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A novel requirement for the X-chromosome in P-M hybrid dysgenesis and the interaction of the garnet and… Wennberg, Richard Arnold 1989

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A NOVEL REQUIREMENT FOR THE X-CHROMOSOME IN P - M HYBRID DYSGENESIS AND THE INTERACTION OF THE GARNET AND ENHANCER OF GARNET LOCI IN DROSOPHILA MELANOGASTER by RICHARD ARNOLD WENNBERG B.Sc, The University of B r i t i s h Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Genetics) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1988 (c) Richard Arnold Wennberg, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology (Genetics) The University of British Columbia Vancouver, Canada Date December. 1988  DE-6 (2/88) ABSTRACT The discovery of extreme mutability around the g. locus of Drosophila melanogaster i n a certain lab s t r a i n , S6-1, led to an investigation of the nature of t h i s phenomenon and any possible relationship i t had with the well described P-M hybrid dysgenesis mutator system. Southern gel analysis showed the S6-1 s t r a i n to contain P element homology and dysgenic crosses involving t h i s P-s t r a i n showed mutability i n the germline of the F-^ . An unexpected finding was the requirement for the presence of the S6-1 X-chromosome i n F-^  dysgenic males i n order to produce the germline mutability. In s i t u hybridization to polytene chromosomes showed the S6-1 s t r a i n to have a number of P s i t e s , the majority of which were on the X-chromosome (app. 20/33) . These r e s u l t s indicated that the only complete P element or elements were confined to the X-chromosome. The presence of an unexpected t h i r d class of orange-eyed males i n the F 2 progeny of the cross: S6-1 males x g_ females led to the i s o l a t i o n of an enhancer of garnet locus, (e(qj), located i n the d i s t a l region of the S6-1 X-chromosome. This study i n c l u d e d an i n v e s t i g a t i o n i n t o the nature of the interaction between the e(g_) locus and the g; locus and other autosomal l o c i . The enhancer was found to be s p e c i f i c to the g_ locus, and, more s p e c i f i c a l l y , to certain spontaneous a l l e l e s located to one end of the fine structure g_ locus map. A description i s given of the i s o l a t i o n and preparation of a P-induced garnet mutation for molecular analysis. i i TABLE OF CONTENTS Page Abstract i i L i s t of Tables i v L i s t of Figures v Acknowledgements v i Chapter 1. X-Chromosome Mutation Induction Associated with P-M Hybrid Dysgenesis and the Isolation of the enhancer of garnet Locus Introduction 2 Materials and Methods 5 Results 7 Discussion 19 Chapter 2. The Interaction Between the garnet and enhancer of garnet Loci Introduction 23 Materials and Methods 28 Results 34 Discussion 41 Summary 44 Bibliography 4 6 Appendix 51 i i i L I S T OF TABLES Table Page Chapter 1 1.1 X-chromosome mutation induction i n the germline of the F-j_ and F 2 male progeny of the cross: S6-1 females x o g. males 9 l . I I X-chromosome mutation induction i n the germline of the F-^  and F 2 male progeny of the cross: g_ females x S6-1 males 11 l . I I I X-chromosome mutation induction i n the germline of the F-j_ male progeny of the cross: S6-1 males x attached-X females 13 1.IV a) X-chromosome mutation induction i n the germline of the F-^  male progeny of the cross: Oregon R males x attached-X females b) X-chromosome mutation induction i n the germline of S6-1 males taken d i r e c t l y from stock 15 i v LIST OF FIGURES Figure Page Chapter 1 1.1 Photomicrograph of polytene chromosome preparation from S6-1 stock showing i n s i t u hybridization of plasmid prr25.1 DNA 17 Chapter 2 2.1 Construction of homozygous stocks mutant at the e(gj locus and the g. locus 30 2.2 Construction of homozygous stocks mutant at the e(o:) locus and at the autosomal l o c i p_r, bw, ry_ and p_, respectively 31 2.3 Scheme for removing the extraneous P elements and for s t a b i l i z i n g the cytotypic background of a P-induced g_ mutation 32 2.4 Hybrid dysgenesis screen for revertants of the gr mutant and subsequent revertant stock formation 33 2.5 Southern analysis showing hybridization of plasmid pn25.1 DNA to complete Bam HI digests of genomic DNA p from the g_ mutant and revertant stocks 39 v ACKNOWLEDGEMENTS I wish to sincerely thank: Dr. Robert Devlin for his advice and willingness to help; Dr. Donald S i n c l a i r for his guidance, encouragement and active and ongoing collaboration throughout the course of t h i s project; and my supervisor, Dr. Thomas G r i g l i a t t i , for his unstinting aid, encouragement and support. v i CHAPTER 1 X-CHROMOSOME MUTATION INDUCTION ASSOCIATED WITH P-M HYBRID DYSGENESIS AND THE ISOLATION OF THE ENHANCER OF GARNET LOCUS 1 I n t r o d u c t i o n Hybrid dysgenesis i s a genetic syndrome marked by the appearance of a variety of aberrant t r a i t s i n the germline of the progeny of c e r t a i n i n t e r - s t r a i n crosses i n Drosophila  melanogaster (28). The symptoms include high rates of i n s e r t i o n a l and chromosomal mutation, male recombination and temperature-sensitive s t e r i l i t y . There exist at least three separate systems of hybrid dysgenesis i n JX. melanogaster: the well-described I-R and P-M systems (6), and a more r e c e n t l y d e s c r i b e d system associated with the transposable element hobo (55). In the P-M system, hybrid dysgenesis results when P-strain males are mated with M - s t r a i n females, but not when the reciprocal cross i s performed, (and not when matings are confined to f l i e s from one s t r a i n or the other) . The causative agent i n t h i s system i s the 2.9 kb P element (45), a DNA transposable element which has an extremely high rate of transposition i n a p a r t i c u l a r maternally-derived c e l l u l a r environment, referred to as the M-cytotype. P elements are present i n P - s t r a i n (P-cytotype) f l i e s , usually as 30-50 copies dispersed over a l l major chromosome arms, but are g e n e r a l l y absent i n M - s t r a i n (M-cytotype) f l i e s (5, 13). The M-cytotype i s dependent upon the absence of f u l l - l e n g t h P elements capable of producing transposase (48; see below). M o l e c u l a r l y , the P element has 31 bp p e r f e c t i n v e r t e d terminal repeats surrounding four open reading frames (38) . A l l four open reading frames are required to encode transposase, the P element gene product which catalyzes the transposition of P 2 elements and other transposable elements. Incomplete P elements with coding region d e f i c i e n c i e s are transposable i n the presence of the trans-acting transposase, providing they possess intact t e r m i n a l repeats (27) . A g e r m l i n e - s p e c i f i c s p l i c e of the P element t r a n s c r i p t i s required for transposase a c t i v i t y ; t h i s forms the basis for the r e s t r i c t i o n of P element transposition to germline tissue (29). The nature of mutation induction associated with P-M hybrid dysgenesis has been e x t e n s i v e l y s t u d i e d . Even before the i s o l a t i o n of the P element, the fact that many of the induced mutations were caused by DNA insertions was evident from t h e i r i n s t a b i l i t y (19, 47) , which was shown to be associated with the presence of the M-cytotype (12). Deletions induced through faulty e x c i s i o n of i n s e r t i o n elements were also recognized (20) . Molecular characterization has implicated insertions of complete or incomplete P elements into a gene or deletions associated with the imprecise excision of P elements resident i n or near a gene as the most common means of dysgenic mutation induction (13, 45). Other mechanisms include induction of chromosome breakage or rearrangement at P element s i t e s of residence along a chromosome, reversal of P element orientation i n a gene, and c a t a l y s i s of transposition of other transposable elements (13, 55). Most spontaneous mutations i n Drosophila are associated with the i n s e r t i o n of transposable elements (30) . In general, such spontaneous mutations a r i s e i n f r e q u e n t l y , but i n h y b r i d dysgenesis systems they can appear at a much higher frequency. Thus, the P-M hybrid dysgenesis system can be used to screen for 3 P-induced mutants at a p a r t i c u l a r gene locus, which can then be cloned f o r molecular c h a r a c t e r i z a t i o n by the method of transposon-tagging (3) . This technique has f a c i l i t a t e d the cloning and characterization of many Drosophila genes i n recent years. Experiments d e s c r i b e d i n t h i s chapter were designed to determine the relationship between an observed high frequency of unstable g_ mutations i n a certain lab s t r a i n and the P-M system of h y b r i d dysgenesis. Two novel f i n d i n g s arose from these experiments: (i) the demonstration of a requirement f o r the presence of the X-chromosome i n a certain P-strain for hybrid dysgenesis mutation i n d u c t i o n ; and ( i i ) the i s o l a t i o n of an enhancer of garnet mutation. 4 M a t e r i a l s and Methods 1. C u l t u r e c o n d i t i o n s : F l i e s were maintained and crosses performed i n quarter-pint milk bottles or s h e l l v i a l s at 2 2°C on medium containing cornmeal, sucrose, yeast and agar. Tegosept (methyl-p-hydroxybenzoate) was included i n the food as a mold i n h i b i t o r . To suppress b a c t e r i a l growth a m p i c i l l i n (50 mg/liter) was used e i t h e r i n combination with t e t r a c y c l i n e or with streptomycin (15 mg/liter each). 2 . Mutant and wild-type s t r a i n s : See Appendix. D e t a i l e d descriptions of most of the mutations used can be found i n Lindsley and G r e l l (31) . Any other strains are described i n the text. 3. Mating schemes for hybrid dysgenesis investigation : For each of the experiments, P-strain males were mated en masse to M-s t r a i n females (15 pairs per b o t t l e ) , or the r e c i p r o c a l . F-^ . males from these matings were crossed to v i r g i n Maxy a / F M 7 females and the female offspring were scored for mutant induction. Similar test crosses were performed with the F 2 male progeny from F-^  sib matings. In these test crosses, groups of 3 males were mated to harems of 10-15 Maxy a / F M 7 females i n s h e l l v i a l s . The f l i e s were transferred to quarter-pint milk bottles a f t e r three days, and thereafter transferred to fresh medium i n bottles every three days to a maximum of four t r a n s f e r s . The parents were subsequently discarded. Mutant females were allowed to mate with t h e i r F M 7 / Y sibs, the only surviving male progeny of the test c r o s s e s . Male l e t h a l mutations were kept as F M 7 - b a l a n c e d stocks while male viable mutants were crossed to v i r g i n females 5 c a r r y i n g a t t a c h e d - X chromosomes (C(l)DX,y_ f/Y) t o o b t a i n a s t o c k . 4. i n s i t u h y b r i d i z a t i o n : L a r v a e f o r s a l i v a r y g l a n d chromosome squashes were r a i s e d a t 18°C and p r e p a r e d a c c o r d i n g t o A t h e r t o n and G a l l ( 1 ) . Squashes were t r e a t e d a t 70°C i n 2xSSC f o l l o w e d by t r e a t m e n t w i t h RNase A (100 ug/ml) as p e r Pardue and G a l l (39). S l i d e s were a c e t y l a t e d a c c o r d i n g t o t h e p r o c e d u r e o f H a y a s h i e t a l . (22) and d e n a t u r e d as p e r Pardue and G a l l . H y b r i d i z a t i o n was c a r r i e d out a t 25°C u s i n g t r i t i u m - l a b e l l e d probe a c c o r d i n g t o t h e p r o c e d u r e d e s c r i b e d by Bingham e t a l . ( 3 ) . A u t o r a d i o g r a p h y was performed as p e r Pardue and G a l l w i t h t h e e x c e p t i o n n o t e d by Bingham e t a l . ( 3 ) . S l i d e s were s t a i n e d f o r 5-6 minutes i n 0.4% t o l u i d i n e b l u e i n 2xSSC a t room t e m p e r a t u r e , r i n s e d w i t h d i s t i l l e d w ater, a l c o h o l d e h y d r a t e d , a i r - d r i e d , and mounted w i t h c o v e r - s l i p s i n Permount. The probe f o r t h e s e h y b r i d i z a t i o n s was p l a s m i d PTT25.1 DNA, c o n t a i n i n g t h e complete P element sequence (38), l a b e l l e d by n i c k t r a n s l a t i o n (Amersham N i c k T r a n s l a t i o n K i t , Amersham C o r p . , O a k v i l l e , Ont.) w i t h JH-dCTP (Amersham Corp.) as a r a d i o a c t i v e p r e c u r s o r . 6 R e s u l t s 1. P element induced X-chromosome mutagenesis: In an EMS screen for paralysis mutants undertaken i n our lab, one s t r a i n was isolated, designated S6-1, which was l a t e r shown to exhibit some unusual phenomena not r e l a t e d to the p a r a l y s i s mutation. In crosses designed to map the induced mutation, the appearance of a high frequency of g mutations was noted. Upon further study, these mutations proved to be highly unstable, suggesting the involvement of a transposable element. Southern hybridization analysis done on the S6-1 s t r a i n and on the gr s t r a i n (which had been used i n the o r i g i n a l mapping experiments and had allowed for the detection of the spontaneous g mutations) showed S6-1 to 9 contain homology to P element DNA and cp to display no P element 9 homology. The S6-1 and gr strains were then tested i n a series of crosses designed to determine i f mutation induction from hybrid dysgenesis was the cause of t h i s unexpected high m u t a b i l i t y around the g locus. Before describing the results of these crosses, one other finding must be noted. In a cross of S6-1/Y males x g^/g^ 9 o females, the F-^  S6-l/g_ and g^/Y progeny were allowed to mate. Surprisingly, i n addition to males with wild-type or garnet-coloured eyes, an unexpected t h i r d class of males with orange eyes showed up i n the F 2- As revealed, a mutation on the S6-1 X-chromosome, mapping d i s t a l l y and around genetic map position 4, interacts with a homozygous or hemizygous g mutation, enhancing the mutant e f f e c t of garnet-coloured eyes. This enhancer locus may or may not be i d e n t i c a l to one previously described (42) and 7 since l o s t ; i t w i l l be referred to hereafter as enhancer of garnet, e(g_) . (The i s o l a t i o n and characterization of e(g_) w i l l be further detailed l a t e r i n t h i s chapter and also i n the next chapter.) To i n v e s t i g a t e the nature of the appearance of the p spontaneous g_ mutations, the S6-1 and g s t r a i n s were re c i p r o c a l l y crossed, and mutation induction on the X-chromosome was looked for i n the germline of the F-^  and F 2 male progeny by outcrossing with v i r g i n females carrying the multiply marked X-chromosome, Maxy a, and balanced over the multiple inversion FM7. The results of these crosses were not e n t i r e l y consistent with the existence of a P-M hybrid dysgenesis system (see Tables 1.1 and l.II) . The most in t e r e s t i n g observation was the complete absence of mutation induction i n the germline of the F-^  male p p p progeny of the cross: S6-1/Y x g /g ; i . e . , the g /Y males from t h i s cross, upon outcrossing to Maxy a/FM7 females, produced no mutant o f f s p r i n g i n 14,784 female progeny scored, a most surprising r e s u l t given the e a r l i e r evidence that S6-1 i s a P-p s t r a i n and gr i s an M-strain. A subsequent cross between S6-1/Y males and attached-X (C(l)DX,y_ f/Y; M cytotype) v i r g i n females produced males that retained the paternal X-chromosome. These F-^  males, upon outcrossing to Maxy a/FM7 v i r g i n s , gave r i s e to induced mutations at a frequency gre a t e r than 3x10"^ j _ n the 16, 027 o f f s p r i n g scored, a result i n d i c a t i v e of a hybrid dysgenesis cross (Table l . I I I ) . Thus i t appeared that mutation induction required the presence of the S6-1 X-chromosome. 8 T a b l e 1.1 X-chromosome mutation induction i n the germline of the p and F 2 male progeny of the cross: S6-1 females x g males. F 1 S6-1/Y progeny; 8,500 females scored over Maxy a or FM7: Mutants No. _L Freo. (x!0~ 4) Mut. events j_ Freq. (x!0~ 4) N (6-1) 6 7.1 1 1.2 p F 2 g /Y progeny; 4, 438 females scored over Maxy a or FM7: Mutants No. j_ Freer. (x!0~ 4) Mut. events j_ Freq. (x!0~ 4) sn 4 9.0 1 2.3 * + ' eye 2 4.5 1 2.3 w 1 2.3 1 2^2. Total: 3 6.8 /Y progeny; 5,061 females scored over Maxy a or FM7: Mutants No. : Freer. (xlO 4) g (2-D 2 4.0 w - N (1) 1 2.0 orange eye (1) 1 2.0 v (s) 1 2.0 2 4.0 1 2.0 1 2.0 1 2.0 Total: 5 10.0 (Cont. next page) 9 (Table 1.1 cont.) orange eye/Y progeny; 4,962 females scored over Maxy a or FM7: Mutants No. j_ Freq. (x!0~^) Mut. events j_ Freq. (xlO w - N (9-1) 9 18.1 5 10.1 x+' eye (1) 1 2.0 1 2.0 Total: 6 12.1 Total number of females scored, a l l classes, i n F 2 experiment 14,461. Total number of mutational events, a l l classes, F 2 experiment 14. (Freq. = 9.7xl0~ 4.) 1 = male l e t h a l s = s t e r i l e 10 T a b l e l . I I X-chromosome mutation induction i n the germline of the F-^  and F 2 male progeny of the cross: a females x S6-1 males. g.z/Y progeny; 14,784 females scored over Maxy a or FM7: Mutants No. j_ Freq. Mut. events j_ Freq. 0 0 0 0 0 **2 3. /Y progeny; 5,701 females scored over Maxy a or FM7: Mutants No. j. Freq. (xlO 4) Mut. events j_ Freq. (xlO ) 5.3 1 1.8 3.5 2 3^ 5. Total: 3 5.3 A + '/Y progeny; 4, 247 females scored over Maxy a or FM7: Mutants No . : F r e q . ( x l O - 4 ) Mut. events : Freq.(xlO w - N (2-1) 2 4.7 2 4.7 a (2-1) 2 4.7 2 4.7 orange eye 2 4.7 1 2.4 sn 2 4.7 1 2.4 w 9. ~ sn 1 2.4 1 2.4 orange eye - ct 1 2.4 1 2.4 Total: 8 18.8 (Cont. next page) sn sn w 3 2 11 (Table l . I I cont.) orange eye/Y progeny; 4,64 9 females scored over Maxy a or FM7: Mutants N (9-1) w - N (6-1) sn No. i 9 6 3 Freq.(xlO 19.4 12.9 6.5 Mut• events I. Freq. (xlO Total: 3 6 J L 10 6.5 12.9 2.2 21.5 Total number of females scored, a l l classes, i n F 2 experiment 14,597. Total number of mutational events, a l l classes, F 2 experiment 21. (Freq. = 1.4xl0~ 3.) 1 = male l e t h a l s = s t e r i l e 12 Table 1.III X-chromosome m u t a t i o n i n d u c t i o n i n t h e g e r m l i n e o f t h e F male progeny o f t h e c r o s s : S6-1 males x a t t a c h e d - X f e m a l e s . Mutants No. : F r e a . ( x l O 4 ) Mut . e v e n t s : F r e q . ( x l O orange eye 34 21.2 21 13.1 a (1-1) 23 14.4 11 6.9 w - N (17-1/3-s) 20 12.5 15 9.4 r a s (4-1) 4 2.5 4 2.5 v (4-s) 4 2.5 3 1.9 sn 2 1.2 2 1.2 w 1 0.6 1 0.6 Y_ 1 0.6 1 0.6 T o t a l : 58 36.2 T o t a l number o f females s c o r e d over Maxy a or FM7 : = 16,027 (8,040/Maxy a; 42/89 mutants) 1 = male l e t h a l s = s t e r i l e 1 3 As controls, Oregon R wild-type males (M-strain) and S6-1 males taken d i r e c t l y from stock were crossed with Maxy a/FM7 v i r g i n s and the F-^  female progeny scored f o r X-chromosome mutation induction. In the Oregon R experiment, no mutations were seen i n 20,332 f l i e s scored. In the S6-1 experiment, a mutational event frequency of greater than 4x10"^ was seen i n 18,737 f l i e s scored (Table 1.IV). This frequency i s approximately 1/10 that seen with the F^ males from the S6-1/Y x attached-X dysgenic cross, a result consistent with a P-M hybrid dysgenesis system. (Mut. event = a presumed single mutational event giving r i s e to one mutant or a cl u s t e r of mutants i s o l a t e d from the same v i a l or bottle as progeny of the 3 male parents. See Materials and  Methods.) The types of mutations induced by dysgenic males i n a l l of the crosses were c h a r a c t e r i s t i c of P-M hybrid dysgenesis: e.g., a high frequency of N and w - N deletions, presumably r e s u l t i n g from imprecise excision of the P element resident at 3C i n the S6-1 s t r a i n (see below), and a similar (total) frequency of presumptive i n s e r t i o n mutation events at a number of v i s i b l e l o c i on the X-chromosome. Interestingly, i n l i g h t of the existence of the e(g.) mutation on the S6-1 X-chromosome, as well as an e a r l i e r f i n d i n g of a g_ t s mutation (mutant at 29°C) on the same chromosome, the most frequently induced mutations were those showing e i t h e r garnet or orange eyes. A m i n o r i t y of these mutations were male l e t h a l , suggesting imprecise excision of the P element resident i n the cr region of the S6-1 X-chromosome (see below). The majority, however, were male viable, and subject to further mutation at the locus, suggesting P element in s e r t i o n or 14 T a b l e l . i v a) X-chromosome mutation induction i n the germline of the F-^  male progeny of the cross: Oregon R males x attached-X females. Mutants No. j_ Freq. Mut. events j_ Freq. 0 0 0 0 0 Total number of females scored over Maxy a or FM7 = 20,332. b) X-chromosome mutation induction i n the germline of S6-1 males taken d i r e c t l y from stock. Mutants No. : Freer. (xlO 4) Mut. events : Freq.(xlO w - N (3-1) 3 1.6 3 1.6 a (1-1) 2 1.1 2 1.1 orange eye 1 0.5 1 0.5 w 1 0.5 1 0.5 v (s) 1 0.5 1 0.5 ras (1) 1 0.5 1 0.5 Total: 9 4.8 Total number of females scored over Maxy a or FM7 = 18,737. -4. (9,400/Maxy a; 5/9 mutants) 1 = male l e t h a l s = s t e r i l e 15 a l t e r a t i o n of the resident P element's s i t e , structure or orientation as the cause of the mutation. 2. In s i t u hybridization of P element DNA to polytene  chromosomes: In s i t u h y b r i d i z a t i o n to polytene chromosome preparations of S6-1 individuals showed the S6-1 s t r a i n to have a number of P s i t e s , the majority of which were on the X-chromosome (approximately 20/33 per i n d i v i d u a l ) . There was P homology i n both the 3C (w - N) and 12C (g_) regions (Figure 1.1). There was no homology to P element DNA seen i n polytene chromosome preparations of either gr or Oregon R individuals (not shown). The i n s i t u hybridization r e s u l t s , combined with the results from the genetic crosses, indicated that the observed e f f e c t s were l i k e l y due to P element transposition i n dysgenic crosses, with the only complete P element or elements i n the S6-1 s t r a i n being present on the X-chromosome, and perhaps immobilized there (e.g. by a mutated inverted repeat sequence). 3. Isolation of the e(g) locus: Crude two-point genetic mapping placed e(g) i n the v i c i n i t y of p o s i t i o n 4 on the genetic map, at the d i s t a l end of the X-chromosome. In the absence of the g2 mutation (or even i n the presence of heterozygous g?) the e(g) mutation showed no phenotype e i t h e r as a homozygote or hemizygote. In addition, the mutant e{g) phenotype was rescued by a duplication for the region which contained an e ( g j + a l l e l e (w+Y chromosome i n males) , suggesting that e (g_) i s a hypomorph or amorph. In females, heterozygous e(g) i n the presence of homozygous g resulted i n a mildly enhanced phenotype i n newly 16 Figure 1.1 Photomicrograph of polytene chromosome preparation from S6-1 stock showing i n s i t u hybridization of plasmid pn25.1 DNA. 17 eclosed f l i e s , which quickly darkened i n the f i r s t 24 hours to overlap garnet. Thus, the modifying ef f e c t of e(qj i s almost s t r i c t l y a recessive one. The presence of d i s t a l P s i t e s on the S6-1 X-chromosome (Figure 1.1) suggested that e(g;) might represent a P element-mediated i n s e r t i o n mutation. A small scale reversion study was done i n which e(g_) gr/Y males (from a stock formed from a single male a r i s i n g from a recombination event i n an S6-l/g female) were crossed to attached-X (C(l)DX,y_ f/Y; M-cytotype) v i r g i n females; the F-j_ progeny were allowed to mate and the F 2 male offspring were scored for garnet-eyed revertants of e.(g_) . No revertants were i s o l a t e d i n 5,693 f l i e s scored. This may indicate that the e.(g) mutation i s not a s s o c i a t e d with an i n s e r t i o n element but may instead be a deletion or a point mutation. It might also be a deletion associated with the previous imprecise e x c i s i o n of a transposable element (e.g. P element) and a tr u n c a t e d P element could s t i l l r e s i d e at the e_(o_) l o c u s . Further, the recombination event that formed the &(g) g?/Y stock may have crossed o f f the S6-1 complete P element or elements, leaving the desired dysgenic F-^  males i n t h i s study incapable of mobilizing a mutated P element from the e(g_) s i t e . 18 D i s c u s s i o n The lab s t r a i n S6-1 was shown to behave as a P-strain i n certain crosses between S6-1 males and M-strain females. Mutation induction on the X-chromosome was examined as an indicator of the occurrence of dysgenic effects i n the germline of the F-^  progeny of such crosses. Crosses of S6-1 (P) males with attached-X M-s t r a i n females produced F-^  germline mutation rates greater than 10 J ; t h i s i s a ten-fold greater frequency of mutation induction p than that seen with either g_ (M-strain) male x S6-1 female crosses or S6-1 i n t r a - s t r a i n crosses, frequencies i n d i c a t i v e of P-M hybrid dysgenesis (45). An unexpected finding was the complete absence of mutation induction i n the Fj progeny of a cross between S6-1 (P) males and p homozygous gr (M) females. This f i n d i n g i n d i c a t e s that the presence of the paternal S6-1 X-chromosome i n the germline of the F^ progeny i s a requirement for mutation induction i n dysgenic crosses involving the S6-1 s t r a i n . This i s a requirement which was met i n the crosses involving attached-X M-strain females, accounting for the high mutation rates observed i n those crosses (as described above). In s i t u hybridization showed S6-1 to possess approximately 30 P element copies, the majority of which lay along the X-chromosome. The evidence suggests that the only complete P element(s) capable of producing transposase i n the S6-1 s t r a i n i s (are) located on the X-chromosome, perhaps immobilized there, as by a mutated inverted repeat sequence. The nature of the mutations induced along the X-chromosome was c o n s i s t e n t w i t h a system o f P-M h y b r i d d y s g e n e s i s (13, 19, 20) . A h i g h f r e q u e n c y o f p u t a t i v e d e l e t i o n m u t a t i o n s was o b s e r v e d , e s p e c i a l l y around t h e w - N and g r e g i o n s (presumably a s s o c i a t e d w i t h i m p r e c i s e e x c i s i o n e v e n t s o f t h e P e l e m e n t s r e s i d e n t i n t h o s e a r e a s ) , a l o n g w i t h a s i m i l a r t o t a l f r e q u e n c y o f p r e s u m p t i v e i n s e r t i o n m u t a t i o n s a t a v a r i e t y o f v i s i b l e l o c i . M u t a t i o n f r e q u e n c i e s i n t h e g e r m l i n e o f t h e F 2 progeny o f t h e r e c i p r o c a l S6-1 x g c r o s s e s were i n t e r m e d i a t e between t h e f r e q u e n c y o b s e r v e d i n t h e o f t h e d y s g e n i c a t t a c h e d - X c r o s s and t h e f r e q u e n c i e s seen i n t h e F-^  o f t h e gy male x S6-1 female c r o s s and t h e S6-1 i n t r a - s t r a i n c r o s s . The f r e q u e n c i e s were somewhat h i g h e r i n t h e F 2 progeny o f t h e (P) male x (M) female c r o s s t h a n i n t h e F 2 o f t h e r e c i p r o c a l (P) female x (M) male c r o s s , i n d i c a t i n g a h i g h e r l e v e l o f P element t r a n s p o s i t i o n i n t h e former c y t o t y p i c background. Among t h e F 2 progeny, a h i g h e r m u t a t i o n r a t e was o b s e r v e d i n t h o s e c l a s s e s p o s s e s s i n g s u b s t a n t i a l p o r t i o n s o f t h e S6-1 X-chromosome P + '/Y, orange e y e / Y ) , p r e s u m a b l y due t o d e l e t i o n i n d u c t i o n a t s i t e s o f P element r e s i d e n c e . p The o c c u r r e n c e o f two s e p a r a t e r e v e r t a n t s o f g i n t h e F 2 e x p e r i m e n t s (Table 1.1) i s p a r t i c u l a r l y i n t e r e s t i n g g i v e n t h a t g_2 i s an M - s t r a i n w h i c h shows a complete absence o f P elements by i n s i t u h y b r i d i z a t i o n . These e v e n t s may r e p r e s e n t t h e P - i nduced t r a n s p o s i t i o n o f a n o t h e r t r a n s p o s a b l e element a s s o c i a t e d w i t h t h e p p spontaneous g a l l e l e . As g i s one o f t h r e e spontaneous g_ a l l e l e s m o d i f i e d by e(g) , d e t e r m i n a t i o n o f t h e m o l e c u l a r s t r u c t u r e o f t h e s e r e v e r t a n t s may p r o v e i n f o r m a t i v e i n t h e 2 0 elucidation of the nature of spontaneous mutation at the g_ locus and the inte r a c t i o n between g_ and e(g_) (see Chapter 2 ) . The e(g.) mutation, present i n the d i s t a l region of the S6-1 X-chromosome, was i d e n t i f i e d as a r e c e s s i v e enhancer of the mutant phenotype of homozygous or hemizygous g_ mutants. The e(o_) mutant has no phenotype i n the absence of the g mutation, and i t appears to function as a hypomorphic or amorphic mutation. 2 1 CHAPTER 2 THE INTERACTION BETWEEN THE GARNET AND ENHANCER OF GARNET LOCI 22 Introduction Enhancer or suppressor mutations act to modify the phenotypic e f f e c t of unrelated mutations located at distant s i t e s i n the genome. Several a l l e l e - s p e c i f i c suppressor l o c i have been d e s c r i b e d i n Drosophila which can reverse the phenotypes of p a r t i c u l a r mutant a l l e l e s at other l o c i . In addition, some of these suppressor l o c i have been noted to behave as enhancers i n interactions with certain of the suppressible a l l e l e s , increasing the mutant phenotype. Most a l l e l e - s p e c i f i c m o d i f i e r l o c i exert t h e i r e f f e c t s through d i r e c t interactions with spontaneous mutations associated with transposable element i n s e r t i o n s . The i n s e r t i o n s are invariably elements of the retrotransposon class, s p e c i f i c a l l y gypsy, copia and 412 (14, 36, 44) , which are composed of int e r n a l DNA segments varying i n size from 5 to 9 kb with dir e c t long terminal repeats (LTRs) several hundred nucleotides long at each end (15) . Other members of t h i s family are the Ty elements of yeast (43) and the vertebrate retroviruses (54). Contained within the LTRs are t r a n s c r i p t i o n i n i t i a t i o n and termination s i g n a l s . DNA sequences adjacent to the Drosophila retrotransposon LTRs show homology to the polypurine t r a c t and tRNA primer binding s i t e of retroviruses, and polyadenylated t r a n s c r i p t s have been i d e n t i f i e d which encode p u t a t i v e gene products homologous to r e t r o v i r a l protease, endonuclease and reverse transcriptase (33). There have been f i v e retrotransposon-mediated suppressor systems d e s c r i b e d i n D_j_ melanogaster, a l l of which r e q u i r e 23 homozygous mutations at the suppressor l o c i : (i) suppressor of Hairy-wing (su(Hw)) suppresses mutations associated with gypsy insertions at a number of d i f f e r e n t l o c i (including Hw, f, lz_, y_/ ct, h and bx) (36); ( i i ) suppressor of forked (su(f)) suppresses the same forked and lozenge a l l e l e s caused by gypsy ins e r t i o n that are suppressed by su (Hw) (40) ; ( i i i ) suppressor of white-apricot (su(w a)) suppresses the wa mutation, which i s associated with a copia i n s e r t i o n (4); (iv) suppressor of purple (su(pr)) suppresses p_r and p r ^ w , associated with 412 insertions (46) ; and (v) suppressor of sable (s_u(s_)), which suppresses mutations (including p_r and p r b w ) at d i f f e r e n t l o c i associated with 412 insertions (46) . Three of the above suppressor l o c i are involved i n enhancing interactions: (i) su(p_r) enhances Hw (31); ( i i ) su(s.) enhances three mutant a l l e l e s at the lz. and b_x l o c i associated with gypsy insertions (36); and ( i i i ) s u ( f ), which enhances wa (18). Transposon-associated suppressor systems have a l s o been described i n yeast (43) , maize (34) and mice (52) . In each case they consist of a transposable receptor element inserted near to, and having a c i s - e f f e c t upon a p a r t i c u l a r gene, and a trans-acting regulatory element which either a l t e r s the s t a b i l i t y of the receptor element or the expression of the adjacent gene. The molecular mechanisms u n d e r l y i n g the Drosophila transposon-associated suppressor systems have been the subject of much investigation. The nature of suppressible a l l e l e s has been e x t e n s i v e l y s t u d i e d and, more r e c e n t l y , molecular characterization of the su(s) and su(wa) l o c i has begun (7, 57). 24 There are certain factors which determine s u p p r e s s i b i l i t y , as not a l l r e t rotransposon induced mutant a l l e l e s at suppressible l o c i are affected by the suppressor l o c i . There i s no requirement for the complete DNA sequence of the inserted element (25) , nor i s there an absolute requirement for a s p e c i f i c orientation of the element r e l a t i v e to d i r e c t i o n of t r a n s c r i p t i o n of the mutant gene (2, 26) (cf. certain yeast suppressors, where Ty element o r i e n t a t i o n i s a requirement (43)). Evidence has accumulated t h a t two important f a c t o r s are the l o c a t i o n of transposon in s e r t i o n with respect to coding sequences and the developmental time (and/or tissue s p e c i f i c i t y ) of expression of the inserted element and the affected gene. The s u p p r e s s i b l e a l l e l e s behave as leaky mutants (hypomorphs) (2, 53, 56), which would suggest that the transposons are inserted into nontranslated regions. DNA sequence analysis of the wa mutation places the copia i n s e r t i o n within an intron (30) , and preliminary molecular analyses of suppressible a l l e l e s at the f, y_ and v l o c i indicate gypsy or 412 i n s e r t i o n into introns (17, 40, 46). Experiments involving gypsy in s e r t i o n mutations suppressible by su(Hw) and/or su(f) at the forked and yellow l o c i have shown gypsy, forked and yellow mRNA to have a similar pattern of temporal expression (40, 41) . This may form the basis of a mechanism of suppression v i a recessive mutation at a distant locus. If the reduced l e v e l of normal tra n s c r i p t s seen with the gypsy-caused f and y_ mutants i s due to a type of t r a n s c r i p t i o n a l interference by the gypsy element on the recipient f or y_ l o c i , s i m ilar to that previously observed i n retroviruses (10, 11), a 25 decrease i n gypsy t r a n s c r i p t i o n could lead to an increase i n the l e v e l of f or y_ mRNA. As the suppressible mutations are a l l hypomorphs, an increase i n gene product l e v e l need not be extreme to reverse the phenotype. Thus, the wild-type function of su(Hw) and su(f) may be to produce trans-acting co-factors necessary f o r the t r a n s c r i p t i o n of the gypsy element (40) . This i s consistent with the decreased l e v e l of gypsy t r a n s c r i p t seen i n homozygous su(Hw) mutants (41). Experiments with the wa mutant have demonstrated the accumulation of abnormal white t r a n s c r i p t s terminated or i n i t i a t e d within one LTR of the copia element inserted into the second intron of the white gene. In t h i s case, su(w a) seems to act to decrease the frequency of white t r a n s c r i p t s that are t r u n c a t e d w i t h i n copia, or, a l t e r n a t i v e l y , to i n c r e a s e the e f f i c i e n c y of c o r r e c t s p l i c i n g of the rare normal white tr a n s c r i p t s found i n wa mutants (30). The mechanism by which the three suppressor l o c i function as enhancers i s not clear. It i s i n t e r e s t i n g to note, however, that each locus enhances mutations a s s o c i a t e d with a d i f f e r e n t transposable element than the one associated with the mutations i t suppresses. Like the suppressor mutations, e(g.) has no d i r e c t e f f e c t on i t s wild-type target locus. Rather, homozygous (or hemizygous) e.(o_) i s an a l l e l e - s p e c i f i c enhancer of c e r t a i n spontaneous mutations at the q_ locus. The garnet locus [1/44.4] has been extensively studied g e n e t i c a l l y at the f i n e s t r u c t u r e l e v e l (8, 9, 24) . 26 Biochemically, the eyes of g_ mutants show de f i c i e n c i e s for both pteridine and ommochrome pigments, and the mutation i s autonomous in eye disc transplants (31). These observations have led to the suggestion that o_ belongs to a class of transport mutants (which includes w and p_) unable to transport eye pigment precursors into c e l l s possessing the enzymatic machinery to complete pigment synthesis (51). It i s i n t e r e s t i n g to note that the white product has recently been found to share amino acid sequence homology with a group of ATP-binding b a c t e r i a l transport proteins (23) which had previously been shown to share s i g n i f i c a n t amino acid homology with the mammalian multidrug r e s i s t a n c e gene, a l s o thought to be a mutation i n a c e l l membrane transport mechanism (21) . 27 Materials and Methods 1. Culture conditions: As per Chapter 1. 2. Mutant and wild-type s t r a i n s : As per Chapter 1. 3. Construction of stocks: i) In constructing stocks homozygous for both the e(g) mutation and another mutant locus crosses involving P-strain males and M-strain females were avoided to prevent the dysgenic mobilization of P elements within the S6-1 P s t r a i n , which contains the mutant e(g) locus. Figures 2.1 and 2.2 i l l u s t r a t e the construction of such stocks mutant at the g locus and at d i f f e r e n t autosomal l o c i (p_r, bw, ry_, PJ.) , respectively. i i ) To construct a stock of a P-induced g mutant r e l a t i v e l y free of extraneous P elements, one of the g_ mutations induced i n the attached-X dysgenic screen from Chapter 1 was selected (g p) . This was a male viable mutant (kept i n stock with attached-X females) which had subsequently received the X-chromosome markers y_, cv, v, and f v i a selection of a double recombinant F 2 male offspring from the cross: g P/Y (P) x y_ QV v f/y_ cv v f (M) . In s i t u and Southern h y b r i d i z a t i o n s demonstrated 6-7 P s i t e s remaining i n t h i s stock, including a s i t e i n the g region. This stock was used i n a dysgenic screen for revertants of the P-induced g mutation and was subsequently further recombined and repeatedly outcrossed to M-strain C(l)DX,y_ f/Y v i r g i n females to remove more of the extraneous P elements and to obtain the g p mutation i n a stable M-cytotype (Figure 2.3). Figure 2.4 shows the revertant screen ca r r i e d out over the severe g 2 a l l e l e and the subsequent recombinational cleaning and M-strain outcrossing of the i s o l a t e d revertants. 2 8 4. I_n s i t u h y b r i d i z a t i o n ; As p e r Chapter 1, w i t h t r i t i u m - l a b e l l e d (by n i c k t r a n s l a t i o n ) gypsy o r c o p i a DNA used as probe where n o t e d . 5. I s o l a t i o n o f DNA f o r Southern g e l a n a l y s i s : The p r o c e d u r e used f o r DNA i s o l a t i o n was a m o d i f i c a t i o n o f t h a t d e s c r i b e d by G e h r i n g e t a l . (16). A p p r o x i m a t e l y 300 f l i e s were homogenized i n a 1.8 ml m i c r o f u g e t u b e w i t h a g l a s s p e s t l e i n 600 u l o f a b u f f e r c o n t a i n i n g 0.1M N a C l , 30mM T r i s - H C l ; p H 8.5, lOmM EDTA, lOmM b e t a -m e r c a p t o e t h a n o l and 0.5% T r i t o n X-100. An e q u a l amount o f a b u f f e r c o n t a i n i n g lOOmM N a C l , lOOmM T r i s - H C l ; p H 8.5 and 20mM EDTA was added and mixed w i t h t h e homogenate. 1% SDS and 100 ug/ml p r o t e i n a s e K were added and t h e m i x t u r e was i n c u b a t e d a t 65°C f o r 1.5-2 h o u r s . A s i n g l e p h e n o l : c h l o r o f o r m e x t r a c t i o n was f o l l o w e d by a s i n g l e c h l o r o f o r m e x t r a c t i o n . RNA was d i g e s t e d w i t h 100 ug/ml RNase A f o r 1 hour a t 42°C. The DNA was e x t r a c t e d v e r y g e n t l y 3X w i t h p h e n o l , p r e c i p i t a t e d 2X w i t h 95% e t h a n o l , r i n s e d once w i t h 70% e t h a n o l and resuspended i n TE b u f f e r (lOmM T r i s -HCl ;pH 8.0, 1 mM EDTA). 6. Southern g e l a n a l y s i s : Southern g e l a n a l y s i s was pe r f o r m e d as d e s c r i b e d i n M a n i a t i s e t a l . (32). The h y b r i d i z a t i o n probe was p l a s m i d prr25.1 DNA (3) l a b e l l e d by n i c k t r a n s l a t i o n (Amersham N i c k T r a n s l a t i o n K i t , Amersham Corp., O a k v i l l e , Ont.) w i t h 3 2 p -dATP (New E n g l a n d N u c l e a r Corp., B o s t o n , Mass.) as a r a d i o a c t i v e p r e c u r s o r . 29 Figure 2.1 Construction of homozygous stocks mutant at the e(gj locus and. the a locus. 0.(3.) /0.(3.) (P) x FM6/Y (M) a J e(a)/FM6 x a*/Y (M) b S(a)/a x O.R. males (M) c J e(a) a /Y (recombinant) x FM6/M(l)of J e(a) a /FM6 x e(a) a /Y (backcross) I STOCK N.B. V i r g i n females selected for each cross. a e(a)/e(a) = S6-1 stock. b * = a a l l e l e s 1, 2, 3, 50-e and 53-d. c O.R. = Oregon R wild-type. 30 Figure 2.2 Construction of homozygous stocks mutant at the e_(g_) locus and at the autosomal l o c i p_r, bw, ry_ and p_, respectively. e (a) /e (a) , +/+ (P) x FM6/Y, +/+ (M) a J FM6/e(a), +/+ x FM6/Y, rjr*/p_r* (M) b I FM6/e(a), Er*/+ x e(g_)/Y, rjr*/ + I £(a)/£(fl)/ x e_(a)/Y, pr . * /px* STOCK N.B. V i r g i n females selected for each cross. a e(o:)/e(a) = S6-1 stock. D Protocol for p_r i l l u s t r a t e d , where * = p_r a l l e l e s 1 and bw. Same protocol was used for the bw locus a l l e l e s 1, 2b and 75; ry_ locus a l l e l e s 1, 2, 8, 26 and 41; and p. locus a l l e l e s 1 and p. 31 Figure 2.3 Scheme f o r removing the extraneous P elements and f o r s t a b i l i z i n g the cytotypic background of a P-induced g_ mutation (see Materials and Methods). y_ cv v g.p f/Y (P) x O.R. v i r g i n females (M) a J y_ cv v g_p f/+ v i r g i n females x O.R. males (M) J g_p f/Y (recombinant) x O.R. v i r g i n females (M) J g_p f/+ v i r g i n females x O.R. males (M) J x J X I g.p/Y (recombinant) C(1)DX/Y v i r g i n females (M) a_p/Y x C(1)DX/Y v i r g i n females (M) STOCK O.R. = Oregon R wild-type. 32 F i g u r e 2.4 Hybrid dysgenesis screen for revertants of the mutant and subsequent revertant stock formation (see Materials and Methods). y_ cv v g_p f/Y (P) x C(1)DX/Y v i r g i n females (M) J y_ cv v g f/Y (dysgenic) x g /g v i r g i n females (M) I Y_ cv v c j . r e v f/g,2 (revertant) x g_2/Y J g r e v f/Y (recombinant) x C(1)DX/Y v i r g i n females (M) J 2 r e v f/Y x C(1)DX/Y v i r g i n females (M) J g r e v f/Y x C(1)DX/Y v i r g i n females (M) I STOCK N.B. Five separate revertants (1-5) were i s o l a t e d i n t h i s way. 33 R e s u l t s 1. Analysis of the modifying ef f e c t of ela) on mutations at other l o c i : To test the ef f e c t of e(g_) on various g mutants, stocks were c o n s t r u c t e d which were homozygous f o r both the e(gj mutation and a mutation at the g locus (Figure 2.1; [modifying 1 7 effects measured v i s u a l l y ] ) . Five g a l l e l e s were used: g , g , 9. r g and c jr J . A l l of these are spontaneous mutations and have been shown by intragenic recombination mapping to l i e i n the following order along the X-chromosome: g53d-g2_g50e_glfg3 ^ 24) . The phenotypes of the mutants g and a_J were unaffected by the presence of homozygous e(g) . However, the mutant phenotypes of g i g and gr a l l became more severe ( i . e . less pigmented) in combination with homozygous e (g_) . As noted i n Chapter 1, the S6-1 stock contains a temperature-sensitive g mutation and shows P element homology by in s i t u hybridization to the g_ region on polytene chromosomes. This g^-a mutation i s not v i s i b l e when homozygous, but when placed over a more severe a l l e l e (g ) or a deficiency for the g region (Df (l)g. 1 f B) the mutation can be scored i n f l i e s raised at 29°C. To check for a modifying ef f e c t of e(g) on the g_ t s mutation, the S6-1 X-chromosome, containing both e(g) and g. t s, was tested over X-chromosomes co n s t r u c t e d to cont a i n both the e_(cr_) and o_2 mutations or the e(g) mutation and the deficiency for the g region. In both instances, at 2 9°C, female f l i e s showed the same mild garnet phenotype seen i n the absence of homozygous e(g_) . Thus there i s no e(g_) modifying e f f e c t on the g_ t s mutation present i n the S6-1 stock. The e f f e c t o f e(g.) on m u t a t i o n s a t o t h e r l o c i was t e s t e d by c o n s t r u c t i n g s t o c k s homozygous f o r b o t h t h e e(g) m u t a t i o n and t h e m u t a t i o n i n q u e s t i o n ( F i g u r e 2.2). The autosomal eye c o l o u r l o c i p r , bw, ry_ and p_ were t e s t e d i n t h i s f a s h i o n ; t h e a l l e l e s used r e p r e s e n t e d b o t h spontaneous and x - r a y i n d u c e d m u t a t i o n s : p_r , p _ r b w , bw 1, bw 2 b, bw 7 5, ry. 1, ry_ 2, ry_ 8, r y _ 2 6 , r y _ 4 1 , p.1 and p_P. R e s u l t s showed t h e p h e n o t y p i c e x p r e s s i o n o f none o f t h e s e mutant l o c i t o be a f f e c t e d by t h e pr e s e n c e o f homozygous (or hemizygous) e(g) . 2. I n s i t u h y b r i d i z a t i o n t o p o l y t e n e chromosomes: To dete r m i n e whether t h e enhanceable g a r n e t a l l e l e s g 2 , g^Oe a n d g-53d w e r e a s s o c i a t e d w i t h gypsy o r c o p i a i n s e r t i o n s , p o l y t e n e chromosome p r e p a r a t i o n s from t h e s e s t o c k s were s e p a r a t e l y h y b r i d i z e d i n s i t u w i t h t r i t i u m - l a b e l l e d gypsy o r c o p i a DNA. There was no homology t o e i t h e r o f t h e s e t r a n s p o s a b l e elements i n t h e 12C (g) r e g i o n (not shown). 3. P r e p a r a t i o n o f s t o c k s f o r c l o n i n g t h e g l o c u s : To b e s t a n a l y s e many o f t h e q u e s t i o n s p e r t a i n i n g t o t h e i n t e r a c t i o n between t h e g and e(g) l o c i i t would be d e s i r a b l e t o o b t a i n a c l o n e o f t h e g gene f o r m o l e c u l a r s t u d y . A m o l e c u l a r s t u d y o f t h e g l o c u s i t s e l f would a l s o be o f g r e a t i n t e r e s t , as i t was one o f t h e f i r s t l o c i i n D r o s o p h i l a t o be a n a l y s e d a t t h e f i n e s t r u c t u r e l e v e l u s i n g g e n e t i c t e c h n i q u e s , and as i t s m o l e c u l a r s t r u c t u r e and b i o c h e m i c a l f u n c t i o n remain u n e l u c i d a t e d . F u r t h e r m o r e , i t would be o f i n t e r e s t t o dete r m i n e t h e s t r u c t u r e o f t h e g l o c u s i n t h e S6-1 s t r a i n and t o examine any a l t e r a t i o n s a t t h e DNA l e v e l a s s o c i a t e d w i t h g m u t a t i o n s i n d u c e d i n t h e d y s g e n i c s c r e e n s from 35 Chapter 1. po o One of these induced a mutants (a ) , picked up over the a a l l e l e present on the Maxy a chromosome, appeared wild-type i n hemizygous males but mutant when heterozygous with a severe a l l e l e i n females. In stock, t h i s mutation underwent a further mutational event producing orange eyes. These orange-eyed males, when crossed with g /a females, produced female progeny with garnet eyes. When the same orange-eyed males were crossed with e(a) a /e(a) a females, a l l female offspring had orange eyes. It would seem then that t h i s new orange mutation could represent a new e(a)-a mutation in t e r a c t i o n . This orange mutation, while i n stock, subsequently underwent a further mutational event producing a a mutation expressed i n both males and females. Crossed with both g 2 / g 2 and e(a) a2/s.(a) o a females the female progeny were garnet-eyed. In s i t u hybridization to polytene chromosomes showed a l l three mutant li n e s to have P element homology i n the a region. Clearly, an a b i l i t y to follow t h i s mutational sequence at the molecular l e v e l would be invaluable i n uncovering the nature of mutation at the a locus and the mechanism of int e r a c t i o n between the e(a) and a l o c i . The mutant chosen for the molecular study of the garnet region was a presumptive P element i n s e r t i o n mutation (aP) i s o l a t e d i n the attached-X screen from Chapter 1. This mutation was i s o l a t e d over the F M 7 chromosome and was subsequently kept as a male stock mated to C(l)DX,y_ f/Y females (see Materials and Methods, Chapter 1). g r males crossed with homozygous a females p produced female progeny with garnet eyes, a males crossed with 36 females homozygous for both e(g_) and g gave r i s e to daughters with a m i l d l y enhanced garnet phenotype ( i . e . orange) upon eclosion, which quickly darkened to garnet within the f i r s t 24 9 9 hours. This i s the same result as seen with g/e(g) of- f l i e s (see Results, Chapter 1) ; one may conclude that e (g) , present on the S6-1 and, therefore, g_p X-chromosome, has no modifying ef f e c t p on the g_ mutation. The i n i t i a l removal of the majority of extraneous P element p s i t e s from the g stock was accomplished with the selection of a double recombinant y_ cv v f/Y male i n the F 2 offspring of a cross involving gf males and homozygous y_ cv v f females (see Materials and Methods). This new s t r a i n , containing 6-7 P s i t e s , was kept as a male stock mated to attached-X females. Males from t h i s s t r a i n were used i n a dysgenic screen for revertants of the a_p mutation (Figure 2.4). Five such revertants were is o l a t e d (g XXJ- J) from f i v e independent reversion events. Stocks of these s t r a i n s were formed as diagrammed i n Fi g u r e 2.4, crossing o f f the X-chromosome d i s t a l to the g locus i n an attempt to further remove extraneous P elements, and outcrossing three times to M-strain attached-X females, both to remove P s i t e s and to e s t a b l i s h the revertants i n a stable M-cytotype. Eye colour i n each of the revertants was v i s u a l l y undifferentiable from wild-type i n either hemizygous males or heterozygous females over the 9 g_ a l l e l e . To remove the remaining extraneous P element s i t e s from the y_ cv v a P f/Y s t r a i n , as well as to establish a stable M-cytotype, f l i e s from t h i s stock were successively outcrossed to 37 M-strain Oregon R wild-type f l i e s , single recombinants being selected on either side of the g_ locus i n the t h i r d and f i f t h generations (see Figure 2.3). In s i t u hybridization to polytene p chromosome preparations from t h i s new g s t r a i n , using plasmid PTT25.1 DNA as probe, showed two s i t e s of P element homology: one strong s i t e i n the 12C (g_) region, and another, weaker, s i t e two bands proximal to i t on the X-chromosome. This l a t t e r s i t e was present, as was homology i n the g region, on the o r i g i n a l S6-1 X-chromosome (cf. Figure 1.1). Southern gel analysis was performed on complete Bam HI d i g e s t s of genomic DNA from the above g^ s t r a i n and i t s progenitor, the y_ cv v g.p f s t r a i n , as well as the f i v e a; + R s t r a i n s , with l a b e l l e d pn-25.1 DNA as probe (Figure 2.5) . Surprisingly, the of s t r a i n showed only one band, 12-13 kb i n size, with P element homology. Presumably, the other s i t e seen with i n s i t u hybridization resides either i n a Bam HI fragment which co-migrates at 12-13 kb or i s so small as to have run o f f the end of the g e l . A l t e r n a t i v e l y , the extent of P element sequence homology at the one s i t e may be so minimal that the amount of probe bound was i n s u f f i c i e n t for detection i n the Southern analysis. (The other p o s s i b i l i t y , that one s i t e resides in a fragment which co-migrates with the 1.8 kb non-P-specific band, [which correlates with DNA from the c y t o l o g i c a l region 17C, present on the prr25.1 plasmid (50)], was disproved by washing and r e h y b r i d i z i n g the same f i l t e r with a g e l - p u r i f i e d , nick translated, Hind III r e s t r i c t i o n fragment containing solely P element DNA, and detecting hybridization only at the 12-13 kb l e v e l i n the g lane.) The same 12-13 kb band was present 38 F i g u r e 2 .5 Southern analysis showing hybridization of plasmid pr[25.1 p DNA to complete Bam HI digests of genomic DNA from the g mutant and revertant stocks. 1 2 3 4 5 6 7 Size markers were Hind III fragments from phage lambda DNA. a Hybridization to DNA from c y t o l o g i c a l region 17C by DNA sequences flanking complete P element i n pn-25.1. Lane 1 = q_p. Lanes 2-6 = g_ + R l~ 5. Lane 7 = y_ cv v a;p f. 39 i n the y_ cv v gf f lane, i n addition to the other s i t e s of P homology present i n that s t r a i n . The revertant lanes showed multiple bands, but upon close examination hybridization was absent at the 12-13 kb l e v e l i n each revertant s t r a i n . These results indicate that the 12-13 kb band present i n the g? s t r a i n represents DNA from the g region (with P element insertion) and that the absence of t h i s band i n the r e v e r t a n t s represents e x c i s i o n of P element DNA as the mechanism of reversion. The determination of the size of the garnet region DNA fragment i n a complete Bam HI digest of g_p genomic DNA enables one to remove t h i s fragment from a g e l f o r use i n the construction of a fragment-enriched plasmid DNA l i b r a r y (37) . This has f a c i l i t a t e d the recent cloning of the g locus, performed i n our lab by D.A.R. S i n c l a i r , using the technique of P element transposon-tagging (3). 40 D i s c u s s i o n The enhancer of garnet was shown to be an a l l e l e - s p e c i f i c modifier locus. Homozygous (or hemizygous) e(g) enhances the phenotypes of the spontaneous garnet mutations gz, gp®e and g?^^, i ^ but does not enhance the spontaneous a l l e l e s g and gJ or two new mutations, g and g . Most spontaneous mutations i n Drosophila are associated with the i n s e r t i o n of transposable elements (30), e i t h e r i n t o 5' tr a n s c r i p t i o n regulatory sequences (35, 49), or into RNA coding sequences (30, 40). Also, i t i s transposon induced mutations which have been shown to be modifiable by recessive mutations at distant l o c i (36, 40) . S i m i l a r i t i e s between the e(g)-g interaction and the well-described transposon-mediated suppressor systems i n Drosophila were sought. When tested with other l o c i , e(g_) was shown not to modify the phenotype of mutant a l l e l e s at the purple, brown, rosy and pink l o c i (including the p_r and p r b w mutations, which are associated with 412 insertions and suppressible by su(p_r) and su(s_) ) . The mutations su(p_r), su(s) and su(f_) can enhance the phenotype of certain mutant a l l e l e s associated with gypsy or copia insertions, however, i n s i t u hybridization demonstrated the absence of gypsy or copia DNA sequences i n the garnet region of the g_ a l l e l e s enhanceable by e(g) . The r e c e s s i v e nature of the e(g) mutation and i t s interaction with spontaneous enhanceable a l l e l e s suggest that t h i s system may be analogous to the transposon-mediated a l l e l e -s p e c i f i c suppressor systems d e s c r i b e d p r e v i o u s l y (36, 40) . 41 However, the mechanism by which the e(g)-g i n t e r a c t i o n occurs i s not clear. Because i t i s an enhancing interaction, release from t r a n s c r i p t i o n a l interference or reduction i n abnormal tr a n s c r i p t accumulation are not appropriate mechanisms. That wild-type e(g.) could produce a gene product which acts d i r e c t l y upon g sequences regulating t r a n s c r i p t i o n , or a l t e r n a t i v e l y , that i t could produce some necessary co-factor for g gene product a c t i v i t y , i s unl i k e l y given the a l l e l e - s p e c i f i c i t y of e(g) and i t s lack of ef f e c t on the wild-type garnet locus. A l b e i t i n the absence of molecular data, i t i s tempting to invoke a t ransposon-mediated mechanism f o r the e.(g_)-cf i n t e r a c t i o n . Given that the enhanceable g mutations are known to be located at one end of the fine structure g locus map, a possible mechanism i s suggested: Transposable element ins e r t i o n into the promoter or 5 ' region of the g gene would disrupt normal t r a n s c r i p t i o n (essentially eliminating t r a n s c r i p t i o n from the normal sta r t site) . The g gene a c t i v i t y which i s observed i n the hypomorphic enhanceable mutants may result from t r a n s c r i p t i o n a l read-through i n i t i a t e d from a promoter w i t h i n an i n s e r t i o n element and continuing into the g gene. In t h i s scenario, the mutant garnet phenotype r e f l e c t s less e f f e c t i v e t r a n s c r i p t i o n of the g locus, or the production of a modified gene product. I f tr a n s c r i p t i o n of the inse r t i o n elements were dependent on wild-type a c t i v i t y of the e(g) gene (analogous to su(Hw) and gypsy), mutations at e(g_) would block read-through into the g gene and result i n the appearance of an enhanced mutant phenotype. To determine the tr u e nature of t h i s system of gene 42 i n t e r a c t i o n a molecular a n a l y s i s i s needed. A P-induced g_ mutant (o.p) i s o l a t e d i n the experiments described i n Chapter 1 was prepared for the molecular analysis of the garnet locus. The cloning of g w i l l enable the elucidation of the structure and location of the putative i n s e r t i o n elements i n the spontaneous g mutants, and w i l l also f a c i l i t a t e the study of a p o t e n t i a l l y informative sequence of successive mutations at the g locus seen in a d i f f e r e n t P-induced g mutant (.g ) (see Results) . cDNA i s o l a t i o n and Northern gel analysis of the t r a n s c r i p t s produced by g a l l e l e s i n the presence and absence of e.(a_) w i l l be informative i n the determination of the molecular structure of the g locus and the nature of the int e r a c t i o n between the g and e(g_) l o c i . 43 SUMMARY The nature of extreme mutability around the g locus i n the lab s t r a i n S6-1 was determined to be associated with P-M hybrid dysgenesis. S6-1 i s a P-strain: males of t h i s stock, when crossed to M-strain v i r g i n females, give r i s e to F-^  progeny showing the germline mutability t y p i c a l of the P-M system. When tested over a multiply marked X-chromosome, mutation induction was observed at a number of X-chromosome l o c i . The preponderance of induced mutations associated with the g_ locus may be accounted for by the presence of a P element s i t e of residence i n the cr region i n the S6-1 s t r a i n , as shown by i n s i t u h y b r i d i z a t i o n to polytene chromosome preparations. New mutations may thus arise from P element i n s e r t i o n or a change associated with the resident P element, e.g. r e v e r s a l of o r i e n t a t i o n , rearrangement, or imprecise excision. A novel s i t u a t i o n was the requirement for the presence of the S6-1 X-chromosome i n F-^  dysgenic males i n order to produce the germline mutability. The results suggest that the only complete P element or elements, ( i . e . those capable of allowing transposition) , are confined to the X-chromosome i n the S6-1 s t r a i n . The existence of a mutation, present i n the d i s t a l region of the S6-1 X-chromosome, which enhances certain mutations at the g locus i s reported. This e(g) mutation, which may or may not be i d e n t i c a l with a previously described, and subsequently l o s t , garnet modifier, acts as a recessive mutation to enhance the phenotype of the spontaneous garnet mutants g 2, g 5 ^ e and g 5 ^ d , a l l of which map to one end of the fine structure g locus map. e(g_) did not modify a P-induced g_ mutant, a temperature-sensitive g_ mutant, or the spontaneous garnet a l l e l e s g_ and g_J. Also, e(g_) was shown to have no ef f e c t on the phenotype of a number of mutant a l l e l e s at the l o c i p_r, bw, ry_ and p_. This enhancing system may represent a v a r i a t i o n on the previously described transposon-mediated suppression systems i n D_s_ melanogaster. 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Yannopoulos, G., Stamitis, N., Monastirioti, M., Hatzopoulos, P. and Louis, C. (1987) hobo i s responsible for the induction of hybrid dysgenesis by strains of Drosophila melanogaster bearing the male recombination factor 23.5MRF. C e l l 49:487-495. Yim, J.J., G r e l l , E.H. and Jacobson, K.B. (1977) Mechanism of suppression i n Drosophila: control of sepiapterin synthesis at the purple locus. Science 198:1168-1170. Zachar, Z., Garza, D., Chou, T., Goland, J. and Bingham, P.M. (1987) Molecular cloning and genetic analysis of the suppressor-of-white-apricot locus from Drosophila  melanogaster. Mol. C e l l . B i o l . 7:2498-2505. 50 APPENDIX D e s c r i p t i o n s of s p e c i a l chromosomes and mutations (see (31) or t e x t f o r more d e t a i l e d d e s c r i p t i o n s ) : C(l)DX,y_ f.: Compound (1) Double X. M o n o c e n t r i c a t t a c h e d - X chromosomes which e x i s t i n XX/Y females. Such females produce sons t h a t i n h e r i t a p a t e r n a l X and maternal Y, and daughters t h a t i n h e r i t the maternal attached-X's and a p a t e r n a l Y. C(l)DX/0 i s l e t h a l . Marked with the r e c e s s i v e a l l e l e s y_ and f . FM7: Balancer X-chromosome i n v e r s i o n w i t h markers y 3 l d s_c® w a yO O f 4 sn F 2. and B. Male v i a b l e and f e r t i l e ; homozygous female v i a b l e but s t e r i l e . Suppresses c r o s s i n g over i n the X-chromosome. Maxy a: M u l t i p l y marked X-chromosome b e a r i n g t h e r e c e s s i v e a l l e l e s y_ c 4 sc. 8 s c j 5 1 pjn w ec rb cm c t ^ sn^ r a s 2 g_2 f os and c a r . Homozygous or hemizygous Maxy a not v i a b l e . bw 1' 2 b ' 7 5 : Recessive a l l e l e s at the brown locu s [2/104.5]. bw 1' 2 b = spontaneous; bw 7 5 = x-ray induced. ^ x , J, j u e , JOU. Spontaneous r e c e s s i v e a l l e l e s at the garnet l o c u s [1/44.4], L o c a l i z e d t o p o l y t e n e chromosome r e g i o n 12B9-12C7. N: Dominant mutation (wing t i p phenotype) at the Notch lo c u s [1/3.0]. L o c a l i z e d t o p o l y t e n e chromosome band 3C7. P; Spontaneous recessive a l l e l e s at the pink locus [3/48.0] px 1' b w : Spontaneous recessive a l l e l e s at the purple locus [2/54.5]. r v 1 ' 2 ' 8 ' 2 6 ' 4 1 : Recessive a l l e l e s at the rosy locus [3/52.0] r y x ' ^ = spontaneous/ ry°' yj- = x-ray induced. w and wa: Spontaneous recessive a l l e l e s at the white locus [1/1.5]. Localized to polytene chromosome band 3C2. 52 

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