UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A genetic analysis of Region 31 on Chromosome 2 of Drosophila melanogaster Brock, Jo-Ann Karen 1989

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1989_A6_7 B76.pdf [ 5.35MB ]
Metadata
JSON: 831-1.0097463.json
JSON-LD: 831-1.0097463-ld.json
RDF/XML (Pretty): 831-1.0097463-rdf.xml
RDF/JSON: 831-1.0097463-rdf.json
Turtle: 831-1.0097463-turtle.txt
N-Triples: 831-1.0097463-rdf-ntriples.txt
Original Record: 831-1.0097463-source.json
Full Text
831-1.0097463-fulltext.txt
Citation
831-1.0097463.ris

Full Text

A GENETIC ANALYSIS OF REGION 31 ON CHROMOSOME 2 OF DROSOPHILA MELANOGASTER By JO-ANN KAREN BROCK .Sc., The U n i v e r s i t y o f B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Zoology) We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a ndard: THE UNIVERSITY OF BRITISH COLUMBIA June 1989 (5) Jo-Ann Karen Brock In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of "3101-0 The University of British Columbia Vancouver, Canada Date JUA/ET [2. , /?g<? DE-6 (2/88) ABSTRACT G e n e s t h a t a r e s u b j e c t t o p o s i t i o n - e f f e c t v a r i e g a t i o n ( P E V ) a r e l o c a t e d a t n e w l y f o r m e d 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 s o f c h r o m o s o m a l r e a r r a n g e m e n t s . V a r i e g a t e d e x p r e s s i o n o f t h e s e g e n e s i s b e l i e v e d t o r e s u l t f r o m s t r u c t u r a l c h a n g e s i n t h e i r n o r m a l c h r o m a t i n c o n f o r m a t i o n . S e v e r a l g e n e t i c m o d i f i e r s o f P E V h a v e b e e n i d e n t i f i e d w h i c h c a n s u p p r e s s v a r i e g a t e d p h e n o t y p e s . T h e w i l d - t y p e p r o d u c t s o f t h e s e g e n e s a r e t h o u g h t t o b e i n v o l v e d i n f o r m a t i o n o r m a i n t e n a n c e o f c h r o m a t i n s t r u c t u r e , e i t h e r a s s t r u c t u r a l c o m p o n e n t s , o r a s a s s e m b l y o r m o d i f y i n g f a c t o r s . S e v e r a l d o m i n a n t s u p p r e s s o r s o f v a r i e g a t i o n ( S u ( v a r ) s ) h a v e b e e n l o c a l i z e d t o t h e l e f t a r m o f c h r o m o s o m e 2 ( r e g i o n 3 1 ) o f D r o s o p h i l a m e l a n o q a s t e r . A n u m b e r o f i n t e r g e n i c e f f e c t s a r e n o t e d w h e n s u p p r e s s o r m u t a t i o n s o f t h i s r e g i o n , m a p p i n g t o s e p a r a t e l o c i , a r e c o m b i n e d i n t r a n s . T h e s e i n c l u d e m a l e l e t h a l i t y , f e m a l e s t e r i l i t y a n d a t l e a s t t w o v i s i b l e p h e n o t y p i c a n o m a l i e s : c h a n g e s i n e y e c o l o u r ( p o s s i b l y r e s u l t i n g f r o m t h e a l t e r e d e x p r e s s i o n o f t h e h e t e r o c h r o m a t i c l i g h t g e n e ) a n d a b n o r m a l w i n g m o r p h o l o g y . T h e s e e f f e c t s may o c c u r t h r o u g h a d d i t i v e o r s y n e r g i s t i c i n t e r a c t i o n s o f m u t a n t S u ( v a r ) p r o d u c t s w h i c h a l t e r t h e i n t e g r i t y o f h e t e r o c h r o m a t i n t o s u c h a n e x t e n t t h a t e x p r e s s i o n o f h e t e r o c h r o m a t i c g e n e s i s a f f e c t e d . T h i s h y p o t h e s i s i s s u p p o r t e d b y t h e f a c t t h a t a l t e r i n g t h e h e t e r o c h r o m a t i c c o n t e n t o f t h e c e l l a f f e c t s t h e e x p r e s s i o n o f t h e s e a b n o r m a l p h e n o t y p e s . D e l e t i o n o r a d d i t i o n o f a Y c h r o m o s o m e ( w h i c h p r e s u m a b l y a c t s a s a s i n k f o r n o r m a l S u ( v a r ) ii p r o d u c t s ) i s a b l e t o , r e s p e c t i v e l y , a m e l i o r a t e o r e x a c e r b a t e t h e i n t e r g e n i c e f f e c t s . M o s t o f t h e s u p p r e s s o r g e n e s i n v e s t i g a t e d i n t h i s s t u d y a r e h e m i z y g o u s l e t h a l o r f e m a l e s t e r i l e . A n a d d i t i o n a l f o r t y -t w o E M S - i n d u c e d m u t a n t s w e r e g e n e r a t e d i n a s c r e e n t o i s o l a t e m u t a t i o n s o f e s s e n t i a l g e n e s ( i n c l u d i n g s u p p r e s s o r l o c i ) i n r e g i o n 3 1 . T h e s e n e w m u t a n t s w e r e c h a r a c t e r i z e d g e n e t i c a l l y . W h i l e n o n e o f t h e m a r e d o m i n a n t s u p p r e s s o r s o f p o s i t i o n - e f f e c t v a r i e g a t i o n , s e v e r a l a l l e l e s o f a t l e a s t o n e S u ( v a r ) l o c u s w e r e r e c o v e r e d . T h i s r e s u l t s u p p o r t s t h e s u g g e s t i o n t h a t a s p e c i a l e v e n t ( a n t i m o r p h i c m u t a t i o n ) i s n e c e s s a r y t o e l i c i t a s u p p r e s s i n g p h e n o t y p e f r o m t h e S u ( v a r ) l o c i l o c a t e d i n r e g i o n 3 1 . TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i GENERAL INTRODUCTION 1 CHAPTER 1. ANALYSIS OF SUPPRESSORS OF VARIEGATION ON CHROMOSOME 2 OF DROSOPHILA  MELANOGASTER I. I n t r o d u c t i o n 9 I I . M a t e r i a l s and Methods 12 I I I . R e sults 19 IV. D i s c u s s i o n 38 CHAPTER 2. ISOLATION AND CHARACTERIZATION OF HEMIZYGOUS LETHAL MUTATIONS IN REGION 31 OF DROSOPHILA MELANOGASTER I. I n t r o d u c t i o n 4 6 I I . M a t e r i a l s and Methods 48 I I I . R e sults 57 IV. D i s c u s s i o n 77 REFERENCES 84 APPENDIX A. EFFECT OF DEFICIENCIES OF REGION 31 ON POSITION-EFFECT VARIEGATION 88 APPENDIX B. EFFECT OF MATERNAL-EFFECT MUTANTS ON VARIEGATION 91 APPENDIX C 95 APPENDIX D 97 i v LIST OF TABLES Table Page 1-1 2L C l u s t e r Suppressors 13 1-2 Complementation A n a l y s i s of the 2L C l u s t e r of Suppressors 20 1-3 Ovary Morphology of Transheterozygous Suppressor Females 27 1-4 Summary of Phenotypes Observed i n C e r t a i n Transheterozygous Combinations of Suppressors 28 1-5 I n t e r a c t i o n s of Suppressors w i t h Df27 31 1-6 Summary of the E f f e c t of It"1- on V i a b i l i t y of C e r t a i n Transheterozygous Combinations of Suppressors 33 1-7 E f f e c t of the I t 1 A l l e l e on Phenotypic I n t e r a c t i o n s of C e r t a i n Transheterozygous Combinations of Suppressors 34 1- 8 E f f e c t of Y Chromosome on Phenotypic I n t e r a c t i o n s E x h i b i t e d by C e r t a i n Transheterozygous Suppressors 36 2- 1 Test f o r Hemizygous L e t h a l i t y Using Df2 58 2-2 Summary of D e f i c i e n c y Mapping of Hemizygous L e t h a l Mutants i n Region 31 63 2-3 I n t e r se Complementation of Hemizygous L e t h a l Mutations i n 31C 69 2-4 I n t e r se Complementation of Hemizygous L e t h a l Mutants i n Region 31D 70 V 2-5 Complementation Between 23-127 and Hemizygous L e t h a l Mutants i n 31E 71 2-6 I n t e r se Complementation of Hemizygous L e t h a l Mutations i n Region 31F1-2 73 2-7 E f f e c t of Hemizygous L e t h a l Mutations i n Region 31 on V a r i e g a t i o n 74 A - l E f f e c t of D e f i c i e n c i e s i n Region 31 on V a r i e g a t i o n of w™ 4 89 B - l E f f e c t of M a t e r n a l - e f f e c t Mutants on V a r i e g a t i o n of w m 4 92 B-2 E f f e c t of hup and wdl on V a r i e g a t i o n of w™ 4 93 C-l V i a b i l i t y of 2L C l u s t e r Suppressors w i t h Df27 94 C-2 . E f f e c t of I t 1 on V i a b i l i t y of C e r t a i n Transheterozygous Combinations of Suppressors 95 D-l D e f i c i e n c y Mapping of Hemizygous L e t h a l Mutations i n Region 31 97 LIST OF FIGURES Figure ( Page 1-1 Mating Scheme used to Generate a ^Suppressor Mutant (213 Tft) i n an Attached XY Background 17 1- 2 Complementation Map of the 2L C l u s t e r of Suppressors Based on Female S t e r i l i t y 24 2- 1 C y t o l o g i c a l Extent of the D e f i c i e n c i e s used i n t h i s Study 49 2-2 I s o l a t i o n of Hemizygous L e t h a l Mutations i n Region 31 52 2-3 Mating P r o t o c o l f o r P l a c i n g Mutations i n w1114 Background 56 2-4 Schematic Representation of D e f i c i e n c y Mapping of Hemizygous L e t h a l Mutations i n Region 31 65 2-5 Summary of Complementation A n a l y s i s of Hemizygous L e t h a l Mutations i n Region 31 78 ACKNOWLEDGEMENTS My t h a n k s t o Tom G r i g l i a t t i , f o r p r o v i d i n g a n i n d e p e n d e n t w o r k i n g e n v i r o n m e n t , f o r a l w a y s b e i n g r e a d y t o d i s c u s s t h e d a t a , a n d f o r a l l t h e a d m i n i s t r a t i v e d e t a i l s t h a t w e r e t a k e n c a r e o f o n my b e h a l f . I a l s o t h a n k D o n S i n c l a i r f o r m a n y h e l p f u l s u g g e s t i o n s a n d d i s c u s s i o n s o f my w o r k . F o r t h e i r f r i e n d s h i p s a n d e n c o u r a g e m e n t t h r o u g h o u t t h e p a s t t h r e e y e a r s , I am h a p p y t o h a v e k n o w n Amy H e d r i c k , D a r y l H e n d e r s o n a n d M e g K e n n a . F i n a l l y , I w o u l d l i k e t o t h a n k my m o t h e r , M a r g a r e t , w h o s e s u p p o r t a n d e n c o u r a g e m e n t h a s m a d e a l l o f t h i s p o s s i b l e . GENERAL INTRODUCTION The study of P o s i t i o n - e f f e c t v a r i e g a t i o n (PEV) began w i t h M u l l e r ' s discovery (1930) of c e r t a i n r a d i a t i o n - i n d u c e d mutations i n Drosophila, termed "e v e r s p o r t i n g displacements". These mutations are t y p i f i e d by the v a r i e g a t e d expression of a gene i n a given t i s s u e or s t r u c t u r e , and occurs when a chromosomal rearrangement places a gene normally found i n euchromatin, near a heterochromatic breakpoint. The mosaicism which r e s u l t s i s b e l i e v e d due t o t r a n s c r i p t i o n a l i n a c t i v a t i o n of the gene i n a subset of c e l l s i n the v a r i e g a t i n g t i s s u e ( for reviews see Baker, 1968; Spofford, 1976). PEV can a l s o occur when genes normally a s s o c i a t e d w i t h heterochromatin are r e l o c a t e d t o a euchromatic environment. For over h a l f a century, PEV has been a subject of extensive i n v e s t i g a t i o n , however, the mechanism r e s p o n s i b l e f o r t h i s phenomenon s t i l l remains unclear. 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 has been found i n many eukaryotes since i t s f i r s t d iscovery, but has been s t u d i e d most e x t e n s i v e l y i n Drosophila melanoaaster. Baker (1968) was the f i r s t to note a cis-dominant r e l a t i o n s h i p between the v a r i e g a t i o n - i n d u c i n g breakpoint and the responding gene. A po l a r , or spreading e f f e c t of the gene i n a c t i v a t i o n a s s o c i a t e d w i t h PEV, was f i r s t observed by Demerec and S l i z y n s k a (1937). In the T (JL; w m258 -18 rearrangement, the roughest gene (rst) i s c l o s e r t o the breakpoint than the white gene (w), and i s observed t o v a r i e g a t e more f r e q u e n t l y . Moreover, patches 1 m a n i f e s t i n g a roughest phenotype are always l a r g e r than, and encompass patches of white t i s s u e . This i n d i c a t e s t h a t i n a c t i v a t i o n of the w gene i s r e s t r i c t e d to clones i n which the r s t gene has already been i n a c t i v a t e d and demonstrates the importance of p r o x i m i t y of the v a r i e g a t i n g gene t o the breakpoint. I t has a l s o been found that a d d i t i o n a l rearrangements, which r e l o c a t e the v a r i e g a t i n g gene i n t o a euchromatic environment,.can l e a d to resumption of normal gene expression, and that a d d i t i o n of a second heterochromatic breakpoint near the gene r e s u l t s i n more extreme v a r i e g a t i o n (Panshin, 1938). Further s t u d i e s demonstrate t h a t gene i n a c t i v a t i o n a s s o c i a t e d w i t h PEV does not r e s u l t from gene mutation (Judd, 1955), nor somatic gene l o s s (Henikoff, 1979). A l t o g e t h e r , these f i n d i n g s suggest t h a t PEV i s caused by the e f f e c t s of a l t e r e d chromosome s t r u c t u r e on gene expression and not by mutation of the v a r i e g a t i n g gene. The most reasonable molecular explanation of gene i n a c t i v a t i o n a s s o c i a t e d w i t h PEV i s th a t the v a r i e g a t i n g gene becomes packaged as heterochromatin. Zuckerkandl (1974) f i r s t proposed t h a t molecules a s s o c i a t e d w i t h heterochromatin may sometimes spread past the heterochromatic/euchromatic boundary, r e s u l t i n g i n heterochromatization of euchromatin, and t r a n s c r i p t i o n a l i n a c t i v a t i o n of the v a r i e g a t i n g gene. Support f o r such a model was f i r s t evident i n polytene chromosomes of va r i e g a t e d s t r a i n s of Drosophila. In these f l i e s , normally euchromatic regions, which have been s i t u a t e d next t o heterochromatin, o f t e n acquire a heterochromatic morphology 2 (Hartmann-Goldstein, 1967; Hartmann-Goldstein and Wargent, 1975) . The p r o p o r t i o n of n u c l e i showing t h i s heterochromatization i s c o r r e l a t e d w i t h the degree of phenotypic v a r i e g a t i o n observed. More r e c e n t l y , Zhimulev et a l (1986) have shown t h a t , i n s a l i v a r y gland c e l l s , the i n a c t i v a t i o n of a v a r i e g a t i n g gene i s always accompanied by i t s formation i n t o a compact block of heterochromatin. Although i t has not been demonstrated, i t i s probable t h a t the chromatin conformation i n these polytene chromosomes i s a r e f l e c t i o n of what occurs i n the chromosomes of m i t o t i c c e l l s . 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 has been p a r t i c u l a r l y amenable to study because the v a r i e g a t i n g phenotype may be a l t e r e d by a number of environmental and genetic f a c t o r s (see Spofford, 1976) . Several such m o d i f i e r s of PEV have been i d e n t i f i e d . One of the f i r s t environmental m o d i f i e r s to be i n v e s t i g a t e d i n d e t a i l was temperature (Gowan and Gay, 1933). In general, lower temperatures are found t o r e s u l t i n enhancement of v a r i e g a t i o n (that i s , the v a r i e g a t i n g gene i s i n a c t i v a t e d i n a greater p r o p o r t i o n of c e l l s ) . Higher temperatures have the opposite e f f e c t . A d d i t i o n a l s t u d i e s have revealed that heterochromatization i s a l s o enhanced at lower temperatures (Hartmann-Goldstein, 197 6), supporting the theory th a t i n a c t i v a t i o n of the v a r i e g a t i n g genes r e s u l t s from packaging the normally euchromatic segment of DNA as heterochromatin. The developmental p e r i o d during which temperature e l i c i t s i t s e f f e c t was examined f o r the expression 3 of a v a r i e g a t i n g white a l l e l e (Baker, 1963; 1967). These stu d i e s i n d i c a t e d that two d i s t i n c t temperature s e n s i t i v e periods e x i s t f o r t h i s gene: one at the end of the f i r s t l a r v a l i n s t a r ; and a second during the pupal stage, at the time of pigment d e p o s i t i o n . These two periods may correspond to the processes of determination ( s e t t i n g the f a t e of the v a r i e g a t i n g a l l e l e ) and d i f f e r e n t i a t i o n (gene expression) r e s p e c t i v e l y . Another potent m o d i f i e r of PEV i s the Y chromosome, f i r s t r eported by Gowan and Gay (1934). I t was found that the presence of an a d d i t i o n a l Y chromosome suppressed v a r i e g a t i o n , such that the v a r i e g a t i n g gene i s a c t i v e and expressed i n a greater p r o p o r t i o n of c e l l s . Conversely, removal of the Y chromosome ( i n X/0 males) r e s u l t e d i n an enhancement of v a r i e g a t i o n . The time at which the Y chromosome exerts i t s e f f e c t was determined by Becker and Janning (1977). By inducing somatic recombination at v a r i o u s developmental stages i n a white-mottled genetic background, they found t h a t the Y chromosome has i t s most dramatic e f f e c t at the end of the f i r s t l a r v a l i n s t a r ( i . e . at the time of c l o n a l i n i t i a t i o n of the eye imaginal d i s c ) . As suggested by the t e m p e r a t u r e - s e n s i t i v i t y study, t h i s p e r i o d may correspond to the a c q u i s i t i o n of determination of the v a r i e g a t i n g white a l l e l e . Heterochromatic elements other than the Y chromosome can a l s o modify PEV. For example, d e f i c i e n c i e s of heterochromatin i n the X chromosome can enhance v a r i e g a t i o n (Panshin, 1938; Noujdin, 1944). In a d d i t i o n , Df(2R)MS-2 1 0, a d e f i c i e n c y of n e a r l y a l l the centromeric heterochromatin i n the r i g h t arm of 4 chromosome 2, i s a strong enhancer of v a r i e g a t i o n (Morgan et a l . , 1941). These f i n d i n g s , t h a t a d d i t i o n a l blocks of heterochromatin suppress, while a l o s s of such elements enhances PEV, suggest that heterochromatic elements act as a s i n k f o r f a c t o r s i n v o l v e d i n the formation of heterochromatin. The amount of genomic DNA normally packaged as heterochromatin may i n f l u e n c e the a v a i l a b i l i t y or concentration of these heterochromatic c o n s t i t u e n t s and thus c o n t r o l or i n f l u e n c e the extent to which heterochromatin extends i n t o the normally euchromatic region i n PEV. Histones are the major p r o t e i n s which a s s o c i a t e w i t h chromatin, and are suspected t o play a r o l e i n the formation of heterochromatin. S t r a i n s of Drosophila which are h a p l o i d f o r the histone gene c l u s t e r e x h i b i t a marked suppression of v a r i e g a t i o n (Moore et a l . , 1979). A m o d i f i e r of h i s t o n e s , n-butyrate, has a l s o been found to suppress v a r i e g a t i o n (Mottus et a l . , 1980) . I t i s suggested t h a t n-butyrate acts by i n h i b i t i n g h istone deacetylase enzymes, l e a d i n g to hyper-a c e t y l a t e d histones (Candido et a l . , 1978). Since m u l t i a c e t y l a t e d histones are known to be a s s o c i a t e d w i t h t r a n s c r i p t i o n a l l y a c t i v e chromatin (Blumenfeld et a l . , 1978), i t i s p o s s i b l e t h a t , i n t h i s form, the histones have a reduced a f f i n i t y f o r DNA, and are thus l e s s able t o p a r t i c i p a t e i n the formation of heterochromatin. These f i n d i n g s are c o n s i s t e n t w i t h the hypothesis t h a t gene i n a c t i v a t i o n a s s o c i a t e d w i t h PEV r e s u l t s from heterochromatization of normally euchromatic 5 sequences. The degree of heterochromatization i s dependent on the histone concentration and a v a i l a b i l i t y t o the c e l l at the time of DNA r e p l i c a t i o n . A number of other genetic m o d i f i e r s of PEV a l s o e x i s t . One of the f i r s t t o be w e l l c h a r a c t e r i z e d was a mutation of the suppressor of v a r i e g a t i o n , or Su(var) locus, which mapped to the l e f t arm of chromosome 3, at p o s i t i o n 41.4 (Spofford, 1967). I t was found t h a t t h i s mutation suppressed the gene i n a c t i v a t i o n a s s o c i a t e d w i t h v a r i e g a t i o n , such that f u l l pigment expression was reached i n f l i e s homozygous f o r the Su(var) l o c u s . More r e c e n t l y , a number of dominant suppressors of v a r i e g a t i o n have been i s o l a t e d i n two separate s t u d i e s . In the f i r s t , Reuter and Wolff (1981), i s o l a t e d 126 such mutants using both X - i r r a d i a t i o n and ethylmethane s u l f o n a t e (EMS). Although these mutations were o r i g i n a l l y i d e n t i f i e d by t h e i r a b i l i t y to suppress v a r i e g a t i o n of the white-mottled phenotype, they were subsequently found to suppress PEV a s s o c i a t e d w i t h the brown and scute l o c i as w e l l (Reuter et a l . , 1982). The study of these m o d i f i e r s suggests t h a t a l l forms of euchromatic PEV have s i m i l a r molecular mechanisms. In another study, 51 EMS-induced dominant suppressors of v a r i e g a t i o n were i s o l a t e d by S i n c l a i r et a l . , (1983). In a d d i t i o n t o white-mottled genes, these mutants a l s o suppress gene expression of v a r i e g a t i n g rearrangements a s s o c i a t e d w i t h brown and Stubble. I t i s of p a r t i c u l a r i n t e r e s t t h a t these mutants were found to be arrayed i n d i s c r e t e c l u s t e r s , mapping to chomosomes 2 and 3. Using standard mapping procedures, 16 6 of these have been mapped to chromosome 2 (8 are found i n one major c l u s t e r on the l e f t arm of the chromosome), while 28 of t h i r t y have been l o c a l i z e d to three d i s t i n c t c l u s t e r s on the r i g h t arm of chromosome 3. This type of o r g a n i z a t i o n may i n d i c a t e the existence of a f u n c t i o n a l r e l a t i o n s h i p among suppressor l o c i i n the same c l u s t e r , as w e l l as f u n c t i o n a l d i f f e r e n c e s between d i f f e r e n t c l u s t e r s . Supporting t h i s suggestion i s the observation t h a t suppressors l o c a t e d on the second chromosome are more e f f e c t i v e at suppressing bw v P e^ than t h i r d chromosome suppressors. In a d d i t i o n , a m a j o r i t y of the suppressors on chromosome 2 ( i n c l u d i n g those i n the c l u s t e r ) are r e c e s s i v e l e t h a l , whereas most of those on chomosome 3 are homozygous v i a b l e . I t i s thought t h a t the normal gene products i d e n t i f i e d by many of the suppressors may be i n v o l v e d i n the formation of chromatin, e i t h e r as s t r u c t u r a l components, or i n an enzymatic f a s h i o n . A study of these mutants, t h e r e f o r e , may be instrumental i n enhancing our understanding of the molecular d e f i n i t i o n of chromatin, and may a l s o serve as an inroad to understanding the r o l e of chromatin s t r u c t u r e i n gene expression. The work i n t h i s t h e s i s encompasses an i n v e s t i g a t i o n of suppressors and other l o c i l o c a t e d i n region 31 of the cytogenetic map on chromosome 2 of Drosophila  melanoaaster. 7 CHAPTER ONE ANALYSIS OF SUPPRESSORS OF VARIEGATION ON CHROMOSOME 2 OF DROSOPHILA MELANOGASTER 8 INTRODUCTION: Most of the suppressors of v a r i e g a t i o n (Su(var)s) i s o l a t e d by S i n c l a i r et a_l. (1983) , map to d i s t i n c t c l u s t e r s along the second and t h i r d chromosomes. One of these, the 2L c l u s t e r , i s l o c a l i z e d t o region 31 of the cytogenetic map of chromosome 2. This c l u s t e r i s comprised of eight suppressors which occupy a 3.4 map u n i t i n t e r v a l (from m e i o t i c map p o s i t i o n 32 to 35.4) on the l e f t arm of the chromosome. Seven of these were EMS-induced while the remaining one (Su(var)209) was spontaneous i n o r i g i n . The suppressors i n t h i s c l u s t e r and others on the second chromosome d i f f e r from those on chromosome 3 i n that many of them appear to be homozygous l e t h a l or female s t e r i l e , suggesting t h a t they encode e s s e n t i a l f u n c t i o n s , whereas those on the t h i r d chromosome are homozygous v i a b l e . C h a r a c t e r i z a t i o n of the 2L c l u s t e r of suppressors has been f a c i l i t a t e d by the e x i s t e n c e of a number of Jammed-derived d e f i c i e n c i e s which span region 31 (Mange and Sandler, 1973; Sandler, 1977; Salas and Lengyel, 1984). Several of these d e f i c i e n c i e s have a l s o been shown to suppress v a r i e g a t i o n , whereas a d u p l i c a t i o n of region 31 was found to enhance PEV. In a d d i t i o n , f l i e s which are heterozygous f o r a d e f i c i e n c y of the region and the 2L suppressor mutations o f t e n e x h i b i t l e t h a l i t y and/or female s t e r i l i t y . These f i n d i n g s i n d i c a t e the existence of d o s e - s e n s i t i v e suppressor genes i n t h i s c y t o l o g i c a l region which correspond t o the g e n e t i c a l l y mapped 2L c l u s t e r mutations. 9 Further analyses of these suppressors , which i n c l u d e p h y s i c a l mapping, and cytogenetic s t u d i e s w i t h the Jammed-der i v e d d e f i c i e n c i e s , revealed the existence of three d i s t i n c t Su(var) l o c i i n t h i s r egion: one i n 31E, d e f i n e d by Su(var)216; one i n 31C, c o n t a i n i n g Su (var) 204. and 2 07; and at l e a s t one gene i n 31A/B, where the remaining four suppressors map. (Su(var)209 could not be mapped i n t h i s manner as i t i s homozygous v i a b l e and f e r t i l e , and has no phenotype other than suppression as a hemizygote.) I t has been proposed t h a t many of these mutants define a l t e r e d f u n c t i o n (antimorphs) r a t h e r than reduced f u n c t i o n l e s i o n s . This i s suggested by the f a c t t h a t some of the 2L c l u s t e r suppressors are l e s s v i a b l e as homozygotes than as hemizygotes, and t h a t the p o i n t mutants suppress PEV more s t r o n g l y than do d e l e t i o n s c o n t a i n i n g the r e s p e c t i v e Su(var) genes. In a d d i t i o n , suppressor-bearing f l i e s which contain a d u p l i c a t i o n of region 31 are l e s s suppressed than those which possess only a s i n g l e copy, arguing against a neomorphic e f f e c t . The antimorphic nature of these suppressors suggest t h a t t h e i r gene products are subunits of a l a r g e r macromolecular complex or s t r u c t u r e . While s i n g l e subunits can be mutated to l o s s of s p e c i f i c f u n c t i o n w i t h i n such a complex, i t i s p o s s i b l e that they r e t a i n the a b i l i t y t o i n t e r a c t w i t h other (non-mutant) subunit members. By doing so, they may reduce or e l i m i n a t e the a b i l i t y of the e n t i r e complex to f u n c t i o n 10 n o r m a l l y . I t h a s a l r e a d y b e e n s u g g e s t e d t h a t s u p p r e s s o r s b e l o n g i n g t o i n d i v i d u a l c l u s t e r s m a y b e f u n c t i o n a l l y r e l a t e d , a n d f u r t h e r , t h a t t h e y may i n t e r a c t t o f o r m c o m p l e x e s i n v o l v e d i n h e t e r o c h r o m a t i n f o r m a t i o n . I n d e e d , i n i t i a l s t u d i e s h a v e r e v e a l e d t h a t c e r t a i n h e t e r o z y g o u s c o m b i n a t i o n s o f t h e 2 L c l u s t e r s u p p r e s s o r m u t a n t s w h i c h a r e c y t o g e n e t i c a l l y s e p a r a b l e , a r e i n v i a b l e , o r d e m o n s t r a t e f e m a l e s t e r i l i t y . T h i s s t u d y e n c o m p a s s e s a d e t a i l e d e x p l o r a t i o n o f t h e c o m p l e m e n t a t i o n a n a l y s i s o f t h e 2 L c l u s t e r o f s u p p r e s s o r s , d e v o t i n g s p e c i a l a t t e n t i o n t o s e v e r a l p h e n o t y p i c i n t e r a c t i o n s w h i c h t h e y e x h i b i t . 11 MATERIALS AND METHODS  Strains For a d e t a i l e d d e s c r i p t i o n of v i s i b l e mutations and s p e c i a l chromosomes used, r e f e r to L i n d s l e y and G r e l l (1968). The suppressor s t r a i n s used i n t h i s study are: Su(var)204, Su (var) 207, Su (var) 209, Su (var) 210, Su (var) 213., Su (var) 214, Su(var)215. and Su(var)216, and w i l l subsequently be r e f e r r e d t o by t h e i r r e s p e c t i v e numbers only. For d e t a i l s of t h e i r map p o s i t i o n s and phenotypes, see Table 1-1. A l l but 209 were o r i g i n a l l y i s o l a t e d on an i s o g e n i c b I t r l chromosome and thus c a r r y these r e c e s s i v e v i s i b l e markers. The suppressor mutants were maintained i n stock balanced over the m u l t i p l y i n v e r t e d chromosome In(2LR)CyO. This i n v e r s i o n , h e r e a f t e r r e f e r r e d to as CyO, i s i d e n t i f i e d by the dominant Curly wing marker. I t contains wild-type a l l e l e s of the second chromosome suppressor l o c i , and has v i r t u a l l y no e f f e c t on expression of the v a r i e g a t i n g genes used i n t h i s study. Su(var)209 was spontaneous i n o r i g i n and t h e r e f o r e does not e x i s t on a marked chromosome. I t i s a l s o balanced over the CyO chromosome. A number of recombinant suppressor chromosomes were generated f o r use i n t h i s study. These inc l u d e 210 b, 213 b, 214 b, 215 b and 216 b. An a d d i t i o n a l recombinant i s o l a t e of 214 (214 Tft) i n which a second s i t e l e t h a l had been removed was al s o used. A l l these chromosomes are maintained i n stock balanced over the CyO chromosome. An a d d i t i o n a l suppressor mutation, Su (var2) 1 ^ , was al s o provided through the generosity of Dr. Reuter. This mutant, 12 TABLE 1-1 2L C l u s t e r Suppressors Suppressor Homozygous Phenotype Map P o s i t i o n C y t o l o g i c a l L ocation 204 v i a b l e and f e r t i l e 33.8+1.6 31C 207 l e t h a l 32 . 0 ^ . 4 31C 209 v i a b l e and f e r t i l e 35.4+2.2 210 l e t h a l 34.8+1.8 31A/B 213 l e t h a l 32.9+2.0 31A/B 214 s e m i - l e t h a l female s t e r i l e 34.9+1.8 31A/B 215 s e m i - l e t h a l female s t e r i l e 32.9+1.5 31A/B 216 l e t h a l 34.2±1.6 31E S u ( v a r 2 ) l 0 1 s e m i - l e t h a l 40.5 31A/B 13 which arose spontaneously, maps to m e i o t i c p o s i t i o n 40.5 (Reuter et a l . , 1982). Subsequent a n a l y s i s using d e f i c i e n c i e s , places i t i n the c y t o l o g i c a l region of 31A-B. Df(2L)J-der27, h e r e a f t e r r e f e r r e d t o as Df27, i s a d e f i c i e n c y generated by r e v e r s i o n of the neomorphic Jammed mutation. I t spans region 31C-31F (Mange and Sandler, 1973; Sandler, 1977). This s t r a i n was generously provided by Dr. L. Sandler. A l e t h a l a l l e l e of the heterochromatic l i g h t gene ( I t 1 ) was k i n d l y s u p p l i e d by Dr. D. Holm. This i s a homozygous l e t h a l mutation recovered by H i l l i k e r (1976) i n a screen f o r EMS-induced mutations i n the heterochromatic region of chromosome 2. Culture Conditions F l i e s were r a i s e d on standard cornmeal-sucrose Drosophila medium to which tegosept (0.04%) was added as an i n h i b i t o r of mould growth. Crosses were performed at 25°C unless i n d i c a t e d otherwise. Complementation Analysis The i n t e r se complementation a n a l y s i s was c a r r i e d out among the suppressors of the 2L c l u s t e r , based on r e c e s s i v e l e t h a l i t y and/or female s t e r i l i t y . The suppressors used in c l u d e the eig h t p r e v i o u s l y i d e n t i f i e d from t h i s l a b , and one 01 ' ( S u ( v a r 2 ) I " 1 ) as a r e p r e s e n t a t i v e a l l e l e of the locus i d e n t i f i e d by Reuter et al (1982). Heterozygous Su/CyO v i r g i n 14 females of a given suppressor were mated to heterozygous Su/CyO males of another i n a l l p a i r - w i s e combinations. For most of these crosses, r e c i p r o c a l matings were done. The r a t i o of Curly to non-Curly winged f l i e s i n the F^ progeny was observed, and the r e s u l t i n g straight-winged animals, when they occurred, were examined f o r f e r t i l i t y . F e r t i l i t y t e s t i n g of males was done by p l a c i n g 3-5 males of a given genotype w i t h 5-10 wild-type v i r g i n females (Oregon-R) i n v i a l s at 22°C. Between 10 and 20 males of each genotype t e s t e d , were examined. In t e s t i n g f e r t i l i t y i n females, approximately 5-7 females of a given genetic c o n s t i t u t i o n were placed i n v i a l s , along w i t h 3-5 f e r t i l e (Oregon-R) males. Scoring was measured simply as f e r t i l e or s t e r i l e , depending on the presence or absence of o f f s p r i n g from such matings. T o t a l numbers of o f f s p r i n g were not determined. In a d d i t i o n , the ovaries of transheterozygous females were examined. They were c l a s s i f i e d i n t o one of 4 groups, based on the f o l l o w i n g morphologies: Class 1: ovaries absent Class 2: rudimentary ovaries - o v a r i o l e s present, but no obvious egg production Class 3: abnormal ovaries - ovaries s m a l l , some eggs present but often developed only up to stage 8 (King, 1970) Class 4: w e l l developed ovaries - o f t e n i n d i s t i n g u i s h a b l e from wild-type Females were aged at l e a s t 3-5 days before ovaries were d i s s e c t e d , and approximately 20-30 ovaries of each genotype were examined. 15 Interactions of Suppressors with Df27 Df27/CyO v i r g i n females were mated to w™ 4 males wi t h v a r i o u s Su/CyO chromosome 2 c o n s t i t u t i o n s . The r e l a t i v e v i a b i l i t y of r e s u l t a n t F^ progeny bearing both Df27 and a suppressor was determined. These f l i e s were a l s o examined f o r phenotypic abberations. Interactions with l i g h t - l e t h a l Recombinants between the I t 1 P i n chromosome and four of the suppressors were recovered t o give the f o l l o w i n g new chromosomes: 210. b I t 1 , 2 1 3 b I t 1 , 215 b I t 1 and 216 b I t 1 . Heterozygous males of these mutants, balanced over the CyO chromosome, were mated to various heterozygous Su/CyO v i r g i n females (actual combinations generated are as i n d i c a t e d i n the r e s u l t s ) . Thus, animals that are transheterozygous f o r two suppressors are a l s o heterozygous f o r the I t 1 a l l e l e . The v i a b i l i t y and/or other phenotypic t r a i t s of such transheterozygotes were compared to c o n t r o l s which were g e n o t y p i c a l l y s i m i l a r , yet lacked a mutant I t a l l e l e . R e c i p r o c a l crosses were not done. E f f e c t of Y Chromosome on 2L Cluster Interactions A stock of the suppressor chromosome 213 T f t , contained i n an attached-XX and attached-XY background, was generated as shown i n Figure 1-1. Crosses were performed by mating XYf Y. B/0; 213 Tft/CyO males t o suppressor-bearing v i r g i n 16 FIGURE 1-1 Mating Scheme used to Generate a Suppressor Mutant (213 Tft) i n an Attached XY Background. XY Gla CyO XY Gla — — cfcf O CyO XY 213 T f t CyO w"14 213 T f t x — 9 9 w ™4 CyO XY 213 T f t _ x --• 99 w*1.4. CyO XY 213 T f t 9 9 w"14 CyO STOCK 17 f e m a l e s . T h e r e s u l t a n t t r a n s h e t e r o z y g o t e s ( w h i c h i n c l u d e X / 0 A m a l e s a n d X Y / X f e m a l e s ) w e r e e x a m i n e d f o r r e l a t i v e v i a b i l i t i e s a n d p h e n o t y p i c e f f e c t s . 18 RESULTS Results of the p a i r - w i s e complementation t e s t i n g are shown i n Table 1-2. When p o s s i b l e , the crosses were performed using suppressor chromosomes from which the r e c e s s i v e markers I t and r l had been removed (the only exception i s 207, i n which such a recombinant was not generated). This was done i n order to avoid reductions i n v i a b i l i t y , or other d e t r i m e n t a l e f f e c t s which may r e s u l t from the homozygosity of these mutant a l l e l e s . In f a c t , e a r l i e r observations i n d i c a t e d that t a k i n g these steps d i d r e s u l t i n an increase i n the r e l a t i v e v i a b i l i t y of transheterozygous animals (other e f f e c t s were a l s o observed, see l a t e r ) . I t should be noted that r e c i p r o c a l crosses were performed f o r each p a i r - w i s e combination, although no s i g n i f i c a n t d i f f e r e n c e s were detected. For t h i s reason, the data from r e c i p r o c a l crosses has been pooled. Male v i a b i l i t y For easy comparison, the numbers of c u r l y to s t r a i g h t winged f l i e s have been converted t o a r a t i o . A value of 0.50 (representing a r a t i o of 2:1) i s expected f o r f u l l transheterozygote v i a b i l i t y . As i n d i c a t e d i n Table 2-1, complete l e t h a l i t y i n e i t h e r sex, f o r any transheterozygous combination of suppressors was never observed. This i s perhaps not s u r p r i s i n g , as a number of the 2L c l u s t e r suppressors themselves have been shown to be homozygous v i a b l e (204 and 209) or only s e m i - l e t h a l (214 and 215). 19 TABLE 1-2 Complementation A n a l y s i s of the 2L C l u s t e r of Suppressors C r o s s a F-j_ males b F^ females 204 X 204 68:31 (0.46) F c 63:32 (0.51) F 204 X 207 422:132 (0.31) F 407:102 (0.25) F 204 X 209 237:124 (0.52) F 191:131 (0.69) F 204 X 210 576:220 (0.38) F 561:149 (0.27) F 204 X 213 332:128 (0.39) F 334:127 (0.38) F 204 X 214 339:163 (0.48) F 286:144 (0.50) F 204 X 215 247:88 (0.36) F 219:67 (0.31) F 204 X 216 524:187 (0.36) F 556:205 (0.37) F 207 X 207 174:0 (0.0) 170:0 (0.0) 207 X 209 260:189 (0.73) F 252:184 (0.73) F 207 X 210 100:25 (0.25) F 116:43 (0.37) S 207 X 213 405:113 (0.28) F 358:100 (0.28) S 207 X 214 636:250 (0.39) F 676:215 (0.32) S 207 X 215 582:116 (0.22) F 634:197 (0.31) S 207 X 216 442:131 (0.30) F 485:157 (0.32) S 209 X 209 64:0 75:0 209 X 210 236:127 (0.54) 247:130 (0.53) F 209 X 213 345:210 (0.61) 319:205 (0.64) F 209 X 214 259:186 (0.72) 229:187 (0.82) F 209 X 215 286:146 (0.51) 270:156 (0.58) F 209 X 216 275:148 (0.54) 304:190 (0.63) F 210 X 210 300:0 (0.0) 341:0 (0.0) 210 X 213 1276:143 (0.11) F 1261:391 (0.31) S 210 X 214 898:162 (0.18) F . 808:242 (0.30) S 210 X 215 1444:566 (0.39) 1455:667 (0.46) F 210 X 216 691:148 (0.21) 769:191 (0.25) S a: see M a t e r i a l s and Methods f o r a more d e t a i l e d d e s c r i p t i o n of crosses performed. b: numbers given i n d i c a t e the t o t a l number of Cy:Cy + scored i n the F^ generation. Figure i n parentheses i s the r a t i o of s t r a i g h t t o c u r l y winged animals. c: F and S r e f e r to f e r t i l i t y and s t e r i l i t y of transheterozygous animals, r e s p e c t i v e l y . 20 Table 1-2 continued Cross males F-^  females (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 01 01 01 01 01 01 01 01 213 397:0 (0 .0) 475: 0 (0 .0) 214 949:26 (0 .03) 909: 250 (0 .28) S 215 1743:431 (0 .25) 1719: 558 (0 .32) F 216 1055:65 (0 .06) 1063: 369 (0 .35) S 214 460:9 (0 .02) F 489: 100 (0 .20) S 215 1809:579 (0 .32) F 1773: 631 (0 .36) F 216 1092:71 (0 .07) F 1116: 344 (0 .31) S 215 153:0 (0 .0) 135: 0 (0 .0) 216 2135:878 (0 .41) F 2199: 951 (0 .43) F 216 338:0 (0 .0) 376: 0 x 204 251:128 (0 .51) 275: 119 (0 .43) F x 207 205:28 (0 .14) F 195 : 105 (0 .54) S x 209 245:115 (0 .47) F 275: 149 (0 .54) F x 210 226:13 (0 .06) F 254: 82 (0 .32) F x 213 480:1 (0 • 0) 385: 177 (0 .46) S x 214 289:20 (0 .07) F 320: 146 (0 .45) S x 215 144:55 (0 .38) 172 : 55 (0 .32) F x 216 228:52 (0 .22) F 284: 125 (0 .44) S 21 In s e v e r a l cases, however, the v i a b i l i t y of transheterozygous males i s severely reduced. The most notable examples are 213/214, 213/216 and 214/216, i n which the numbers of s u r v i v o r s are l e s s than 20% of expected ( r a t i o below 0.10). These values are comparable to those found f o r homozygosis of the s e m i - l e t h a l suppressor, 214. Other combinations of suppressors i n which reduced male v i a b i l i t i e s were demonstrated (a r a t i o of l e s s than 0.25) i n c l u d e 210/213, 210/214 and 0 1 210/216. The independently i s o l a t e d suppressor, Su(var2)1°, a l s o r e s u l t e d i n decreased transheterozygote male v i a b i l i t y i n conjunction w i t h some of the 2L c l u s t e r mutants. The most notable are 210, 213 and 214, at frequencies of l e s s than 20% of expected, while others i n c l u d e 207 and 216. These r e s u l t s , together w i t h the d e f i c i e n c y mapping i s c l e a r evidence t h a t n 1 Su(var2)1 does belong to the 2L c l u s t e r of suppressors. With the exception of 207/215, a l l heterozygous combinations i n v o l v i n g 204, 207, 209 or 215 d i d not r e s u l t i n marked reductions of male v i a b i l i t y (defined here as r a t i o s of l e s s than 0.25, or 50% of expected v i a b i l i t y ) . In c o n t r a s t to the preceding observations, the l e v e l s of s u r v i v i n g transheterozygous females f o r any of these suppressor combinations appear to be unaffected. Female s t e r i l i t y While some of the 2L c l u s t e r suppressors are known to be v i a b l e as homozygotes, at l e a s t two of these (214 and 215) are homozygous female s t e r i l e . Therefore, as p a r t of the complementation study, the f e r t i l i t y of transheterozygous animals ( p a r t i c u l a r l y females) was examined. Two of the suppressors, 204 and 209, which are themselves homozygous female f e r t i l e , a l s o generate f e r t i l e females when combined i n tran s w i t h a l l other suppressor mutants. Among the remaining seven suppressors, a complex assortment of i n t e r a c t i o n s i s evident. One (207) demonstrates female s t e r i l i t y i n a l l combinations. Another (215) r e s u l t s i n f e r t i l e females w i t h a l l suppressors but 207. Except f o r one combination o i ( 2 1 0 / S u ( v a r 2 ) l u x ) , the remaining f i v e suppressors (210, 213, 214, 216 and S u ( v a r 2 ) l ^ 1 ) produce female s t e r i l i t y i n any pa i r w i s e combination among themselves, as w e l l as i n combination w i t h 207. I n t e r e s t i n g l y , a l l combinations of male transheterozygotes t e s t e d , even those w i t h extremely reduced v i a b i l i t i e s ( i n c l u d i n g 214 homozygotes), have proven t o be f e r t i l e . One c o r r e l a t i o n which i s evident from the r e s u l t s presented so f a r i s t h a t , w i t h one exception (210/Su (var2) 1UJ-) , a l l transheterozygous combinations of suppressors which r e s u l t i n reduced male v i a b i l i t y are a l s o female s t e r i l e . Using female s t e r i l i t y as a phenotype, a complementation map can be generated as shown i n Figure 1-2. This map d i f f e r s somewhat from t h a t reported e a r l i e r ( S i n c l a i r et a l . , 1983), although the i n t e r a c t i o n s of mutants w i t h d i s t i n c t l y d i f f e r e n t c y t o l o g i c a l l o c a t i o n s i s s t i l l e vident. Although complementation was determined by the v i a b i l i t y and f e r t i l i t y of r e s u l t i n g transheterozygous animals, some 23 FIGURE 1-2 C o m p l e m e n t a t i o n M a p o f t h e 2 L C l u s t e r o f S u p p r e s s o r s B a s e d o n F e m a l e S t e r i l i t y 2 4 204 209 207 (2)101 210 215 a t t e n t i o n was al s o given i n determining the b a s i s of s t e r i l i t y i n females when i t occurred. This was accomplished by examining such females f o r t h e i r a b i l i t y to l a y eggs, and by examining t h e i r o v a r i e s . The r e s u l t s are shown i n Table 1-3. As f a r as was i n v e s t i g a t e d , s t e r i l i t y appears to be d i r e c t l y c o r r e l a t e d w i t h the i n a b i l i t y t o l a y eggs. In no genotype was s t e r i l i t y accompanied by the production of eggs, suggesting th a t the s t e r i l e phenotype i s not a consequence of maternal-e f f e c t l e t h a l i t y . In examining the o v a r i e s , i t was found t h a t those of f e r t i l e females ranged from c l a s s 2, w i t h no egg production (see M a t e r i a l s and Methods f o r d e s c r i p t i o n of classes) t o w i l d - t y p e . Of the s t e r i l e females, ovaries were never seen developed beyond c l a s s 2, and i n one case (213/216), ovaries could not be detected at a l l . Phenotypic Interactions An i n t r i g u i n g and unexpected f i n d i n g i n t h i s study i s the ma n i f e s t a t i o n of c e r t a i n phenotypic anomalies a s s o c i a t e d w i t h many of the transheterozygous combinations of suppressors (Table 4-1). One of these i s a wing de f e c t . I t i s most s t r i k i n g i n males. In i t s most severe form, as seen i n 214/214 homozygous males, the wings are he l d s t r a i g h t out from the body, i n a h o r i z o n t a l f a s h i o n , and are u s u a l l y misshapen (improperly u n f u r l e d ) . This phenotype i s evident i n a l l males which c o n t a i n any two of the f o l l o w i n g suppressors: 210, 213, 214 and 216. There appears to be a c o r r e l a t i o n between t h i s phenotypic e f f e c t , and reduced male v i a b i l i t y observed e a r l i e r . 26 TABLE 1-3 Ovary Morphology of Transheterozygous Suppressor Females Ovary C l a s s i f i c a t i o n Genotype a Eggs*3 204/214 (F) + 0 0 5 10 207/214 (S) - 0 22 0 0 210/213 (S) - 0 22 0 0 210/214 (S) - 0 19 0 0 210/215 (F) + 0 8 32 5 210/216 (S) - 0 37 0 0 213/214 (S) - 0 23 0 0 213/215 (F) + 0 13 24 2 213/216 (S) - 28 0 0 0 214/214 (S) - 0 8 0 0 214/215 (F) + 0 5 23 5 214/216 (S) - 0 17 0 0 215/216 (F) + 0 3 26 3 a: l e t t e r s i n parentheses i n d i c a t e f e r t i l i t y (F) and s t e r i l i t y (S) of the genotypic females. b: the presence and absence of eggs are i n d i c a t e d by + and -r e s p e c t i v e l y . c: see M a t e r i a l s and Methods f o r a d e s c r i p t i o n of c l a s s e s . 27 TABLE 1-4 Summary of Phenotypes Observed i n C e r t a i n Transheterozygous Combinations of Suppressors males females Genotype e y e a wing" eye wing 210/213 mod ext none none 210/214 mod ext mod mod 210/215 mod none none none 210/216 ext ext none none 213/214 mod ext mod mod 213/215 mod none none none 213/216 ext ext ext none 214/214 ext ext ext mod 214/215 ext mod none mod 214/216 ext ext ext mod 215/216 mod none none none 204/any none none none none 209/any none none none none a: mod= moderate eye colour phenotype (orange/brown) ext= extreme eye colour phenotype ( l i g h t orange) none : = no abnormal eye colour b: mod= moderate wing abnormality (wings splayed) ext= extreme wing abnormality (held out and misshapen) none: = no wing abnormality 28 A l l suppressor combinations i n Table 1-4 w i t h t h i s wing abnormality a l s o d i s p l a y reduced male v i a b i l i t y , and are female s t e r i l e ( r e f e r back to Table 1-2). In l e s s severe cases, the wings maintain a normal shape, but remain s l i g h t l y spread out. This milder form of wing phene i s u s u a l l y noted i n females, p a r t i c u l a r l y combinations i n v o l v i n g 214, but i s a l s o seen i n male transheterozygotes of genotype 214/215. There appears to be no c o r r e l a t i o n between suppressor combinations i n which females demonstrate t h i s wing phenotype and female s t e r i l i t y . A l l combinations i n v o l v i n g e i t h e r 204 or 209 f a i l e d to manifest any type of wing anomaly. Su(var)207 i s not i n c l u d e d i n t h i s a n a l y s i s , as the chromosome s t i l l contains the I t and r l a l l e l e s , which may r e s u l t i n exacerbation of t h i s and other phenotypes (see l a t e r ) . Another t r a i t e x h i b i t e d by some transheterozygous animals i s an abnormal eye co l o u r . Although they contain two suppressors of v a r i e g a t i o n , the pigment l e v e l s of these f l i e s , upon v i s u a l examination, appear to be lower than those seen f o r s i b l i n g s bearing the r e s p e c t i v e i n d i v i d u a l suppressors. In a d d i t i o n , the pigment i s evenly d i s t r i b u t e d , and not v a r i e g a t e d i n the f a s h i o n t y p i c a l of w m 4. The r e s u l t i s a l i g h t e r eye colour, s i m i l a r to that seen i n f l i e s which express the l i g h t mutation. L i k e the wing t r a i t , t h i s abnormal eye phenotype v a r i e s i n i t s i n t e n s i t y from one genotype to another, and i s u s u a l l y more severe i n males than t h e i r female counterparts (see Table 1-4). When w m 4 i s taken out of the background of transheterozygous suppressor f l i e s , the phenotype p e r s i s t s (data not shown), i n d i c a t i n g i t t o be a p r o p e r t y o f t h e s u p p r e s s o r s and not t h e abnormal w h i t e gene. W h i l e a l l male c o m b i n a t i o n s i n v o l v i n g 210, 213, 214, 215 and 216 demonstrate t h i s eye phenotype t o some degree, t h e r e appears t o be no s t r o n g c o r r e l a t i o n t o e i t h e r t h e r e d u c e d v i a b i l i t y o b s e r v e d i n males, o r t h e female s t e r i l i t y phenotype. T h i s c o n t r a s t s w i t h t h e wing phenotype, which shows a c o r r e l a t i o n w i t h b o t h male v i a b i l i t y and female s t e r i l i t y . I n a d d i t i o n , p r e s e n c e o r s e v e r i t y o f t h e eye phenotype i s not c o r r e l a t e d w i t h p r e s e n c e or absence o f t h e wing phenotype, e i t h e r i n males o r f e m a l e s . T h i s i s p a r t i c u l a r l y n o t e d by t h e f a c t t h a t some females (214/215) have h e l d out wings w i t h o u t t h e eye phenotype, w h i l e o t h e r s show t h e eye but not t h e wing e f f e c t (213/216). In males, some c o m b i n a t i o n s o f s u p p r e s s o r s w i t h a moderate eye phenotype (210/215, 213/215 and 215/216) have normal wings, w h i l e o t h e r s have an extreme wing phenotype (210./213, 210/214 and 213/214) . Df27 i s a d e f i c i e n c y w h i c h i n c l u d e s t h e l o c u s f o r 216, but none o f t h e o t h e r 2L c l u s t e r s u p p r e s s o r s . To d e t e r m i n e whether a n u l l a l l e l e o f 216 has t h e same e f f e c t as t h e p o i n t m u t a t i o n , f l i e s b e a r i n g b o t h Df27 and one o f t h e o t h e r s u p p r e s s o r s were examined f o r v i a b i l i t y e f f e c t s and p h e n o t y p i c i n t e r a c t i o n s . The r e s u l t s , as i n d i c a t e d i n T a b l e 1-5, c l e a r l y show t h a t Df27 i s not e q u i v a l e n t t o 216. I n a l l c a s e s , male v i a b i l i t y i s i n c r e a s e d i n f l i e s b e a r i n g Df27 r e l a t i v e t o t h o s e w i t h 216. In a d d i t i o n , t h e wing and eye phenotypes w h i c h were e v i d e n t w i t h 30 TABLE 1-5 I n t e r a c t i o n s of Suppressors w i t h Df27 F-^  males F-^  females C r o s s a Cy:Cy* eye/wing Cy:Cy + eye/wing r a t i o " phenotype r a t i o phenotype Df27 X 207 0.72 (0.30) c none 0.46 (0.32) none Df27 X 210 0.54 (0.21) none 0.25 (0.25) none Df27 X 213 0.44 (0.06) none 0.38 (0.35) none Df27 X 215 0.56 (0.41) none 0.50 (0.43) none Df27 X 216 0.0 0.0 a: d e t a i l s of the crosses performed are described f u l l y i n M a t e r i a l s and Methods. b: f o r a c t u a l data, see Appendix C, Table C - l . c: numbers i n parentheses are v i a b i l i t y r a t i o s obtained using 216 i n place of Df27. These f i g u r e s are taken from Table 1-1. 31 216 are not seen when suppressor-bearing f l i e s c o n t a in Df27 i n i t s p l a c e . Interaction with l t ^ -The f a c t t h a t the eye colour changes i n transheterozygous f l i e s resembled that of the l i g h t gene mutation prompted f u r t h e r i n v e s t i g a t i o n of t h i s phenomenon. A l e t h a l a l l e l e of the l i g h t gene was crossed on t o a number of the suppressor chromosomes, as described e a r l i e r ( M a t e r i a l s and Methods). The i n t e n t was t o see what e f f e c t , i f any, h e t e r o z y g o s i t y f o r t h i s l i g h t a l l e l e would have on l e t h a l i t y and/or the eye colour i n t e r a c t i o n i n transheterozygous f l i e s . Table 1-6 shows r e s u l t s of v i a b i l i t y . Whereas some combinations of suppressors are unaffected i n t h i s regard (210/213, 210/215 and any transhetero.zygotes i n v o l v i n g 214) , some do demonstrate increased male l e t h a l i t y i n the presence of one I t 1 a l l e l e (213/215 and 215/216). I n t e r e s t i n g l y , two suppressor combinations (210/216 and 213/216) show what appears to be a maternal e f f e c t . For these combinations, r e d u c t i o n of male v i a b i l i t y only occurs when 216 i s c o n t r i b u t e d by the female parent. In examining the wing and eye i n t e r a c t i o n s (Table 1-7) , i t appears t h a t , i n males, transheterozygous combinations i n which I t 1 had no e f f e c t on male v i a b i l i t y , these phenotypes were a l s o unaffected. Others d i d show a l t e r a t i o n s i n both the eye and wing phenotypes when I t 1 was present. I n t e r e s t i n g l y , the 32 TABLE 1-6 Summary of the E f f e c t of I t x on V i a b i l i t y of C e r t a i n Transheterozygous Combinations of Suppressors C r o s s 3 F n males F-, females 210 X 213 0 .04 (0 .06) 0 .40 (0 .28) 213 X 210 i t 1 0 .05 (0 .08) 0 .33 (0 .14) 210 X 216 0 .28 (0 .23) 0 .46 (0 .32) 216 X 210 i t 1 0 .07 (0 .20) 0 .26 (0 .34) 213 X 216 0 .05 (0 .02) 0 .45 (0 .35) 216 X 213 i t 1 0 .0 (0 .13) 0 .36 (0 .45) 210 X 215 x t i 0 .55 (0 .30) 0 .50 (0 .63) 215 X 210 i t 1 0 .34 (0 .42) 0 .39 (0 .47) 213 X 215 0 . 14 (0 .24) 0 .32 (0 .45) 215 X 213 i t 1 0 .27 (0 .47) 0 .35 (0 .43) 216 X 215 0 .22 (0 .36) 0 .28 (0 .38) 215 X 216 i t 1 0 .25 (0 .41) 0 .31 (0 .39) 215 X 215 i t 1 0 .19 0 .28 214 Tft x 210 I t * 0 .27 (0 .19) 0 .36 (0 .29) 214 Tft x 213 l t ^ 0 .00 (0 .03) 0 .21 (0 .26) 214 Tft x 215 I t l 0 .20 (0 .38) 0 .22 (0 .37) 214 Tf t x 216 l t X 0 .02 (0 .02) 0 .12 (0 .21) a: i n the crosses i n d i c a t e d / the suprressor c o n t r i b u t e d by the female parent i s l i s t e d f i r s t . b: the numbers i n d i c a t e the r a t i o of Cy to Cy + (transheterozygote) f l i e s . Those contained i n parentheses are c o n t r o l values. Refer to Appendix C, Table C-2 f o r a c t u a l numbers of animals scored. 33 TABLE 1-7 E f f e c t of the I t 1 A l l e l e on of C e r t a i n Transheterozygous Suppressors Phenotypic I n t e r a c t i o n s Combinations of males females Genotype 3 eye wing eye wing 210/213 l t 1 mod ext mod none 213/210 l t 1 mod ext mod none 213/210 mod ext none none 214 Tft/210 I t 1 ext mod 210/214 mod mod mod mod 210/215 l t 1 mod none none none 215/210 I t 1 mod mod none none 210/215 mod none none none 210/216 I t 1 none none 216/210 I t 1 ext none 210/216 none none 214 Tft/213 I t 1 ext 213/214 mod ext mod mod 213/215 I t 1 ext mod mod none 215/213 l t 1 ext mod mod none 213/215 mod none none none 213/216 I t 1 ext none 213/216 ext ext ext none 215/216 I t 1 ext mod mod none 216/215 I t 1 ext ext mod none 215/216 mod none none none a: i n the genotypes i n d i c a t e d , the chromosome c o n t r i b u t e d by the female parent i s l i s t e d f i r s t . 34 e f f e c t i s not r e s t r i c t e d t o j u s t the eye phenotype. With the exception of some combinations i n v o l v i n g 215, transheterozygous females which are a l s o heterozygous f o r I t 1 demonstrate the eye phenotype t o some extent. This occurs even when the o r i g i n a l chromosomes f a i l e d to r e v e a l such an e f f e c t (for instance, combinations of 210/213, 213/215 and 215/216). Moreover, at l e a s t one combination (213/214) which was p r e v i o u s l y shown to possess t h i s i n t e r a c t i n g eye phenotype i n females, was found t o manifest i t t o a greater extent when one copy of I t . 1 i s present i n the genome. One genotype (210/216, heterozygous f o r I t 1 ) appears to demonstrate a maternal e f f e c t f o r the eye phenotype i n females as i t d i d f o r v i a b i l i t y i n males. When 216 i s c o n t r i b u t e d by the female parent, the r e s u l t i s a severe eye phenotype, whereas when 210 comes from the female, there i s no detectable e f f e c t . I t should be noted here t h a t males and females of the same genotype respond d i f f e r e n t l y t o the a d d i t i o n of I t 1 . For instance, i n the transheterozygote 210/214, males develop a more severe eye phenotype, while females do not. A l t e r n a t i v e l y , i n 210/213, females, but not males, are a f f e c t e d . E f f e c t o f t h e Y Chromosome Removal and a d d i t i o n of the Y chromosome was done i n order to determine what e f f e c t changes i n heterochromatin content would have on the v a r i o u s i n t e r a c t i o n s d i s p l a y e d among the 2L c l u s t e r of suppressors. As seen i n Table 1-8, absence of the Y 35 TABLE 1-8 E f f e c t of Y Chromosome on Phenotypic I n t e r a c t i o n s E x h i b i t e d by C e r t a i n Transheterozygous Suppressors F l males (X/O) F l females (X/XY) C r o s s a Cy :Cy + r a t i o eye wing Cy:Cy + r a t i o eye wing 213 Tft X 210 0.40 (108:43) b none none 0.02 (62:1) none mod 213 T f t X 215 0.40 (151:61) none none 0.03 (75:2) ext ext 213 T f t X 216 0.46 (225:104) none none 0.02 (56:1) n. d. c n. i a: r e f e r t o M a t e r i a l s and Methods f o r a f u l l d e s c r i p t i o n of the crosses performed. b: numbers i n parentheses are a c t u a l numbers of f l i e s scored, c: n.d. = not determined. 36 chromosome i n males a l l e v i a t e s a l l i n t e r a c t i o n s which occur i n the transheterozygous animals examined. R e l a t i v e male v i a b i l i t y of transheterozygotes increases t o a normal l e v e l , w hile the wing and eye phenotypes are no longer evident. A l t e r n a t i v e l y , when females contain an a d d i t i o n a l Y chromosome, the e f f e c t i s reversed. R e l a t i v e v i a b i l i t y of transheterozygotes decreases d r a m a t i c a l l y . In a d d i t i o n , the few such s u r v i v o r s a l s o demonstrate the eye (213/215) and wing (213/210 and 213/215) phenotypes where they were not present before. These females die d before f e r t i l i t y t e s t i n g could be done. 37 DISCUSSION Several l i n e s of evidence suggest that the 2L c l u s t e r suppressors are antimorphic i n nature. One i s t h a t s e v e r a l d e f i c i e n c i e s which contain suppressor l o c i are l e s s e f f e c t i v e at suppressing v a r i e g a t i o n than the corresponding EMS-induced mutations which they delete (see appendix A). In a d d i t i o n , homozygosity f o r a s i n g l e suppressor p o i n t mutation i s o f t e n more d e l e t e r i o u s than the hemizygous c o n d i t i o n . F i n a l l y , two doses of the w i l d type suppressor a l l e l e s can ameliorate, but does not t o t a l l y e l i m i n a t e , the e f f e c t of the suppressor mutants. I n i t i a l complementation a n a l y s i s of the 2L c l u s t e r suppressors has p r e v i o u s l y been reported ( S i n c l a i r et a l . , 1983). The r e s u l t s i n d i c a t e d t h a t these mutants i d e n t i f i e d the existence of at l e a s t three and perhaps four genetic l o c i . Subsequent s t u d i e s however, p o s i t i o n i n g the mutants v i a d e f i c i e n c y mapping, revealed an apparent i n c o n g r u i t y between the two i n v e s t i g a t i o n s . In p a r t i c u l a r , 216, mapping to region 31E, had p r e v i o u s l y been shown unable t o complement suppressors i n 31A-B. In a d d i t i o n , 207, which was l o c a l i z e d t o 31C, was a l s o i n e f f e c t i v e i n complementing mutants i n the 31A-B region. These r e s u l t s r e a f f i r m e d the suggestion t h a t suppressors belonging t o a s i n g l e c l u s t e r are f u n c t i o n a l l y i n t e r - r e l a t e d , and prompted a re-examination of the complementation p a t t e r n e x h i b i t e d by t h i s p a r t i c u l a r group of suppressors. Since a number of suppressors which r e s i d e i n the 2L c l u s t e r demonstrate homozygous l e t h a l i t y and/or female . 3 8 s t e r i l i t y , complementation a n a l y s i s was accomplished using these phenotypes as a b a s i s . Figure 1-2 (Results) i s a rep r e s e n t a t i o n of the complementation p a t t e r n revealed using these c r i t e r i a . While four complementation groups are i d e n t i f i e d by the nine suppressors, i t i s important t o r e c a l l t h a t these are dominant mutations, many of which demonstrate antimorphic p r o p e r t i e s . Thus, the r e s u l t s cannot be i n t e r p r e t e d i n the same manner as i f they were deri v e d from simple hypomorphic or amorphic mutations. In f a c t , i t i s c l e a r that the r e s u l t s here do not r e v e a l the d i s t i n c t i o n of separate genes per se. C e r t a i n transheterozygous combinations of 207 w i t h suppressors which map to regions p h y s i c a l l y d i s t i n c t from i t s e l f r e s u l t i n female s t e r i l i t y . In a d d i t i o n , while 216 i s a separate locus from a l l other suppressors i n t h i s c l u s t e r , i t f a i l s to complement s e v e r a l of them f o r female f e r t i l i t y . This r e s u l t p e r s i s t s even when recombinant suppressor chromosomes are used t o preclude complications which may r e s u l t from second s i t e mutations. In c o n t r a s t , when Df27 (an e f f e c t i v e n u l l mutation of 216) i s used i n t h i s a n a l y s i s i n place of the EMS-induced 216 poi n t mutant, the same i n t e r a c t i o n s are not generated. This r e s u l t suggests t h a t i t i s the antimorphic nature of p a r t i c u l a r suppressor mutations which causes the observed i n t e r g e n i c e f f e c t s . I t i s al s o c l e a r that s i m p l e , complementation a n a l y s i s of the suppressor poi n t mutations i s 3 9 not i n f o r m a t i v e i n separating the mutants i n t o d i s c r e t e p h y s i c a l e n t i t i e s . I t appears l i k e l y t hat the above a n a l y s i s i s complicated by the exis t e n c e of a d d i t i v e or s y n e r g i s t i c i n t e r a c t i o n s which r e s u l t when c e r t a i n combinations of suppressors are contained w i t h i n the same genome. This phenomenon i s not unusual, and se v e r a l other examples of i n t e r g e n i c i n t e r a c t i o n s have been reported. For instance, heterozygous combinations of c e r t a i n n o n - l e t h a l mutations (Dobzhansky, 1945) were found to r e s u l t i n what i s termed s y n t h e t i c l e t h a l i t y , and other v i s i b l e morphological e f f e c t s . Further s t u d i e s suggested t h a t these types of i n t e r a c t i o n s occur among mutants of f u n c t i o n a l l y r e l a t e d gene products (Lucchesi, 1968). More r e c e n t l y , an examination of genes a f f e c t i n g muscle development i n Drosophila have a l s o revealed f u n c t i o n a l i n t e r a c t i o n s (Homyk and Emerson, 1988). I t i s more than l i k e l y t hat the mutants under i n v e s t i g a t i o n i n t h i s study are f u n c t i o n a l l y r e l a t e d , and may i n t e r a c t at the molecular l e v e l . In a d d i t i o n to red u c t i o n i n male v i a b i l i t y and production of female s t e r i l i t y , c e r t a i n transheterozygous combinations among 210, 213, 214, 215 and 216 a l s o demonstrate abnormal wing and eye phenotypes. None of the combinations i n v o l v i n g 204 or 209 demonstrate such an e f f e c t ( r e c a l l t h a t they d i d not r e s u l t i n decreased male l e t h a l i t y or female s t e r i l i t y e i t h e r ) . o i Although not examined xn t h i s work, S u ( v a r 2 ) l u x has a l s o been found t o manifest abnormal eye and wing phenotypes w i t h s e v e r a l of these mutants (D. S i n c l a i r , pers. comm.). E f f e c t s on wing morphology have a l s o been reported to e x i s t between c e r t a i n combinations of suppressors which map to chromosome 3 (Reuter, 1986; Hedrick, 1989), although these have been i n t e r p r e t e d as r e s u l t i n g from i n t r a g e n i c , or a l l e l i c , r a t h e r than i n t e r g e n i c e f f e c t s . Although the m a n i f e s t a t i o n of these phenotypic i n t e r a c t i o n s i s v a r i a b l e , they are r e s t r i c t e d to suppressor combinations which a l s o demonstrate female s t e r i l i t y and reduced male v i a b i l i t y . This underscores the f a c t that complex i n t e r a c t i o n s occur among t h i s subset of 2L c l u s t e r suppressors. I f , as p r e v i o u s l y suggested, these mutations i d e n t i f y f a c t o r s i n v o l v e d i n the formation and/or maintainance of heterochromatin, i t i s p o s s i b l e t h a t they act as u n i t s of a multimeric p r o t e i n complex. The a b i l i t y of these mutants to suppress v a r i e g a t i o n , then, may be explained i n s e v e r a l ways. For instance, the l o s s of s t o i c h i o m e t r i c a l l y balanced r a t i o s of c e r t a i n subunits may l e a d t o i n e f f i c i e n t formation of heterochromatin, and greater expression of the v a r i e g a t i n g gene. A l t e r n a t i v e l y , a l t e r a t i o n s i n c e r t a i n subunits may cause them to e x h i b i t a lowered a f f i n i t y f o r chromatin, while they r e t a i n the a b i l i t y to i n t e r a c t w i t h other gene products. An e f f e c t such as t h i s would e x p l a i n both the antimorphic p r o p e r t i e s of the suppressor mutants, and the compounding i n t e r a c t i o n s which are noted when mutations e x i s t i n more that one gene (or subunit member). One p o s s i b l e e xplanation f o r the p l e i o t r o p i c e f f e c t s 41 e x h i b i t e d b y t r a n s h e t e r o z y g o u s s u p p r e s s o r s i s t h e i n a p p r o p r i a t e e x p r e s s i o n o f a n u m b e r o f d i f f e r e n t g e n e s w h i c h r e s u l t s w h e n c h r o m a t i n s t r u c t u r e i s d i s r u p t e d . W h i l e o n e s u p p r e s s o r may d e c r e a s e t h e i n t e g r i t y o f h e t e r o c h r o m a t i n e n o u g h t o a l l o w g r e a t e r e x p r e s s i o n o f v a r i e g a t i n g g e n e s ( l o c a t e d c l o s e t o h e t e r o c h r o m a t i c / e u c h r o m a t i c b o u n d a r i e s ) , t h e p r e s e n c e o f t w o ( o r m o r e ) s u p p r e s s o r s may a l t e r c h r o m a t i n s t r u c t u r e t o s u c h a d e g r e e t h a t g e n e s w h i c h n o r m a l l y r e s i d e i n o r n e a r h e t e r o c h r o m a t i n a r e a f f e c t e d . T o i n v e s t i g a t e t h i s f u r t h e r , t r a n s h e t e r o z y g o u s a n i m a l s w e r e g e n e r a t e d i n w h i c h o n e c o p y o f t h e h e t e r o c h r o m a t i c l i g h t g e n e a l l e l e , I t 1 , w a s a l s o p r e s e n t . T h e f a c t t h a t t h e e y e p h e n o t y p e w a s e x a c e r b a t e d i n a n u m b e r o f s u c h s u p p r e s s o r c o m b i n a t i o n s s u g g e s t s t h e i n v o l v e m e n t o f t h e l i g h t g e n e i n c a u s i n g t h i s e y e a b n o r m a l i t y ( f l i e s w h i c h a r e h e t e r o z y g o u s f o r I t 1 w i t h o u t t h e p r e s e n c e o f a n y s u p p r e s s o r s h a v e a n o r m a l e y e c o l o u r ) . I t a p p e a r s , t h e n , t h a t t h e p o s s e s s i o n o f c e r t a i n c o m b i n a t i o n s o f s u p p r e s s o r m u t a n t s c a n a l t e r l i g h t e x p r e s s i o n , p r e s u m a b l y t h r o u g h c h a n g e s i n c h r o m a t i n s t r u c t u r e . O n e w a y t o f u r t h e r t e s t t h i s h y p o t h e s i s i s t o e x a m i n e t h e t r a n s c r i p t l e v e l s o f t h e l i g h t g e n e i n f l i e s w i t h v a r i o u s c o m b i n a t i o n s o f m u t a n t s u p p r e s s o r g e n e s . S u c h a n e x p e r i m e n t s h o u l d b e p o s s i b l e n o w t h a t t h e l i g h t g e n e h a s b e e n c l o n e d . T h e o b v i o u s s e x u a l s p e c i f i c i t y o f t h e t r a n s h e t e r o z y g o u s i n t e r a c t i o n s s u g g e s t s t h a t t h e h e t e r o c h r o m a t i c Y c h r o m o s o m e may b e i n v o l v e d . I n f a c t , s e v e r a l s t u d i e s h a v e p r e v i o u s l y s h o w n t h e 2L c l u s t e r s u p p r e s s o r s t o b e s e n s i t i v e t o a l t e r a t i o n s i n 42 the amount of genomic heterochromatin (Mottus, 1983; Brock, 1986), wherein d e l e t i n g heterochromatic elements r e s u l t s i n reduction of the suppressed phenotype. I t i s thought that t h i s occurs by the subsequent increase i n the a v a i l a b l e heterochromatic p r o t e i n s r e l a t i v e to heterochromatic DNA which must be packaged. Consistent w i t h previous f i n d i n g s , d e l e t i o n of the Y chromosome i n transheterozygous males t o t a l l y a l l e v i a t e s a l l i n t e r a c t i o n s , while the a d d i t i o n of a Y chromosome t o such females e l i c i t s t h e i r expression (decreased v i a b i l i t y and eye and wing phenotypes). The f a c t t h a t changing the amount of heterochromatin can r e s u l t i n these e f f e c t s s t r o n g l y supports the suggestion that the suppressors encode products which are i n v o l v e d i n heterochromatin s t r u c t u r e . In examining the i n t e r a c t i o n s which e x i s t among combinations of suppressor mutations, s e v e r a l r e l a t i o n s h i p s are evident which a l l o w a rough o r g a n i z a t i o n of these mutants i n t o genetic l o c i . Su(var)216 maps separately from the others and th e r e f o r e defines one d i s t i n c t gene, l o c a t e d i n 31E. Of the suppressors i n 31A-B, 213 and 214 are almost i d e n t i c a l i n t h e i r i n t e r a c t i o n s w i t h other suppressors, suggesting them t o be a l l e l i c . In a d d i t i o n , 210 al s o demonstrates many, but not a l l of the same responses (unl i k e 213 and 214, i t i s not s t e r i l e i n combination w i t h S u ( v a r 2 ) 1 Q 1 ) , suggesting t h a t i t may be a weaker a l l e l e of the same gene. A l t e r n a t i v e l y , 215 responds very d i f f e r e n t l y than any of the other suppressors i n 31A-B, suggesting i t to be a separate lo c u s . The f a c t t h a t i t i s more 43 s e n s i t i v e t o reduced male v i a b i l i t y i n combination w i t h 207 than any other suppressors argues against i t being simply a D1 . weaker a l l e l e of another l o c u s . S u ( v a r 2 ) l u , which a l s o maps to 31A-B v i a d e f i c i e n c y mapping, appears to be a l l e l i c to the 213/214/210 locu s , although i t i s a l s o p o s s i b l e that t h i s mutant defines another gene i n the r e g i o n . In 31C, at l e a s t one suppressor locus i s evident. The two mutants which map here (204 and 207) have no s i m i l a r i t i e s beyond the f a c t that they both suppress PEV. While t h i s may i n d i c a t e t h a t two gene l o c i e x i s t i n the region, i t should be noted t h a t the homozygous v i a b l e 204 demonstrates p r o p e r t i e s which suggest i t t o be a hypomorph r a t h e r than an antimorph (D. S i n c l a i r , pers. comm.). This may e x p l a i n why i t f a i l s to demonstrate i n t e r a c t i o n s w i t h the other suppressors, and a l s o suggests t h a t i t may not be i n f o r m a t i v e i n i d e n t i f y i n g gene l o c i . The remaining suppressor, 209, since i t i s both homozygous and hemizygous v i a b l e , and shows no i n t e r a c t i o n s w i t h the other suppressors, cannot be placed i n t h i s scheme. Al t o g e t h e r , there appears to be at l e a s t two (perhaps three) gene l o c i i n 31A-B, one (or two) i n 31C and one i n 31E, f o r a t o t a l of between four and s i x suppressor genes. A more d e f i n i t i v e genetic i d e n t i f i c a t i o n of these suppressors may be accomplished w i t h the generation of n u l l mutations of the r e s p e c t i v e genes. 44 CHAPTER 2 ISOLATION AND CHARACTERIZATION OF HEMIZYGOUS LETHAL MUTATIONS IN REGION 31 OF DROSOPHILA MELANOGASTER 45 INTRODUCTION The cytogenetic region of 31-32 on the l e f t arm of chromosome 2 contains a number of genetic l o c i which are capable of suppressing a va r i e g a t e d phenotype. The o r i g i n a l 2L c l u s t e r of dominant Su(var) mutations appears to be a h i g h l y complex i n t e r a c t i n g set of genes (Chapter 1). Six of these dominant suppressors, as w e l l as the dominant Su(var)1 s e r i e s , i s o l a t e d by Reuter and Wolff (1981) are l o c a l i z e d t o region 31A-C. At l e a s t two l o c i , i d e n t i f i e d by d e f i c i e n c y mapping, and perhaps as many as f i v e (see Chapter 1 Results and Discussion) are i d e n t i f i e d by these mutants. The remaining dominant suppressor maps to region 31E. In a d d i t i o n t o the dominant mutations, two r e c e s s i v e suppressors of v a r i e g a t i o n have been found t o map i n 31F-32A (see Appendix B). Both of these i d e n t i f y separate l o c i , g i v i n g a minimum of f i v e to seven l o c i i n the region which can suppress PEV. The two r e c e s s i v e suppressors, male-female s t e r i l e (mfs48) and w a v o i d - l i k e (wdl), have p r e v i o u s l y been i d e n t i f i e d as belonging t o a c l u s t e r of m a t e r n a l - e f f e c t mutations (Sandler, 1977; L i n d s l e y et a l . , 1980) which i n t e r a c t w i t h sex chromosome heterochromatin. I t i s i n t e r e s t i n g that a number of the suppressors, i n c l u d i n g those i n the 2L c l u s t e r , have a l s o been shown to respond to a l t e r a t i o n s i n Y chromosome heterochromatin (Mottus, 1983; Brock, 1986) . This, as w e l l as the f a c t t h a t some of them can suppress v a r i e g a t i o n , f u r t h e r suggests a f u n c t i o n a l s i m i l a r i t y between these m a t e r n a l - e f f e c t mutants, i and the dominant suppressors i n t h i s region. Only four other mutations, besides the group of maternal-e f f e c t genes, are known to map w i t h i n or near the region occupied by the 2L c l u s t e r suppressors. Two of these are z y g o t i c l e t h a l mutations, pimple and basket (Nusslein-Volhard et a l . , 1984) which map t o 31C-31F, as defined by Df J-der27 (see f i g u r e 2-1). Another i s trunk (Schupbach and Weischaus, 1986) which has a m e i o t i c map p o s i t i o n of 2-36, s i m i l a r to those of the suppressor mutants (see Chapter 1, Table 1-2). An exact c y t o l o g i c a l p o s i t i o n f o r t h i s mutant, using d e f i c i e n c i e s , has not been determined. F i n a l l y , the neomorphic Jammed mutation a l s o maps to t h i s region at a p o s i t i o n of 2-41.0. Reversion of Jammed has been instrumental i n generating the d e f i c i e n c i e s which e x i s t i n region 31. In an attempt to f u r t h e r c h a r a c t e r i z e t h i s region of the genome, a la r g e s c a l e genetic a n a l y s i s was undertaken. The approach to i d e n t i f y i n g l o c i i n the region was t o i s o l a t e hemizygous l e t h a l mutations u t i l i z i n g the d e f i c i e n c y D f ( 2 L ) J -der2 ( r e f e r to Figure 2-1). Since many of the suppressor mutants l o c a l i z e d t o t h i s region are homozygous l e t h a l , a d d i t i o n a l Su(var) mutations could a l s o be i d e n t i f i e d i n t h i s manner. The f o l l o w i n g work describes the implementation of a screen f o r hemizygous l e t h a l mutations, and the r e s u l t s of some of the i n i t i a l g e netic analyses of the mutants obtained. 47 MATERIALS AND METHODS  Strains For a d e t a i l e d d e s c r i p t i o n of the v i s i b l e mutations and s p e c i a l chromosomes used, r e f e r to L i n d s l e y and G r e l l (1968). S i x Jammed-derived d e f i c i e n c y s t r a i n s , l o c a t e d i n region 31 of chromosome arm 2L (Mange and Sandler, 1973; Sandler, 1977; Salas and Lengyel, 1984) were used i n t h i s study. Three were k i n d l y provided by Dr. L. Sandler: Df(2L)J-der2, extending from 31A-32A; Df(2L)J-der27 which uncovers region 31C-31F; and Df(2L)J-der39. Three a d d i t i o n a l such mutants s u p p l i e d through the generosity of Dr. J . Lengyel are: Df(2L)J-der222, a small d e f i c i e n c y i n 31E; Df(2L)J-der77, uncovering 31C-31E; and Df(2L ) J - d e r l 0 6 , extending from 31E-31F (Salas and Lengyel, 1984). Henceforward, these mutations w i l l be r e f e r e d t o as Df2; Df27; Df39; Df222: Df77; and Dfl06 r e s p e c t i v e l y . In a d d i t i o n to i t s chromosomal abberation i n 31, Df2 al s o c a r r i e s a small d e f i c i e n c y at the t i p of 2L. Refer to Figure 2-1 f o r a graphic r e p r e s e n t a t i o n of these d e f i c i e n c i e s . A l l these d e f i c i e n c i e s except Df39 were kept i n stock balanced over the m u l t i p l y i n v e r t e d chromosomes In(2LR)CvSM5 (Df27, Df77, Dfl06, and Df222) or In(2LR)CvO (Df2) , r e f e r r e d t o as SM5 and CyO r e s p e c t i v e l y . (These chromosomes are i d e n t i f i e d by the dominant Curly wing phenotype.) Df39 i s a dominant female s t e r i l e chromosome which a l s o manifests a Minute phenotype. I t i s kept i n stock by o u t c r o s s i n g males of genotype Df39/SM5 or Df39/bw v D e 2 to v i r g i n bw v D e 2/SM5 females each generation. 48 FIGURE 2-1 C y t o l o g i c a l E x t e n t o f t h e D e f i c i e n c i e s u s e d i n t h i s S t u d y A m b i g u i t y i n t h e p r e c i s e b r e a k p o i n t s o f s o m e d e f i c i e n c i e s i s d e n o t e d b y d o t t e d l i n e s . 49 CM Q 0) CO «•— Q Q CN o Q 0 5 0 One f u r t h e r d e f i c i e n c y i n t h i s region, designated Df(2D Sco, was k i n d l y provided by Dr. G. Reuter. L i k e Df39, t h i s d e f i c i e n c y d i s p l a y s a Minute phenotype and shows p a r t i a l l y dominant female s t e r i l i t y . I t i s kept i n stock i n a manner s i m i l a r t o tha t described above f o r Df39. C u l t u r e C o n d i t i o n s F l i e s were r a i s e d on standard sucrose cornmeal medium. Tegosept (0.04%) was added as an i n h i b i t o r of mould growth. Crosses were c a r r i e d out at the temperatures i n d i c a t e d i n the ensuing d e s c r i p t i o n s . Mutant I s o l a t i o n The i s o l a t i o n of hemizygous l e t h a l mutations i n region 31 of 2L was accomplished as o u t l i n e d i n Figure 2-2. Adult males, i s o g e n i c f o r chromosome 2 marked wi t h the re c e s s i v e v i s i b l e mutations cn and bw, were fed e t h y l methanesulphonate (EMS, 0.24% v/v) according to the method of Lewis and Bacher (1968), and mated to Gla/SM5 females at 25°C. Sin g l e F-^  males of the genetic c o n s t i t u t i o n cn bw /Gla (where * i n d i c a t e s a chromosome exposed to EMS) were then mated i n v i a l s t o four or f i v e Df2/CyO females. This cross was c a r r i e d out at 2 9°C i n order to i s o l a t e p u t a t i v e temperature s e n s i t i v e mutants. A f t e r four days, these f l i e s were t r a n s f e r r e d i n t o f r e s h v i a l s and kept at 18°C, while the o r i g i n a l v i a l s remained at 29°C. The F 2 generation, developed at 29°C was then examined f o r the presence of s t r a i g h t winged, non-Gla progeny (genotype FIGURE 2-2 I s o l a t i o n of Hemizygous L e t h a l Mutations i n Region 31 EMS cn bw cn bw Gla _ _ x — 99 SM5 cn bw single X Gla Cfcf 29°C D f 2 Cyo 99 cn bw cn bw D f 2 CyO D f 2 subculture at 18°C Gla Gla CyO present absent discard cn bw CyO Cfcf and 99 STOCK 5 2 Df2/cn bw ). When an absence of such f l i e s ( i n d i c a t i n g hemizygous l e t h a l i t y ) i n e i t h e r sex was found, a stock was generated by c o l l e c t i n g and mating males and v i r g i n females of the genotype cn bw /SM5 from the d u p l i c a t e c u l t u r e s maintained at 18°C. Three t r i e t h y l e n e melamine (TEM) and 18 gamma-induced mutants i s o l a t e d p r e v i o u s l y v i a a p r o t o c o l s i m i l a r to tha t j u s t described, were in c l u d e d i n the subsequent analyses. The complementation a n a l y s i s and some of the d e f i c i e n c y mapping of these mutants had been done p r i o r t o t h i s i n v e s t i g a t i o n by K. Kafer and D. S i n c l a i r . The three TEM induced mutants are designated 1-44, 2-119, and 3-50, while the gamma induced mutants inc l u d e those numbered 12-114, 13-47, 13-83, 13-117, 14-95, 14-140, 16-165, 17-83. 19-153, 23-127, 24-127, 25-159, 27-168, 29-142, 29-185, 30-18, 33-24, and 33-161. Three of these mutants, 14-95, 25-159 and 33-24 are homozygous v i a b l e . I d e n t i f i c a t i o n o f T e m p e r a t u r e - s e n s i t i v e Mutations Males of each mutant s t r a i n were crossed i n v i a l s t o 15-20 Df2/CyQ females at 29°C and t r a n s f e r r e d every day to f r e s h v i a l s t o obtai n a t o t a l of 8 successive subcultures. Four of these were l e f t to develop at 2 9°C while the others were allowed to devolop at 25°C. (25°C was used as the permissive temperature i n order t o allow good penetrance of the c u r l y phenotype.) Each set was then examined f o r the presence of s t r a i g h t winged f l i e s of e i t h e r sex (cn bw /Df2) at each temperature. D e f i c i e n c y Mapping Males of each mutant s t r a i n (cn bw /SM5) were crossed t o v i r g i n females of Df27, Df77, Dfl06 and Df222. The r e s u l t a n t progeny were then scored f o r absence of a s t r a i g h t winged phenotypic c l a s s (cn bw /Df), i n d i c a t i n g a f a i l u r e to complement the s p e c i f i c d e f i c i e n c y . Except f o r Df222, a l l mutant s t r a i n s i s o l a t e d were t e s t e d w i t h each of the above l i s t e d d e f i c i e n c i e s . Df222 was used only i n mapping those mutations which f i r s t showed a f a i l u r e t o complement each of the other d e f i c i e n c i e s . Since Df39 and Df(2L)Sco are dominant female s t e r i l e , the mapping crosses were done by mating d e f i c i e n c y - b e a r i n g males (Df/SM5) to v i r g i n females of each mutant s t r a i n . These d e f i c i e n c i e s were used only i n mapping those mutants which had p r e v i o u s l y been shown t o complement Df27, Df77 and Df106. A l l d e f i c i e n c y mapping was c a r r i e d out at 29°C. I n t e r - s e Complementation Three of the groups defined by d e f i c i e n c y mapping were f u r t h e r i n v e s t i g a t e d by performing i n t e r - s e complementation analyses. This was done by making p a i r - w i s e crosses of a l l p o s s i b l e combinations of mutants w i t h i n a p a r t i c u l a r group. Since the mutants were a l l i s o l a t e d on a cn bw chromosome, f a i l u r e to complement was i n d i c a t e d by the absence of homozygous cn bw/cn bw (white eyed) f l i e s . Crosses were c a r r i e d out at 29°C. 54 E f f e c t on V a r i e g a t i o n Each mutant was outcrossed i n t o a wm^ background as o u t l i n e d i n Figure 2-3. Males of each mutant s t r a i n were mated en masse to w v i r g i n females which were heterozygous f o r a standard second chromosome marked w i t h the dominant Tuft (Tft) mutation and the m u l t i p l y i n v e r t e d chromosome, CyO. Non-Tuft F-^  i n d i v i d u a l males (w^/Y; cn bw*/CyO) were then crossed t o wm^/wm^; Tft/CyO females, and non-Tuft F2 progeny were s e l e c t e d to e s t a b l i s h balanced stocks. Three to f i v e l i n e s , each deri v e d from a s i n g l e F-j_ male, were generated f o r each mutant s t r a i n . The l e v e l of eye pigment present i n these f l i e s , when r a i s e d at 22°C was measured as described p r e v i o u s l y (Chapter 1, M a t e r i a l s and Methods). 55 F I G U R E 2 - 3 Mating P r o t o c o l f o r P l a c i n g Mutations i n w1 Background X c n b w CyO Cfcf w ™ 4 T f t w ™ 4 CyO w ™ 4 cn b w CyO s i n g l e X T f t 99 w ™ 4 CyO w ™ 4 c n b w CyO c T c f x w " 1 4 w " 1 4 c n b w CyO 99 STOCK 56 RESULTS An EMS-induced screen f o r hemizygous l e t h a l or strong s e m i - l e t h a l mutations i n the region d e f i n e d by Df2 y i e l d e d 53 mutants from a t o t a l of 1848 chromosomes t e s t e d . One of these (G5_4) upon r e t e s t i n g , was found to i d e n t i f y a dominant temperature s e n s i t i v e (DTS) mutant, d i s p l a y i n g l e t h a l i t y at 29°C, and dominant female s t e r i l i t y at a l l temperatures. No f u r t h e r c h a r a c t e r i z a t i o n of t h i s mutant has been done. Of the remaining 52 mutants, eight (A63, A65, B100, D121, E24, F15, G25 and H95) were found to demonstrate a leaky, or s e m i - l e t h a l hemizygous phenotype at 29°C (Table 2-1), while the remainder appear t o be completely l e t h a l . A number of these mutants were al s o found t o demonstrate secondary c h a r a c t e r i s t i c s which are described here. One of these i s G83. F l i e s bearing t h i s mutation d i s p l a y a Minute phenotype at a l l temperatures. In a d d i t i o n , females are p a r t i a l l y s t e r i l e (a c h a r a c t e r i s t i c of Minutes). Two of the s e m i - l e t h a l mutants, A65 and G25, demonstrate an abnormal wing phenotype i n the hemizygous c o n d i t i o n . The wings of these animals remain spread out (at approximately a 45° angle) even when at r e s t . I t i s curious that t h i s resembles a m i l d form of the abnormal wing phenotype observed w i t h c e r t a i n combinations of the 2L c l u s t e r Su(var) mutations (Chapter 1). Several of the EMS-induced mutants are v i a b l e as homozygotes (Table 2-1), and can thus be c l a s s i f i e d as hypomorphs (Muller, 1932), or more s p e c i f i c a l l y , as haplo-TABLE 2-1 Test f o r Hemizygous L e t h a l i t y Using Df2 Mutant 2 9°C 25°C S t r a i n males females males females A8 68:0 a 59:0 21:0 13:0 A43 77:0 147:0 32:15 32:3 A61 92:0 91:0 77:0 88:0 A63 47:0 48:1 51:6 66:0 A65 107:0 130:2 53:23 75:12 A7 6 29:0 28:0 32:6 20:0 A102 23:0 41:0 24:0 33:0 A141 36:0 40:0 8:0 8:0 A156 25:0 49:0 26:0 36:0 B2 6 44:0 65:0 55:0 57:0 B47 16:0 19:0 37:0 35:0 B35 56:0 41:0 44:0 58:0 B100 66:0 76:1 51:1 76:0 B106 67:0 81:0 72:0 95:0 B149 21:0 25:0 28:0 39:0 *C25 17:0 23:0 27:0 36:0 C35 70:0 77:0 58:0 98:0 C36 28:0 45:0 39:0 35:0 C70 37:0 50:0 30:0 58:0 C93 80:0 63:0 75:3 83:0 C98 66:0 91:0 51:0 88:0 C104 15:0 37:0 38:1 45:0 C131 32:0 51:0 66:0 75:0 C133 31:0 71:0 60:5 69:1 D22 96:0 140:0 65:0 98:0 D57 63:0 82:0 57:0 55:3 D121 75:1 85:0 62:3 87:0 D155 71:0 81:0 47:0 50:0 * represents mutations which are homozygous v i a b l e . a: numbers represent t o t a l c u r l y : n o n - c u r l y f l i e s i n the progeny. Crosses were performed as described i n M a t e r i a l s and Methods. 58 Table 2-1 continued Mutant 2 9°C 25°C S t r a i n males females males females 132:0 164 :0 101:10 109:0 E6 74:0 106:0 37:0 34:0 *E10 24:0 44:0 53:0 84:0 E15 42:0 48:0 50:0 47:0 *E20 35:0 30:0 45:14 46:15 E24 94:1 122:0 84:5 86:0 *E34 22:0 19:0 16:0 17:0 E56 38:0 57:0 25:4 46:0 E73 68:0 114:0 64:0 83:0 E113 125:0 166:0 57: 6 95:0 F15 75:1 120:0 106:16 162:1 F40 124:0 174:43 102:0 133:18 F75 73:0 61:0 68:0 50:0 F133 30:0 92:0 35:0 35:0 Gl 82:0 24:0 101:2 124:5 G21 60:0 84:0 60:0 69:0 G25 91:4 132:7 26:4 36:2 G55 40:0 68:0 35:0 40:0 G78 101:0 143:0 69:5 99:1 G83 2:0 9:0 19:0 27:0 ^H30 126:0 167:0 47:0 51:0 H95 158:0 132:2 71:0 58:2 H113 46:0 67:0 35:1 37:0 59 s p e c i f i c l e t h a l s (Nash and Janca, 1983). Although a systematic study has not yet been completed, at l e a s t one of these homozygous v i a b l e mutations (C25) a l s o appears .to be homozygous male and female s t e r i l e . Others, however, are f e r t i l e , and can be maintained i n stock as homozygotes. The remaining mutants a l l appear to be homozygous as w e l l as hemizygous l e t h a l . I t should be noted that the chromosomes on which the hemizygous l e t h a l phenotype was recovered have not undergone recombination replacement of the chromosome arms. Thus the p o s s i b i l i t y t h a t any of these a d d i t i o n a l phenotypes r e s u l t from the e x istence of a second s i t e mutation cannot be discounted. The 52 mutants i d e n t i f i e d by t h i s screen, together w i t h the 21 gamma ray and TEM-induced mutants p r e v i o u s l y i s o l a t e d , were subject t o the f o l l o w i n g genetic analyses. Sexual Dimorphism Only one of the 52 EMS-induced mutants (F4Q; see Table 2-1) was found t o e x h i b i t sexual dimorphism. With t h i s mutation, l e t h a l i t y occurs only i n hemizygous males. G e n o t y p i c a l l y s i m i l a r females are v i a b l e . These s u r v i v i n g females, however, are m o r p h ologically reduced i n s i z e , and s t e r i l e . An examination of the ovaries of these f l i e s demonstrates t h a t the s t e r i l i t y i s a r e s u l t of undeveloped o v a r i e s . O v arioles are present, but no egg production i s apparent. 60 T e m p e r a t u r e - s e n s i t i v i t y Thirteen of the EMS-induced mutants are temperature-s e n s i t i v e l e t h a l s (Table 2-1). Three of these, A43, A65 and E20, have the most obvious e f f e c t , r e s u l t i n g i n both male and female v i a b i l i t y of hemizygotes at 25°C where at 29°C, they are v i r t u a l l y complete l e t h a l s . The abnormal wing phenotype of hemizygous A65 s u r v i v o r s which was observed at 2 9°C (see above), i s not apparent when the same genotype i s produced at 25°C. The f a c t that both l e t h a l i t y and the wing phenotype are temperature-sensitive i n d i c a t e s t h a t they may r e s u l t from the same mutation. The remaining ten temperature-sensitive mutants are not as s t r i k i n g i n t h e i r e f f e c t . Four (C133. F15, Gl and G78) demonstrate only a minor increase i n v i a b i l i t y of hemizygotes at the permissive temperature (25°C). The remaining s i x appear to show s e x - s p e c i f i c l e t h a l i t y at 25°C. F i v e , A63, A7 6, C93, E3 and E24, all o w s u r v i v a l of only hemizygous males, while one, D57, r e s u l t s i n hemizygous female v i a b i l i t y . In one of these mutants, C93, the few males which s u r v i v e at 25° a l s o demonstrate the same abnormal wing phenotype described p r e v i o u s l y . The number of hemizygous animals which s u r v i v e at 25°C i s considerably l e s s than expected. Thus, although these mutants are t emperature-sensitive, they do not d i s p l a y a completely wild-type phene at 25°C. These r e s u l t s may i n d i c a t e t h a t leaky expression of the gene products f o r these mutants occurs at 61 25°C. In f a c t , t h i s may be expected, since the temperature d i f f e r e n c e between the r e s t r i c t i v e and permissive c o n d i t i o n s i s s l i g h t (29°C versus 25°C). I t i s p o s s i b l e that a more dramatic d i f f e r e n c e would be noted i f the permissive temperature was lowered to 22°C (25°C was needed f o r expression of the Curly phenotype). A l t e r n a t i v e l y , i t i s p o s s i b l e that these r e s u l t s are somewhat spurious, given t h a t s c o r i n g the Curly phenotype i s often d i f f i c u l t even i n f l i e s which are r a i s e d at 25°C. This problem may be overcome i n f u t u r e by progeny t e s t i n g the apparent hemizygote s u r v i v o r s . D e f i c i e n c y Mapping and Complementation A n a l y s i s P h y s i c a l mapping of these mutants was performed using a number of d e f i c i e n c i e s which overlap Df2. The r e s u l t s are summarized i n Table 2-2, and depicted g r a p h i c a l l y i n Figure 2-4. The breakpoints of f i v e of these d e f i c i e n c i e s (Df27, Df77, Df106, Df39 and Df(2DSco) l o c a t e d i n region 31 allowed the s u b d i v i s i o n of Df2 i n t o nine d i s t i n c t regions. (Df222, a small d e f i c i e n c y l o c a t e d i n region 31E, f a i l e d to uncover any of the mutants t e s t e d , and i s t h e r e f o r e not shown on the d e f i c i e n c y map i n Figure 2-4.) Ten of the EMS-induced and four of the gamma-induced mutants map to the t i p d e f i c i e n c y of Df2 and were thus ommitted from f u r t h e r genetic a n a l y s i s . Six mutants (3 EMS, 1 TEM and 2 gamma) map to a region i n 31A/B, de f i n e d by the d i s t a l breakpoint of Df2 and the d i s t a l breakpoint of Df39. This region i s a l s o known to contain four of the 2L c l u s t e r suppressors (210, 213, 214 and 215). Three 62 TABLE 2 -2 Summary of D e f i c i e n c y Mutants i n Region 31 Mapping of Hemizygous L e t h a l Mutant Df27 Dfl06 Df77 Df39 Df (2L) Sco Region A8 31E A4 3 + + + + + t i p A61 + - + 31F A63 - + - 31D A65 + + + - + 32A A7 6 - - - 31E A102 + + + + - 31A/B A141 - + - 31D A156 + + + + + t i p B2 6 — — — 31E B47 - - - 31E B35 - - - 31E B100 + + + - - 31B B106 - - - 31E B149 + + - 31C C25 + + + + + t i p C35 - + - 31D C36 - - - 31E C70 - - - 31E C93 - - - 31E C98 + + + + - 31A/B C104 + + + - 31A/B C131 + + + + + t i p C133 + + + + + t i p D22 — — — 31E D57 - - - 31E D121 + + + - + 32A D155 + + + + + t i p E3 + - + 31F E6 + + + + + t i p E10 - - - - - 31E E15 - + - 31F E20 - - - 31E E24 + + + - + 32A E34 - - - 31E E56 - + - 31F E73 + + + - + 32A E113 - - - 31E 63 T a b l e 2-2 c o n t i n u e d M u t a n t Df27 D f l 0 6 Df77 Df39 Df (2L) SCO R e g i o n F15 + + + _ + 32A F40 + + + - + 32A F75 - - - 31E F133 + + + — - 31B G l + + + — + 32A G21 + + + - + 32A G25 + + + + + t i p G55 + + + + + t i p G78 - - - 31E G83 + + + n d n d n d H30 - + - 31D H95 + + + - + 32A H133 - - - 31E 1-44 + + + — 31A/B 2-119 + + - 31C 3-50 - + - 31D 12-114 + + + + t i p 13-47 - - 31E 13-83 - - - 31E 13-117 - - - 31E 14-95 + - + 31F 14-140 + + - 31C 16-165 + + + - 31A/B 17-83 + + + + t i p 19-153 + + - 31C 23-127 - - 31D-31E 24-127 + + - 31C 25-159 - - 31D 27-168 + + + - 31A/B 29-142 - + - 31D 29-85 - - + 31F 30-18 + + + + t i p 33-24 + + + + t i p 33-161 - - 31E n . d . = n o t d e t e r m i n e d . ( f o r a c t u a l d a t a , r e f e r t o A p p e n d i x D) 64 FIGURE 2-4 Schematic Representation of D e f i c i e n c y Mapping of Hemizygous L e t h a l Mutations i n Region 31 D e f i c i e n c i e s are represented by the h o r i z o n t a l l i n e s . This map i s i n c l u d e d only to demonstrate the r e l a t i v e p o s i t i o n s of the d e f i c i e n c y breakpoints. The extent of each d e l e t i o n i s not n e c e s s a r i l y drawn to s c a l e . Df(2L)Sco i s shown by a dotted l i n e to i n d i c a t e the u n c e r t a i n t y i n i t s breakpoints r e l a t i v e t o the others. The approximate c y t o l o g i c a l extent of each subregion i s a l s o i n d i c a t e d . 65 31AB 31B 31C 31D CTi CT> A I M ftlOO B149 M 3 C98 ^133 A M I C104 C3J U - 1 4 2 £56 19-153 H30 1-44 24-127 16-165 2-119 27-168 31E 31F1 31F 32A Df39 Df77 Df27 Df106 Df 2 L S c o A8 29-85 A61 A 6 5 A76 t.3 0121 8 2 6 E l 5 C24 035 F 7 3 $47 F15 &106 14-95 F40 C36 01 C70 Q21 093 H95 D22 057 £10 120 E.34 £113 F75 H113 13-47 13- 83 13-117 33-161 EMS-induced mutants ( i n c l u d i n g the Minute, G83), map to 31B/C, between the d i s t a l breakpoints of Df39 and Df77, where two suppressors (204 and 207) have a l s o been l o c a l i z e d . Complementation between the suppressors and the mutants i d e n t i f i e d i n t h i s study which map w i t h i n the same cytogenetic i n t e r v a l s has not yet been done. The region i n 31C between the d i s t a l breakpoint of Df77 and that of Df27, contains f i v e hemizygous l e t h a l mutations (1 EMS, 1 TEM and 3 gamma). I n t e r se complementation among these mutants has been completed. The mutants i n t h i s group are defined by B149, 2-119, 14-140, 19-153 and 24-127 (Figure 2-4). Complementation among the four gamma and TEM-induced mutants was done p r e v i o u s l y (D. S i n c l a i r ) . As i n d i c a t e d i n Table 2-3, each of these mutants were shown to represent separate l o c i . The r e s u l t s of complementation between these mutants and B149 i s a l s o shown i n Table 2-3. This mutant c l e a r l y f a i l s to complement the TEM-induced 2-119, and one of the gamma-induced mutants, 19-153. Thus, i t appears th a t at l e a s t four l o c i e x i s t i n region 31C, between the d i s t a l breakpoint of Df77 and the d i s t a l breakpoint of Df27. Nine mutants (5 EMS, 1 TEM and 3 gamma) can be l o c a l i z e d t o a region i n 31D, defined by the d i s t a l breakpoints of Df27 and Df106. The i n t e r se complementation of t h i s group has been p a r t i a l l y completed. Previous r e s u l t s demonstrated two of the three gamma-induced mutants i n t h i s region (23-127 and 29-142) to be a l l e l i c , w hile 25-159 and the TEM-induced 3-50 i d e n t i f i e d 67 TABLE 2-3 I n t e r se Complementation of Hemizygous L e t h a l Mutations i n 31C 2-119 14-140 19-153 24-127 B149 - + - + ( l l l : 0 ) a (133:34) (103:0) (133:33) 2-119 + b + + 14-140 + + 19-153 + a: numbers represent Cy:Cy + f l i e s i n the F-^  progeny. b: r e s u l t s without accompanying data were determined p r e v i o u s l y by D. S i n c l a i r . 68 two a d d i t i o n a l l o c i . S u r p r i s i n g l y , 23-127 was a l s o found unable to complement a t h i r d gamma-induced mutant (13-117) which i s l o c a l i z e d t o a c y t o l o g i c a l l y d i s t i n c t region from 2 9-142. This suggests t h a t 23-127 may be a small d e f i c i e n c y . Complementation among the f i v e EMS-induced mutants i n t h i s region demonstrates a more complex r e l a t i o n s h i p (Table 2-4). Although i t i s i t s e l f homozygous v i a b l e and f e r t i l e , E56 i s unable to complement three other mutants (A141, C35 and H30), a l l of which f a i l e d to complement each other. The other, A63, was capable of complementing only E56. Thus, these f i v e mutants define at l e a s t two l o c i . Although complementation between the gamma and TEM mutants i n t h i s region and the EMS-induced mutants has not been c a r r i e d out e x t e n s i v e l y , 23-127 was able to complement a l l f i v e EMS mutants, d e f i n i n g at l e a s t one a d d i t i o n a l l o c u s . These r e s u l t s i n d i c a t e that as few as three, and as many as f i v e separably mutable l o c i e x i s t i n the region of 31D, as defined by these mutants. Twenty-three mutants (18 EMS and 5 gamma) map to region 31E, between the d i s t a l breakpoint of Df106 and the proximal breakpoint of Df77. This region a l s o contains the 2L c l u s t e r suppressor Su(var)216. Complementation has been done between these mutants and 23-127 (Table 2-5). The r e s u l t s f a i l to re v e a l any a d d i t i o n a l a l l e l e s of t h i s mutant. Further i n t e r se complementation of t h i s group i s c u r r e n t l y underway. The small region bounded by the proximal breakpoints of Df27 and Df77, 31F1-2, contains only one gamma-induced mutant (29-85). Four mutants (3 EMS and 1 gamma) are l o c a l i z e d to TABLE 2-4 I n t e r se Complementation of Hemizygous L e t h a l Mutants i n Region 31D A141 C35 E56 H30 23-127 A63 467:0 a (-) 220:0 (-) 168:56 ( + ) 207:0 (-) + b A141 234:0 (-) 313:0 (-) 253:0 (-) 15: 9 ( + ) C35 260:0 (-) 213:0 (-) 53:17 ( + ) E56 257:0 (-) 65:41 ( + ) H30 61:23 ( + ) a: numbers represent t o t a l Cy:Cy f l i e s i n the progeny, b: t h i s r e s u l t was determined separately by N. Clegg. 70 TABLE 2-5 Complementation Between 23-127 and Hemizygous L e t h a l Mutants i n 31E F-j_ Progeny Cross c y Cy + Complementation 23-127 X A8 37 20 + 23-127 X A7 6 39 16 + 23-127 X B26 58 28 + 23-127 X B35 43.' 23 + 23-127 X B47 67 34 + 23-127 X B106 27 8 + 23-127 X C36 42 21 + 23-127 X C70 18 13 + 23-127 X C93 52 9 + 23-127 X D22 46 13 + 23-127 X D57 67 16 + 23-127 X E34 37 19 + 23-127 X E113 38 10 + 23-127 X F75 55 30 + 23-127 X G7 8 52 28 + 23-127 X H113 58 19 + 71 31F3-32A, the region d e f i n e d by the proximal breakpoints of Df27 and Df106. The i n t e r se complementation a n a l y s i s of t h i s group has been completed. The r e s u l t s of t h i s study, shown i n Table 2-6, i d e n t i f y at l e a s t three l o c i i n the region, one of which i s represented by two a l l e l e s (the EMS-induced A61 and the gamma-induced 14-95). F i n a l l y , eight EMS-induced mutants map to region 32A, from the proximal breakpoint of Df106 to the proximal breakpoint of Df2. This i s the same region t o which the m a t e r n a l - e f f e c t mutants ( i n c l u d i n g the two which act as suppressors) have been l o c a l i z e d . Suppressor A n a l y s i s To determine whether any of the new mutations i d e n t i f y a d d i t i o n a l suppressor genes, or a l l e l e s of p r e - e x i s t i n g suppressor l o c i , the mutants i s o l a t e d i n t h i s study were t e s t e d f o r t h e i r a b i l i t y to suppress v a r i e g a t i o n . To do t h i s , each EMS, TEM and gamma-induced mutant was placed i n a w™ 4 background (Figure 2-2, M a t e r i a l s and Methods). As i n d i c a t e d by Table 2-7, none of the mutants t e s t e d appear to be strong suppressors. Two of them, however, show a notable e f f e c t (an increase i n pigment l e v e l s of more than 25%) which appears to be male s p e c i f i c . One of these, 16-165, maps to the same i n t e r v a l as four p r e v i o u s l y i d e n t i f i e d suppressors (210, 213, 214. 215). The second, 23-127, i s the same mutant tha t was p r e v i o u s l y defined as a small d e f i c i e n c y i n region 72 TABLE 2-6 I n t e r se Complementation of Hemizygous L e t h a l Mutations i n Region 31F1-2 E3 E15 14-95 A61 + . + (90:61) a (105:41) (186:0) E3 + + (62:30) (80:26) E15 + (83:29) a: numbers represent t o t a l Cy:Cy f l i e s i n F q progeny. 73 TABLE 2-7 E f f e c t of Hemizygous L e t h a l Mutations i n Region 31 on V a r i e g a t i o n Pigment L e v e l s 3 Mutant males females A102 12 + 1 15 + 3 C98 17 + 3 21 + 4 C104 10+2 19+4 1-44 30 + 5 21 + 2 16-165 52+3 31 + 9 27-168 28 + 3 18+2 B100 6+2 12+2 F133 12 + 3 12+2 B149 10+2 12+2 2-119 19+1 11+2 14-142 42 + 3 2 6+6 19-153 29+2 15+2 24-127 4 9+7 33+2 A63 18+3 21 + 3 A141 13 + 4 18+2 C35 8+2 17 + 7 E56 10+2 13 + 1 H30 24 + 3 18+2 3-50 32+5 29+3 23-127 64+5 29+4 25-159 35+5 24+3 29-142 33±4 16+3 A8 30 + 4 43+5 A7 6 27 + 8 23 + 9 B2 6 19+4 20+3 B35 19+4 32 + 3 B47 9+2 11+2 B106 17 + 3 16+2 C36 18+2 34 + 12 C70 10+2 15+2 C93 12 + 3 24 + 4 D22 9 + 1 20+2 D57 41 + 6 24 + 6 E10 24 + 8 27 + 6 E20 10+2 20 + 3 E34 9+3 19+6 E113 12 + 3 17+2 74 F75 5±2 Table 2-7 continued Pigment Levels Mutant males females H113 17 + 4 30 + 4 13-47 48 + 6 35 + 9 13-83 27 + 3 16+2 13-117 41 + 6 23+5 33-161 4 0±3 2 9+8 29-85 34 + 3 22+2 A61 11+2 19+3 E3 25+2 16+4 14-95 36+8 27 + 6 A65 8 + 1 14+2 E24 10 + 2 21 + 1 E73 19+3 25 + 3 F40 9±2 20+2 F15 14+2 26+4 Gl 17+3 23+4 G21 12+2 20 + 6 H95 14+2 18 + 3 cnbw c o n t r o l 27+2 30+5 a: pigment l e v e l s are e x p r e s s e d as p e r c e n t of w i l d - t y p e (Oregon-R). 75 31D/E (see D e f i c i e n c y Mapping and Complementation A n a l y s i s , above). This mutant may define an as yet u n i d e n t i f i e d Su(var) mutation, or may be an a l l e l e of Su(var)216, which i s l o c a t e d i n 31E. 76 DISCUSSION Many of the suppressor mutations i n the 2L c l u s t e r are hemizygous l e t h a l . This property, which i d e n t i f i e s them as e s s e n t i a l genes, has a l s o enabled these suppressors to be c h a r a c t e r i z e d c y t o l o g i c a l l y . P h y s i c a l mapping has i d e n t i f i e d a minimum of three separate l o c i which occupy a lar g e region (31A-31E, approximately 33 bands) of chromosome 2. The i n t e n t of t h i s study was t o i d e n t i f y a d d i t i o n a l a l l e l e s of suppressor mutations and other e s s e n t i a l genes l o c a t e d i n region 31. A t o t a l of 42 EMS-induced hemizygous l e t h a l or strong s e m i - l e t h a l mutations i n region 31 of chromosome 2 were generated. These mutants were l o c a l i z e d t o s p e c i f i c subregions v i a d e f i c i e n c y mapping. They were found t o map f a i r l y randomly throughout the region, and are not c l u s t e r e d i n t o d i s c r e t e s i t e s near the suppressor l o c i . Thus, a number of e s s e n t i a l genes, i n a d d i t i o n to the known suppressors, e x i s t i n t h i s r egion. Many of these mutants are homozygous as w e l l as hemizygous l e t h a l . Complementation a n a l y s i s was used as a means of determining the number of genes i n s e v e r a l subregions of 31A-F using homozygous l e t h a l i t y as a d i a g n o s t i c phenotype. The r e s u l t s of t h i s a n a l y s i s are summarized i n Figure 2-5. In region 31C, f i v e mutants define as many as four genetic l o c i . A somewhat complex p a t t e r n e x i s t s i n that one of these mutants f a i l s to complement two mutants which complement each other. A p o s s i b l e explanation f o r such a r e s u l t i s discussed more f u l l y below. 77 FIGURE 2-5 Summary of Complementation.Analysis of Hemizygous L e t h a l Mutations i n Region 31 78 U J 79 The n i n e m u t a t i o n s i n 31D i d e n t i f y between t h r e e and f i v e c omplementation groups. Some a m b i g u i t y e x i s t s because a l l p o s s i b l e c o m b i n a t i o n s have not been t e s t e d (see Table 2-4 and R e s u l t s ) . Complementation among o n l y t h e EMS-induced mutants . i n t h i s group shows them t o d e f i n e an a p p a r e n t l y complex l o c u s w i t h two s e p a r a b l y mutable c i s t r o n s (E56 and A63). T h i s i s c o n s i s t e n t , f o r i n s t a n c e , w i t h a gene p r o d u c t which has more t h a n one f u n c t i o n a l domain. Note, however, t h a t a t l e a s t one o f t h e s e mutants, E5_6, i s i t s e l f homozygous v i a b l e , i d e n t i f y i n g i t as a h a p l o - s p e c i f i c l e t h a l m u t a t i o n (Nash and J a n c a , 1983). I t s a b i l i t y t o complement A63 may s i m p l y be a r e f l e c t i o n o f t h i s f a c t (see b e l o w ) . I n any c a s e , t h e complementation groups d e f i n e d by t h e EMS-induced mutants i n 31D are d i s t i n c t from a t l e a s t one o t h e r l o c u s i n t h e r e g i o n , which i s i d e n t i f i e d by 23- 127 and 29-142. The o t h e r two mutants i n t h i s r e g i o n 3-50 and 25-159, w h i l e s e p a r a t e from each o t h e r and t h e 23-127/29-142 l o c u s , have not been t e s t e d a g a i n s t t h e EMS-induced mutants. F i n a l l y , i n 31F, f o u r m u t a t i o n s i d e n t i f y t h r e e s e p a r a t e complementation groups. In e x a m i n i n g and i n t e r p r e t i n g t h e complementation p a t t e r n s e x h i b i t e d by t h e s e m u t a t i o n s , an i m p o r t a n t c o n s i d e r a t i o n must be kept i n mind. I t i s w e l l known t h a t EMS i s an e f f e c t i v e mutagen which o f t e n g e n e r a t e s independent l e t h a l m u t a t i o n s on t h e p a r t i c u l a r chromosome under i n v e s t i g a t i o n . I f a second s i t e l e t h a l m u t a t i o n i s p r e s e n t i n a d d i t i o n t o t h e i d e n t i f i e d m u t a t i o n i n r e g i o n 31, t h e n h a p l o - s p e c i f i c l e t h a l s (hemizygous 80 l e t h a l m u t a t i o n s which a re homozygous v i a b l e ) may be i n c o r r e c t l y i d e n t i f i e d as homozygous l e t h a l m u t a t i o n s . The e x i s t e n c e o f l e t h a l m u t a t i o n s a t a second s i t e s h o u l d not r e s u l t i n t h e w r o n g f u l i d e n t i f i c a t i o n o f a l l e l i c m u t a t i o n s (except i n t h e u n l i k e l y event t h a t t h e second s i t e m u t a t i o n s o f two d i f f e r e n t chromosomes a r e a l l e l i c ) . However, t h e y may r e s u l t i n t h e s p u r i o u s i d e n t i f i c a t i o n o f i n t e r a l l e l i c c o mplementation. T h i s would l e a d t o b o t h an o v e r e s t i m a t i o n o f the number o f l o c i t h a t e x i s t i n t h e r e g i o n , and undue c o m p l e x i t i e s i n complementation p a t t e r n s . I n f a c t , c o m p l i c a t i o n s o f t h i s t y p e were e v i d e n t i n a stu d y i n v o l v i n g h a p l o - s p e c i f i c l e t h a l m u t a t i o n s i n a r e g i o n o f t h e X chromosome (Nash and Ja n c a , 1983). Thus i t i s p r o b a b l y p r u d e n t i n any f u r t h e r i n v e s t i g a t i o n o f t h e s e mutants, t o e x c l u d e t h e p o s s i b i l i t y o f e x t r a n e o u s l e t h a l s ( f o r i n s t a n c e , by r e c o m b i n a t i o n o f chromosome arms). The gamma and TEM-induced m u t a t i o n s examined i n t h i s s t u d y were o r i g i n a l l y g e n e r a t e d t o o b t a i n d e f i c i e n c i e s and t r a n s l o c a t i o n s i n t h e r e g i o n which would cause s u p p r e s s i o n o f v a r i e g a t i o n and t h u s f a c i l i t a t e t h e c l o n i n g o f t h e 2L c l u s t e r s u p p r e s s o r s . Based on d e f i c i e n c y and complementation mapping (D. S i n c l a i r ) , one such mutant (23-127) appears t o have been g e n e r a t e d . I t spans t h e d i s t a l b r e a k p o i n t o f Df106. T h i s mutant s u p p r e s s e s w1114 v a r i e g a t i o n e x t e n s i v e l y i n males ( i t s e f f e c t i s comparable o r g r e a t e r t h a n t h a t o f Df77, Df27 and Df106, see appendix A ) . I n a d d i t i o n , one o t h e r gamma i n d u c e d mutant, 16-165, was found t o e x h i b i t m a l e - s p e c i f i c dominant 81 suppression of v a r i e g a t i o n . I t maps to the same region as four other dominant suppressors of v a r i e g a t i o n , and may be an a l l e l e . The male s p e c i f i c i t y of the suppressing phenotype of these mutants i s probably a f u n c t i o n of the heterochromatic Y chromosome. Several mutants i n region 31, i n c l u d i n g the suppressors and m a t e r n a l - e f f e c t mutants have been shown to be responsive t o a l t e r a t i o n s i n heterochromatic elements (Mottus, 1983; P i m p i n e l l i et a l . , 1985; Brock, 1986). In a d d i t i o n , a number of phenotypes e l i c i t e d by suppressor mutations are more severe i n males than g e n o t y p i c a l l y comparable females (see Chapter 1). This sexual dimorphism i s a l s o s e n s i t i v e to d e l e t i o n s and a d d i t i o n s of the Y chromosome i n males and females, r e s p e c t i v e l y . None of the EMS-induced mutants are strong suppressors of PEV, a r e s u l t which i s not s u r p r i s i n g . In a previous screen f o r dominant suppressors of v a r i e g a t i o n i n t h i s region ( S i n c l a i r et al.., 1983), the frequency of recovery was l e s s than one i n 3000. Only 1848 chromosomes were screened i n t h i s study. This does not n e c e s s a r i l y imply that suppressor l o c i were not i d e n t i f i e d . As discussed i n Chapter 1, most of the suppressors i n t h i s region demonstrate p l e i o t r o p i c e f f e c t s (homo- and hemizygous male l e t h a l i t y and female s t e r i l i t y i n a d d i t i o n to suppression). The suppressing phenotypes of these mutants are b e l i e v e d to be antimorphic i n nature. I t i s p o s s i b l e t h a t the genes represented by these suppressors can 82 a l s o be mutated to an hypo- or amorphic form, i n which a suppressor phenotype would not be apparent. Indeed, complementation a n a l y s i s has shown th a t three of the hemizygous l e t h a l mutants i n region 31E are a l l e l i c t o Su(var)216 (Clegg and Whitehead, unpublished observations) although they do not suppress v a r i e g a t i o n . Further i n v e s t i g a t i o n of the mutants i n t h i s region (besides completion of complementation a n a l y s i s ) should i n c l u d e an examination of r e l a t i o n s h i p s between these and the suppressor mutations. This would inc l u d e complementation and a search f o r phenotypic i n t e r a c t i o n s such as those e x h i b i t e d among the suppressor mutations (see Chapter 1). In a d d i t i o n , the e f f e c t of Y chromosome on hemizygous l e t h a l i t y should be i n v e s t i g a t e d . I f some of these mutants i d e n t i f y a l l e l e s of suppressor l o c i , or other genes which may be i n v o l v e d i n the i n t e g r i t y of heterochromatin, then a l t e r a t i o n s of heterochromatic elements i n the genome (such as the Y chromosome) may a f f e c t the phenotypes which they e x h i b i t . 83 REFERENCES Baker, W. K. (1963) Genetic c o n t r o l of pigment d i f f e r e n t i a t i o n i n somatic c e l l s . Am. Zool. 3: 57-69. Baker, W. K. (1967) A c l o n a l system of d i f f e r e n t i a l gene a c t i v i t y i n Drosophila. Developmental Biology 16: 1-17. Baker, W. K. (1968) P o s i t i o n - e f f e c t v a r i e g a t i o n . Adv. Genet. 14: 133-169. Becker, H. J . and W. Janning (1977) Heterochromatin of the Drosophila melanoqaster Y chromosome as m o d i f i e r of p o s i t i o n - e f f e c t v a r i e g a t i o n : the time of i t s a c t i o n . Mol. Gen. Genet. 151: 111-114. Blumenfeld, M., J . W. Orf, B.J. Sina, R.A. Kreber, M.A. Callahan, J . I . M u l l i n s and L.A. Snyder (1978) C o r r e l a t i o n between phosphorylated HI histones and s a t e l l i t e DNA's i n Drosophila v i r i l i s . Proc. N a t l . Acad. S c i . USA 75: 866-870. Brock, J.K. (1986) The e f f e c t of heterochromatic d e f i c i e n c i e s on p o s i t i o n - e f f e c t v a r i e g a t i o n and suppressors of p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila melanoqaster. B.Sc. Thesis, U n i v e r s i t y of B r i t i s h Columbia. Candido, E.P.M., R. Reeves, and J.R. Davie (1978) Sodium butyrate i n h i b i t s h istone d e a c e t y l a t i o n i n c u l t u r e d c e l l s . C e l l 14: 105-113.. Demerec, M. and H. S l i z y n s k a (1937) Mottled w h i t e 2 5 8 " 1 8 of Drosophila melanoqaster. Genetics 50: 237. Dobzhansky, T. (1946) Genetics of n a t u r a l p o p u l a t i o n s . X I I I . recombination and v a r i a b i l i t y i n populations of Drosophila  pseudoobscura. Genetics 31: 269-290. Gowan, J.W. and E.H. Gay (1933) E f f e c t of temperature on ev e r s p o r t i n g eye colour i n Drosophila melanoqaster. Science 77 312. Gowan, J.W. and E.H. Gay (1934) Chromosome c o n s t i t u t i o n and behaviour i n e v e r - s p o r t i n g and m o t t l i n g i n Drosophila  melanoqaster. Genetics 19: 189-208. Hartmann-Goldstein, I . J . (1967) On the r e l a t i o n s h i p between heterochromatization and v a r i e g a t i o n i n Drosophila, w i t h s p e c i a l reference to temperature-sensitive p e r i o d s . Genet. Res. 10: 143-159. 84 Hartmann-Goldstein, I . J . and J.M. Wargent (1975) C y t o l o g i c a l observations on the i n t e r a c t i o n between two i n v e r s i o n s r e s p o n s i b l e f o r p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila  melanogaster. Chromosoma 52: 349-362. Hedrick, A.L. (1989) C h a r a c t e r i z a t i o n of a c l u s t e r of dominant suppressors of p o s i t i o n - e f f e c t v a r i e g a t i o n i n c l u d i n g e f f e c t s on heterochromatic v a r i e g a t i n g rearrangments i n Drosophila melanogaster. M.Sc. Thesis. U n i v e r s i t y of B r i t i s h Columbia. Henikoff, S. (1979) Does p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila r e s u l t from somatic gene l o s s ? In Eukaryotic Gene Regulation, R. A x e l , T. M a n i a t i s and C F . Fox, eds. (New York: Academic P r e s s ) , pp.123-132. H i l l i k e r , A.J. (1976) Genetic a n a l y s i s of the c i n t r o m e r i c heterochromatin of chromosome 2 of Drosophila  melanogaster: d e f i c i e n c y mapping of EMS-induced l e t h a l complementation groups. Genetics 83:765. Homyk, T. and C P . Emerson (1988) F u n c t i o n a l i n t e r a c t i o n s between u n l i n k e d muscle genes w i t h i n h a p l o i n s u f f i c i e n t regions of the Drosophila genome. Genetics 119: 105-121. Judd, B. (1955) D i r e c t proof of a variegated-type p o s i t i o n e f f e c t at the white locus i n Drosophila melanogaster. Genetics 196: 739-744. King, R.C. (1970) Ovarian development i n Drosophila  melanogaster. Academic Press, New York. Lewis, E.B. and F. Bacher (1968) Method of feeding e t h y l methanesulphonate (EMS) to Drosophila males, Drosophila Inform. Serv. 43:193. L i n d s l e y , D.L. and E.H. G r e l l (1968) Genetic v a r i a t i o n s of Drosophila melanogaster. Carnegie I n s t i t u t e of Washington P u b l i c a t i o n . No. 627. L i n d s l e y , D.E., L. G o l d s t e i n and L. Sandler (1980) DIS 55: 84. Lucchesi, J.C. (1968) Sy n t h e t i c l e t h a l i t y and s e m i - l e t h a l i t y among f u n c t i o n a l l y r e l a t e d mutants of Drosophila  melanogaster. Genetics 59: 37-44. Mange, A.P. and L. Sandler (1973) A note on the mat e r n a l - e f f e c t mutants daughterless and abnormal oocyte i n Drosophila  melanogaster. Genetics 73: 73-86. Moore, G., J . Procunier, D. Cross and T. G r i g l i a t t i (1979) Histone gene d e f i c i e n c i e s and p o s i t i o n - e f f e c t v a r i e g a t i o n i n D rosophila. Nature 282: 312-314. 85 Morgan, T.H., J . Schultz and V. Curry (1941) I n v e s t i g a t i o n s of the c o n s t i t u t i o n of the germinal m a t e r i a l i n r e l a t i o n to h e r e d i t y . Carnegie I n s t . Yearbook 40, 282-287. Mottus, R. (1983) P o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila  melanoqaster: chemical m o d i f i c a t i o n and mutational analysis.B.Sc. Thesis, U n i v e r s i t y of B r i t i s h Columbia. Mottus, R., R. Reeves and T. G r i g l i a t t i (1980) Butyrate suppression of p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila  melanoqaster. Mol. Gen. Genet. 178: 465-469. M u l l e r , H.J. (1930) Types of v i s i b l e v a r i a t i o n s induced by X-rays i n Drosophila. J . Genetics 22: 299-334. M u l l e r , H.J. (1932) Further s t u d i e s on the nature and cause of gene mutations. In: Proceedings of the S i x t h I n t e r n a t i o n a l Congress of Genetics pp. 213-255. Nash, D. and C. Janca (1983) Hypomorphic l e t h a l mutations and t h e i r i m p l i c a t i o n s f o r the i n t e r p r e t a t i o n of l e t h a l complementation s t u d i e s i n Drosophila. Genetics 105 957-968. Noujdin, N.I. (1944) The r e g u l a r i t i e s of heterochromatin i n f l u e n c e on mosaicism. Zh. Obsh. B i o l . 5 357-388. Nusslein-Volhard, C , E. Wieschaus and H. Kluding (1984) Mutations a f f e c t i n g the p a t t e r n of the l a r v a l c u t i c l e i n Drosophila melanoqaster 1. Zygotic l o c i on the second chromosome. Roux's Arch. Dev. B i o l . 193: 267-282. Panshin, I.B. (1938) The cytogenetic nature of the p o s i t i o n -e f f e c t of the genes white (mottled) and cubitus i n t e r r u p t u s . B i o l . Zh. (Mosk) 7: 837-868. P i m p i n e l l i , S., W. S u l l i v a n , M. Prout and L. Sandler (1985) On b i o l o g i c a l f u n c t i o n s mapping to the heterochromatin of Drosophila melanoqaster. Genetics 109: 701-724. Reuter, G. and I. Wolff (1981) I s o l a t i o n of dominant suppressor mutations f o r p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila  melanoqaster. Mol. Gen. Genet. 182: 516-519. Reuter, G., W. Werner and H.J. Hoffmann (1982) Mutants a f f e c t i n g p o s i t i o n - e f f e c t heterochromatization i n Drosophila melanoqaster. Chromosoma 85: 539-551. Reuter, G., R. Dorn, G. Wustmann, B. Fr i e d e and G. Rauh (1986) T h i r d chromosome suppressors of p o s i t i o n e f f e c t v a r i e g a t i o n l o c i i n Drosophila melanoqaster Mol. Gen. Genet. 202: 481-487. 86 Salas, F., and J.A. Lengyel (1984) DIS 60: 243. Sandler, L. (1977) Evidence f o r a set of c l o s e l y l i n k e d autosomal genes that i n t e r a c t w i t h sex-chromosome heterochromatin i n Drosophila melanogaster. Genetics 86: 567-582. Schupbach, T. and E. Wieschaus (1986) M a t e r n a l - e f f e c t mutations a l t e r i n g the a n t e r i o r - p o s t e r i o r p a t t e r n of the Drosophila embryo. Roux's Arch. Dev. B i o l . 195: 302-317. S i n c l a i r , D.A.R., R.C. Mottus and T. G r i g l i a t t i (1983) Genes which suppress p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila  melanogaster are c l u s t e r e d . Mol. Gen. Genet. 86: 567-582. Spofford, J.B. (1967) S i n g l e locus m o d i f i c a t i o n of p o s i t i o n -e f f e c t v a r i e g a t i o n i n Drosophila melanogaster. I. White v a r i e g a t i o n . Genetics 57: 751-766. Spofford, J.B. (1976) P o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila. In: Ashburner and Novitsky (eds) The genetics and b i o l o g y of Drosophila, v o l . IC. Academic Press, New York, pp. 955-1018. Zhimulev, I . , E. Belyaeva, 0. Fomina, M. Protopopov and V. Bolshakov (198 6) Cytogenetic and molecular aspects of p o s i t i o n - e f f e c t v a r i e g a t i o n i n Drosophila melanogaster. Chromosoma (Berl) 94: 492-504. Zuckerkandl, E. (1974) Recherces sur l e s p r o p r i e t e s et l ' a c t i v i t e b i o l o g i q u e de l a chromatine. Biochimie 56 937-954. 87 APPENDIX A E f f e c t of Deficiencies of Region 31 on Posi t i o n -e f f e c t Variegation The Jammed-derived s e r i e s of d e f i c i e n c i e s which were used i n mapping the 2L c l u s t e r suppressor mutants often delete one or more of the Su(var) l o c i . Several of these d e f i c i e n c i e s , t h e r e f o r e , were examined f o r t h e i r a b i l i t y to modify 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 . V i s u a l observations of outcrosses had revealed that s e v e r a l were capable of suppressing v a r i e g a t i o n of w to var i o u s extents. Three of the d e l e t i o n s , Df27, Df77 and Df106 , were placed i n a w m 4 background. Crosses we're c a r r i e d out as f o l l o w s : v i r g i n females of w^/w™ 4; Df/CyO were outcrossed to w m 4/Y; +/+ males, and the r e s u l t i n g progeny (w m 4/Y; Df/+ and w m 4/Y; CyO/+ males and w m 4/w m 4; Df/+ and w m 4/w m 4; CvO/+ females) were assayed f o r pigment l e v e l s . To t e s t f o r maternal e f f e c t s , r e c i p r o c a l crosses were done. w m 4/Y; Df/CyO males and w m 4/w m 4; Df/CyO females were a l s o assayed. The crosses f o r Df39 and Df(2L)Sco, which are dominant female s t e r i l e , were performed as f o l l o w s : w m 4/w m 4; +/+ v i r g i n females were mated t o w m 4/Y; Df/CyO males. The r e s u l t i n g progeny (w m 4/Y; Df/+ and w m 4/Y; CyO/+ males, and w m 4/w m 4; Df/+ and w m 4/w m 4; CyO/+ females) were assayed. R e c i p r o c a l crosses of these experiments could not be done. The r e s u l t s , shown i n Table A - l , r e v e a l t h a t Df(2D Sco acts as a strong suppressor of v a r i e g a t i o n . Three of the d e f i c i e n c i e s , Df27, Df77 and Df106, show a moderate degree of suppression, an e f f e c t t h a t i s more notable i n males. For two 88 TABLE A - l E f f e c t of D e f i c i e n c i e s i n Region 31 on V a r i e g a t i o n C r o s s a males Pigment L e v e l s 1 3 females Df/ + Cy/ + Df/Cy Df/ + Cy/ + Df/Cy Dfl06/Cy x +/+ 25±5 10+2 32 + 5 24+5 +/+ x Dfl06/Cy 27+2 11 + 4 44±5 29±7 Dfl06/Cy x Dfl06/Cy 25+3 36±3 Df77/Cy x + / + 21 + 4 8 + 3 29±4 15+2 +/+ x Df77/Cy 60 + 6 36+1 36±6 25+7 Df77/Cy x Df77/Cy 51+5 33 + 4 Df27/Cy x + / + 19+2 6+1 2 6±2 12 + 3 +/+ x Df27/Cy 53 + 3 21 + 4 43 + 4 30 + 8 Df27/Cy x Df27/Cy 42 + 9 27 + 3 +/+ x Df39/Cy 23+2 30+3 14+4 21+4 +/+ x Df(2L)Sco/Cy 7 6+4 14 + 4 81 + 7 10+2 a: genotype on the l e f t i s the second chromosome c o s t i t u t i o n of the female parent, t h a t on the r i g h t i s the male. A l l i n d i v i d u a l s c o n tain w m 4 X chromosomes. b: pigment l e v e l s are expressed as percent of wild-type (Oregon-R) 89 of these, Df27 and Df77, there appears to be a paternal effect. Pigment levels are higher in both experimental and control animals when the deficiency-bearing chromosome is contributed by the male parent. This effect was seen in two additional repeats involving Df27. A possible explanation of this phenomenon is that the wm4 stock used in these crosses may contain a low level maternal-effect modifier of variegation the effect of which is amplified in the presence of deficiency for wild type suppressor products. In fact, spontaneous suppressors of variegation are often noted in variegating strains. An alternate hypothesis is that deficiency-bearing females may undergo some type of amplification or overproduction of wild-type suppressor product (for instance, in response to stress induced by abnormal heterochromatin formation). These products would be transmitted to the egg during oogenesis and result in a less suppressed phenotype in the progeny. Interestingly, Df39 shows no suppression of wm4 phenotype. Since i t has a more proximal breakpoint than the other deficiencies, this result could be due to the deletion of an enhancer of variegation which is not identified by the other deficiencies. An alternate explanation could be the need for stoichiometric ratios of certain suppressor products. While Df27. Df77 and Df106 delete only 216, Df39 uncovers this suppressor as well as those in 31B/C (214 and 207), restoring their particular stoichiometric balance. 90 APPENDIX B E f f e c t of Maternal-effect Mutants on P o s i t i o n -e f f e c t Variegation Region 32A of chromosome 2 contains s e v e r a l maternal-e f f e c t mutants which are s e n s i t i v e to a l t e r a t i o n s i n sex chromosome heterochromatin (Sandler, 1977). Suppressors of the 2L c l u s t e r which maps to region 31 have a l s o been found t o respond to changes i n Y chromosome heterochromatin (Brock, 1986). This s i m i l a r i t y , as w e l l as the c l o s e p r o x i m i t y of both sets of mutants, prompted the i n v e s t i g a t i o n of the c a p a c i t y f o r the m a t e r n a l - e f f e c t mutations t o act as m o d i f i e r s of v a r i e g a t i o n . The mutants t e s t e d here are: l e t h a l (2) 54 (.1 (2.) 54.)/ male- female s t e r i l e 48 (mfs48), abnormal oocyte (abo), daughterless- abnormaal o o c y t e - l i k e ( d a l ) , daqhterless (da), hold-up (hup) and w a v o i d - l i k e (wdl). Each mutation was i s o l a t e d i n t o a w m 4 background. Crosses were performed by o u t c r o s s i n g mutant-bearing w m 4 males and females separately t o w™ 4; +/+ f l i e s of the appropriate sex ( i . e . r e c i p r o c a l crosses were done). The A r e s u l t i n g progeny (w animals c o n t a i n i n g e i t h e r a mutant or Curly chromosome 2) were c o l l e c t e d and assayed f o r pigment l e v e l s . In a d d i t i o n , heterozygotes and homozygotes (when po s s i b l e ) from the mutant stock maintained i n a w™ 4 background were a l s o assayed. Two of the mutants (hup andwdl) were t e s t e d at both 22° and 25°C, while the remainder were done only at 25°C. As i n d i c a t e d i n Tables B - l and B-2, mfs48 and wdl are the only two mutants of t h i s group which demonstrate c o n s i s t e n t 91 TABLE B-l E f f e c t of M a t e r n a l - e f f e c t Mutants on V a r i e g a t i o n of T7m4 w Genotype 3 males females 1(2)54/+ 15+3 28+3 Cy/+ 11+3 32+9 +/1(2)54 10+2 43±4 + /Cy 9+2 55+7 1(2)54/Cy 9+2 33+4 mfs48/+ 17+2 32+4 Cy/+ 5+2 20+4 +/mfs4 8 16+3 31+3 +/Cy 7+3 23+3 mfs48/Cy 13+2 23+6 mfs48/mfs48 v i s u a l l y suppressed^ v i s u a l l y suppressed abo/+ 36+8 60+5 Cy/+ 24+5 50+6 +/abo 4 6+3 44+2 +/Cy 53+3 4 0+4 abo/Cy 66+6 63+3 abo/abo 65+3 57+5 dal/+ 22+2 33+7 Cy/+ 21+7 42+5 +/dal 20+2 38+5 +/Cy 15+3 44+7 dal/Cy 15+2 34+6 d a l / d a l 20+5 31+4 da/+ 22+4 32+6 Cy/+ 18+6 24+3 +/da 38+5 36+5 +/Cy 27+3 30+5 da/Cy 4 6+6 30+5 da/da 45+3 31+7 a: the f i r s t chromosome represented i s de r i v e d from the female parent. b: t h i s genotype i s v i s u a l l y more suppressed that the corresponding heterozygotes. Too few f l i e s were generated f o r an assay. 92 TABLE B-2 E f f e c t of hup and wdl on V a r i e g a t i o n of w™ 4 Genotype 22°C 25°C males females males females hup/ + 9+3 31+4 10+3 26+7 Cy/+ 16+2 23 + 3 8+2 18+3 + /hup 7 + 4 19+3 7 + 3 20+3 + /Cy 5+2 21 + 4 8 + 1 29+3 hup/Cy 8+2 22 + 6 9+2 17 + 3 hup/hup 10+2 2 9+5 7+2 14 + 3 wdl/ + 26+5 30 + 4 29+9 46+7 Cy/+ 16+3 16+4 16+8 26+5 + /wdl 17 + 3 41 + 9 21+5 56+5 + /Cy 10 + 4 23+5 9+2 30 + 8 wdl/Cy 19+3 42 + 4 18 + 3 53+4 wdl/wdl 44 + 6 82 + 9 53 + 4 69+6 93 suppression. This e f f e c t i s more notable i n homozygotes than heteroygotes f o r both of these mutants, suggesting that they define hypomorphic suppressor mutations. 94 APPENDIX C TABLE C-l V i a b i l i t y of 2L C l u s t e r Suppressors w i t h Df27 males F-^  females Cross Cy:Cy + Cy:Cy + r a t i o r a t i o Df27 X 207 22:16 26:12 Df27 X 210 89:43 128:32 Df27 X 213 105:46 129:49 Df27 X 215 122:68 141:71 Df27 X 216 135:0 131:0 95 TABLE C-2 E f f e c t of l t x on V i a b i l i t y of C e r t a i n Transheterozygous Combinations of Suppressors Cross F 1 males F 1 females 210 X 213 111:7 110:31 210 X 213 I t 1 220:9 146:58 210 X 215 136:40 109:69 210 X 215 I t 1 150:82 171:86 210 X 216 154:35 139:44 210 X 216 L t 1 80:22 99:46 213 X 210 224:18 254:34 213 X 210 I t 1 333:18 326:107 213 X 215 211:50 217:98 213 X 215 I t 1 209:30 194:63 213 X 216 180:4 191:67 213 X 216 I t 1 80:4 100:45 214 T f t x 210 338:65 373:107 214 T f t x 210 i t 1 74:20 . 81:29 214 T f t x 213 406:14 436:114 214 T f t x 213 I t 1 211:1 179:37 214 T f t x 215 382 :147 356:130 214 T f t x 215 I t 1 245:49 250:55 214 T f t x 216 217:5 281:59 214 Tf t x 216 I t 1 228:4 252:29 215 X 210 375:156 348:162 215 X 210 I t 1 201:69 209:81 215 X 213 284:133 315:136 215 X 213 231:63 260:90 215 X 215 i t 1 98:19 141:39 215 X 216 171:70 156:61 215 X 216 I t 1 148:32 144:41 216 X 210 126:25 114:39 216 X 210 I t 1 128:9 183:47 216 X 213 66:9 62:28 216 X 213 i t 1 53:0 58:21 216 X 215 354:129 367:141 216 X 215 i t 1 112:28 120:37 96 APPENDIX D TABLE D--1 D e f i c i e n c y Mapping o f Hemizygous L e t h a l M u t a t i o n s i n Region 31 Mutant Df27 D f l 0 6 Df77 Df39 Df (2L) Sco A8 m a 3 9 : 0 b 48:0 87:1 f 19:0 50:0 114:0 A43 m 40:13 30:14 77:36 144:16 68:0 f 34:16 30:21 89:48 132:9 76:1 A61 m 150:87 169:0 93:29 f 149:81 193:0 123:49 A63 m 58:0 52:15 185:0 f 3 7 : 0 5 0 : 2 6 164:0 A65 m 89:29 110:34 92:22 51:0 14:1 f 108:35 91:26 114:29 32:0 25:0 A7 6 m 79:0 78 : 0 113 :0 f 4 7 : 0 56:0 117:0 A102 m 23:9 83:35 53:21 47:7 19:0 f 40:12 71:57 45:6 48:3 36:0 A141 m 42:0 67:37 51:0 f 46:0 46:22 40:0 A156 m 173:126 145:85 142:75 34:12 27:11 f 207:113 120:67 151:43 58:9 32 :7m B2 6 m 89:0 83:0 123:0 f 100:1 89:0 95:0 B47 m 70:0 74:0 26:0 f 98:0 76:0 36:0 B35 m 48:0 80:0 141:0 f 35:0 69:0 149:0 B100 m 10:12 30:5 179:69 47 : 0 20:0 f 11:5 27:5 184 :47 87:0 29:0 B106 m 40:0 101:0 150:0 f 32:0 105:0 220:0 B149 m 149:120 34:22 122:0 f 260:164 40:17 176:0 C25 m 14:12 43:19 31:17 44:2 59:15 f 35: 6 31:20 39:9 52:2 63:8 C35 m 78:0 101:44 131:0 f 49:0 72:47 183:0 a : m and. f d e s i g n a t e male and female progeny, r e s p e c t i v e l y . b: f i g u r e s r e p r e s e n t t o t a l numbers o f c u r l y : s t r a i g h t winged f l i e s 97 Table D-l continued Mutant Df27 Dfl06 Df77 Df39 Df(2L)Sco C36 m 53:0 76:0 f 57:0 69:0 C70 m 49:1 70:0 f 48:2 75:0 C93 m 51: 0 84:0 f 42:0 72:0 C98 m 80:19 28:12 f 47:10 33:17 C104 m 25:12 64:13 f 25:4 69:15 C131 m 25:19 53:23 f 17:18 86:33 C133 m 23:15 47:13 f 30 :25 49:17 D22 m 104:13 120:1 f 105:3 117:0 D57 m 52:0 119:0 f 48:0 128:0 D121 m 149:69 69:22 f 161:39 59:26 D155 m 111:74 84:48 f 114:81 67:43 E3 m 50:33 227:0 f 48:25 210:0 E6 m 45:23 66:14 f 47:17 43:16 E10 m 52:0 60:0 f 66:0 64:0 E15 m 51:0 75:32 f 32:0 73:25 E20 m 40:0 81:3 f 15:0 101:3 E24 m 89:33 41:23 f 85:33 39:16 E34 m 26:0 92:0 f 10:0 89:0 E56 m 33:0 23:8 f 50 :0 46:22 E73 m 28:5 39:13 f 35:10 40:18 E113 m 42:0 184:0 f 46:0 190:0 105:0 96:0 74:0 109:0 80:0 114:0 79:11 68:5 69:0 94:34 81:5 118:0 40:14 92:1 57:0 42:18 103:1 68:0 76:25 90:9 55:5 68:28 122:9 43:4 87:34 49:7 84:0 100:32 59:1 100:1 98:0 92:0 99:0 76:0 190:61 103:0 52:3 197:67 135:0 55: 6 165:66 101:5 50:0 233:79 142:9 62:2 257:109 273:116 79:32 12:1 21:5 75:40 10:0 17:2 82:0 28:0 31:0 88:0 18:0 26:0 136:0 131:0 62:0 86:1 70:27 111:0 65:6 73:37 116:0 71:5 67:0 65:0 76:0 84:0 89:28 84:0 45: 6 113:49 111:0 44:7 96:0 108:0 98 Table D-l continued Mutant Df27 D f l 0 6 Df77 Df39 Df(2L)Sco F15 m 37:24 33:17 f • , 51:12 42:19 F40 m 41:21 15:10 f 52:28 18:22 F75 m 21:0 148:0 f 26:0 85:0 F133 m 76:85 77:32 f 94:75 87:44 Gl m 63:23 35:8 f 49:14 44:12 G21 m 42:7 61:23 f 78:22 65:38 G25 m 103:49 40:22 f 97:43 46:21 G55 m 37:13 67:16 f 30:12 65:27 G78 m 47:1 77:0 f 45:0 116:0 G83 m 43:24 57:43 f 67:40 64:61 H30 m 42:0 27:13 f 14 :0 33:12 H95 m 133:46 82:29 f 156:63 110:18 H113 m 39:0 155:0 f 21:0 133:0 24-127 m 105:13 f 133:16 25-159 m 99:12 f 106:21 19-153 m 74:38 f 89:41 3-50 m 124:7 f 133:8 2-119 m 56:25 f 81:38 14-140 m 43:18 f 55:18 29-142 m 93:17 f 115:4 105:20 79:0 58:15 98:37 100:0 64:14 29:16 69:0 73:1 39:10 111:12 63:1 67:0 83:0 92:87 80:0 85:0 106:42 107:0 63:0 64:25 133:0 34:1 59:18 144:0 34:4 60:1 17:0 73:1 56:6 18:0 59:5 98:8 113:12 17:1 112:5 153:12 20:1 77:13 105:1 35:11 91:18 129:2 24:3 68:0 68:0 9:17 3:0 16:18 5:0 16:0 13:0 196:99 65:0 40:9 223:92 80:0 42:7 50:0 45:0 89:1 122:1 43:5 49:2 99 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0097463/manifest

Comment

Related Items