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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. H e d r i c k B.Sc,  P o r t l a n d S t a t e 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 in THE FACULTY OF GRADUATE STUDIES (Zoology)  We a c c e p t 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. H e d r i c k , 198 9  In  presenting this  degree freely  at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  available for reference and study. I further  copying  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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  DE-6 (2/88)  15 April 1989  ii  ABSTRACT  The mosiac, cell-autonomous expression of genes r e s u l t i n g from chromosomal rearrangement and relocation next to broken heterochromatin i s termed position e f f e c t variegation (PEV).  Since the gene i s inactivated due to  chromatin changes, t h i s system allows the genetic study of chromatin structure and function using mutations which rescue the mosaic phenotype.  These mutations c a l l e d  suppressors of variegation, Su(var)s, must influence chromatin structure.  The genetic characterization of  several groups of Su(var)s has been undertaken i n t h i s study using Drosophila  melanogaster.  Variegation of the light  gene, located i n  heterochromatin, i s enhanced by several Su(var) mutations on chromosome two.  This opposite e f f e c t suggests that products  of these Su(var)s are essential f o r functioning heterochromatin and deleterious f o r euchromatic environments.  Other Su(var)s have s l i g h t or no e f f e c t s 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 i n t e r se complementation based on newly established  iii 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, i n combination with mapping data, suggests that four seperate l o c i make up t h i s group of Su(var)s. Eight of nine Su(var)s are extremely sensitive to heterochromatic deletions as shown by t h e i r responses to loss of 2R heterochromatin, as well as the Y chromosome. In contrast, Su(var)A130 i s insensitive to both forms of heterochromatic d e f i c i e n c i e s .  Su(var)s show complicated  reactions to maternal verses paternal source e f f e c t s . Six of nine Su(var)s show a female-specific sensitive maternal e f f e c t .  temperature  Some maternal and paternal  e f f e c t s are observed at 22 C. l e t h a l and suppressed at 29 C.  Su(var)A57  i s maternal semi-  This characterization has  better defined these mutants, making them ammenable to molecular study.  iv  TABLE OF CONTENTS Page  Abstract  i i  Table of Contents  iv  L i s t of Tables  vi  L i s t of Figures. .  .viii  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  E f f e c t on Su(var)s on light  variegation  ...9  Fluorometric assay of eye pigment  13  Statistics  15  Results.  16  Discussion Chapter 2 - Characterization of a proximal c l u s t e r of Su(var) mutations on chromosome three  33 38  Introduction  38  Materials and Methods  41  Stocks  41  Culture Conditions  41  Mapping the 3R proximal c l u s t e r Su(var)s  41  Effect of heterochromatin d e f i c i e n c i e s on Su (var) a c t i v i t y  45  i)  Loss of Y chromosome  48  V  ii)  Loss of 2R heterochromatin  Maternal e f f e c t s  48 48  V i a b i l i t y and f e r t i l i t y studies/ complementations tests Statistics. Results.  52 .56 57  Deficiency mapping  57  Compound mapping  60  Effect of heterochromatin loss on Su(var) a c t i v i t y  63  Maternal e f f e c t s . . . . .  67  Homozygous v i a b i l i t y / complementation analysis  74  Discussion  78  Bibiliography  87  Appendix  92  vi  LIST  OF TABLES Page  Table 1: Suppressor o f p o s t i o n e f f e c t mutations used i n t h i s study Table 2: D e s c r i p t i o n rearrangements  o f light  variegation 10  variegating 11  Table 3: B a s a l light v a r i e g a t i n g c o n t r o l pigment l e v e l s from t h r e e seperate t r i a l s Table 4:  E f f e c t s o f S u ( v a r ) I I I mutations  on t h r e e light Table 5:  17  variegating  strains  Su(var)s which enhance light  26 variegation  31  Table 6: D e f i c i e n c i e s used t o map 3R proximal Su(var)s Table 7: Marked stocks e s t a b l i s h e d f o r Su (var) I I I mutations  55  Table 8: E f f e c t s o f d e f i c i e n c i e s i n the 3R proximal r e g i o n on wm4 v a r i e g a t i o n  58  Table 9: R e s u l t s o f complementation a n a l y s i s w i t h d e f i c i e n c i e s i n t h e 3R proximal r e g i o n and 3R Su(var) mutations...  59  Table 10: Summary o f new compound progeny r e c o v e r e d from gamma r a d i a t i o n screen  61  Table 11: R e s u l t s o f progeny t e s t i n g f o r new compound females heterozygous f o r wm4; t e s t f o r presence o f absence o f Su(var)B143  62  Table 12: E f f e c t s o f l o s s o f Y chromosome on Su(var) mutations o f chromosome 3.  64  Table 13:  42  E f f e c t s o f l o s s o f 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 o f maternal and p a t e r n a l e f f e c t s o f wm4;TM3 progeny o f Su(var) parents at 22 and 29 C Table 17: Maternal e f f e c t s o f Su(var)A57 at d i f f e r e n t developmental temperatures  71 73  vii  Table 18: Homzygous phenotypes o f p r o x i m a l 3R Su (var) s  75  Table 19: Complementation a n a l y s i s o f 3R p r o x i m a l Su(var) m u t a t i o n s  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  viii LIST OF FIGURES  Page  F i g u r e 1: C o n s t r u c t i o n of stocks f o r t e s t i n g t h i r d chromosome Su(var)s with v a r i e g a t i n g rearrangements  light  12  F i g u r e 2: Crosses t o t e s t e f f e c t s of S u ( v a r ) I I I mutants on light v a r i e g a t i n g rearrangements  14  F i g u r e s 3a-f: E f f e c t s of S u ( v a r ) I I mutations on light variegation  19  F i g u r e 4: Cytology of 3R proximal r e g i o n i n c l u d i n g d e f i c i e n c i e s used to map Su (var) I I I mutations  43  F i g u r e 5: Strategy used t o map u s i n g new compound formation  46  Su(var)s  F i g u r e 6: P r o t o c o l t o determine presence or absence of Su(var) i n newly formed compound females heterozygous f o r wm4  47  F i g u r e 7: P r o t o c o l used t o determine s e n s i t i v i t y of Su(var)s to l o s s of the Y chromosome  49  F i g u r e 8: P r o t o c o l used to determine s e n s i t i v i t y of Su(var)s t o the l o s s of 2R c e n t r i c heterochromatin  50  F i g u r e 9: R e c i p r o c o l c r o s s e s used t o determine maternal e f f e c t s f o r 3R Su(var)s  51  FigurelOa: Protocol for constructing a m u l t i p l y marked s t r a i n with TM3 b a l a n c e r l a c k i n g Stubble  53  F i g u r e 10b: P r o t o c o l f o r c o n s t r u c t i o n of dominantly marked TM3 balanced s t r a i n l a c k i n g Stubble  53  F i g u r e 11: Protocol for constructing marked stocks of Su (var) mutations  54  F i g u r e 12: Complementation groups based on t r a n s heterozygous phenotypes and p h y s i c a l mapping  83  F i g u r e A:  93  Oregon-R pigment value l i n e a r i t y assay  F i g u r e C: Recombination data f o r Su(var)s and A130  A160  100  ACKNOWLEDGEMENTS  I thank my supervisor, Tom G r i g l i a t t i 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 f l y 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  Position effect variegation  (PEV) r e s u l t s when a gene  i s moved v i a chromosome rearrangement  from i t s u s u a l  l o c a t i o n t o a p o s i t i o n next t o a newly formed e u c h r o m a t i c h e t e r o c h r o m a t i c boundary.  The r e a r r a n g e d 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 o t h e r s . T h i s o n / o f f 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, gene.  r e s u l t i n g i n mosaic e x p r e s s i o n o f t h e  Both e u c h r o m a t i c and h e t e r o c h r o m a t i c genes are  s u b j e c t t o t h i s phenomena, but t h e m a j o r i t y o f work has been done w i t h e u c h r o m a t i c v a r i e g a t i n g genes. above was  PEV as d e f i n e d  f i r s t d e s c r i b e d by S t u r t e v a n t (1925).  most work has been done i n Drosophila  melanogaster  v i r t u a l l y e v e r y gene t e s t e d i s s u b j e c t t o PEV see Lewis 1950; Baker 1968;  Since then, where  (for reviews,  S p o f f o r d 1976) .  A l t h o u g h much p r o g r e s s has been made s i n c e 1925, m o l e c u l a r mechanism o f PEV i s s t i l l unknown.  Judd  the  (1955)  has p r o v i d e d e v i d e n c e a g a i n s t m u t a t i o n r e s u l t i n g i n i n a c t i v a t e d genes, and H e n i k o f f (1979) has r u l e d out gene l o s s as a cause o f mosaic e x p r e s s i o n .  T h i s suggests  t h a t changes i n c h r o m a t i n s t r u c t u r e are t h e cause o f variegating position effects. Zuckerkandl elements  somatic  (1974) proposed t h a t h e t e r o c h r o m a t i c  s p r e a d beyond t h e d i s r r u p t e d h e t e r o c h r o m a t i c  boundary, c a u s i n g 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 o f  2 neighbouring euchromatic l o c i through a l t e r e d chromatin structure.  There i s e v i d e n c e t o support such a model.  F i r s t , PEV shows a s p r e a d i n g 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 t o 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 u n l e s s o t h e r 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;  S c h u l t z 1941; f o r an e x c e p t i o n  see Chovnick and C l a r k 1986).  This supports the idea that  c o n d e n s i n g m o l e c u l e s s p r e a d outward from h e t e r o c h r o m a t i n and i n a c t i v a t e e u c h r o m a t i c genes.  Second, H a r t m a n - G o l d s t e i n  (1967) and more r e c e n t l y Zhimulev e t al.  (1986) have  c o r r e l a t e d h e t e r o c h r o m a t i n - l i k e morphology o f s a l i v a r y g l a n d chromosomes w i 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 o f t h i s s p r e a d i n g has been i n v e s t i g a t e d by T a r t o f et al.  (1984).  By c l o n i n g t h e h e t e r o c h r o m a t i c  j u n c t i o n s o f t h r e e s t a n d a r d v a r i e g a t i n g rearrangements  (wm4,  wm51b, wmMc) t h e y have shown t h a t t h e e u c h r o m a t i c h e t e r c h r o m a t i c boundary i s f l a n k e d by m o b i l e e l e m e n t - l i k e sequences.  These sequences  a l o n e are not c a p a b l e o f  inducing chromatin condensation.  T h i s suggests t h a t  h e t e r o c h r o m a t i z a t i o n i s i n i t i a t e d f a r away from t h e breakpoint, w i t h i n the heterochromatin. al.  However, R e u t e r e t  (1985) have found complete p h e n o t y p i c 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 m u t a t i o n s o f  PEV.  They c o n c l u d e t h a t t h e e u c h r o m a t i c - h e t e r o c h r o m a t i c j u n c t i o n f l a n k e d by one or more o f t h e s e sequences cause v a r i e g a t i o n .  i s sufficient to  3 PEV and i t s a s s o c i a t e d c h r o m a t i n changes can be i n v e s t i g a t e d due t o t h e e x i s t a n c e o f s e v e r a l p o t e n t m o d i f i e r s o f t h e PEV phenotype.  A l l v a r i e g a t i n g phenotypes  are suppressed by h i g h developmental temperature,  so t h a t  t h e v a r i e g a t i n g gene i s e x p r e s s e d i n a g r e a t e r number o f cells.  Low developmental temperature enhances v a r i e g a t i o n ,  c a u s i n g t h e gene t o be i n a c t i v a t e d i n more c e l l s Gay  1933).  (Gowan and  Another s t a n d a r d m o d i f i e r o f PEV i s  heterochromatin content of the c e l l .  Variegation i s  enhanced by l o s s o f h e t e r o c h r o m a t i n such as t h e Y-chromosome (Gowan and Gay 1934) and t h e c e n t r i c h e t e r o c h r o m a t i n d e l e t i o n Df(2R) M-S210 (Morgan e t a l . 1941). e x t r a Y-chromosome suppresses PEV.  C o n v e r s l y , an  These o b s e r v a t i o n s  suggest t h a t t h e presence o f h e t e r o c h r o m a t i n i n a c e l l a c t s as a s i n k f o r h e t e r o c h r o m a t i c elements.  Extra  h e t e r o c h r o m a t i n a t t r a c t s more o f t h e s e 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 r e a r r a n g e d gene i n a e u c h r o m a t i c environment, l e s s extreme mutant phenotype.  resulting i n a  Loss o f h e t e r o c h r o m a t i n  f r e e s t h e s e elements and a l l o w s f o r m a t i o n o f h e t e r o c h r o m a t i n a t t h e b r e a k p o i n t , c a u s i n g a more severe mutant phenotype. T h i s s i n k e f f e c t may be due t o h e t e r o c h r o m a t i n c o n t e n t , o r to s p e c i f i c regions (binding sites) i n heterochromatin. Brock  (1986) r e p o r t s t h a t d i f f e r e n t r e g i o n s o f t h e 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 d i s c o v e r e d m o d i f i e r s o f PEV i n c l u d e h i s t o n e gene m u l t i p l i c i t y , and b u t y r a t e , an i n h i b i t o r o f histone de-acetylases. demonstrated  Moore et al.  (1979;  1983)  t h a t v a r i e g a t i n g phenotypes are s u p p r e s s e d by a  histone deficiency.  T h i s suggests t h a t a decrease  h i s t o n e s a v a i l a b l e t o the chromatin l i m i t s  of  transcriptional  i n a c t i v a t i o n due t o h e t e r c h r o m a t i c p a c k a g i n g , thus a l l o w i n g a more w i l d t y p e phenotype.  In a d d i t i o n ,  Mottus et  (1980) have found t h a t sodium b u t y r a t e can  al.  suppress  v a r i e g a t i o n , p r o b a b l y s i n c e h i s t o n e d e - a c e t y l a s e s are inhibited, resulting i n acetylated histones.  These f i n d i n g s  support t h e h e t e r o c h r o m a t i z a t i o n mechanism f o r PEV  gene  inactivation. G e n e t i c m o d i f i e r s o f PEV are perhaps t h e most u s e f u l investigative tools.  S p o f f o r d (1967) i d e n t i f i e d t h e  dominant s u p p r e s s o r o f PEV Reuter and W o l f f  (Su(var)) m u t a t i o n .  (1981) and S i n c l a i r e t al.  i n d e p e n d e n t l y i s o l a t e d over 150 X-ray or EMS dominant Su(var) m u t a t i o n s . t h e i r e f f e c t s on t h e white wm4,  The  first  Since then,  (1983) have induced  S u ( v a r ) s were s e l e c t e d f o r  gene v a r i e g a t i o n rearrangement,  but show g e n e r a l e f f e c t s , s u p p r e s s i n g b o t h brown and  scute  v a r i e g a t i o n (Reuter and W o l f f 1981)  Stubble  and brown and  v a r i e g a t i o n ( S i n c l a i r et a l . 1983).  G e n e t i c mapping r e v e a l e d t h a t many o f t h e 50 S u ( v a r ) s o f 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  clusters  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 t h r e e centimorgan  distance.  Reuter and W o l f f  (1981) have  5 suggested t h i s l a b e l i s unnecessary, but f o r the purposes of d i s c u s s i o n these Su(var)s w i l l be r e f e r r e d t o as the 2L c l u s t e r e d and n o n c l u s t e r e d S u ( v a r ) s , and the 3R p r o x i m a l , middle and d i s t a l  clusters.  C h a r a c t e r i z a t i o n of Su(var) mutations this thesis.  i s the s u b j e c t of  F i r s t , i n t e r a c t i o n s between Su(var)s and  heterochromatic v a r i e g a t o r s , heterochromatic l o c i t h a t v a r i e g a t e as a consequence of r e l o c a t i o n near t o euchromatin,  are i n v e s t i g a t e d t o determine  the  between heterochromatin and gene i n a c t i v a t i o n .  relationship Second, a 3R  proximal c l u s t e r i s c h a r a c t e r i z e d w i t h regard t o standard m o d i f i e r s of PEV  and g e n e t i c r e l a t i o n s h i p of the Su(var)s i n  t h i s c l u s t e r , t o p r o v i d e a necessary knowledge base f o r p o s s i b l e molecular e x p e r i m e n t a t i o n .  6 CHAPTER 1 - The e f f e c t o f Su(var) m u t a t i o n s on light variegation.  gene  INTRODUCTION Q u e s t i o n s about c h r o m a t i n p a c k a g i n g are fundamental u n d e r s t a n d i n g gene e x p r e s s i o n .  to  Position effect variegation  (PEV) causes h e t e r o c h r o m a t i n i z a t i o n o f t h e DNA 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  that i s also  (see g e n e r a l  i n t r o d u c t i o n ) . Thus, PEV p r o v i d e s a model system f o r s t u d y i n g h e t e r o c h r o m a t i n and i t s r o l e i n gene r e g u l a t i o n . Both e u c h r o m a t i c and h e t e r o c h r o m a t i c l o c i are s u b j e c t t o PEV.  U s i n g e u c h r o m a t i c and h e t e r o c h r o m a t i c 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 t h e mechanisms o f gene i n a c t i v a t i o n can be s t u d i e d . T h i s study u t i l i z e s a h e t e r o c h r o m a t i c gene, light which  (It)  e x h i b i t s a mosaic phenotype when moved away from  c e n t r i c h e t e r o c h r o m a t i n and r e l o c a t e d next t o d i s t a l euchromatin  ( S c h u l t z and Dobzhansky 1934).  of I t appears  Light  variegation  i n many ways t o be r e c i p r o c a l t o s t a n d a r d  e u c h r o m a t i c PEV gene.  The  (euPEV), such as v a r i e g a t i o n o f t h e  white  v a r i e g a t i o n , t h e n , i s one example o f  h e t e r o c h r o m a t i c PEV  (hPEV).  (See S p o f f o r d 197 6 f o r r e v i e w  of PEV and Hannah 1 9 5 1 ; H i l l i k e r e t al.  1980,  f o r reviews of  heterochromatin). Few examples o f hPEV are documented, p r o b a b l y s i n c e so few genes have been mapped t o h e t e r o c h r o m a t i n .  Three  h e t e r o c h r o m a t i c genes are known t o v a r i e g a t e : peach i n D. virilus,  Baker  melanogaster,  (1953); cubitus Khvostov  interruptus  (1939); and light,  (ci) i n D. a l s o i n D.  7 melanogaster, H e s s l e r been o b s e r v e d . variegate, be  (1958).  i nthe d i s t a l  o f t h e e u c h r o m a t i c b r e a k p o i n t must  t w o - t h i r d s o f t h e chromosome  h e t e r o c h r o m a t i n which  i s then d i s r u p t e d ) .  euchromatin  A gene t h a t  light  hPEV, i t does  (1968)  Baker  i srelocated to  cannot be reached by t h i s  h e t e r o c h r o m a t i n and, t h e r e f o r e , While  (or i n other  a r e p a i r mechanism s p r e a d s f r o m t h e  undisturbed heterochromatin. distal  f o r hPEV h a s  I n o r d e r f o r a h e t e r o c h r o m a t i c gene t o  the position  suggests that  One r e q u i r e m e n t  variegation  demonstrate  repair  competent  cannot take  follows this  place.  requirement f o r  Light  some i r r e g u l a r t i e s . xv  variegating  rearrangements  1958).  (Hessler  (lt s)  Some r e a r r a n g e m e n t s  m o t t l e d e y e , h a v i n g many light cells.  Others  show v a r i o u s result  ommatidia  phenotypes  i n a pale-  a n d few w i l d - t y p e  show a d a r k - m o t t l e d e y e , w h i c h  appears  w i l d - t y p e eye i n t e r s p e r s e d w i t h d a r k e r ommatidia. rearrangements extremes.  g i v e phenotypes  Hessler  as a  Some  i n t e r m e d i a t e between t h e s e  (1958) f o u n d 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 a n d any one p h e n o t y p e . All  It p h e n o t y p e s  such as temperature  heterochromatin  respond t o standard m o d i f i e r s  and t h e p r e s e n c e o r absence o f  (Baker 1968).  Light  suppressed by e l e v a t e d temperature variegation.  However, a d d i t i o n  variegation i s  as i s euchromatic  o f a Y chromosome ( n o r m a l l y  a s u p p r e s s o r o f PEV) a c t s a s a n e n h a n c e r This  observation  o f PEV,  sets  It a p a r t  o f It v a r i e g a t i o n .  from o t h e r h e t e r o c h r o m a t i c  8  variegators (ie; peach and ci) and suggests some aspects of It variegation are reciprocal to euPEV. If the mechanism of It variegation i s reciprocal to euchromatic variegation, i t s movement into distal euchromatin i s 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 c e l l 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 i s 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:  M u t a t i o n s 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 T a b l e s 1 and 2.  All  s u p p r e s s o r s o f v a r i e g a t i o n , S u ( v a r s ) , were i s o l a t e d as d e s c r i b e d by S i n c l a i r et al., rearrangements  (1983).  Light  variegating  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 ( p e r s o n a l communications). Since the  t h i r d chromosome S u ( v a r ) s do not c a r r y It  m u t a t i o n s , i t was n e c e s s a r y t o c o n s t r u c t two s t o c k s , diagrammed i n F i g u r e 1. background  These s t o c k s p r o v i d e a It  so t h a t I t v a r i e g a t i o n can be d e t e c t e d i n t h e  presence o f t h e s e S u ( v a r ) s .  CULTURE CONDITIONS:  F l i e s were r a i s e d at 22 C ( u n l e s s  o t h e r w i s e s t a t e d ) on s t a n d a r d c o r n m e a l - s u c r o s e medium.  Tegosept was  Drosophila  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 t h e e f f e c t of  second chromosome s u p p r e s s o r s  ( S u ( v a r ) I I ) s on I t  v a r i e g a t i o n , t h e f o l l o w i n g c r o s s was performed. t h e genotype ltxv/Gla  wm4;Su(var), b It rl/CyO  or Itxv/ltxv  virgins.  Males  were c r o s s e d t o  of +/+;  F l h e t e r o z y g o t e s 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 b e l o w ) .  TABLE 1:  SUPPRESSOR OF POSITION EFFECT VARIEGATION USED IN THIS STUDY.  Mutations  Alternate Designation  Map p o s i t i o n (+ 95% c o n f i d e n c e levels)  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 CLUSTERED  2L NONCLUSTERED 5. 7 + 1 .3  Su (var) 208  Su(var)T44  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  Su (var) 310  Su (var)A63  54. 4 + 0 .7  Su (var) 316  Su (var)A48  47. 4  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  Su(var)323  Su (var)A130  3L  Su(var)303  Su (var)AlSO  3R PROXIMAL  0 .8  3L ARM see appendix  3L  11  TABLE 2:  DESCRIPTION OF LIGHT  LIGHT REARRANGEMENT (abrieviation)  VARIEGATING REARRANGEMENTS  PHENOTYPE  CYTOLOGY  pale-mottled  T (2;3)40?, 94D + T(2;3)32C, 64  T (2;3) ItxS (ltx6)  moderately-mottled  insertional translocation of 63-74 t o 36h*  T(2;3)ltx2 (2tx2)  moderately-mottled  In (2L)25D5, 40A + In(2LR)40A, 53A1,2  T (2;3) l t l 3 (ltl3)  dark-mottled' homozygous v i a b l e  T(2;3)37h; 97D2  T(2;3)ltx24 (ltx24)  dark-mottled,  T(2;3)37,38h; 61F  T(2;3)ltx4 (ltx4)  dark-mottled  T(2;3) 36,37h;97Dl  In (2L) (ltxl8)  ltxl8  *h i n d i c a t e s heterochromatic c y t o l o g y u n i t s i n 2L heterochromatin (unpublished map).  61D3-  12  FIGURE 1:  CONSTRUCTION OF STOCKS FOR TESTING THIRD CHROMOSOME SU(VAR)S WITH LIGHT VARIEGATING REARRANGEMENTS  To p r o v i d e b,lt,rl  background:  wm4 +  wm4 CyO; +  Ly  i / Y  /  + TM3,Sb,Ser  + b,lt,rl  -;  /  ;- x  Y b,lt,rl  wm4 Tft +  wm4 CyO Ly wm4 +  x  + wm4  Y CyO  +  Su(var)Ills:  x  TM3,Sb Ser  x (  +  wm4 +  cr« 9  Y  TM3,Sb Ser  +  r  wm4 Tft Ly  r  wm4 Tft  +  wm4 + Ly  wm4 CyO +  wm4 +  b,lt,rl  s e l e c t CyO,Ly f l i e s each generation  wm4 Tft +  siblings: wm4 CyO  Tft  Ly  b,lt,rl  To mark the second chromosome o f  wm4 Tft +  +  wm4  cr«9  FI  wm4 CyO +  +  Y  +  +  wm4 +  Ly TM3, Sb Ser  x  r  Y  +  Su(var)III TM3,Sb,Ser  wm4 Tft Su (var) III +  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 a l l e l e was required i n the Su(var)III s t r a i n s .  This was  accomplished with the protocol shown i n Figure 2. Itxv;Su(var) F l progeny were collected 0-3 days posteclosion, 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 i n glass tubes at -70 C.  F l i e s were decapitated  immediately after removal from -70 C by firmly banging the tubes.  A t o t a l of 50 heads for each genotype were placed i n  1.5 ml Ependorf tubes, 5 heads per tube, males and females separated at c o l l e c t i o n .  Pigment was extracted by  sonication of heads i n 30 u l of 0.25M 2-mercaptoethanol i n 1% aqueous NH4OH.  After b r i e f centrifugation, samples were  placed i n the dark for approximately 1 hour at room temperature. The head fragments were precipitated by centrifugation at 12,000 G for 2 minutes.  5 u l of the  supernatant from each sample was pippetted onto Whatman #3 f i l t e r paper fixed to a microscope s l i d e so that each s l i d e contained f i v e 5 u l aliquots of one genotype. The amount of pigment was quantified by flourescence at >500 um linear range of the instrument) using a Zeiss  (in the  14  FIGURE 2:  CROSSES TO TEST EFFECTS OF SU(VAR)III LIGHT VARIEGATING REARRANGEMENTS  MUTANTS ON  Experimental: wm4  CyO  Ly  wm4 Tft Su (var) III  x  wm4 b , l t , r l +  4  Y  + Itxv  +  x  wm4  Tft  + Gla  +  Y  b lt,rl  experimental:  +  Itxv  +  TM3, Sb, Ser Su(var)III Ly  r  Su(var)III  *' b , l t , r l '  + pigment assay  internal control:  +  Itxv  Ly  b,lt,rl  +  Control: + Itxv f  + Gla  + I  +  wm4 A  CyO f  Y  Ly f  b , l t , r l .+  V  + Itxv + —; ;— * b lt,rl +  pigment assay  r  *Chromosome i s e i t h e r an X c a r r y i n g wm4 o r a Y.  15 microfluoremeter.  Pigment l e v e l s are e x p r e s s e d as a  percentage of w i l d type  STATISTICS:  (Oregon-R) pigment  levels.  F l u o r e s c e n c e v a l u e s e x p r e s s e d as p e r c e n t a g e s  Oregon-R c o n t r o l  of  pigment l e v e l s , which form a b i n o m i a l  d i s t r i b u t i o n , were t r a n s f o r m e d t o t h e i r a r c s i n e v a l u e s t o approximate  a normal d i s t r i b u t i o n .  compared w i t h c o n t r o l m u l t i p l e range t e s t  S u ( v a r ) I I v a l u e s were  v a l u e s by ANOVA f o l l o w e d by (Zar 1984).  D i f f e r e n c e s between  S u ( v a r ) I I I v a l u e s and t h e i r i n t e r n a l  control  v a l u e s were  d e t e r m i n e d by u n p a i r e d t - t e s t s . The s t a t i s t i c a l significance  was t a k e n as P<0.05.  Dunnett's  limit  of  16  RESULTS  To measure effects of Su(var)s on It variegation, i t i s f i r s t necessary to analyze strengths of each It variegating strain.  A l l 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 i s so great that pigment values are often significantly different between t r i a l s . Even so, i t i s possible to group these variegators into 3 categories of variegating strength. F i r s t , ltxl8  i s a very strong  variegator (pale mottled) often showing lt/lt levels, indicating that the lights  pigment  gene i s 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: GENOTYPE  BASAL LIGHT VARIEGATING CONTROL PIGMENT LEVELS FROM THREE SEPERATE TRIALS TRIAL 1 TRIAL 2 (mean + 2 S.E.)  TRIAL 3  It/It male female  32.5 + 1.5 31.2+2.3  30.3 + 1.2 33.5 + 1.2  33.0 + 2.0 *34.2 + 1.0  ItxlQ male female  26.9 + 1.4 *31.0 ± 3.6  26.7 + 2.5 *24.6 + 3.9  31.6 + 1.9 *40.9 + 6.0  ltx6 male female  64.1 + 8.2 *50.1 ± 4.9  53.3 + 4.6 42.1 + 3.4  54.1 + 8.1 41.8 + 3.5  ltx2 male female  54.6 +10.9 *57.0 + 5.5  46.2 + 9.5 *47.0 + 7.3  45.2 + 3.7 *38.5 ± 2.0  ltxl3 male female  84.4 + 4.7 66.1 + 5.2  *91.5 ± 7.0 *105.0 + 7.1  80.0 + 4.7 70.7 + 0.9  ltx24 male female  *102.5 + 2.5 82.3±5.7  77.5 +10.5 89.8±4.0  81.8 + 3.3 *7 6 . 0 ± 2 . 7  ltx4 male female  118.7 + 2.7 88.2 ± 2.6  93.4 + 9.6 91.7 + 4.6  77.1 + 6.3 *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 o t h e r c o n t r o l v a l u e s w i t h i n genotype and sex.  that  18 Su(var)s would probably be undetectable i n these strains since higher pigment levels may be indistinguishable from basal Itxv l e v e l s . Enhancement, or reduced pigment could be readily detected. Su(var)s used i n these experiments can also be grouped into categories:  clustered and nonclustered chromosome two  suppressors and t h i r d chromosome suppressors (see Table 1) Results of the effects of both groups of Su(var)II suppressors on It variegation are shown i n Figures 3a-f (actual pigment values are l i s t e d i n the appendix).  Two  t r i a l s were completed for most combinations of ltxvs  with  Su(var)II mutants.  T r i a l I was assayed i n one  sitting,  while t r i a l II was s p l i t into 2 assays, each having i n d i v i d u a l 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 s t r a i n  from each category of It variegation strength was chosen for analysis of the t h i r d chromosome Su(var)s. these crosses are shown i n Table 4.  Pigment data for  Each Itxv;Su(var)  pigment l e v e l i s shown with an internal control value taken from ltxv;Ly  siblings.  Differences between these values  indicate effects of the Su(var) mutation versus a nonsuppressor 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 d i f f e r e n t l y to a  19  FIGURE 3a-f: E f f e c t s o f S u ( v a r ) I I m u t a t i o n s on light variegation. Shaded a r e a s i n d i c a t e b a s a l light variegating pigment l e v e l s . Open b a r s i n d i c a t e p i g m e n t l e v e l s o f Su(var)II/Itxv h e t e r o z y g o t e s . V a l u e s a r e mean + 2 S.E.  % Oregon—R pigment  33 O  males  females  o o o o o o o o o o o o o o o o o o o o o 1  1  1  1  1  1  1  1 1  d  m  —1—1—1—1—1—1—1—1—r—  o  5 >  wmmh * • I'-'J. .„, T3F31  ••  TI  3 (D r-r-  *>tt  t,  TTJ.  < C/)  CD oo CD  CO c Q  6  wmmm «+•  o Oi  00 zr  X  CD  CD r-t-  CD  CD  a> o  x-xvWvXyXiXM u 1 •  to o  r+-  (D CO  03  % Oregon—R pigment females - > N U * t f l O ) s l 0 D ( O  males - ' l O O l A U l O l ^ a i D O  23  O d  m  o o o1 o1 o1 oI o 1 o I—1—ro o o o1 o I o 1 o I o 1 o 1 o 1 o 1o I o  •• 5  TJ cQ*  > Cn  3  CD  CD~ <  3 &  CO  CO  CO c  Q  5 i  n  II X CD  CD  X  Oi CO  •m .  DEP  '**Hfr$Ji5r*'*i • nn . :  CO  ' rni  1  r-rCD ~1  O N O  r-t-  CD OO  13  % Oregon—R pigment males  females o  - ' S ) O I > U I O ) N | 0 9 0 o o o o o o o o o o  I  I  I  I  I  o  IO U ^ Ul O) N l a ID o o o o o o o o o o  I  I  I  ZB£?S5QSK  u  s  I  JJ  O C  m o •• TJ  -  — •  cQ  >  mmmmm^ 3 i »  3 0  <  cp_ c/)  00 CO  o  —h CO  c Q  3H o  rcsi .  Ol  LtLLI  1  NJ  X CO  ro =r  0  r+Oi CO  CD -\  o N  v:  cQ O  rift  22  % Oregon—R pigment males  females o  - » r o o i £ o i a » N j ( » < o o o o o o o o o o o  I  1  I  I  I  I  I  I  I  o  - t r o G i £ a i o > N i t » < o Q o o o o o o o o o  I  I  I  I  I  I  I  I  I  3j CD cz X3 m CL  ••  5  TJ  > Ol  3  o cn o  3-  NI  t  cp_  3-  CD  00  co  gg-i  101.3 J L . 2.1  5  2 Ol  co  O —H  CO c  Q  X  Ol  CO  CO  3  o  Oi  CD CD" <  If  2  3  GJ •:v:ai -  i£3P  D"  CD  r-t-  CD  O N ^< cQ O  r-t-  CD CO  % Oregon—R pigment  o  o  I  o 1  o  I  o  o  o  o  <z  males  females o  o  o  I 1  o  o  I  o  I  o 1  o  I  o  I  o  I  o  I  o  m  o  I  1  CD  *  n  ^  a  —  o o  •  >  3 *  |.VJj.;. ;.;.v.|  1  <  o_ c  t  CD CO  1  I.V.TXN'.V.TT  o a ro  1  3  CD Zl  r-t-  <  U  CO I |'»X*I*.y.'.l  *  N) In  CD  c 113.3 .±9.1  (5  JI  Ol vi  110.3 ± 4 . 4 •  X  Oi  X ho  3" (D *-+-  CD  CO  Q  •i•  O  Oi  *  3 3 *  o 1 3  N O  r-t-  CD CO  fr3  S3  TABLE 4: EFFECTS OF SU(VAR)III MUTATIONS ON THREE LIGHT VARIEGATING STRAINS t-value (mean + 2 S.E.)  GENOTYPE  MALE  ltxl3;  86.4 10.8  +  ltxl3;B94  70.5  control  73.9  6.2  ltxl3;B76  76.9  6.1  control  80.3  ltxl3;C119  73.7 5.4  control  82.8 2.8  ltxl3;A63  83.4 5.8  control  82.5 6.6  ltxl3;A48  60.8 8.5  control  68.0 1.1  ltxl3;A57  74.8 5.0  control  76.5 3.4  t(8)=2.31  71.4 4.0  4.4  5.5  t=0.98  t=0.82  t=1.90  t=0.20  t=2.96*  t=0.62  t(7)=2.37  control=internal  t-value  FEMALE  63.0  2.6  57.9  4.2  70.2  3.0  57.2  6.1  70.5 71.3 63.9 59.7 59.3 61.6  t=4.31*  2.5 t=0.36 3.9 4.7 t=1.17 5.6 4.8 t=0.86 2.7  62.1  1.8 t=2.28* 58.7 2.4  *=significant  control,  t=2.14  ie;ltxv;Ly  difference  27 TABLE 4:  continued  GENOTYPE  MALE  ltx6;+  45.8  2.0  ltx6;B94  36.3  7.9  t-value (mean + 2 S.E.)  FEMALE  t-value  35.2  8.2  31.4  1.3  30.3  0.7  35.2  1.0  32.3  2.8  33.5  2.0  39.7  8.6  34.6  6.0  30.0  1.3  24.6  0.8  21.8  2.9  27.8  1.8  24.2  2.5  t=1.92 control  43.6  3.8  ltx6;B76  36.2  3.7  t=1.4  t=2.67* control  41.3  2.3  ltx6;C119  36.6  2.9  t=2.27  t=2.42* control  43.6  5.5  ltx6;A63  49.8  6.0  t=2.09  t=1.01 control  47.4  1.7  ltx6;A48  39.9  3.8  control  43.1  4.4  ltx6;A57  32.6  2.9  t=1.14  t=2.06  t=2.64*  t=2.33* control  38.2  3.9  t=2.41*  28  TABLE 4: c o n t i n u e d GENOTYPE  MALE  t-value (mean + 2 S.E.)  ltx2;+  33.1 3.4  ltx2;B94  29.36.1  control  32.4  ltx2;B76  23.9 2.3  control  27.7 2.7  ltx2;C119  35.7 2.0  control  37.3 3.4  ltx2;A63  33.0 2.4  control  35.2 7.7  ltx2;A48  28.7 1.3  control  36.3 3.1  ltx2;A57  33.0  control  36.6  5.0  t-value  27.9 0.7  24.41.4 t=0.84  t=2.06  t=0.86  t=0.51  t=5.42*  4.8 4.8  FEMALE  t=2.68*  25.0 30.1 36.7 30.3 31.6 33.1 29.9  1.7  t=0.55  1.9 t=2.86* 5.3 3.9 t=0.45 4.3 5.1 t=1.32 2.4  30.8 3.0 29.8  t=0.47 2.9  30.1  3.4  25.3  0.8  t=4.08*  29 particular Su(var), these effects are not consistent between t r i a l s 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 s t a t i s t i c a l techniques were applied to determine significant changes from basal It variegating levels due to Su(var) action.  However, given  the i n t e r - t r i a l variability of control basal It variegating levels, pigment differences between Su(var)II/Itxv f l i e s and control Itxv levels were considered biologically significant i f two c r i t e r i a were met:  1) s t a t i s t i c a l l y significant  differences were consistent between t r i a l s and 2) the direction of change (i.e. enhancement or suppression) was the same for each t r i a l . controls were used.  For Su(var)III crosses, internal  This eliminates i n t e r - t r i a l 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 variegation. i n Table 5.  Enhancing e f f e c t s o f Su(var)s are  summarized  Many S u ( v a r ) s a r e c a p a b l e o f enhancing It  v a r i e g a t i o n , but S u ( v a r ) s T44 and M43 most g e n e r a l enhancers.  are the strongest  and  T h e i r 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 o r more p e r c e n t a g e u n i t drops i n pigment  from c o n t r o l v a l u e s .  Su(var)M43 s t r o n g l y  a l l Itxvs tested, excluding  ltxl8  s t r o n g l y enhances a l l ltxvs  with the exception of  rearrangements.  males.  enhances  Su(var)T44  Su(var)B89 a l s o has s t r o n g  ltxl8  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 o n l y ltx2 ltx24  females and ltx6  males and f e m a l e s .  males,  A l l three of  t h e s e s t r o n g enhancers b e l o n g t o t h e u n c l u s t e r e d  group o f 2L  Su (var) s. S u ( v a r ) s H69, A151 and M59  a r e more moderate enhancers,  c a u s i n g a p p r o x i m a t e l y 10 p e r c e n t a g e 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 ,  a f f e c t i n g l e s s t h a n h a l f of t h e v a r i e g a t o r s  usually  tested,  (see  Table 5) and i n most c a s e s , t h e y have no e f f e c t on It variegation.  S u ( v a r ) s B76, C119, A57 and A48 are a l l  l o c a t e d on t h e t h i r d chromosome and are v e r y weak enhancers o f It v a r i e g a t i o n , never c a u s i n g pigment l e v e l drops o f even 10 p e r c e n t a g e u n i t s . The moderate I t v a r i e g a t o r s ,  ltx6" and ltx2  s u s c e p t a b l e t o enhancement by S u ( v a r ) s ,  a r e t h e most  showing reduced  pigment w i t h 7 and 9 S u ( v a r ) s t e s t e d , r e s p e c t i v e l y . s t r o n g v a r i e g a t i n g rearrangement, ltxl8 s u s c e p t a b l e , showing l i t t l e r e d u c t i o n  The  i s the l e a s t i n pigment and no  31  TABLE 5:  SU(VAR)S WHICH ENHANCE LIGHT VARIEGATION  LIGHT VARIEGATING STRAIN  SU(VAR)S WHICH ENHANCE LIGHT VARIEGATION  APPROXIMATE PERCENTAGE UNIT DROP IN PIGMENT (refer to figures 3a-f)  ItxlS  A151 M43  ( n o n c l u s t e r e d 2L) "  females o n l y , 10 females o n l y , 15  M59  ( c l u s t e r e d 2L)  females o n l y , 9  ltx6  ltx2  ltxl3  T44 ( n o n c l u s t e r e d 2L) M43 " B89 "  15-25 20-25 15  H69 ( c l u s t e r e d 2L)  females o n l y , 10  B76 (3R) C119 " A57 "  males o n l y , 5 males o n l y , 7 males o n l y , 5  T44 ( n o n c l u s t e r e d 2L) A151 " M43 " B89 "  15 males o n l y , 10 20 males o n l y , 15  H69 ( c l u s t e r e d 2L) M59 "  10-15 10-15  A48 (3R) A57 " B76 "  males o n l y , 7 males o n l y , 3 females o n l y , 6  T44 ( n o n c l u s t e r e d 2L)  45-50  M43  15  A48 ltx24 ltx4  " (3R)  males o n l y , 8  T44 ( n o n c l u s t e r e d 2L) M43 " B89 " T44 ( n o n c l u s t e r e d 2L) M43 "  40 20-25 females o n l y , 15 40-50 20  M59  10  ( c l u s t e r e d 2L)  32 response t o most S u ( v a r ) s . The weak v a r i e g a t o r s ltxl3, and ltx4  ltx24  were s t r o n g l y a f f e c t e d by S u ( v a r ) s T44 and M43, b u t  u n r e s p o n s i v e t o most o t h e r S u ( v a r ) s . S u p p r e s s i o n o f I t v a r i e g a t i o n by S u ( v a r ) s i s much l e s s frequent.  Itxl8  Su(var)C157. Su(var)B76.  males a r e m o d e r a t e l y s u p p r e s s e d by  Itxl3 Itx2  females a r e m o d e r a t e l y s u p p r e s s e d by females a r e v e r y weakly s u p p r e s s e d by  S u ( v a r ) s B7 6 and A57 w i t h pigment i n c r e a s e s o f a p r o x i m a t e l y 5 p e r c e n t a g e u n i t s over c o n t r o l v a l u e s .  Similarly,  females a r e v e r y weakly s u p p r e s s e d by S u ( v a r ) A 4 8 .  ltx6 No  c o r r e l a t i o n between s u p p r e s s i o n by S u ( v a r ) s and t y p e o f I t x v rearrangements i s apparent.  33  DISCUSSION  The r e s u l t s indicate that dominant suppressors of (Su(var)s)  PEV  are capable of s i g n i f i c a n t l y enhancing the  e f f e c t s of heterochromatic variegating rearrangements (Figures 3 a - 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 t h i r d chromosome has been suggested based on p o s i t i o n only ( S i n c l a i r , 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  nonclustered 2L Su(var) and two suppressors. enhance hPEV.  one  of the clustered 2L  These groups overlap i n t h e i r a b i l i t i e s to In f a c t , one of the 2L clustered Su(var)s i s  capable of suppressing It variegation.  Four of the 3R  Su(var)s can enhance It variegation, but t h e i r e f f e c t s are extremely weak. Although clear differences i n 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 e f f e c t . 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 i n t h e i r e f f e c t s , enhancing a l l It variegating rearrangements tested with the exception of I t x l S .  It seems l i k e l y that t h e i r mechanism of action i s  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 t h e case o f  ltxl8,  d e t e c t i o n o f f u r t h e r i n a c t i v a t i o n t h r o u g h a reduced pigment phenotype i s d i f f i c u l t . variegator: ommatidia. an a l r e a d y  t h e light  Itxl8  i s an e x t r e m e l y s t r o n g  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  T h e r e f o r e , t h e a d d i t i o n o f a Su(var) m u t a t i o n t o d r a s t i c a l l y p e r t u r b e d 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  locus.  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 reduced pigment i s not an i s s u e .  of  I t i s s u r p r i s i n g then, t o  see t h a t t h e 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 t h a n a r e moderate variegators  (ltx6,  ltx2) .  The a b i l i t y o f S u ( v a r ) s t o  enhance I t v a r i e g a t i o n must a l s o be dependent upon t h e p a r t i c u l a r variegator present. enhanced  ltx4,  ltx2  and ltxl8  o t h e r rearrangements t e s t e d .  F o r example  Su(var)M59  v a r i e g a t i o n , but none o f t h e The p a t t e r n o b s e r v e d i s  d i f f i c u l t t o a s s o c i a t e w i t h s t r e n g t h o f 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 Differences  (Hessler  1958).  between v a r i e g a t o r s must be due not t o t h e  r e l a t i v e p o s i t i o n , but t h e n a t u r e o f t h e  breakpoints.  Perhaps t h e S u ( v a r ) 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. S e v e r a l p o s s i b l e machanisms may  e x p l a i n t h e enhancing  c a p a b i l i t i e s o f s p e c i f i c Su(var) m u t a t i o n s .  First, a  s i m p l i s t i c model o f I t v a r i e g a t i o n would assume a s e r i e s o f e v e n t s r e c i p r o c a l t o e u c h r o m a t i c 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 i s 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 c e l l s .  The actual role of a suppressor mutation in this case, i s 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 i s  dependent upon homolog (locus to locus) pairing.  When one  It locus is rearranged, i t becomes topologically d i f f i c u l t to pair with i t s homolog, thus causing variable expression from c e l l 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 f i r s t  36 ( r e c i p r o c a l ) model i s f a v o r e d  f o r the f o l l o w i n g r e a s o n .  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 i s homozygous (such as ltxl3)  rearrangement which  s h o u l d not be a  v a r i e g a t o r , or perhaps s h o u l d not v a r i e g a t e shown by b a s a l ltxl3 i n d e e d weak.  A  strong  at a l l .  As  pigment l e v e l s , t h i s v a r i e g a t o r i s  However, i t responds d r a m a t i c a l l y t o  several  S u ( v a r ) s showing s i g n i f i c a n t l y d e c r e a s e d pigment l e v e l s i n the p r e s e n c e o f S u ( v a r ) s T44  and M43.  This i n t e r f e r e n c e  by  the Su(var) m u t a t i o n must not be a r e s u l t o f homolog p a i r i n g problems, s i n c e 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.  T h e r e f o r e , a more l i k e l y r o l e of  Su(var) l o c i i s t h a t of h e t e r o c h r o m a t i c s t r u c t u r e  or  maintanence. Su(var);It  c o m b i n a t i o n s 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) p r o d u c t s appear u n i m p o r t a n t t o hPEV.  These Su(var) genes may  p r o d u c t s which have a more g e n e r a l  code f o r  f u n c t i o n , or  functions  s p e c i f i c t o e u c h r o m a t i c PEV.  T h i s r e s u l t a l s o suggests t h a t  hPEV and euPEV are not s i m p l y  r e c i p r o c a l events.  various  Certainly,  S u ( v a r ) s are s p e c i f i c t o euPEV and are not  involved  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 e x p r e s s i o n  of a  d i s p l a c e d h e t e r o c h r o m a t i c gene. C o n t r a r y t o the models proposed above, f i v e o f s u p p r e s s i o n o f It v a r i e g a t i o n by S u ( v a r s ) s t h i s study.  instances  do e x i s t i n  None o f t h e s e cases i n v o l v e pigment  increases  o f more t h a n 13 p e r c e n t a g e u n i t s over c o n t r o l v a l u e s ; cases i n v o l v e <=6  p e r c e n t a g e u n i t changes.  No  3 of 5  Su(var) i s  37 able t o suppress one  genotype,  visually. enhances  It variegation  and  i n no  case  ( i n m a l e s ) ltxl3 on  is a relatively  inconsistent  event.  o p p o s i t e t o any In  rare,  If this  of the proposed  c o n c l u s i o n , i f light  in  rearrangements  By  introducing  limit  i t s e x p r e s s i o n may  which cannot  maintain that  a s u p p r e s s o r o f PEV,  observed  for several  that  function  dominant  that due  environment. to  t o a more e x t r e m e This effect  Su(var)s, strongly  is  suggesting  f o r the  appropriate expression of heterochromatic l o c i , loci.  be d i s r u p t e d  gene i s e x p e c t e d  o f t h e s e genes i s e s s e n t i a l  f o r euchromatic  genes.  e x p r e s s i o n i s d e p e n d e n t upon a  showing enhanced v a r i e g a t i o n  inhibitory  function  f u n c t i o n s f o r Su(var)  on t h e h e t e r o c h r o m a t i c e n v i r o n m e n t .  the  suppression  than a l l other Su(var)s t e s t e d here; that i s ,  h e t e r o c h r o m a t i c environment,  suffer,  has  Therefore,  a r e a l phenomenon, t h e s e S u ( v a r ) s must have a  much d i f f e r e n t  of  and  Su(var)A57 a l s o  rearrangements.  suppression of I t variegation e x t r e m e l y weak and  ( i n females)  variegation.  ltx2  females  i s the suppression d e t e c t a b l e  Su(var)B75 b o t h s u p p r e s s e s  contrasting effects  is  i n b o t h m a l e s and  but i s  38 CHAPTER 2 - C h a r a c t e r i z a t i o n o f a p r o x i m a l c l u s t e r o f Su(var) m u t a t i o n s on chromosome t h r e e . INTRODUCTION The  i n a c t i v a t i o n o f genes due t o p o s i t i o n e f f e c t  variegation  (PEV)  can be a t t r i b u t e d t o c h r o m a t i n 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 t h e c h r o m a t i n l e v e l can be s t u d i e d by d e t e r m i n i n g The  the mechanisms o f  i n v e s t i g a t i o n o f dominant m o d i f i e r s o f PEV  s t r a t e g y used t o study t h i s p r o c e s s .  PEV.  i s one  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 t h e p r o c e s s w i l l 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 p r o v i d e i n f o r m a t i o n towards m o l e c u l a r genes.  valuable  c h a r a c t e r i z a t i o n of these  This information w i l l contribute to  understanding  our  o f gene c o n t r o l at t h e c h r o m a t i n l e v e l .  Many dominant s u p p r e s s o r s  of PEV,  S u ( v a r ) 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 t o v a r y i n g degrees 1967; al.  R e u t e r and W o l f f 1981; 1986;  R e u t e r et al.  S i n c l a i r et al.  1987).  i s o l a t e d 51 dominant s u p p r e s s o r s  o f PEV,  1983;  R e u t e r et  (1983)  Su(var)s.  These  f e l l i n t o c l u s t e r e d and  groups on chromosomes two  d e s c r i b e d i n the general i n t r o d u c t i o n . mutants on t h e l e f t arm  (Spofford,  S i n c l a i r et al.  mutants were mapped g e n e t i c a l l y and nonclustered  give  and t h r e e The  o f chromosome two  as  clustered (2L) were found t o  be homozygous l e t h a l , whereas c l u s t e r s on t h e  third  chromosome were i n t i a l l y d e s c r i b e d as b e i n g homozygous viable.  The  l a t t e r S u ( v a r ) s make up t h r e e d i s c r e t e c l u s t e r s  ( p r o x i m a l , m i d d l e and d i s t a l ) on t h e r i g h t arm three  (3R).  o f chromosome  39  Second chromosome S u ( v a r ) s have been a n a l y z e d w i t h respect t o standard modifiers of v a r i e g a t i o n  (temperature  and h e t e r o c h r o m a t i n l o s s ) , as w e l l as b u t y r a t e s e n s i t i v i t y and p o s s i b l e m a t e r n a l e f f e c t s .  Clustered  S u ( v a r ) s on t h e  l e f t arm o f chromosome two (2L) a r e i n s e n s i t i v e t o t e m p e r a t u r e , show l o s s o f v i a b i l i t y butyrate  when t r e a t e d w i t h  ( L l o y d 1986) and show no m a t e r n a l e f f e c t .  In  c o n t r a s t , n o n - c l u s t e r e d 2L mutants a r e temperature s e n s i t i v e , a r e i n s e n s i t i v e t o b u t y r a t e 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 t o  c l u s t e r e d 2L S u ( v a r ) s .  A l l Su(var)s t e s t e d are s e n s i t i v e t o  Y-chromosome (heterochromatin) l o s s Harden 1984).  ( S i n c l a i r e t al. 1983;  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  n o n - c l u s t e r e d S u ( v a r ) s have been suggested based on t h e s e characteristics. R e u t e r e t al. (1986) c h a r a c t e r i z e d 63 i n d e p e n d e n t l y i s o l a t e d X-ray and ethylmethane Su(var) m u t a t i o n s .  s u l f o n a t e (EMS) i n d u c e d  These m u t a t i o n s have been a s s i g n e d t o 12  s e p a r a t e l o c i mapping t o chromosome t h r e e . show v a r i o u s degrees o f homozygous v i a b i l i t y i n a d d i t i o n t o b u t y r a t e and h e t e r o c h r o m a t i n s p e c i f i c t o each o f t h e 12 l o c i .  These S u ( v a r ) s and f e r t i l i t y , sensitivity  I n f a c t , Reuter e t al.  (1986) suggest t h a t c l u s t e r s o f homozygous v i a b l e S u ( v a r ) s on chromosome t h r e e r e p o r t e d by S i n c l a i r e t al. (1983) a r e a l l e l i c t o s i m i l a r l o c i r e p o r t e d i n R e u t e r e t al. (1986): S u - v a r ( 3 ) l , S u - v a r ( 3 ) 2 and S u - v a r ( 3 ) 9 .  40 T h i s study w i l l examine t h e p r e v i o u s l y u n c h a r a c t e r i z e d 3R p r o x i m a l c l u s t e r o f Su(var) m u t a t i o n s .  It will  n i n e Su(var) m u t a t i o n s o r i g i n a l l y a s s i g n e d t o t h i s  include cluster,  mapping between 4 6.4 and 54.2 and some o f which may be a l l e l i c t o S u - v a r ( 3 ) l and/or S u - v a r ( 3 ) 2 .  These dominant  m u t a t i o n s have been p h y s i c a l l y mapped u s i n g new compound chromosome f o r m a t i o n and d e f i c i e n c i e s . l o s s of heterochromatin  and m a t e r n a l  determined, and homozygous v i a b i l i t y established.  The s e n s i t i v i t y t o  e f f e c t s has been and f e r t i l i t y have been  C h a r a c t e r i z a t i o n u s i n g t h e above c r i t e r i a  better defined t h i s cluster into genetic l o c i  has  and has  provided information t o f u r t h e r i n v e s t i g a t e the functions of these c l u s t e r e d Su(var)s.  41 MATERIALS AND METHODS  Stocks A l l 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 i s either a very small deletion, or a point mutation  (A. Dutta).  s  C(3L)ri;C(3R)e i s 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) i s 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  Df(3L)78A3; 79E1,2  DIS 65, p.47  ,pP  Df(3R)83E3; 84A4,5  Hazelrigg and Kaufman (1983)  , rede  Df(3R)84A4,5; 84C1,2  II  Df (3'R) 84B1,2; 84D1,2  II  Df(3R)84Bl;2 + 84D5;84F4  II  1. D f O L J P C ^  2. Df (3R)  Dfd  w+Rx2  3. D f ( 3 R ) S c x 4. D f ( 3 R ) S c x 5. Df  +Rxl3  w+Rx4  (3R)Hu  ,rede  +Rxl  30  6. Df (3R) p ,rede al. 62  Df(3R)84F4-6; 85D3-5  Kemphues et (1983)  7. Df (3R) by , rede  Df(3R)85D11-14; 85F6 + Dp of Ant-C + more on Y  8. Df (3R)E-075  •Df (3R) 86E20; 87B8,9  9. Df(3R)E-078 10. Df (3R) 125c 11. D f ( 3 R ) k a r  sz11  *point mutation Df(3R)87El; F12,13 Df(3R)87C7/8; 87E5,6  II  Reuter et al. (1987) Ashish Dutta Reuter et al. (1987) 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. 6O0  466  47.0  AMAI *WA»|**4tU»lllh*»U  84185  82*83  2. Df (3R) DfdfRxl^ipp 4. Df (3R) Scx  v+Rx4  ,  +Rxl  {3K)Scx  w+Rx2  ,rede  Mi !Miffr1l»l)I^E7f/f0fl(ltttligi WWWl 1.  AMI AMMNIUI MA HI IIHAIAMIAIIII A> Al IAAM B  C  D  E  A F 8 6 87  62  7. Df OK) by , rede  rede  5. Df {ZK)Hu 3. Df  30  6. Df QK) p , rede  t H!!MH M l  tlAMA 1 1 1 I I I IM'IIHUAHIHIIII iLlUMA* M t l i M l M U Wi"l>a A F C [ P |E B 87 88  8. D f ( 3 R ) £ - 0 7 9  z  11. Df (3R) kar* **>  9. Df(3R)E-078  10. Df(3R)126c " '  MIMIILMIIII » m m B  1  C  44 p r o x i m a l t o t h e centromere on 3R s i n c e g e n e t i c 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 r e b a l a n c e d w i t h TM3,e,Ser to  facillitate  scoring.  D e f i c i e n c y b e a r i n g males were f i r s t  o u t c r o s s e d t o wm4 v i r g i n s t o check f o r s u p p r e s s i n g w h i c h would suggest locus.  ability  a s t r a i n d e f i c i e n t f o r a suppresssor  Pigment assays were p e r f o r m e d as d e s c r i b e d  previously  (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 s o u r c e s were n o t t e s t e d by t h i s method. I n s t e a d , t h e a f f e c t o f t h e Df on wm4 was s c o r e d v i s u a l l y i n b a l a n c e r s i b l i n g s r e s u l t i n g from c r o s s e s d e s c r i b e d below. V i r g i n S u ( v a r ) s were mated t o Df/TM3 males and t r a n s f e r r e d t o new food every 2-3 days. S e v e r a l c r o s s e s were done a t 22, 25 and 29 C, b u t r e m a i n i n g c r o s s e s were c u l t u r e d at  25 C s i n c e no e f f e c t o f temperature  was observed.  Complementation was s c o r e d by c o u n t i n g Df/Su(var) 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 .  I n most c a s e s ,  a minimum o f 100 f l i e s were s c o r e d where 1/3 were t o be t h e d i a g n o s t i c c l a s s .  flies  expected  I n non-complementation c a s e s ,  t h e c r o s s e s were r e p e a t e d so t h a t no l e s s than 100 f l i e s were s c o r e d .  ii) was  A compound chromosome t h r e e s t r a i n ,  C(3L)ri;C(3R)e  s  used t o map S u ( v a r ) s r e l a t i v e t o t h e centromere o f  chromosome t h r e e .  Two S u ( v a r ) s , B143 and A63,  w i t h t h i s method.  These mutants were chosen based on t h e i r  were mapped  45 genetic map positions as being most l i k e l y to define the outermost positions i n the c l u s t e r .  A summary of the  protocol and strategy i s given i n Figure 5. wm4; Su(var)/TM3 v i r g i n females were collected every four hours and subjected to 2500 rads of gamma i r r a d i a t i o n . Approximately 3300 treated v i r g i n s 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 r e s u l t i n g i n new compound chromosome formation and nondysjuction events w i l l contribute to viable F l progeny.  Almost a l l other events  w i l l result i n genetically unbalanced, and therefore inviable embryos. Since the compound stock d i d not have a wm4 rearrangement, only males show presence or absence of a suppressor d i r e c t l y .  New compound females were tested for  presence or absence of the Su(var) mutation by backcrossing to patroclinous males as shown i n Figure 6.  Of the r i  progeny recovered, one h a l f w i l l show a wm4 phenotype i f the suppressor i s not present.  E f f e c t of heterochromatin deficiencies on Su(var) a c t i v i t y  Two tests were used to determine what effects a l t e r a t i o n i n 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, Sb Ser  x  + ;C (3L) r i ; C (3R) es  r  wm4;C(3l>) ;C (3R)  NO COMPOUND \  ri > /  3 L  3L ri  3 L /  COMPOUND RIGHT  \/  new compound formation  COMPOUND LEFT  NONDYSJUNCTION Su(var)/TM3 or 0  3R  3 L / \ 3 R  3R  3L\ / 3 R  3 R  3 L / \ 3 R  e ( s  \  NONDYSJUNCTION:  MATROCLINOUS  AND PATROCLINOUS  PROGENY  3 L \ / 3 R  , \/ 3L  3R  3L/13R  3L / \ 3 R 3L\ /3L  0 3 R / \ 3 R s  *patroclinous progeny, r i , e phenotype l i k e male parent **matroclinous progeny, Su(var)/TM3 phenotype l i k e 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? + new compound virgin female  x  wn?4;C(3L) ri;C(3R)e Y patroclinous male  EXPECTED PROGENY CLASSES PHENOTYPE:  r i only **wm4;  C(3L)ri;C(3R)Su  +;C(3L)ri;C(3R)Su?  e  s r  ri  wm4;C (3L) ri;C (3R) e  + ;C (3L) ri;C (3R) e  s  s  *This newly formed compound chromosome possibly carries a Su(var) mutation, masked by wild type allele for white. **If the Su(var) i s present on C(3R), a l l of these progeny w i l l be red-eyed, due to suppression of wm4. If some wm4 progeny are found, Su(var) must be on 3L.  s  48 i)  Loss o f Y chromosome:  By u s i n g a s t r a i n w i t h t h e  a t t a c h e d - X chromosome, C(l)RM,pn, c r o s s e s were performed t o produce males c o n t a i n i n g no Y chromosome (see F i g u r e 7 ) . These males were compared t o males which d i d r e c e i v e a Y chromosome from an a t t a c h e d - X background.  B o t h genotypes  were s u b j e c t e d t o pigment a n a l y s i s as d e s c r i b e d 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 t h e b a l a n c e r homolog s e r v e as i n t e r n a l controls.  ii)  Loss o f 2R h e t e r o c h r o m a t i n :  f o r 2R h e t e r o c h r o m a t i n o n l y .  Df(2R) MS-210 i s d e f i c i e n t  The e f f e c t o f removing t h i s  s p e c i f i c segment o f h e t e r o c h r o m a t i n on t h e e x p r e s s i o n o f t h e Su(var) phenotype was examined u s i n g t h e p r o t o c o l shown i n F i g u r e 8.  F l i e s were r a i s e d a t 25 C t o ensure a c c u r a t e  s c o r i n g o f t h e Curly  wing phenotype.  Maternal E f f e c t s  To check f o r m a t e r n a l e f f e c t s i n each o f t h e mutants, r e c i p r i c a l c r o s s e s 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 t h e c r o s s e s a t 18, 22 and 29 C.  Su(var) and b a l a n c e r s i b l i n g s  from m a t e r n a l and p a t e r n a l c r o s s e s r a i s e d a t 22 and 29 C were compared u s i n g pigment a s s a y s as b e f o r e . r a i s e d a t 18 C were s c o r e d v i s u a l l y .  Crosses  FIGURE 7:  PROTOCOL USED TO DETERMINE SENSITIVITY OF SU(VAR)S TO LOSS OF THE Y CHROMOSOME  EXPERIMENTAL: A *XX + 0  wm4  x  +  Y  wm4 Su(var)  Su(var) TM3,SJb, Ser  wm4  and  pigment assay 0  0  TM3,Sb Ser r  CONTROL: A  wm4 Su(var)  XX +  Y  x  +  **wm4 Su(var) ;  Y wm4  and  pigment assay Y  **ST0CK CONSTRUCTION: A XX X 0 A XX  TM3,Sb,Ser  (Oregon-R)  x A XY  A XY  x *^X=C (1) RM  TM3,Sjb,Ser  0 A XX —  STOCK  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  wm4 + Su (var)  • __  • __ • __________________  +  CyO  |  Y  + TM3, 'Sb,Ser  wm4 Df (2R) M-S210 Su (var)  • —___—_——_______—_____ • ____________  99.dy  "  +  +  pigment assay  wm4 CyO Su(var) + CONTROL: wm4  CyO  +  « ^  wm4 + +  •  \T  «  wm4 Df (2R) M-S210 +  99 * o"o"  w  m  4 D  f (  2  R  )  Y  M  -  5  2  1  °  +  +  «  + +  pigment assay  FIGURE 9:  RECIPROCOL CROSSES USED TO DETERMINE EFFECTS FOR 3R SU(VAR)S  MATERNAL  PATERNAL CROSS:  wm4 +  wm4 S u ( v a r ) x  wm4 +  Y  TM3,S2>, S e r  wm4 TM3,Sb,Ser  wm4 Su (var) and  ;  +  MATERNAL  +  pigment assay  CROSS: wm4 Su (var) wm4 TM3,  wm4 +  Sb, Ser  as  above  x  Y  +  pigment  assay  52  Viability and f e r t i l i t y studies/complementation tests  The f i r s t step in characterizing the cluster was to construct marked stocks of each Su(var).  This was  accomplished by f i r s t 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 f a l l s approximately 6 map  units from Glued on the left and 8 map units from Stubble its right.  to  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 v i a b i l i t y and f e r t i l i t y .  Each M-Su(var)  was crossed back to i t s 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 f e r t i l e .  If  no larvae resulted, homozygous s t e r i l i t y was assumed. Once homozygous phenotypes had been established,  inter  se complementation tests were done. Since i t 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  wm4  x  wm4  GI Sb H  wm4  x  wm4  +  GI Sb H TM3  x  wm4 TM3S£>Ser  TmSbSer  Ly  FIGURE 10b:  Ly  wm4  + M(3)w  -;  Y TM3eSer  Ly  TM3 e Ser  STOCK  e Ser  PROTOCOL FOR CONSTRUCTION OF DOMINANTLY MARKED TM3 BALANCED STRAIN LACKING STUBBLE  wm4  GI Sb H  wm4  TM3 e Ser  wm4  x  wm4  ;  Ly  TM3 e Ser  Y  Ly TM3 Sb Ser  cfc? * QQ  54  FIGURE 11:  PROTOCOL FOR CONSTRUCTING MARKED STOCKS OF SU(VAR) MUTATIONS wm4 GI Sb  wm4 S u ( v a r ) Y ' T M 3 Sb Ser wm4  wm4 TM3  + Su (var) +  wm4 GI  wm4  x  Sb  wm4  x •i  + Su(var) +  Y  +  +  Ly  Ser  Sb  wm4 GI  TM3 e S e r  Sb  wm4  TM3 e Ser wm4  e Ser  TM3 e  TM3 e Ser wm4  H  GI  Su(var) + cross over between GI and S u ( v a r )  TM3 e S e r  + Su (var) Sb  wm4  GI + + cross over between Sb and S u ( v a r )  /  'TM3  e Ser  TM3 e S e r  TO ESTABLISH MARKED STOCKS: wm4 + S u ( v a r ) Sb  TM3 e S e r  wm4  x  parental  Ly TM3  e Ser  wm4 + S u ( v a r ) Sb ; MARKED STOCK TM3 e S e r  55  TABLE 7:  MARKED STOCKS ESTABLISHED FOR S U ( V A R ) I I I  M - Su (var)A63;  C - A63, Sb A B D E F  M - Su (var)A57:  -  GI, GI, GI, GI, GI,  M - Su (var)B76:  A63 A63 A63 A63 A63  A - A57, Sb B - A57, Sb  D - B94, Sb B - GI, B94 E - GI, B94  M - Su (var) C119:  A - Cll9, C - Cll9,  Sb Sb  A - GI, C119 C - GI, Cll9 M  Su (var)B143i  B - B143, Sb C - B143, Sb F - B143, Sb A - GI, B143 B - GI, B143 E - GI, B143  M - Su (var) A48,  B C D E  -  A48, A48, A48, A48,  Sb Sb Sb Sb  A - GI, A48 B - GI, A48 C - GI, A48  A - B76, Sb A C D E F  M -  Su(var)A130  B - GI, A57 M - Su (var)B94,  MUTATIONS  M - Su (var)A160  -  GI, GI, GI, GI, GI,  B76 B76 B76 B76 B76  A130 GI Sb A130 GI + A130 + Sb C - A160 Sb D - A160 Sb  56 t h a t second s i t e l e t h a l s would map t o t h e same p o s i t i o n s i n d i f f e r e n t S u ( v a r ) s , o r i g i n a l Su(var)/TM3 s t o c k s were used. F l i e s were mated i n each p a i r w i s e c o m b i n a t i o n . 150 f l i e s were s c o r e d f o r each c r o s s .  A minimum o f  F a i l u r e t o complement  i s seen i f l e s s t h a n 10% e x p e c t e d progeny e c l o s e and s u r v i v e , o r i f t r a n s h e t e r o z y g o t e s show i n f e r t i l i t y .  Statistics  S t a t i s t i c a l t e s t s between two mean v a l u e s were c a r r i e d out u s i n g an u n p a i r e d t - t e s t  (Zar 1984).  Differences  between 3 o r more means were t e s t e d u s i n g a n a l y s i s o f variance test  (ANOVA) f o l l o w e d by t h e Neuman-Keuls m u l t i p l e range  (Zar 1984).  V a l u e s e x p r e s s e d as p e r c e n t a g e s were f i r s t  t r a n s f o r m e d t o t h e i r a r c s i n e v a l u e , 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 , b e f o r e s t a t i s t i c a l t e s t s were done. V a l u e s a r e e x p r e s s e d as mean +/- s t a n d a r d e r r o r . t a k e n as t h e l i m i t o f s i g n i f i c a n c e .  P<0.05 was  RESULTS  D e f i c i e n c y mapping  Deficiencies  capable o f s u p p r e s s i n g 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 siblings.  Out  wm4;balancer/+  o f 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 t h e centromere on 3R o n l y Df(3R)Sex  2 + R x 2  a suppressor  one,  , r e d e shows a pigment l e v e l c l o s e t o t h a t o f (see Table 8 ) .  However, i t s b a l a n c e r  shows t h e same amount o f s u p p r e s s i o n .  The  o n l y case where  t h e e f f e c t s o f a d e f i c i e n c y and i t s b a l a n c e r significantly different i s Df(3R)Dfd  + r x  ^3.  siblings  are i  n  this  case,  t h e d e f e c i e n c y 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 t h a n i t s b a l a n c e r s i b , s u g g e s t i n g t h a t D f ( 3 R ) D f d may l o c u s w h i c h enhances PEV, 079,  an En(var)  E-078, k a r s z l l and 126c  Reuter  delete a  l o c u s . D e f i c i e n c i e s E-  show s u p p r e s s i o n a c c o r d i n g t o  e t a l . (1987), making t h e s e d e f i c i e n c i e s good  c a n d i d a t e s f o r mapping S u ( v a r ) 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 s u p p r e s s i n g a b i l i t y when observed D f / b a l a n c e r s i b l i n g s o f Df/Su(var) heterozygotes Two Su(var)s  flies.  in  Df/Su(var)  show no abnormal eye phenotype.  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 o f t h e (see Table 9 ) .  S u ( v a r ) s A57  t h e p r e s e n c e o f Df(3R)E-079.  and A63  are l e t h a l i n  These S u ( v a r ) s l i k e l y map  to  t h e r e g i o n spanned by t h i s d e f i c i e n c y . Df(3R)£-078, w h i c h  58  TABLE 8:  EFFECTS OF DEFICIENCIES ON WM4 VARIEGATION  DEFICIENCY GENOTYPE (wm4 background)  IN THE 3R PROXIMAL REGION  % OREGON-R PIGMENT + S.D.  SIGNIFICANT DIFFERENCE, p<.05  control: wm4/Y; + / +  7.9  Df(3R)Scx  w + R x 4  ,rede/+  TM3/+  +  0.9 no  0.0  Df(3R)Dfd  + R x l 3  ,pP/+  TM3/+  Df(3R)Scx TM3/+  w + R x 2  ,rede/+  •  Df ( 3 R ) # u * +i  xl  Df (3R) p ,rede/ 30  9.0  +  1.9  19.4 +  3.9  4 8.1  +  43.7 +  TM3/+  TM3/ +  1.7  +1.6  +  yes  5.3 5.7  1.3  +  1.3  2.4  +  0.7  4.8  +  2.1  4.1  +  1.3  no  no  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 c o r r e s p o n d 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 c o m p l e t e l y 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 m u t a t i o n f a i l s t o complement w i t h Su (var) A57,  b u t not A63,  are separable l o c i .  s u g g e s t i n g t h a t t h e s e two Su (var) s  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 , c r o s s e s were r e p e a t e d w i t h marked s t o c k s o f S u ( v a r ) s A57 and  A63. A63  I d e n t i c a l r e s u l t s were found, s u g g e s t i n g t h a t S u ( v a r ) s and A57 a c t u a l l y map i n t h e r e g i o n o f 8 6E-87B.  Compound Mapping New compound chromosome f o r m a t i o n was undertaken a s s i g n l o c a t i o n s f o r Su(var)A53 the centromere.  to  and B143 t o e i t h e r s i d e o f  F l i e s w i t h e i t h e r the e  s  o r r i phenotype,  but not b o t h , i n d i c a t e d a new compound f o r m a t i o n .  Since the  marked compound s t o c k d i d not c a r r y a wm4 rearrangement, t h e presence  o r absence o f a s u p p r e s s o r c o u l d be seen d i r e c t l y  o n l y i n t h e males. homozygous l e t h a l t h e occurance  S u ( v a r ) s t e s t e d were e f f e c t i v e l y ( p o s s i b l y due t o second s i t e l e t h a l s ) so  o f a wm4 male w i t h a new compound must be used  i n d i r e c t l y t o a s s i g n t h e Su(var) t o 3L o r 3R. Su(var)B143 c r o s s e s y e i l d e d 5 new compound (see Table 10) i n c l u d i n g one r i male which was informative.  formations immediately  S i n c e t h e male was wm4; C ( 3 R ) ; C ( 3 L ) r i and  shows a wm4 phenotype, Su(var)B143 i s not on 3R.  A  female  o f t h e same phenotype was progeny t e s t e d and found t o be Su(var)+, 11).  c o n f i r m i n g t h a t Su (var) B14.3 maps t o 3L (see Table  Three e  s  females r e c o v e r e d had t h e genotype wm4/+;  61  TABLE 10:  SUMMARY OF NEW COMPOUND PROGENY RECOVERED FROM GAMMA RADIATION SCREEN  SU(VAR) SUBJECTED TO GAMMA TREATMENT  GENOTYPE OF NEW COMPOUND PROGENY C(3L);C(3R)e  s  LOCATION  C(3L)ri;C(3R)  4 males, wm4 phenotype no progeny  Su(var)A63 TM3 Sb Ser  3R  5 females, w i l d type eye non-virgins  1 male, wm4 phenotype Su(var)B143 :  — TM3 Sb Ser  3 females, sterile  *progeny t e s t r e s u l t s shown i n Table 11  3L 1 female, w i l d type eye *progeny t e s t e d  62  TABLE 11: RESULTS OF PROGENY TESTING FOR NEW COMPOUND FEMALES HETEROZYGOUS FOR WM4; TEST FOR PRESENCE OR ABSENCE OF  GENETIC CROSS:  SU(VAR)B143.  wm4;C(3L)ri;C(3R) x wm4;C (3D r i ; C (3R) e + Y ( p a t r o c l i n o u s male) s  RESULTING PROGENY PHENOTYPE:  r i only  wm4;C (3L) r i ; C (3R)  3 wm4 males 2 wm4 females +;C(3L)ri;C(3R) 6 males 3 females  ri,  e  k  wm4;C(3L) ri;C(3R)  e  s  4 wm4 males 5 wm4 females + ;C (3L) r i ; C ( 3 R ) e  s  4 males 3 females  I f Su(var) i s p r e s e n t on C(3R), a l l r i o n l y progeny w i l l have r e d eyes. I f some r i o n l y progeny a r e p r e s e n t w i t h wm4 eyes, t h e Su(var) must n o t be p r e s e n t . S i n c e wm4, ri progeny r e s u l t from t h e c r o s s , Su(var)B143 p r e s e n t on C(3R) and must be on 3L.  i s not  s  C(3L) B143;C (3R) e .  They were a l l s t e r i l e ,  based on spontaneous Nine new  B143 homozygotes o b s e r v e d i n s t o c k .  compound f o r m a t i o n s were r e c o v e r e d from  Su(var)A6"3 c r o s s e s (see T a b l e 1 0 ) . genotype  which i s e x p e c t e d  wm4;C(3L)/C(3R)e . s  A l l males were o f t h e  S i n c e t h e y were a l l  white  m o t t l e d , S u ( v a r ) A 5 3 can be a s s i g n e d t o t h e r i g h t arm o f chromosome t h r e e .  Females r e c o v e r e d 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 o f h e t e r o c h r o m a t i n l o s s on Su(var)  i) to  C(l)RM,pn/0  activity  females were c r o s s e d t o Su(var)/TM3  males  produce male progeny d e f i c i e n t f o r t h e Y-chromosome.  Pigment X/O;  l e v e l s f o r t h e s e f l i e s are shown i n T a b l e 12.  Su(var) male f l i e s a r e compared t o X/Y;  Su(var)  from a c r o s s which c o n t r o l s f o r any e f f e c t s o f  These flies  C(l)RM,pn  background. The l o s s o f a Y chromosome has a d r a m a t i c and s i g n i f i c a n t e f f e c t on 8 o f 9 S u ( v a r ) s t e s t e d .  Su(var)A130  i s t h e o n l y s t r a i n which shows no enhancement o f v a r i e g a t i o n due t o l o s s o f t h e Y chromosome. mutants  found t o map  I t i s a l s o one o f t h e  o u t s i d e the c l u s t e r  (see a p p e n d i x ) .  However, t h e o t h e r n o n - c l u s t e r e d s u p p r e s s o r t e s t e d , Su(var)A2 60 shows a l a r g e d e c r e a s e i n pigment, almost down to  wm4  levels.  A l l o t h e r S u ( v a r ) s a r e s t r o n g l y a f f e c t e d by  l o s s o f a Y-chromosome.  These r e s u l t s a r e 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 4.9  2.5 ± 0.3  4.6 + 1.1  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  103.8 + 2.9  0.3  wm4/0  wm4;Su(var)  A130 A160  103.3 ±2.3 14.4 ± 3.0  74.8  13.8  10.8  Values given are mean + S.D.;c r i t i c a l t--value, df(8)=2.31  65  those r e p o r t e d by S i n c l a i r et al.(1983)  and Harden  (1984)  f o r a l l S u ( v a r ) s s t u d i e d , e x c e p t i n g Su(var)A130.  ii)  Df(2R)M-S210 i s known t o enhance v a r i e g a t i o n  (Morgan  et al. 1941) and i s d e f i c i e n t f o r centromeric heterochromatin on the r i g h t arm o f chromosme two and Holm 1975).  (Hilliker  Although t h i s d e f i c i e n c y i s a l s o Minute i n  phenotype, t h i s mutation has been shown t o have no s i g n i f i c a n t e f f e c t on v a r i e g a t i o n o f wm4 (Harden  1984).  F l i e s were r a i s e d 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 h i g h wm4 c o n t r o l v a l u e s shown i n Table 13. Df(2R) MS-210 e f f e c t s wm4 v a r i e g a t i o n at t h i s temperature, c a u s i n g an enhanced phenotype or r e d u c t i o n i n pigment l e v e l s .  In a d d i t i o n , Df(2R) MS-210 s i g n i f i c a n t l y  reduces pigment i n 8 o f 9 suppressors t e s t e d . c a b a b i l i t y o f Df(2R)MS-210  The enhancing  i s much weaker than t h a t caused  by l o s s o f the Y-chromosome, but i s c o n s i s t e n t throughout. A g a i n , only Su(var)A130 i s completely u n a f f e c t e d by l o s s o f heterochromatin.  In g e n e r a l , both males and females are  a f f e c t e d by Df(2R) MS-210, but females appear more s u s c e p t a b l e t o the heterochromatin l o s s .  Where both sexes  are i n f l u e n c e d , females i n v a r i a b l y show a l a r g e r d i f f e r e n c e i n comparing Minute siblings.  individuals to their  CyO-balancer  The e x c e p t i o n 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 b a l a n c e r s i b s . These r e s u l t s are c o n s i s t e n t w i t h the f i n d i n g s o f 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) v a l u e s are % Oregon-R pigment + S.D.  + 0.5  17.4 + 1.7  t (8) =12.3  9.9 + 1.0  40.3 + 2.7  t (8) =23.7  male  69.4 + 4.5  89.9 + 3.8  female  72.5 + 6.8  83.0 + 6.8  t (7) =2.0*  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  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  male  67.0  81.3  t (8) =3.2  wm4; +male female  B76  C119  A63  B143  A48  A57  B94  A160  A130  t-value  9.6  7.8  6.6  ' t (8)= 6.6  female  41.7 + 5.1  76.7 + 3.5  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  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  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  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  male  69.6 + 1.5  73.9 + 4.8  t (8) = 1.5*  female  70.5  72.1  ±  t (8 =0.6*  3.6  3.6  t (8) =11.8  t - v a l u e s are given with degrees o f freedom i n ( ) ; c r i t i c a l t v a l u e , df(8)=2.31; df(7)=2.37 * i n s i g n i f i c a n t d i f f e r e n c e 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. the crosses are presented in Tables 14 and 15.  Results of 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 (wm4 background) B143/+  TM3/+ B76/ +  TM3/ + A57/ +  TM3/ + A48/ +  TM3/ + B94/ +  TM3/ + A63/+  TM3/ + C119/+  TM3/ + A130/+  TM3/ + A160/ +  TM3/ +  PATERNAL CROSS MATERNAL CROSS t-value values are % Oregon-R pigment + S.D.  male female male female  54.9 32.1 19.8 5.8  + + + +  5.4 7.9 3.3 1.7  43.4 47.9 7.6 13.0  + + + +  5.1 6.4 1.7 3.3  t (8)=3.1* t (8)=3.1* t (7)= 6.6* t (8)=4.2*  male female male female  75.5 75.6 4.4 3.5  + + + +  9.0 4.4 1.5 2.4  82.6 70.7 9.2 7.9  + + + +  4.4 10.1 1.6 2.5  t (8)=1.5 t (7)=0.9 t (8)=4.5* t (7)=2.4*  male female male female  69.1 54.1 20.5 15.8  + + + +  4.9 9.5 5.0 5.6  36.8 52.5 8.8 14.8  + + + +  2.9 5.1 3.0 3.3  t (8)=12.3* t (8)=0.4 t (8)=3.8* t (7)= 0.2  male female male female  76.3 54.8 15.7 9.3  + + + +  3.1 6.8 1.4 3.4  43.8 56.9 5.3 17.4  + + + +  6.1 2.5 1.8 2.7  t (8)=10.2* t (8)= 0.7 t (5)= 6.9* t (8)=3.9*  male female male female  75.1 57.3 15.8 6.7  + + + +  2.9 5.8 4.7 3.8  55.6 68.1 7.8 37.7  + + + +  2.8 4.7 5.9 6.0  t (8)=5.9* t (8) =3.1* t (8) =0.9 t (8) = 8.6*  male female male female  71.4 52.5 10.9 5.1  + + + +  9.1 7.7 2.4 2.6  50.9 64.8 11.7 15.2  + + + +  5.4 3.2 3.7 2.4  t (8) =4.3* t (8) =3.4* t (8) =1.2 t (8) =5.8*  male female male female  65.6 50.7 15.6 5.7  + + + +  5.2 5.2 3.2 1.1  67.5 67.3 14.8 27 .2  + + + +  2.4 3.1 1.2 5.9  t (8) =0.8 t (8) =5.6* t (8) =0.5 t (8) =10.4*  male female male female  79.4 72.1 9.4 3.3  + + + +  1.2 7.6 0.9 1.4  65.4 71.8 5.4 14.0  + 6.6 + 8.3 + 1.5 ± 2.0  t (8) =7.0* t (8) =0.0 t (8) =4.8* t (8) =8.6*  male female male female  66.7 50.8 18.1 12.4  + + + +  5.8 5.7 3.3 3.4  62.7 58.9 8.4 10.9  ± 3.7 ± 8.0 ± 2.1  t (8 =1.2 t (8 =1.7 t (8 >=5.0* t (8 1=3.0  wm4 control values: males, 7.0 t-values are given with degrees values: df(8)=2.31, df(7)=2.37, * s i g n i f i c a n t difference between  + 3.0  + 2.2; females, 9.2 + 2.3. of freedom i n (); c r i t i c a l t df(5) = maternal and paternal progeny  69 TABLE 15:  MATERNAL EFFECTS MEASURED AT 29 C  GENOTYPE SEX (,wm4 background) B143/+  TM3/ + B76/ +  TM3/ + A57/ +  TM3/ + A48/ +  TM3/ + B94/ +  TM3/ + A63/ +  TM3/ + C119/+  TM3/ + A130/+  TM3/ + Al60/+  TM3/ +  PATERNAL CROSS MATERNAL CROSS t - v a l u e v a l u e s are % Oregon--R pigment + S.D.  male female male female  50.5 58.7 19.7 20.5  + + + +  4.8 4.5 6.3 3.6  47.8 56.1 13.2 34.1  + + + +  3.2 6.2 4.7 9.9  t t t t  male female male female  82.2 91.4 9.7 32.4  + + + +  4.4 6.2 1.4 4.2  93.0 96.0 17.3 55.0  + + + +  3.5 4.7 3.5 5.9  t (8) =3.5* t (8) =1.3 t(8) =4.9* t (8)=5.7*  male female male female  (8) =0.9 (8) =0.7 (8) =1.6 (8) =3.2*  see Table 17  male female male female  59.0 73.4 16.0 24.6  + + + +  7.8 5.8 4.6 2.5  63. 9 85.4 16.3 41.1  + + + +  6.2 6.1 2.7 6.1  t t t t  (8) =5.4* (8) =2.2 (8)=0.2 (8)=5.6*  male female male female  60.7 67.4 19.6 26.0  + + + +  1.9 6.2 1.7 6.5  78.8 95.0 35.8 71.8  + + + +  2.0 1.6 6.1 6.9  t t t t  (8) =13.4 (6) = 9.2* (7) =7.0 (8) = 9.0*  male female male female  79.9 91.3 25.0 34.9  + + + +  5.0 5.2 3.1 5.4  94.8 102.2 28.4 67.3  + + + +  4.6 1.2 6.6 4.6  t t t t  (8) =4.4* (8) = 6.9* (8) = 1.0 (7) =8.3*  male female male female  89.5 100.0 29.7 36.5  + + + +  4.8 2.0 5.4  80.2 97.5 35.6 62.0  + + + +  10.6 1.2 5.8 7.4  t t t t  (8) =1.8 (8) =3.5* (8) =1.3 (8) =5.7*  male female male female  77.2 80.7 14.5 21.5  + + + +  4.6 6.0 2.4 5.5  84.6 86.4 15.4 47.9  + + + +  4.3 3.7. 2.3 6.4  t t t t  (7) =2.1 (8) =1.6 (8) =0.6 (8) = 6.2*  male female male female  59.3 78.4 32.0 53.0  + + + +  5.1 10.6 9.1 4.4  36.9 70.8 14.7 36.0  + + + +  2.2 3.9  t t t t  (8) = 9.2* (8) = 1.9 (8)=3.7* (8) =4.2*  6.8  4.8 7.2  wm4 c o n t r o l v a l u e s : males, 12.0 + 3.4; females, 46.6 + 4.9. t - v a l u e s are given w i t h degrees o f freedom i n ( ) ; c r i t i c a l t v a l u e s : df(8)=2.31, df(7)=2.37, df(6)=2.45 * s i g n i f i c a n t d i f f e r e n c e between maternal and p a t e r n a l progeny  70 F l i e s 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 s i g n i f i c a n t .  Seven of nine maternally  derived female wm4;+/TM3 progeny show higher pigment levels than t h e i r paternally derived counterparts. recognized as a maternal e f f e c t .  This i s  The maternal effect i s  observed only i n the female progeny except for the case of Su(var)B76", i n which both males and females show a maternal effect.  Su(var)s B94 and C119 are most sensitive to  maternal e f f e c t s , 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 t h i s temperature. Males show a different trend.  Five out of nine  Su(var)s show wm4;+/TM3 males from paternal crosses with pigment levels s i g n i f i c a n g l y higher than analogous maternally derived males.  The suppression seen i n these  Su(var)+ males i s l a b e l l e d a paternal e f f e c t .  The paternal  effects observed are weaker than maternal effects seen i n females; Su+ females show r e l a t i v e l y high pigment levels compared to Su+ males and females of paternal crosses, as well as to wm4 control values at t h i s 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 s i g n i f i c a n t l y 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) PARENT  MALE  A57  22 C  29 C  FEMALE  MALE  FEMALE  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 t h e i r maternal analogs.  In t h i s case, both  males and females exhibit a maternal e f f e c t . At 29 C, 8 of 9 Su(var) crosses have females which demonstrate a maternal e f f e c t .  The strongest e f f e c t s with  respect to pigmentation are observed i n 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 l e v e l s . The most interesting case i s that of Su(var)A57. I n i t i a l l y , t h i s suppressor was labeled temperature sensitive maternal l e t h a l 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 i s lowest for progeny maintained  at 2 9 from oogenesis through pupation. A strong maternal effect i s observed i n females, but only when oogenesis takes place at 2 9 C.  These females appear completely suppressed.  (Flies were scored v i s u a l l y since pigment assays require a minimum of 25 f l i e s per genotype). Su(var)A2 60 i s an exception to the female s p e c i f i c maternal e f f e c t .  Both males and females i n t h i s reciprocal  cross exhibit a paternal e f f e c t .  This i s f a i r l y consistent  73  TABLE 1.7: MATERNAL EFFECTS OF SU (VAR) A57 AT DIFFERENT DEVELOPMENTAL TEMPERATURES  DEVELOPMENTAL TEMPERATURE  PATERNAL  TM3/+ MALE FEMALE GROUP I (4 v i a l s ) no. o f progeny: phenotype: GROUP I I (8 v i a l s ) no. o f progeny: phenotype:  30+  30+  wm4  30+ 30+  phenotype:  A57/ + MALE FEMALE  30+  30+  suppressed  30+ 30+  wm4  GROUP I I I (4 v i a l s ) no. o f progeny: 30+  MATERNAL  30+ 30+  30+ 30+  suppressed  30+  wm4  30+  30+  suppressed  TM3/+ MALE FEMALE  3  3 wm4  4 3  5 6  A57/ + MALE FEMALE  3  10  suppressed  4 0  9 16  wm4 suppressed suppressed (80%)  0  2  1  1  supressed suppressed (80%)  GROUP I: Oogenesis @ 22 C, food @ 22 C, developmental temp. 29 C GROUP I I : Oogenesis @ 29 C, food @ 22 C, developemntal temp. 29 C GROUP III:Oogenesis through p u p a t i o n , 29 C. Phenotypes were s c o r e d v i s u a l l y , e s t i m a t e d s u p p r e s s i o n given i n 0 .  74 with observations from 22 C where males showed a paternal effect and females showed no e f f e c t .  Males tend to show no  susceptability to parental effects at 2 9 C.  Exceptions i n  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 e f f e c t .  Homozygous viability/complementation analysis Premliminary experiments suggested that many of the t h i r d chromosome proximal cluster of Su(var)s were homozygous v i a b l e .  The l e t h a l i t y observed was l i k e l y due to  second s i t e l e t h a l mutations.  However, no homozygous l i n e s  were established, suggesting that homozygotes while v i a b l e , 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 o f f any second s i t e l e t h a l s .  In the  process of establishing recombinant l i n e s , the Su(var) mutations were remapped.  It became apparent that two of the  Su(var)s were wrongly assigned to the c l u s t e r . and A160 map to the l e f t of Glued and Stubble, more d i s t a l location on 3L (see appendix).  Su(var)A130 that i s i n a  These mutants  were characterized along with the proximal c l u s t e r , and used as a comparison for nonclustered verses clustered Su(var)s. These marked stocks (refer to Table 7) were crossed to o r i g i n a l Su(var)/TM3,Sb,Ser stocks to look for homozygous viable and f e r t i l e progeny.  Results are given i n Table 18.  TABLE 18: HOMOZYGOUS PHENOTYPES OF PROXIMAL 3R SU(VAR)S  SU(VAR)  *LETHAL  A63  X  A48  X  VIABLE  STERILE  X  B143  X  B76  C119  X  A57  X  B94  X  A160  X  A130  SEMI-LETHAL  X  *Lethality, semi-lethality and s t e r i l i t y 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, f i v e remained homozygous  l e t h a l , even with both chromosome arms crossed o f f . Of the three homozygous viable Su(var)s, only one, Su(var)B76" i s fertile. stock.  This Su(var) s t r a i n i s maintained as a homozygous Su(var)J3143 i s semi-lethal. Few  f l i e s survive (20%  of expected based on Su(var)/TM3 siblings) and these are sterile.  This i s consistent with the observation that  homozygotes which appear within t h i s TM3-balanced stock are sterile  (personal observations).  Su(var)A230 i s completely  viable when homozygous, but i s s t e r i l e .  Su(var)A57 i s  homozygous l e t h a l , except for one s t r a i n , B-Gl A57. Surviving f l i e s are  fertile.  Since many Su(var)s exhibit phenotypes such as homozygous or hemizygous l e t h a l i t y or s t e r i l i t y , inter complementation tests were done. every pairwise combination. 19.  se  Matings were set up i n  Results are summarized i n Table  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 t h i s region, characterized by Reuter et al. (1986) and for 2L Su(var) trans-heterozygotes  (personal communication, Jo-Ann Brock).  TABLE 19:  SU (VAR) STRAIN  COMPLEMENTATION ANALYSIS OF 3R PROXIMAL SU(VAR) MUTATIONS  A63  A48  B143 B76  C119  A57  B94  A160  +  +  +  +  A130  A63  +  +  +  A48  --  FS,W  +'  FS, W +  FS,W  +  +  B143  +  FS,W  +  FS,W  +  +  B76  —  +  +  +  +  +  —  +  FS,W  +  +  —  +  +  +  —  +  +  C119 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 p r o x i m a l c l u s t e r o f Su(var) m u t a t i o n s maps between 4 6.4 + 1.1 and 54.4 + 0.7 map u n i t s . centromere i s a s s i g n e d a map p o s i t i o n o f 4 6.0  Since the ( L i n d s l e y and  G r e l l 1968) i t was p o s s i b l e t h a t t h e s e m u t a t i o n s spanned t h e centromere. Mapping by compound autosome f o r m a t i o n 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 a t l e a s t two l o c i w i t h one on 3L and t h e o t h e r on 3R. D e f i c i e n c y mapping extends t h e 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 t o complement w i t h Df(3R)E-079, b u t  complements Df(3)E-078 w h i l e Su(var)A57 i s l e t h a l w i t h b o t h deficiencies.  As mentioned p r e v i o u s l y , a c c o r d i n g t o  c y t o g e n e t i c 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 point mutation.  T h e r e f o r e , Su(var)A6"3 and  Su(var) A57 p r o b a b l y r e p r e s e n t two s e p a r a t e l o c i .  Their  p o s i t i o n s c o r r e s p o n d r o u g h l y t o t h o s e o f Su-var(3)13  var(3)6  i s o l a t e d and mapped by R e u t e r et al.  and Su-  (1986, 1987).  Su-var(3) 6 and 13 a r e r e c e s s i v e l e t h a l s as a r e b o t h Su(var)A6\3 and A57, s t r o n g l y s u g g e s t i n g t h e s e f o u r suppressors are a l l e l i c p a i r s .  T h i s c o u l d be p r o v e d w i t h  complementation t e s t s between t h e s e Su(var) a l l e l e s .  Such  t e s t s would a l s o c o n f i r m t h a t l e t h a l i t y i s not due t o second s i t e m u t a t i o n s , s i n c e t h e s e would n o t l i k e l y map t o i d e n t i c l e l o c i among t h e s e mutants. Loss o f t h e Y-chromosome s i g n i f i c a n t l y reduces i n a l l Su(var)s previously tested  pigment  ( S i n c l a i r e t al. 1983/  79 Harden 1984).  In e i g h t o f nine Su(var)s t e s t e d i n t h i s  study, v a r i e g a t i o n was enhanced d r a m a t i c a l l y by Y-chromosome l o s s 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 a f f e c t e d by d e f i c i e n c y o f 2R h e t e r o c h r o m a t i n .  This i s i n contrast to  the behaviour o f d i s t a l l y c l u s t e r e d Su(var) no s e n s i t i v i t y t o 2R heterocromatin l o s s Reuter  l o c i which show  (Harden 1984).  et al. (1983) r e p o r t e d a t h i r d chromosome s u p p r e s s o r ,  Su(var)c , 1001  Su(var)clOOl  which was g r e a t l y a f f e c t e d by Df(2R) maps t o 46.7, w i t h i n the 3R proximal  MS-2 . 10  cluster,  s u g g e s t i n g a l l e l i s m t o one or more o f the Su(var) genes i n t h i s study. their  A g a i n , complementation t e s t s c o u l d  determine  relationships. Su(var)A230 i s a s u r p r i s i n g e x c e p t i o n t o the h i g h  heterochromatin  s e n s i t i v i t y e x h i b i t e d by most S u ( v a r ) s .  T h i s suppressor i s not a f f e c t e d by l o s s o f Y chromosome or 2R h e t e r o c h r o m a t i n .  I t i s a n o n - c l u s t e r e d suppressor which  maps near the t i p o f 3L. heterochromatin  This i n s e n s i t i v i t y to  suggests a f u n c t i o n a l d i f f e r e n c e between  Su(var)A130 and other Su(var) genes.  I t has been  h y p o t h e s i z e d t h a t l o s s o f the heterochromatic Y chromosome f r e e s heterochromatic elements which are then a v a i l a b l e t o i n a c t i v a t e genes through 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 a h i g h e r p r o p o r t i o n o f c e l l s , thus enhancing (Zuckerkandl 1974).  variegation  Su(var)A130 may be so s t r o n g t h a t no  amount o f f r e e heterochromatic elements can make up f o r the d e f i c i e n c y o f Su(var)A230 p r o d u c t . may produce a n o n - s t r u c t u r a l product  Alternately,  Su(var)A130  i n v o l v e d i n the c o n t r o l  80  of gene i n a c t i v a t i o n i n PEV. act  For example, t h i s product may  to maintain s t r u c t u r a l decisions.  white  I f early on, the  gene was packaged as heterochromatin, but no signal  (suppressor product) was available to maintain t h i s f a c u l t a t i v e packaging, the white  gene i n a c t i v a t i o n may  be  reversed and that decision c l o n a l l y maintained, r e s u l t i n g i n 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 e f f e c t s .  These suppressors  must act early i n development, before zygotic t r a n s c r i p t i o n begins, and produce a protein product, subject to heat denaturation ( i n a c t i v a t i o n ) .  By doing s h i f t s from  permissive to r e s t r i c t i v e temperatures, developmental a c t i v i t y of the Su(var)s can be determined.  It has already  been determined that Su(var)A57 acts extremely e a r l y , during oogenesis.  Eggs l a i d at 22, but s h i f t e d to 29 C after 24  hours do not show the maternal suppression or the l e t h a l i t y observed i n t h i s s t r a i n 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 i n t h i s genetic background, and therefore exhibit a Su(var) +  phenotype even i f they are genotypically S u ( v a r ) .  81 Genetic characterization i s c r u c i a l to understanding the functions of suppressor l o c i .  By establishing  homozygous v i a b i l i t y , l e t h a l i t y and s t e r i l i t y , i t becomes possible to determine the genetic makeup of the c l u s t e r . 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 l e t h a l or s t e r i l e as marked stocks. fertile.  Only Su(var)B75 i s homozygous viable and  The p o s s i b i l i t y exists that second s i t e lethals  may remain on those portions of chromosome not replaced by recombination.  However, i t seems r e l a t i v e l y unlikely that  a l l f i v e homozygous l e t h a l stocks carry a second s i t e l e t h a l 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)  l o 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 l e t h a l i t y observed in the o r i g i n a l balanced stocks. The assumption of second s i t e lethals has been discussed by Nash et al. (1983).  Their concern i s that EMS  mutagenesis i s quite e f f e c t i v e at inducing "many" extraneous l e t h a l mutations which may mask a haplo-specific l e t h a l mutation by exhibiting homozygous l e t h a l i t y not due to the mutation i n question. They define a locus as being haplos p e c i f i c l e t h a l i f i t i s l e t h a l over a deficiency , but not l e t h a l as a homozygote.  Two of the 3R Su(var) l o c i have  demonstrated l e t h a l i t y over Df(3R)E-079, one of which shows  82 semi-lethality i n one recombinant s t r a i n , B - G1,A57.  If  t h i s semi-lethality i s due to crossing o f f a second l e t h a l , or even haplo-specific l e t h a l , i t may represent a locus such as Nash et a l .  (1983) suggest.  Although t h i s s t r a i n i s not  completely v i a b l e , survivors are f e r t i l e , suggesting that i n t h i s case, an extraneous phenotype observed.  l e t h a l was involved i n the l e t h a l  This p o s s i b i l i t y should be of concern  to investigators of dominant recessive l e t h a l 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 i n a l l inter Since Su{var)B143  se combinations.  maps to 3L, a l l 4 a l l e l e s of t h i s locus  can be t e n t a t i v e l y assigned to t h i s l o c a t i o n . (1986) i d e n t i f i e d a locus, Su-var(3)3, and c y t o l o g i c a l l y  Reuter et a l .  which maps genetically  (via new compound formation) near the  centromere on 3L. They report that mutants assigned to t h i s 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 i n B143 homozygotes exactly, strongly suggesting a l l e l i s m to Reuter's Su-var  (3) 3.  Su(var) trans-heterozygotes i n the complementation group observed i n t h i s study are completely female s t e r i l e with no eggs produced.  They are also semi-lethal i n 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 transheterozygous phenotypes and physical mapping.  recombination units 0 10 20 30  40  61 c y t o l o g i c a l map  GI 80  —  A160  A130  46 50  x  86-87 Sb  Xf-  B143 B76 A57 A63 A48 B94 Cll 9  x=centromere  60  100  84  Su(var)B143.  When studying dominant mutations, i t i s always possible that trans-heterozygous phenotypes are a result of interactions between separate l o c i .  In f a c t , t h i s has been  observed for Su(var)s i n the 2L group (personal communication, Jo-Ann Brock).  However, the observed  behavior i s best attributed to i n t e r - a l l e l i c f a i l u r e to complement for the following reasons.  F i r s t , a l l four  mutations i n question have genetic map positions with overlapping 95% confidence l e v e l s , strongly suggesting one locus.  Second, no interaction phenotypes were observed for  any other Su(var)s tested, four of which are p h y s i c a l l y separated from t h i s complementation group: A63  Su(var)A5 7 and  by cytogenetic analysis and Su(var)A130 and Al60  genetic mapping.  by  This question could be further addressed  by mapping the Su(var) l o c i i n question by compound-autosome formation.  This could be done by capturing the newly formed  compound with a compound s t r a i n that carries a wm4 rearrangement (see appendix).  A result showing 3L locations  would strongly support the hypothesis that these Su(var) l o c i are a l l e l i c . Of the remaining Su(var) mutants, only one i s a none s s e n t i a l locus, Su (var) B76".  It i s homozygous viable and  f e r t i l e and shows no interactions with the clustered or nonclustered Su (var) s tested.  Su(var)B76" does, however, react  to heterochromatin l o s s , just as the essential l o c i do, suggesting a s i m i l a r function.  85  Locke et al. (1988) have h y p o t h e s i z e d t h a t a l l dominant m o d i f i e r s of PEV  are dosage s e n s i t i v e and f a l l  two  C l a s s I m o d i f i e r s are h a p l o - i n s u f f i c i e n t ,  categories.  i n t o one  s u p p r e s s i n g when hemizygous over a d e f i c i e n c y and when t r i p l o i d f o r the l o c u s .  of  enhancing  C l a s s II m o d i f i e r s are r a r e  and have the o p p o s i t e e f f e c t s t o dosage, s u p p r e s s i n g when t r i p l o i d and enhancing Su(var) mutations  over a d e f i c i e n c y .  They suggest  the  r e p o r t e d by Reuter et al. (1981) and  S i n c l a i r et al. (1983) are c l a s s I m o d i f i e r s and t h e r e f o r e hypomorphic or amorphic l o c i .  T h i s p o s s i b i l i t y has not been  d i s p r o v e n , s i n c e d e f i c i e n c y s t u d i e s were u n i n f o r m a t i v e i n the most proximal 3R r e g i o n .  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  p l a u s i b l e based on the dosage s e n s i t i v i t y r e p o r t e d by et  al. (1987) and Locke et al.  (1988).  Probable f u n c t i o n s f o r these l o c i have been by S i n c l a i r et al. (1983) and others 1986;  Locke et al. 1988).  Reuter  suggested  (Reuter et al.  1981,  Based on phenotypes of  s u p p r e s s i o n , r e c e s s i v e 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 c o n t r o l chromatin  condensation  or  c o n t r i b u t e s t r u c t u r a l l y t o formation and or maintenance  of  heterochromatin.  T h i s h y p o t h e s i z e d f u n c t i o n i s supported  by recent work of James and E l g i n i s o l a t e d a non-histone heterochromatin.  (198 6).  They have  chromosomal p r o t e i n s p e c i f i c t o  Through in situ  h y b r i d i z a t i o n , they have  86 mapped i t s cDNA to a locus very close to or the same as Su(var)M43, a suppressor locus i s o l a t e d by S i n c l a i r et al. (1983) . Genetic characterization of these mutants provides functional information and a s o l i d background for molecular characterization.  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LINEARITY CURVE  To determine i f the microfluoremeter i s reading pigment amounts i n a l i n e a r fashion, the following protocol was used: Pigment from Oregon-R heads was extracted as described i n Materials and Methods, Chapter 1. Dilutions were made to represent 1, 2, 3, 4 and 5 heads. For example, 2 u l supernatent + 8 u l 2-mercaptoethanol = 20% of Oregon-R pigment or the equivalent of 1 head (1/5=20%). Fluorescence of each sample was measured and r e s u l t i n g pigment levels are plotted i n Figures A-1,2 and 3. B.  LTX2/SU(VAR)II DATA TO ACCOMPANY GRAPHS IN FIGURES 3A-F.  Pigment values are given i n Tables Ba-f. Values are mean + standard deviations. Asterisks indicate values s t a t i s t i c a l l y d i f f e r e n t from basal levels for each variegator tested, s p e c i f i c 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 v i r g i n females were recovered from the compound autosome mapping study reported i n Chapter 2 that made i t possible to construct a compound 3 s t r a i n carrying wm4: wml;C (3L) r i ; C (3R) e Y  x  s  wwA;C (3L) r i ; C (3R) e +  i wm4;C(3L)ri;C(3R)e  s  progeny  This i s a valuable stock since the presence o f a S u ( v a r ) c a n b e d e t e c t e d i n a l l v i a b l e new compound f o r m a t i o n s r e s u l t i n g f r o m a s c r e e n s u c h a s t h e one u s e d i n t h i s study.  s  % Oregon-R pigment  % Oregon-R pigment  % Oregon-R pigment  21 O  c —m  gi o ? 8 8 S S 8 3 8 8 8 S g p 1g 18 1$ 18 18 13 18 1S 18 1S 1o o § 8 £ 8 8 S 8 8 8 S g t%> *i Q < l — I — I — I — I — I — I — I — I — I — I — 1 — l _ _ J 1 7__7 7 7 7 7 7 7 , , •  1  1  1  1  1  1  1  1  1  1 1  1  1  1  1  1  1  1  1  1  1 1  —1—1—1—1  < O  ft*  • f  3 C  a>  ac o>  09  TJ C D -1  ±  • V  r+- 01 +  C  1  1—1—1—1—  \ •  O 3 ta o  1 '  3 10 cr  CL  1  _!  Q  a  1  =1 I  X  TJ CO  .  3  0)  £ \/  C O  5" a> o  cr  a CO  94 TABLE B.a: SU(VAR) MALE  LTX18/SU(VAR)  PIGMENT VALUES  TRIAL I 26.5 + 1.4  TRIAL I I *35.5 + 2.9  T44 FEMALE  *21.0  + 1.3  *34.0 + 3.6,  21.7 + 3.1  34.6 +.1.3  FEMALE  *20.6 + 2.1  *33.5 + 1.3  MALE  *19.1 + 2.1  27.3 + 1.9  FEMALE  *16.0  + 1.4  *26.8 +1.3  MALE A151  M43 MALE  28.9 + 0.6  N.D.  FEMALE  29.2 + 1.8  N.D.  MALE  27.3 + 2.2  *37.9 + 3.9  FEMALE  *24.5 + 2.8  *40.6 + 3.6  MALE  *38.7 + 1.2  *40.5 + 4.8  35.0 + 4.4  *34.5 + 1.3  B89  A24  C157 FEMALE MALE  *22.1 + 3.9  N.D.  FEMALE  *24.7 + 0.6  N.D.  H69 MALE  28.9 + 4.9 •  *37.4 + 2.9  M5 9 FEMALE  *22.5 + 3.5  *32.9 + 0.6  95 TABLE B.b:  SU(VAR) T44  A151  M43  B89  A24  C157  H69  M59  LTX6/SU(VAR)  PIGMENT VALUES  TRIAL I  TRIAL I I  MALE  *33.0 + 1.2  *30.5 + 1.8  FEMALE  *31.7 + 0.5  *31.6 + 2 . 0  56.3 + 6.3  *45.4 + 1.4  FEMALE  *35.9 + 3.4  42.4 + 2.2  MALE  * 2 9 . 8 + 1.6  * 3 4 . 3 + 2.5  FEMALE  *26.0 + 0.7  * 2 6 . 9 + 1.6  MALE  *48.4 + 2 . 3  *40.8 + 3.5  FEMALE  *32.1 + 1.8  *32.5 + 1.7  63.9 + 3.5  52.1 + 8.3  FEMALE  *41.0 + 2.2  43.1 ± 5.4  MALE  *52.4 + 8.2  5 6 . 9 + 6.2  FEMALE  *35.5 ±2.4  MALE  *51.3 + 6.7  FEMALE  *32.7 + 0.7  *37.1 + 2.3  MALE  *47.0 + 0.9  48.1 + 7.5  FEMALE  *34 .4 ± 2.6  *36.3 + 3.2  MALE  MALE  45.2 + 5.4 46.2 + 10.5  96  TABLE B.c: LTX2/SU(VAR) PIGMENT VALUES SU(VAR)  TRIAL I  TRIAL II  MALE  *36.1  ±  6.3  *28.2  + 1.4  FEMALE  *36.5  ±  2.1  *31.8  ± 1.2.  MALE  *28.5  ±  1.7  *38.0  ± 3.2  FEMALE  *35.0  ±  2.3  39.8  ± 4.2  MALE  *32.5  ±  2.6  *25.8  ± 2.4  FEMALE  *31.0  ±  1.2  *21.9  + 0.8  MALE  *29.1  ±  1.8  *34.1 ± 6 . 9  FEMALE  *28.6  ±  1.4  *34.9  ± 3.7  MALE  *41.7  ±  3.2  49.3  ± 6.2  FEMALE  *37.0  ±  1.5  44.8  ± 2.4  57.8  ±  4.2  50.6  ± 6.2  FEMALE  *51.0  ±  2.2  41.9  ±  MALE  *43.2  ±  3.0  FEMALE  *43.5  +  2.4  MALE  *35.7  +  2.6  FEMALE  *37.1  +  2.5  T44  A151  M43  B89  A24  MALE C157  .  3.1  *35.6  ± 4.7  *28.6  ± 2.0  H69  N.D.  M59 *31.4  ± 1.4  97 TABLE B.d: SU (VAR) T44  A151  M43  B89  A24  C157  H69  M59  LTX13/SU(VAR)  PIGMENT VALUES  TRIAL I  TRIAL I I  MALE  *40.8 + 2.4  *35.0 + 0.8  FEMALE  *32.4 + 0.9  *39.8 + 2.9  MALE  84.8 + 5.0  70.9 + 5.9  FEMALE  65.0 + 0.8  68.5 + 3.1  MALE  *64.3 + 3.6  *65.7 + 7.2  FEMALE  *47.3 + 1.2  *53.0 + 3.5  MALE  84.5 + 4.7  86.7 + 4.9  FEMALE  68.4 + 3.0  *81.2 + 4.0  MALE  101.3 + 2.1  94.6 + 1.5  FEMALE  *77.0 + 3.4  *93.7 + 2.3  MALE  *74.9 + 6.2  73.8 + 9.0  64.9 + 4.7  72. 6 + 3.4  *93.9 + 3.0  91.8 + 2.3  FEMALE  67.6 + 2.8  *82.5 + 3.9  MALE  80.7 + 2.7  *67.0 + 7.0  FEMALE  68.6 + 0.7  *61.7 + 6.8  FEMALE MALE  98 TABLE B.e: SU(VAR)  TRIAL I  TRIAL II  MALE  *54.3 ± 2.4  *36.7 + 3.4  FEMALE  *40.2 + 2.3  *45.8 ± 2.4  MALE  94.9 ± 7.8  *65.2 + 5.5  FEMALE  68.3 ± 5.8  *60.1 + 4.7  T44  A151 MALE  *77.9 ± 7.2  *67.0 ± 6.8  FEMALE  *48.8 ± 5.1  *54.9 + 6.7  M43 MALE  96.1 ± 4.1  FEMALE  66.7 ± 2.3  *71.9 + 3.6  MALE  113.4 ± 9.1  *97.6 + 1.3  FEMALE  81.6  ± 3.1  93.7 + 2.3  B89  A24  C157  85.1 ± 8.8  MALE  85.4 ± 1.9  N.D.  FEMALE  71.7 ± 0.9  N.D.  MALE H69  M59  LTX24/SU(VAR) PIGMENT VALUES  110.3 ± 4.4  96.1 + 3.1  FEMALE  69.5 + 3.4  *79.5 ± 7.1  MALE  83. 9 + 3.1  83.9 + 2.9  FEMALE  68.0 + 3.1  72.8 ± 4 . 1  99 TABLE B . f :  SU(VAR)  PIGMENT VALUES  TRIAL I  TRIAL II  MALE  *71.5 ±13.0  *35.8 + 2.3  FEMALE  *38.9 ±1.2  *40.6 + 3.0  MALE  106.0 ±4.5  *68.5 + 4.9  FEMALE  *79.5 + 8.1  68.6 + 4.8  T44  A151  LTX4/SU(VAR)  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  FEMALE  82.7 + 5.2  *72.2 + 9.2  MALE  117.8 + 3.2  92.5 + 1.4  FEMALE  91.6  + 5.1  88.7 + 3.4  MALE  *89.2 + 2.4  83.1 + 6.0  FEMALE  *72.5 + 1.5  75.6 + 3.6  MALE  109.6 ±8.8  *83.5 + 5.4  81.0 ±6.6  *80.3 + 5.1  MALE  *90.9 ±6.8  *67.5 + 6.2  FEMALE  *76.4 + 3.9  *64.0 + 3.3  B89  A24  C157  H69  M59  FEMALE  FIGURE C:  RECOMBINATION DATA FOR SU (VAR)S A160 AND A130  CROSS:  wm4 ;  wm4  Su x  wm4 GI Sb (H)  Y  TM3 e Ser  A130  OFFSPRING  A160  GI Sb H  PARENTAL:  Su SCO I:  DCO:  +  (+)  +  +  +  GI + (+)  Su +  Sb (H)  +  Sb (H)  +  Su GI +  GI I  916  39  401  41  154  47  (+)  TOTAL Su  270 ++  Su GI Sb (H)  SCO I I :  order:  Ly  351  Sb  1,518  H  II  A130:  <—11.4  ><  12.0  >  A160:  <—29.5  ><  13.2  >  aproximate distances  

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