b i n a t i o n i s a l t e r e d by 325 i n d i c a t e s t h a t 3 2 5 most l i k e l y a c t s upon e u c h r o m a t i c sequences, s i n c e m e i o t i c recombination i s very r a r e i n hetero-c h r o m a t i n , and r a t h e r i n f r e q u e n t i n t h e r e g i o n of t h e t r a n s i t i o n zone. 53 CONCLUSIONS In d y s g e n i c c r o s s e s i n v o l v i n g the P s t r a i n s T-007 and OKI, r a r e l y , i f ever, does h e t e r o c h r o m a t i c exchange o c c u r on chromosome two. I t was p o s t u l a t e d that t h i s l a c k of exchange r e f l e c t e d s t e r i c c o n s t r a i n t s due to the t i g h t p a c k a g i n g of h e t e r o c h r o m a t i n . T h i s h y p o t h e s i s was t e s t e d by e x p o s i n g dysgenic h y b r i d males from e i t h e r s t r a i n to .15M b u t y r i c a c i d , a presumed m o d i f i e r of c h r o m a t i n s t r u c t u r e . In both P s t r a i n s , the b u t y r i c a c i d induced s i g n i f i c a n t l e v e l s of male r e c o m b i n a t i o n i n the h e t e r o c h r o m a t i c l_t to r_l i n t e r v a l of chromosome two. Thus i t appears that chromatin s t r u c t u r e can i n f l u e n c e the i n s e r t i o n and/or e x c i s i o n of the e l e m e n t s and t h i s i s r e f l e c t e d i n male r e c o m b i n a t i o n . D i f f e r e n c e s i n r e s p o n s e t o b u t y r a t e by t h e two P s t r a i n s may r e f l e c t d i f f e r e n c e s i n the s i t e s of r e s i d e n c e of P e l e m e n t s i n each s t r a i n . The same dysgenic c r o s s e s were performed i n combination w i t h two Su(var) genes: 3 2 5 and 318. N e i t h e r produced any heterochromatic recombination, which suggests that t h e i r modes of a c t i o n may be q u i t e d i f f e r e n t from, and more s p e c i f i c than, the r a t h e r general e f f e c t s of b u t y r a t e . 325 caused an i n c r e a s e i n the recombination frequency i n the i n t e r v a l c o n t a i n i n g the l e f t heterochromatin/euchromatin 54 boundary of chromosome two of the OKI s t r a i n . I t i s suggested that the 325 mutant most l i k e l y i n t e r a c t s with the euchromatin (although the p o s s i b i l i t y of an i n t e r a c t i o n with heterochro-m a t i n i s a l s o d i s c u s s e d ) i n the t r a n s i t i o n zone, and a c t s to r e l e a s e some sequences from t h e i r h i g h l y condensed s t a t e to a l e s s compressed form. In areas where newly exposed sequences c o n t a i n e i t h e r r e c o g n i t i o n sequences f o r P e l e m e n t s , or the e l e m e n t s t h e m s e l v e s , d y s g e n i c r e c o m b i n a t i o n may take p l a c e . The f a c t t h a t the r i g h t boundary of chromosome two does not act s i m i l a r l y and that the T-007 s t r a i n experiences no s i m i l a r i n c r e a s e i n male recombination may be e x p l a i n e d by d i f f e r i n g s i t e s of r e s i d e n c e of the P elements i n d i f f e r e n t chromosome l o c a t i o n s , and between s t r a i n s . The most important o b s e r v a t i o n i s that a l t e r i n g chromatin s t r u c t u r e does not appear to i n f l u e n c e dysgenic recombination and thus (by i n f e r e n c e ) the m o b i l i t y of the P e l e m e n t s . 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S i t e - s p e c i f i c b r e a k s i n d u c e d by the male r e c o m b i n a t i o n f a c t o r 23.5 MRF i n D r_ o_ s^ .ojpji _i _1 _a melanogaster. Mutation Res. 108: 185-202. 64 APPENDIX I I p e r f o r m e d an a d d i t i o n a l e x p e r i m e n t w i t h the P s t r a i n OKI. A r e c i p r o c a l cross (one i n which the maternal parent was of the P s t r a i n , and the p a t e r n a l p a r e n t was the M s t r a i n hearing the m u l t i p l y marked second chromosome) was set up and t r e a t e d with 0.15M butyrate, as o u t l i n e d i n the MATERIALS AND METHODS s e c t i o n . T h i s was done to a s c e r t a i n that butyrate did not a c t o u t s i d e of the r e g u l a r d y s g e n i c mechanisms ( t o derepress the P elements, f o r example, d e s p i t e t h e i r being i n a P c y t o t y p e ) . 10,183 F2 p r o g e n y were s c o r e d , and 13 recombinant events were observed (36 recombinant i n d i v i d u a l s ) , 5 (7 r e c o m b i n a n t i n d i v i d u a l s ) i n i n t e r v a l 2 and 8 (29 i n d i v i d u a l s ) i n i n t e r v a l 4. There was no r e c o m b i n a t i o n observed i n any other i n t e r v a l . A c o n t r o l ( t h e r e c i p r o c a l c r o s s , n o t t r e a t e d w i t h b u t y r a t e ) was not p e r f o r m e d , but a n a l o g o u s e x p e r i m e n t s have been p e r f o r m e d numerous t i m e s (see B r e g l i a n o and K i d w e l l , 1983, f o r review). These s t u d i e s suggest that the r e c i p r o c a l c r o s s s h o u l d y i e l d male r e c o m b i n a t i o n at f r e q u e n c i e s about t e n - f o l d l o w e r than those of the d y s g e n i c c r o s s . I f the r e c i p r o c a l c r o s s had been p e r f o r m e d and had y i e l d e d f r e q u e n c i e s f i f t e e n times lower than the dysgenic cross, there would have been no s i g n i f i c a n t (G t e s t , p = .05) d i f f e r e n c e 65 between t h i s c r o s s and the r e c i p r o c a l c r o s s t r e a t e d w i t h b u t y r a t e . Thus, b u t y r a t e a p p e a r s not to o p e r a t e o u t s i d e of t h e d y s g e n i c s y s t e m . M o s t i m p o r t a n t , i t c a u s e no heterochromatic recombination o u t s i d e of the dysgenic system. However, because the c o n t r o l (untreated r e c i p r o c a l ) c r o s s was not a c t u a l l y performed, I decided to place t h i s o b s e r v a t i o n i n an appendix, r a t h e r than i n the t e x t .
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Zoology
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The effect of chromatin structure on P element-induced male recombination in Drosophila melanogaster
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