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Analysis of Y-linked temperature-sensitive mutations in Drosophila melanogaster causing male sterility… Suchowersky, Oksana 1975

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ANALYSIS OF Y-LINKED TEMPERATURE-SENSITIVE MUTATIONS IN DROSOPHILA MELANOGASTER, CAUSING MALE STERILITY or Great A s p i r a t i o n s by OKSANA SUCHOWERSKY B . S c , U n i v e r s i t y o f A l b e r t a , 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n GENETICS We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1975 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion. •"Department of ^ ^^^^uu^f C^d-^ The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1WS Date - i i -ABSTRACT A number of EMS-induced h e a t - s e n s i t i v e and c o l d - s e n s i t i v e male s t e r i l e mutations were recovered on an XY chromosome. A l l mutations which recovered f e r t i l i t y i n the presence of a f r e e Y chromosome were c l a s s i f i e d as Y - l i n k e d . Of the Y-l i n k e d hs mutations, a l l were f e r t i l e w i t h a f r e e Y L fragment, g w h i l e the cs mutations were f e r t i l e w i t h a Y fragment. Ten of the hs Y - l i n k e d mutations were s t u d i e d i n d e t a i l ; L e i g h t o f these were shown to be unambiguously l o c a t e d on Y , and had a temperature s e n s i t i v e p e r i o d c o i n c i d i n g w i t h spermatid d i f f e r e n t i a t i o n . Two of the hs mutant stocks were double mutations and a t l e a s t one of these s t o c k s had an X - l i n k e d mu-tation.a which i n t e r a c t e d w i t h the Y chromosome. T h i s stock had a t s p which, although e x e r t i n g i t s e f f e c t d u r i n g spermio-L g e n e s i s , was p r i o r t o t h a t o f the mutations l o c a t e d on Y . Of the 5 cs mutations c l a s s i f i e d as Y - l i n k e d , a t l e a s t t h r e e were shown to be mutations on the X chromosome which S i n t e r a c t e d w i t h Y . The t s p ' s o f these mutations are e i t h e r pre-or e a r l y p o s t - m e i o t i c . Thus, no t s mutations were S recovered on Y . These r e s u l t s suggest t h a t : L 1) the f e r t i l i t y f a c t o r s on Y are p r e s e n t i n s i n g l e c o p i e s and produce products which are d i r e c t l y i n v o l v e d i n spermiogenesis f g 2) the genes on Y co u l d have a r e g u l a t o r y f u n c t i o n d u r i n g spermatogenesis, and e i t h e r do not produce products which are t h e r m o - l a b i l e , or are p r e s e n t i n m u l t i p l e c o p i e s , 3) t h e r e are a c l a s s o f genes on the X chromosome which a f f e c t spermatogenesis through i n t e r a c t i o n w i t h the Y chromosome. - i i i -S e v e r a l models were proposed to account f o r t h i s X-Y chromosome i n t e r a c t i o n . - i v -TABLE OF CONTENTS INTRODUCTION METHODS AND MATERIALS RESULTS DISCUSSION SUMMARY BIBLIOGRAPHY - v -LIST OF FIGURES Figure Page 1. Dravring of adult testes with accessory organs. 2 2. Diagramatic representation of spermatogenesis within the testes. 3 3. Diagramatic representation of Y chromosome of D. melanoga ster. 9 4. Results of rec i p r o c a l temperature s h i f t s of hs-cs double mutations with si m i l a r phenotypes. -^5 5. Diagram of attached -XY chromosomes used i n th i s study. ^6 6. Procedure for the synthesis of a cs-hs double mutant Y chromosome i n an attached -XY chromosome. 17 7. Screening protocol for i s o l a t i o n of hs and cs s t e r i l e mutations on an XY chromosome. 21 8. Length of developmental stages at 17°, 22° and 28°, i n days. 23 9. Protocol for comrjlementation tests of ts s t e r i l e mutations on an XY chromosome with normal (sc 8) Y. 25 10. Protocol for comrjlementation tests of ts s t e r i l e mutations on an XY chromosome with a Y fragment or Y deficiency chromosome. 45 11. Daily mean f e r t i l i t y of 502, 598, and 1216 at 22° following a 3-day heat pulse at 28 u i n the adult and controls. 4g 12. Daily mean f e r t i l i t y of 1312/ 1608 and 3269 at 22° following a 3-day heat pulse at 28° i n the adults, and controls. 47 13. Daily mean f e r t i l i t y of 56_ at 22°, grown at 28° during various stages of development. 43 14. Daily mean f e r t i l i t y of 56 : at 22° grown at 28° for PI, PII, of PIII, ancPfor entire l i f e cycle except PII. 49 15. Daily mean f e r t i l i t y of 887 at 22°, grown at 28° during various stages of development. 50 - v i -F i g u r e Page 16. D a i l y mean f e r t i l i t y o f 887 a t 2 2 °, grown 51 a t 2 8 ° f o r PI, P I I , o r P H I , and f o r e n t i r e l i f e c y c l e except P I I , 17. D a i l y mean f e r t i l i t y o f 631 a t 2 2 ° , grown a t 2 8 ° d u r i n g v a r i o u s stages o f development. 52 18. D a i l y mean f e r t i l i t y of 631 a t 2 2 ° , grown a t 2 8 ° f o r PI, P I I , o r P H I . 19. D a i l y mean f e r t i l i t y o f 2225 a t 2 2 ° , grown a t 2 8 ° d u r i n g v a r i o u s stages o f development. 53 20. D a i l y mean f e r t i l i t y of 2225 a t 2 2 ° , grown a t 2 8 ° f o r PI, P I I , or P H I . 54 21*- Tsp's of hs and cs mutations. 55 22. D a i l y mean f e r t i l i t y o f 150 a t 2 2 ° , grown a t 1 7 ° d u r i n g v a r i o u s stages of development. 5 8 23. D a i l y mean f e r t i l i t y o f 2661 a t 2 2 ° , grown a t 1 7 ° d u r i n g v a r i o u s stages of development. 59 24. D a i l y mean f e r t i l i t y o f 2661 a t 2 2 ° , grown a t 1 7 ° d u r i n g v a r i o u s stages o f development. 60 25. D a i l y mean f e r t i l i t y o f 3480 a t 2 2 ° , grown a t 1 7 ° d u r i n g v a r i o u s stages o f development. 61 26. D a i l y mean f e r t i l i t y o f 3480 a t 2 2 ° , grown a t 1 7 ° d u r i n g v a r i o u s stages o f development. 62 27. D a i l y mean f e r t i l i t y o f A208 a t 2 2 ° , grown a t 1 7 ° d u r i n g v a r i o u s stages o f development. 63 - v i i -LIST OP TABLES Table Page 1. Timing of spermatogenesis ixr D. melanogaster at 25° i n adults and l a r v a l stages. 5 2. Isolation and pretests of ts male s t e r i l e mutations on XY chromosomes. 33 3. Complementation patterns of Y-linked ts mutations with sc 8Y, Y s, and Y L. 36 4. Complementation patterns of Y-linked ts mutations with Y deficiency chromosomes. 38 5. Recombinational analysis of Y-linked ts mutations. 40 - v i i i -ACKNOWLEDGEMENTS My s i n c e r e s t thanks t o : Dr. David Suzuki f o r encouragement; Dr. Tom Kaufman f o r guidance; a l l the members of the l a b f o r camaraderie; and my f l i e s f o r almost making i t p o s s i b l e . INTRODUCTION Spermatogenesis has been widely studied by both developmental b i o l o g i s t s and geneticists because i t i s an autonomous system which proceeds i n an orderly, well-defined manner within one s p e c i f i c organ, the t e s t i s . In Drosophila melanogaster, the process of normal spermatogenesis has been extensively characterized c y t o l o g i c a l l y both with the l i g h t microscope (Cooper, 1950; Hannah-Alava, 1965) and with the electron microscope (Perotti, 1969; Stanley et a l . , 1972; Peacock e t a l . , 1972; Tokuyasu et a l . , 1972a,b; Tokuyasu, 1974a,b, 1975). A b r i e f review of spermatogenesis i n D. melanogaster w i l l now be given. In the adult f l y , the primary spermatogonia are located at the a p i c a l t i p of the testes. They divide asynchronously to produce secondary spermatogonia which, through four mitotic d i v i s i o n s , produce a bundle of sixteen spermatocytes i n t e r -connected by cytoplasmic bridges and enclosed i n a cyst (Hannah-Alava, 1965). The prophase of the f i r s t meiotic d i v i s i o n l a s t s approximately four days during which the c e l l s undergo a large increase i n volume. As the c e l l s divide and mature, they migrate down the sides of the anterior half of the testes so that the approximate stage of the c e l l s can be determined by t h e i r p o s i t i o n (Cooper, 1950) (Figures 1,2). After a rapid meiotic d i v i s i o n , the cyst, now containing 64 spermatids s t i l l embedded within a common cytoplasm, i s extruded into the lumen. Here, the spermatids within a cyst synchronously undergo a process of complex d i f f e r e n t i a t i o n leading to i n d i v i d u a l i z e d , motile sperm. This process can be - 2a -Figure 1. Drawing of adult testes with accessory organs. t, testes; sv, seminal vescicle; pa, paragonia; sp, sperm pump; de, ductus ejaculacorius. From Bodenstein (1950), p. 308. Upper diag-ram, enlargement of section through apical tip of testis show-ing ing developing sperm cel l s . Spermatogonia, g, are found at tip and move down the testis wall as they divide and mature as spermatocytes, c. Elongating spermatids, m, are found in the lumen. From Cooper (1950), p. 6. - 2b -- 3a -Figure 2. Diagramatic representation of spermatogenesis within the testis. 1) spermatogonia, which move down the testis wall as they un-dergo mitosis; 2) 16 primary spermatocytes enclosed in cyst; 3) after meiosis, 64 spermatids connected by cytoplasmic brid-ges are found in the cyst; 4) elongating spermatids, extruded into the lumen, head (H), moving posteriorly; 5) f u l l y elon-gated sperm bundles starting individualization at head region, excess material being removed in cystic bulge (CB); 6): cystic bulge (CB) moving in an anterior direction, leaving individu-alized sperm behind i t ; 7) completely individualized spermatids, excess material being sloughed off in waste bag (WB); 8) sperm undergoing coiling with heads (H) embedded in the terminal epi-thelium (TE). From Peacock et a l . , (1972). -3b 2-H 3-H ICB-\|) - 4 -d i v i d e d i n t o f o u r stages (Peacock e t a l . f 1972) (Figure 2 ) : a) E l o n g a t i o n - The spermatids elongate b i d i r e c t i o n a l l y w i t h a c o n c u r r e n t condensation o f chromatin and f o r m a t i o n o f axoneme and two m i t o c h o n d r i a l d e r i v a t i v e s . b) I n d i v i d u a l i z a t i o n - The i n t e r c o n n e c t i n g c y t o p l a s m i c b r i d g e s are removed by a c y s t i c bulge which t r a v e l s p o s t e r i o r l y . The minor m i t o c h o n d r i a l d e r i v a t i v e i s much reduced i n s i z e . c) Entrapment and c o i l i n g - The spermatid heads are embedded i n the e p i t h e l i a l c e l l s a t the t e r m i n a l end of the t e s t i s and c o i l i n g o ccurs by the r e t r a c t i o n o f the t a i l s from the a p i c a l r e g i o n . d) Release of m o t i l e sperm i n t o the seminal v e s i c l e . Mature sperm are 1.8 mm. i n l e n g t h , of which the head r e g i o n c o n s t i t u t e s o n l y 10 microns (Cooper, 1950). No RNA s y n t h e s i s i s seen p o s t - m e i o t i c a l l y ( O l i v i e r i and O l i v i e r i , 1965; Gould-Somero and H o l l a n d , 1974) although p r o t e i n s y n t h e s i s does occur (Brink, 1968). In a d u l t f l i e s , the process of spermatogenesis takes about 10 days a t 25°, 5 f o r pre-meiotic and 5 f o r p o s t - m e i o t i c develop-ment (Chandley and Bateman, 1962). Newly e c l o s e d males c o n t a i n m o t i l e sperm i n t h e i r seminal v e s i c l e s , and the stage of development of t h i s f i r s t c o h o r t of sperm can be c o r r e l a t e d w i t h the stage o f the t e s t e s and l a r v a l development (Hannah-A l a v a , 1965) (Table 1). L i n d s l e y and L i f s c h y t z (1972) estimate t h a t about 750 genes i n the D r o s o p h i l a genome are i n v o l v e d i n male f e r t i l i t y . T h i s i s probably an over estimate of the number i n v o l v e d i n sperm development s i n c e any a b n o r m a l i t i e s which prevent sperm - 5 -Table 1 Timing of spermatogenesis in D. melanogaster at 25° in adult and l a r v a l stages* Day in Adult Larval Stage Stage in Spermiogenesis v i 1 Egg, 1st instar Spermatogonia 2 2nd instar 1° spermatocyte 3 3rd inst a r 4 3 Me ios is 6" Pupa Spermatid elongation and d i f f e r e n t i a t i o n 7 8-9 'Coiling 10 4lc 1 os ion M o t i l e sperm * based on tables from Chandley and Bateman (1962), Lindsley and L i f s c h y t z (1972), and Kiefer (1973). - 6 -t r a n s f e r w i l l be i n c l u d e d i n t h i s c l a s s o f mutations. Male s t e r i l e mutations s p e c i f i c a l l y a f f e c t i n g sperm development have been induced by EMS, X-rays and ot h e r mutagens on the Y (Neuhaus, 1939; Brosseau, 1960; W i l l i a m s o n , 1968; A y l e s e t a l . , 1973) , the X (Berg, 1937; L i n d s l e y e t a l . , 1960), the second (Romrell e t a l . , 1972a,b), and the t h i r d (Wilkinson e t a l . , 1974) chromosomes. I t should be mentioned t h a t the male and female germinal c y c l e s appear t o be r e g u l a t e d by separate genes. Thus, f o r example, mutations c a u s i n g male s t e r i l i t y seldom cause female s t e r i l i t y and v i c e v e r s a , although m e i o s i s I I i n both sexes may be a f f e c t e d by the same l o c i (Davis, 1971; Sandler e t a l . , 1968). However, a mutation has been i s o l a t e d which prevents c y t o k i n e s i s i n m e i o s i s I of both males and females (Romrell e t a l . , 1972a). The Y chromosome i s unique i n t h a t i t s major f u n c t i o n appears to be c o n f i n e d t o spermatogenesis. I t has been known f o r over 60 years t h a t f l i e s which l a c k a Y chromosome (symbolized as X/O) are s t e r i l e , but p h e n o t y p i c a l l y i n d i s t i n g u i s h a b l e from w i l d type (X/Y) males (Bridges, 1916). Examination of t e s t e s from X/O males shows t h a t although some sperm e l o n g a t i o n has o c c u r r e d , m o t i l e sperm are never found ( S a f i r , 1920). In somatic c e l l s , the presence or absence of the Y chromosome has l i t t l e e f f e c t on t h e i r phenotype. F l i e s w i t h one X chromosome are male, w h i l e those w i t h two X chromosomes female, whether they c a r r y the Y chromosome or not. (Bridges, 1925). However, an e x t r a dose of the Y chromosome ( i n X/Y/Y males and X/X/Y females) can produce some e f f e c t s such as su p p r e s s i o n of v a r i e g a t i o n (Gowen and Gay, 1934; Cooper, 1956) - 7 -and of some l e t h a l i t y due to X chromosome aberrations (Lindsley et a l . , I960), and a v a r i e t y of minor phenotypic changes, such as an increase i n the number of stenopleural b r i s t l e s and i n the length of the t h i r d l o ngitudinal wing vein (Hannah, 1951). The whole Y chromosome, divided into two unequal arms by the centromere, i s heterochromatic i n somatic c e l l s : i t i s p o s i t i v e l y heteropycnotic, l a t e r e p l i c a t i n g , and f a i l s to synthesize m-RNA (Brown, 1966). However, i t does have regions of specialized function. Different regions of i t suppress variegation to d i f f e r e n t degrees (Cooper, 1956; Hess, 1970b), and there i s a region of homology between the short arm of the Y chromosome and the proximal (heterochromatic) part of the X chromosome, for a high proportion of exchanges involving the X and Y chromosomes occur i n t h i s region (Lindsley, 1955; Lucchesi, 1965). Both chromosomes carry the nucleolus organizer (the genes coding for ribosomal RNA) i n t h i s region of homology (Ritossa and Spiegelman 1965; Ritossa et. a l . , 1966), both X and Y NO regions produce i d e n t i c a l RNA (Maden and T a r t o f f , 1974). However, a f l y requires only one complete NO region on either chromosome i n order to be v i a b l e . Thus, the only indispensible function of the Y chromosome i s i n spermatogenesis, to produce motile sperm. Brosseau (1960) used X-ray induced Y - s t e r i l e mutations to show that the Y chromosome consists of 7 complementation regions involved i n spermatogenesis, 2 on the short arm (Y ) and 5 on the long arm (Y L) (Figure 3). A l l 7 of these factors have to be present i n at l e a s t one wild type dose for a male to produce motile sperm. - 8 -Deficiencies i n one or more of these factors r e s u l t s i n sperm that reach various stages of elongation and c o i l i n g , but then s t a r t to degenerate. Unfortunately, i t has not been possible to correlate a s p e c i f i c v i s i b l e defect i n spermiogenesis with the loss of any s p e c i f i c locus. A l l mutant-Y bearing males show i r r e g u l a r i t i e s i n axoneme formation, development of mitochondrial derivatives, and i n d i v i d u a l i z a t i o n . Although c e r t a i n mutants may show more of one type of abnormality than another, i t i s d i f f i c u l t to categorize the mutants even on a quantitative basis since a single mutant male w i l l show differences i n degree of maturation, type of abnormalities found, and degeneration among the spermatids examined (Kiefer, 1968, 1973). In D. hydei, the process of spermatogenesis appears to be very similar to the one i n D.melanogaster, although the sperm develop i n bundles of 32, and sperm development i n X/O males stops before meiosis (Hess and Meyer, 1968). In the prophase of the primary spermatocyte i n X/Y D. hydei males, the nucleus i s f i l l e d with large loops which incorporate H-uridine and are reve r s i b l y disintegrated by actinomycin D and X-rays, i n d i c a t i n g RNA synthesis. That these loops are formed by the Y chromosome can be shown by the fac t that no such loops are seen i n X/O males, and X /Y/Y males produce two complete sets of loops (Hess and Meyer, 1968). Furthermore, 50% of a l l RNA (excluding r-RNA) synthesized i n the primary spermatocyte i s complementary to Y-DNA (Hennig, 1968). Six pairs of loops, each corresponding to a f e r t i l i t y f actor, are seen i n the nucleus, and a l l s i x have to be - 9a -Figure 3. Diagramatic representation of the Y chromosome of D. melano-gaster showing r e l a t i v e positions of f e r t i l i t y factors on short S L arm (Y ) of Y chromosome ( k s l , ks2), and on long arm (Y ) of Y chromosome ( k l l - 5 ^ , and the nucleolus organizer (NO). V ro CO O to i - 10 -present i n at le a s t one dose to produce motile sperm. If XY translocations are used to make various combinations of Y loops only those f l i e s which receive a f u l l complement are f e r t i l e . (Hess, 1967). Deficiencies of one or more loops r e s u l t i n immotile sperm i n various stages of elongation. As i n D. melanogaster the disappearance of a p a r t i c u l a r sperm organelle cannot be correlated with a s p e c i f i c loop deficiency. (Meyer, 1969). Another important factor i s the state of the loops. I f , due to a translocation or treatment with chemical agents, the loops are prevented from unfolding, spermatid d i f f e r e n t i a t i o n i s arrested (Hess, 1970a). Each of the six pairs of loops i s s t r u c t u r a l l y unique and i t s shape depends only the Y chromosome, "from which i t a r i s e s . When a hybrid X/Y/Y male i s made carrying one Y chromosome from D. neohydei (which also produces Y-loops), and the second from D. hydei, the two Y chromosomes within the same nucleus each produce a set of loops c h a r a c t e r i s t i c of i t s species, regardless of genetic background (Hess and Meyer, 1968). Hennig et a l . , (1973) have estimated that about 1/12 of the DNA i n the Y chromosome pa r t i c i p a t e s i n loop formation and r-RNA synthesis. No function has yet been found for the rest of the DNA. In D. melanogaster, some Y-loop formation i s seen i n the primary spermatocyte but i n d i v i d u a l loops are not distinguishable, and correlations between cytology and gene function comparable to the D. hydei studies cannot be made. However, because spermatogenesis i n these 2 related species i s s i m i l a r , conclu-sions reached from research i n D. hydei can be used to int e r p r e t - l i -the genetic data i n D. melanogaster. For example, i n D. hydei, RNA-DNA hybridization studies have shown that RNA complementary to Y-DNA (excluding r-DNA) i s transcribed only i n the prophase of the primary spermatocyte (Hennig, 1968). Since Y-loops are also present i n D. melanogaster at t h i s time, i t i s believed that they are ac t i v e l y synthesizing RNA (Hess and Meyer, 1968). In the spermatocyte nuclei i n X/0 D. melanogaster males, the formation of c r y s t a l needles i s seen, presumably due to an accumulation of protein unusable without the presence of the Y chromosome (Meyer et aJL., 1961). When spermatids from such males are examined under the electron microscope, a l l the necessary s t r u c t u r a l components are present but appear disorganized (Kiefer, 1966). Therefore, the Y chromosome does not seem to code for any of the st r u c t u r a l components of the sperm, but rather, i t i s needed to regulatetheir assembly. Furthermore, the Y chromosome has to be present i n the gonial c e l l s themselves to d i r e c t sperm maturation, and the genotype of the testes and n u t r i t i v e c e l l s i s immaterial i n t h i s process (Stern and Hadorn, 1938: Williamson, 1970a). Also, once the spermatocytes have undergone meiosis, the haploid spermatids develop and pa r t i c i p a t e i n f e r t i l i z a t i o n normally regardless of gross chromosome duplications and d e f i c i e n c i e s , i f the d i p l o i d genotype was complete (McCloskey, 1966; Lindsley and G r e l l , 1969). Since there i s no post-meiotic RNA synthesis ( O l i v i e r i and O l i v i e r i , 1965,; Gould-Somero and Holland, 1974), and chromosomes are dispensible i n the spermatids, a l l d i r e c -tions necessary for spermiogenesis must be made i n the primary spermatocyte and stored as long-lived RNA or protein i n the - 12 -c e l l s (Lindsley and G r e l l , 1969). Thus, the Y chromosome has a s p e c i f i c s i t e of action, namely the male germ l i n e c e l l s (being t o t a l l y dispensible i n somatic c e l l s ) . Furthermore, i t has a s p e c i f i c time of tr a n s c r i p t i o n , i n the primary spermatocyte, and has a s p e c i f i c function, to organize the developing spermatids into f u l l y functioning sperm. Although Y-dependent RNA t r a n s c r i p t i o n occurs i n the primary spermatocyte, the time of action of the Y chromosome gene products i n the sperm c e l l s remains to be determined. The use of conditional mutants for t h i s purpose has proved invaluable i n other systems because i t allows the detection of the temperature sens i t i v e period (tsp) of the gene product. (Hartwell et al_., 1970; Jarvik and Botstein, 1973; Suzuki, 1970, 1974). In previous work i n t h i s lab, 8 EMS-induced heat sens i t i v e Y-linked male s t e r i l e mutations were is o l a t e d (Ayles et a l . , 1973). Since males grown at 28° for 2 days showed a reduction i n f e r t i l i t y 5-6 days following the heat pulse, i t seemed that the pre-meiotic stages of spermatogenesis were being affected by the mutation (see Table 1). Further studies on two of these mutants showed that one mutation had i t s tsp i n the primary spermatocyte, while the other was i n spermiogenesis (according to t h e i r interpretation of the data). Also, a leaky cold sensitive Y-linked mutation has been analyzed which has a post-meiotic tsp (Frankel, 1973). From the above, i t was suggested that there are two types of genes on the Y chromosome a f f e c t i n g s t e r i l i t y : those involved with Y-dependent RNA t r a n s c r i p t i o n , which have a pre-meiotic tsp, and - 13 -those d i r e c t i n g sperm assembly, having a post-meiotic tsp (Sanders and Ayles, 1973). To analyze more extensively the properties of the s t e r i l i t y factors on the Y chromosome, i t was decided to i s o l a t e a large number of heat-sensitive (hs) and cold-sensitive (cs) mutations. The hs mutationswould be s t e r i l e at 28° (the r e s t r i c t i v e temperature) but f e r t i l e at 22° (the permissive temperature), while the cs mutationswould be s t e r i l e at 17° but f e r t i l e at 22°. Furthermore, since i t has not been possible to d i s t i n g u i s h among the Y-linked mutations on a c y t o l o g i c a l basis, i t was hoped, through the use of genetic techniques and the hs and cs mutations, to do so on a developmental basis. This has been previously shown to be possible i n bacteriophage P22 (Jarvik and Botstein, 1973). A series of hs and cs mutants which i n t e r f e r r e d with phage morphogenesis were i s o l a t e d , and by performing temperature s h i f t s on the hs-cs double mutations, i t was possible to determine the sequence i n which the genes affected were expressed i n the phage assembly pathway. For example, i f 2 ts lesions a f f e c t i n g phage assembly, one hs and one cs, were present i n the organism, growth at either r e s t r i c t i v e temperature would r e s u l t i n defective p a r t i c l e s . If these two lesions were i n the same developmental pathway, growth of the organism f i r s t at the low temperature, then at the high temperature would produce a d i f f e r e n t r e s u l t from that of the r e c i p r o c a l s h i f t . In one case, wild-type phage would be produced, i n the other case only defective p a r t i c l e s would be present, depending on which gene product was needed f i r s t (Figure 4b,c). On the - 14 -o t h e r hand, i f the 2 gene products were r e q u i r e d s i m u l t a n e o u s l y , or a c t e d independently, the r e c i p r o c a l s h i f t s would produce e i t h e r d e f e c t i v e or w i l d - t y p e phage i n both s h i f t s (Figure 4a). In t h i s way, i t i s p o s s i b l e t o o r d e r the time of a c t i o n of v a r i o u s gene products i n the same developmental pathway, and a s i m i l a r approach was proposed to d e a l w i t h a number of hs-cs double mutations i n the f e r t i l i t y f a c t o r s . The s y n t h e s i s of a double mutant i n d i v i d u a l to perform these experiments i n D. melanogaster posed a problem s i n c e c r o s s i n g over does not occur i n males, and the Y chromosome i t s e l f recombines very i n f r e q u e n t l y (Brosseau, 1958). For t h i s reason, an attached-XY'(XY) chromosome ( L i n d s l e y and N o v i t s k i , 1959) was used (Figure 5) . The two arms of the Y chromosome are separated by an e n t i r e X chromosome w i t h i n which c r o s s i n g over can take p l a c e i n females. By i n d u c i n g the t s mutations on 3 d i f f e r e n t l y marked XY chromosomes, c r o s s i n g over can combine two d i f f e r e n t Y - l i n k e d t s mutations (Figure 6) . Thus, the i s o l a t i o n and c a t e g o r i z a t i o n of a l a r g e number of hs and cs male s t e r i l e mutations on an XY chromosome was undertaken both as a v e r i f i c a t i o n o f e a r l i e r work, and to p r o v i d e new i n f o r m a t i o n : 1) to compare f r e q u e n c i e s and l o c a t i o n s of the hs and cs mutations on the Y chromosome ( s i n c e an XY chromosome was being used, t h i s a l s o allowed the i s o l a t i o n o f mutations causing male s t e r i l i t y , and comparison of t h e i r frequency to t h a t of the Y - l i n k e d mutations) 2) t o determine the t s p ' s of the Y - l i n k e d hs and cs mutations (and t h e r e f o r e determine when the gene products f u n c t i o n - 15a -Figure 4. Results of reciprocal temperature shifts of hs and cs double mutations with similar mutant phenotypes. A. both mutations are affecting genes which act in independent pathways. Recip-rocal shifts yield either mutant phenotype or wild-type pheno-type in both cases. B. cs mutation acts before hs mutation in same pathway. Growing the double mutant f i r s t at the high temperature, then at the low temperature produces a wild-type phenotype. The reverse shift produces a mutant phenotype. G. hs mutations acts before cs mutation in same pathway. Reverse result from that in B. is obtained. cs o — H cs - 16a -Figure 5. Diagram of the attached-XY chromosomes used i n this s t u d y . y, yellow body c o l o r ; f , f o r k e d b r i s t l e s ; v, V e r m i l l i o n eye c o l o r ; B, bar eye; In(l)EN, i n v e r s i o n spanning the e n t i r e X chromosome. Hatched bars represent heterochromatin; s o l i d l i n e represents euchromatin; c i r c l e represents centromere. - 16b -j 1 ir- S UJ X Uj X QQ 0 3 CO CO CO ^ 1 - 17a -Figure 6. Procedure for the synthesis of a cs-hs double mutant Y chromo-some in an attached-XY chromosome. cs, cold-sensitive muta-tion; hs, heat-sensitive mutation; FM6, multiply inverted X chromosome used as a balancer. Solid bar represents hetero-chromatin; line represents euchromatin; circle represents centromere. - 17b -- 18 -during spermatogenesis) 3) to determine i f the f e r t i l i t y factors are act ing i n the same developmental pathway, and i f so, to order the time of ac t ion of t h e i r gene products through the use of r e c i p r o c a l sh i f t s with the hs-cs double mutants. - 19 -MATERIALS AND METHODS 1. Mutant Stocks and Media Three XY chromosomes marked d i f f e r e n t l y were used for i s o l a t i o n of t s s t e r i l i t y mutations: Y 5 X ' Y L , I N ( 1 ) E N , y v f B ' y + (referred to as XY,y y f B 'y + ) . Y 5 X • Y L , In (1) E N , y + y (referred to as X Y , y + y ); and Y S X ' Y L ' T n ( T ) E N , y B (referred to as XY,y_B) . The phenotype and genetic p o s i t i o n of the markers i s seen i n Figure 5. These males were kept i n stock with XX,C(T)RM,y v bb females (referred to as XX,y v / 0 ) . Deta i led descr ip t ions of the mutations and chromosomes used i n these experiments can be found i n L inds ley and G r e l l (1968). I t should be noted that the X Y , y + y chromosome i s l i s t e d i n L inds ley and G r e l l (1968) as y * y + . However, the recombination data c o l l e c t e d i n using t h i s chromosome (part 4) , revealed that the d u p l i c a t i o n i n our q stock i s a c t u a l l y on Y as shown i n Figure 5. In a d d i t i o n , these f l i e s no longer exh ib i t the hairy wing phenotype c h a r a c t e r i s t i c df t h i s d u p l i c a t i o n . For the complementation tests (part 3), females of stocks XX,C( l )DX,y f b b " / y + Y * (referred to as XX,y, f / y + Y * ) , were used, where the y^Y represents the te s t chromosomes y_ + (sc 8 )Y, y + Y L , v + Y k s l - , . ^ 5 2 " , y > k 1 1 " , y V 1 2 " , y + Y k 1 3 " 4 " + k l5 — and y Y . Since the females are bb, they cannot survive without the Y chromosome (which i s b b + ) . In a d d i t i o n , each Y c a r r i e d a y_^  dup l i ca t ion which permits detect ion of i t s S presence by i t s ha iry wing phenotype. The Y tes t fragment was kept i n XX,C(I)RM,y/Y S females (referred to as X X , y / Y S ) . Because large number of v i r g i n females were necessary for - 20 -the many p a i r matings and complementation te s t s , the XX,y v / O , XX,y f / y + Y * and X X f y / Y S females were kept i n stock with X Y , y / 0 ; X Y , y / y + Y * : X Y , v / Y S (Parker and McCrone, 1958) males r e s p e c t i v e l y , in to which a s h i t s ^ mutation ( G r i g l i a t t i et a l . , 1973) had been inserted by cross ing over. Thus, when these stocks are shi f ted to 2 8 ° a l l male f l i e s , at any stage of development, are immediately paralyzed and die within 2 days, thereby ensuring ec los ion of only females. F l i e s were ra i sed e i ther i n s h e l l v i a l s or h p i n t mi lk bot t l e s on standard (cornmeal, sucrose, agar, yeast) Drosophila medium. 2. I so l a t ion of Mutants The screening procedure used for i s o l a t i n g Y - l i n k e d ts male s t e r i l e mutations i s seen i n Figure 7. The r e s t r i c t i v e temperature chosen for hs mutations was 2 8 ° and for cs mutation was 1 7 ° ; the permissive temperature for both was 2 2 ° . Day o ld X Y , y + y and XY,y B males were fed a 0.0125 M so lut ion of e thy l methanesulfonate (EMS) d i s so lved i n 1% sucrose so lu t ion (Lewis and Bacher, 1968) for 24 hours at 2 2 ° . The treated males were allowed to recover for one day i n medium-containing bot t le s and then mated to XX,y v/O v i r g i n females (25 males: 25 females per b o t t l e ) . Every three days these parents were transferred to fresh bot t le s for 3 successive broods af ter which they were discarded. Indiv idua l F^ males, each of which c a r r i e d a d i f f e r e n t EMS-treated XY chromosome, were mated to 5 XX,y v/O v i r g i n females i n v i a l s for 5 days, then discarded. Progeny from f e r t i l e - 21a -F i g u r e 7. S c r e e n i n g p r o c e d u r e f o r the i s o l a t i o n of hs and cs male s t e r i l e m u t a t i o n s on an XY chromosome. - 21b -XY/O c/d" x XX/O ?? (EMS-treated)[ 22° XY*/0 cf x XX/O ?? 22° XY*/0 (?& x XX/O $? (25 pairs per bottle) (ld":5?? per vial) ( f e r t i l e matings transferred en masse) F 2 progeny 22° score for presence of progeny F 2 progeny .17° F 2 progeny score for absence of progeny 28° ^ denotes mutagenized chromosome - 22 -matings were transferred en masse to fresh v i a l s , l e f t at 22° for 3 days, subcultured to fresh v i a l s for 7 days at 17°, the parents discarded, and the progeny l e f t to develop at the temperatures s p e c i f i e d . Upon eclosion, the progeny were transferred, en masse, to new v i a l s and checked for f e r t i l i t y at the temperature at which they had been grown. F l i e s i n the r e p l i c a t e v i a l s which were s t e r i l e at 17° but f e r t i l e at 22° were kept as putative cs male s t e r i l e mutations. Using males of genotype XY,y v f B»y and the method outlined above (Figure 7) M. Kiess (1974) is o l a t e d a number of hs and cs mutations. She also i s o l a t e d several cs mutations on the XY,y y chromosome. The conditional male s t e r i l e mutations i s o l a t e d By M. Kiess and myself were retested several times at t h e i r non-permissive temperatures to confirm the ts s t e r i l i t y . This o ^ involved mating f i v e males from each stock at 22 to 10 XX,y v/O v i r g i n females for 3 days at 28° or.: 7 days at 17° a f t e r which time they were discarded. The F^ f l i e s were transferred en masse to fresh v i a l s at the non-permissive temperature and the v i a l s scored for the presence or absence of progeny. In the case of the cs mutations (since the f l i e s take 26 days to develop at 17°) (Figure 8), the F^ parents were discarded af t e r 7 days and v i a l s brought up to 22° to speed early development. Of the hs mutations, only those which consistently showed complete s t e r i l i t y at the r e s t r i c t i v e temperature were retained for further study. However, owing to the small number of cs mutations recovered, these were retained i f they - 23a -Figure 8. Length of developmental stages at 17°, 22°, and 28°, in days. l ' s t , 2*nd, and 3'rd are l a r v a l instars; pp, pre-pupa; Age of Culture in Days Temperature 1 2 3 4 5 6 7 8 9 10 II 12" 13 14 15 16 17 18 19 2 0 21 22 23 2 4 25 26 Egg )'st 2'hd 3 ' r d pp Pupa /'st p Egg 2'nd 3'rd p Pupa 3 Egg 1st 2'nd 3 ' rd Pupa produced 15 or fewer progeny per v i a l . 3. Complementation Males carrying confirmed ts mutations were then tested for the location of the s t e r i l i t y mutation. Males of each stock /\ 8 were mated to XX,y f/sc Y females, (5 males: 10 females per v i a l ) , l e f t for 3 days at 22°, then transferred to fresh v i a l s at the r e s t r i c t i v e temperature. The hs bearing mutants were mated f o r 3 days at 28° while the cs bearing mutants were mated at 17° for 7 days, and the parents discarded. A l l F^ males i n thi s cross received a free Y element from t h e i r female parent which allowed a complementation test for male f e r t i l i t y . The F-^  f l i e s were transferred to fresh food, mated to 10 v i r g i n females per v i a l , and the v i a l s scored for the presence of progeny (Figure 9). F e r t i l i t y of males carrying a sc Y chromosome at the r e s t r i c t i v e temperature indicated the Y location of the ts s t e r i l i t y mutation on the XY chromosome. Each mutation . showing recovery of f e r t i l i t y with sc Y was tested i n a si m i l a r manner with Y' ,y_ Y , and a series of Y chromosomes thought to be d e f i c i e n t for one or more of the 7 f e r t i l i t y f actors: : £ ? Y k s 1 - , y^Y* 5 2", y^Y* 1 1-,; y ^ " yV13"4-+ kl5~ r y^ -Y (Figure 10), to allow l o c a l i z a t i o n of the mutation to a p a r t i c u l a r f e r t i l i t y factor. For example, i f f e r t i l i t y was 8 + L recovered i n the presence of a sc Y, y_.Y and a l l deficiency chromosomes except .^Y^H , the l e s i o n was considered to be i n k l l . At least 3 r e p l i c a t e v i a l s were tested for each cross with every mutant stock and instead of the F^ being transferred en masse to fresh v i a l s as i n part 2, 5 males showing the hairy - 25a -Figure 9. Protocol for complementation tests of ts s t e r i l e mutations on an XY chromosome with a normal (sc ) Y chromosome. Figure 10. Protocol for complementation tests of ts s t e r i l e mutations on an XY chromosome with a Y fragment or Y deficiency chro-mosome. XY t S/0 cf x XX/sc Y $ 22° 28° or 17° XY t S / s c 8 Y cf * XX/O ?? It XY t S/sc 8Y cf ".V22° 28° or 17° score for presence of progeny ,ts XY /0 cf x XX/Y* ? 22c 28° or 17° XYtS/Y* cT x' XX/O ?? x XYtS/Y* cf 22° 28° or 17° score for presence score for presence or absence of progeny of progeny S + L deno.tes a Y fragment (Y or y_ Y ), or Y deficiency chromosome ,'/+$si" ,+vks2" +lsl l" +J<12" -t- kl3 * V + vkl5~ (y_ Y , y_ Y , y_ Y , y_ Y y_ Y , y_ Y ) - 26 -wing phenotype were chosen from each cross and mated to 10 v i r g i n females i n each v i a l , to standardize the f e r t i l i t y t e s t . The v i a l s were then scored as s t e r i l e (0 progeny), semi-sterile (less than 15 progeny per v i a l ) , or f e r t i l e (more than 15 progeny per v i a l ) . On the basis of these r e s u l t s , 10 hs and 4 cs mutant stocks were selected for further study. 4. Single Arm Analysis To corroborate the complementation data which l o c a l i z e d the male s t e r i l e mutations to the Y chromosome, the two Y chromosome arms from each mutation, were i n d i v i d u a l l y combined with a wild type complement through recombination on the X chromosome. For the hs mutations, non-ts bearing XX,y_y_ males were mated to FM6/M(l)o f females and FM6/XY,y y female progeny c o l l e c t e d . These were then mated to the XY,y v f B"y mutant males, XY,y y/ XY,y v f B*y females were c o l l e c t e d , crossed to XY,y y males, and progeny examined. Recombinants of several types could be distinguished: singles i n the y-v region, v-f region, and d i s t a l to B (see Figures 5,6). Markers on the recombinant chromosome showed which chromosome S L carr i e d Y or Y from the mutant stock. In addition, double crossovers spanning the v region could be recovered. Ten male recombinants i n each of the three regions (5 i n each re c i p r o c a l c l a s s ) , and a l l doubles were tested at 28° and 22° for f e r t i l i t y , as explained i n part 2, along with 5 males from each of the two noncrossover parental classes as controls. As mentioned before, the y duplication on the XY,y y - 27 -chromosome had no hairy wing phenotype. Therefore, the XY,y y*y recombinant chromosome could be distinguished from the s£Y,y+y parental chromosome on t h i s basis. The same procedure was used to select recombinants for the cs s t e r i l e s . However, some of the recombinant classes were d i f f e r e n t because the mutants were induced on the XY,y +y and XN XY,y B chromosomes. ^* Double Mutant Analysis Double mutants containing both a hs and cs l e s i o n on the same XY chromosome were constructed i n a manner i d e n t i c a l to the one i n the previous section, (see Figure 6). Five of each r e c i p r o c a l recombinant class were co l l e c t e d and tested for s t e r i l i t y at 28°, 22°, and 17°. Those which were s t e r i l e at 28° and 17° but f e r t i l e at 22° were kept for further study. Since two of the cs mutations (2661 and 3480) were induced on a chromosome with the same markers as the hs mutations, stocks developed from recombinants i s o l a t e d i n the single arm analysis x\ J . + were used to allow detection of recombination. The XY,y y'y recombinants from hs mutants 56, 1312, 1608, and 3269 were used to synthesize a double mutant with 3480 and 2661. An XY,y f B recombinant of 3480 was used to construct a double mutant with 631 and 2225. 6. M o t i l i t y A simple test for the presence of motile sperm i n the testes of males at permissive and r e s t r i c t i v e temperatures was performed on each ts stock. Testes and accessory organs from newly eclosed males were excised i n Ephrussi-Beadle (1936) - 28 -Ringer ' s s o l u t i o n , placed on a glass s l i d e i n a drop of the s o l u t i o n , and covered gently with a c o v e r s l i p . The testes were f i r s t examined under a phase contract microscope for the presence of any obvious abnormalit ies i n the appearance and loca t ion of the gonia l c e l l s or te s tes . Then the c o v e r s l i p was tapped l i g h t l y with forceps to release the c e l l s i n s i d e , and the mature sperm were then checked for m o t i l i t y . Care was taken to include the seminal v e s i c l e with the testes for t h i s i s where the moti le sperm are found. Testes from males grown at both 2 2 ° and the r e s t r i c t i v e temperature, with and q without a sc Y were compared. F ive males were used i n each sample. 7. Determination of Temperature Sens i t ive Period The temperature sens i t ive period (tsp) i s defined as the developmental i n t e r v a l during which exposure to the r e s t r i c t i v e temperature would produce the mutant phenotype (Tarasoff and Suzuki , 1968; Suzuki , 1970). Determination of the tsp of the male s t e r i l e mutations can be done i n 2 ways: a) by exposing various l a r v a l stages to the r e s t r i c t i v e temperature to see when such a "pulse" would produce s t e r i l i t y i n the newly eclosed male, b) by subject ing adult males from 2 2 ° cu l tures to several days of the r e s t r i c t i v e temperature and noting the day on which a drop i n f e r t i l i t y occurs (Ayles e t a l . , 1973). Since sperm mature at a s p e c i f i c r a te , i t i s poss ib le to extrapolate i n e i ther case from the time at which s t e r i l i t y occurs to the stage of gonia l c e l l af fected (Table 1) . For - 29 -example, i f a pulse to the adult causes s t e r i l i t y 2-3 days l a t e r , post-meiotic sperm development i s affected. In such a case, a pulse during days 8-9 of the developmental cycle (at 25°) should produce s t e r i l e males on eclosion. Thus, the two tests provide i n t e r n a l checks for the v a l i d i t y of each experiment. Examination of s t e r i l i t y by both methods required that males be brooded. This involved mating 10 newly eclosed males i n d i v i d u a l l y from each experimental pulse, to 5 females i n v i a l s and tran s f e r r i n g the males to a new harem of 5 females every 24 hours. The males were transferred without etherization (by aspiration) to reduce any detrimental e f f e c t s . This procedure was car r i e d out for 10 consecutive days a f t e r which the males were discarded. Since sperm production was continuous, t h i s method was employed to exhaust d a i l y the sperm produced. The v i r g i n females used were at lea s t 2 days old to ensure t h e i r receptiveness to mating. Since females were capable of storing sperm and laying f e r t i l i z e d eggs for over 2 weeks a f t e r mating (Lefevre and Jonsson, 1962), they were l e f t i n the v i a l s a f t e r removal of the male. Twelve to fourteen days a f t e r mating, the number of pupae i n each v i a l was recorded, a) heat sensitive s t e r i l e mutations Ten newly eclosed males from each of the hs stocks selected (56,502, 631, 887, 898, 1216, 1312, 1608, 2225, 3269) were brooded for 3 days at 28°, then 7 days at 22°. O r i g i n a l l y , brooding was done every 12 hours. However, i t was found that the a t t r i t i o n rate for males was very high from excessive - 30 -handling and did not, i n any event, provide a much better resolution of the time of onset of s t e r i l i t y than did 24 hour broods. Therefore, a l l broods are given i n 24 hour in t e r v a l s i n t h i s report. To further confirm the tsp, f l i e s were allowed to lay eggs on p e t r i plates containing Drosophila medium and plates were changed every 2 hours. The medium was scratched with a probe and salted with yeast to enhance egg layin g . One of the plates was grown at 28° u n t i l pupation, then shifted down to 22° u n t i l eclosion. A second plate was grown at 22° u n t i l pupation, then shifted up to 28° u n t i l eclosion. Males were brooded at 22° as described above. Since the tsp's were found to be pupal i n a l l cases, further studies were undertaken to delineate the i n t e r v a l more c l o s e l y . The pupal stage at 22° was approximately twice as long as at 28° (Figure 8). Therefore, a 48 hour i n t e r v a l during the pupal stage at 22° was equated to a 24 hour i n t e r v a l at 28°. Obviously, t h i s was a generous assumption as the rate of development of d i f f e r e n t discs and c e l l types may be d i f f e r e n t i a l l y affected at the temperatures studied. The 6 day pupal stage (at 22°) was subdivided into 3 parts (called p i , p l l , and p i l l ) . Three plates were c o l l e c t e d for each stock. The f i r s t was grown for p i at 28° (the f i r s t 24 hours of pupation), the second plate for p l l , and the t h i r d for p.III. The plates were kept at 22° for the res t of the l i f e cycle and the males brooded at 22°. If the 24 hour heat pulse did not confer t o t a l s t e r i l i t y , longer heat pulses were t r i e d at various times during the pupal stage to see - 31 -which would produce t o t a l s t e r i l i t y i n the newly eclosed male. Conversely, i f a 24 hour pulse did confer t o t a l s t e r i l i t y , shorter periods of time at 28° were t r i e d to determine the minimum length of heat pulse required to produce s t e r i l i t y . Several types of controls were performed: males which had been grown at 22° were brooded at 22° to allow comparison of normal f e r t i l i t y with mutant patterns; and males were grown at 28° for t h e i r entire developmental time except for the days of t h e i r d iscrete tsp, then brooded at 22°. b) cold s e n s i t i v e s t e r i l e mutations The r e s u l t s of brooding newly eclosed males at 17° for 3 days, then at 22° for 7 days were not as clear cut as they were for the hs mutations. Therefore, cultures from eggs co l l e c t e d i n a 2 hour period were grown at 17° during the following stages of the l i f e cycle: egg and f i r s t i n s t a r ; egg-second i n s t a r ; egg-third i n s t a r ; second and t h i r d i n s t a r s ; t h i r d instar; t h i r d i n s t a r and pupa; and pupa. The l a r v a l stages were determined by the morphology of t h e i r mouth parts (Bodenstein, 1950) (Figure 8). Newly eclosed males were brooded at 22° as previously described. Also, males grown at 22° were brooded at 22°. - 32 _ RESULTS 1. I s o l a t i o n of Mutants A temperature s e n s i t i v e male s t e r i l e mutation on the XY chromosome which r e c o v e r s f e r t i l i t y i n the presence of a f r e e Y chromosome was c l a s s i f i e d as a Y - l i n k e d mutation. By t h i s d e f i n i t i o n , a t o t a l o f 26 hs and 5 cs Y - l i n k e d mutations was recovered (Table 2). These a r e , of course r e c e s s i v e mutations and those c l a s s i f i e d as X - l i n k e d t s male s t e r i l e s c o u l d a c t u a l l y be dominant Y - l i n k e d mutations. Of the t s mutations induced on the XY,y v f B'y chromosome, the frequency of Y - l i n k e d hs mutations (0.46%) i s more than ten times h i g h e r than t h a t of the Y - l i n k e d cs mutations (0.036%). The frequency of the cs mutations on the o t h e r chromosomes used cannot be c a l c u l a t e d s i n c e the t o t a l number of p a i r matings was not recorded. A y l e s e t a l . (1973) recovered .0.4%, hs mutations u s i n g a f r e e , w i l d type Y chromosome. The s i m i l a r mutation r a t e suggests t h a t s e p a r a t i o n o f the Y arms and t h e i r attachment to the X chromosome does not a f f e c t the m u t a b i l i t y o f the Y l o c i . An i n t e r e s t i n g phenomenon which can be seen from Table 2 i s t h a t of the spontaneous r e c o v e r y o f f e r t i l i t y o f both hs and cs Y - l i n k e d mutations w i t h time. For example, between the second and t h i r d r e t e s t s , a p e r i o d o f about 6 months, h a l f of the Y - l i n k e d t s mutations recovered f e r t i l i t y . T h i s i s not the case however, w i t h the X - l i n k e d t s mutations. Of 48 mutations i n i t i a l l y i s o l a t e d , 39 have remained s t e r i l e upon subsequent r e t e s t . Loss o f the mutant phenotype i n t s Stock Table 2 Is o l a t i o n and retests of ts male s t e r i l e mutations on XY chromosomes RETEST NUMBER Number of f e r t i l e chromosomes tested I II III hs cs hs cs hs cs IV hs cs y v f B«y 1,620 132 16 X Y 39 63 2 14 X 32 32 X Y 32 26 + • y y 2,808 25 X Y X Y 4 2 X Y 4 2 y B 317 Y X Y X Y Total 4,745 132 49 X Y 39 9 63 23 X 32 32 10 X Y 32 26 34 -mutations has been noted before i n Habrobracon (Smith, 1968) and Drosophila (Kaufman and Suzuki, 1974). Recovery of f e r t i l i t y for both X and Y-linked ts male s t e r i l e mutants has also been reported i n D. Lindsley's lab (R. Denell, personal communication), and could be a c h a r a c t e r i s t i c of some ts mutations. However, the Y-linked mutations which were s t e r i l e at ret e s t 4 have consistently retained t h i s phenotype for over a year. The 8 hs Y-linked mutations f i r s t i s o l a t e d i n t h i s lab (Ay les' e t a l . , 1973) over 6 years ago were also a l l s t e r i l e when tested at 28° i n June, 1975. The frequency of the hs X41inked mutations (0.57%) i s again much larger than that of the cs mutations (0.036%), but both are comparable to those of the stable Y-linked ts mutations (Table 2). This i s surprising i n view of the fa c t that the Y chromosome i s estimated to possess only 7 f e r t i l i t y locio (Brosseau, 1960). Perhaps the X chromosome has few genes involved i n spermiogenesis, the Y chromosome i s p a r t i c u l a r l y mutable, or the Y-linked l o c i are very large. A l l 26 hs Y-linked mutations are completely s t e r i l e when grown at 28°, whereas only one of the cs mutations, 3480, i s completely s t e r i l e at 17° (although i t was semi-sterile i n retest 2). A208, A527, and 2661 produce less than 5 progeny per v i a l . A527 and A208 also have a s l i g h t l y longer develop-mental period at 17° than the other cs mutations. No mutation was found which was both cs and hs. 2. Complementation A l l of the cs Y-linked mutations recover f e r t i l i t y with Y S - 35 -while the hs mutations dp so only with (Table 3). Even the cs mutations which are leaky when tested at 17° are completely s t e r i l e with Y L but have wild type f e r t i l i t y with S Y . Thus, a previous report (Ayles e t a l . , 1973) of the Y L position of a l l 8 hs mutations i s reconfirmed i n our study. L S The confinement of hs mutations to Y and cs mutations to Y cannot be a fortuitous sampling error and must r e f l e c t a fundamental difference between the function of the l o c i i n each arm. By using Y chromosomes d e f i c i e n t for various f e r t i l i t y l o c i , i t was hoped to l o c a l i z e the p a r t i c u l a r factor on Y" L or Y i n which the mutation had occurred. However, these data proved impossible to i n t e r p r e t . I n i t i a l complementation re s u l t s with a l l 26 mutants were odd i n that some of the mutations appeared to be deletions of more than one complemen-ta t i o n group and yet were t s . A t r i v i a l explanation for the above r e s u l t s was that the Y chromosome deficiency stocks used as standards were not what they were purported to be. Indeed, when tested among themselves for complementation, these deficiency chromosomes did not produce the expected r e s u l t s (T. C. Kaufman, personal communication). Stocks which were supposed to be complementary yielded s t e r i l e males i n X/Y/Y individuals and some were i n the wrong arm (e.g. a stock ksl"" L l a b e l l e d Y acted l i k e a l e s i o n i n Y •.;).,. Thus, i n i t i a l reports of the Y-linked mutations being i n several l o c i and i n both arms, which led to speculation of EMS-induced d e f i c i e n c i e s , or mutations i n genes c o n t r o l l i n g several l o c i (Williamson, 1970; Suchowersky et a l . , 1974) are c e r t a i n l y open for reappraisal. Table 3 8 S L Complementation patterns of Y-linked ts mutations with sc Y, Y and Y COLD-SENSITIVE STERILES HEAT-SENSITIVE STERILES F e r t i l i t y Pattern Stock Mutant s;c8Y YP YL y y J A208 F F S ,* y y A527 F F S y> B 7 150 F F s + y v f B«y 7 2661 F F s + y'v f B • y 7 3480 F F s F = f e r t i l e S = s t e r i l e SS= s e m i - s t e r i l e y= chosen f or further analysis Stock y v f B.y F e r t i l i t y Pattern Mutant sc 8Y 7 56 F S F 453 ix"-.-' 502 F S F y F S F y 631 F S F 696 F S F 794 F . S F 873 F SS F y 887 F S F y • 898 F S F y 1216 F S F 1265 F S F y 1312 F S F 1531 F S F y 1608 F S F 1731 F S F 17:55 F S F 1761 F SS F 1800 F SS SS 1805 F S F 2115 F S F 2212 F S F y 2225 F S F 2522 F •  S F 2986 F s F y 3269 F S F 3491 F S SB cn -37- -Another problem encountered i n these experiments was that although i n most cases the r e s u l t s were reproducible, some mutants showed large variations among r e p l i c a t e v i a l s . k l 2 " For example, when 1312 was tested for f e r t i l i t y with Y , one v i a l was s t e r i l e , another had 3 progeny, the t h i r d 10 progeny, and the fourth 40 progeny. The cs mutations were es p e c i a l l y prone to show these v a r i a t i o n s . This was at le a s t p a r t i a l l y due to the leakiness i n the cs mutants themselves and perhaps more penetrant s t e r i l e stocks could be reselected from i n d i v i d u a l males. Another factor could be differences i n the v i a b i l i t y of the stocks themselves. Therefore, 10 hs and 4 cs mutations showing d i f f e r e n t patterns of complementation were chosen and a l l complementation tests repeated i n quadruplicate under standardized conditions. A complementation map of the Y deficiency / chromosomes used i s not available at t h i s time, but regardless of the f i n a l map, the Y-linked mutants s t i l l f a l l into several d i s t i n c t categories (Table 4). Among the hs mutations, there are 4 k l l ~ k l 2 ~ patterns: s t e r i l e with Y and Y (56, 631, 1216): kl "?"4" kl5-with Y (502, 1312, 3269); with Y (887, 1608); and with a l l 6 Y deficiency chromosomes (898, 2225) . Among the ks2~ cs mutations, two patterns emerge: s t e r i l i t y with Y (A527, 3480, 150), or with both Y k s l and Y k s 2 (A208, 2661). Thus, most of the ts Y-linked lesions seem to have occurred in d i f f e r e n t l o c i along the Y chromosome and when the comple-mentation map of the Y deficiency chromosomes i s completed, i t should be possible to subdivide the above classes further. Only 2 mutant stocks (898, 2225) behave strangely i n that they - 38 -Table 4 Complementation patterns of Y-linked ts mutations with Y deficiency chromosomes Y Deficiency Chromosome Mutant ksl" ks2~ k l l " kl2~ kl34" kl5" hs 56 F F s S F F 631 F F s S F F 1216 F F s S F F 502 F F F F S F 1312 SS F SS :ss S F 3269 F F F F s F 887 F F F F F S 1608 SS F SS SS SS S 898 S S S S S SS 2225 S S s S S s cs AA527 SS s SS SS F F 5480 ss s F F F F 150 F s F F F F A208 s s F F F F 2661 S s F F F F S = sterile SS= semi-sterile F = f e r t i l e - 39 -recover f e r t i l i t y with Y L, (Table 3), yet are s t e r i l e with k s l - ks2~" L Y and Y which carry a normal Y (Table 4). These 2 stocks could carry multiple s i t e mutations, or a mutation i n a regulatory region. 3. Single Arm Analysis Recombination analysis confirmed the complementation data (Tables 3 and 4) for eight of the hs mutations (56, 502, 631, 887, 1216, 1312, 1608, 3269)which placed t h e i r lesions i n Y L (Table 5). The recombination analysis involving 2225 suggested that i t consists of 2 mutations as suggested above (part 2), but that these lesions are on the X chromosome, one i n the y-v region, and the second d i s t a l to B (on the inversion chromosome). Both mutations have to be present for t o t a l s t e r i l i t y : e i t h e r one alone allows p a r t i a l f e r t i l i t y . I t i s S possible that the mutation d i s t a l to B i s ac t u a l l y on Y since k s l ~ ks2~ S " 2225 i s s t e r i l e with Y and Y . However, a Y fragment does not restore even p a r t i a l f e r t i l i t y to 2225 and i n the presence of Y L, i t i s completely f e r t i l e . The r e s u l t s for 898 also suggest some in t e r a c t i o n L between 2 mutant l o c i , one of which i s on Y , but the data are less extensive and no conclusions can be reached. Examining the single arm analysis data for the cs mutations A208, 2661, and 3480, i t can be seen that i n a l l S cases, the recombinant males carrying Y from the mutant chromosome are not s t e r i l e as expected from the complemen-tatio n r e s u l t s (due to i t s leakiness, the data on 150 were inconclusive). Recombination data show that a l l 3 cs Table 5 Recombinational analysis of Y-linked ts mutations RECOMBINATIONAL CLASS PARENTAL CLASS Mutant YL s Mutant Y Mutant + y y y v f B«y + + y y + '+ , + •y y y v v y y v f B y f B y hs 56 F S s s F F F 502 F S s S ?F F F 631 F S S s F F F 887 , F S S ss F F F 898 F S ss ss F F F 1216 F S s s F F F 1312 F S s s F F F 1608 F S s s F F F 2225 F S ss ss s ss ss 3269 F S s s F F F c's 2661 F S S-F ss S-F F F 3480 F S F F 'Mutant YS s s Mutant YL F A208 S F F F S-F s FV S = s t e r i l e , SS = s e m i - s t e r i l e , F = f e r t i l e - 41 -mutations tested were act u a l l y on the X chromosome: 2661 and A208 i n the y^ -v region, and 3'480 d i s t a l to v (on the inversion S chromosome). These mutations must be inter a c t i n g with Y . Both 2661 and A280, which are i n the same region, show the same complementation pattern, while 3480 has both a genetic po s i t i o n and complementation pattern d i f f e r e n t from the other two mutations. An;interesting point i s that a l l 3 X-linked mutations i n the y-v region (2661, A208 and 2225) are leaky. 4. Double Mutant Synthesis Recombination data c o l l e c t e d while synthesizing the double mutants served to corroborate the evidence presented i n the previous section which placed 2661 and one l e s i o n of 2225 i n the y-v region, and '3480 d i s t a l to v. Several hs-cs double mutant stocks are now available for s h i f t studies. A p a r t i c u l a r l y i n t e r e s t i n g double mutant i s 3480/3269 which i s f e r t i l e at 22° but immediately s t e r i l e at 17° or 28°, and produces no o f f s p r i n g whatsoever at the two r e s t r i c t i v e temperatures. 5. M o t i l i t y Using the phase contract microscope, sperm m o t i l i t y i s re a d i l y seen. When the testes of a normal male are broken open on a s l i d e i n Ringer's solution and examined under the microscope, the f i e l d of view i s soon covered by c i r c u l a r , pulsating strands of mature sperm. Many spermatocytes and spermatid bundles are also seen. In the testes of an X/O male, motile sperm are never observed, and the number of - 42 -spermatid bundles i s reduced. Most of the ts s t e r i l e mutations appear to be between these two extremes. Of the 10 hs s t e r i l e mutations chosen for detailed o study, 8 showed no motile sperm at 28 . Two, 631 and 887 showed a s l i g h t m o t i l i t y i n a few strands. Masses of degenerating material were also seen i n the 10 mutations. Ci o 8 A l l showed wild type m o t i l i t y at 22 , and at 28 with a sc Y. The other 16 hs s t e r i l e mutations also produced non-motile sperm at the r e s t r i c t i v e temperature. The cs mutations showed a si m i l a r pattern: A208, 2661 and 3480 produced non-motile sperm when grown at 17°, but motile sperm at 22°. 150 showed some m o t i l i t y at 17° which correlated well with i t s leakiness i n the r e t e s t s . With a 8 sc Y, 150 and 3480 gave wild type m o t i l i t y , while A208 and 2661 had p a r t i a l m o t i l i t y . No differences could be seen between the X and Y-linked mutations. A l l mutant stocks had normal testes, with spermatocytes and spermatid bundles, and normal g e n i t a l i a . 6. Temperature Sensitive Periods a) heat sensitive s t e r i l e mutations A 3-day heat pulse applied to newly eclosed adult males i n 9 of the mutant stocks (56, 502, 631, 887, 898, 1216, 1312, 1608, 3269) resulted i n f e r t i l i t y u n t i l the fourth day, at which time the males become s t e r i l e (Figures 11, 12, 13d, 15d, 17d). Only one, 2225 remains f e r t i l e u n t i l day 7 (Figure 19d). If spermatid d i f f e r e n t i a t i o n l a s t s 5 days at 25° (Table 1), these f i r s t 9 mutations must be a f f e c t i n g post-meLotic development -43 -(since development i s s l i g h t l y slower at 22°)',. The l a t e onset of s t e r i l i t y i n 2225 suggests an e f f e c t i n pre-meiotic or early post-meiotic spermatogenesis. Since testes of adult f l i e s comprise a heterogeneous population of maturing sperm a heat pulse i n the adult cannot be considered the most r e l i a b l e method of determining the tsp. More accurate r e s u l t s are obtained by pulsing l a r v a l stages as the f i r s t cohort of sperm develop synchronously (Hannah-Alava, 1965). When the hs mutations are grown at 28° only during the pupal stage, males i n a l l stocks (including 2225) are s t e r i l e upon eclosion (Figures 13c, 15c, 17c, 1 9 c ) . On the other hand, growth of these stocks at 28° for the r e s t of the l i f e cycle has no e f f e c t on f e r t i l i t y (Figures 13ab, 15ab, 17ab, 19ab). This shows that the tsp of a l l 10 mutations i s post-meiotic, but the tsp of 2225 could be p r i o r to that of the other hs mutations. By applying heat pulses for selected periods of the pupal stage (see Methods and Materials), i t has been possible to show that i n a l l mutations except 2225, the tsp occurs during the middle i n t e r v a l of the pupal stage (Figures 21, and 14abc, 16abc, 18abc). This coincides with the period of intense spermatid d i f f e r e n t i a t i o n (Lindsley and L i f s c h y t z , 1972). A minimum of a 24 hour heat pulse during p l l of the pupal stage i s required to induce t o t a l s t e r i l i t y of 56, 502, 631, 898, and 1216 males at eclosion, whereas a 48 hour heat pulse i s required for s t e r i l i t y of 887, 1312 and 1608 males. To confirm that t h i s was indeed the discrete tsp, the f l i e s were grown at 28° for the entire developmental period, except p l l . Upon eclosion, the males of both 56 and 887 were f e r t i l e " 44 " (Figures 14d, 16d). Although a heat p u l s e i n days 2-5 of the pupal stage i s necessary to produce t o t a l s t e r i l i t y i n 887, a p u l s e which i n c l u d e s a p o r t i o n of these days produces a r e d u c t i o n i n f e r t i l i t y ( Figure 16abc). The d i f f e r e n c e i n l e n g t h o f heat p u l s e to the pupal stage which produces s t e r i l i t y can be c o r r e l e t a t e d w i t h r e c o v e r y o f f e r t i l i t y i n males s u b j e c t e d to a heat p u l s e as a d u l t s . For example, 1312 (Figure 12a) and 887 (Figure 15d) both r e q u i r e a 48 hour heat p u l s e and are s t e r i l e f o r 3 days f o l l o w i n g a 3-day a d u l t heat p u l s e ( a f t e r which they recover f e r t i l i t y ) . In c o n t r a s t , 1216 (Figure 11c) and 56_ (Figure 13d) , which r e q u i r e o n l y a 24 hour pupal heat p u l s e , are s t e r i l e f o r 5-6 days a f t e r the 3-day a d u l t heat p u l s e . Thus, the mutations can be d i v i d e d i n t o those which have a more severe e f f e c t (56, 502, 631, 898, 1216) and a l e s s severe e f f e c t (887, 1312, 1608) on f e r t i l i t y . Mutant.:\i s t o c k s 887 and 1608 which are i n k l 5 " the l e s s severe category, are both s t e r i l e w i t h Y k l 5 " Brosseau ( c i t e d by F r a n k e l , 1973) has r e p o r t e d t h a t the Y mutations tend to be l e a k y . Mutant 2225 i s i n a c l a s s by i t s e l f : i t s t s p i s i n p i (Figure 20abc), i t i s s t e r i l e w i t h every d e f i c i e n c y chromosome (Table 4), and a c t u a l l y maps t o the X chromosome (Table 5). Since the t s p ' s o f the Y1, mutations are i n the middle o f the pupal stage, they appear to be d i r e c t l y i n v o l v e d i n spermatid assembly. 2225, being X - l i n k e d and having an e a r l i e r t s p than the o t h e r mutations, c o u l d be a mutation i n a r e g u l a t o r y r e g i o n . T h i s w i l l be f u r t h e r e x p l o r e d i n the D i s c u s s i o n . - 45a -Figure 11. Daily mean f e r t i l i t y of 10 i n d i v i d u a l l y mated males, brooded for 10 days (5 females per brood, each brood = 24 h r s . ) . Dashed l i n e : males brooded at 22° for 10 days. S o l i d l i n e : males brooded at 28° for f i r s t 3 days and at 22° for 7 days. Bars indicate 95% confidence l i m i t s . a. 502, b. 898, c. 1216. - 45to-01 I ^ " / 2 3 4 5 6 7 8 9 / BROOD NUMBER - 46a -Figure 12. Daily mean f e r t i l i t y of -10 individually mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Dashed line: males brooded at 22° for, 10 days. Solid line: males brooded at 28° for f i r s t 3 days, then at 22° for 7 days. Bars indicate 95% confidence limits. a. 1312, b. 1608, c. 3269. - 46b -BROOD NUMBER - 47a -Figure 13. Daily mean f e r t i l i t y of _56 grown at 28° during various deve-lopmental stages. a. entire l i f e cycle at 22°, brooded at 22°; b. egg-3rd instar at 28°, pupa at 22°, brooded at 22°; c. egg-3rd instar at 22°, pupa at 2&§, brooded at 22°; d. la r v a l stages at 22°, brooded f i r s t 3 days at 28°, then 7 days at 22°. Each point represents mean of 10 ind i v i d u a l l y .mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence l i m i t s . - 48a -Figure 14. Daily mean f e r t i l i t y of J56 grown at 28° for various times during pupal stage and brooded at 22°. a. pjSI at 28? (days 1,2 of pupal stage); b. p l l at 28° (days 3,4,of pupal stage) c. r>III at 28° (days 5,6 of pupal stage); d. grown at 28° for entire larval cycle, except piI. Each point represents mean of 10 individually mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence limits. - 48b -BROOD NUMBER - 49a -Figure 15. Daily mean f e r t i l i t y of 887.grown at 28° during various deve-lopmental stages. a. entire l i f e cycle at 22°, brooded at 22°; b. egg-3rd instar at 28°, pupa at 22°, brooded at 22°; c. egg-3 instar at 22°, pupa at 28°, brooded at 22°; d. larval stages at 22°, brooded f i r s t 3 days at 28°, then 7 days at 22°. Each point represents mean of 10 individually mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence limits. - 4 9 b -BROOD NUMBER - 50a -Figure 16. Daily mean f e r t i l i t y of 887 grown at 28° for various times during the pupal stage and brooded at 22°. a. pi at 28° (days 1,2,3 of the pupal stage); b. p l l at 28° (days 2,3, 4,5 of the pupal stage); c. p i l l at 28° (days 4,5,6 of the pupal stage); d. grown at 28° for entire larval cycle ex-cept p l l . Each point represents mean of 10 individually mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence limits. - 50b -- 51a -Figure 17. Daily mean f e r t i l i t y of 631 grown at 28'£ during various deve-lopmental stages. a. entire l i f e cycle at 22°, brooded at 22°; b. egg-3rd instars at 28°, pupa at 22°r,\ brooded at 22°; c. egg-3rd instars at 22°, pupa at 28°, brooded at 22°; d. la r v a l stages at 22<S, brooded f i r s t 3 days at 28°, then 7 days at 22°. Each point represents mean of 10 indi v i d u a l l y mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence l i m i t s . - 52 a -Figure 18. Daily mean f e r t i l i t y of 631 grown at 28° for various timgss during the pupal stage and brooded at 22°. a. pi at 28° (days 1,2 of the pupal stage); b. pi I at 28° (days 3,4 of the pupal stage); c. p i l l at 28° (days 5,6,of the pupal stage). Each point represents mean of 10 individually mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence limits. 2 3 4 5 / 2 3 4 5 / 2 3 4 5 B.ROOD NUMBER - 53a -Figure 19. Daily f e r t i l i t y of 2225 grown at 28° for various stages du-i t s development, a. entire l i f e cycle at 22°, brooded at 22°; b. egg-3rd instars at 28°, pupa at 22°, brooded at 22°; c. egg-3rd instars at 22°, pupa at 28°, brooded at 22°; d. l a r v a l stages at 222, brooded f i r s t 3 days at 28°, then 7 days at 22°. Each point represents mean of 10 i n d i -v i d u a l l y mated males brooded for 10 days (5 females per brood, each brood = 24 h r s . ) . Bars indicate 95% confidence l i m i t s . - 5 4a -Figure 20. Daily mean f e r t i l i t y of 2225 grown at 28° for various times during pupal development, and brooded at 22°. a. pi at 28° (days 1,2 of pupal stage); b. p l l at 28° (days 3,4,of pupal stage); c. p i l l at 28° (days 5,6 of pupal stage). Each point represents mean of 10 i n d i v i d u a l l y mated males brooded for 10 days (5 females per brood, each brood = 24 t i E s s ) . Bars indicate 95% confidence l i m i t s . - w -- 55a -Figure 21. Temperature sensitive periods of hs and cs Y-linked male ste-r i l e mutations. Hatched bars represent minimal amount of t&mesrequired at the restricitve temperature to cause ster-i l i t y in newly-eclosed males. Open bars indicate stages which wi l l increase length of s t e r i l i t y in newly-eclosed males when grown at the restrictive temperature in combination with the stage covered by the hatched bar, but have no effect on fer-t i l i t y alone at the restrictive temperature. -.5 5b -Larval Instar Mutant Egg 1st 2 nd 3 rd Pupa hs 56 '////A 502 '////A 631 '/////, 887 //////////// 898 V//// 1216 '/////, 1312 '/////////// 160 8 •///////////, 2225 cs 150 V///////, 2661 '////////. 3 4 80 - 56 -b) cold-sensi t i v e s t e r i l e mutations As seen from Figure 21, the tsp's of the cs mutations 150, 2661, and 3480 occur i n the t h i r d i n s t a r . At t h i s stage, the f i r s t cohort of sperm should have formed primary sperm-atocytes i n the testes (Hannah-Alava, 1965). However, care has to be taken i n interpreting these r e s u l t s , because the experiments to determine t h i s c o r r e l a t i o n were performed at 25° (Table 1). I t i s possible that testes do not develop at the same rate r e l a t i v e to the l a r v a l stages at 17°. Indeed, evidence to support such caution was reported by Frankel (1973). She found testes of f l i e s grown at 19° contain a stage of spermatid d i f f e r e n t i a t i o n l a t e r than expected at the beginning of the pupal stage (although 25° controls for comparison were not done). In v i t r o organ cultures show a faster rate of growth of testes at 17° that the other organs (R. Denell, personal communication). Thus, the tsp of the cs mutations could a c t u a l l y be i n early post-meiotic c e l l s . Nevertheless, the behaviour of the cs mutations c l e a r l y d i f f e r s from that of the hs mutations. F i r s t , as mentioned above, the tsp i s pre- or early post-meiotic. F l i e s grown at 17° u n t i l the end of the second instar show wild type f e r t i l i t y upon eclosion (Figures 22ab, 23ab, 25ab), while f l i e s grown at 17° from the t h i r d i n s t a r show a long-lasting s t e r i l i t y on eclosion (Figures 23c, 25c), and those grown for just the pupal stage at 17°, i n i t i a l l y show f e r t i l i t y on eclosion (Figures 22c, 24c, 26c). ( a l l the hs mutations are s t e r i l e on eclosion when grown for the pupal stage at 28°). Secondly, the t o t a l length of time the organism i s grown at 17° (including stages preceding the tsp) a f f e c t s the duration of s t e r i l i t y of males after eclosion. Growth of just the t h i r d (or second and third) instars at 17° produces a shorter period of s t e r i l i t y . than growth of the egg to pupal stages at 17°, even though i t can be shown that the egg-second instar stages have no e f f e c t on f e r t i l i t y (Figures 24ab, 26ab). In a l l 3 mutations, growing the pupal stage at 17° does not produce an i n i t i a l wild type burst of f e r t i l i t y as expected i f the tsp was pre-meiotic (Figures 22d, 24c, 26c). Instead, f e r t i l i t y i s reduced thereby suggesting there i s some post-meiotic temperature s e n s i t i v i t y during sperm development, or the stages of d i f f e r e n t i a t i o n within the gonad have become heterogeneous as a consequence of the changes i n temperature. F e r t i l i t y of A208 i s low even at 22°, and t h i s renders the s h i f t studies d i f f i c u l t to inte r p r e t (Figure 27). The o mutation seems to be exerting i t s e f f e c t at 22 , and growth at the lower temperature i s only serving to enhance the phenotype. - 58a -Figure 22. Daily mean f e r t i l i t y of 150 grown during various developmen-tal stages at 17°, and brooded at 22°. a. entire l i f e cycle at 22°; b. egg-2nd instars at 17°; c. 2nd and 3rd instars at 17°; d. pupa at 17°. The rest of the l i f e cycle was grown at 22°. Each point represents mean of 10 individually mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence limits. NUMBER of PROGENY/VIAL - 59a -Figure 23. Daily mean f e r t i l i t y "of 2661 grown at 17° during various de-velopmental stages and brooded at 22°. a. enti r e l i f e c y c l e ' at 22°; b. egg-2nd instars at 17°; c. 3rd instar and pupa at 17°; Rest of l i f e cycle grown at 22°. Each point represents mean of 10 i n d i v i d u a l l y mated males brooded for 10 days (5 fe-males per brood, each brood = 24 hr s . ) . Bars indicate 95% confidence l i m i t s . - 59 b -- 6 6a -Figure 24. Daily mean f e r t i l i t y of 2661 grown at 17° during various de-velopmental stages and brooded at 22°. a. 3rd instar at 17°; b. egg-3rd instars at 17°; c. pupa at 17°. Rest of l i f e cycle grown at 229,. Each point represents mean of 10 indi-vidually mated males brooded for 10 days (5 females per brood, ,each brood = 24 hrs.). Bars indicate 95% confidence limits. - 6 Ob -BROOD NUMBER - 6ia -Figure 25. Daily mean f e r t i l i t y of 3480 grown at 17° during various de-velopmental stages and brooded at 22°. a. e n t i r e l i f e cycle at 22°; b. egg-2nd instars at 17°; c. 3rd -instar and pupa at 17°. Rest of l i f e cycle grown at 22°. Each point represents mean of 10 i n d i v i d u a l l y mated males brooded for 10 days (5 females per brood, each brood = 24 h r s . ) . Bars indicate 95% confidence!imits. 50 0 2 3 4 5 6 7 8 BROOD NUMBER 9 10 - 6 2a -Figure 26. Daily mean f e r t i l i t y of 3480 grown at 17° during various de-velopmental stages. a. 3rd instar at 17°j b. egg-3rd in-stars at 17°; c. pupa at 17°. Rest of l i f e cycle grown at 22°. Each point represents mean of 10 individually mated males brooded for 10 days.(5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence limits. - 63a -Figure 27. Daily mean f e r t i l i t y of A208 grown at 17° for various deve-lopmental stages. a. entire l i f e cycle at 22°; b. egg-3rd instars at 17°; c. pupa at 17°. Rest of l i f e cycle grown at 22°, and brooded at 22°. Each point represents mean of 10 individually mated males brooded for 10 days (5 females per brood, each brood = 24 hrs.). Bars indicate 95% confidence limits. - 63b-- 64 -DISCUSSION The usefulness of temperatures-sensitive (ts) mutations i n studying gene function has been demonstrated i n a wide var i e t y of organisms; they are easy to recover and maintain, and allow determination of time of gene product function through temperature s h i f t s (Epstein e t a l , , 1963; Igarashi, 1966; Hartwell et a l . , 1970; Jarvik and Botstein, 1973; Suzuki, 1970, 1974). In microorganisms, the basis for temperature-s e n s i t i v i t y has been shown to be the r e s u l t of an amino acid substitution which confers a thermo-labile property to the protein (Jockusch, 1966). Since i n Drosophila ts mutations can be i s o l a t e d i n genes coding for enzymes (Camfield and Suzuki, 1972; Vigue and Sofer, 1974), and are predominantly recessive, (Tasaka and Suzuki, 1973) i t would seem that here too, ts mutations represent lesions i n genes coding for proteins,resulting i n amino acid substitutions. Comparison of heat-sensitive (hs) and cold-sensitive (cs) mutations shows that several s t r i k i n g differences e x i s t between these two classes of ts mutations i n t h e i r genetic d i s t r i b u t i o n s and protein functions i n both microorganisms and Drosophila. Whereas hs mutations map extensively throughout the genome (Edgar and L i e l a s i s , 1964; Igarashi, 1966; Suzuki, 1970), cs mutations occur at a lower frequency and tend to be clustered in a few genes (Scotti, 1968; Cox and Strack, 1971; Rosenbluth et a l . , 1971; Mayoh and Suzuki, 1973). Furthermore, while hs mutants a f f e c t both s t r u c t u r a l and control genes i n micro-organisms, cs mutations appear to a f f e c t p r e f e r e n t i a l l y genes - 65 -with control functions, such as self-assembly of ribosomes (Guthrie £t a l . , 1969; Tai et ai l . , 1969; Nashimoto et a l . , 1971) or regulatory enzymes (Condon and Ingraham, 1967). The present study allows another comparison of hs and cs mutations. About 0.4% of a l l EMS-treated XY chromosomes carri e d a hs mutation on the Y chromosome; a l l of these were on Y L (note that 'this i s the frequency of recessive Y-linked hs mutations, as a l l mutations which did not recover f e r t i l i t y i n x\ XY/Y males would be c l a s s i f i e d as X-linked). In a previous study (Ayles et a l . , 1973), i t was also found that a l l 8 hs Y-linked mutations were located on Y L. The hs Y L mutations were not confined to a single complementation group, but were found i n at lea s t 3 d i f f e r e n t l o c i (Table 3). S h i f t studies on these mutations showed that t h e i r tsp's were post-meiotic. Figure 21). In contrast to the hs mutations, the cs mutations occurred at a much lower frequency, and although they recovered s S f e r t i l i t y with Y , they were found to be located on the X chromosome i n the mapping experiments (Table 5). Thus, no cs mutations were recovered on the Y chromosome (unless some of those c l a s s i f i e d as X-linked are dominant Y-linked mutations). The tsp's of the cs mutations also d i f f e r e d from those of the hs Y mutations, occupying pre- or early post-meiotic: stages, p r i o r to the e f f e c t of hs Y L mutations. These studies corroborate other workers i n showing that cs mutations generally d i f f e r from hs mutations i n three properties: frequency, lo c a t i o n , and function. But what does t h i s t e l l us about the actual nature of the genes affected? - 66 -RNA-DNA hybridization studies i n p. hydei have shown that the Y-loops are composed of r e p e t i t i v e sequences (Hennig et a l . , 1973), However, EMS-induced mutations i n the f e r t i l i t y factors of D, melanogaster were e a s i l y recoverable. Since EMS predominantly causes point mutations (Krieg, 1963; Suzuki, 1970), these mutations most probably have occurred i n genes present i n single copies. This i s further strengthened by the fact that some of these mutations are temperature-se n s i t i v e . Williamson (1972) also reached the conclusion that the f e r t i l i t y factors are present i n single copies, and suggests that they may be amplified during t r a n s c r i p t i o n . The 8 Y L mutations examined i n t h i s study show 3 d i f f e r e n t complementation patterns, which suggests that these lesions are i n d i f f e r e n t f e r t i l i t y factors along Y L. Their tsp's are a l l i n the pupal stage, which coincides with spermatid d i f f e r e n t -i a t i o n i n the testes. Thus, we can conclude that several d i f f e r e n t f e r t i l i t y factors on Y are present i n single copies and that they are d i r e c t l y involved i n spermiogenesis. Previous suggestions that EMS-induced hs mutations on Y L are i n l o c i which control t r a n s c r i p t i o n of the f e r t i l i t y factors (present i n several copies), which would be indicated by a pre-meiotic tsp (Ayles et a l . , 1973; Sanders and Ayles, 1973; Kiefer, 1973) would now appear to be incorrect. Their temperature s h i f t s (Ayles et a l . , 1973) did not include the proper controls to discount t h e i r conclusions i n 7 of t h e i r 8 mutations. Tsp's defined by s h i f t studies can delineate two periods: the time of t r a n s l a t i o n (temperature-sensitive synthesis), or the time that the protein a c t u a l l y functions (Hartwell, et al_., - 6 7 -1970; Jarvik and Botstein, 1973). In the f i r s t case, once formed at the r e s t r i c t i v e temperature, the protein would be i r r e v e r s i b l y inactive; i n the second case, the mutant protein configuration could be reversed when the organism i s shi f t e d to the permissive temperature. In t h i s system, because of the long heat pulses necessary to cause s t e r i l i t y , i t i s not possible to di s t i n g u i s h between these two processes. However, the observation that some of the hs mutations require a 48 hour pupal heat pulse to confer s t e r i l i t y and recover f e r t i l i t y i n a few days afte r an adult heat pulse suggests a reversib l e e f f e c t of heat on the gene product, whereas those which require a 24 hour pupal heat pulse, and take a longer time to recover f e r t i l i t y a f t e r the adult heat pulse (see Results, part 6a) could be the r e s u l t of a non-reversible e f f e c t . Another p o s s i b i l i t y i s that the mutations causing s t e r i l i t y a f t e r a 24 hour pupal pulse could be a f f e c t i n g gene products which are required at a s p e c i f i c stage during sperm assembly. If the spermatids pass through t h i s stage when the gene product i s inacti v e , they w i l l be immobile. Conyersly, the mutations requiring a 48 hour pupal pulse could be a f f e c t i n g gene products which may act at one of several points over a longer period of time. Therefore, a longer heat pulse i s required to induce s t e r i l i t y . The Y L mutations can be distinguished both by t h e i r discrete tsp's (Figure 21) and by complementation data (Table 4). 887 and 1608, both i n k!5, require a 48 hour heat pulse i n the pupal stage for s t e r i l i t y on eclosion. 56_, 631, and 1216, a l l i n k l l , 2 , have a 24 hour heat pulse requirement i n - 68 -the pupal stage. The tsp of the 3 mutations i n k!3 4, 502, 1312, and 3269 are not s i m i l a r to each other. 502 and 3269 require a 24 hour heat pulse, while 1312 requires a 48 hour heat pulse i n the pupal stage, for s t e r i l i t y . Perhaps these mutations are ac t u a l l y i n 2 d i f f e r e n t l o c i , 502 and 3269 i n one and 1312 i n another. S No hs mutations have been is o l a t e d on Y . In f a c t , no S ts mutations of either kind were is o l a t e d on Y from over 10,000 mutagenized chromosomes. I t i s possible that the genes i n t h i s region are very small and therefore r a r e l y mutate, do not produce products which are thermo-labile, are present i n multiple copies, or only mutate to dominant a l l e l e s . However, non-ts EMS-induced mutations on Y can be i s o l a t e d (Williamson, 1970b, 1972). The use of the XY chromosome has led to the discovery of an e x c i t i n g new c l a s s of mutations, X-linked male s t e r i l i t y lesions which are suppressed by the presence of an extra Y chromosome. Other workers studying male s t e r i l i t y screened for mutations either on a free X or free Y chromosome and either discarded or missed mutations of the type we recovered. In t h i s study, at l e a s t one hs and 3 cs mutations of t h i s type have been i s o l a t e d . However, i t i s not known at t h i s point how many d i f f e r e n t l o c i on the X chromosome are actually being affected. These studies revealed at le a s t 2 genes on the X chromosome which interact with d i f f e r e n t regions on Y to produce s t e r i l i t y . These are the cs mutations 2661, and A208 i n the y-v region, and 3480, d i s t a l to v. The mutations i n the two regions show a - 69 -d i f f e r e n t complementation pattern with Y s. An extra Y chromosome lacking k s l and/or ks2 does not restore f e r t i l i t y i n 2661 and A208, while one lacking ks2 has no e f f e c t i n 3480 (Table 4). Although no ts lesions were found on Y , i t c l e a r l y does in t e r a c t with genes on the X chromosome. There-S fore, i t i s possible that the Y gene products are not d i r e c t l y involved i n spermatid d i f f e r e n t i a t i o n ; rather, they may be purely regulatory and i n t e r a c t with the X chromosome and Y L during spermatogenesis. As would be expected i f t h i s were the case, the tsp's of these mutations are p r i o r to those exhibited by the Y L mutations. Further, Kiefer (1973) g reports that lesions i n Y' show the same severity of defects c h a r a c t e r i s t i c of X/O males, whereas Y L d e f i c i e n c i e s permit more extensive sperm development. The X-linked hs mutation of t h i s type, 2225, interacts with Y L i n a f f e c t i n g f e r t i l i t y , and also has a tsp p r i o r to that of the Y L mutations. Interactions between the X and Y chromosomes during spermatogenesis have been reported i n other organisms. In D. hydei, an- X-linked mutation has been found which prevents unfolding of the Y-loops (Lifschytz, 1974). A l l loops are affected to the same degree, and 2 doses of Y chromosome does not correct the s t e r i l i t y . In the grasshopper, i t has been shown that the X chromosome becomes heteropycnotic i n the primary spermatocyte (Lima-de-Faria and Jawarska, 1966). I t has been known for a long time that most X:autosome trans-locations i n D. melanogaster r e s u l t i n male s t e r i l i t y , except where the X chromosome breakpoints are near the t i p or the centromere (Lindsley and L i f s c h y t z , 1972). L i f s c h y t z and - 70 -Lindsley (1972) postulated that a s i t u a t i o n s i m i l a r to that i n grasshopper exists i n Drosophila, that i s , the X chromosome i s inactivated i n primary spermatocytes and t h i s must occur before the Y chromosome can function. Further, s t r u c t u r a l continuity of the X chromosome i s a prerequisite for i n a c t i v a t i o n of the entire X chromosome at the required time. Thus, a trans-location involving the X chromosome r e s u l t s i n male s t e r i l i t y because the chromosome i s not kept i n t a c t and the i n a c t i v a t i o n mechanism can no longer operate. Two models can account for the re s u l t s reported i n these experiments., '' • In the f i r s t model, several genes on the X chromosome produce repressors which prevent the Y chromosome from functioning i n most c e l l s by int e r a c t i n g with S Y . In the primary spermatocyte, these repressor genes are themselves shut o f f when the X chromosome i s inactivated, S L thereby releasing Y to activate Y , which starts synthesizing RNA. The cs mutations would be lesions i n the X chromosome which produce "super-repressors" which bind i r r e v e r s i b l y to S S Y at the r e s t r i c t i v e temperature, thereby preventing Y from functioning at the required time. Two doses of Y would restore f e r t i l i t y because of a threshold e f f e c t , that i s , there S i s not enough repressor to inactivate both Y fragments. The second model i s s l i g h t l y more complex. Here, a gene on the X chromosome produces a repressor which binds to a s i t e L S on Y . In the primary spermatocyte, Y produces a substance which i n i t i a t e s i n a c t i v a t i o n of the X chromosome by acting on several genes along i t s length. By v i r t u e of the X chromosome in a c t i v a t i o n , the repressor acting on Y^ would not be produced, - 71 -and Y L would be free to synthesize RNA. In t h i s case, the cs mutations would be genes or prote ins which have a reduced a f f i n i t y for the Y i n a c t i v a t o r s , while 2225 could be a mutation . producing a "super-repressor" which binds to Y L . S Two doses of Y i n the cs mutations would overcome the problem of reduced a f f i n i t y of the genes affected by producing twice the amount of repressor , and two doses of Y would restore f e r t i l i t y i n the hs mutation because of the threshold e f fec t mentioned i n model 1. Obviously, both models are very s i m p l i s t i c and heavi ly dependent on models of regula t ion from microorganisms. In view of the unique aspects of m u l t i c e l l u l a r eukaryotic developmental processes, these models may prove to be without bas i s , but nevertheless serve as f o c i for carry ing out further tests and a l so to generate tes table p r e d i c t i o n s . The data c o l l e c t e d so far f i t both models equal ly w e l l . Model 1 has advantages because the cs mutations are proposed to be a f f ec t ing proteins which do have thermo-labi le p roper t i e s , and lower temperatures are known to s t a b i l i z e c e r t a i n types of bonds. Model 2 has advantages because i t could expla in the behaviour of 2225. The tsp data suggest that the cs mutations are act ing before the Y L mutations, while 2225 acts a f ter the cs mutations but p r i o r to the Y L l o c i (Figure 21). Several c r i t i c a l experiments must be c a r r i e d out (some of these are already i n progress) , to provide more information and to allow the construct ion of a better model which incorporates a l l of the data: - 7 2 " 1) the Y-suppressed X-linked mutations have to be mapped accurately. It i s not known at t h i s point whether A2 08, 2661, and 2225 are a l l e l e s or i n d i f f e r e n t genes; 2) a l l the above mutants and 3480 and 150 must be crossed out onto a free X chromosome and tested for s t e r i l i t y with one and two free Y chromosomes to see i f they behave i n the same manner. 3) the r e s u l t s of the double mutant s h i f t s are important. A male carrying both a cs, and a hs mutation on Y L grown u n t i l pupation at 28°, then sh i f t e d to 17° u n t i l eclosion, S L should be f e r t i l e i f Y does act before Y . In the r e c i p r o c a l s h i f t , the male should be s t e r i l e . If 2225 i s indeed acting on Y L as suggested by model 2, then r e c i p r o c a l s h i f t s on indivi d u a l s containing a cs mutation and 222 5 should show that the tsp of the cs mutations i s p r i o r to that of 2225. 4) once mapped, the cs mutations and 2225 can be tested for precise c y t o l o g i c a l positions by complementation tests with a series of duplications. From model 1, i t can be predicted that a duplication could restore f e r t i l i t y at the r e s t r i c t i v e temperature, depending on the type of competition between the normal and mutant repressors. In model 2, f e r t i l i t y would not be restored because continuity of the X chromosome i s needed for i n a c t i v a t i o n . The function of the Y chromosome during spermatogenesis and i t s close i n t e r a c t i o n with the X chromosome poses an i n t r i g u i n g problem which cannot be solved by the simple recovery of cs and hs mutations on the Y chromosome as i n i t i a l l y suggested. Results from these and future experiments on male s t e r i l i t y w i l l lead to an insight into the complex interactions of genes and chromosomes during development. SUMMARY The main f u n c t i o n o f the Y chromosome i n D r o s o p h i l a  me 1 anogaster i s t o a l l o w development o f m o t i l e sperm. Seven f e r t i l i t y f a c t o r s have been shown t o be p r e s e n t on the Y chromosome, each o f which has to be p r e s e n t i n a t l e a s t one w i l d type dose f o r normal sperm development. To c h a r a c t e r i z e the f u n c t i o n of the f e r t i l i t y f a c t o r s more e x t e n s i v e l y , and to determine the time o f a c t i o n of t h e i r gene p r o d u c t s , a number of EMS-induced heat- and c o l d - s e n s i t i v e Y - l i n k e d male s t e r i l e mutations were i s o l a t e d on an attached-XY chromosome. A t o t a l o f 26 hs and 5 cs mutations were i s o l a t e d which recovered f e r t i l i t y i n the presence of a f r e e Y chromosome, and these were l a b e l l e d as Y - l i n k e d . A l l the hs Y - l i n k e d mutations recovered f e r t i l i t y w i t h Y L, and a l l the cs mutations w i t h Y . However, recombination data showed t h a t w h i l e most of the hs mutations examined were l e s i o n s on Y L, a t l e a s t 3 of the cs mutations and one of the hs mutations were a c t u a l l y l o c a t e d on the X chromosome. Furthermore, the t s p ' s of the hs mutations on Y L were p o s t - m e i o t i c c o i n c i d i n g w i t h spermatid d i f f e r e n t i a t i o n , w h i l e the t s p ' s o f the X - l i n k e d mutations were pr e - or e a r l y p o s t - m e i o t i c , p r i o r t o those of the hs L mutations on Y . From the above, i t seems t h a t the f e r t i l i t y f a c t o r s on Y L are p r e s e n t i n s i n g l e c o p i e s and code f o r products which are d i r e c t l y i n v o l v e d i n spermiogenesis. Because no t s S mutations were recovered on Y , but i t i n t e r a c t s w i t h X-l i n k e d mutations, i t c o u l d have r e g u l a t o r y f u n c t i o n s d u r i n g spermatogenesis. The f a c t t h a t t h e r e are X - l i n k e d male s t e r i l e mutations which r e c o v e r f e r t i l i t y w i t h 2 doses of Y chromosome shows t h a t X-Y chromosome i n t e r a c t i o n s are important d u r i n g spermatogenesis, and occur b e f o r e the products of the Y L f e r t i l i t y f a c t o r s can f u n c t i o n i n sperm mat u r a t i o n . - 76 -LITERATURE CITED A y l e s , G. B., Sanders, T. G., K i e f e r , B. I . , and Suzuki, D. T. (1973). T e m p e r a t u r e - s e n s i t i v e mutations i n D r o s o p h i l a melanogaster. XI. 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