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Characterization of a cluster of dominant suppressors of position effect variegation including effects… Hedrick, Amy L. 1989

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CHARACTERIZATION OF A CLUSTER OF DOMINANT SUPPRESSORS OF POSITION EFFECT VARIEGATION INCLUDING EFFECTS ON HETEROCHROMATIC VARIEGATING REARRANGEMENTS IN DROSOPHILA MELANOGASTER By Amy L. Hedrick B . S c , P o r t l a n d State U n i v e r s i t y , 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1989 © A m y L. Hedrick, 198 9 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology  The University of British Columbia Vancouver, Canada Date 15 April 1989  DE-6 (2/88) i i ABSTRACT The mosiac, cell-autonomous expression of genes resulting from chromosomal rearrangement and relocation next to broken heterochromatin is termed position effect variegation (PEV). Since the gene is inactivated due to chromatin changes, this system allows the genetic study of chromatin structure and function using mutations which rescue the mosaic phenotype. These mutations called suppressors of variegation, Su(var)s, must influence chromatin structure. The genetic characterization of several groups of Su(var)s has been undertaken in this study using Drosophila melanogaster. Variegation of the light gene, located in heterochromatin, is enhanced by several Su(var) mutations on chromosome two. This opposite effect suggests that products of these Su(var)s are essential for functioning heterochromatin and deleterious for euchromatic environments. Other Su(var)s have slight or no effects on the same variegating rearrangements, demonstrating functional differences, among the Su(var)s tested. A group of Su(var)s located within 4 map units near.the centromere of chromosome three was characterized using deficiency mapping, new compound autosome formation and inter se complementation based on newly established i i i homozygous phenotypes. Two Su(var)s mapped to 87B on 3R, while one Su(var) maps to 3L according to compound mapping. Inter se complementation, in combination with mapping data, suggests that four seperate loci make up this group of Su(var)s. Eight of nine Su(var)s are extremely sensitive to heterochromatic deletions as shown by their responses to loss of 2R heterochromatin, as well as the Y chromosome. In contrast, Su(var)A130 is insensitive to both forms of heterochromatic deficiencies. Su(var)s show complicated reactions to maternal verses paternal source effects. Six of nine Su(var)s show a female-specific temperature sensitive maternal effect. Some maternal and paternal effects are observed at 22 C. Su(var)A57 is maternal semi-lethal and suppressed at 29 C. This characterization has better defined these mutants, making them ammenable to molecular study. iv TABLE OF CONTENTS P a g e Abstract i i Table of Contents iv List of Tables v i List of Figures. . . v i i i Acknowledgements ix General Introduction ...1 Chapter 1 - The effect of Su(var) mutations on light gene variegation 6 Introduction 6 Materials and Methods 9 Stocks 9 Culture conditions 9 Effect on Su(var)s on light variegation ...9 Fluorometric assay of eye pigment 13 Statistics 15 Results. 16 Discussion 33 Chapter 2 - Characterization of a proximal cluster of Su(var) mutations on chromosome three 38 Introduction 38 Materials and Methods 41 Stocks 41 Culture Conditions 41 Mapping the 3R proximal cluster Su(var)s 41 Effect of heterochromatin deficiencies on Su (var) activity 45 i) Loss of Y chromosome 48 V i i ) Loss of 2R heterochromatin 48 Maternal effects 48 Via b i l i t y and f e r t i l i t y studies/ complementations tests 52 Statist i c s . .56 Results. 57 Deficiency mapping 57 Compound mapping 60 Effect of heterochromatin loss on Su(var) activity 63 Maternal effects..... 67 Homozygous v i a b i l i t y / complementation analysis 74 Discussion 78 Bibiliography 87 Appendix 92 v i L I S T OF T A B L E S P a g e Table 1: Suppressor of postion e f f e c t variegation mutations used i n t h i s study 10 Table 2: Description of light variegating rearrangements 11 Table 3: Basal light variegating control pigment leve l s from three seperate t r i a l s 17 Table 4: Eff e c t s of Su(var)III mutations on three light variegating strains 26 Table 5: Su(var)s which enhance light variegation 31 Table 6: Deficiencies used to map 3R proximal Su(var)s 42 Table 7: Marked stocks established for Su (var) III mutations 55 Table 8: Eff e c t s of de f i c i e n c i e s i n the 3R proximal region on wm4 variegation 58 Table 9: Results of complementation analysis with d e f i c i e n c i e s i n the 3R proximal region and 3R Su(var) mutations... 59 Table 10: Summary of new compound progeny recovered from gamma radiation screen 61 Table 11: Results of progeny t e s t i n g for new compound females heterozygous for wm4; test for presence of absence of Su(var)B143 62 Table 12: Eff e c t s of loss of Y chromosome on Su(var) mutations of chromosome 3. 64 Table 13: Eff e c t s of loss of 2R heterochromatin on Su(var) mutations on chromosome 3 .66 Table 14: Maternal e f f e c t s measured at 22 C 68 Table 15: Maternal e f f e c t s measured at 29 C 69 Table 16: Summary of maternal and paternal ef f e c t s of wm4;TM3 progeny of Su(var) parents at 22 and 29 C 71 Table 17: Maternal effects of Su(var)A57 at d i f f e r e n t developmental temperatures 73 v i i Table 18: Homzygous phenotypes of proximal 3R Su (var) s 75 Table 19: Complementation a n a l y s i s of 3R proximal Su(var) mutations 77 TablesBa-f: L t x 2 / S u ( v a r ) I I data t o accompany graphs i n f i g u r e s 3a-f 92 v i i i LIST OF FIGURES Page Figure 1: Construction of stocks for t e s t i n g t h i r d chromosome Su(var)s with light variegating rearrangements 12 Figure 2: Crosses to test e f f e c t s of Su(var)III mutants on light variegating rearrangements 14 Figures 3a-f: E f f e c t s of Su(var)II mutations on light variegation 19 Figure 4: Cytology of 3R proximal region including d e f i c i e n c i e s used to map Su (var) III mutations 43 Figure 5: Strategy used to map Su(var)s using new compound formation 46 Figure 6: Protocol to determine presence or absence of Su(var) i n newly formed compound females heterozygous for wm4 47 Figure 7: Protocol used to determine s e n s i t i v i t y of Su(var)s to loss of the Y chromosome 49 Figure 8: Protocol used to determine s e n s i t i v i t y of Su(var)s to the loss of 2R ce n t r i c heterochromatin 50 Figure 9: Reciprocol crosses used to determine maternal e f f e c t s for 3R Su(var)s 51 FigurelOa: Protocol for constructing a multiply marked s t r a i n with TM3 balancer lacking Stubble 53 Figure 10b: Protocol for construction of dominantly marked TM3 balanced s t r a i n lacking Stubble 53 Figure 11: Protocol for constructing marked stocks of Su (var) mutations 54 Figure 12: Complementation groups based on trans-heterozygous phenotypes and physical mapping 83 Figure A: Oregon-R pigment value l i n e a r i t y assay 93 Figure C: Recombination data for Su(var)s A160 and A130 100 ACKNOWLEDGEMENTS I thank my supervisor, Tom Grigliatti for allowing me independence of research, and for always taking time to help with advice and encouragement. Thanks also to Don Sinclair for his support and input throughout this study and Hugh Brock for spending time to read the rough drafts. Especially, I would like to thank my husband, Michael and my fellow fly pushers, Jo-Ann Brock and Joanie McKeon for invaluble discussion, advise, encouragment and support. Finally, thanks to a l l the zoology geneticists who made graduate school enjoyable. 1 GENERAL INTRODUCTION P o s i t i o n e f f e c t v a r i e g a t i o n (PEV) r e s u l t s when a gene i s moved v i a chromosome rearrangement from i t s usual l o c a t i o n t o a p o s i t i o n next to a newly formed euchromatic-heterochromatic boundary. The rearranged gene i s i n a c t i v a t e d i n some c e l l s , but remains a c t i v e i n others. This on/off d e c i s i o n i s made e a r l y i n development and i s c l o n a l l y propogated, r e s u l t i n g i n mosaic expression of the gene. Both euchromatic and heterochromatic genes are subject to t h i s phenomena, but the m a j o r i t y of work has been done w i t h euchromatic v a r i e g a t i n g genes. PEV as d e f i n e d above was f i r s t d escribed by Sturtevant (1925). Since then, most work has been done i n Drosophila melanogaster where v i r t u a l l y every gene t e s t e d i s subject t o PEV (for reviews, see Lewis 1950; Baker 1968; Spofford 1976) . Although much progress has been made since 1925, the molecular mechanism of PEV i s s t i l l unknown. Judd (1955) has provided evidence against mutation r e s u l t i n g i n i n a c t i v a t e d genes, and Henikoff (1979) has r u l e d out somatic gene l o s s as a cause of mosaic expression. This suggests t h a t changes i n chromatin s t r u c t u r e are the cause of v a r i e g a t i n g p o s i t i o n e f f e c t s . Zuckerkandl (1974) proposed t h a t heterochromatic elements spread beyond the d i s r r u p t e d heterochromatic boundary, causing t r a n s c r i p t i o n a l i n a c t i v a t i o n of 2 neighbouring euchromatic l o c i through a l t e r e d chromatin s t r u c t u r e . There i s evidence to support such a model. F i r s t , PEV shows a spreading or p o l a r a f f e c t . Genes are i n a c t i v a t e d i n order of t h e i r p r o x i m i t y to the heterochromatic breakpoint. Genes f a r t h e r away w i l l not v a r i e g a t e unless other genes between are a l s o v a r i e g a t i n g (Demerec and S l i z y n s k a 1937; Schultz 1941; f o r an exception see Chovnick and C l a r k 1986). This supports the idea t h a t condensing molecules spread outward from heterochromatin and i n a c t i v a t e euchromatic genes. Second, Hartman-Goldstein (1967) and more r e c e n t l y Zhimulev et al. (1986) have c o r r e l a t e d h eterochromatin-like morphology of s a l i v a r y gland chromosomes wi t h v a r i e g a t i n g phenotypes. The i n i t i a t i o n of t h i s spreading has been i n v e s t i g a t e d by T artof et al. (1984). By c l o n i n g the heterochromatic j u n c t i o n s of three standard v a r i e g a t i n g rearrangements (wm4, wm51b, wmMc) they have shown th a t the euchromatic-heterchromatic boundary i s f l a n k e d by mobile element-like sequences. These sequences alone are not capable of inducing chromatin condensation. This suggests that heterochromatization i s i n i t i a t e d f a r away from the breakpoint, w i t h i n the heterochromatin. However, Reuter et al. (1985) have found complete phenotypic r e v e r t a n t s which r e t a i n s e n s i t i v i t y t o dominant enhancer mutations of PEV. They conclude t h a t the euchromatic-heterochromatic j u n c t i o n f l a n k e d by one or more of these sequences i s s u f f i c i e n t t o cause v a r i e g a t i o n . 3 PEV and i t s a s s o c i a t e d chromatin changes can be i n v e s t i g a t e d due to the existance of s e v e r a l potent m o d i f i e r s of the PEV phenotype. A l l v a r i e g a t i n g phenotypes are suppressed by high developmental temperature, so th a t the v a r i e g a t i n g gene i s expressed i n a greater number of c e l l s . Low developmental temperature enhances v a r i e g a t i o n , causing the gene to be i n a c t i v a t e d i n more c e l l s (Gowan and Gay 1933). Another standard m o d i f i e r of PEV i s heterochromatin content of the c e l l . V a r i e g a t i o n i s enhanced by l o s s of heterochromatin such as the Y-chromosome (Gowan and Gay 1934) and the c e n t r i c heterochromatin d e l e t i o n Df(2R) M-S210 (Morgan et a l . 1941). Conversly, an e x t r a Y-chromosome suppresses PEV. These observations suggest t h a t the presence of heterochromatin i n a c e l l acts as a si n k f o r heterochromatic elements. E x t r a heterochromatin a t t r a c t s more of these elements away from the f a c u l t a t i v e spreading heterochromatin, thus l e a v i n g the rearranged gene i n a euchromatic environment, r e s u l t i n g i n a l e s s extreme mutant phenotype. Loss of heterochromatin frees these elements and allows formation of heterochromatin at the breakpoint, causing a more severe mutant phenotype. This sink e f f e c t may be due t o heterochromatin content, or to s p e c i f i c regions (binding s i t e s ) i n heterochromatin. Brock (1986) rep o r t s that d i f f e r e n t regions of the Y-chromosome have an enhancing e f f e c t on v a r i e g a t i o n which i s not c o r r e l a t e d w i t h s i z e . 4 More r e c e n t l y discovered m o d i f i e r s of PEV i n c l u d e histone gene m u l t i p l i c i t y , and butyrate, an i n h i b i t o r of hi s t o n e de-acetylases. Moore et al. (1979; 1983) demonstrated that v a r i e g a t i n g phenotypes are suppressed by a hi s t o n e d e f i c i e n c y . This suggests that a decrease of histones a v a i l a b l e to the chromatin l i m i t s t r a n s c r i p t i o n a l i n a c t i v a t i o n due t o heterchromatic packaging, thus a l l o w i n g a more w i l d type phenotype. In a d d i t i o n , Mottus et al. (1980) have found t h a t sodium butyrate can suppress v a r i e g a t i o n , probably since histone de-acetylases are i n h i b i t e d , r e s u l t i n g i n a c e t y l a t e d h i s t o n e s . These f i n d i n g s support the heterochromatization mechanism f o r PEV gene i n a c t i v a t i o n . Genetic m o d i f i e r s of PEV are perhaps the most u s e f u l i n v e s t i g a t i v e t o o l s . Spofford (1967) i d e n t i f i e d the f i r s t dominant suppressor of PEV (Su(var)) mutation. Since then, Reuter and Wolff (1981) and S i n c l a i r et al. (1983) have independently i s o l a t e d over 150 X-ray or EMS induced dominant Su(var) mutations. The Su(var)s were s e l e c t e d f o r t h e i r e f f e c t s on the white gene v a r i e g a t i o n rearrangement, wm4, but show general e f f e c t s , suppressing both brown and scute v a r i e g a t i o n (Reuter and Wolff 1981) and brown and Stubble v a r i e g a t i o n ( S i n c l a i r et a l . 1983). Genetic mapping revealed that many of the 50 Su(var)s of S i n c l a i r et al. (1983) f a l l i n t o d i s c r e t e c l u s t e r s d e f i n e d as mutants o r i g i n a l l y mapping w i t h i n a three centimorgan d i s t a n c e . Reuter and Wolff (1981) have 5 suggested t h i s l a b e l i s unnecessary, but for the purposes of discussion these Su(var)s w i l l be referred to as the 2L clustered and nonclustered Su(var)s, and the 3R proximal, middle and d i s t a l c l u s t e r s . Characterization of Su(var) mutations i s the subject of t h i s t h e s i s . F i r s t , interactions between Su(var)s and heterochromatic variegators, heterochromatic l o c i that variegate as a consequence of relocation near to euchromatin, are investigated to determine the relat i o n s h i p between heterochromatin and gene i n a c t i v a t i o n . Second, a 3R proximal clu s t e r i s characterized with regard to standard modifiers of PEV and genetic relationship of the Su(var)s i n t h i s c l u s t e r , to provide a necessary knowledge base for possible molecular experimentation. 6 CHAPTER 1 - The e f f e c t of Su(var) mutations on light gene v a r i e g a t i o n . INTRODUCTION Questions about chromatin packaging are fundamental t o understanding gene expression. P o s i t i o n e f f e c t v a r i e g a t i o n (PEV) causes h e t e r o c h r o m a t i n i z a t i o n of the DNA t h a t i s a l s o c o r r e l a t e d w i t h gene i n a c t i v a t i o n (see general i n t r o d u c t i o n ) . Thus, PEV provides a model system f o r studying heterochromatin and i t s r o l e i n gene r e g u l a t i o n . Both euchromatic and heterochromatic l o c i are subject t o PEV. Using euchromatic and heterochromatic l o c i which v a r i e g a t e , d i f f e r e n c e s and s i m i l a r i t i e s i n the mechanisms of gene i n a c t i v a t i o n can be s t u d i e d . This study u t i l i z e s a heterochromatic gene, light (It) which e x h i b i t s a mosaic phenotype when moved away from c e n t r i c heterochromatin and r e l o c a t e d next to d i s t a l euchromatin (Schultz and Dobzhansky 1934). The v a r i e g a t i o n of I t appears i n many ways to be r e c i p r o c a l to standard euchromatic PEV (euPEV), such as v a r i e g a t i o n of the white gene. Light v a r i e g a t i o n , then, i s one example of heterochromatic PEV (hPEV). (See Spofford 197 6 f o r review of PEV and Hannah 1 9 5 1 ; H i l l i k e r et al. 1980, f o r reviews of heterochromatin). Few examples of hPEV are documented, probably since so few genes have been mapped to heterochromatin. Three heterochromatic genes are known to v a r i e g a t e : peach i n D. virilus, Baker (1953); cubitus interruptus (ci) i n D. melanogaster, Khvostov (1939); and light, a l s o i n D. 7 melanogaster, H e s s l e r (1958). One requirement f o r hPEV has been observed. In order f o r a heterochromatic gene t o v a r i e g a t e , the p o s i t i o n of the euchromatic b r e a k p o i n t must be i n the d i s t a l t w o - t h i r d s of the chromosome (or i n other heterochromatin which i s then d i s r u p t e d ) . Baker (1968) suggests t h a t a r e p a i r mechanism spreads from the un d i s t u r b e d heterochromatin. A gene t h a t i s r e l o c a t e d t o d i s t a l euchromatin cannot be reached by t h i s competent heterochromatin and, t h e r e f o r e , r e p a i r cannot take p l a c e . While light v a r i e g a t i o n f o l l o w s t h i s requirement f o r hPEV, i t does demonstrate some i r r e g u l a r t i e s . Light v a r i e g a t i n g rearrangements (ltxvs) show v a r i o u s phenotypes (Hessler 1958). Some rearrangements r e s u l t i n a p a l e -m o t t l e d eye, having many light ommatidia and few w i l d - t y p e c e l l s . Others show a dark-mottled eye, which appears as a wil d - t y p e eye i n t e r s p e r s e d w i t h darker ommatidia. Some rearrangements g i v e phenotypes i n t e r m e d i a t e between these extremes. H e s s l e r (1958) found no c o r r e l a t i o n between c y t o l o g i c a l b r e a k p o i n t s and any one phenotype. A l l It phenotypes respond t o standard m o d i f i e r s o f PEV, such as temperature and the presence or absence of heterochromatin (Baker 1968). Light v a r i e g a t i o n i s suppressed by e l e v a t e d temperature as i s euchromatic v a r i e g a t i o n . However, a d d i t i o n of a Y chromosome (normally a suppressor o f PEV) a c t s as an enhancer o f It v a r i e g a t i o n . T h i s o b s e r v a t i o n s e t s It apart from other heterochromatic 8 variegators (ie; peach and ci) and suggests some aspects of It variegation are reciprocal to euPEV. If the mechanism of It variegation is reciprocal to euchromatic variegation, its movement into distal euchromatin is likely associated with chromatin changes which are detrimental to proper expression of the gene. The response of It variegation to the heterochromatic Y chromosome implies sensitivity to the amount of heterochromatin in the cell which is opposite to that of euchromatic variegation. Dominant modifiers of PEV can be employed to investigate the response of heterochromatic variegators to alterations in chromatin. Dominant suppressors of PEV (Su(var)s) are able to completely reverse the inactivation of genes which results from abnormal proximity to heterochromatin. It is likely that these Su(var) genes encode products involved with heterochromatin (see main introduction). If light variegation is mechanically reciprocal, one can expect Su(var)s to act as enhancers of light variegation, due to further stress on heterochromatin in the c e l l . This study examines the effect of several dominant suppressors of euchromatic PEV on various light variegating rearrangements. 9 MATERIALS AND METHODS STOCKS: Mutations used i n t h i s study not l i s t e d i n L i n d s l e y and G r e l l (1968) are l i s t e d i n Tables 1 and 2. A l l suppressors of v a r i e g a t i o n , Su(vars), were i s o l a t e d as described by S i n c l a i r et al., (1983). Light v a r i e g a t i n g rearrangements were i s o l a t e d and c h a r a c t e r i z e d by Barbara Wakimoto (personal communications). Since the t h i r d chromosome Su(var)s do not c a r r y It mutations, i t was necessary to construct two stocks, diagrammed i n Figure 1. These stocks provide a It background so t h a t I t v a r i e g a t i o n can be detected i n the presence of these Su(var)s. CULTURE CONDITIONS: F l i e s were r a i s e d at 22 C (unless otherwise stated) on standard cornmeal-sucrose Drosophila medium. Tegosept was added as a mould i n h i b i t o r . EFFECT OF SU(VAR)S ON LIGHT VARIEGATION: To t e s t the e f f e c t of second chromosome suppressors ( S u ( v a r ) I I ) s on I t v a r i e g a t i o n , the f o l l o w i n g cross was performed. Males of the genotype wm4;Su(var), b It rl/CyO were crossed t o +/+; ltxv/Gla or Itxv/ltxv v i r g i n s . F l heterozygotes were c o l l e c t e d 0-3 days p o s t - e c l o s i o n , aged 5 days, and pigment a n a l y s i s was performed (see below). TABLE 1: SUPPRESSOR OF POSITION EFFECT VARIEGATION USED IN THIS STUDY. Mutations Alternate Designation 2L CLUSTERED Su (var) 214 Su (var)A24 34. 9 + 1 .8 Su (var) 210 Su(var)C157 34. 8 + 1 .8 Su (var) 207 Su (var)H69 32. 0 + 1 .4 Su(var)216 Su (var) M59 34. 2 + 1 .6 2L NONCLUSTERED Su (var) 208 Su(var)T44 5. 7 + 1 .3 Su (var) 206 Su (var)A151 51. 3 + 0 .9 Su (var) 205 Su(var)M43 30. 8 + 1 .6 Su (var) 201 Su (var)B89 45. 9 + 3 .5 3R PROXIMAL Su (var) 310 Su (var)A63 54. 4 + 0 .7 Su (var) 316 Su (var)A48 47. 4 0 .8 Su(var)308 Su(var)A57 49. 2 + 1 .2 Su (var) 307 Su (var)B94 47. 4 + 0 . 9 Su(var)304 Su (var)B143 46. 4 + 1 .1 Su(var)321 Su(var)Cll9 47. 6 + 1 .3 Su (var) 319 Su(var)B76 48. 6 + 1 .3 3L ARM Map pos i t i o n (+ 95% confidence levels) Su(var)323 Su(var)303 Su (var)A130 Su (var)AlSO 3L 3L see appendix 11 TABLE 2: DESCRIPTION OF LIGHT VARIEGATING REARRANGEMENTS LIGHT REARRANGEMENT (abrieviation) PHENOTYPE CYTOLOGY In (2L) ltxl8 (ltxl8) T (2;3) ItxS (ltx6) T(2;3)ltx2 (2tx2) T (2;3) l t l 3 (ltl3) T(2;3)ltx24 (ltx24) T(2;3)ltx4 (ltx4) pale-mottled moderately-mottled moderately-mottled dark-mottled' homozygous viable dark-mottled, dark-mottled T (2;3)40?, 94D + T(2;3)32C, 64 in s e r t i o n a l translocation of 63-74 to 36h* In (2L)25D5, 40A + In(2LR)40A, 53A1,2 T(2;3)37h; 97D2 T(2;3)37,38h; 61D3-61F T(2;3) 36,37h;97Dl *h indicates heterochromatic cytology units i n 2L heterochromatin (unpublished map). 12 FIGURE 1: CONSTRUCTION OF STOCKS FOR TESTING THIRD CHROMOSOME SU(VAR)S WITH LIGHT VARIEGATING REARRANGEMENTS To provide b,lt,rl background: wm4 + Ly wm4 CyO; + i / / / Y + TM3,Sb,Ser wm4 Tft + wm4 CyO Ly wm4 + + x + b,lt,rl + -; ;- x Y b,lt,rl + wm4 Tft wm4 CyO + wm4 Tft + Y b,lt,rl + cr«9 wm4 CyO Ly b,lt,rl + select CyO,Ly f l i e s each generation To mark the second chromosome of Su(var)Ills: wm4 Tft + wm4 CyO + FI s i b l i n g s : wm4 CyO TM3,SbrSer wm4 + + wm4 Tft Ly x ( wm4 + TM3, Sbr Ser wm4 + Ly x Y + TM3,SbrSer wm4 Tft Ly Y + + wm4 + Su(var)III x cr« 9 wm4 Tft Su (var) III + TM3,Sb,Ser Y + TM3,Sb,Ser select Tft;Su(var) f l i e s each generation 13 To test the effect of Su(var)Ills on It variegation (chromosome 2) a second chromosome containing a It mutant allele was required in the Su(var)III strains. This was accomplished with the protocol shown in Figure 2. Itxv;Su(var) Fl progeny were collected 0-3 days post-eclosion, aged 5 days and subjected to pigment analysis (see below). FLUOROMETRIC ASSAY OF EYE PIGMENT: To measure the effect of the Su(var)s on It variegation, eye pigments were extracted and analyzed as follows. Adults aged 5-8 days post-eclosion were frozen in glass tubes at -70 C. Flies were decapitated immediately after removal from -70 C by firmly banging the tubes. A total of 50 heads for each genotype were placed in 1.5 ml Ependorf tubes, 5 heads per tube, males and females separated at collection. Pigment was extracted by sonication of heads in 30 ul of 0.25M 2-mercaptoethanol in 1% aqueous NH4OH. After brief centrifugation, samples were placed in the dark for approximately 1 hour at room temperature. The head fragments were precipitated by centrifugation at 12,000 G for 2 minutes. 5 ul of the supernatant from each sample was pippetted onto Whatman #3 f i l t e r paper fixed to a microscope slide so that each slide contained five 5 ul aliquots of one genotype. The amount of pigment was quantified by flourescence at >500 um (in the linear range of the instrument) using a Zeiss 14 FIGURE 2: CROSSES TO TEST EFFECTS OF SU(VAR)III MUTANTS ON LIGHT VARIEGATING REARRANGEMENTS Experimental: wm4 CyO Ly wm4 b , l t , r l + + Itxv + + Gla + experimental: i n t e r n a l c o n t r o l : x 4 x wm4 Tft Su (var) III Y + TM3, Sb, Ser wm4 Tft Su(var)III Y b r l t , r l Ly + Itxv Su(var)III *' b , l t , r l ' + pigment assay + Itxv Ly b , l t , r l + C o n t r o l : + Itxv + wm4 CyO Ly f I A f f + Gla + Y b , l t , r l .+ V + Itxv + —; ;— pigment assay * b r l t , r l + *Chromosome i s e i t h e r an X c a r r y i n g wm4 or a Y. 15 microfluoremeter. Pigment l e v e l s are expressed as a percentage of w i l d type (Oregon-R) pigment l e v e l s . STATISTICS: Fluorescence values expressed as percentages of Oregon-R c o n t r o l pigment l e v e l s , which form a binomial d i s t r i b u t i o n , were transformed to t h e i r a r c s i n e values to approximate a normal d i s t r i b u t i o n . S u ( v a r ) I I values were compared w i t h c o n t r o l values by ANOVA followed by Dunnett's m u l t i p l e range t e s t (Zar 1984). D i f f e r e n c e s between S u ( v a r ) I I I values and t h e i r i n t e r n a l c o n t r o l values were determined by unpaired t - t e s t s . The s t a t i s t i c a l l i m i t of s i g n i f i c a n c e was taken as P<0.05. 16 RESULTS To measure effects of Su(var)s on It variegation, i t is first necessary to analyze strengths of each It variegating strain. A ll control crosses for Su(var)II experiments were performed three separate times and the results are listed in Table 3. Notice that lt/lt pigment levels are very consistent, ranging from 30.3+1.2 to 34.2+1.0 percent of Oregon-R pigment. As expected, basal levels of It variegating strains are more variable; in fact, the variability is so great that pigment values are often significantly different between tr i a l s . Even so, i t is possible to group these variegators into 3 categories of variegating strength. First, ltxl8 is a very strong variegator (pale mottled) often showing lt/lt pigment levels, indicating that the lights gene is inactivated in approximately 100% of the ommatidia. Suppression of variegation by Su(var)s may be observed in this strain since increases in pigment could be easily detected. ltxe> and ltx2 are moderate variegators, producing between 40 and 65% of Oregon-R pigment levels. Suppression or enhancement may be detected in these strains since their pigment levels can range upward or downward from basal variegating levels. Finally, ltxl3, ltx24 and ltx4 are very weak variegators (dark mottled) usually producing pigment between 70 and 100% of Oregon-R pigment levels. Therefore, suppression by 17 TABLE 3: BASAL LIGHT VARIEGATING CONTROL PIGMENT LEVELS FROM THREE SEPERATE TRIALS GENOTYPE TRIAL 1 TRIAL 2 TRIAL 3 (mean + 2 S.E.) It/It male 32.5 + 1.5 female 31.2+2.3 30.3 + 1.2 33.0 + 2.0 33.5 + 1.2 *34.2 + 1.0 ItxlQ male 26.9 + 1.4 26.7 + 2.5 31.6 + 1.9 female *31.0 ± 3.6 *24.6 + 3.9 *40.9 + 6.0 ltx6 male 64.1 + 8.2 53.3 + 4.6 54.1 + 8.1 female *50.1 ± 4.9 42.1 + 3.4 41.8 + 3.5 ltx2 male 54.6 +10.9 46.2 + 9.5 45.2 + 3.7 female *57.0 + 5.5 *47.0 + 7.3 *38.5 ± 2.0 ltxl3 male 84.4 + 4.7 *91.5 ± 7.0 80.0 + 4.7 female 66.1 + 5.2 *105.0 + 7.1 70.7 + 0.9 ltx24 male *102.5 + 2.5 77.5 +10.5 81.8 + 3.3 female 82.3±5.7 89.8±4.0 *7 6.0±2.7 ltx4 male 118.7 + 2.7 93.4 + 9.6 77.1 + 6.3 female 88.2 ± 2.6 91.7 + 4.6 *70.6 + 2.9 * s i g n i f i c a n t l y d i f f e r e n t from other c o n t r o l values w i t h i n t h a t genotype and sex. 18 Su(var)s would probably be undetectable in these strains since higher pigment levels may be indistinguishable from basal Itxv levels. Enhancement, or reduced pigment could be readily detected. Su(var)s used in these experiments can also be grouped into categories: clustered and nonclustered chromosome two suppressors and third chromosome suppressors (see Table 1) Results of the effects of both groups of Su(var)II suppressors on It variegation are shown in Figures 3a-f (actual pigment values are li s t e d in the appendix). Two t r i a l s were completed for most combinations of ltxvs with Su(var)II mutants. Trial I was assayed in one s i t t i n g , while t r i a l II was split into 2 assays, each having individual control Itxv levels measured. Results of the effects of Su(var)III mutants were more d i f f i c u l t to obtain because of stock v i a b i l i t y problems. Therefore, one strain from each category of It variegation strength was chosen for analysis of the third chromosome Su(var)s. Pigment data for these crosses are shown in Table 4. Each Itxv;Su(var) pigment level is shown with an internal control value taken from ltxv;Ly siblings. Differences between these values indicate effects of the Su(var) mutation versus a non-suppressor chromosome marked with Lyre. Pigment levels are measured for males and females independently, to account for any sexually dimorphic properties common among Su(var) mutants. Although many cases show that males and females react differently to a 19 FIGURE 3a-f: E f f e c t s o f S u ( v a r ) I I mutations on light v a r i e g a t i o n . Shaded areas i n d i c a t e b a s a l light v a r i e g a t i n g pigment l e v e l s . Open bars i n d i c a t e pigment l e v e l s of Su(var)II/Itxv h e t e r o z y g o t e s . Values are mean + 2 S.E. % Oregon—R pigment females males o o o o o o o o o o o o o o o o o o o o o 5 > t, CD oo CD 6 o O i X CD CD CD 1 1 1 1 1 1 1 1 1 —1—1—1—1—1—1—1—1—r— wmmh * • I'-'J. .„, T3F 31 *>tt TTJ. wmmm «+• x-xvWvXyXiXM u 1 • 33 O d m o • • T I 3 (D r-r-< C/) CO c Q 00 z r CD r-t-a> o to o r+-(D CO 03 5 > Cn CO 5 X Oi CO CO % Oregon—R pigment fema les males - > N U * t f l O ) s l 0 D ( O - ' l O O l A U l O l ^ a i D O o o o o o o o o o o o o o o o o o o o o o 1 1 1 I 1 I—1—r-3 & • m . D E P '**Hfr$J:i5r*'*i • nn . ' r n i 1 1 I 1 I 1 1 1 1 I i n II 23 O d m • • TJ cQ* 3 CD CD~ < CO CO c Q X C D CD r-r-CD ~1 O N O r-t-CD OO 13 % Oregon—R pigment f ema les males - ' S ) O I > U I O ) N | 0 9 0 IO U ^ Ul O) N l a ID o o o o o o o o o o o o o o o o o o o o o o > 00 CO o Ol NJ X CO Oi CO I I I I I u s -mmmmm^ 3 i » 3 H rcsi . LtLLI 1 I I I I ZB£?S5QSK J J O C m o • • T J — • cQ 3 0 < cp_ c/) o —h CO c Q ro = r 0 r+-CD -\ o N v: cQ O ri-ft 22 % Oregon—R pigment females males - » r o o i £ o i a » N j ( » < o - t r o G i £ a i o > N i t » < o Q o o o o o o o o o o o o o o o o o o o o o 5 > Ol 2 t CD 00 co 5 o Ol Oi CO 2 Ol co I 1 I I I I I I I 3 o cn o If NI 3 •:v:ai -i£3P I I I I I I I I I 3-3-gg-i 101.3 J L . 2.1 3j CD cz X3 m C L • • TJ 3 CD CD" < cp_ C O O —H CO c Q X GJ D " CD r-t-CD O N <^ cQ O r-t-CD C O > t CD CO CD (5 O Ol v i X Oi CD Oi CO % Oregon—R pigment fema les males o o o o o o o o o o o o o o o o o o o o o I 1 I I 1 1 I.V.TXN'.V.TT1 I |'»X*I*.y.'.l * • i • 3 3 * I I 1 I I I I 1 I n ^ a — • * |.VJj.;. ;.;.v.| 1 113.3 .±9.1 * J I 110.3 ± 4 . 4 • 1 3 * o o 3 < o_ c a U o ro N) In <z m CD 3 CD Zl r-t-< CO c Q X ho 3" (D *-+-o N O r-t-CD CO fr3 S3 TABLE 4: EFFECTS OF SU(VAR)III MUTATIONS ON THREE LIGHT VARIEGATING STRAINS GENOTYPE MALE t-value FEMALE t-value (mean + 2 S.E.) ltxl3; + 86.4 10.8 71.4 4.0 ltxl3;B94 70.5 4.4 63.0 2.6 t=0.98 t=2.14 control 73.9 6.2 57.9 4.2 ltxl3;B76 76.9 6.1 70.2 3.0 t=0.82 t=4.31* control 80.3 5.5 57.2 6.1 ltxl3;C119 73.7 5.4 70.5 2.5 t=1.90 t=0.36 c o n t r o l 82.8 2.8 71.3 3.9 ltxl3;A63 83.4 5.8 63.9 4.7 t=0.20 t=1.17 c o n t r o l 82.5 6.6 59.7 5.6 ltxl3;A48 60.8 8.5 59.3 4.8 t=2.96* t=0.86 c o n t r o l 68.0 1.1 61.6 2.7 ltxl3;A57 74.8 5.0 62.1 1.8 t=0.62 t=2.28* c o n t r o l 76.5 3.4 58.7 2.4 t(8)=2.31 t(7)=2.37 * = s i g n i f i c a n t d i f f e r e n c e c o n t r o l = i n t e r n a l c o n t r o l , i e ; l t x v ; L y 27 TABLE 4: continued GENOTYPE MALE t-value FEMALE t-value (mean + 2 S.E.) ltx6;+ 45.8 2.0 35.2 8.2 ltx6;B94 36.3 7.9 31.4 1.3 t=1.92 t=1.4 control 43.6 3.8 30.3 0.7 ltx6;B76 36.2 3.7 35.2 1.0 t=2.67* t=2.27 control 41.3 2.3 32.3 2.8 ltx6;C119 36.6 2.9 33.5 2.0 t=2.42* t=2.09 control 43.6 5.5 39.7 8.6 ltx6;A63 49.8 6.0 34.6 6.0 t=1.01 t=2.06 control 47.4 1.7 30.0 1.3 ltx6;A48 39.9 3.8 24.6 0.8 t=1.14 t=2.64* control 43.1 4.4 21.8 2.9 ltx6;A57 32.6 2.9 27.8 1.8 t=2.33* t=2.41* control 38.2 3.9 24.2 2.5 28 TABLE 4: continued GENOTYPE MALE t-value FEMALE t-value (mean + 2 S.E.) ltx2;+ 33.1 3.4 27.9 0.7 ltx2;B94 29.36.1 24.41.4 t=0.84 t=0.55 control 32.4 5.0 25.0 1.7 ltx2;B76 23.9 2.3 30.1 1.9 t=2.06 t=2.86* c o n t r o l 27.7 2.7 36.7 5.3 ltx2;C119 35.7 2.0 30.3 3.9 t=0.86 t=0.45 c o n t r o l 37.3 3.4 31.6 4.3 ltx2;A63 33.0 2.4 33.1 5.1 t=0.51 t=1.32 c o n t r o l 35.2 7.7 29.9 2.4 ltx2;A48 28.7 1.3 30.8 3.0 t=5.42* t=0.47 c o n t r o l 36.3 3.1 29.8 2.9 ltx2;A57 33.0 4.8 30.1 3.4 t=2.68* t=4.08* control 36.6 4.8 25.3 0.8 29 particular Su(var), these effects are not consistent between trials for Su(var)lis and differences are small (t-values are low) for Su(var)III mutations tested. The variability associated with PEV, as observed in basal It variegating pigment levels, complicates analysis of these data. Several factors may contribute to this variation, including the inherent variability of these strains, the pigment assay system and pipetting error. Linearity tests (see appendix) have shown that higher amounts of pigment approach the end of the linear range of the instrument. Pipetting errors also potentially contribute to the overall error since very small amounts of pigment are used. In this study, standard statistical techniques were applied to determine significant changes from basal It variegating levels due to Su(var) action. However, given the inter-trial variability of control basal It variegating levels, pigment differences between Su(var)II/Itxv flies and control Itxv levels were considered biologically significant i f two criteria were met: 1) statistically significant differences were consistent between trials and 2) the direction of change (i.e. enhancement or suppression) was the same for each t r i a l . For Su(var)III crosses, internal controls were used. This eliminates inter-trial error and minimizes variability due to the assay system. Using this analysis, each Su(var) can be characterized as having an enhancing, suppressing or no effect on It 30 v a r i e g a t i o n . Enhancing e f f e c t s of Su(var)s are summarized i n Table 5. Many Su(var)s are capable of enhancing It v a r i e g a t i o n , but Su(var)s T44 and M43 are the strongest and most general enhancers. Their e f f e c t s can be seen v i s u a l l y and always i n v o l v e 15 or more percentage u n i t drops i n pigment from c o n t r o l values. Su(var)M43 s t r o n g l y enhances a l l I t x v s t e s t e d , e x c l u d i n g ltxl8 males. Su(var)T44 s t r o n g l y enhances a l l ltxvs w i t h the exception of ltxl8 rearrangements. Su(var)B89 al s o has strong enhancing e f f e c t s , but i s more s p e c i f i c , a f f e c t i n g only ltx2 males, ltx24 females and ltx6 males and females. A l l three of these strong enhancers belong to the un c l u s t e r e d group of 2L Su (var) s. Su(var)s H69, A151 and M59 are more moderate enhancers, causing approximately 10 percentage u n i t drops from c o n t r o l pigment l e v e l s . Their e f f e c t s are very s p e c i f i c , u s u a l l y a f f e c t i n g l e s s than h a l f of the v a r i e g a t o r s t e s t e d , (see Table 5) and i n most cases, they have no e f f e c t on It v a r i e g a t i o n . Su(var)s B76, C119, A57 and A48 are a l l l o c a t e d on the t h i r d chromosome and are very weak enhancers of It v a r i e g a t i o n , never causing pigment l e v e l drops of even 10 percentage u n i t s . The moderate I t v a r i e g a t o r s , ltx6" and ltx2 are the most susceptable t o enhancement by Su(var)s, showing reduced pigment w i t h 7 and 9 Su(var)s t e s t e d , r e s p e c t i v e l y . The strong v a r i e g a t i n g rearrangement, ltxl8 i s the l e a s t susceptable, showing l i t t l e r e d u c t i o n i n pigment and no 31 TABLE 5: SU(VAR)S WHICH ENHANCE LIGHT VARIEGATION LIGHT SU(VAR)S WHICH APPROXIMATE VARIEGATING ENHANCE LIGHT PERCENTAGE UNIT STRAIN VARIEGATION DROP IN PIGMENT (r e f e r t o f i g u r e s 3a-f) I t x l S A151 (nonclustered 2L) females only, 10 M43 " females only, 15 M59 ( c l u s t e r e d 2L) females only, 9 ltx6 T44 (nonclustered 2L) 15-25 M43 " 20-25 B89 " 15 H69 ( c l u s t e r e d 2L) females only, 10 B76 (3R) males only, 5 C119 " males only, 7 A57 " males only, 5 ltx2 T44 (nonclustered 2L) 15 A151 " males only, 10 M43 " 20 B89 " males only, 15 H69 ( c l u s t e r e d 2L) 10-15 M59 " 10-15 A48 (3R) males only, 7 A57 " males only, 3 B76 " females only, 6 ltxl3 T44 (nonclustered 2L) 45-50 M43 " 15 A48 (3R) males only, 8 ltx24 T44 (nonclustered 2L) 40 M43 " 20-25 B89 " females only, 15 ltx4 T44 (nonclustered 2L) 40-50 M43 " 20 M59 ( c l u s t e r e d 2L) 10 32 response t o most Su(var)s. The weak v a r i e g a t o r s ltxl3, ltx24 and ltx4 were s t r o n g l y a f f e c t e d by Su(var)s T44 and M43, but unresponsive t o most other Su(var)s. Suppression of I t v a r i e g a t i o n by Su(var)s i s much l e s s frequent. Itxl8 males are moderately suppressed by Su(var)C157. Itxl3 females are moderately suppressed by Su(var)B76. Itx2 females are very weakly suppressed by Su(var)s B7 6 and A57 wi t h pigment increases of aproximately 5 percentage u n i t s over c o n t r o l values. S i m i l a r l y , ltx6 females are very weakly suppressed by Su(var)A48. No c o r r e l a t i o n between suppression by Su(var)s and type of I t x v rearrangements i s apparent. 33 DISCUSSION The results indicate that dominant suppressors of PEV (Su(var)s) are capable of significantly enhancing the effects of heterochromatic variegating rearrangements (Figures 3a-f). Since not a l l Su(var)s are able to enhance It variegating phenotypes, there appear to be functional differences among the Su(var)s tested. A functional difference between clustered and nonclustered mutants on the second and third chromosome has been suggested based on position only (Sinclair, et al., 1983). The strongest enhancers of It variegation are a l l nonclustered Su(var)s located on 2 L . Moderate enhancers of It include one nonclustered 2L Su(var) and two of the clustered 2L suppressors. These groups overlap in their a b i l i t i e s to enhance hPEV. In fact, one of the 2L clustered Su(var)s is capable of suppressing It variegation. Four of the 3R Su(var)s can enhance It variegation, but their effects are extremely weak. Although clear differences in function cannot be attributed to clustered groups, the 2L mutants as a whole have an a b i l i t y to enhance hPEV while 3R Su(var)s are very weak enhancers or show no enhancing effect. Detection of enhancement of It variegation may be dependent upon the strength of the variegator being tested. Su(var)s M43 and T44 are general in their effects, enhancing a l l It variegating rearrangements tested with the exception of ItxlS. It seems likely that their mechanism of action is 34 s i m i l a r f o r a l l It v a r i e g a t o r s , but i n the case of ltxl8, d e t e c t i o n of f u r t h e r i n a c t i v a t i o n through a reduced pigment phenotype i s d i f f i c u l t . Itxl8 i s an extremely strong v a r i e g a t o r : the light locus i s i n c a t i v a t e d i n v i r t u a l l y a l l ommatidia. Therefore, the a d d i t i o n of a Su(var) mutation to an already d r a s t i c a l l y perturbed c e l l may not cause f u r t h e r i n a c t i v a t i o n of t h i s l o c u s . In moderate and weak I t v a r i e g a t o r s , d e t e c t i o n of reduced pigment i s not an i s s u e . I t i s s u r p r i s i n g then, t o see th a t the weak v a r i e g a t o r s , ltxl3, ltx24 and ltx4 are l e s s s u s c e p t i b l e t o enhancement than are moderate v a r i e g a t o r s (ltx6, ltx2) . The a b i l i t y of Su(var)s t o enhance I t v a r i e g a t i o n must al s o be dependent upon the p a r t i c u l a r v a r i e g a t o r present. For example Su(var)M59 enhanced ltx4, ltx2 and ltxl8 v a r i e g a t i o n , but none of the other rearrangements t e s t e d . The p a t t e r n observed i s d i f f i c u l t to a s s o c i a t e w i t h s t r e n g t h of v a r i e g a t i o n which i n t u r n does not c o r r e l a t e w i t h p h y s i c a l ( c y t o l o g i c a l ) d i f f e r e n c e s between It rearrangements (Hessler 1958). D i f f e r e n c e s between v a r i e g a t o r s must be due not t o the r e l a t i v e p o s i t i o n , but the nature of the breakpoints. Perhaps the Su(var)s are a c t i n g i n a sequence dependent f a s h i o n , s p e c i f i c t o each rearrangement. Several p o s s i b l e machanisms may e x p l a i n the enhancing c a p a b i l i t i e s of s p e c i f i c Su(var) mutations. F i r s t , a s i m p l i s t i c model of I t v a r i e g a t i o n would assume a s e r i e s of events r e c i p r o c a l t o euchromatic v a r i e g a t i o n such as wm4. 35 That i s , the It gene is inactivated due to spreading of euchromatin across some boundary, or that heterochromatin is no longer maintained once a break occurs proximal to some boundary. This reciprocal model assumes that the It locus requires heterochromatic packaging for proper expression. The addition of a Su(var) mutation to the variegating strain may perturb the heterochromatic environment further, causing light to be inactivated in a higher proportion of cells. The actual role of a suppressor mutation in this case, is to make variegation more extreme by providing fewer or aberrant elements, structural or enzymatic, necessary for normal packaging, structure and/or maintenance of heterochromatin. A second mechanism for It variegation and related Su(var) activity assumes a transvection-like model (see Lewis 1954), which involves synapse-dependent complementation of alleles. In this model, It expression is dependent upon homolog (locus to locus) pairing. When one It locus is rearranged, i t becomes topologically difficult to pair with its homolog, thus causing variable expression from cell to c e l l . The role of a suppressor gene may be to facilitate proper pairing, via DNA-binding proteins or possibly through indirect (e.g. enzymatic) means. The addition of a Su(var) mutation to the variegating rearrangement would further diminish pairing, thus causing a more extreme light phenotype. The results obtained do not distinguish between these models for enhancement by Su(var)s, but the first 36 ( r e c i p r o c a l ) model i s favored f o r the f o l l o w i n g reason. A t r a n s v e c t i o n model p r e d i c t s t h a t a light rearrangement which i s homozygous (such as ltxl3) should not be a strong v a r i e g a t o r , or perhaps should not v a r i e g a t e at a l l . As shown by b a s a l ltxl3 pigment l e v e l s , t h i s v a r i e g a t o r i s indeed weak. However, i t responds d r a m a t i c a l l y t o s e v e r a l Su(var)s showing s i g n i f i c a n t l y decreased pigment l e v e l s i n the presence of Su(var)s T44 and M43. This i n t e r f e r e n c e by the Su(var) mutation must not be a r e s u l t of homolog p a i r i n g problems, since t o p o l o g i c a l c o n s t r a i n t s are not a f a c t o r f o r t h i s p a r t i c u l a r mutant. Therefore, a more l i k e l y r o l e of Su(var) l o c i i s that of heterochromatic s t r u c t u r e or maintanence. Su(var);It combinations t h a t do not show enhanced v a r i e g a t i o n p o i n t out t h a t many Su(var) products appear unimportant to hPEV. These Su(var) genes may code f o r products which have a more general f u n c t i o n , or f u n c t i o n s s p e c i f i c t o euchromatic PEV. This r e s u l t a l s o suggests t h a t hPEV and euPEV are not simply r e c i p r o c a l events. C e r t a i n l y , v a r i o u s Su(var)s are s p e c i f i c t o euPEV and are not i n v o l v e d i n s t r u c t u r e s or f u n c t i o n s c r u c i a l t o the expression of a d i s p l a c e d heterochromatic gene. Contrary to the models proposed above, f i v e instances of suppression of It v a r i e g a t i o n by Su(vars)s do e x i s t i n t h i s study. None of these cases i n v o l v e pigment increases of more than 13 percentage u n i t s over c o n t r o l values; 3 of 5 cases i n v o l v e <=6 percentage u n i t changes. No Su(var) i s 37 able t o suppress I t v a r i e g a t i o n i n both males and females of one genotype, and i n no case i s the s u p p r e s s i o n d e t e c t a b l e v i s u a l l y . Su(var)B75 both suppresses ( i n females) and enhances ( i n males) ltxl3 v a r i e g a t i o n . Su(var)A57 a l s o has c o n t r a s t i n g e f f e c t s on ltx2 rearrangements. Therefore, s u p p r e s s i o n of I t v a r i e g a t i o n i s a r e l a t i v e l y r a r e , extremely weak and i n c o n s i s t e n t event. I f t h i s s u p p r e s s i o n i s a r e a l phenomenon, these Su(var)s must have a f u n c t i o n much d i f f e r e n t than a l l other Su(var)s t e s t e d here; t h a t i s , o p p o s i t e t o any of the proposed f u n c t i o n s f o r Su(var) genes. In c o n c l u s i o n , i f light e x p r e s s i o n i s dependent upon a heterochromatic environment, i t s e x p r e s s i o n may be d i s r u p t e d i n rearrangements which cannot maintain t h a t environment. By i n t r o d u c i n g a suppressor of PEV, t h a t gene i s expected to s u f f e r , showing enhanced v a r i e g a t i o n due to a more extreme l i m i t on the heterochromatic environment. T h i s e f f e c t i s observed f o r s e v e r a l dominant Su(var)s, s t r o n g l y s u g g e s t i n g t h a t the f u n c t i o n of these genes i s e s s e n t i a l f o r the a p p r o p r i a t e e x p r e s s i o n of heterochromatic l o c i , but i s i n h i b i t o r y f o r euchromatic l o c i . 38 CHAPTER 2 - C h a r a c t e r i z a t i o n of a proximal c l u s t e r of Su(var) mutations on chromosome three. INTRODUCTION The i n a c t i v a t i o n of genes due t o p o s i t i o n e f f e c t v a r i e g a t i o n (PEV) can be a t t r i b u t e d to chromatin changes. I t f o l l o w s t h a t c o n t r o l of gene r e g u l a t i o n at the chromatin l e v e l can be s t u d i e d by determining the mechanisms of PEV. The i n v e s t i g a t i o n of dominant m o d i f i e r s of PEV i s one s t r a t e g y used to study t h i s process. Genetic c h a r a c t e r i z a t i o n of genes i n v o l v e d i n the process w i l l give i n f o r m a t i o n on s p e c i f i c f u n c t i o n s and provide v a l u a b l e i n f o r m a t i o n towards molecular c h a r a c t e r i z a t i o n of these genes. This in f o r m a t i o n w i l l c o n t r i b u t e t o our understanding of gene c o n t r o l at the chromatin l e v e l . Many dominant suppressors of PEV, Su(var)s, have been i s o l a t e d and c h a r a c t e r i z e d to v a r y i n g degrees (Spofford, 1967; Reuter and Wolff 1981; S i n c l a i r et al. 1983; Reuter et al. 1986; Reuter et al. 1987). S i n c l a i r et al. (1983) i s o l a t e d 51 dominant suppressors of PEV, Su(var)s. These mutants were mapped g e n e t i c a l l y and f e l l i n t o c l u s t e r e d and nonclustered groups on chromosomes two and three as described i n the general i n t r o d u c t i o n . The c l u s t e r e d mutants on the l e f t arm of chromosome two (2L) were found to be homozygous l e t h a l , whereas c l u s t e r s on the t h i r d chromosome were i n t i a l l y d escribed as being homozygous v i a b l e . The l a t t e r Su(var)s make up three d i s c r e t e c l u s t e r s (proximal, middle and d i s t a l ) on the r i g h t arm of chromosome three (3R). 39 Second chromosome Su(var)s have been analyzed w i t h respect t o standard m o d i f i e r s of v a r i e g a t i o n (temperature and heterochromatin l o s s ) , as w e l l as butyrate s e n s i t i v i t y and p o s s i b l e maternal e f f e c t s . C l u s t e r e d Su(var)s on the l e f t arm of chromosome two (2L) are i n s e n s i t i v e t o temperature, show l o s s of v i a b i l i t y when t r e a t e d w i t h butyrate (Lloyd 1986) and show no maternal e f f e c t . In c o n t r a s t , non-clustered 2L mutants are temperature s e n s i t i v e , are i n s e n s i t i v e t o butyrate and show a s l i g h t maternal e f f e c t . The 3R d i s t a l c l u s t e r r e a c t s s i m i l a r l y to c l u s t e r e d 2L Su(var)s. A l l Su(var)s t e s t e d are s e n s i t i v e to Y-chromosome (heterochromatin) l o s s ( S i n c l a i r et al. 1983; Harden 1984). F u n c t i o n a l d i f f e r e n c e s between c l u s t e r s and non-clustered Su(var)s have been suggested based on these c h a r a c t e r i s t i c s . Reuter et al. (1986) c h a r a c t e r i z e d 63 independently i s o l a t e d X-ray and ethylmethane s u l f o n a t e (EMS) induced Su(var) mutations. These mutations have been assigned to 12 separate l o c i mapping to chromosome three. These Su(var)s show va r i o u s degrees of homozygous v i a b i l i t y and f e r t i l i t y , i n a d d i t i o n t o butyrate and heterochromatin s e n s i t i v i t y s p e c i f i c t o each of the 12 l o c i . In f a c t , Reuter et al. (1986) suggest t h a t c l u s t e r s of homozygous v i a b l e Su(var)s on chromosome three reported by S i n c l a i r et al. (1983) are a l l e l i c t o s i m i l a r l o c i reported i n Reuter et al. (1986): S u - v a r ( 3 ) l , Su-var(3)2 and Su-var(3)9. 40 This study w i l l examine the p r e v i o u s l y uncharacterized 3R proximal c l u s t e r of Su(var) mutations. I t w i l l i n c l u d e nine Su(var) mutations o r i g i n a l l y assigned t o t h i s c l u s t e r , mapping between 4 6.4 and 54.2 and some of which may be a l l e l i c to S u - v a r ( 3 ) l and/or Su-var(3)2. These dominant mutations have been p h y s i c a l l y mapped using new compound chromosome formation and d e f i c i e n c i e s . The s e n s i t i v i t y t o l o s s of heterochromatin and maternal e f f e c t s has been determined, and homozygous v i a b i l i t y and f e r t i l i t y have been e s t a b l i s h e d . C h a r a c t e r i z a t i o n using the above c r i t e r i a has b e t t e r d e f i n e d t h i s c l u s t e r i n t o genetic l o c i and has provided i n f o r m a t i o n t o f u r t h e r i n v e s t i g a t e the f u n c t i o n s of these c l u s t e r e d Su(var)s. 41 MATERIALS AND METHODS  Stocks All mutations have been previously described with the exception of the following. Deficiencies used to map Su(var)s and their breakpoints are listed in Table 6, followed by a cytological map of the 3R proximal region (see Figure 4). Df(3R)e-078 was provided by Dr. Reuter and cytologically analyzed in our lab. No deletion loops were detected, so this mutation is either a very small deletion, or a point mutation (A. Dutta). C(3L)ri;C(3R)es is a compound chromosome strain obtained from Dr. Holm, used to map Su(var)s with respect to the centromere. Df(2R)M-S210 (Lindsley and Grell 1968; Hilliker and Holm, 1975) is deficient for 2R heterochromatin and was used to test heterochromatic sensitivity of the Su(var)s. Culture Conditions As descrobed in Chapter 1. Mapping the 3R proximal cluster Su(var)s Attempts to map 3R proximal Su(var)s involved two methods. i) To map the Su(var)s into discrete regions of the chromosome, deficiencies were chosen to cover the region 42 TABLE 6: DEFICIENCIES USED TO MAP 3R PROXIMAL SU(VAR)S DEFICIENCY CYTOLOGY REFERENCE 1. DfOLJPC^ Df(3L)78A3; 79E1,2 DIS 65, p.47 2. Df (3R) Dfd+Rxl3,pP Df(3R)83E3; 84A4,5 Hazelrigg and Kaufman (1983) 3. Df(3R)Scxw + R x 2, rede Df(3R)84A4,5; 84C1,2 II 4. Df(3R)Scxw + R x 4,rede Df (3'R) 84B1,2; 84D1,2 II 5. Df (3R)Hu+Rxl Df(3R)84Bl;2 + 84D5;84F4 II 6. al. Df (3R) p30,rede Df(3R)84F4-6; 85D3-5 Kemphues et (1983) 7. Df (3R) by62, rede Df(3R)85D11-14; 85F6 + Dp of Ant-C + more on II Y 8. Df (3R)E-075 •Df (3R) 86E20; 87B8,9 Reuter et al. (1987) 9. Df(3R)E-078 *point mutation Ashish Dutta 10. Df (3R) 125c Df(3R)87El; F12,13 Reuter et al. (1987) 11. Df(3R)kars z 1 1 Df(3R)87C7/8; 87E5,6 II ^originally isolated as a deficiency, this mutant was given to our lab through Dr. Speirer; cytology was done in our lab by Ashish Dutta Gupta. FIGURE 4; CYTOLOGY OF 3R PROXIMAL REGION INCLUDING DEFICIENCIES USED TO LOCALIZE SU(VAR) MUTATIONS. 47.0 466 6O0 AMAI *WA»|**4tU»lllh*»U 82*83 2. Df (3R) DfdfRxl^ipp 4. Df (3R) Scxv+Rx4, rede 5. Df {ZK)Hu+Rxl 84185 6. Df QK) p30, rede 7. Df OK) by62, rede 3. Df {3K)Scxw+Rx2,rede WWWl Mi !Miffr1l»l)I^ E7f/f0fl(ltttligi t H!!MH M l AMI AMMNIUI B C MA HI II D HAIAMIAIIII A> E Al IAAM F 86 A 87 1. tlAMA 1 1 1 III B IM'IIHUAHIHIIII iLlUMA* C [ P | E MtliMlMU F 8 7 Wi"l>a A 8 8 MIMIILMIIII » m m B 1 C 8. Df(3R)£-079 11. Df (3R) kar*z**> 10. Df(3R)126c 9. Df(3R)E-078 " ' 44 proximal to the centromere on 3R since genetic mapping l o c a l i z e d them t o t h i s area. Most d e f i c i e n c i e s used were rebalanced w i t h TM3,e,Ser to f a c i l l i t a t e s c o r i n g . D e f i c i e n c y bearing males were f i r s t outcrossed t o wm4 v i r g i n s t o check f o r suppressing a b i l i t y which would suggest a s t r a i n d e f i c i e n t f o r a suppresssor lo c u s . Pigment assays were performed as described p r e v i o u s l y (see M a t e r i a l s and Methods, Chapter 1) on wm4;Df f l i e s and t h e i r wm4;TM3 s i b s . D e f i c i e n c i e s acquired l a t e r from v a r i o u s sources were not t e s t e d by t h i s method. Instead, the a f f e c t of the Df on wm4 was scored v i s u a l l y i n balancer s i b l i n g s r e s u l t i n g from crosses described below. V i r g i n Su(var)s were mated t o Df/TM3 males and t r a n s f e r r e d to new food every 2-3 days. Several crosses were done at 22, 25 and 29 C, but remaining crosses were c u l t u r e d at 25 C since no e f f e c t of temperature was observed. Complementation was scored by counting Df/Su(var) f l i e s r e l a t i v e t o t h e i r Su(var)/balancer s i b l i n g s . In most cases, a minimum of 100 f l i e s were scored where 1/3 were expected to be the d i a g n o s t i c c l a s s . In non-complementation cases, the crosses were repeated so tha t no l e s s than 100 f l i e s were scored. i i ) A compound chromosome three s t r a i n , C ( 3 L ) r i ; C ( 3 R ) e s was used t o map Su(var)s r e l a t i v e t o the centromere of chromosome three. Two Su(var)s, B143 and A63, were mapped wi t h t h i s method. These mutants were chosen based on t h e i r 45 genetic map positions as being most likely to define the outermost positions in the cluster. A summary of the protocol and strategy is given in Figure 5. wm4; Su(var)/TM3 virgin females were collected every four hours and subjected to 2500 rads of gamma irradiation. Approximately 3300 treated virgins of each Su(var) genotype were mated after 3 days to compound males. Adults were transferred to new food three times and then discarded. Theoretically, only mutational events resulting in new compound chromosome formation and nondysjuction events w i l l contribute to viable F l progeny. Almost a l l other events wi l l result in genetically unbalanced, and therefore inviable embryos. Since the compound stock did not have a wm4 rearrangement, only males show presence or absence of a suppressor directly. New compound females were tested for presence or absence of the Su(var) mutation by backcrossing to patroclinous males as shown in Figure 6. Of the r i progeny recovered, one half w i l l show a wm4 phenotype i f the suppressor is not present. Effect of heterochromatin deficiencies on Su(var) activity Two tests were used to determine what effects alteration in the amount of genomic heterochromatin may have on the Su(var) phenotype. 46 FIGURE 5: STRATEGY USED TO MAP SU(VAR)S USING NEW COMPOUND FORMATION 2500 rads gamma radiation wm4;Su(var)III TM3, SbrSer x + ;C (3L) ri;C (3R) es wm4;C(3l>) ;C (3R) new compound formation NO COMPOUND COMPOUND COMPOUND NONDYSJUNCTION RIGHT LEFT Su(var)/TM3 or 0 \ 3 L r i > / 3 L r i3 L \ / 3 R 3 L / \ 3 R / 3 R es ( \ 3 R 3 L \ / 3 R 3 L / \ 3 R N O N D Y S J U N C T I O N : M A T R O C L I N O U S A N D P A T R O C L I N O U S P R O G E N Y 3 L \ / 3 R , 3 L\/ 3 R 3 L / \ 3 R 3 L / 1 3 R 0 3 L \ / 3 L 3 R / \ 3 R *patroclinous progeny, r i , es phenotype like male parent **matroclinous progeny, Su(var)/TM3 phenotype like female parent 47 FIGURE 6: PROTOCOL TO DETERMINE PRESENCE OR ABSENCE OF SU(VAR) IN NEWLY FORMED COMPOUND FEMALES, HETEROZYGOUS FOR WM4 wm4;C(3L)ri;*C(3R)Su? x wn?4;C(3L) ri;C(3R)es + Y new compound patroclinous virgin female male EXPECTED PROGENY CLASSES PHENOTYPE: r i only esr ri **wm4; C(3L)ri;C(3R)Su wm4;C (3L) ri;C (3R) es +;C(3L)ri;C(3R)Su? + ;C (3L) ri;C (3R) es *This newly formed compound chromosome possibly carries a Su(var) mutation, masked by wild type allele for white. **If the Su(var) is present on C(3R), a l l of these progeny will be red-eyed, due to suppression of wm4. If some wm4 progeny are found, Su(var) must be on 3L. 48 i ) Loss of Y chromosome: By using a s t r a i n w i t h the attached-X chromosome, C(l)RM,pn, crosses were performed to produce males c o n t a i n i n g no Y chromosome (see Figure 7). These males were compared to males which d i d r e c e i v e a Y chromosome from an attached-X background. Both genotypes were subjected to pigment a n a l y s i s as described p r e v i o u s l y . S i b l i n g s i n h e r i t i n g the balancer homolog serve as i n t e r n a l c o n t r o l s . i i ) Loss of 2R heterochromatin: Df(2R) MS-210 i s d e f i c i e n t f o r 2R heterochromatin only. The e f f e c t of removing t h i s s p e c i f i c segment of heterochromatin on the expression of the Su(var) phenotype was examined using the p r o t o c o l shown i n Figure 8. F l i e s were r a i s e d at 25 C t o ensure accurate s c o r i n g of the Curly wing phenotype. Maternal E f f e c t s To check f o r maternal e f f e c t s i n each of the mutants, r e c i p r i c a l crosses were made as shown i n f i g u r e 9. Temperature s e n s i t i v i t y was measured by c a r r y i n g out the crosses at 18, 22 and 29 C. Su(var) and balancer s i b l i n g s from maternal and p a t e r n a l crosses r a i s e d at 22 and 29 C were compared using pigment assays as before. Crosses r a i s e d at 18 C were scored v i s u a l l y . FIGURE 7: PROTOCOL USED TO DETERMINE SENSITIVITY OF SU(VAR)S TO LOSS OF THE Y CHROMOSOME EXPERIMENTAL: A *XX + 0 + x wm4 Su(var) 0 and wm4 Su(var) Y TM3,SJb, Ser wm4 0 TM3,SbrSer pigment assay CONTROL: A XX + Y + x **wm4 Su(var) ; and wm4 Su(var) Y TM3,Sjb,Ser wm4 Y TM3,Sb,Ser pigment assay **ST0CK CONSTRUCTION: X (Oregon-R) A XX x 0 A XX A XY A XY x 0 A XX — STOCK *^X=C (1) RM 50 FIGURE 8: PROTOCOL USED TO DETERMINE SENSITIVITY OF SU(VAR)S TO THE LOSS OF 2R CENTRIC HETEROCHROMATIN. EXPERIMENTAL CROSS: wm4 Df (2R) M-S210 + wm4 + Su (var) * • __ • __ • __________________ wm4 CyO + | Y + TM3, 'Sb,Ser wm4 Df (2R) M-S210 Su (var) • —___—_——_______—_____ • ____________ 9 9 . d y + " + wm4 CyO Su(var) pigment assay + CONTROL: wm4 CyO + wm4 + + « ^ • \T « « wm4 Df (2R) M-S210 + Y + + 99 * o"o" w m 4 D f ( 2 R ) M - 5 2 1 ° + pigment assay + FIGURE 9: RECIPROCOL CROSSES USED TO DETERMINE MATERNAL EFFECTS FOR 3R SU(VAR)S PATERNAL CROSS: wm4 + wm4 Su(var) x wm4 + Y TM3,S2>, Ser wm4 Su (var) and + wm4 TM3,Sb,Ser ; pigment + assay MATERNAL CROSS: wm4 Su (var) x wm4 TM3, Sb, Ser wm4 + Y + as above pigment assay 52 Viability and fert i l i t y studies/complementation tests The first step in characterizing the cluster was to construct marked stocks of each Su(var). This was accomplished by first constructing a reliable balanced mapping strain, wm4; GI Sb H/1M3,e,Ser and a strain with a dominant marker Lyre and an appropriate balancer as shown in Figures 10a and b. The cluster falls approximately 6 map units from Glued on the left and 8 map units from Stubble to its right. These strains were then used to obtain recombinant Su(var) strains with one chromosome arm marked by a dominant mutation as shown in Figure 11. Several stocks for each Su(var) were established and are listed in Table 7. These marked Su(var) strains (M-Su(var)) were used to test for homozygous viability and f e r t i l i t y . Each M-Su(var) was crossed back to its original Su(var)/TM3,Sb,Ser stock. Strains were labeled homozygous lethal i f no progeny resulted from the backcross. Semi-lethality was assumed i f less that 25% of expected progeny resulted from the backcross. Surviving homozygotes resulting from this cross were allowed to mate with strains known to be fertile. If no larvae resulted, homozygous sterility was assumed. Once homozygous phenotypes had been established, inter se complementation tests were done. Since it is not likely 53 FIGURE 10a: PROTOCOL FOR CONSTRUCTING A MULTIPLY MARKED STRAIN WITH TM3 BALANCER LACKING STUBBLE + GI, Sb, H Y In (3L) Payne wm4 Ly x wm4 TmSbSer wm4 GI Sb H Ly x wm4 Ly + M(3)w x -; wm4 TM3S£>Ser Y TM3eSer wm4 Ly + TM3 e Ser wm4 GI Sb H TM3 e Ser STOCK FIGURE 10b: PROTOCOL FOR CONSTRUCTION OF DOMINANTLY MARKED TM3 BALANCED STRAIN LACKING STUBBLE wm4 GI Sb H wm4 Ly x wm4 TM3 e Ser Y TM3 Sb Ser wm4 Ly ; cfc? * QQ TM3 e Ser 54 FIGURE 11: PROTOCOL FOR CONSTRUCTING MARKED STOCKS OF SU(VAR) MUTATIONS wm4 Su(var) Y ' T M 3 Sb Ser wm4 + Su (var) + wm4 GI Sb H x wm4 GI Sb x • i wm4 + Su(var) + TM3 e Ser wm4 + + Sb TM3 e Ser wm4 + Su (var) Sb ' T M 3 e Ser wm4 TM3 e Ser wm4 Ly Y TM3 e Ser wm4 GI Sb TM3 e Ser wm4 GI Su(var) + TM3 e Ser wm4 GI + + / TM3 e Ser p a r e n t a l c r o s s over between GI and Su(var) c r o s s over between Sb and Su(var) TO ESTABLISH MARKED STOCKS: wm4 + Su(var) Sb TM3 e Ser x wm4 Ly T M 3 e Ser wm4 + Su(var) Sb ; MARKED STOCK TM3 e Ser 55 TABLE 7: MARKED STOCKS ESTABLISHED FOR SU(VAR)III MUTATIONS M - Su (var)A63; C - A63, Sb M - Su (var)B76: A - B76, Sb M - Su (var)A57: M - Su (var)B94, M - Su (var) C119: M Su (var)B143i M - Su (var) A48, A -B -D -E -F -A -B -B -D -B -E -A -C -A -C -B -C -F -A -B -E -B -C -D -E -GI, A63 GI, A63 GI, A63 GI, A63 GI, A63 A57, Sb A57, Sb GI, A57 B94, Sb GI, B94 GI, B94 Cll9, Sb Cll9, Sb GI, C119 GI, Cll9 B143, Sb B143, Sb B143, Sb GI, B143 GI, B143 GI, B143 A48, Sb A48, Sb A48, Sb A48, Sb M - Su(var)A130 M - Su (var)A160 A C D E F - GI, B76 - GI, B76 - GI, B76 - GI, B76 - GI, B76 A130 GI Sb A130 GI + A130 + Sb C - A160 Sb D - A160 Sb A - GI, B - GI, C - GI, A48 A48 A48 56 t h a t second s i t e l e t h a l s would map t o the same p o s i t i o n s i n d i f f e r e n t Su(var)s, o r i g i n a l Su(var)/TM3 stocks were used. F l i e s were mated i n each p a i r w i s e combination. A minimum of 150 f l i e s were scored f o r each c r o s s . F a i l u r e to complement i s seen i f l e s s than 10% expected progeny eclose and su r v i v e , or i f transheterozygotes show i n f e r t i l i t y . S t a t i s t i c s S t a t i s t i c a l t e s t s between two mean values were c a r r i e d out using an unpaired t - t e s t (Zar 1984). D i f f e r e n c e s between 3 or more means were t e s t e d using a n a l y s i s of variance (ANOVA) followed by the Neuman-Keuls m u l t i p l e range t e s t (Zar 1984). Values expressed as percentages were f i r s t transformed to t h e i r a r c s i n e value, which converts b i n o m i a l l y d i s t r i b u t e d data t o values c l o s e l y approximating a normal d e s t r i b u t i o n , before s t a t i s t i c a l t e s t s were done. Values are expressed as mean +/- standard e r r o r . P<0.05 was taken as the l i m i t of s i g n i f i c a n c e . RESULTS D e f i c i e n c y mapping D e f i c i e n c i e s capable of suppressing wm4 v a r i e g a t i o n are c h a r a c t e r i z e d by comparing wm4;Df/+ and wm4;balancer/+ s i b l i n g s . Out of f i v e d e f i c i e n c i e s chosen f o r t h e i r p o s i t i o n c l o s e t o the centromere on 3R only one, D f ( 3 R ) S e x 2 + R x 2 , r e d e shows a pigment l e v e l c l o s e t o t h a t of a suppressor (see Table 8). However, i t s balancer s i b l i n g s shows the same amount of suppression. The only case where the e f f e c t s of a d e f i c i e n c y and i t s balancer are s i g n i f i c a n t l y d i f f e r e n t i s Df(3R)Dfd + r x ^ 3. i n t h i s case, the defeciency s i b l i n g has s i g n i f i c a n t l y l e s s pigment than i t s balancer s i b , suggesting that Df(3R)Dfd may delete a locus which enhances PEV, an En(var) l o c u s . D e f i c i e n c i e s E-079, E-078, k a r s z l l and 126c show suppression according t o Reuter et a l . (1987), making these d e f i c i e n c i e s good candidates f o r mapping Su(var)s. A l l other d e f i c i e n c i e s t e s t e d showed no suppressing a b i l i t y when observed i n Df/balancer s i b l i n g s of Df/Su(var) f l i e s . Df/Su(var) heterozygotes show no abnormal eye phenotype. Two d e f i c i e n c i e s f a i l e d t o complement w i t h any of the Su(var)s (see Table 9). Su(var)s A57 and A63 are l e t h a l i n the presence of Df(3R)E-079. These Su(var)s l i k e l y map t o the region spanned by t h i s d e f i c i e n c y . Df(3R)£-078, which 58 TABLE 8: EFFECTS OF DEFICIENCIES IN THE 3R PROXIMAL REGION ON WM4 VARIEGATION DEFICIENCY % OREGON-R PIGMENT SIGNIFICANT GENOTYPE + S.D. DIFFERENCE, (wm4 background) p<.05 c o n t r o l : wm4/Y; + / + 7.9 +1.6 D f ( 3 R ) S c x w + R x 4 , r e d e / + 1.7 + 0.9 TM3/+ 0.0 no Df(3R)Dfd + R x l 3,pP/+ 9.0 + 1.9 TM3/+ 19.4 + 3.9 yes D f ( 3 R ) S c x w + R x 2 , r e d e / + 4 8.1 + 5.3 TM3/+ • 43.7 + 5.7 no Df (3R)#u + i* x l 1.3 + 1.3 TM3/+ 2.4 + 0.7 no Df (3R) p30,rede/ + TM3/ + 4.8 + 2.1 4.1 + 1.3 no 59 TABLE 9: RESULTS OF COMPLEMENTATION ANALYSIS WITH DEFICIENCIES IN THE 3R PROXIMAL REGION AND 3R SU(VAR) MUTATIONS DEFICIENCY* Su(var) 1 2 3 4. 5 6 7 8 9 10 11 B143 + + + + + + + + + + + A48 + + + + + + + + + + + B94 + + + + + + + + + + + C119 + + + + + + + + + + + B76 + + + + + + + + • + + + A57 + + + + + + + - - + + A63 + + + + + + + - + + .+ A160 ND ND ND ND ND ND + + + ND + A130 ND ND ND + ND + + + + ND ND ^numbers 1-11 correspond t o d e f i c i e n c i e s l i s t e d i n Table 6 +=Df/Su(var) progeny v i a b l e and f e r t i l e -=Df/Su(var) progeny completely l e t h a l ND=not done 60 i s a c t u a l l y a p o i n t mutation f a i l s to complement w i t h Su (var) A57, but not A63, suggesting that these two Su (var) s are separable l o c i . Since these d e f i c i e n c i e s p o s s i b l y f a i l e d t o complement w i t h a second s i t e l e t h a l , crosses were repeated w i t h marked stocks of Su(var)s A57 and A63. I d e n t i c a l r e s u l t s were found, suggesting t h a t Su(var)s A63 and A57 a c t u a l l y map i n the region of 8 6E-87B. Compound Mapping New compound chromosome formation was undertaken t o assign l o c a t i o n s f o r Su(var)A53 and B143 t o e i t h e r s ide of the centromere. F l i e s w i t h e i t h e r the es or r i phenotype, but not both, i n d i c a t e d a new compound formation. Since the marked compound stock d i d not c a r r y a wm4 rearrangement, the presence or absence of a suppressor could be seen d i r e c t l y only i n the males. Su(var)s t e s t e d were e f f e c t i v e l y homozygous l e t h a l ( p o s s i b l y due t o second s i t e l e t h a l s ) so the occurance of a wm4 male wi t h a new compound must be used i n d i r e c t l y to assign the Su(var) t o 3L or 3R. Su(var)B143 crosses y e i l d e d 5 new compound formations (see Table 10) i n c l u d i n g one r i male which was immediately i n f o r m a t i v e . Since the male was wm4; C(3R);C(3L)ri and shows a wm4 phenotype, Su(var)B143 i s not on 3R. A female of the same phenotype was progeny t e s t e d and found t o be Su(var)+, confirming t h a t Su (var) B14.3 maps to 3L (see Table 11). Three e s females recovered had the genotype wm4/+; 61 TABLE 10: SUMMARY OF NEW COMPOUND PROGENY RECOVERED FROM GAMMA RADIATION SCREEN SU(VAR) GENOTYPE OF NEW COMPOUND PROGENY LOCATION SUBJECTED TO GAMMA TREATMENT C(3L);C(3R)es C(3L)ri;C(3R) 4 males, wm4 phenotype Su(var)A63 no progeny 3R TM3 Sb Ser 5 females, wild type eye non-virgins 1 male, wm4 phenotype Su(var)B143 3 females, :— s t e r i l e 3L TM3 Sb Ser 1 female, wild type eye *progeny tested *progeny test results shown i n Table 11 62 TABLE 11: RESULTS OF PROGENY TESTING FOR NEW COMPOUND FEMALES HETEROZYGOUS FOR WM4; TEST FOR PRESENCE OR ABSENCE OF SU(VAR)B143. GENETIC CROSS: wm4;C(3L)ri;C(3R) x wm4;C (3D r i ; C (3R) e s + Y (patroc l i n o u s male) RESULTING PROGENY PHENOTYPE: r i only ri, e k wm4;C (3L) r i ; C (3R) wm4;C(3L) ri;C(3R) es 3 wm4 males 2 wm4 females 4 wm4 males 5 wm4 females +;C(3L)ri;C(3R) + ;C (3L) ri;C(3R) e s 6 males 3 females 4 males 3 females I f Su(var) i s present on C(3R), a l l r i only progeny w i l l have red eyes. I f some r i only progeny are present w i t h wm4 eyes, the Su(var) must not be present. Since wm4, ri progeny r e s u l t from the cross, Su(var)B143 i s not present on C(3R) and must be on 3L. C(3L)B143;C(3R)es. They were a l l s t e r i l e , which i s expected based on spontaneous B143 homozygotes observed i n stock. Nine new compound formations were recovered from Su(var)A6"3 crosses (see Table 10). A l l males were of the genotype wm4;C(3L)/C(3R)es. Since they were a l l white mottled, Su(var)A53 can be assigned t o the r i g h t arm of chromosome three. Females recovered were not v i r g i n s , so progeny t e s t i n g was not done. E f f e c t of heterochromatin l o s s on Su(var) a c t i v i t y i ) C(l)RM,pn/0 females were crossed to Su(var)/TM3 males to produce male progeny d e f i c i e n t f o r the Y-chromosome. Pigment l e v e l s f o r these f l i e s are shown i n Table 12. These X/O; Su(var) male f l i e s are compared to X/Y; Su(var) f l i e s from a cross which c o n t r o l s f o r any e f f e c t s of C(l)RM,pn background. The l o s s of a Y chromosome has a dramatic and s i g n i f i c a n t e f f e c t on 8 of 9 Su(var)s t e s t e d . Su(var)A130 i s the only s t r a i n which shows no enhancement of v a r i e g a t i o n due t o l o s s of the Y chromosome. I t i s a l s o one of the mutants found to map outside the c l u s t e r (see appendix). However, the other non-clustered suppressor t e s t e d , Su(var)A2 60 shows a l a r g e decrease i n pigment, almost down to wm4 l e v e l s . A l l other Su(var)s are s t r o n g l y a f f e c t e d by l o s s of a Y-chromosome. These r e s u l t s are c o n s i s t e n t w i t h TABLE 12: EFFECTS OF LOSS OF Y CHROMOSOME ON SU(VAR) MUTATIONS OF CHROMOSOME 3 X/O; SU(VAR) X/Y; SU(VAR) t-value values are % of Oregon-R pigment levels wm4/0 2.5 ± 0.3 4.6 + 1.1 4.9 wm4;Su(var) B76 69.0 ± 7.8 105.1 + 3.2 11.4 A48 12.0 ± 1.8 71.0 + 6.3 21.2 B94 20.9 ± 5.0 89.9 + 7.9 11.7 C119 13.7 ± 0.5 83.2 + 9.5 .31.1 A63 5.1 ± 0.2 49.4 + 12.1 32.5 A57 14.1 ± 1.6 58.9 + 10.4 13.7 B143 16.7 ± 2.4 60.3 + 5.2 15. 9 A130 103.3 ±2.3 103.8 + 2.9 0.3 A160 14.4 ± 3.0 74.8 13.8 10.8 Values given are mean + S.D.; critical t--value, df(8)=2.31 65 those reported by S i n c l a i r et al.(1983) and Harden (1984) for a l l Su(var)s studied, excepting Su(var)A130. i i ) Df(2R)M-S210 i s known to enhance variegation (Morgan et al. 1941) and i s def i c i e n t for centromeric heterochromatin on the right arm of chromosme two ( H i l l i k e r and Holm 1975). Although t h i s deficiency i s also Minute i n phenotype, t h i s mutation has been shown to have no s i g n i f i c a n t e f f e c t on variegation of wm4 (Harden 1984). F l i e s were raised at 25 C and t h i s i s r e f l e c t e d by the r e l a t i v e l y high wm4 control values shown i n Table 13. Df(2R) MS-210 effects wm4 variegation at t h i s temperature, causing an enhanced phenotype or reduction i n pigment l e v e l s . In addition, Df(2R) MS-210 s i g n i f i c a n t l y reduces pigment i n 8 of 9 suppressors tested. The enhancing c a b a b i l i t y of Df(2R)MS-210 i s much weaker than that caused by loss of the Y-chromosome, but i s consistent throughout. Again, only Su(var)A130 i s completely unaffected by loss of heterochromatin. In general, both males and females are affected by Df(2R) MS-210, but females appear more susceptable to the heterochromatin l o s s . Where both sexes are influenced, females invariably show a larger difference in comparing Minute individuals to t h e i r CyO-balancer s i b l i n g s . The exception i s Su(var)B76", where only males are s i g n i f i c a n t l y d i f f e r e n t than t h e i r CyO balancer s i b s . These results are consistent with the findings of Reuter et al. (1983) which show the suppressors on 66 TABLE 13: EFFECTS OF LOSS OF 2R HETEROCHROMATIN ON SU(VAR) MUTATIONS ON CHROMOSOME 3 Df(2R) M-5210;Su(var) CyO;Su(var) t-value values are % Oregon-R pigment + S.D. wm4; + male 9 . 6 + 0.5 17.4 + 1.7 t (8) =12.3 female 9.9 + 1.0 40.3 + 2.7 t (8) =23.7 B76 male 69.4 + 4.5 89.9 + 3.8 ' t (8) = 6.6 female 72.5 + 6.8 83.0 + 6.8 t (7) =2.0* C119 male 56.9 + 4.1 70.1 + 3.4 t (8) =5.0 female 50.3 + 1.4 71.6 + 4.6 t (8) =10.8 A63 male 61.8 + 5.5 91.1 + 6.1 t (8) = 6.5 female 58.8 + 4.0 85.8 + 4.7 t (8) = 8.6 B143 male 67.0 7.8 81.3 6.6 t (8) =3.2 female 41.7 + 5.1 76.7 + 3.5 t (8) =11.8 A48 male 58.6 + 10.0 85.3 ± 8.9 t (8) =4.1 female 41.1 + 4.2 81.1 ± 6 . 6 t (8) =10.1 A57 male 70.7 + 4.4 108.3 ± 10.0 t (8) = 9.0 female 54.7 + 6.4 96.1 ± 6.6 t (8) =8.2 B94 male 59.4 + 7.4 94.6 ± 8.9 t (8) =5.4 female 45.9 + 5.3 88.1 ± 2.1 t (8) =16.3 A160 male 52.1 + 5.8 90.2 ± 5.7 t (8) =7.9 female 36.9 + 2.9 80.2 ± 9.5 t (8) =10.6 A130 male 69.6 + 1.5 73.9 + 4.8 t (8) = 1.5* female 70.5 3.6 72.1 ± 3.6 t (8 =0.6* t-values are given with degrees of freedom i n (); c r i t i c a l t -value, df(8)=2.31; df(7)=2.37 * i n s i g n i f i c a n t difference between Df(2R)M-S210 and Cyo progeny. 67 chromosome 2 and 3 are affected by removal of 2R heterochromatin. However, the distal cluster Su(var)s on chromosome 3 have been tested (Harden, 1984) and show no enhancement by this same deficiency. Maternal effects To determine whether the Su(var) mutations had any maternal effect on variegation, reciprocal crosses were made. The variegating non-Su(var) offspring from each cross were examined for the amount of eye pigment. Pre-zygotic expression of maternal RNAs may be detected as a suppressed phenotype in wra4;+/TM3 (Su+) progeny of Su(var) female parents. To test for temperature sensitivity of the product, reciprocal crosses were done at 18, 22 and 29 C. Results of the crosses are presented in Tables 14 and 15. Control wm4 variegating levels are normal for 22 and surprisingly higher at 29 C. Females are more susceptable to suppression caused by high temperature in this wm4 strain (also observed by Harden, 1984). Although wm4;+/TM3 levels are often lower than control values, any influence of the TM3 balancer does not interfere with comparison of maternal and paternal progeny, since both carry the balancer. Su(var)/+ progeny have pigment values within the normal ranges observed. At 18 C, visual observations suggested no differences between maternal and paternal crosses (data not shown). 68 TABLE 14: MATERNAL EFFECTS MEASURED AT 22 C GENOTYPE SEX PATERNAL CROSS MATERNAL CROSS t-value (wm4 background) values are % Oregon-R pigment + S.D. B143/+ male 54.9 + 5.4 43.4 + 5.1 t (8) =3.1* female 32.1 + 7.9 47.9 + 6.4 t (8) =3.1* TM3/+ male 19.8 + 3.3 7.6 + 1.7 t (7) = 6.6* female 5.8 + 1.7 13.0 + 3.3 t (8) =4.2* B76/ + male 75.5 + 9.0 82.6 + 4.4 t (8) =1.5 female 75.6 + 4.4 70.7 + 10.1 t (7) =0.9 TM3/ + male 4.4 + 1.5 9.2 + 1.6 t (8) =4.5* female 3.5 + 2.4 7.9 + 2.5 t (7) =2.4* A57/ + male 69.1 + 4.9 36.8 + 2.9 t (8) =12.3* female 54.1 + 9.5 52.5 + 5.1 t (8) =0.4 TM3/ + male 20.5 + 5.0 8.8 + 3.0 t (8) =3.8* female 15.8 + 5.6 14.8 + 3.3 t (7) = 0.2 A48/ + male 76.3 + 3.1 43.8 + 6.1 t (8) =10.2* female 54.8 + 6.8 56.9 + 2.5 t (8) = 0.7 TM3/ + male 15.7 + 1.4 5.3 + 1.8 t (5) = 6.9* female 9.3 + 3.4 17.4 + 2.7 t (8) =3.9* B94/ + male 75.1 + 2.9 55.6 + 2.8 t (8) =5.9* female 57.3 + 5.8 68.1 + 4.7 t (8) =3.1* TM3/ + male 15.8 + 4.7 7.8 + 5.9 t (8) =0.9 female 6.7 + 3.8 37.7 + 6.0 t (8) = 8.6* A63/+ male 71.4 + 9.1 50.9 + 5.4 t (8) =4.3* female 52.5 + 7.7 64.8 + 3.2 t (8) =3.4* TM3/ + male 10.9 + 2.4 11.7 + 3.7 t (8) =1.2 female 5.1 + 2.6 15.2 + 2.4 t (8) =5.8* C119/+ male 65.6 + 5.2 67.5 + 2.4 t (8) =0.8 female 50.7 + 5.2 67.3 + 3.1 t (8) =5.6* TM3/ + male 15.6 + 3.2 14.8 + 1.2 t (8) =0.5 female 5.7 + 1.1 27 .2 + 5.9 t (8) =10.4* A130/+ male 79.4 + 1.2 65.4 + 6.6 t (8) =7.0* female 72.1 + 7.6 71.8 + 8.3 t (8) =0.0 TM3/ + male 9.4 + 0.9 5.4 + 1.5 t (8) =4.8* female 3.3 + 1.4 14.0 ± 2.0 t (8) =8.6* A160/ + male 66.7 + 5.8 62.7 ± 3.7 t (8 =1.2 female 50.8 + 5.7 58.9 ± 8.0 t (8 =1.7 TM3/ + male 18.1 + 3.3 8.4 ± 2.1 t (8 >=5.0* female 12.4 + 3.4 10.9 + 3.0 t (8 1=3.0 wm4 control values: males, 7.0 + 2.2; females, 9.2 + 2.3. t-values are given with degrees of freedom in (); c r i t i c a l t-values: df(8)=2.31, df(7)=2.37, df(5) = *significant difference between maternal and paternal progeny 69 TABLE 15: MATERNAL EFFECTS MEASURED AT 29 C GENOTYPE SEX PATERNAL CROSS MATERNAL CROSS t-value (,wm4 background) values are % Oregon--R pigment + S. D. B143/+ male 50.5 + 4.8 47.8 + 3.2 t (8) =0.9 female 58.7 + 4.5 56.1 + 6.2 t (8) =0.7 TM3/ + male 19.7 + 6.3 13.2 + 4.7 t (8) =1.6 female 20.5 + 3.6 34.1 + 9.9 t (8) =3.2* B76/ + male 82.2 + 4.4 93.0 + 3.5 t (8) =3.5* female 91.4 + 6.2 96.0 + 4.7 t (8) =1.3 TM3/ + male 9.7 + 1.4 17.3 + 3.5 t(8) =4.9* female 32.4 + 4.2 55.0 + 5.9 t (8) =5.7* A57/ + male female see Table 17 TM3/ + male female A48/ + male 59.0 + 7.8 63. 9 + 6.2 t (8) =5.4* female 73.4 + 5.8 85.4 + 6.1 t (8) =2.2 TM3/ + male 16.0 + 4.6 16.3 + 2.7 t (8) =0.2 female 24.6 + 2.5 41.1 + 6.1 t (8) =5.6* B94/ + male 60.7 + 1.9 78.8 + 2.0 t (8) =13.4 female 67.4 + 6.2 95.0 + 1.6 t (6) = 9.2* TM3/ + male 19.6 + 1.7 35.8 + 6.1 t (7) =7.0 female 26.0 + 6.5 71.8 + 6.9 t (8) = 9.0* A63/ + male 79.9 + 5.0 94.8 + 4.6 t (8) =4.4* female 91.3 + 5.2 102.2 + 1.2 t (8) = 6.9* TM3/ + male 25.0 + 3.1 28.4 + 6.6 t (8) = 1.0 female 34.9 + 5.4 67.3 + 4.6 t (7) =8.3* C119/+ male 89.5 + 4.8 80.2 + 10.6 t (8) =1.8 female 100.0 + 2.0 97.5 + 1.2 t (8) =3.5* TM3/ + male 29.7 + 6.8 35.6 + 5.8 t (8) =1.3 female 36.5 + 5.4 62.0 + 7.4 t (8) =5.7* A130/+ male 77.2 + 4.6 84.6 + 4.3 t (7) =2.1 female 80.7 + 6.0 86.4 + 3.7. t (8) =1.6 TM3/ + male 14.5 + 2.4 15.4 + 2.3 t (8) =0.6 female 21.5 + 5.5 47.9 + 6.4 t (8) = 6.2* Al60/+ male 59.3 + 5.1 36.9 + 2.2 t (8) = 9.2* female 78.4 + 10.6 70.8 + 3.9 t (8) = 1.9 TM3/ + male 32.0 + 9.1 14.7 + 4.8 t (8) =3.7* female 53.0 + 4.4 36.0 + 7.2 t (8) =4.2* wm4 control values: males, 12.0 + 3.4; females, 46.6 + 4.9. t-values are given with degrees of freedom i n (); c r i t i c a l t -values: df(8)=2.31, df(7)=2.37, df(6)=2.45 * s i g n i f i c a n t difference between maternal and paternal progeny 70 Flies raised at 22 C show sexual dimorphisms among wm4; TM3/+ (Su+) progeny (see Table 16 for summary). Differences between maternally and paternally derived progeny are small, but s t a t i s t i c a l l y significant. Seven of nine maternally derived female wm4;+/TM3 progeny show higher pigment levels than their paternally derived counterparts. This is recognized as a maternal effect. The maternal effect is observed only in the female progeny except for the case of Su(var)B76", in which both males and females show a maternal effect. Su(var)s B94 and C119 are most sensitive to maternal effects, with maternally derived +/TM3 females having pigment levels greater than 20 percentage units over paternally derived females. Su(var)Al50 and A57 females are insensitive to maternal effects at this temperature. Males show a different trend. Five out of nine Su(var)s show wm4;+/TM3 males from paternal crosses with pigment levels significangly higher than analogous maternally derived males. The suppression seen in these Su(var)+ males is labelled a paternal effect. The paternal effects observed are weaker than maternal effects seen in females; Su+ females show relatively high pigment levels compared to Su+ males and females of paternal crosses, as well as to wm4 control values at this temperature. Su(var)B34, A63 and C119 males are not affected by paternal factors. An exception, Su(var)S76" crosses show Su(var) + paternally derived males with significantly lower pigment 71 TABLE 16: SUMMARY OF MATERNAL AND PATERNAL EFFECTS OF P7M4;TM3 PROGENY OF SU (VAR) PARENTS AT 22 AND 29 C SU(VAR) 22 C 29 C PARENT MALE FEMALE MALE FEMALE A57 PA NE SEE FIGURE 17 A48 PA MA NE MA* B94 NE MA* MA* MA* A63 NE MA NE MA* C119 NE MA* NE MA* B143 PA MA NE MA B76 MA MA MA MA* A130 PA MA NE MA* A160 PA NE PA PA* PA=paternal effect, MA=maternal effect, NE=no effect. *difference between maternal and paternal progeny was greater than 15 percentage units. 72 levels than their maternal analogs. In this case, both males and females exhibit a maternal effect. At 29 C, 8 of 9 Su(var) crosses have females which demonstrate a maternal effect. The strongest effects with respect to pigmentation are observed in Su(var)s A130, B94, A63 and C119, with pigment differences greater than 25 percentage units between maternally and paternally derived Su+ females. Maternal effects are noticebly stronger at 29 C, often with pigment levels approximately 10 percentage units over 22 C levels. The most interesting case is that of Su(var)A57. I n i t i a l l y , this suppressor was labeled temperature sensitive maternal lethal since no maternally derived progeny were recovered at 29 C after two attempts. The crosses were repeated with egg lays at 22 and 29 C, but rearring at 29 C. Special care was taken to avoid dessication and overcrowding. Maternally derived progeny can survive, but at much lower frequencies than paternally derived progeny (see Table 17). Survival is lowest for progeny maintained at 2 9 from oogenesis through pupation. A strong maternal effect is observed in females, but only when oogenesis takes place at 2 9 C. These females appear completely suppressed. (Flies were scored visually since pigment assays require a minimum of 25 f l i e s per genotype). Su(var)A2 60 is an exception to the female specific maternal effect. Both males and females in this reciprocal cross exhibit a paternal effect. This is f a i r l y consistent 73 TABLE 1.7: MATERNAL EFFECTS OF SU (VAR) A57 AT DIFFERENT DEVELOPMENTAL TEMPERATURES DEVELOPMENTAL PATERNAL MATERNAL TEMPERATURE TM3/+ A57/ + TM3/+ A57/ + MALE FEMALE MALE FEMALE MALE FEMALE MALE FEMALE GROUP I (4 v i a l s ) no. of progeny: 30+ 30+ 30+ 30+ 3 3 3 10 phenotype: wm4 suppressed wm4 suppressed GROUP II (8 v i a l s ) no. of progeny: 30+ 30+ 30+ 30+ 4 5 4 9 30+ 30+ 30+ 30+ 3 6 0 16 phenotype: wm4 suppressed wm4 suppressed suppressed (80%) GROUP III (4 v i a l s ) no. of progeny: 30+ 30+ 30+ 30+ 0 2 1 1 phenotype: wm4 suppressed supressed suppressed (80%) GROUP I: Oogenesis @ 22 C, food @ 22 C, developmental temp. 29 C GROUP II : Oogenesis @ 29 C, food @ 22 C, developemntal temp. 29 C GROUP III:Oogenesis through pupation, 29 C. Phenotypes were scored v i s u a l l y , estimated suppression given i n 0 . 74 with observations from 22 C where males showed a paternal effect and females showed no effect. Males tend to show no susceptability to parental effects at 2 9 C. Exceptions in addition to Su (var) Al 60 include Su (var) B76", where both males and females show maternal effects as at 22 C; and Su(var)B94 where males show a maternal effect. Homozygous viability/complementation analysis Premliminary experiments suggested that many of the third chromosome proximal cluster of Su(var)s were homozygous viable. The lethality observed was likely due to second site lethal mutations. However, no homozygous lines were established, suggesting that homozygotes while viable, may have been very weak or s t e r i l e . To determine homozygous v i a b i l i t y and f e r t i l i t y , marked stocks were constructed with the intent of crossing off any second site lethals. In the process of establishing recombinant lines, the Su(var) mutations were remapped. It became apparent that two of the Su(var)s were wrongly assigned to the cluster. Su(var)A130 and A160 map to the left of Glued and Stubble, that is in a more distal location on 3L (see appendix). These mutants were characterized along with the proximal cluster, and used as a comparison for nonclustered verses clustered Su(var)s. These marked stocks (refer to Table 7) were crossed to original Su(var)/TM3,Sb,Ser stocks to look for homozygous viable and f e r t i l e progeny. Results are given in Table 18. TABLE 18: HOMOZYGOUS PHENOTYPES OF PROXIMAL 3R SU(VAR)S SU(VAR) *LETHAL SEMI-LETHAL VIABLE STERILE A63 X A48 X B143 X B76 X C119 X A57 X B94 X A160 A130 X X *Lethality, semi-lethality and sterility are defined in Materials and Methods, p.52. **one recombinant strain, B - GI, A57, showed 3 female survivors out of 194 A57/TM3 siblings. These females were fertile. 76 /Among the 9 Su(var)s tested, five remained homozygous lethal, even with both chromosome arms crossed off. Of the three homozygous viable Su(var)s, only one, Su(var)B76" is f e r t i l e . This Su(var) strain is maintained as a homozygous stock. Su(var)J3143 is semi-lethal. Few f l i e s survive (20% of expected based on Su(var)/TM3 siblings) and these are s t e r i l e . This is consistent with the observation that homozygotes which appear within this TM3-balanced stock are sterile (personal observations). Su(var)A230 is completely viable when homozygous, but is s t e r i l e . Su(var)A57 is homozygous lethal, except for one strain, B-Gl A57. Surviving f l i e s are f e r t i l e . Since many Su(var)s exhibit phenotypes such as homozygous or hemizygous lethality or s t e r i l i t y , inter se complementation tests were done. Matings were set up in every pairwise combination. Results are summarized in Table 19. Su(var)s making up the 3L complementation group show a spread-wing phenotype as trans-heterozygotes, along with complete s t e r i l i t y . This wing phenotype has been reported for Su(var) homozygotes mapping to this region, characterized by Reuter et al. (1986) and for 2L Su(var) trans-heterozygotes (personal communication, Jo-Ann Brock). TABLE 19: COMPLEMENTATION ANALYSIS MUTATIONS OF 3R PROXIMAL SU(VAR) SU (VAR) STRAIN A63 A48 B143 B76 C119 A57 B94 A160 A130 A63 + + + + + + + + A48 -- FS,W +' FS, W + FS,W + + B143 + FS,W + FS,W + + B76 — + + + + + C119 — + FS,W + + A57 — + + + B94 — + + A160 + A130 — +=full complementation, FS=female s t e r i l e , W=spreadwing phenotype in male trans-heterozygotes. 78 DISCUSSION The 3R proximal c l u s t e r of Su(var) mutations maps between 4 6.4 + 1.1 and 54.4 + 0.7 map u n i t s . Since the centromere i s assigned a map p o s i t i o n of 4 6.0 (Lindsley and G r e l l 1968) i t was p o s s i b l e t h a t these mutations spanned the centromere. Mapping by compound autosome formation has s p l i t t h i s g e n e t i c c l u s t e r i n t o at l e a s t two l o c i w i t h one on 3L and the other on 3R. D e f i c i e n c y mapping extends the c l u s t e r d i s t a l l y , out t o 87B. Su(var)A63 f a i l s to complement wi t h Df(3R)E-079, but complements Df(3)E-078 while Su(var)A57 i s l e t h a l w i t h both d e f i c i e n c i e s . As mentioned p r e v i o u s l y , according t o cytogenetic a n a l y s i s c a r r i e d out i n t h i s l a b , Df(3)E-078 i s a c t u a l l y a p o i n t mutation. Therefore, Su(var)A6"3 and Su(var)A57 probably represent two separate l o c i . Their p o s i t i o n s correspond roughly t o those of Su-var(3)13 and Su-var(3)6 i s o l a t e d and mapped by Reuter et al. (1986, 1987). Su-var(3) 6 and 13 are r e c e s s i v e l e t h a l s as are both Su(var)A6\3 and A57, s t r o n g l y suggesting these four suppressors are a l l e l i c p a i r s . This could be proved w i t h complementation t e s t s between these Su(var) a l l e l e s . Such t e s t s would a l s o confirm t h a t l e t h a l i t y i s not due to second s i t e mutations, s i n c e these would not l i k e l y map to i d e n t i c l e l o c i among these mutants. Loss of the Y-chromosome s i g n i f i c a n t l y reduces pigment i n a l l Su(var)s p r e v i o u s l y t e s t e d ( S i n c l a i r et al. 1983/ 79 Harden 1984). In eight of nine Su(var)s tested i n t h i s study, variegation was enhanced dramatically by Y-chromosome loss and was s i m i l a r i l y and s i g n i f i c a n t l y affected by deficiency of 2R heterochromatin. This i s i n contrast to the behaviour of d i s t a l l y clustered Su(var) l o c i which show no s e n s i t i v i t y to 2R heterocromatin loss (Harden 1984). Reuter et al. (1983) reported a t h i r d chromosome suppressor, Su(var)c1001, which was greatly affected by Df(2R) MS-210. Su(var)clOOl maps to 46.7, within the 3R proximal c l u s t e r , suggesting a l l e l i s m to one or more of the Su(var) genes i n t h i s study. Again, complementation tests could determine t h e i r r e l a t i o n s h i p s . Su(var)A230 i s a surprising exception to the high heterochromatin s e n s i t i v i t y exhibited by most Su(var)s. This suppressor i s not affected by loss of Y chromosome or 2R heterochromatin. It i s a non-clustered suppressor which maps near the t i p of 3L. This i n s e n s i t i v i t y to heterochromatin suggests a functional difference between Su(var)A130 and other Su(var) genes. It has been hypothesized that loss of the heterochromatic Y chromosome frees heterochromatic elements which are then available to inactivate genes through p o s i t i o n e f f e c t variegation i n a higher proportion of c e l l s , thus enhancing variegation (Zuckerkandl 1974). Su(var)A130 may be so strong that no amount of free heterochromatic elements can make up for the deficiency of Su(var)A230 product. A l t e r n a t e l y , Su(var)A130 may produce a non-structural product involved i n the control 80 of gene inactivation in PEV. For example, this product may act to maintain structural decisions. If early on, the white gene was packaged as heterochromatin, but no signal (suppressor product) was available to maintain this facultative packaging, the white gene inactivation may be reversed and that decision clonally maintained, resulting in a suppressed phenotype. This type of developmental model could be investigated i f temperature sensitive phenotypes were established. Reciprocal crosses among the Su(var)s revealed that some (including Su(var)A230) have female-specific, temperature sensitive maternal effects. These suppressors must act early in development, before zygotic transcription begins, and produce a protein product, subject to heat denaturation (inactivation). By doing shifts from permissive to restrictive temperatures, developmental activity of the Su(var)s can be determined. It has already been determined that Su(var)A57 acts extremely early, during oogenesis. Eggs la i d at 22, but shifted to 29 C after 24 hours do not show the maternal suppression or the lethality observed in this strain when raised at constant (2 9 C) temperature. Paternal and maternal effects could also be due to some sort of chromosome imprinting. Chromosomes from paternal or maternal Su(var) parents could be pre-programmed by being in this genetic background, and therefore exhibit a Su(var) phenotype even i f they are genotypically Su(var)+. 81 Genetic characterization is crucial to understanding the functions of suppressor l o c i . By establishing homozygous v i a b i l i t y , lethality and s t e r i l i t y , i t becomes possible to determine the genetic makeup of the cluster. Homozygous v i a b i l i t y tests show that most of the Su(var)s mapping to the cluster are homozygous lethal or sterile as marked stocks. Only Su(var)B75 is homozygous viable and f e r t i l e . The possibility exists that second site lethals may remain on those portions of chromosome not replaced by recombination. However, i t seems relatively unlikely that a l l five homozygous lethal stocks carry a second site lethal mutation between the 2 markers Glued and Stubble. This region makes up an absolute maximum of 15% of the chromosome, i f the recombination breakpoints were located exactly proximal to either marker. Furthermore, two Su(var) lo c i are homozygous viable or semi-lethal, but s t e r i l e . This s t e r i l i t y must be separate from the lethality observed in the original balanced stocks. The assumption of second site lethals has been discussed by Nash et al. (1983). Their concern is that EMS mutagenesis is quite effective at inducing "many" extraneous lethal mutations which may mask a haplo-specific lethal mutation by exhibiting homozygous lethality not due to the mutation in question. They define a locus as being haplo-specific lethal i f i t is lethal over a deficiency , but not lethal as a homozygote. Two of the 3R Su(var) l o c i have demonstrated lethality over Df(3R)E-079, one of which shows 82 semi-lethality in one recombinant strain, B - G1,A57. If this semi-lethality is due to crossing off a second lethal, or even haplo-specific lethal, i t may represent a locus such as Nash et a l . (1983) suggest. Although this strain is not completely viable, survivors are f e r t i l e , suggesting that in this case, an extraneous lethal was involved in the lethal phenotype observed. This possibility should be of concern to investigators of dominant recessive lethal mutations. Inter se complementation tests suggest that 2 or more complementation groups make up the 3L-3R proximal cluster (see Figure 12). Su(var)s B143, A48, B94 and C119 are s t e r i l e as trans-heterozygotes in a l l inter se combinations. Since Su{var)B143 maps to 3L, a l l 4 alleles of this locus can be tentatively assigned to this location. Reuter et a l . (1986) identified a locus, Su-var(3)3, which maps genetically and cytologically (via new compound formation) near the centromere on 3L. They report that mutants assigned to this locus are homozygous semi-lethal and s t e r i l e , with females producing no eggs. Males show a spread wing phenotype. This description matches the phenotypes observed in B143 homozygotes exactly, strongly suggesting allelism to Reuter's Su-var (3) 3. Su(var) trans-heterozygotes in the complementation group observed in this study are completely female sterile with no eggs produced. They are also semi-lethal in males, which show a spread-wing phenotype. However, Su(var)C119, A48 and B94 do not show the same homozygous phenotypes as 83 FIGURE 12: Complementation groups based on trans-heterozygous phenotypes and physical mapping. recombination units 0 10 20 30 40 46 50 60 100 x 61 GI 80 86-87 Sb cytological map A160 A130 — Xf-B143 A48 B94 C l l 9 B76 A57 A63 x=centromere 84 Su(var)B143. When studying dominant mutations, i t is always possible that trans-heterozygous phenotypes are a result of interactions between separate l o c i . In fact, this has been observed for Su(var)s in the 2L group (personal communication, Jo-Ann Brock). However, the observed behavior is best attributed to i n t e r - a l l e l i c failure to complement for the following reasons. F i r s t , a l l four mutations in question have genetic map positions with overlapping 95% confidence levels, strongly suggesting one locus. Second, no interaction phenotypes were observed for any other Su(var)s tested, four of which are physically separated from this complementation group: Su(var)A5 7 and A63 by cytogenetic analysis and Su(var)A130 and Al60 by genetic mapping. This question could be further addressed by mapping the Su(var) l o c i in question by compound-autosome formation. This could be done by capturing the newly formed compound with a compound strain that carries a wm4 rearrangement (see appendix). A result showing 3L locations would strongly support the hypothesis that these Su(var) lo c i are a l l e l i c . Of the remaining Su(var) mutants, only one is a non-essential locus, Su (var) B76". It is homozygous viable and f e r t i l e and shows no interactions with the clustered or non-clustered Su (var) s tested. Su(var)B76" does, however, react to heterochromatin loss, just as the essential l o c i do, suggesting a similar function. 8 5 Locke et al. (1988) have hypothesized that a l l dominant modifiers of PEV are dosage sensitive and f a l l into one of two categories. Class I modifiers are h a p l o - i n s u f f i c i e n t , suppressing when hemizygous over a deficiency and enhancing when t r i p l o i d for the locus. Class II modifiers are rare and have the opposite e f f e c t s to dosage, suppressing when t r i p l o i d and enhancing over a def i c i e n c y . They suggest the Su(var) mutations reported by Reuter et al. (1981) and S i n c l a i r et al. (1983) are class I modifiers and therefore hypomorphic or amorphic l o c i . This p o s s i b i l i t y has not been disproven, since deficiency studies were uninformative i n the most proximal 3R region. Neither i s i t possible to d i s t i n g u i s h between the suppressors as h a p l o - i n s u f f i c i e n t l o c i or antimorphs. Both c l a s s i f i c a t i o n s f i t the data presented i n t h i s t h e s i s . Hypomorphic mutations are more plausible based on the dosage s e n s i t i v i t y reported by Reuter et al. (1987) and Locke et al. (1988). Probable functions for these l o c i have been suggested by S i n c l a i r et al. (1983) and others (Reuter et al. 1981, 1986; Locke et al. 1988). Based on phenotypes of suppression, recessive l e t h a l i t y , s t e r i l i t y and wing phenotypes, Su(var)s l i k e l y control chromatin condensation or contribute s t r u c t u r a l l y to formation and or maintenance of heterochromatin. This hypothesized function i s supported by recent work of James and E l g i n (198 6). They have i s o l a t e d a non-histone chromosomal protein s p e c i f i c to heterochromatin. Through in situ hybridization, they have 86 mapped i t s cDNA to a locus very close to or the same as Su(var)M43, a suppressor locus isolated by Sinclair et al. (1983) . 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I n v e s t i g a t i o n s on the c o n s t i t u t i o n of the germinal m a t e r i a l i n r e l a t i o n t o h e r e d i t y . Carnegie I n s t . Yearbook 40, 282-287. Mottus, R., Reeves, R. and T. G r i g l i a t t i (1980). butyrate suppression of p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila melanogaster. Mol. Gen. Genet. 178: 4 65-469. M u l l e r , H.J. (1930). Types of v i s i b l e v a r i a t i o n s induced by X-rays i n Drosophila. J . Genetics 22: 299-334. Nash, D. and C. Janca (1983). Hypomorphic l e t h a l mutations and t h e i r i m p l i c a t i o n s f o r the i n t e r p r e t a t i o n of l e t h a l complementation s t u d i e s i n Droshophila. Genetics 105, No4: 957-968. 90 Reuter, G. and I. Wolff (1981). Isolation of dominant suppressor mutations for position-effect variegation in Drosophila melanogaster. Mol. Gen. Genet. 182: 516-519. Reuter, G., Dorn, R., Wustmann, G., Friede, B. and G. Rauh (1986) . 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Zhimulev, I., Belyaeva, E., Fomina, 0. Protopopov, M. and V. Bolshakov (1986). Cytogenetic and molecular aspects of position-effect variegation in Drosophila melanogaster. Chromosoma (Berl) 94: 492-504. Zuckerkandl, J. (1974). A possible role of "inert" heterochromatin in c e l l differentiation. Action and competition for "locking" molecules. Biochimie 46; 937-954. 92 A P P E N D I X A. LINEARITY CURVE To determine i f the microfluoremeter is reading pigment amounts in a linear fashion, the following protocol was used: Pigment from Oregon-R heads was extracted as described in Materials and Methods, Chapter 1. Dilutions were made to represent 1, 2, 3, 4 and 5 heads. For example, 2 ul supernatent + 8 ul 2-mercaptoethanol = 20% of Oregon-R pigment or the equivalent of 1 head (1/5=20%). Fluorescence of each sample was measured and resulting pigment levels are plotted in Figures A-1,2 and 3. B. LTX2/SU(VAR)II DATA TO ACCOMPANY GRAPHS IN FIGURES 3A-F. Pigment values are given in Tables Ba-f. Values are mean + standard deviations. Asterisks indicate values s t a t i s t i c a l l y different from basal levels for each variegator tested, specific to each t r i a l . C. RECOMBINATION DATA FOR SU(VAR)S A169 AND A130 See Figure C. D. COMPOUND WM4 STRAIN Patroclinous males and virgin females were recovered from the compound autosome mapping study reported in Chapter 2 that made i t possible to construct a compound 3 strain carrying wm4: wml;C (3L) ri;C (3R) es x wwA;C (3L) r i ; C (3R) es Y + i wm4;C(3L)ri;C(3R)es progeny T h i s i s a v a l u a b l e stock s i n c e the presence o f a Su(var) can be de t e c t e d i n a l l v i a b l e new compound formations r e s u l t i n g from a screen such as the one used i n t h i s study. % Oregon-R pigment % Oregon-R pigment % Oregon-R pigment gi o ? 8 8 S S 8 3 8 8 8 S g p g 8 $ 8 8 3 8 S 8 S o o § 8 £ 8 8 S 8 8 8 S g > * i Q < l — I — I — I — I — I — I — I — I — I — I — 1 — l _ _ J 1 1 1 1 1 1 1 1 1 1 1 1 7__7 7 7 7 7 7 7 , , t% 21 O c — m • 1 1 1 1 1 1 1 1 1 1 1 ft* 1 ' • V 1          1 X . —1—1—1—1 1 1 1 1—1—1—1— _! \/ \ • < Q O • f 3 C 3 10 cr a> ac o> a CL ± 09 TJ CD -1 r+- 01 + C cr O 3 ta o =1 I TJ CO 3 0) £ C O 5" a> o a CO 94 TABLE B.a: LTX18/SU(VAR) PIGMENT VALUES SU(VAR) TRIAL I TRIAL II MALE 26.5 + 1.4 *35.5 + 2.9 T44 FEMALE *21.0 + 1.3 *34.0 + 3.6, MALE 21.7 + 3.1 34.6 +.1.3 A151 FEMALE *20.6 + 2.1 *33.5 + 1.3 MALE *19.1 + 2.1 27.3 + 1.9 M43 FEMALE *16.0 + 1.4 *26.8 +1.3 MALE 28.9 + 0.6 N.D. B89 FEMALE 29.2 + 1.8 N.D. MALE 27.3 + 2.2 *37.9 + 3.9 A24 FEMALE *24.5 + 2.8 *40.6 + 3.6 MALE *38.7 + 1.2 *40.5 + 4.8 C157 FEMALE 35.0 + 4.4 *34.5 + 1.3 MALE *22.1 + 3.9 N.D. H69 FEMALE *24.7 + 0.6 N.D. MALE 28.9 + 4.9 • *37.4 + 2.9 M5 9 FEMALE *22.5 + 3.5 *32.9 + 0.6 95 TABLE B.b: LTX6/SU(VAR) PIGMENT VALUES SU(VAR) TRIAL I TRIAL I I MALE *33.0 + 1.2 *30.5 + 1.8 T44 FEMALE *31.7 + 0.5 *31.6 + 2 . 0 MALE 56.3 + 6.3 *45.4 + 1.4 A151 FEMALE *35.9 + 3.4 42.4 + 2.2 MALE *29 . 8 + 1.6 *34 . 3 + 2.5 M43 FEMALE *26.0 + 0.7 *26 . 9 + 1.6 MALE *48.4 + 2 . 3 *40.8 + 3.5 B89 FEMALE *32.1 + 1.8 *32.5 + 1.7 MALE 63.9 + 3.5 52.1 + 8.3 A24 FEMALE *41.0 + 2.2 43.1 ± 5.4 MALE *52.4 + 8.2 56 . 9 + 6.2 C157 FEMALE *35.5 ±2.4 45.2 + 5.4 MALE *51.3 + 6.7 46.2 + 10.5 H69 FEMALE *32.7 + 0.7 *37.1 + 2.3 MALE *47.0 + 0.9 48.1 + 7.5 M59 FEMALE *34 .4 ± 2.6 *36.3 + 3.2 9 6 TABLE B.c: LTX2/SU(VAR) PIGMENT VALUES SU(VAR) TRIAL I TRIAL II M A L E * 3 6 . 1 ± 6 . 3 * 2 8 . 2 + 1 . 4 T 4 4 F E M A L E * 3 6 . 5 ± 2 . 1 * 3 1 . 8 ± 1 . 2 . M A L E * 2 8 . 5 ± 1 . 7 * 3 8 . 0 ± 3 . 2 A 1 5 1 F E M A L E * 3 5 . 0 ± 2 . 3 3 9 . 8 ± 4 . 2 M A L E * 3 2 . 5 ± 2 . 6 * 2 5 . 8 ± 2 . 4 M 4 3 F E M A L E *31.0 ± 1 . 2 * 2 1 . 9 + 0 . 8 M A L E * 2 9 . 1 ± 1 . 8 * 3 4 . 1 ± 6 . 9 B 8 9 F E M A L E * 2 8 . 6 ± 1 . 4 * 3 4 . 9 ± 3 . 7 M A L E * 4 1 . 7 ± 3 . 2 4 9 . 3 ± 6 . 2 A 2 4 F E M A L E * 3 7 . 0 ± 1 . 5 4 4 . 8 ± 2 . 4 M A L E 5 7 . 8 ± 4 . 2 5 0 . 6 ± 6 . 2 C 1 5 7 F E M A L E * 5 1 . 0 ± 2 . 2 4 1 . 9 ± 3 . 1 M A L E * 4 3 . 2 ± 3 . 0 . * 3 5 . 6 ± 4 . 7 H 6 9 F E M A L E * 4 3 . 5 + 2 . 4 * 2 8 . 6 ± 2 . 0 M A L E * 3 5 . 7 + 2 . 6 N.D. M 5 9 F E M A L E * 3 7 . 1 + 2 . 5 * 3 1 . 4 ± 1 . 4 97 TABLE B.d: LTX13/SU(VAR) PIGMENT VALUES SU (VAR) TRIAL I TRIAL I I MALE *40.8 + 2.4 *35.0 + 0.8 T44 FEMALE *32.4 + 0.9 *39.8 + 2.9 MALE 84.8 + 5.0 70.9 + 5.9 A151 FEMALE 65.0 + 0.8 68.5 + 3.1 MALE *64.3 + 3.6 *65.7 + 7.2 M43 FEMALE *47.3 + 1.2 *53.0 + 3.5 MALE 84.5 + 4.7 86.7 + 4.9 B89 FEMALE 68.4 + 3.0 *81.2 + 4.0 MALE 101.3 + 2.1 94.6 + 1.5 A24 FEMALE *77.0 + 3.4 *93.7 + 2.3 MALE *74.9 + 6.2 73.8 + 9.0 C157 FEMALE 64.9 + 4.7 72. 6 + 3.4 MALE *93.9 + 3.0 91.8 + 2.3 H69 FEMALE 67.6 + 2.8 *82.5 + 3.9 MALE 80.7 + 2.7 *67.0 + 7.0 M59 FEMALE 68.6 + 0.7 *61.7 + 6.8 98 TABLE B.e: LTX24/SU(VAR) PIGMENT VALUES SU(VAR) TRIAL I TRIAL II MALE *54.3 ± 2.4 *36.7 + 3.4 T44 FEMALE *40.2 + 2.3 *45.8 ± 2.4 MALE 94.9 ± 7.8 *65.2 + 5.5 A151 FEMALE 68.3 ± 5.8 *60.1 + 4.7 MALE *77.9 ± 7.2 *67.0 ± 6.8 M43 FEMALE *48.8 ± 5.1 *54.9 + 6.7 MALE 96.1 ± 4.1 85.1 ± 8.8 B89 FEMALE 66.7 ± 2.3 *71.9 + 3.6 MALE 113.4 ± 9.1 *97.6 + 1.3 A24 FEMALE 81.6 ± 3.1 93.7 + 2.3 MALE 85.4 ± 1.9 N.D. C157 FEMALE 71.7 ± 0.9 N.D. MALE 110.3 ± 4.4 96.1 + 3.1 H69 FEMALE 69.5 + 3.4 *79.5 ± 7.1 MALE 83. 9 + 3.1 83.9 + 2.9 M59 FEMALE 68.0 + 3.1 72.8 ±4.1 99 TABLE B.f: LTX4/SU(VAR) PIGMENT VALUES SU(VAR) TRIAL I TRIAL II MALE *71.5 ± 13.0 *35.8 + 2.3 T44 FEMALE *38.9 ± 1.2 *40.6 + 3.0 MALE 106.0 ± 4.5 *68.5 + 4.9 A151 FEMALE *79.5 + 8.1 68.6 + 4.8 MALE N.D. *43.4 + 4.9 M43 FEMALE *59.7 + 2.2 *49.8 + 3.7 MALE 99.7 + 2.8 *83.2 + 5.8 B89 FEMALE 82.7 + 5.2 *72.2 + 9.2 MALE 117.8 + 3.2 92.5 + 1.4 A24 FEMALE 91.6 + 5.1 88.7 + 3.4 MALE *89.2 + 2.4 83.1 + 6.0 C157 FEMALE *72.5 + 1.5 75.6 + 3.6 MALE 109.6 ± 8.8 *83.5 + 5.4 H69 FEMALE 81.0 ± 6.6 *80.3 + 5.1 MALE *90.9 ± 6.8 *67.5 + 6.2 M59 FEMALE *76.4 + 3.9 *64.0 + 3.3 FIGURE C: RECOMBINATION DATA FOR SU (VAR)S A160 AND A130 CROSS: wm4 Su wm4 Ly ; x wm4 GI Sb (H) Y TM3 e Ser OFFSPRING A130 A160 PARENTAL: GI Sb H Su + + 270 916 SCO I: Su GI Sb (H) + + + (+) 39 401 SCO II: + GI + (+) Su + Sb (H) 41 154 DCO: + + Sb (H) Su GI + ( + ) 47 TOTAL 351 1,518 order: Su GI Sb H I II A130: <—11.4 >< 12.0 > aproximate A160: <—29.5 >< 13.2 > distances 

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