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The genetic analysis of the heterochromatin of chromosome 3 of Drosophila melanogaster Marchant, Gary Elvin 1986

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THE GENETIC ANALYSIS OF THE HETEROCHROMATIN OF CHROMOSOME 3 OF DROSOPH1LA HELAHQGASTER by GARY ELVIN MARCHANT B . S c , Un ivers i ty of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept th i s thes i s as conforming to the required s-tandard THE UNIVERSITY OF BRITISH COLUMBIA Ju ly , 1986 © G a r y E lv in Marchant, 1986 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 i t 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 Z o o l oo, The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 3oU <C i t 9 g ( . DE-6 (2/79) ABSTRACT The heterochromatin of the t h i r d chromosome i s the largest uncharacterized region of the D r o s o p h i l a we 1 a n o g a s t e r genome, and the la s t major block of D. we 1anogaster heterochromatin to be thoroughly analyzed. In the present study, t h i s region was g e n e t i c a l l y dissected by generating and analyzing a ser ies of attached, detached and reattached t h i r d chromosomes. Separate detachment experiments were conducted for a l l twelve poss ib le combinations of four newly synthesized s i s t e r - s t r a n d compound-3L's and three newly synthesized s i s t e r - s t r and compound-3R' s. A t o t a l of 443 recess ive l e tha l detachment products carrying putat ive heterochromatic d e f i c i e n c i e s were tested for complementation in a several-stage complementation ana ly s i s . The r e s u l t s revealed the presence of seven separable v i t a l regions in the heterochromatin of chromosome three. Attempts to reattach d e f i c i e n c y - c a r r y i n g detachment products es tabl i shed that six of these v i t a l regions are on the l e f t arm, and only one i s on the r ight arm. An analys i s of the types and frequencies of detachment product d e f i c i e n c i e s generated in each detachment experiment permitted the genetic charac te r i za t ion of the progenitor compounds. It was also poss ib le to determine the proximal-d i s t a l o r i e n t a t i o n of the genes on each arm, and to i d e n t i f y poss ib le breakpoints for each l e tha l detachment product produced. Seventy-f ive EMS-induced l e t h a l a l l e l e s of detachment product d e f i c i e n c i e s were also recovered and tested for complementation. Four add i t iona l genes in t h i r d chromosome heterochromatin were revea led , three on the l e f t arm and one on the r ight arm. At least three of the EMS-induced l e t h a l s were small d e f i c i e n c i e s . The i n t e r - a l l e i i c complementation observed between some EMS-induced l e t h a l s , as well as the recovery of a temperature s ens i t i ve mutation of a heterochromatic gene, provided further evidence that there are s ing le-copy, t ranscr ibed v i t a l genes in t h i r d chromosome heterochromatin. F i n a l l y , a c y t o l o g i c a l ana lys i s of three of the detachment product d e f i c i e n c i e s by L. Sandler and S. P i m p i n e l l i (personal i i communication) provided evidence that at least some of the genes uncovered in t h i s study are located in the most d i s t a l segments of t h i r d chromosome heterochromatin. The re su l t s suggest that the eleven v i t a l genes discovered in the heterochromatin of the t h i r d chromosome are not randomly d i s t r i b u t e d between, nor w i t h i n , the heterochromatic blocks of the l e f t and r ight arms. Th study provides further evidence that Drosophila heterochromatin i s gene t i ca l ly heterogeneous, with v i t a l genes being present in some heterochromatic segments but not others . TftBLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENT v i i INTRODUCTION 1 MATERIALS AND METHODS 23 RESULTS 29 DISCUSSION 73 LITERATURE CITED 77 i v LIST OF TABLES TABLE PAGE I. The number of detachment products recovered from each detachment experiment 30 II. The r e s u l t s of l e t h a l i t y tes t s with the detachment products from each detachment experiment . .32 III . S i s te r - s t rand compound t h i r d s recovered from reattachment of selected detachment exper iment s . . . . 40 IV. Dupl icat ions and d e f i c i e n c i e s ca r r i ed by progenitor compound th i rd s 46 V. Rescue of homozygous de f i c i en t s i s t e r - s t r and compound-3's by progenitor compounds 48 VI. Recovery of EMS-induced l e tha l a l l e l e s of d e f i c i e n c i e s c a r r i ed by t h i r d chromosome detachment products 54 VII . Results of complementation analys i s of twenty EMS-induced l e tha l a l l e l e s of L(3L)4B 63 v LIST OF FIGURES FIGURE PAGE 1. Models showing the synthesis of the four poss ible types of compound autosomes 18 2. Model showing the generation of four poss ible types of proximal d e f i c i e n c i e s c a r r i ed by detachment products 20 3. Accumulated complementation map D f putat ive d e f i c i e n c i e s ca r r i ed by detachment products from a l l twelve detachment experiments 35 4. Revised complementation map incorporat ing r e s u l t s of recombination mapping and reattachment experiments 44 5. D i s t r i b u t i o n of genes in heterochromatin of chromosome three 50 6. Complementation pattern of d e f i c i e n c i e s and EMS-induced l e t h a l s of proximal r ight arm 56 7. Complementation pattern of d e f i c i e n c i e s and EMS-induced l e tha l s of genes L (3D 1, L(3L)2, and L(3L)3 59 8. Complementation pattern of d e f i c i e n c i e s and EMS-induced l e t h a l s of 8A-80 de f ic iency 61 9. Complementation pattern of d e f i c i e n c i e s and EMS-induced l e t h a l s of L(3L)5, L(3L)6, L(3L)7, and L(3L)8 regions 65 10. Chromosome banding analys i s of three detachment product d e f i c i e n c i e s 69 vi ACKNOWLEDGEMENT I g r e a t l y a p p r e c i a t e t h e g u i d a n c e , a s s i s t a n c e and s u p p o r t o f Dr. D a v i d Holm i n c a r r y i n g o u t t h i s work. I am a l s o i n d e b t e d t o t h o s e w o r k i n g i n Dr. Holm's l a b o r a t o r y , p a r t i c u l a r l y K a r e n M o r i n and Bob D e v l i n . F i n a l l y , I would l i k e t o t h a n k B r o c k Rhone and e s p e c i a l l y J e n n i f e r K i n l o c h M a r c h a n t f o r t h e i r g e n e r o u s a s s i s t a n c e i n p r e p a r i n g t h e f i g u r e s and f i n a l d r a f t of t h i s t h e s i s . v i i INTRODUCTION Since f i r s t described by the German inves t iga tor Emil Heitz (Hei tz , 1928), heterochromatin has both puzzled and fascinated researchers in many f i e l d s of biology for over half a century. Although s i g n i f i c a n t quant i t ie s of heterochro-matin have been observed in the genomes of almost a l l higher organisms (Brown, 1966), no function has ever been c l e a r l y demonstrated for these chromosomal regions . Much remains unknown about the nature, composit ion, and propert ies of heterochromatin. Furthermore, the s i gn i f i c ance of what i s known i s often not r ead i ly apparent. Exceptions have been found to almost every rule or genera l i za -t ion about heterochromatin. To paraphrase Winston C h u r c h i l l , heterochromatin appears to be "a r i d d l e wrapped in a mystery ins ide an enigma". Heterochromatin was o r i g i n a l l y defined by i t s d i f f e r e n t i a l s t a in ing pattern or "heteropycnosis" (Hei tz , 1928). Unlike the other regions of the genome known as euchromatin, heterochromatin remains condensed and dark-s ta in ing throughout most of the c e l l c y c l e . However, there are problems with def ining heterochro-matin so le ly by the property of heteropycnosis . It i s now known that there are several types of condensed, heteropycnotic chromatin. In some cases, such as the inac t iva ted nucleated erythrocytes of b i r d s , f i s h , and r e p t i l e s , the ent i re genome becomes permanently condensed. This type of condensed chromatin i s not properly c l a s s i f i e d as heterochromatin, s ince i t involves the general conden-sation of the ent i re genome rather than s p e c i f i c chromosomes or chromosomal regions . Condensed chromatin that i s true heterochromatin can be of two types -c o n s t i t u t i v e or f a c u l t a t i v e heterochromatin (Brown, 1966). Cons t i tu t ive hetero-chromatin i s present at i d e n t i c a l pos i t ions on homologous chromosomes, and can 1 c o n s i s t o f an e n t i r e c h r o m o s o m e o r a s p e c i f i c r e g i o n o f a c h r o m o s o m e . F a c u l -t a t i v e h e t e r o c h r o m a t i n i s f o r m e d by t h e c o n d e n s a t i o n o f o n l y o n e o f a p a i r o f h o m o l o g o u s c h r o m o s o m e s . A w e l l - k n o w n e x a m p l e o f f a c u l t a t i v e h e t e r o c h r o m a t i n i s t h e r a n d o m i n a c t i v a t i o n a n d c o n d e n s a t i o n o f o n e o f t h e X c h r o m o s o m e s i n m o s t f e m a l e m a m m a l i a n c e l l s f o r t h e p u r p o s e s o f d o s a g e c o m p e n s a t i o n ( L y o n , 1 9 6 2 ) . S i n c e a g i v e n X c h r o m o s o m e i s h e t e r o c h r o m a t i c i n some c e l l s b u t a n o r m a l e u c h r o -m a t i c X i n o t h e r s , f a c u l t a t i v e h e t e r o c h r o m a t i n i s c o n s i d e r e d t o b e a s p e c i a l c o n d e n s e d s t a t e o f e u c h r o m a t i n . C o n s t i t u t i v e h e t e r o c h r o m a t i n , on t h e o t h e r h a n d , c a n be d i s t i n g u i s h e d f r o m b o t h e u c h r o m a t i n a n d f a c u l t a t i v e h e t e r o c h r o m a t i n by t h e C - b a n d i n g t e c h n i q u e w h i c h o n l y s t a i n s c o n s t i t u t i v e h e t e r o c h r o m a t i n ( P a r d u e a n d G a l l , 1 9 7 0 ; A r r i g h i a n d H s u , 1 9 7 1 ) . A k e y d i s c o v e r y t h a t c l e a r l y d e m o n s t r a t e d t h a t c o n s t i t u t i v e h e t e r o c h r o m a t i n i s c h e m i c a l l y a n d s t r u c t u r a l l y d i s t i n c t f r o m b o t h e u c h r o m a t i n a n d f a c u l t a t i v e h e t e r o c h r o m a t i n was t h e f i n d i n g t h a t c o n s t i t u t i v e h e t e r o c h r o m a t i n i s g r e a t l y e n r i c h e d i n h i g h l y r e p e a t e d s a t e l l i t e DNA ( Y a s m i n e h a n d Y u n i s , 1 9 6 9 ; R a e , 1 9 7 0 ; J o n e s a n d R o b e r t s o n , .1970; P a r d u e a n d G a l l , 1 9 7 0 ; P e a c o c k et a i . , 1 9 7 3 ; G a l l a n d A t h e r t o n , 1 9 7 4 ) . S a t e l l i t e DNA c o n s i s t s o f v e r y s h o r t s e q u e n c e s p r e s e n t i n h u n d r e d s o f t h o u s a n d s o f c o p i e s p e r g e n o m e . P e a c o c k e t al. ( 1 9 7 3 ; 1 9 7 7 a ) h a v e s h o w n t h a t t h e c o n s t i t u t i v e h e t e r o c h r o m a t i n o f Drosophila m e l a n o g a s t e r a n d o t h e r o r g a n i s m s i s a l m o s t e n t i r e l y c o m p o s e d o f l o n g , h o m o g e n e o u s b l o c k s o f i n d i v i d u a l s a t e l l i t e DNA s e q u e n c e s t a n d e m l y a r r a n g e d i n a c h r o m o s o m e - s p e c i f i c o r d e r . T h e e s t i m a t e d a m o u n t o f s a t e l l i t e DNA i n e a c h b l o c k o f h e t e r o c h r o m a t i n a l m o s t c o m p l e t e l y a c c o u n t s f o r t h e t o t a l a m o u n t o f DNA p r e s e n t ( P e a c o c k et al., 1 9 7 7 b ) , s u g g e s t i n g t h a t t h e r e a r e f e w , i f a n y , o t h e r t y p e s o f DNA s e q u e n c e s w i t h i n D. m e l a n o g a s t e r h e t e r o c h r o m a t i n . T h i s v i e w was s u p p o r t e d by t h e d i s c o v e r y o f j u n c t i o n DNA m o l e c u l e s t h a t c o v a l e n t l y l i n k a d j a c e n t b l o c k s o f s a t e l l i t e DNA ( B r u t l a g et al., 1 9 7 7 ) . W h i l e v e r y f e w n o n - s a t e l l i t e s e q u e n c e s a p p e a r t o b e i n h e t e r o c h r o m a t i n , o n l y t r a c e a m o u n t s o f s a t e l l i t e DNA a r e f o u n d o u t s i d e o f 2 heterochromatin (Peacock e* a i . , 1977a, Cohen and Bowman, 1979). The c lose correspondence between c o n s t i t u t i v e heterochromatin and s a t e l l i t e DNA has important impl i ca t ions for the poss ib le function of heterochromatin. S a t e l l i t e DNA i s almost c e r t a i n l y g e n e t i c a l l y i n e r t . Most s a t e l l i t e sequences would code for very odd prote ins ; and others contain such a high proportion of t r a n s l a t i o n a l stop s ignals that i t i s very u n l i k e l y they could code for a protein (Southern, 1970; Bostock, 1980). More important ly , no RNA has been found to hybr id ize to s a t e l l i t e DNA (Flamm, Walker and McCallum, 1969; Woodcock and S i b a t i , 1975; Bostock, 1980). The only known example of s a t e l l i t e DNA t ran-s c r i p t i o n i s the apparently accidental "run-on" t r a n s c r i p t i o n of some s a t e l l i t e DNA from newt lampbrush chromosomes (Baldwin and MacGregor, 1985; Var ley , Macgregor and Erba, 1980). The high concentrat ion of s a t e l l i t e DNA found in c o n s t i t u t i v e heterochro-matin i s consis tent with e a r l i e r evidence that heterochromatin i s g e n e t i c a l l y i n a c t i v e . Heitz (1929) was the f i r s t to suggest that the condensed nature of heterochromatin implies that these regions are of l imi ted genetic importance. Drosophila g e n e t i c i s t s discovered long ago a s t r i k i n g c o r r e l a t i o n between regions of the genome with l i t t l e or no genetic a c t i v i t y and regions of hetero-chromatin. For example, Bridges (1916) observed that XO males in D r o s o p h i l a n e l a n o g a s t e r were phenotypica l ly normal but s t e r i l e , and therefore concluded that the heterochromatic Y chromosome was devoid of any v i t a l genes. S i m i l a r l y , very few mutations were mapped to the X chromosomal and autosomal heterochro-matin of t h i s species (Muller and Pa in ter , 1952; Kaufman, 1934). As well as having low mutation frequencies , heterochromatic regions are almost t o t a l l y exempt from meiotic crossing-over (Muller and Pa inter , 1932). Baker (1954) l a ter demonstrated that the absence of cross ing-over i s a general property of regions of c o n s t i t u t i v e heterochromatin. 3 The genetic i n a c t i v i t y of heterochromatin i s further supported by the frequent polymorphic d i s t r i b u t i o n of heterochromatin. In many species , the amount of heterochromatin on cer ta in chromosomes var ies considerably between i n d i v i d u a l s , with no apparent effect on phenotype or v i a b i l i t y (eg. Cra ig -Holmes, Moore and Shaw, 1975; Miklos and John, 1979; K u r n i t , 1979; Gustafson, Lukasewski and Bennett, 1983; Maresca, Singer and Lee, 1984). In D. ae l a / i o -gaster, females with between one and four doses of the heterochromatic region of the X chromosome are phenotypicai 1 y normal (Yamamoto and Mik los , 1977). Biochemical studies have also suggested that heterochromatin i s g e n e t i c a l l y i n e r t . Hsu (1967) pu l s e - l abe l l ed mouse c e l l s with t r i t i a t e d u r i d i n e , and observ-ed no rad ioac t ive grains over the heterochromatic regions . S ieger , Pera and Schwarzacher (1970) obtained s imi la r r e su l t s using N i c r o t u s a g r e s t i s . A l l t h i s evidence led to the widely held b e l i e f that c o n s t i t u t i v e heterochromatin i s g e n e t i c a l l y i n e r t . However, i t should be noted that none of the evidence excludes the p o s s i b i l i t y that heterochromatin possesses a low leve l of genetic ac t i vi ty . Since c o n s t i t u t i v e heterochromatin ex i s t s in s i g n i f i c a n t quant i t ie s in the genomes of almost a l l higher organisms and does not seem to code for appreciable amounts of prote in or RNA, i t i s reasonable to expect that heterochromatin performs some other funct ion . A number of functions have been proposed for heterochromatin, although there i s no conclus ive evidence to prove or disprove any of the functions that have been suggested. Several researchers have speculated that the primary function of hetero-chromatin i s to "protect " or " s t a b i l i z e " centromeres, nucleolar organizer regions , and telomeres (Brown, 1966; Yunis and Yasmineh, 1971; Hsu, 1975). Although the exact mechanism by which heterochromatin would provide protect ion i s usual ly not s p e c i f i e d , i t may be that heterochromatin acts as a passive spacer region that p h y s i c a l l y separates d i f f e rent regions of the chromosome. 4 A l t e r n a t i v e l y , heterochromatin may help to prevent spec i a l i zed chromosomal regions , such as the tandemly arranged ribosomal RNA genes at nucleolar organi-zer regions , -from being disrupted by meiotic cross ing-over . Heterochromatin does have a non-random d i s t r i b u t i o n in the genome and i s usual ly found adjacent to centromeres, nucleolus organizers , and telomeres. However, there are exceptions, such as the large V chromosome in D r o s o p h i l a n a s u t o i d s which i s completely heterochromatic except for the centromeric region (Cordeiro et a l . , 1975). A l s o , there are a few higher organisms, such as the Norwegian r a t , that do not have detectable amounts of heterochromatin and/or s a t e l l i t e DNA (Gosden et a l . , 1975; Timberlake, 1978; John and Miklos , 1979; Miklos , Wil lcocks and Baverstock, 1980; Sealy et a l . , 1981). If heterochromatin does function to protect or s t a b i l i z e cer ta in regions of the chromosome, then in at least some organisms such protec t -ion i s not requ i red . A second function proposed for heterochromatin i s that i t i n i t i a t e s the synapsis of homologous chromosomes during meiosis (Goldr ing , Brutlag and Peacock, 1975; Peacock et a l . , 1977a; Beauchamp et a l . , 1979). The s p e c i f i c pattern of s a t e l l i t e DNA blocks in the heterochromatin of each chromosome could be the basis of recogni t ion for proper homologue p a i r i n g . It has been shown that there are s p e c i f i c sequences, c a l l e d "co l 1ochores", in the heterochromatin of the X and Y chromosomes of D r o s o p h i l a nelaiiogaster that serve as X-Y pa i r ing s i t e s in males (Cooper, 1964; Yamamoto and Miklos , 1977; A u l t , L in and Church, 1982; Appels and H i l l i k e r , 1982). However, a var ie ty of chromosomal manipula-t ions in D. $ e l a n o g a s t e r have demonstrated that heterochromatin in general i s not necessary for proper meiotic pa i r ing (Yamamoto and Miklos , 1977; Yamamoto, 1979a; H i l l i k e r , Holm and Appels, 1982). Further , c y t o l o g i c a l s tudies have detected no evidence that heterochromatin f a c i l i t a t e s meiotic pa i r ing (Maguire, 1972; John, 1976). F i n a l l y , the fact that some organisms contain no observable 5 amounts of heterochromatin i s d i f f i c u l t to explain i f heterochromatin i s required for meiotic p a i r i n g . A t h i r d proposed function for heterochromatin i s the i n d i r e c t control of c e l l s ize and the rate of c e l l growth and d i v i s i o n (Bennett, 1972; C a v a l i e r -Smith, 1978; Cava l ie r -Smith , 1980). Important c h a r a c t e r i s t i c s such as c e l l and nuclear volume and the length of the c e l l cycle are i n d i r e c t l y cont ro l l ed by the amount of chromatin in the nucleus, known as the "nucleotype" . In t ra spec i f i c v a r i a t i o n s in heterochromatin content provides a poss ible means of a l t e r i n g the nucleotype and thus modifying important c e l l c h a r a c t e r i s t i c s such as c e l l s i ze and generation time. Var i a t ion in the amount of heterochromatin within a popula-t ion may help a species adjust to a new environment. Although a nucleotypic function for heterochromatin i s pos s ib le , there i s no experimental evidence that heterochromatin performs such a function in nature. At least two d i f fe rent ro les have been suggested for heterochromatin in f a c i l i t a t i n g spec i a t ion . One poss ible ro le i s that heterochromatic regions are p r e f e r e n t i a l l y involved in the i n i t i a t i o n or f i x a t i o n of chromosomal rearrange-ments which re su l t in the formation of new species (Baimai, 1975; Hatch et a l . , 1976 a ,b ) . According to t h i s hypothesis , the more heterochromatin a species has, the more frequently i t w i l l be involved in spec ia t ion events. There i s some supporting evidence that the amount of heterochromatin in a species corre la te s with the number of recognizable subspecies (Mazrimas and Hatch, 1972) and that there i s a good c o r r e l a t i o n between high rates of spec ia t ion and chromosomal evolut ion (Bush et a l , 1977). However, there i s also some contradic tory evidence. For example, a pocket gopher species group with a very small amount of heterochromatin has a very high frequency of chromosomal rearrangements (Patton and Sherwood, 1982). A second way heterochromatin may f a c i l i t a t e spec ia t ion i s by d i r e c t l y funct ioning as a s t e r i l i t y bar r ie r (Corneo, 1978; Fry and Sa l ser , 1977). If 6 substant ia l heterochromatic polymorphisms within a population disrupted meiosis , i t may re su l t in the e f f e c t i v e genetic i s o l a t i o n of subgroups d i f f e r i n g in amounts of heterochromatin, leading to sympatric spec i a t ion . There are many s t r i k i n g examples from a var ie ty of organisms of c lo se ly re la ted species whose only v i s i b l e d i f ference in chromosome structure i s the loss or gain of blocks of heterochromatin (Pathak, Hsu and A r r i g h i , 1973; Holmquist, 1975; Rangonath, Schmidt and Hagele, 1982; Sen and Sharma, 1983). The pa i r ing behaviour in the wheat-rye hybrid t r i t i c a l e shows that heterochromatin can disrupt meiotic p a i r i n g . A large block of heterochromatin on one of the rye chromosomes disrupts the pa i r ing between the wheat and rye chromosomes in meiosis and re su l t s in chromosome bridges (Bennett, 1977; Bedrook, O 'Del l and F l a v e l l , 1980). However, there are also examples of subspecies with large di f ferences in heterochromatin content that form v iab le and f e r t i l e hybrids (Blumenfeld, 1978 et a i . ; Patton and Sherwood, 1982). The most substantiated of a l l the functions proposed for heterochromatin i s that i t modifies the amount and/or d i s t r i b u t i o n of cross ing-over . Although numerous studies have confirmed the quant i ta t ive and q u a l i t a t i v e inf luence of heterochromatin on recombination (John, 1973; Miklos and N a n k i v e l l , 1976; Rhoades, 1978; Yamamoto, 1979b; John and King, 1982), i t i s not c lear that heterochromatin evolved and ex i s t s in the genome for t h i s purpose. Although many functions have been suggested for c o n s t i t u t i v e heterochro-matin, i t i s u n l i k e l y that the function(s) of heterochromatin w i l l be c l e a r l y understood u n t i l some key questions about the nature of heterochromatin have been reso lved . For example, i s the function of s a t e l l i t e DNA equivalent to the function of heterochromatin? Are there DNA sequences other than s a t e l l i t e DNA in heterochromatin? If so, what ro le do these sequences have in determining the general propert ies and functions of heterochromatin? Do a l l DNA sequences within 7 a block of heterochromatin perform the same function? Does a l l c o n s t i t u t i v e heterochromatin have the same composit ion, propert ies and function? To quote Carlson and Brutlag (1978a), "the dearth of knowledge about the n o n s a t e l l i t e sequences present in heterochromatin c l e a r l y stands as a bar r ie r to our under-standing of the functions of heterochromatin". There i s growing evidence that heterochromatin i s not uniform in composi-t ion and exhib i t s a var ie ty of molecular and genetic c h a r a c t e r i s t i c s . For example, there are exceptions to the general ru le that heterochromatin i s p r imar i ly composed of s a t e l l i t e DNA. Some blocks of heterochromatin have been reported to contain l i t t l e or no s a t e l l i t e DNA (Hennig, 1972; A r r i g h i et a i . , 1974,; Cordeiro et a l . , 1975; Holmquist, 1975; Wheeler et a i . , 1978 Ranganath, Schmidt and Hagele,1982). As w e l l , some middle r e p e t i t i v e and unique sequences have been detected in blocks of heterochromatin that do contain mostly highly repeated s a t e l l i t e DNA (Comings and Mattoccia , 1972). The heterochromatin of Peromyscus c e l l s contains s a t e l l i t e DNA sequences covalent ly l inked to nonrepet-i t i v e sequences <Kuo and Hsu, 1978). Type I Insert ion Sequences, o r i g i n a l l y discovered within the rDNA genes, have been found in Drosphiia w e l a n o g a s t e r heterochromatin outside of the nucleolus organizer region (Peacock et a i . , 1981; Dawid et a l . , 1981; Appels and H i l l i k e r , 1982; H i l l i k e r and Appels, 1982; Cantu and Gay, 1984). Other moderately r e p e t i t i v e DNA sequences have been detected in s i g n i f i c a n t quant i t ie s in Drosophila heterochromatin (Renkawitz, 1978; Mukherjee and Lakhot ia , 1979; Leigh Brown and Ish-Horowicz, 1981; L i s , Ish-Horowicz and P i n c h i n , 1981; Spradl ing and Rubin, 1981). A moderately repeated DNA sequence that binds a 1.6 ki lobase RNA i so la ted from embryos has been discovered adjacent to a cloned Drosophila welanogaster s a t e l l i t e sequence (Carlson and Brut lag , 1978b). It i s now c lear that n o n - s a t e l l i t e DNA seqences are present in c o n s t i t u -t i v e heterochromatin, although in l imi ted amounts. Unt i l more i s known about the ro le and genetic c h a r a c t e r i s t i c s of these sequences i t i s premature to assume 8 that the propert ies and functions of s a t e l l i t e DNA automatical ly correspond to those of heterochromatin. Most attempts to g e n e t i c a l l y character ize heterochromatin have used the f r u i t f l y Drosophila melanogaster. This organism offers several important advantages for the study of c o n s t i t u t i v e heterochromatin. It has a low chromo-some number <2n=8) and a r e l a t i v e l y large heterochromatin content, comprising about 287. of the to ta l genome (Peacock et a l , , 1973). The heterochomatin i s present in a small number of e a s i l y observed large blocks which account for the proximal half of the acrocentr ic X chromosome, the ent i re Y chromosome, the proximal quarters of the large second and t h i r d metacentric autosomes, and most of the small fourth autosome. Other advantages of D. a e 1 a n o g a s t e r include i t s short generation time, the ease with which i t can be handled and r a i s e d , and the large number of mutations and chromosomal rearrangements that have been i so l a ted and succes s fu l ly mapped using polytene chromosomes. Furthermore, a l l the hetero-chromatin of D. ne lanogaster is c o n s t i t u t i v e and so there i s no confusion between c o n s t i t u t i v e and f a c u l t a t i v e heterochromatin. A number of genetic functions have been mapped to the heterochromatin of Drosophila aelanogaster (reviewed by H i l l i k e r , Appels and Schalet , 1980). Ritossa and Spiegelman (1965) determined that the tandemly repeated genes coding for rRNA are located at the nucleolus organizer regions present in both the X and Y heterochromatin. A l a ter study showed that a p a r t i a l de le t ion of the rRNA genes r e s u l t s in the bobbed ibb) phenotype (Ritossa , Atwood, and Spiegelman, 1966). Although the rRNA genes are present at large secondary c o n s t r i c t i o n s in the heterochromatin, these genes re ta in many of the propert ies of heterochro-matin even when separated from the surrounding blocks of heterochomatin ( H i l l i -ker and Appels , 1982). Male f e r t i l i t y in D. nelanogaster requires the presence of six d i s t i c t 9 f e r t i l i t y factors on the heterochromatic Y chromosome (Brousseau, 1960; Kenni-son, 1981; Haze l r i gg , F o r n i t i and Kaufman, 1982; Gatt i and P i m p i n e l l i , 1983). Each f e r t i l i t y factor i s involved in a s p e c i f i c step in sperm development (Hardy, Tokoyasu and L i n d s l e y , 1981) and seems to code for a d i f f e rent prote in required for sperm development and f e r t i l i t y (Golds te in , Hardy and L inds l ey , 1982). The recovery of EMS-induced (Will iamson, 1970,1972) and temperature-sens i t ive (Ayles et a i . , 1973) mutations of the male f e r t i l i t y factors suggest that they are s ing le-copy, t ranscr ibed genes. However, on the basis of an exhaustive break-point ana ly s i s , Gatt i and P i m p i n e l l i (1983) have suggested that at least some of the Y f e r t i l i t y factors may have an enormous phys ica l s i z e , each containing up to several thousand ki lobases of DNA. In addi t ion to the six f e r t i l i t y factors there i s another s p e c i f i c segment on the long arm of the Y chromosome that i s required for normal sperm development. The de le t ion of t h i s segment re su l t s in c r y s t a l formation in primary spermatocytes, but not s t e r i l i t y (Hardy et a i . , 1984; L ivak , 1984). Several other genetic propert ies have been mapped to s p e c i f i c regions of D. nelanogaster heterochromatin, although i t i s not c lear whether t ranscr ibed genes are involved . The chromosomal regions responsible for nucleolar dominance (Durica and K r i d e r , 1978) and compensation response (Procunier and Tar to f , 1978) are located in the X chromosome heterochromatin. A segment of the X heterochro-matin also in te rac t s with a family of recess ive maternal mutations inc luding abnormal oocyte (Sandler, 1970, 1972, 1977; Parry and Sandler, 1974; P i m p i n e l l i et a i . , 1985). Extra doses of a s p e c i f i c segment of X heterochromatin can p a r t i a l l y compensate for the maternal mutant defect in abnormal oocyte progeny. Two segmants in the Y heterochromatin have a s imi l a r ef fect (Sandler, 1970; P impine l l i et a i . , 1985). F i n a l l y , elements involved in the Segregation D i s t o r t -ion (SD) phenomenon have been mapped to the heterochromatin of chromosome two (Ganetsky, 1977; Br i t tnacher and Ganetsky, 1984; Sharp, H i l l i k e r and Holm, 10 1985). Thorough genetic d i s sec t ions of the heterochromatin of the X (Shalet and Lefevre, 1973; H i l l i k e r and Appels, 1982) and the Y (Kennison, 1981; Gatt i and P i m p i n e l l i , 1983) chromosomes have f a i l e d to detect any addi t iona l genetic functions in these regions . By comparison, very l i t t l e was known about the genetic content of the autosomal heterochromatin of D. melanogaster u n t i l the recent development of an experimental procedure for s e l e c t i v e l y producing d e f i c i e n c i e s of the proximal heterochromatin of the major autosomes. The detachment of compound autosomes can be used to generate proximal d e f i c i e n c i e s r e s t r i c t e d to heterochromatin. A compound autosome i s a chromosomal rearrangement which has two homologous autosomal arms attached to the same centromere. For example, a compound-3R, designated C(3R), has two r ight arms of chromosome 3 attached to a s ing le centromere in a reversed metacentric conf ig -ura t ion . To maintain d i p l o i d y , f l i e s carrying a given compound autosome (such as a compound-3R) usual ly also carry the complementary compound autosome (in t h i s case a compound-3L). The o r i g i n and meiotic behaviour of compound autosomes has been examined in an extensive review by Holm (1976). Compound autosomes are formed by a t rans-l o c a t i o n - l i k e event, usual ly induced by r a d i a t i o n , between two breakpoints on opposite s ides of the centromere of any two chromatids in a tetrad (Rasmussan, 1960; Leigh and Sobels , 1970; Holm, 1976). Rejoining between a cen t r i c and acentr ic fragment from d i f f e rent chromatids w i l l re su l t in the formation of a new compound autosome. This model for compound autosome formation predic t s that both s i s t e r and non-s i s ter strand attachments can occur. This p red ic t ion was confirmed by experiments showing that compound autosomes homozygous for a reces-sive v i s i b l e mutation could be synthesized in heterozygous females (Leigh and Sobels, 1970; Holm, 1976). One s ix th of a l l compounds synthesized from a stan-11 dard pair o-f autosomes w i l l be a s i s t e r - s t r a n d attachment between the two chro-matids of a given homologue. Compound autosomes can be detached to form standard chromosomes by a s i m i -lar two-hi t , t r ans loca t ion-1 ike event. Detachment occurs when f l i e s carrying a pair of complementary autosomes are i r r a d i a t e d and separate break points are induced on both the l e f t and r ight compound chromosomes. Rejoining of the cen t r i c fragment from one compound chromosome with the acentr ic fragment from the complementary compound w i l l re su l t in a reconst i tuted standard chromosome, known as a detachment product. Two important aspects of compound autosome synthesis and detachment make i t a useful procedure for screening for genetic l o c i in the proximal heterochro-matin of autosomes. The f i r s t i s that the formation of compound autosomes and detachments seems to involve only breakpoints in heterochromatin ( H i l l i k e r and Holm, i9755 Gibson, 1977). Secondly, the t r an loca t ion-1 ike event in both the synthesis and detachment of compounds i s asymmetrical with respect to the cen-tromere. The breaks occur to one side of the centromere or the other , so that r e j o i n i n g can re su l t in d e f i c i e n c i e s and dupl ica t ions of heterochromatic seg-ments in the newly formed compound autosomes and detachment products. If a detachment product c a r r i e s a de f ic iency for a v i t a l gene in heterochromatin, i t w i l l be l e tha l when homozygous. For these reasons, the technique of compound autosome formation and detachment of fers a r e l a t i v e l y easy procedure for generating a ser ie s of proximal d e f i c i e n c i e s of autosomal heterochromatin. The compound detachment procedure was f i r s t applied to chromosome three of D, nelanogaster (Baldwin and Suzuki, 1971). Females carry ing one of seven d i f fe rent coropound-3L chromosomes paired with the same compound-3R were i r r a d i a t e d and mated to males carrying standard, balancer t h i r d chromosomes. A to ta l of 162 f e r t i l e progeny carrying recons t i tu ted , standard t h i r d chromosomes were recovered. S ix ty- s ix of these detachment products behaved as recess ive 12 l e tha l s and were i d e n t i f i e d as putat ive de le t ions of v i t a l heterochromatic l o c i . These chromosomes were tested for complementation in a l l inter se combinations. Thirteen of the l e t h a l s complemented a l l others , i n d i c a t i n g that the l e t h a l i t y was a re su l t of a random t h i r d - h i t elsewhere on the chromosome. The remaining 52 l e tha l s f e l l into 6 complementation groups, which formed a complementation pattern suggesting four d i s t i n c t funct ional u n i t s . Mapping experiments demon-strated that the l e t h a l s of a l l four complementation uni t s were between the most proximal markers known on the l e f t and r ight arms of chromosome three. However, i t was not poss ib le to assign these l e t h a l s to heterochromatin with complete c e r t a i n t y . H i l l i k e r and Holm <1975) undertook a s imi l a r but more extensive study of the proximal heterochromatin of the second chromosome of D. s e 1 a n o g a s t e r . Their analys i s was f a c i l i t a t e d by the existence of several known c y t o l o g i c a l and genetic markers present in the chromosome two centromeric reg ion . A secondary c o n s t r i c t i o n serves as a c y t o l o g i c a l landmark for the euchromatic-heterochro-matic junction on the l e f t arm, and the previous ly i so l a ted de le t ion HS2-10 removes the ent i re block of heterochromatin on the r ight arm. As w e l l , the recess ive mutations r o l l e d and light were bel ieved to res ide in second chromo-some heterochromatin. H i l l i k e r and Holm (1975) recovered a to ta l of 253 detachments from three compound-2 bearing s t r a i n s . Of these, 122 behaved as recess ive l e tha l s and were tested for complementation in a l l poss ib le i n t e r se combinations. As w e l l , the le tha l detachment products were tested against HS2-10, r o l l e d , l i g h t and some other known proximal d e f i c i e n c i e s on chromosome two. The complementation r e s u l t s suggested that at least f i ve v i t a l genetic l o c i were present in the heterochro-matin of chromosome two. Using the r e s u l t s of the complementation tes t s with MS2-10, H i l l i k e r and Holm were able to assign three of the l o c i to the l e f t arm 13 heterochromatin and the other two to the r ight arm. Rolled and light were pseudo-dominant over d e f i c i e n c i e s of the r ight and the l e f t arm, r e s p e c t i v e l y , confirming the heterochrmatic locat ion of these markers. Cyto log ica l examination of polytene chromosmes carry ing the proximal d e f i c -ienc ies f a i l e d to detect any extension of the de let ions into the euchromatin. Further , complementation te s t ing demonstrated that the detachment d e f i c i e n c i e s were proximal to pre-ex i s t ing de let ions that extended from the euchromatin into the heterochromatin, proving that the d e f i c i e n c i e s were r e s t r i c t e d to hetero-chromatin . H i l l i k e r (1976) extended the analys i s of second chromosome heterochromatin by c o l l e c t i n g EMS-induced l e tha l s which f a i l e d to complement the detachmant product d e f i c i e n c i e s . Complementation te s t ing of the EMS-induced l e t h a l s i d e n t i -f ied at least th i r teen genetic l o c i in chromosome two heterochromatin, seven on the l e f t arm and six on the r ight arm. None of the EMS l e t h a l s behaved as a d e f i c i e n c y , and extensive i n t e r - a l 1 e l i c complementation was observed. The genetic ana lys i s of heterochromatin of chromosome two of D. n e l a n o g a s t e r i s the strongest evidence yet that s ing le-copy, v i t a l genes do res ide in c o n s t i t u t i v e heterochromatin. However, the euchromatin-1ike genes shown to be in the chromosome two heterochromatin do not seem to exis t in the heterochromatin of the X or Y chromosme of the same organism (reviewed by H i l l i k e r , Appels and Schalet , 1980). These f indings r a i s e the p o s s i b i l i t y that heterochromatin in D. we 1anogaster may be heterogeneous with respect to genetic a c t i v i t y . The heterochromatin of the sex chromosomes has a very d i f fe rent genetic composition than the heterochro-matin of at least one of the major autosomes. It would be of s i g n i f i c a n t in teres t to determine i f the genetic content of chromosome three , the other major autosome, conforms to t h i s dichotomous pat tern . The re su l t s of Baldwin and Suzuki (1971) suggesting that chromosome three heterochromatin does contain 14 essent ia l genes i s strengthened by the -finding that chromosome two detachment product d e f i c i e n c i e s are r e s t r i c t e d to heterochromatin ( H i l l i k e r and Holm, 1975). However, there i s i n s u f f i c i e n t information about the number, d i s t r i -but ion , repe t i t ivenes s and nature of genes in chromosome three heterochromatin to make a v a l i d comparison between the heterochromatic gene content of the two major autosomes. There i s some evidence which hints that the genetic composition of chromo-some two and chromosome three heterochromatin may d i f f e r . For example, s a t e l l i t e DNA accounts for c lose to 100% of the DNA in the heterochromatic blocks on the second, X and Y chromosomes; but a s i g n i f i c a n t port ion of chromosome three heterochromatin does not appear to be composed of these highly repeated sequences (Peacock et a l . , 1977b). Banding studies also suggest d i f ferences between the heterochromatic blocks of chromosomes two and three. C-banding of the D. Be lanogaster genome reveals that the heterochromatin of the X chromosome, both arms of the second chromo-some, and one arm of the t h i r d chromosome i s subdivided by a number of l i g h t l y stained bands. However, the Y chromosome and heterochromatin on the other arm of chromosome three does not appear to.be subdivided (Hsu, 1971). P i m p i n e l l i , Gatt i and De Marco (1975) also observed a heterogeneous response of Drosophila hetero-chromatin to treatment with 33258 Hoechst, an agent known to decondense mouse heterochromatin. In D. n e l a n o g a s t e r , the heterochromatin of the r ight arm of chromosome three , both arms of the second chromosome, and part of the X chromo-some was not inf luenced by t h i s treatment. The l e f t arm heterochromatin of chromosome three , the remainder of the X chromosome heterochromatin, and most of the Y chromosome was decondensed by Hoechst. The d i f ferences in molecular s t ructure and banding behaviour between chromosome two and three heterochromatin may r e f l e c t d i f ferences in gene con-15 tent . An extensive genetic analys i s Df t h i r d chromosome heterochromatin i s needed to determine i f each heterochromatic block in D. n e 1 a n o g a s t e r i s genet i -c a l l y unique or i f there are d i f fe rent c lasses (such as autosomal and sex heterochromatin) of g e n e t i c a l l y act ive Drosophila heterochromatin. No accurate genera l i za t ion about the genetic a c t i v i t y of Drosophila n e l a r i o g a s t e r heterochro-matin can be made u n t i l t h i s las t major uncharted block of heterochromatin in the genome of th i s species i s analyzed. A thorough genetic ana lys i s of t h i r d chromosome heterochromatin i s now feas ib le with improvements and extensions of the experimental procedures used by Baldwin and Suzuki (1971). The key di f ference i s that the present study w i l l u t i l i z e several d i f fe rent compound t h i r d s for each arm, and a l l poss ib le combinations of these w i l l be detached. Furthermore, the detachment products from each pair of compound t h i r d s w i l l be analyzed seperate ly . The advantage of t h i s approach i s that i t allows the charac ter i za t ion of the progenitor compound autosomes used to generate each detachment. A compound autosome can carry a dup l i ca t ion of a v i t a l region of the opposite arm, a de f ic iency of a v i t a l region on the same arm, or both or ne i ther . (Figure 1) Each type of compound autosome w i l l produce a d i f fe rent pattern of detachment products. There are many poss ib le types of detachment products, and those with a de f ic iency can carry a po lar , non-polar , centromere-spanning, or complex d e l e t i o n . (Figure 2) The types and frequencies of de let ions generated depends on the c la s s of the progenitor compounds. For example, only polar d e f i c i e n c i e s w i l l be generated from compound autosomes carrying no dup l i ca t ions or d e f i c i e n c i e s . Polar d e f i c i e n c i e s can also be ' obtained from other c lasses of progenitor compounds. Non-polar d e f i c i e n c i e s w i l l only be recovered from progenitor compounds carrying a dup l i ca t ion or d e f i c -iency; and centromere-spanning d e f i c i e n c i e s should only re su l t from progenitors hemizygous for at least one heterochromatic locus . 16 FIGURE 1 Models showing the synthesis of the four poss ib le types of compound autosomes. C(3L) chromosomes formed by s i s t e r - s t r and attachments are used as examples. Balancer chromosomes are shown as wavy l i n e s , i r r a d i a t i o n - i n d u c e d breaks as s o l i d arrows, 3L heterochromatin as cross-hatched b locks , and 3R heterochromatin as p l a in blocks . The symbols a, b, and c represent regions found in wild-type l e f t heterochromatin; while d, e, and f designate regions in wild-type r ight heterochromatin. TOP LEFT: Synthesis of a C(3L) chromosome carrying no proximal d e f i c i e n c i e s or dup l i ca t ions of v i t a l l o c i . TOP RIGHTs Synthesis of a C(3L) chromosome carrying a proximal dup l i ca t ion of 3R heterochromatin. BOTTOM LEFT: Synthesis of a C(3L) chromosome carrying a proximal def ic iency of a 3L heterochromatic reg ion. BOTTOM RIGHT: Synthesis of a C<3L) chromosome carrying both a proximal 3R dup l i ca t ion and 3L de f i c i ency . 17 1 8 FIGURE 2 Models showing the generation of four possible types of proximal d e f i c i e n c i e s carried by detachment products. Irradiation-induced breaks are shown as s o l i d arrows, 3L heterochromatin as cross-hatched blocks, and 3R heterochromatin as plai n blocks. The symbols a, b, and c represent regions found in wild-type l e f t heterochromatin; while d, e, and f designate regions in wild-type right heterochromatin. TOP LEFT: Production of a polar deficiency of the l e f t arm from progenitor compounds carrying no duplications or d e f i c i e n c i e s of v i t a l l o c i . TOP RIGHT: Production of a non-polar deficiency of the l e f t arm from progenitor compounds carrying a duplication Df l e f t arm heterochromati n. BOTTOM LEFT: Production of a centromere-spanning deficiency from progenitor compounds carrying a deficiency of l e f t arm heterochromati n. BOTTOM RIGHT: Production of a more complex deficiency from progenitor compounds carrying duplications and d e f i c i e n c i e s . 19 20 The •frequency of l e tha l detachment products generated i s also p a r t i a l l y determined by the c la s s of the progenitor compounds. For each detachment event, there i s a r ec iproca l detachment which would be expected to occur at an equal frequency. Each time a compound autosome carry ing a de f ic iency for a given region i s detached, one of the two r e c i p r o c a l detachment products w i l l carry the de f i c i ency . Therefore, a minimum of 50% of the detachment products from a d e f i c i e n c y - c a r r y i n g compound w i l l be de f i c i en t for the same reg ion. If no de f ic iency i s c a r r i ed by the progenitor compounds, the proport ion of detachment products de f i c i en t for a s p e c i f i c region cannot exceed 50%. The percentage of detachment products carrying any def ic iency w i l l also be p a r t i a l l y dependent on whether the progenitor compounds are carrying d e f i c i e n c i e s and/or d u p l i c a t i o n s . Addi t iona l information about the d i s t r i b u t i o n of genes in the proximal heterochromatic region can be obtained from a detachment experiment i f one knows the nature of the progenitor compounds. However, i t i s not poss ib le to deter-mine immediately to which of the four poss ible c lasses a p a r t i c u l a r compound autosome belongs. Any def ic iency ca r r i ed by a compound autosome i s masked by the other arm of the same chromosome. It i s assumed that progeny with compounds carrying a dup l i ca t ion are also phenotypical1y normal. A compound autosome can only be character ized by working backwards from the pattern of detachment products generated. Since the frequency and type of l e tha l detachments produced from a given compound autosome pair i s a function of the i n t e r a c t i o n of the c lasses of both compounds in a p a i r , each compound autosome must be detached in several d i f f e rent combinations before i t can be properly charac ter i zed . In the present study, these ob ject ives are met by undertaking separate detachment experiments for a l l 12 poss ib le combinations of three d i f f e rent COR) chromo-somes and four d i f f e rent C(3L) chromosomes. It was necessary to analyze a r e l a -t i v e l y large number of detachment products to ensure a representat ive sample from each of the twelve detachment experiments. 21 Another improvement in the procedure used here was to detach only newly synthesized s i s t e r - s t r a n d compound t h i r d s . Evidence suggests that compound chromosomes may accumulate spontaneous recess ive l e t h a l s , e s p e c i a l l y near the centromere where homozygosis i s infrequent (Parker, 1954). To avoid th i s problem, newly generated isogenic s i s t e r - s t r a n d attachments are used to ensure that the progenitor compounds are free of any contaminating l e tha l mutations. One further refinement of the compound autosome synthesis and detachment procedure deserves mention here. Unlike chromosome two, there are no known d e f i c i e n c i e s of chromosome three heterochromatin that can be used to determine on which side of the centromere newly discovered proximal genes re s ide . To overcome t h i s disadvantage, an attempt was made to reattach newly synthesized detachment products carrying d e f i c i e n c i e s . New s i s t e r - s t r and attachments w i l l only be recovered from the chromosome arm not carrying the d e f i c i e n c y . Using t h i s new approach, i t was poss ib le to assign each def ic iency to e i ther the r ight or l e f t arm heterochromatin of chromosome three . Other improvements in the procedure are described l a t e r . The procedural improvements described above have made poss ib le an extensive genetic i n v e s t i g a t i o n of the proximal heterochromatin of chromosome three, extending the r e s u l t s of Baldwin and Suzuki (1971). The chromosome three hetero-chromatin i s the largest uncharacterized region of the D. welanogaster genome, and the l a s t major block of D. ntelanogaster heterochromatin to be thoroughly analyzed. The r e s u l t s of t h i s study provide a deta i led genetic map of chromosome three heterochromatin, and shed new l i g h t on the genetic nature and heterogen-e i ty of Drosophila heterochromatin. 22 MATERIALS and METHODS Drosophila Stocks Several s t r a in s of Drosophila ne1anoqaster with mutated or rearranged t h i r d chromosomes were used in t h i s study. A l l new compound t h i r d chromosomes and detachment products that were generated car r i ed the proximal recess ive markers radius incovpletus on the l e f t arm and pink-peach on the r ight arm. A l l detach-ment products were balanced over the I n ( 3 L R ) T H 3 , y * ri p" se b x 3 * a e* chromosome (hereafter re ferred to as 7W3), which suppresses cross ing-over on chromosome three. Further information on a l l the mutations and rearranged chromosomes used in t h i s study i s given in L inds ley and G r e l l (1968). Synthesis of New Comoound-3 Chromosomes Only newly synthesized isogenic s i s t e r - s t r a n d attachments were used for detachment. An isogenic ri pp s t r a in was obtained by mating a s ing le r i p"/TH3 male to TH3/Ly females, and then mating s i b l i n g s of the r i pp/TH3 progeny to i s o l a t e a homozygous isogenic ri p" stock. Isogenic ri p p males were c o l l e c t e d and mated to v i r g i n TM3/Ly females. Approximately 1500 v i r g i n F l r i pp/TH3 females were c o l l e c t e d and treated with 2000 rads of gamma rad ia t ion from a * ° C o source. The i r r a d i a t e d females were mated in groups of 25 to C(3L)V71,se; C(3R)VKl,e' males in p i n t - s i z e d b o t t l e s . The parents were cleared after ten days. New compounds generated in females hetero-zygous for TH3 could only be formed by the attachment of s i s t e r chromatids of the non-inverted r i pp homologue. The only surv iv ing progeny ca r r i ed a newly formed compound t h i r d , or were a rare nondis junct ional o f f sp r ing . A l l r i e" progeny car r i ed a newly formed s i s t e r - s t r a n d C(3L) chromosome, while se pp 23 progeny ca r r i ed a new s i s t e r - s t r and C(3R) chromosome. Separate stocks were establ i shed for each poss ible combination of newly synthesized compound t h i r d s . Recovery of Detachment Products A separate detachment experiment was performed for each of twelve l i n e s carrying a d i f f e rent combination of new C(3L) and C(3R) chromosomes. In each experiment, 2000 v i r g i n compound-3 females were treated with 2200 rads of gamma rad ia t ion and mated in groups of f i f t y to TH3/Ly males in p i n t - s i z e d b o t t l e s . The parents were cleared after 10 days, and of f spr ing were c o l l e c t e d at 1 8 ° for the next 20 days. V i r g i n females and males carrying detachment products recovered over TH3, and males carrying detachment products recovered over the iy chromosome, were es tabl i shed in stock over 7W3. L e t h a l i t y Tests Each detachment product was tested for recess ive l e t h a l i t y by mating males and females carrying the same detached ri p" chromosome balanced over TN3. A l l l e t h a l i t y tes t s were ca r r i ed out in dupl ica te in she l l v i a l s with two pairs of f l i e s per v i a l . If the detachment ca r r i ed a recess ive l e t h a l , no homozygous r i , p p progeny were produced. If the detachment product was homozygous v i a b l e , then ri pp/TH3 heterozygotes and homozygous ri pp f l i e s were recovered in approximately a 2:1 r a t i o . A l l progeny in each v i a l were counted to ensure that any semi- lethals would be detected. Detachment products carrying recess ive l e tha l s were maintained in stock over TH3. Approximately 150 non-lethal detach-ment products were also saved. Complementation Tests To make poss ib le the analys i s of a large number of detachment products without requ i r ing an astronomical number of crosses , complementation te s t ing was done in several stages. F i r s t , the l e tha l detachment products from f ive of the 24 twelve detachment experiments were tested in a l l inter se combinations with a l l other l e tha l detachments from the same experiment. From these t e s t s , a separate complementation map was produced for each of the f ive detachment experiments. The second stage of the complementation analys i s was to test between complementation groups of the f ive detachment experiments. Several detachment products were selected from every complementation group in each of the f ive experiments. These chromosomes were then pooled and tested in a l l inter se combinations, producing an accumulated complementation map for the f ive o r i g i n a l experiments. Further complementation tes t s were carr ied out within complementa-t ion groups on the accumulated map wherever necessary to resolve any ambigu-i t i e s . The t h i r d stage of the complementation analys i s was to test a l l l e tha l detachment products from the seven remaining experiments against a tester chromosome from each complementation group on the accumulated map. From the re su l t s of these crosses , a l l the detachment products of the seven remaining experiments were assigned to a complementation group on the overa l l map. F i n a l l y , some random complementation tests were conducted within each comp-lementation group to ensure that none of the complementation groups could be further subdivided. In each stage of the complementation ana lys i s described above, a l l tes ts involved cross ing a pair of males carry ing one l e tha l balanced over TH3 to a pair of females carrying a second l e tha l over TM3. A l l crosses were carr ied out in she l l v i a l s at 2 5 ° . Reciprocal crosses were performed for most complement-ation te s t s . Crosses between two non-complementing l e tha l s produced only r i pp/TH3 progeny. In crosses between complementing l e t h a l s , ripp/TH3 and ri pp/ri p p progeny were recovered in an approximate r a t i o of 2 :1 . 25 Recombination Happing The proximal pos i t ion of the putat ive de let ions ca r r i ed by the detachment products was confirmed by a cross-over ana ly s i s . A tester detachment chromosome from each complementation c las s was tested in two recombination mapping exper i -ments. The f i r s t experiment sought to determine i f the de f ic iency on the tester chromosomes were to the l e f t or r ight of eagle, a very proximal marker on the l e f t arm of chromosome three. Females carrying a detachment product over the marker chromosome st eg Ki were mated to homozygous st in ri eg males. The F l progeny was screened for crossovers between ri at map pos i t ion 47.0 and eg at map pos i t ion 47.3. One cross-over product, recovered in progeny with a radius incompletus, eagle, Kinked phenotype, included almost the ent i re l e f t arm euchromatin of the o r i g i n a l detachment chromosome. Progeny with a scar le t pheno-type car r i ed the r e c i p r o c a l recombinant product which includes the ent i re r ight arm and the heterochromatin of the l e f t arm of chromosome three. Both classes of recombinant products were recovered and tested for l e t h a l i t y over the o r i g i n a l detachment product. If the recombinant chromosome recovered in the scar le t progeny f a i l s to survive over the detachment product, then the def ic iency car-r ied by the detachment product i s to the r ight of the ri-eg reg ion. Conversely, i f the recombinant product recovered in radius incompletus, eagle, Kinked progeny f a i l s to surv ive , then the def ic iency i s in the l e f t arm euchromatin. A s imi l a r experiment was conducted for the r ight arm. Females carrying the detachment product over the marker chromosome eg Ki were mated to homozygous ri pp males. Cross-overs in the region between Kinked at map pos i t ion 47.6 and pink-peach at map pos i t ion 48.0 were recovered. Progeny with a Kinked, pink-peach phenotype car r i ed a recombinant chromosome containing most of the r ight arm euchromatin whereas progeny with a radius incompletus phenotype carr ied the rec iproca l recombinant product containing the ent i re l e f t arm and the r ight arm heterochromatin. If the recombinant chromosome which includes the ent i re l e f t 26 arm and the heterochromatin of the r ight arm i s l e tha l over the o r i g i n a l detach-ment product, then the detachment product def ic iency i s to the l e f t of the Ki pp region. If the recombinant product with only the r ight arm euchromatin f a i l s to survive over the complete detachment product, then the de f ic iency i s d i s t a l to the Ki pp region on the r ight arm. A putat ive detachment product def ic iency can be assigned to the proximal region of chromosome three with cer ta in ty i f i t maps to the r ight of the ri eg region and to the l e f t of the Ki pp reg ion . Reattachments To determine whether proximal d e f i c i e n c i e s were on the l e f t or r ight arm of chromosome three , an attempt was made to synthesize new s i s t e r - s t r a n d reat tach-ments of l e tha l detachment products from several complementation c lasses . V i r g i n females carry ing the tester detachment product over Tti3 were i r r a d i a t e d and mated to C(3L)VH3,st; C3R)SH19,r males. For each detachment product tes ted , 2500 females were treated with 2500 rads and mated, and in addi t ion 1250 females were treated with 4000 rads and mated. Progeny with a radius incompletus phenotype carr ied a newly formed s i s t e r - s t r and C < 3 D ; and those with a s c a r l e t , pink-peach phenotype car r i ed a new s i s t e r - s t r and C<3R). Only C(3L) ' s should be recovered from detachment products with a r ight-arm d e f i c i e n c y , and v ice versa . EMS Mutagenesis Newly eclosed males from the isogenic ri p" stock were aged two days and then fed with a 0.025 M EMS in IX sucrose so lu t ion for IS hours as described by Lewis and Bacher <1968). After treatment, the males were allowed to recover for one day on standard medium and then mated to v i r g i n TH3/Ly females. Male progeny with a mutagenized ri pp chromosome recovered over Ly were c o l l e c t e d and tested for a l l e l i s m with detachment product d e f i c i e n c i e s of the l e f t and r ight 27 arm. Each male was s ing le pair mated at 2 9 ° in she l l v i a l s to females car-rying a detachment product def ic iency of the l e f t arm balanced over TM3. After four days, the parents were removed, and the males re-mated to females carrying a de le t ion of the r ight arm heterochromatin. In a l l , two d i f f e rent d e f i c i e n c i e s of the l e f t arm and two of the r ight arm were used; although each mutagenized chromosome was only tested against one def ic iency of each arm. The progeny of each cross was examined for the presence or absence of ri pp/ri p p and ri p"/TH3 progeny. V i a l s that contained no ri p"tri p" progeny were scored as putat ive l e tha l a l l e l e s of the detachment product de f i c i ency . The absence of ri p"/TH3 progeny indicated a l e tha l a l l e l e of TH3 had been produced. A l l putat ive EMS-lethal a l l e l e s of d e f i c i e n c i e s were re-tes ted at both 2 2 ° and 2 9 ° to confirm the i r l e t h a l i t y and to test for temperature-sensi-t i v i t y . Each confirmed l e tha l was pos i t ioned r e l a t i v e to the de f ic iency comple-mentation map by a ser ies of l e t h a l i t y tests against small d e f i c i e n c i e s of i n d i v i d u a l complementation regions . A l l EMS mutants that mapped to a p a r t i c u l a r complementation group were then complementation tested against each other in a l l inter se combinations. Putat ive EMS-induced l e tha l a l l e l e s of TH3 were also maintained and tested for complementation in a l l inter se combinations. If the rearranged TN3 chromo-some has accumulated any recess ive l e t h a l s in the centromeric reg ion , a l l detachment products carrying a de f ic iency that uncovered the TW3 le tha l would not have been recovered. To test t h i s p o s s i b i l i t y , several of the EMS-induced l e tha l a l l e l e s of TH3 were analyzed by the cross-over procedure described e a r l i e r to determine i f any mapped to the proximal region of the t h i r d chromo-some. 28 RESULTS Synthesis of New Compound-3 Chromosomes Six new s i s t e r - s t r a n d compound-3L and three s i s t e r - s t r and compound-3R chromosomes were recovered from approximately 1500 i r r a d i a t e d females. Each new compound t h i r d was assigned an alpha-numeric code according to the system described by Holm (1976). The three C(3R) chromosomes were designated as C(3R)VH1 ,pp to C(3R)VH3 ,ppi and the six C(3L) chromosomes were designated C(3L)VH1 ,ri to C ( 3 U V H 6 , r i . A l l three C(3R) chromosomes and the f i r s t four C(3L) chromosomes were used for the detachment experiments. Separate l i n e s were establ i shed for a l l twelve poss ib le combinations of these compound chromosomes. Recovery of Detachment Products A separate detachment experiment was undertaken for each of the twelve compound-3 combinations. In each experiment, 2000 v i r g i n females carrying a pair of compound-3's were i r r a d i a t e d and mated to TN3/Ly males. Detachment products were recovered in the progeny over the TH3 and Ly chromosomes. The to ta l number of detachment products recovered in each experiment i s shown in Table I. E x p e r i -ment 6 was repeated because of the low number of detachment products recovered. It i s in te re s t ing to note that the four detachment experiments invo lv ing C(3R)VN2 produced s u b s t a n t i a l l y fewer detachment products than any of the other experiments. To test whether t h i s ef fect was a re su l t of reduced f e r t i l i t y in stocks carrying the C(3R)VH2 chromosome, fecundity tests were ca r r i ed out for each of the twelve compound l i n e s . Two day old v i r g i n females from each l i n e were treated with 2200 rads and mated to males carrying a pair of compound t h i r d s . Six bot t le s of 25 females and 10 males were mated for each compound l i n e . The fecundity of stocks carrying the C(3R)VH2 chromosome was not s i g n i f i -cantly d i f fe rent from the other compound stocks. 29 TABLE I The number of detachment products recovered from each detachment experi ment. Expt. * C(3L) C<3R) # Females I r radi ated Detachments Recovered 1 2 1 2000 218 2 4 2 2000 108 3 3 1 2000 166 4 1 1 2000 201 5 1 2 2000 112 6A 3 2 2000 80 6B 3 2 2000 98 7 4 3 2000 178 8 4 1 2000 144 9 2 2 2000 93 10 3 3 2000 218 11 1 3 2000 163 12 2 3 2000 238 30 Another poss ib le explanation i s that the C(2R)VH2 chromosome c a r r i e s a massive de f ic iency of proximal heterochromatin, and therefore presents a smaller target for gamma rays . The re su l t s reported l a ter in t h i s study do not substan-t i a t e t h i s hypothesis . Nevertheless , the p o s s i b i l i t y that some i n t r i n s i c proper-ty of compound autosomes may inf luence the i r detachment frequencies i s an i n t e r -est ing f i n d i n g . Detachment products recovered in males or females over TH3 and in males over Ly were maintained in stock balanced over 7W3. Each detachment product was assigned a code number which i d e n t i f i e d the experiment and the detachment product. For example, 2-10 i s the tenth detachment product recovered from experiment 2. L e t h a l i t y Tests The r e s u l t s of l e t h a l i t y tests with the detachment products from each experiment are given in Table II. No s i g n i f i c a n t semi-1ethal i ty or s t e r i l i t y was observed. It can be seen from the data that the percentage of 1e tha l -carry ing detachment products varied considerably from experiment to experiment. In three experiments, less than 35X of the detachment products ca r r i ed l e t h a l s ; while in f ive others over 60'/. of the detachment products were homozygous l e t h a l . The other four experiments were within f i v e percentage points of 50% l e t h a l s . These d i f ferences in l e t h a l i t y frequencies l i k e l y r e f l e c t d i f fe rent c lasses of progenitor compounds. At least 25 l e tha l detachment products from each exper i -ment were maintained in stock and used in the complementation ana ly s i s . A t o t a l of approximately 150 non-lethal detachment products were also saved. Complementation Analys i s A to ta l of 443 l e tha l detachment products from the twelve separate exper i -ments were tested for complementation fol lowing the several-step procedure described in the Mater ia l s and Methods s ec t ion . F i r s t , a separate complement-31 TABLE II The r e s u l t s of l e t h a l i t y tests with detachment products from each detachment experiment. The ""/. Le tha l s " column l i s t s the percentage of a l l detachment products in each experiment which c a r r i e d recess ive l e t h a l s . Exp. # C(3L) C<3R) LETHALS NON-LETHALS X LETHALS 1 2 1 40 79 33X 2 4 2 53 31 63X 3 3 1 66 38 637. 4 1 1 38 116 25X 5 1 2 42 47 47X 6 3 2 42 16 72X 7 4 3 62 31 67X 8 4 1 33 109 23X 9 2 2 39 32 55X 10 3 3 62 29 68X 11 1 3 50 51 50X 12 2 3 41 39 51X 32 ation map was produced for experiments 1,2,3,4, and 10 by te s t ing the le tha l detachment products of each experiment in a l l inter se combinations. Testing between complementation groups from the d i f f e rent experiments produced an accumulated complementation map for the f ive experiments. F i n a l l y , an accumu-lated complementation map for a l l twelve experiments was produced by tes t ing a l l the l e t h a l detachment products from the remaining seven experiments against tester chromosomes from each complementation group in the five-experiment complementation map. The accumulated complementation map of the le tha l detachment products from a l l twelve experiments i s shown in Figure 3. The complementation groups are indicated by s o l i d bars, and those which are shown to overlap f a i l to comple-ment. Each complementation group i s designated by a representat ive detachment product, whose code number i s given above the bar. The number under the bar represents the number of detachment products which f a l l into that p a r t i c u l a r complementation group. As Figure 3 shows, there are 18 complementation groups, with as many as 146 and as few as 1 detachment product per group. This i n i t i a l complementation map suggests that at least eight separate v i t a l regions have been uncovered by the putat ive d e f i c i e n c i e s on the detachment chromosomes. Small complementing d e f i c i e n c i e s s p e c i f i c for seven of these regions have been recovered, and the presence of an eighth region between the 3-30 and 3-9 groups i s implied by the complementation pat tern . The two complementation groups shown as two s o l i d blocks joined by a dotted l i n e were interpreted as detachment products that carr ied double d e f i c i e n c i e s , one on each side of the centromere. Such cent-romere-spanning double d e f i c i e n c i e s can only be generated by progenitor compound chromosomes carry ing a de f i c i ency . It should be noted that there i s a cer ta in degree of a r b i t r a r i n e s s in the 33 FIGURE 3 Accumulated complementation map of putat ive d e f i c i e n c i e s c a r r i ed by detachment products from a l l twelve detachment experiments. Each bar represents a d i f fe rent complementation group. Bars that do not overlap represent complementing groups, and v ice versa . The number above each bar designates the detachment product used to represent the complementation group. The number below the bar ind ica te s the number of detachment products assigned to the complementation group. See text for further d e t a i l s . 34 6B-29 10-26 2 10-42 1 10-68 10-39 58 1-166 2-66 81 3-126 4-184 4-134 74 9-56 3-144 6-61 8A-80 3-30 3-9 10-65 1-168 way the complementation map i s drawn in Figure 3. Other variations of t h i s map are also consistent with the r e s u l t s . For example, a d i f f e r e n t position and p o l a r i t y of some of the complementation groups i s possible. These ambiguities w i l l be resolved in later sections. In addition to the detachment chromosomes shown on the complementation map, there were 44 chromosomes that behaved as random h i t s . These chromosomes comple-mented a l l others against which they were tested. Twenty of the putative random hit l e t h a l s from d i f f e r e n t experiments were selected and complementation tested in a l l inter se combinations. A l l crosses resulted in complementation, providing further evidence that these 44 chromosomes carry l e t h a l s elsewhere on the chromosome generated by random t h i r d - h i t s during the detachment event. The only other detachment product included in the complementation analysis but not shown on the complementation map i s le t h a l 11-64, This detachment chromosome i s semi-lethal over a l l d e f i c i e n c i e s of the region defined by le t h a l 3-30, and surviving heterozygotes have s l i g h t l y outspread wings and only a p a r t i a l posterior wing cross-vein. The 11-64 chromosome f u l l y complements detachment products from a l l other complementation groups. Unfortunately, the 11-64 stock was l o s t , making further tests impossible. V i a l s containing detachment products of the 3-9 complementation class also exhibited a phenotype. Rare homozygous adults would survive, but would die within 48 hours of eclosion. The surviving adults had a phenotype resembling rotund, with droopy wings and reduced sex combs in males. However, a l l detach-ment products of t h i s class f u l l y complemented r o t u n d , which has been mapped to band 84D on the right arm of chromosome three (Duncan and Kaufman, 1975). The phenotype expressed by homozygotes of the 3-9 complementation group was desig-nated rotund-like. A number of non-lethal detachment products were also tested for complement-ation with some of the le t h a l complementation groups. If the heterochromatin of 36 chromosome t h r e e c o n t a i n s t a n d e m l y r e p e a t e d g e n e s , i t i s p o s s i b l e t h a t p a r t i a l d e l e t i o n s w i l l be homozygous v i a b l e . However, t h e p a r t i a l d e l e t i o n s may be l e t h a l o v e r more s e v e r e d e f i c i e n c i e s . To t e s t t h i s p o s s i b i l i t y , a p p r o x i m a t e l y 150 n o n - l e t h a l d e t a c h m e n t p r o d u c t s were e a c h c r o s s e d t o l e t h a l s 3-126, 1-166, and 10-65, w h i c h c o l l e c t i v e l y span t h e a c c u m u l a t e d c o m p l e m e n t a t i o n map. However, a l l t h e c r o s s e s r e s u l t e d i n f u l l c o m p l e m e n t a t i o n , and t h e n o n - l e t h a l d e t a c h m e n t p r o d u c t s t o c k s were d i s c a r d e d . As w e l l , t o keep t h e number of s t o c k s m a n a g e a b l e , a maximum of t e n l e t h a l s t o c k s c o n t i n u e d t o be m a i n t a i n e d f o r e a c h c o m p l e m e n t a -t i o n g r o u p . R e c o m b i n a t i o n M a p p i n g The p r o x i m a l p o s i t i o n o f t h e p u t a t i v e h e t e r o c h r o m a t i c d e f i c i e n c i e s was c o n f i r m e d u s i n g t h e r e c o m b i n a t i o n mapping p r o t o c o l d e s c r i b e d i n t h e M a t e r i a l s and M e t h o d s s e c t i o n . E x c h a n g e e v e n t s between t i g h t l y l i n k e d p r o x i m a l m a r k e r s on e i t h e r t h e l e f t o r r i g h t arm were r e c o v e r e d f r o m f e m a l e s h e t e r o z y g o u s f o r a det a c h m e n t p r o d u c t and a marker chromosome. F o r most d e t a c h m e n t p r o d u c t s , t h e l e t h a l d e f i c i e n c y a l w a y s r e m a i n e d w i t h t h e r e c o m b i n a n t p r o d u c t c o n t a i n i n g t h e c e n t r o m e r i c f r a g m e n t of t h e d e t a c h m e n t chromosome. T h i s was t h e c a s e f o r d e t a c h -ment p r o d u c t s 10-65, 10-42, 4-134, 10-39, 6B-29, 3-9, 6-61, and 4-75. T h e s e d e f i c i e n c i e s , w h i c h i n c l u d e most of t h e c o m p l e m e n t a t i o n g r o u p s , a r e now c o n f i r m -ed t o be l o c a t e d i n t h e p r o x i m a l r e g i o n of chromosome t h r e e . However, t h e l e t h a l s on d e t a c h m e n t p r o d u c t s 1-168 and 3-144 b o t h mapped t o t h e r i g h t arm e u c h r o m a t i n . The c o m p l e m e n t a t i o n g r o u p r e p r e s e n t e d by d e f i c i e n c y 1-168 c o n t a i n s f i v e l e t h a l d e t a c h m e n t p r o d u c t s , a l l g e n e r a t e d i n d e t a c h m e n t e x p e r i m e n t 1. The d e f i c i e n c i e s i n t h i s g r o u p complement a l l o t h e r c o m p l e m e n t a -t i o n g r o u p s . I t s h o u l d be n o t e d t h a t t h e t w e l v e compound s t o c k s u s e d f o r t h e d e t a c h m e n t e x p e r i m e n t s were k e p t i n s t o c k f o r c l o s e t o a y e a r b e f o r e b e i n g d e t a c h e d . T h e r e f o r e , i t seems l i k e l y t h a t a s p o n t a n e o u s r e c e s s i v e l e t h a l a r o s e 37 in the r i gh t arm euchromatin and spread within a sub-population of the exper i -ment 1 compound stock. F i f t y percent of the detachment products generated from females bearing the l e tha l mutation would also carry the l e t h a l . This explana-t ion i s consis tent with the f inding that spontaneous recess ive l e t h a l s do accu-mulate on compound chromosomes (Parker, 1954). A s i m i l a r explanation i s l i k e l y for l e tha l 3-144. The three other chromo-somes that do not complement 3-144 are a l l from experiment 3. Two of these l e t h a l s , making up the 3-126 complementation group, also f a i l e d to complement a large group of d e f i c i e n c i e s which were in the proximal reg ion . The same recombi-nation mapping procedure was carr ied out for detachment product 3-J!2<>, and i t was found that t h i s chromosome car r i ed two separable l e t h a l s , one in the p r o x i -mal region and one in the r ight euchromatin that did not complement 3-144. Therefore, i t seems that a spontaneous l e tha l also arose in the r ight arm euchromatin of a sub-population of the compound l i n e used in experiment three. Of the four detachment products recovered carry ing t h i s l e t h a l , two also ca r r i ed a proximal de f ic iency that resul ted from the detachment event. Four of the 44 detachment chromosomes that had been c l a s s i f i e d as random h i t s were also subjected to the same recombination mapping ana ly s i s . Three of the four l e t h a l s mapped outside of the proximal reg ion, but the four th , des ig-nated 10-33, mapped to the proximal reg ion . This chromosome was tested against a l l detachment products s t i l l in stock from complementation groups which may poss ib ly extend further than shown on the complementation map. Three of the ten stocks from the 10-39 complementation group and f ive of the ten stocks from the 4-134 group f a i l e d to complement 10-33. Therefore, 10-33 defines a new comple-mentation group that was previous ly undetected because the tester chromosomes used to represent the 10-39 and 4-134 complementation groups did not extend out to include 10-33. The 10-39 and 4-134 groups were i n c o r r e c t l y c l a s s i f i e d as 38 s ingle complementation groups, s ince they both contain some d e f i c i e n c i e s that include the 10-33 region and some that do not. To determine i f any other comple-mentation groups had been overlooked in a s imi la r way, ten other chromosomes c l a s s i f i e d as random h i t s were tested against a l l members of complementation groups that could poss ib ly extend further than shown. However, no addi t iona l complementation groups were discovered and the re su l t s were consis tent with the o r i g i n a l c l a s s i f i c a t i o n of the ten l e t h a l s as random h i t s . Reattachment of Detachment Products The detachment product d e f i c i e n c i e s were assigned to e i ther the l e f t or r ight arm heterochromatin by the r e s u l t s of attempts to reattach the detachment chromosomes. Females heterozygous for TH3 and a tester chromosome from various complementation groups were i r r a d i a t e d and mated to males carrying compound t h i r d s . For each tes ter chromosome, 2500 females were treated with 2500 rads and 1250 females were treated with 4000 rads, and then mated. Except for rare non-dis junct i onal events, the only surv iv ing progeny car r i ed a newly synthesized s i s t e r - s t r a n d compound-3 chromosome. Since the new s i s t e r - s t r and attachments are homozygous for the appropriate chromosomal arm of the detachment chromosome, only new compound-3L's should be recovered from detachment products carrying a def ic iency of the r ight arm, and v ice versa. The re su l t s of the reattachment experiments are summarized in Table III . Only new C<3R)'s were generated from tester chromosomes representing the 3-9, 3-30, 1-16, 1-166 and 2-66 complementa-t ion groups. Therefore, these complementation groups and others that f a i l to complement them are presumed to represent d e f i c i e n c i e s of the l e f t arm hetero-chromatin. As expected, the 10-26 and 10-42 chromosomes f a i l e d to produce any new s i s t e r - s t r a n d attachments of the l e f t or r i gh t arm, confirming that these detachment chromosomes carry double d e f i c i e n c i e s . The 10-65 chromosome reattachment experiment was repeated because of 39 TABLE III S i s t e r - s t r and compound th i rds recovered from reattachment of se lected detachment products. Detachment Females S i s ter-S trand Compounds Recovered Product Treated C(3L) ' s C(3R)'s 3-9 3750 0 5 3-30 3750 0 6 1-16 3750 0 2 1-166 3750 0 4 2-66 3750 0 2 10-26 3750 0 0 10-42 3750 0 0 10-65 3750 5 1 10-65 (repeat) 3750 4 0 1-168 3750 21 0 r i p" (control) 3750 6 40 unusual r e s u l t s obtained in the f i r s t experiment. Five new s i s t e r - s t r a n d C ( 3 L ) ' s and one new C(3R) were recovered. The recovery of compounds from both arms would suggest that the 10-65 chromosome does not carry a l e tha l on e i ther arm. How-ever, i f t h i s were the case, then new C ( 3 L ) ' s and C (3R ) ' s would be expected to be recovered in equal numbers. Further , the 10-65 chromosome d e f i n i t e l y does carry a recess ive l e t h a l . To resolve t h i s anomaly, the 10-65 reattachment experiment was repeated, and t h i s time only new C (3L ) ' s were recovered. It seems that the 10-65 chromosome does carry a l e tha l on the r ight arm} and that the s ingle C<3R) recovered in the f i r s t experiment may have been caused by an experimental e r r o r , such as m i s l a b e l l i n g or contamination of bo t t l e s used in the experi ment. The reattachment r e s u l t s with the 1-168 chromosome are also p e c u l i a r . As explained in the previous s ec t ion , t h i s chromosome c a r r i e s a l e tha l outside the proximal region somewhere in the r ight euchromatin. The recovery of only C ( 3 L ) ' s confirms that the l e tha l i s on the r ight arm. However, the r e l a t i v e l y large number of reattachments recovered i s d i f f i c u l t to exp la in . It appears that t h i s detachment chromosome d i f f e r s s i g n i f i c a n t l y from the other detachment chromo-somes tested in i t s a b i l i t y to undergo further rearrangements. A s imi la r f ind ing was observed in the o r i g i n a l detachment experiment, where one p a r t i c u l a r compound chromosome exhibi ted a s i g n i f i c a n t l y reduced propensity to be detached. These r e s u l t s suggest that t h i r d chromosomes that have previous ly been involved in attachment/detachment events with heterochromatic breakpoints d i f f e r widely in the i r competency to be involved in further such rearrangements. A control reattachment experiment was conducted by reat taching the isogenic ri p" chromosome while balanced over TH3. Since the ri pp chromosome does not carry any recess ive l e t h a l s , i t should produce new s i s t e r - s t r a n d attachments of both the l e f t and r ight arms. As pred ic ted , both C ( 3 L ) ' s and C (3R ) ' s were recovered in approximately equal numbers. 41 Further Analys i s of Compound and Detachment Chromosomes A revised complementation map that includes the r e s u l t s of the recombina-t ion mapping and reattachment experiments i s shown in Figure 4. The revised map suggests the presence of six proximal genes on the l e f t arm and one on the r ight arm. The r ight arm gene i s designated L(3R)1, and the six genes on the l e f t arm are numbered L(3L)1 to L(3L)6. Although the complementation map i s shown with L(3L)1 proximal and L(3L)6 d i s t a l on the l e f t arm, the re su l t s up to th i s point cannot d i s t i n g u i s h between t h i s p o s s i b i l i t y and the opposite o r i e n t a t i o n . The o r i en ta t ion of the d e f i c i e n c i e s on the l e f t arm can be determined by g e n e t i c a l l y charac te r i z ing the progenitor compounds used in the detachment experiments, s ince d e f i c i e n c i e s or dup l i ca t ions carr ied by the compounds w i l l be polar ( i . e . w i l l include the most proximal gene and extend outwards). Individual complementation maps for each of the twelve detachment experiments were produced from the overa l l map, and then analyzed for clues about the genetic make-up of the progenitor compounds. If a progenitor compound i s carry ing a d e f i c i e n c y , over 50X of a l l detach-ment products from that compound w i l l carry a de f ic iency for the same s p e c i f i c reg ion. An ana lys i s of the data shows that over 507. of the detachment chromo-somes produced in a l l four experiments invo lv ing C(3R)VN3 c a r r i ed d e f i c i e n c i e s of L (3R) i . Therefore, C(3R)VH3 also must have a de f ic iency of LOR) 1 on one arm. S i m i l a r l y , two of three experiments using C<3L)VM3 produced over 507. l e t h a l s for a s p e c i f i c region on the l e f t arm, i n d i c a t i n g that C(3L)VH3 c a r r i e s a de f ic iency of l e f t arm heterochromatin. The t h i r d experiment invo lv ing t h i s compound that did not produce over 507. l e t h a l s for any region must involve a C<3R) that ca r r i e s a dup l i ca t ion that compensates for the def ic iency carr ied on C(3L)Vtt3. The centromere-spanning d e f i c i e n c i e s produced only in experiment 10, where C{3R)VN3 i s paired with C(3L)VH3, are diagnost ic of de f i c i ency-car ry ing 42 FIGURE 4 Revised complementation map incorporat ing re su l t s of recombination mapping and reattachment experiments. The number above each bar designates the detachment product used to represent each complement-at ion group; and the number below the bar indicates the number of detachment products assigned to each group. The number of detach-ment products in the 4-134, 1-16, 10-39, and 9-52 complement-at ion groups i s shown in brackets because they are estimates. Estimates were necessary because only some of the detachment pro-ducts in these groups were ava i l ab le for complementation tes t s with the previous ly undetected 10-33 group. 43 6B-29 10-26 2 * 1042 1 9-52 (17) 10-39 (41) 1-166 2-66 81 10-68 1-16 (37) 4-184 compounds and confirms that at least one of these two compounds does carry a de f i c i ency . None of the other progenitor compounds behaved as i f they were carrying a de f i c i ency . Any detachment experiment that does not involve the two de f i c i ency-ca r ry ing compounds and which re su l t s in non-polar detachment d e f i c i e n c i e s must involve a progenitor compound carrying a d u p l i c a t i o n . However, i t i s not poss ib le to ident i fy non-polar d e f i c i e n c i e s without f i r s t knowing the prox imal-d i s ta l o r i e n -ta t ion of the d e f i c i e n c i e s . To solve both unknowns s imultaneously, a set of poss ib le models of the progenitor compound chromosomes was constructed and analyzed to determine i f i t could account for a l l the detachment product c lasses generated in the twelve experiments. The only poss ib le conf igurat ion that could explain a l l 4 4 3 detachment products had the o r i en ta t ion of the l e f t arm shown in Figure 4 and the progenitor compounds described in Table IV. Using t h i s set of progenitor compounds and o r i en ta t ion of the l e f t arm, poss ib le breakpoints for every detachment product were i d e n t i f i e d . No other models could come close to expla ining the o r i g i n of a l l the detachment products. Two ambiguit ies remained about the compound chromsome conf igurat ions described in Table IV. F i r s t , the dup l i ca t ion car r i ed by C(3R)VH3 could extend to L ( 3 L ) 5 or L ( 3 L ) 6 . Goth p o s s i b i l i t i e s are consistent with the data, and cannot be d i s t ingui shed in any other way. The second ambiguity i s that i t i s not known for ce r t a in whether any of the four C ( 3 L ) chromosomes carry d u p l i c a t i o n s . Since there i s only one complementation group on the r ight arm, i t i s not poss ible to ident i fy non-polar d e f i c i e n c i e s that would be diagnost ic for dup l i ca t ions carr ied on C<3L) ' s . This ambiguity was resolved by a reattachment experiment which tested the c a p a b i l i t y of progenitor compounds to rescue homozygous d e f i c -ient s i s t e r - s t r a n d attachments. As described e a r l i e r , no C<3R) s i s t e r - s t r a n d attachments w i l l be recovered from a detachment product carrying a def ic iency on 45 TABLE IV Dupl ica t ions and d e f i c i e n c i e s c a r r i ed by progenitor compound-thirds. Progenitor Compound Dupli c a t i on Def ic iency C(3L)VM1 C(3L)VM2 C<3L)VM3 C(3L)VM4 C(3R)VM1 C <3R)VM2 C<3R)VM3 none none none none L <3L)1-3 none L(3L)1-6 none none L(3L)1-5 none none none L(3R)1 46 the r ight arm, and vice versa. However, when females carrying a detachment product with a de f ic iency on the r ight arm balanced over TH3 are i r r a d i a t e d and mated to males carrying a C(3L) compound with a dup l i ca t ion of the r ight arm, the dup l i ca t ion should be able to rescue homozygous de f i c i en t s i s t e r - s t r and C(3R) ' s . Each of the seven progenitor compounds used in the detachment experiments was tested for the presence of dup l i ca t ions by t h i s method. The re su l t s of the reattachment experiments are shown in Table V. Each of the four C(3L) chromo-somes was tested by mating 8000 i r r a d i a t e d females carrying the 10-65 r ight arm def ic iency balanced over TH3 to males carrying the C(3L) chromosome being tes ted , paired with C(3R)SH19The progeny was screened for the presence of newly synthesized compound-3R's. Since no s i s t e r - s t r and C<3R)'s were recovered, the four progenitor C(3L) chromosomes must not carry dup l i ca t ions of the region defined by the 10-65 de f i c i ency . The three C(3R) chromosomes used in the detachment experiments were tested in a s imi l a r manner. Females carrying the 3-9 l e f t arm def ic iency were treated with 4000 rads and mated to males carrying a progenitor C(3R) chromosome paired with C(3L)VH3,st. Newly synthesized s i s t e r - s t r a n d C(3L) ' s were rescued by C(3R)VH1 and C(3R)VH3, but not C(3R)VH2. These re su l t s confirm that C(3R)VH1 and C(3R)VH3 are the only progenitor compounds carrying d u p l i c a t i o n s . D i s t r i b u t i o n of Putat ive Heterochromatic Loci The breakpoints involved in the formation of the complete set of detachment products were not evenly d i s t r i b u t e d along the complementation map. Some regions of the map had a high frequency of breakpoints whereas in other regions break-points were rare . If the distance between two genes i s assumed to be proport-ional to the number of breakpoints that occur in that reg ion , i t i s poss ible to construct a d i s t r i b u t i o n map of the genes in the t h i r d chromosome heterochro matin (Figure 5) . 47 TABLE V Rescue of homozygous de f i c i en t s i s t e r - s t r a n d compound-3's by progenitor compounds. Progenitor Females Predicted Homozygous Def ic ient Compound Treated Dupl icat ions Compound-3's Rescued C(3R)'s C(3L) ' s C<3L)VM1 8000 none 0 C<3L)VM2 8000 none 0 C(3L)VM3 8000 none 0 C(3L)VM4 8000 none 0 C(3R)VM1 8000 L (3D 1-3 - 5 C(3R)VM2 8000 none - 0 C<3R)VM3 8000 L<3L)l-6 - 6 48 FIGURE 5 D i s t r i b u t i o n o-f genes in heterochromatin o-f chromosome three. Re la t ive map distances are based on the number of r ad ia t ion breakpoints induced in each reg ion . 49 E-H JUNCTION L(3L)5 L(3L)4 L(3L)3 L(3L)2 L(3L)1 CENTROMERE L(3R)1 E-H JUNCTION One d i f f i c u l t y in producing such a map was that in experiments invo lv ing a progenitor compound carrying a dup l i ca t ion or de f i c i ency , there were often two or more poss ib le sets of breakpoints for generating the same detachment product. Therefore, when c a l c u l a t i n g the map distances for e i ther the l e f t or r ight arm, experiments that involved compounds carrying a def ic iency or dup l i ca t ion for the arm being examined were excluded from the ana ly s i s . As a r e s u l t , the only experiments used in the c a l c u l a t i o n s were those where the breakpoints could be assigned to a s p e c i f i c region with complete c e r t a i n t y . For the r ight arm, four experiments involved compounds with a dup l i ca t ion or de f ic iency of the r ight arm and thus had to be excluded from the ana ly s i s . In the remaining eight experiments, only 22 out of a to ta l of 649 l e tha l and non-le tha l detachment products were de f i c i en t for the gene(s) defined by the 10-65 de f i c i ency . If a breakpoint occurrs on the r i gh t arm proximal to the 10-65 reg ion , none of the r e s u l t i n g detachment products w i l l carry a de f ic iency of the 10-65 reg ion . If , on the other hand, the breakpoint occurs d i s t a l to the 10-65 reg ion , then one-half of a l l detachment products w i l l be de f i c i en t for t h i s reg ion. One of the two rec iproca l detachment products that w i l l re su l t from such breakpoints w i l l carry a dup l i ca t ion of the 10-65 reg ion, and the r e c i p r o c a l detachment product w i l l carry a de f i c i ency . Therefore, the actual frequency of breakpoints that occur d i s t a l to a p a r t i c u l a r gene i s twice the frequency of d e f i c i e n c i e s recovered for that gene. If i t i s assumed that detachment product breakpoints occur only in heterochromatin and are randomly d i s t r i b u t e d within the heterochromatic block, then the distance of the 10-65 gene r e l a t i v e to the centromere from the euchromatic-heterochromatic junction i s ca lcula ted as fo i lows : 2 x (# of detachment products de f i c i en t for 10-65) x 100X Total # of detachment products = 2 x 22 x 100 X 649 = 7X 51 Therefore, on the basis of breakpoint frequencies , the 10-65 gene i s located at a pos i t ion 77. of the distance from the euchromatic-heterochromatic junction to the centromere. S imi la r c a l c u l a t i o n s were made for each gene on the l e f t arm. Only data from experiments 2, 5, and 9 were used, since these are the only experiments not invo lv ing compounds carrying dupl i ca t ions or d e f i c i e n c i e s of the l e f t arm. The r e l a t i v e pos i t ions of genes L(3L)1, L(3L)2, L(3L)3, L(3L)4, and L(3L)5 were 91X, 877., 85%, 24% and 23% re spec t ive ly of the distance from the l e f t euchromatic-heterochromatic junct ion to the centromere. The map pos i t ion of L(3L)6 was not ca lcu la ted because of the extremely low number of detachment products tested for a de f ic iency of t h i s gene. However, a very rough estimate would place i t ha l f -way between L(3L)5 and the l e f t euchromatic-heterochromatic junc t ion . The map distances show in Figure 5 are proport ional to the number of r ad ia t ion breakpoints induced in each region. The map distances are proport ional to the actual phys ica l distances between genes i f and only i f r ad i a t ion break-points are randomly d i s t r i b u t e d within the heterochromatic block. Lethal Phases The l e tha l phases of several of the detachment product d e f i c i e n c i e s were determined using egg count experiments. Females carrying a def ic iency over TH3 were mated to s i b l i n g males and then allowed to lay eggs on pe t r i dishes con-ta in ing fresh medium for three successive two-hour per iods . The f i n a l pe t r i dish in each ser ies was examined and counted d a i l y . A l l the d e f i c i e n c i e s tested had l a rva l l e tha l phases, e i ther during the second or during the t h i r d ins tar stage. EMS Induced A l l e l e s EMS-induced a l l e l e s of t h i r d chromosome proximal d e f i c i e n c i e s were i so l a t ed by te s t ing EMS-treated ri pp chromosomes against several d i f fe rent d e f i -c iency-car ry ing detachment products. De f i c i enc ie s 10-65 and 4-75 were used to 52 screen for l e tha l a l l e l e s on the r ight arm; and d e f i c i e n c i e s 1-166 and 1-16 were used for the l e f t arm. The number of chromosomes treated and EMS-induced l e tha l a l l e l e s recovered i s summarized in Table VI. The newly recovered EMS le tha l s were pos i t ioned r e l a t i v e to the accumulated def ic iency complementation map by tests against d e f i c i e n c i e s for each region of the map. The EMS-lethals were then complementation tested against a l l other EMS l e t h a l s and a l l d e f i c i e n c i e s from the same region. Each EMS induced l e tha l i s designated by the def ic iency i t was recovered against , followed by the numerical order in which i t was recovered (e.g. 10-65-2 i s the second le tha l recovered against the 10-65 chromosome). Analys i s of EMS-Induced Lethals on the Right firm Two EMS-induced l e tha l a l l e l e s of L(3R)1 were recovered from 6301 ri p* chromosomes tested against 10-65} and 13 l e tha l s of L < 3R) 1 were recovered from 3342 chromosomes tested against the 4-75 chromosome. The two l e t h a l s recovered using the 10-65 chromosome f a i l e d to complement each other and a l l ten d e f i -c ienc ie s of the 10-65 region retained for t e s t i n g . The l e t h a l s recovered using 4-75 resul ted in a more complex pattern that revealed a second, and poss ib ly a t h i r d , gene on the r ight arm, as shown in Figure 6. Two of the l e t h a l s recovered using 4-75, designated 4-75-12 and 4-75-17, f a i l e d to complement the two EMS l e t h a l s of L(3R)1 recovered using 10-65. Ten of the remaining eleven comp-lemented a l l EMS a l l e l e s of L(3R)1, as well as eight of the ten d e f i c i e n c i e s assigned to the 10-65 c l a s s . These ten l e tha l a l l e l e s of 4-75 did not complement def ic iency 5-53 as well as 4-75 i t s e l f . This complementation pattern c l e a r l y defines a second gene in the r ight arm heterochromatin, designated L(3R)2. When tested against each other , the EMS l e t h a l s of L(3R)2 re su l t in a complementation pattern with two groups. Five l e tha l s f a i l to complement both groups. The two complementation groups probably represent i n t e r - a l l e i i c complementation between le tha l a l l e l e s of a s ing le gene; but could represent complementation between 53 TABLE VI Recovery o-f EMS-induced l e tha l a l l e l e s of d e f i c i e n c i e s ca r r i ed by t h i r d chromosome detachment products . # Mutagenized Chromosomes Screened EMS Lethals Recovered 7368 2170 6301 3342 23 37 2 13 Detachment Product Screened Against Defic iency 1-166 1-16 10-65 4-75 L(3L)1-4 L(3L)4-6 L<3R>1 L(3R)1 54 FIGURE 6 Complementation pattern of d e f i c i e n c i e s and EMS-induced l e t h a l s of proximal r i gh t arm. Def i c i enc ie s are indicated by s o l i d bars and EMS-induced l e tha l s are designated by th in l i n e s . See text for further d e t a i l s . 55 5-53 4-75 10-65 7-53 10-120 7-93 12-68 10-73 12-122 4-75-08 4-75-17 4-75-12 4-75-13 10-65-6 4-75-6 4-75-7 10-65-2 4-75-14 4-75-4 4-75-3 4-75-16 4-75-9 4-75-5 4-75-15 a l l e l e s o-f two d i f f e rent genes. If the l a t t e r was the case, the f ive l e tha l s that f a i l to complement both groups would be d e f i c i e n c i e s . The 4-75-8 EMS-induced l e tha l d e f i n i t e l y i s a d e f i c i e n c y , as i t f a i l s to complement a l l other d e f i c i e n c i e s and EMS-induced l e tha l s of the r ight arm. These re su l t s demonstrate the presence of at least one addi t iona l gene d i s t a l to L(3R)1 in the proximal region of the r ight arm of chromosome three. Analys i s of EMS-Induced Lethals on Left firm Lethal EMS-induced a l l e l e s of L ( 3 D I, L(3L)2 and L(3L)3 were recovered using the 1-166 chromosome. These l e t h a l s produced r e l a t i v e l y simple complement-ation patterns (Figure 7). A l l nine EMS-induced l e tha l s of L(3L)1 f a i l e d to complement each other and a l l d e f i c i e n c i e s that uncover t h i s reg ion . Cur ious ly , only one of the nine EMS l e tha l s exhibi ted the rotund-1ike phenotype observed with a l l d e f i c i e n c i e s of the 3-9 c l a s s . Three l e tha l a l l e l e s of L(3L)2 were recovered, a l l of which f a i l e d to complement each other and a l l d e f i c i e n c i e s uncovering L(3L)2. Two a l l e l e s of L(3L)3 were recovered, and again f a i l e d to complement each other and a l l d e f i c i e n c i e s of L(3L)3. Two addi t iona l EMS-induced l e tha l s behaved as d e f i c i e n c i e s . Lethal 1-166-12 f a i l e d to complement a l l d e f i c i e n c i e s and EMS-induced l e tha l s of both L(3L)2 and L(3L)3. S i m i l a r l y , 1-166-46 did not complement d e f i c i e n c i e s and a l l EMS-induced l e tha l a l l e l e s of the three t i g h t l y l inked genes L(3L)1, L(3L)2 and L(3L)3. The analys i s of EMS-induced l e t h a l s f a i l e d to discover any addi t iona l genes in t h i s reg ion . The complementation analys i s of EMS-induced le tha l a l l e l e s of L(3L)4 produced the most complex complementation pattern of t h i s study. Since the 1-166 and 1-16 d e f i c i e n c i e s overlap at t h i s p o s i t i o n , l e tha l a l l e l e s of L(3L)4 where recovered using both d e f i c i e n c i e s . A t o t a l of 30 EMS-lethals of L(3L)4 were recovered, and tested in a l l inter se combinations as well as against a l l d e f i c i e n c i e s of t h i s reg ion. The r e s u l t i n g complementation map shown in Figure 8 57 FIGURE 7 Complementation pattern of d e f i c i e n c i e s and EMS-induced l e t h a l s of genes L(3L)1, L(3L)2, and L(3L)3. Def i c ienc ie s are indica ted by s o l i d bars and EMS-induced l e t h a l s are designated by th in l i n e s . See text for further d e t a i l s . 58 1-166 4-184 4 A - 4 8 4 - 7 6 9-56 9-10 9-93 9-81 8A-49 2-30 2-1 3-30 3-9 10-32 1-66-46 1-166-12  1-166-1 1-166-2 1-166-29 1-166-37 1-166-3 1-166-34 1-166-38 1-166-33 1-166-22 1-166-40 1-166-39 1-166-13 1-166-27 1-16645 L(3L)3 L(3L)2 L(3L)1 FIGURE 8 Complementation pattern of d e f i c i e n c i e s and EMS-induced l e t h a l s of 8A-80 deficiency. Deficiencies are indicated by s o l i d bars and EMS-induced l e t h a l s are designated by thin l i n e s . A l l ten class A l e t h a l s f a i l to complement each other. The twenty class B l e t h a l s form a complex complementation pattern with considerable apparent i n t e r - a l 1 e l i c complementation. See text for further d e t a i l s . 60 00 i -H vi vo 8 VO CN CO i in O co rt r-l co co 00 in vo 6 °? rt OO CN >0 IT) i - H VO t— o 00 00 VD M 5 (N m in M VO* vo vo vo vo" 00 m » a H H VO vo vo vo v i VO VO VD VO VO — >n to •* in VD VO VO VO VO co T J - in — i cs CN CN CN co co • i i i i VO VO VO VO VO co r- o CN rt • — i CS CN CN \^ \^ \C) ^ ^ti pa (A (A — 0 3 rt CN c-~ o o <—i • — i v i v i vi v i 61 reveals that the 8A-80 d e f i c i e n c y , previous ly thought to define L(3L)4, ac tua l ly uncovers two separate genes. A l l 1-16 c lass d e f i c i e n c i e s uncover both genes; but two out of four 1-166 c lass d e f i c i e n c i e s , inc lud ing 1-166 i t s e l f , only uncover the most proximal of the two genes, now designated L(3L)4A. The other two 1-166 class d e f i c i e n c i e s , designated 6-21 and 9-37, uncovered both genes. The group A l e tha l s in Figure 8 consis t of ten EMS-induced l e tha l a l l e l e s of L(3L)4A, eight of which were recovered against 1-166 and two against 1-16. A l l ten l e tha l s f a i l e d to complement each other. The twenty EMS-induced l e t h a l s of group B in Figure 8 were a l l i so l a ted using def ic iency 1-16, and form a very complex complementation pat tern . The re su l t s of inter se complementation tests between a l l twenty l e tha l s are summarized in Table VII . A l l of the l e t h a l s f a i l to complement most other l e t h a l s in the group, but only 1-16-17 and 1-16-22 don't complement at least one other l e t h a l . The complex complementation pattern suggests i n t e r - a l l e i i c complementation of a l l e l e s of a s ing le gene, designated C<3L)4B. Moving d i s t a l l y along the l e f t arm, f ive EMS-induced l e tha l a l l e l e s of L(3L)5 were i so l a ted using def ic iency 1-16. Four of these l e tha l s f a i l e d to complement each other in a l l combinations. The f i f t h l e tha l complemented a l l of the other four. A l l f i ve l e tha l s did not complement any of the d e f i c i e n c i e s which included the L<3L)5 reg ion . This complementation pattern may represent e i ther i n t e r - a l l e l i c complementation or a l t e r n a t i v e l y complementation between mutations of two separate genes. Two EMS-induced le tha l a l l e l e s of L(3L)6 were also recovered using the 1-16 de f i c i ency . These l e tha l s f a i l e d to complement each other and a l l d e f i c i e n c i e s of the L(3L)6 reg ion . Eight add i t iona l EMS-induced l e t h a l s of the 1-16 de f ic iency were recovered which complemented d e f i c i e n c i e s for a l l the known complementation regions on the l e f t arm. Further te s t ing against d e f i c i e n c i e s of the 1-16 and 9-52 classes revealed that these l e tha l s defined two new genes, designated L<3L)7 and L(3L)8, 62 TABLE VII Results of complementation analys i s of twenty EMS-induced l e tha l a l l e l e s of L(3L )4B . Complementing crosses are indicated by a "+"; and non-complementating crosses are indicated by a r-» l-> 1—» I-1 1—» 1—« h-1 h-1 h-' 1—' i—» h-» t-1 O N O N O N O N O N O N O N O N O N O N O N O N O N O N O N O N | O N | O N 1 O N 1 1 OO 1 h-» 1 1 I-1 to NJ K) K> K) N) w w w •fa. Ji-I-* K) w o \->- W Cn Cn + + + + + + + + - + -+ + + + + + 1-16-4 1-16-7 1-16-8 1-16-11 1-16-12 1-16-13 1-16-17 1-16-20 1-16-21 1-16-22 1-16-23 1-16-24 1-16-25 + - - - - - 1-16-31 - - - - - 1-16-32 - - - - 1-16-35 - - - 1-16-41 - - 1-16-43 1-16-44 1-16-45 FIGURE 9 Complementation pattern of d e f i c i e n c i e s and EMS-induced l e t h a l s of L(3L)5, L<3L)6, L(3L)7, and L(3L)8 regions . De f i c i enc ie s are indica ted by s o l i d bars and EMS-induced l e t h a l s are designated by th in l i n e s . See text for further d e t a i l s . 64 1-16 1-16-37 1-16-29 1-16-36 1-16-2 1-16-10 1-16-30 1-1640 1-16-38 L(3L)8 L(3L)7 9-52 1-104 2-85 5-84 10-58 3-135 1-17 4-134 3-10 9- 7 3-109 6-53 8-68 10- 14 10-39 9-2 3-164 10-33 12-123 6-61 10-210 12-2 1-16-18 1-16-28 1-16-16 1-16-26 1-16-19 1-16-34 1-16-27 L(3L)6 L(3L)5 that were d i s t a l to L(3L)6. The complementation re su l t s for the L(3L)5 to L(3L)8 regions are shown in Figure 9. Four of the d e f i c i e n c i e s from the 1-16 and 9-52 classes uncover both L(3L)7 and L(3L)8, while three others uncover L(3L)7 but not L(3L)8. Five EMS-induced l e tha l a l l e l e s of L(3L)7 were recovered. These l e tha l s form an overlapping complementation pattern that suggests in ter -a l1 e l i c complementation. The three l e tha l s of L(3L)8 that were recovered a l l f a i l e d to complement each other. To summarize these r e s u l t s , a t o t a l of at least nine putat ive genes were i d e n t i f i e d in the heterochromatin of the l e f t arm, and at least two putat ive genes were detected in the r ight arm heterochromatin. A l l EMS-treated chromosomes were o r i g i n a l l y recovered on the basis of the i r l e t h a l i t y with a detachment product def ic iency at 2 9 ° . After r e - t e s t i n g at 2 2 ° , two of the l e t h a l s were c l a s s i f i e d as temperature-sensit ive mutations. The f i r s t , designated 1-166-5, i s a temperature-sensit ive a l l e l e of L<3L)4A, and i s included in the complementation map shown in Figure 8. This mutation i s homozygous le tha l at both the r e s t r i c t i v e and permissive temperatures, but i s l e tha l over a l l d e f i c i e n c i e s and EMS-induced l e tha l a l l e l e s of L(3L)4A only at 2 9 ° . The temperature-sensit ive period of the 1-166-5 mutation was determined using shi f t -up and shift-down experiments. Progeny carrying the 1-166-5 chromo-some over a de f ic iency of L<3L)4A had a very long temperature-sensit ive period that began at the s tar t of the second l a r v a l ins tar stage and extended u n t i l la te pupation. The other temperature-sensit ive mutation i so l a ted i s designated 1-166-4. This mutation i s homozygous non-lethal at both 2 2 ° and 2 9 ° , and i s l e tha l over a l l d e f i c i e n c i e s of L<3L)2 at 2 9 ° but not 2 2 ° . However, 1-166-4 i s only semi- lethal over the EMS-induced a l l e l e s of L(3L)2 at the r e s t r i c t e d temp-erature. Shift-up and shift-down experiments determined that t h i s mutation has a 66 sharply defined temperature-sensit ive period ear ly in the pupal stage. Because of the s e m i - l e t h a l i t y of 1-166-4 with other L(3L)2 EMS-induced l e t h a l s , i t i s not c lear i f t h i s mutation i s an a l l e l e of L(3L)2 or a mutation elsewhere on the chromosome which in te rac t s with l e tha l s of C(3L)2. As explained in the Mater ia l s and Methods s e c t i o n , EMS-induced le tha l a l l e l e s of TH3 were also recovered from the EMS screens and used to test for the presence of proximal l e tha l s on the TH3 chromosome. A t o t a l of 24 l e tha l a l l e l e s of TH3 were recovered and complementation tested against each other in a l l poss ible combinations. The r e s u l t i n g complementation map included one group of eight l e t h a l s , one group of f ive l e t h a l s , two groups of three , one group of two, and three groups with one l e tha l each. One l e tha l from each group with two or more members was chosen and recombination mapped by the same procedure used e a r l i e r for the detachment product d e f i c i e n c i e s . A l l the l e tha l TH3 a l l e l e s mapped outside of the proximal reg ion . These r e su l t s ind ica te that the TM3 chromosome i s l i k e l y not carrying a hidden proximal l e t h a l . Cv to loo ica l Examination Squashes of polytene and mitot ic chromosomes carrying detachment product d e f i c i e n c i e s did not reveal any obvious dup l i ca t ions or d e f i c i e n c i e s of t h i r d chromosome proximal regions . However, Larry Sandler and Sergio P i m p i n e l l i (personal communication) examined three of the detachment product d e f i c i e n c i e s using the sophis t ica ted fluorescence and N-banding techniques developed by Gatt i and P i m p i n e l l i (1983). The re su l t s of the i r analys i s are shown schematical ly in Figure 10. The top chromosome shows a standard Oregon-R wild-type t h i r d chromo-some. The r ight arm heterochromatin c a r r i e s a l arge , i n t e r s t i t i a l non-f luor-escing block that appears as a dark-s ta in ing band in N-banding preparat ions . The l e f t arm heterochromatin i s b r i g h t l y f luoresc ing except for a narrow dark band indicated by a notch in the diagram. 67 FIGURE 10 Chromosome banding analys i s of three detachment product d e f i c i e n c i e s . Schematic representat ions of r e su l t s of c y t o l o g i c a l examination by Sandler and P i m p i n e l l i of Oregon-R chromosome and detachment products 2-85, 9-2, and 7-53 by f luorescence and N-banding techniques. Heterochromatic regions are represented by b locks , and the centromere by an open c i r c l e . B r i g h t l y f luoresc ing segments are represented by unshaded blocks ; and f l u o r e s c e n c e - d u l l , N-banded regions are indicated by s o l i d b locks . The notch on the l e f t arm represents a narrow f luorescence-d u l l gap. See text for further d e t a i l s . 68 • bright fluorescence no fluorescence Oregon-R D.P. 2-85 (Def. L(3L)l-7) D.P. 9-2 (Def. L(3L)l-5) D.P. 7-53 (Def. L(3R)1) Two detachment product d e f i c i e n c i e s of the l e f t arm of chromosome three were examined by Sandler and P i m p i n e l l i . Detachment product 2-85 was de f i c i ent for genes L (3D 1 -7, and detachment product 9-2 was de f i c i en t for genes L ( 3 L ) l - 5 . Both the 9-2 and 2-85 chromosomes were generated from progenitor compounds known to be carry ing no dup l i ca t ions or d e f i c i e n c i e s of v i t a l l o c i . Detachment products carrying d e f i c i e n c i e s of the l e f t arm that are generated from such progenitor compounds must be formed by the j o in ing of a cent r i c fragment from the r ight compound with an acentr ic fragment from the l e f t compound. The cent r i c fragment w i l l carry one complete arm of the C(3R) progenitor chromosome, and in reverse o r i en ta t ion on the opposite side of the centromere, a dup l i ca t ion of proximal heterochromatin from the other arm of the progenitor C(3R) that extends from the centromere to the radia t ion- induced breakpoint. The l e f t acentr ic fragment w i l l carry only a segment of the l e f t heterochromatin b lock , s t a r t ing with the l e f t euchromatic-heterochromatic j u n c t i o n , and extending inwards towards the centromere. For the 9-2 chromosome, the large f luorescence-dul l band present on the l e f t arm indicates that the dup l i ca t ion of r i ght arm heterochromatin accounts for at least the proximal three-quarters of the heterochromatin block on the l e f t arm. At most, only the small segment of heterochromatin d i s t a l to the f luorescence-dul l block on the l e f t arm of 9-2 could poss ib ly be normal l e f t arm heterochromatin. Since 9-2 i s only de f i c i en t for genes LI3.LI1-5, the genes L(3L)6-8 must remain in the 9-2 chromosome heterochromatin. Therefore, geneB L(3L)6-8 must be located within the small segment of heterochromatin that accounts for about the d i s t a l one-eighth of the wild type left-arm heterochro-matic block. Two observations are of s i gn i f i c ance for expla ining the c y t o l o g i c a l analys i s of the 2-85 detachment product. F i r s t , the 2-85 chromosome does not carry a dup l i ca t ion of the large r ight-arm f luorescence-dul l segment, so r i gh t 70 arm heterochromatin can account for a maximum of about one-half of the hetero-chromatin on the l e f t arm of 2-85. Combining these two observat ions , almost half of the heterochromatin on the l e f t arm of 2-85 cannot be accounted for by e i ther d i s t a l l e f t heterochromatin car r i ed by the acentr ic detachment fragment, or r ight-arm heterochromatin. Therefore, t h i s unaccounted heterochromatin must represent a dup l i ca t ion of non-v i ta l proximal l e f t heterochromatin present on the progenitor C(3R) chromosome, and car r i ed by the cen t r i c detachment fragment. The c y t o l o g i c a l ana lys i s of the 2-85 detachment product provides no addi t iona l evidence about the d i s t r i b u t i o n of genes within t h i r d chromosome heterochro-matin . The other chromosome examined in the c y t o l o g i c a l ana lys i s i s detachment product 7-53, which c a r r i e s a de f ic iency of the r ight arm heterochromatin. This chromosome was generated from a set of progenitor compounds carry ing no d u p l i -cat ions or d e f i c i e n c i e s on the l e f t compound and a large dup l i ca t ion of l e f t heterochromatin that includes L (3L) l -6 on the r ight compound. From an ana lys i s of the poss ib le breakpoints that could re su l t in the formation of 7-53, i t i s very l i k e l y that t h i s chromosome c a r r i e s a large dup l i ca t ion of wild-type l e f t heterochromatin on i t s r i ght arm. The c y t o l o g i c a l analys i s confirms t h i s p r e d i c t i o n , showing that at least the proximal two-thirds (and perhaps much more) of the heterochromatin on the r ight arm of 7-53 i s ac tua l ly a dup l i ca t ion of l e f t arm heterochromatin. This f ind ing ind ica tes that gene L(3R)2, which i s not deleted in 7-53, must be present in the small amount of remaining d i s t a l r ight-arm heterochromatin. Summarizing the re su l t s of the c y t o l o g i c a l ana ly s i s , a l l the detachment product d e f i c i e n c i e s examined were missing almost a l l the heterochromatin normally found on the arm to which they had been assigned by the genetic ana ly s i s . However, in each case, the large d e f i c i e n c i e s were p a r t i a l l y or 71 completely compensated -for by dupl i ca t ions of heterochromatin from the opposite arm. The r e s u l t s also ind ica te that at least three genes in the l e f t heterochro-matin and one gene in the r ight heterochromatin are located very d i s t a l l y in the i r respect ive heterochromatic b locks . 72 DISCUSSION In t h i s study, a ser ie s of attached, detached, and re-attached t h i r d chromosomes were generated and used to dissect the heterochromatin of chromosome three of Drosophila m e l a n o g a s t e r . The analys i s of approximately 400 proximal d e f i c i e n c i e s ca r r i ed by detachment products, as well as 75 EMS-induced l e tha l a l l e l e s of the d e f i c i e n c i e s , revealed the presence of at least eleven v i t a l genes in the proximal region of chromosome three . Attempts to re-at tach chromo-somes carrying d e f i c i e n c i e s of these genes establ i shed that nine of the genes were on the l e f t arm and two on the r ight arm. This analys i s re in forces the usefulness of the compound autosome attachment and detachment procedure for analyzing the proximal regions of Drosophi1 a auto-somes. Using t h i s basic technique, i t was poss ib le to generate a de ta i led genetic map of what was u n t i l now the largest uncharted region of the D. welano-gaster genome. Several new modif icat ions of the compound attachment and detach-ment procedure were used in the course of t h i s study. F i r s t , reattachment of de f i c i ency-ca r ry ing detachment products was used to determine which arm of the chromosome the d e f i c i e n c i e s were on. Secondly, the a b i l i t y of compound autosomes to rescue newly synthes ized, homozygous de f i c i en t s i s t e r - s t r and attachments was used to test for the presence of dup l i ca t ions on the compound chromosomes. F i n a l l y , the use of many d i f fe rent combinations of compound chromosomes in detachment experiments permitted the genetic charac te r i za t ion of the progenitor compounds and therefore a de ta i led and useful breakpoint ana lys i s of a l l the r e s u l t i n g detachment products . As was the case with the second chromosome ( H i l l i k e r and Holm, 1975; H i l l i -ker, 1976), the genes uncovered in the proximal region of chromosome three appeared s imi la r to s ingle-copy genes found in the euchromatin. Inter-a l 1 el i c 73 complementation between EMS-induced mutants was observed in the larger comple-mentation groups. No evidence o-f r e p e t i t i v e genes resul ted from the tes t ing o-f non-lethal detachment products over l e tha l detachment product d e f i c i e n c i e s . A l l of the mutants were recess ive l e t h a l s , with the only observed phenotype being the r o t u n d - 1 i k e phenotype displayed by rare surv iv ing adults homozygous for d e f i c i e n c i e s of the 3-9 complementation c l a s s . However, unl ike the H i l l i k e r (1976) study, some of the EMS-induced l e tha l s recovered in t h i s study were c l e a r l y d e l e t i o n s , s ince they uncovered two or more genes separable by detachment product d e f i c i e n c i e s . The EMS-induced d e f i c i e n c i e s are probably quite smal l , as the genes they uncover appear to be t i g h t l y l i n k e d . Di f ferent f indings have been reported about the a b i l i t y of EMS to induce de le-t i o n s . While Lim and Synder (1974) and H i l l i k e r (1976) recovered EMS mutations a f fect ing only one c i s t r o n , others have recovered EMS-induced de le t ions (Will iamson, 1970; Bishop and Lee, 1973; Olson and Lim, 1976). In two of the studies where EMS-induced de let ions were recovered, i t was found that EMS was more l i k e l y to induce de let ions near or in heterochromatin than in euchromatin (Bishop and Lee, 1973; Olson and Lim, 1976). It i s not c lear why EMS-induced de let ions are found in some studies but not others . There i s convincing evidence that the genes uncovered by detachment product d e f i c i e n c i e s , inc lud ing those discovered in t h i s study, are within heterochro-matin. Gibson (1977) tested a large number of newly formed compound-2's for dup l i ca t ions of the complementary arm. Over half of the new C(2L) ' s car r ied a dup l i ca t ion of the r ight heterochromatic marker r i , but never ca r r i ed a d u p l i c a -t ion of the proximal r ight euchromatic gene stw. Further , detachment product d e f i c i e n c i e s analyzed by H i l l i k e r and Holm (1975) were g e n e t i c a l l y proximal to previous ly i so l a ted de le t ions that extended from the euchromatin into the het-erochromatin. S i m i l a r l y , the d e f i c i e n c i e s of the t h i r d chromosome recovered by Baldwin and Suzuki (1971) complemented a l l known proximal euchromatic markers on 74 the t h i r d chromosome. F i n a l l y , careful c y t o l o g i c a l examination of both polytene and mi tot ic chromosomes carrying detachment product d e f i c i e n c i e s f a i l e d to detect any removal of euchromatin ( H i l l i k e r and Holm, 1975). The r e s u l t s of the present study are also consistent with a heterochromatic loca t ion for v i t a l genes uncovered by detachment product d e f i c i e n c i e s . A c y t o l o g i c a l examination of three detachment products demonstrated that putat ive d e f i c i e n c i e s g e n e t i c a l l y mapped to e i ther the l e f t or r i gh t arm correspond to de let ions of most of the heterochromatic block of the appropriate arm. These f indings ve r i fy that funct ioning genes do res ide within the t h i r d chromosome heterochromatin of D. m e l a n o g a s t e r . The analys i s of the three detachment product def ic iences examined both g e n e t i c a l l y and c y t o l o g i c a l l y indica te that at least some of the genes in chromosome three heterochromatin are present in the most d i s t a l segments of centromeric heterochromatin on both arms. Another poss ible source of information about the d i s t r i b u t i o n of genes in the t h i r d chromosome heterochromatin i s the r ad ia t ion map produced from the quant i ta t ive analys i s of detachment product breakpoints (Figure 5). The re su l t s of the r ad i a t ion breakpoint and c y t o l o g i c a l analyses are in agreement with pos i t ions for L(3R)2 in the d i s t a l r i ght hetero-chromatin and L(3L)6-8 in the d i s t a l l e f t heterochromatin. However, the c y t o l o g i c a l r e s u l t s do not ind ica te whether or not the proximal pos i t ion of genes L (3L) l -3 shown in Figure 5 i s accurate. The rad ia t ion map distances shown in Figure 5 are proport ional to the actual phys ica l distances between the genes i f and only i f i t i s assumed that r ad i a t ion breakpoints are evenly d i s t r i b u t e d within the heterochromatic block. There i s some l imi ted experimental evidence that r ad i a t ion induced break-points are not evenly d i s t r i b u t e d within heterochromatin. Visual examinations of a ser ies of rad ia t ion- induced rearrangements with heterochromatic breakpoints 75 detected a preponderance of breakpoints at or near the euchrontatic-heterochro-matic junct ion ( G a t t i , Tanzare l la and O l i v i e r i , 1974; Schubert and Rieger, 1976). Kennison (1981) also observed a s t r i k i n g non-random d i s t r i b u t i o n of t r ans loca t ion breakpoints on the heterochromatic Y chromosome, with most i f not a l l breakpoints within non-fluorescent segments, or near junct ions between d i f f e r e n t i a l l y s t a in ing regions . If r ad i a t ion breakpoints in heterochromatin are c lus tered near the euchro-matic-heterochromatic j u n c t i o n , then the r ad ia t ion breakpoint map in Figure 5 i s skewed towards the centromere, and the real pos i t ions of the genes are more d i s t a l than shown. A more extensive c y t o l o g i c a l analys i s i s required to deter-mine whether the map in Figure 5 i s skewed, and i f so, to what extent. Two important conclusions about the d i s t r i b u t i o n of genes in t h i r d chromo-some heterochromatin have emerged from t h i s study. F i r s t , the r i gh t arm hetero-chromatin of the t h i r d chromosome contains s i g n i f i c a n t l y fewer genes than does the l e f t arm heterochromatin. Second, at least some of the genes present in the heterochromatin of chromosome-3 are located very d i s t a l 1y. L i f schytz (1978) has suggested that the euchromatic-heterochromatic junction (E-H junction) i s a t r a n s i t i o n a l zone, with more and more genes intermingl ing with the heterochro-matic sequences as the heterochromatin ends and the euchromatin begins. This model i s based on the discovery of a l t e rna t ing rad ia t ion "hot spots" and "co ld spots" in the E-H junct ion reg ion . There i s evidence that the E-H junct ion region i s g e n e t i c a l l y ac t ive . The heterochromatic chromocentre of Drosophila polytene chromosomes cons i s t s of a dense inner core which does not incorporate 3 H - u r i d i n e , surrounded by more di f fuse regions near the E-H junctions of each arm c a l l e d beta heterochromatin which do incorporate 3 H - u r i d i n e (Lakhotia and Jacob, 1974). Most mRNA that hybr id izes to the chromocentre bind to the beta heterochromatin (Spradl ing, Penman and Pardue, 1975; Renkawitz, 1978; Gvozdev et a i . , 1980 Young et a i . , 76 1983). fl cloned DNA segment that codes for a 26,000 MW prote in hybr id izes to the E-H junct ion of the l e f t arm of chromosome three of D. nelanogaster (Biessman et a l . , 1981). A l l t h i s evidence suggests that there may be segments of " i n t e r -calary euchromatin" interspersed in the d i s t a l regions of blocks of heterochro-matin. The genes known to res ide in the d i s t a l region of chromosome-3 hetero-chromatin may in fact be present in a segment of interspersed euchromatin in the t r a n s i t i o n a l region between heterochromatin and euchromatin. An extensive c y t o l o g i c a l examination of a large ser ie s of gene t i ca l ly defined proximal d e f i c i e n c i e s i s necessary to further define the d i s t r i b u t i o n of genes in t h i r d chromosome heterochromatin. The f indings of th i s study confirm that the heterochromatin of D. welano-g a s t e r i s g e n e t i c a l l y heterogeneous. Each major heterochromatic block in D. welaTiogaster seems to have a novel gene content and d i s t r i b u t i o n . Unique sequence v i t a l genes are found only in autosomal heterochromatin; and not the heterochromatin of the sex chromosomes (reviewed by H i l l i k e r , Appels and Schalet , 1980). As w e l l , s i g n i f i c a n t l y fewer genes are present in the r ight arm heterochromatin of chromosome three than in the heterochromatin of the l e f t arm of chromosome three or e i ther arms of chromosome two. Even within a s p e c i f i c heterochromatin block, such as the heterochromatin of the l e f t arm of chromosome three, v i t a l genes are not evenly d i s t r i b u t e d . The factors responsible for the r e s t r i c t e d locat ions of v i t a l genes in Drosophila heterochromatin are unknown. It has been proposed that the molecular s t ructure of the surrounding heterochromatin provides an environment es sent ia l for the funct ioning of these l o c i ( H i l l i k e r , Appels and Schalet , 1980; Peacock and Lohe, 1980). Perhaps only some regions of the heterochromatin have the molecular c h a r a c t e r i s t i c s necessary for the presence of funct ioning genes. The f e r t i l i t y factors on the Y-chromosome are s p e c i f i c a l l y associated with 77 segments of heterochromatin that are Hoechst-dul1 and N-banded (Kennison, 1981; Gatt i and P i m p i n e l l i , 1983). As w e l l , within the X heterochromatin and on the heterochromatic Y chromosome, the regions that in terac t with the abnormal oocyte maternal ef fect mutation are also Hoechst-dul1 (P impine l l i et a l . , 1985). The chromosome-2 and chromosome-3 heterochromatic blocks have f ive and three N-bands r epec t ive ly (Gatti and P i m p i n e l l i , 1983). One of the genes defined by H i l l i k e r (1976) in the second chromosome heterochromatin i s deleted by a def ic iency of a s ing le N-band in the r ight-arm heterochromatin of chromosome-2 (Gatti and Pimpi-n e l l i , 1983). Although the molecular nature of N-banded regions i s unknown, i t i s poss ib le that N-bands correspond to blocks of a s p e c i f i c s a t e l l i t e sequence. The presence of the 1.705 g/cm* s a t e l l i t e sequence has been s p e c i f i c a l l y mentioned as the poss ib le basis of N-banding (Dennis and Peacock, 1984). Again, a careful c y t o l o g i c a l ana lys i s of a se r ie s of t h i r d chromosome d e f i c i e n c i e s would be useful for determining whether heterochromatic genes correspond to s p e c i f i c banding regions and/or s p e c i f i c s a t e l l i t e sequences. Although the molecular basis and s i gn i f i cance of the genetic heterogeneity of DroBophila heterochromatin i s uncer ta in , i t i s c lear that genes are r e s t r i c t e d to only some segments of the heterochromatin, and each chromosome in the Drosophila genome i s unique with respect to gene content. One l i m i t a t i o n of the present ana lys i s of chromosome three heterochromatin i s that i t would f a i l to detect any v i t a l genes that are present in the hetero-chromatin of more than one chromosome in the D. nelanogaster genome. It i s known that a number of t ranscr ibed midd le - repe t i t ive DNA sequences hybr id ize to the chromocentre of D. melanoqaster polytene chromosomes, e s p e c i a l l y the more d i s t a l beta heterochromatin (Spradl ing, Penman and Pardue, 1975; Carlson and Brut lag , 1978a; Spradl ing and Rubin, 1981). None of these midd le- repe t i t ive sequences are confined only to the autosomes; and as a rule they are found Dn both the Y chromosome and one or more autosomes (Li f schytz and Hareven, 1982). Therefore, 78 i t i s poss ib le that there are v i t a l genes in the the t h i r d chromosome hetero-chromatin other than those -found in t h i s study. Such genes, i f present, would be r e p e t i t i v e and dispersed to the heterochromatic block of one, or more, of the other chromosomes in the D. nelanogaster genome. Two other f indings of t h i s study are of in teres t but of unknown s i g n i f i -cance. The f i r s t i s that attached or detached chromosomes can d i f f e r widely in the i r propensity to undergo further rearrangements. In the compound detachment experiments, a l l experiments involv ing C(3R)VH2 cons i s tent ly generated s i g n i f i -cantly fewer detachment products than experiments invo lv ing the other compound autosomes. S i m i l a r l y , the 3-126 detachment chromosome was several times more l i k e l y to undergo reattachment than the other detachment products tes ted . The second observation of poss ib le s i gn i f i cance i s that detachment products carrying a large de f ic iency also tended to carry a large d u p l i c a t i o n . A l l three detachment products analyzed by Sandler and P i m p i n e l l i had r e l a t i v e l y large d e f i c i e n c i e s accompanied by large d u p l i c a t i o n s . As a r e s u l t , there was an approximately normal-sized heterochromatic block on both B i d e s of the centromere of each detachment product. H i l l i k e r and Holm (1975) observed a s i m i l a r phenom-enon with second chromosome heterochromatic de le t ions . Although t h e i r detachment products car r ied g e n e t i c a l l y large d e f i c i e n c i e s , the mitot ic chromosomes appeared normal, suggesting that the de le t ions were compensated by accompanying d u p l i c a t i o n s . It i s known that chromosomes with l i t t l e or no heterochromatin on one arm can surv ive . The MS2-10 chromosome, which i s not a detachment product, c a r r i e s a c y t o l o g i c a l l y v i s i b l e def ic iency of most i f not a l l of the r ight arm heterochromatin ( H i l l i k e r and Holm, 1975). Therefore, there may be something i n t r i n s i c about the detachment process which generates o f f s e t t ing dup l i ca t ions and d e f i c i e n c i e s . This study completes the genetic d i s sec t ion of the major heterochromatic 79 blocks in Drosophila welanogaster, the only organism whose genome has been sys temat ica l ly analyzed for the presence of heterochromatic l o c i . However, there are i n d i c a t i o n s that other organisms may also have g e n e t i c a l l y act ive hetero-chromatin. Khush, Rick and Robinson (1964) mapped a gene to the heterochromatin of the tomato genome, although i t i s poss ib le that the gene i s located at a small euchromatic gap in the heterochromatic block. The c o n s t i t u t i v e heterochro-matin on the X chromosome of H i c r o t u s a g r e s t i s i s very s ens i t i ve to DNase I, i s not h ighly condensed at interphase, and appears to have newly synthesized RNA attached to i t (Sperl ing et al., 1985). In the snake Elaphe radiak, segments of s a t e l l i t e - r i c h heterochromatin decondense at s p e c i f i c stages in development and are associated with t r a n s c r i p t i o n , as evidenced by 3 H - u r i d i n e incorporat ion (Singh, Purdom and Jones, 1979). The discovery of genes in at least some heterochromatic segments ra i ses important questions about how and when these genes are expressed. The condensed nature of heterochromatin does not seem compatible with t r a n s c r i p t i o n . However, s ince these regions are access ib le for DNA r e p l i c a t i o n , they may also be access ib le for t r a n s c r i p t i o n . Another p o s s i b i l i t y i s that heterochromatic genes are expressed ear ly in development, before heterochromatic regions condense. The heterochromatin of D. welanogaster does not condense u n t i l blastomere formation (Sonnenblick, 1950); and the heterochromatin of Hicrotus agrestis i s also r e l a t i v e l y uncondensed ear ly in development (Vunis and Yasmineh, 1971). If heterochromatic genes are expressed ear ly in development, then mutants of these genes might be expected to have ear ly l e tha l phases and temperature sens i t ive per iods . The t h i r d chromosome heterochromatic d e f i c i e n c i e s tested in t h i s study had l e tha l phases during the second or t h i r d l a r v a l i n s t a r . H i l l i k e r (1976) found that the EMS-induced l e tha l a l l e l e s of heterochromatic l o c i on the second chromosome had le tha l phases in the la te l a rva l or pupal stages. While these r e s u l t s do not ind ica te that heterochromatic genes are expressed early in 80 development, they do not exclude such a p o s s i b i l i t y . Lethal phases do not repre-sent the stage at which a gene i s expressed, but rather a c r i t i c a l period in development during which the organism i s unable to pass success fu l ly as a re su l t of a mutant gene expressed e a r l i e r in development (Wright, 1970). More precise information about the timing of expression of genes in hetero-chromatin may be revealed by the temperature sens i t ive periods (TSPs) of temper-ature s ens i t i ve (TS) mutations of heterochromatic genes. The only d e f i n i t e TS mutant of a heterochromatic gene recovered in t h i s study (1-166-5) had a very prolonged TSP which began during the ear ly second l a rva l ins tar stage. Tempera-ture s e n s i t i v i t y probably r e s u l t s from s t ruc tura l changes in h e a t - l a b i l e pro-te ins (Jockusch, 1966; Suzuki , 1970), a f ind ing which has two important i m p l i c a -t ions for the r e s u l t s of t h i s study. F i r s t , the recovery of a TS mutation of a heterochromatic gene provides further evidence that such genes do code for prote ins . The second impl i ca t ion i s that a TS mutation must be t ranscr ibed pr io r to the TSP i f a h e a t - l a b i l e protein i s responsible for the temperature sens i -t i v i t y . Therefore, the r e s u l t s from the 1-166-5 TS mutant ind ica te that at least one gene in t h i r d chromosome heterochromatin must be t ranscr ibed before the beginning of the second l a r v a l ins tar stage. The recovery and analys i s of more TS mutations of heterochromatic l o c i may help to answer the important question of when genes in heterochromatin are expressed. There i s evidence from a var ie ty of organisms which suggests that genes present in heterochromatin are expressed in the germ l i n e rather than somatic t i s sues . During po ly ten iza t ion in several t i s sues of Drosophila larvae the heterochromatin i s severely under- rep l i ca ted , and a s imi l a r condit ion occurs in the p o l y p l o i d somatic nuclei of adult t i s sues (Rudkin, 1965; Gal l and Atherton, 1974; Spear, 1977; Lakhot ia , 1984). In many species of nematodes and copepods, s a t e l l i t e DNA-rich heterochromatic segments are s p e c i f i c a l l y removed from 81 somatic c e l l chromosomes during the ear ly cleavage stages (Moritz and Roth, 1976; Beerman, 1977; Streeck, Moritz and Beer, 19B2). The germ l i n e c e l l s of a l l these organisms are d i p l o i d and re ta in a f u l l complement of heterochromatin. In the insect C a i l i p h o r a e r y t h r o c e p h a l a , s a t e l l i t e DNA i s d r a s t i c a l l y under-r e p l i c a t e d in polytene t i s sues of s a l i va ry glands and malphigian tubules , but i s coordinate ly r e p l i c a t e d with the n o n - s a t e l l i t e sequences during po ly ten iza t ion of nurse c e l l nucle i in the germ l i n e (Dover, 1980). Cooper (1977) i r r a d i a t e d and observed cul tured tticrotus a g r e s t i s bone marrow c e l l s , and found many stable rearrangements and de le t ions of the heterochromatin. However, no such delet ions are observed in natural populat ions , suggesting that heterochromatic regions are e s sent ia l for the germ l i n e , but are not required in at least some somatic eel 15. The condensation or under - rep l i ca t ion of heterochromatin may be a mechanism for regulat ing the expression of genes in heterochromatin. P i m p i n e l l i et a l . (1985) have recent ly put forward an i n t r i g u i n g suggestion about the region of D. jielanogaster heterochromatin Dn the X and Y chromosomes that can rescue a se r ie s of maternal mutants inc lud ing a b n o r m a l o o c y t e . Their experimental f indings led them to conclude that heterochromatic regions may carry a dupl ica te set of the euchromatinc maternal effect genes. Based on the timing of rescue of the maternal effect mutations, the heterochromatic gene set appears to be expressed early in development before nuclear migration and the general a c t iva t ion of zygotic gene a c t i o n , while the euchromatic gene set i s expressed after b la s to-derm formation. The authors suggest that one consequence of a heterochromatic loca t ion i s an out-of-phase gene a c t i v i t y . A second example of how heterochromatin may regulate the timing of gene a c t i v i t y comes from the snake E l a p h e r a d i a k (Singh, Purdom and Jones, 1979). In the growing oocytes of t h i s species , the W chromosome s a t e l l i t e - r i c h heterochro-matin decondenses completely and i s associated with a period of 3 H - u r i d i n e 82 incorpora t ion , while the autosomal s a t e l l i t e - r i c h heterochromatin regions remain condensed. Later , the cycle i s reversed and the W chromosome heterochromatin condenses while the autosomal heterochromatin decondenses. These examples suggest that one consequence o-f genes located in heterochromatin i s that the i r expression may be r e s t r i c t e d to cer ta in periods in the development of an organism. What are some of the other impl ica t ions of the presence of funct ioning genes in heterochromatin? It i s u n l i k e l y that the small number of genes in heterochromatin have a s i g n i f i c a n t inf luence on the nature and propert ies of heterochromatin. In f ac t , the low gene density in heterochromatin supports the model that cy to log ica l1y observed heterochromatin i s usual ly a consequence of a high concentrat ion of tandemly repeated s a t e l l i t e DNA sequences (Wollensien, Barsanti and Hearst , 1977; Appels and Peacock, 1978; Bostock, 1980). Since a l l s i g n i f i c a n t concentrat ions of s a t e l l i t e DNA are heterochromatic, and most but not a l l heterochromatic regions contain p r i m a r i l y s a t e l l i t e DNA sequences, s a t e l l i t e DNA seems to be a s u f f i c i e n t but not necessary condi t ion for hetero-chromatin formation. Prote ins that have been shown to s p e c i f i c a l l y bind s a t e l -l i t e DNA <Hsieh and Brut lag , 1979; Levinger and Varshavsky, 1982; Strauss and Varshavsky, 1984) may be responsible for the compact nature of heterochromatin. Although heterochromatic genes are u n l i k e l y to have a s i g n i f i c a n t ef fect on the nature and propert ies of heterochromatin, they may have a profound consequences for the function of heterochromatin. As discussed e a r l i e r , a number of functions have been proposed for heterochromatin, but none have been proven. One of the problems in assigning a function to heterochromatin i s that i t i s so v a r i a b l e , both within and between species . V a r i a b i l i t y i s such a predominant feature of s a t e l l i t e DNA and heterochro-matin that several authors have now proposed that the primary function of heter-83 ochromatin i s to provide organisms with a means to respond rap id ly to environ-mental changes (John and Mik los , 1979; Bostock, 1980; Hiklos and G i l l , 1981). Rapid changes in the amount of heterochromatin may re su l t in changes in the amount and d i s t r i b u t i o n of cross ing-over , an a l tered nucleotype, new f e r t i l i t y b a r r i e r s , and perhaps other unknown e f fec t s . According to th i s hypothesis heterochromatin provides a species with a dimension of f l e x i b i l i t y unavai lable to organisms with only unique sequence DNA. At least one mechanism that seems to be involved in generating var i a t ions in heterochromatic content i s unequal s i s t e r chromatid exchange (John and Miklos , 1979; K u r n i t , 1979; Hiklos and G i l l , 1981). Whatever the mechanism, the rapid expansion or contract ion of heterochromatin could re su l t in the de le t ion or dup l i ca t ion of genes present in heterochromatin (Miklos and G i l l , 1981). Therefore, the presence of funct ioning genes in heterochromatin may tend to r e s t r i c t the v a r i a b i l i t y of heterochromatin. I f , as suggested, the key property of heterochromatin i s i t s f l u i d i t y , then the presence of genes could i n t e r f e r e with the proper funct ioning of heterochromatin. On the other hand, a species which i s well adapted to i t s environment may f ind i t b e n e f i c i a l to have i t s heterochromatin s t a b i l i z e d by the presences of heterochromatic genes. The genes may function as anchors, impeding any f luc tua t ions in heterochromatin. The amount of i n t r a s p e c i f i c heterochromatic va r i a t i on d i f f e r s great ly between species . This d i f ference in f l u i d i t y may r e f l e c t the presence or absence of heterochromatic genes. Heterochromatin in the human genome i s very poly-morphic on some chromosomes but not others (Craig-Holmes et a i . , 1975). Maize heterochromatin i s highly conserved with no observed polymorphisms (Rhoades, 1978); while rye heterochromatin has extensive va r i a t i ons (Jones and F l a v e l l , 1982). In Drosphila s e l a n o g a s t e r , va r i a t ions have been detected in X chromosome heterochromatin (Hal fer , 1981; P i m p i n e l l i et a i . , 1985), which does not contain s ingle-copy v i t a l genes. However, no polymorphisms of autosomal heterochromatin, 84 which does contain unique sequence v i t a l genes, have been detected (Sved and V e r l i n , 1980; H a l f e r , 1981). In a Drosophila velanogaster c e l l l i n e examined for a seven year per iod , the loss or gain o-f sex chromosome heterochromatin, but not autosomal heterochromatin, was observed (HaHer , 1978). These r e s u l t s re-a-f-firm the heterogeneity o-f heterochromatin, as some blocks are more conserved and less f l u i d than other heterochromatic regions . The amount of i n t r a s p e c i f i c hetero-chromatin va r i a t i on may be a good ind ica tor of the presence or absence of heterochromatic genes. In te r spec i f i c va r i a t i ons in heterochromatin between c l o s e l y re la ted species may help to reveal the s i gn i f i c ance of genes in heterochromatin. If i t i s important that genes are located in heterochromatin, then the hetrochromatic pos i t ion of the gene should be conserved between c lo se ly re la ted species . On the other hand, i f the gene's pos i t ion i s unimportant, i t may not be conserved. A useful experiment would be to undertake a compound detachment study with the second or t h i r d autosome of D. s inu ians . D. sinuians and 2?. aelanogaster are s i b l i n g species , and the i r polytene and mitot ic chromosome are almost i d e n t i c a l . The blocks of heterochromatin in the two species are very s imi la r in s ize and p o s i t i o n , but there are dra s t i c d i f ferences in the r e l a t i v e amounts of d i f f e rent s a t e l l i t e sequences present in the heterochromatin of the two species (Peacock et a i . , 1977b; Peacock and Lohe, 1980). The arrangement of sequence order within the heterochromatic blocks of the two species has been conserved, and so l i k e the euchromatin, there have not been any s i g n i f i c a n t rearrangements. However, there have been major sa l ta tory changes in the r e l a t i v e amounts of p a r t i c u l a r DNA sequences. The increase of one sequence in D. s i m i a n s r e l a t i v e to D. melanogaster i s compensated by a r e l a t i v e decrease in a d i f fe rent sequence, so that the t o t a l amount of s a t e l l i t e DNA in the two species remains equal . It would be i n t e r e s t i n g to compare the gene content of , for example, the 85 t h i r d c h r o m o s o m e h e t e r o c h r o m a t i n o f b o t h D. siwulans a n d D. welanogaster. I f t h e h e t e r o c h r o m a t i c g e n e c o n t e n t i s c o n s e r v e d b e t w e e n t h e s e two s p e c i e s d e s p i t e t h e m a j o r c h a n g e s i n t h e s u r r o u n d i n g s a t e l l i t e DNA, i t w o u l d i n d i c a t e t h a t t h e h e t e r o c h r o m a t i c p o s i t i o n o f t h e s e g e n e s i s s i g n i f i c a n t . C o n v e r s e l y , i f t h e h e t e r o c h r o m a t i c g e n e s w e r e n o t c o n s e r v e d b e t w e e n t h e t w o c l o s e l y r e l a t e d s p e c i e s , i t w o u l d s u g g e s t t h a t t h e l o c a t i o n o f h e t e r o c h r o m a t i c g e n e s i s o f l i t t l e c o n s e q u e n c e . To s u m m a r i z e , t h e r e s u l t s o f t h e g e n e t i c a n a l y s i s o f c h r o m o s o m e t h r e e h e t e r o c h r o m a t i n o f Drosophila welanogaster p r o v i d e s f u r t h e r e v i d e n c e t h a t f u n c t i o n i n g g e n e s a r e p r e s e n t i n some h e t e r o c h r o m a t i c r e g i o n s , b u t n o t i n o t h e r s . A t l e a s t some o f t h e g e n e s p r e s e n t i n c h r o m o s o m e - 3 h e t e r o c h r o m a t i n a r e l o c a t e d i n t h e m o s t d i s t a l s e g m e n t s o f h e t e r o c h r o m a t i n on b o t h a r m s . 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