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Molecular analysis of the DPY-14 region of chromosome I in Caenorhabditis elegans Starr, Terence 1989

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MOLECULAR ANALYSIS OF THE DPY-14 REGION OF CHROMOSOME I IN CAENORHABDITIS ELEGANS By TERENCE V.B. STARR B . S c , Simon F r a s e r U n i v e r s i t y , 1983 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1986 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILLMENT OF FOR THE DEGREE OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES GENETICS PROGRAMME We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF Cc) T e r e n c e V.B. BRITISH COLUMBIA 1989 S t a r r , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Medical Genetics The University of British Columbia Vancouver, Canada Date December 28, 1989  DE-6 (2/88) 11 A b s t r a c t T h i s t h e s i s d e s c r i b e s the alignment of c l o n e d DNA with the g e n e t i c map, and the i d e n t i f i c a t i o n of coding elements w i t h i n the a l i g n e d DNA. The r e g i o n of study was the dpy-5 unc-29 i n t e r v a l from chromosome I of the nematode Caenorhabdi t i s elegans, with an emphasis on the r e g i o n surrounding the gene dpy-14. The o b j e c t i v e s of t h i s t h e s i s were: 1) to a l i g n the p h y s i c a l and g e n e t i c maps .of the r e g i o n ; 2) to i d e n t i f y and c h a r a c t e r i z e the coding elements i n the v i c i n i t y of dpy-14; and 3) to c r o s s - h y b r i d i z e the i d e n t i f i e d C. elegans coding elements to mammalian DNA i n an attempt to i d e n t i f y e v o l u t i o n a r i l y conserved genes. Six polymorphisms from the dpy-5 unc-29 i n t e r v a l were mapped with r e s p e c t to the f r e e d u p l i c a t i o n sDp2. The polymorphisms hP5, sPl, and hP9 were found to.be i n s i d e the re g i o n spanned by sDp2 while the polymorphisms hP4, hP6, and hP7 were found to be o u t s i d e t h i s i n t e r v a l . In a d d i t i o n , these s i x polymorphisms were mapped with r e s p e c t to v i s i b l e markers from the dpy-5 unc-29 i n t e r v a l . These a n a l y s e s demonstrated the g e n e t i c order to be dpy-5, hP5, unc-37, (dpy-14, sPl), hP9, unc-13, hP7, (hP4, hP6), unc-29. Lambda phage c o n t a i n i n g the hP5, sPl, and hP6 s i t e s i d e n t i f i e d and anchored cosmid c o n t i g s to the g e n e t i c map. The i n t e r v a l from the l e f t of hP5 to the r i g h t of unc-13 i s c o n t a i n e d i n a s i n g l e c o n t i g of approximately 1400 Kb. The amount of DNA i n Kb a c r o s s the hP5 and unc-13 i n t e r v a l was compared to the g e n e t i c d i s t a n c e i n map u n i t s . The DNA per map u n i t value was I l l found to vary i n t h i s i n t e r v a l with the g r e a t e s t value found between hP9 and unc-13. Seven cosmids r e p r e s e n t i n g 173 Kb of N2 genomic DNA near the gene dpy-14 were i s o l a t e d . Using c r o s s - s p e c i e s h y b r i d i z a t i o n to C. briggsae DNA ten conserved regions were i d e n t i f i e d w i t h i n these seven cosmids. The ten conserved fragments were used to i d e n t i f y seven cDNAs, s i x of which a l s o i d e n t i f i e d RNAs on Northern b l o t s . The r e l a t i v e abundance of the i s o l a t e d cDNAs v a r i e d 250 f o l d with the most abundant having a l e v e l s i m i l a r to that found f o r a c t i n . The f i r s t comprehensive survey of mammalian homologies i n a contiguous set of ten coding r e g i o n s found three coding elements to be. conserved. One was demonstrated to be the sma l l n u c l e a r RNA gene U1. Another shared sequence s i m i l a r i t i e s with the gene S-adenosyl-L-homocysteine h y d r o l a s e . No d e t e c t a b l e homologies were i d e n t i f i e d with the t h i r d . A formaldehyde-induced mutation that f a i l e d to complement the genes unc-37, unc-87, dpy-14, let-83 and let-86 was i s o l a t e d . T h i s mutation appeared to be the r e s u l t of a DNA rearrangement which had one breakpoint w i t h i n the cosmid C14A12. Using the conserved elements i d e n t i f i e d i n t h i s t h e s i s together with the rearrangements and mapped genes from the r e g i o n , a d e t a i l e d p h y s i c a l and g e n e t i c map i n the v i c i n i t y of dpy-14 was c o n s t r u c t e d . IV DEDICATION To my wife Trudy, my sons Darren and Kevin, my daughter Candace, and Mom and Dad. You were always there to h e l p . V ACKNOWEDGEMENTS T h i s work would not have been p o s s i b l e without the support and encouragement of my s e n i o r s u p e r v i s o r s , Dr. A.M. Rose and Dr. S. Wood. I would a l s o l i k e to thank my s u p e r v i s o r y committee, Dr. D.L. B a i l l i e , and Dr. C. Wehrhahn, f o r t h e i r h e l p f u l comments at committee meetings; Dr. A.M. Rose, Dr. S Wood, Dr. D.L. B a i l l i e , Dr. C. Wehrhahn, Dr. A.M. Howell, Dr. S. Prasad, K. McKim, and K. Peters f o r t h e i r h e l p f u l comments i n p r e p a r i n g t h i s manuscript; Dr. D.L B a i l l i e and Dr. S. Prasad f o r t h e i r h e l p with the database comparisons; Dr. A.M. Rose, Dr. A.M Howell, Dr. L H a r r i s , and K. Peters f o r p r o v i d i n g N 2 / B 0 recombinant s t r a i n s ; Dr. A.M. Rose, Dr. S Wood, Dr. D.L. B a i l l i e , Dr. A.M. Howell, Dr. S. Prasad, K. McKim, K. Pe t e r , and J . Ba b i t y f o r t h e i r h e l p f u l d i s c u s s i o n s ; J . McDowall and K. Beckenbach f o r p r o v i d i n g probes f o r the polymorphic s i t e s ; and f i n a l l y to Dr. A.M. Rose, Dr. A.M. Howell, and K. McKim f o r t h e i r p a t i e n c e when h e l p i n g me with g e n e t i c a n a l y s e s . vi TABLE OF CONTENTS A b s t r a c t i i D e d i c a t i o n i v Acknowledgements v L i s t of Tables ix L i s t of F i g u r e s x I. INTRODUCTION 1 1. The C. elegans P h y s i c a l Map 5 2. The Linkage Group I Gene C l u s t e r 8 3. The dpy-14 Region of Linkage Group I 12 I I . M a t e r i a l s and Methods 1. C u l t u r i n g Nematodes 16 2. C o n s t r u c t i o n of Three-Factor Mappings S t r a i n s 16 3. Screening f o r Dpy-14 Mutants 17 4. Complementation T e s t i n g Using hDf8 19 5. Cosmids Used 20 6. DNA I s o l a t i o n 20 1. Nematode Genomic 20 2. Yeast Genomic 21 3. Plasmid and Cosmid 23 1. mini-prep 23 2. l a r g e s c a l e 24 4. Phage 25 7. Agarose Gel E l e c t r o p h o r e s i s 27 8. Subcloning 28 9. Southern T r a n s f e r and H y b r i d i z a t i o n 29 vii 10. Northern T r a n s f e r and H y b r i d i z a t i o n 30 11. H y b r i d i z a t i o n C r i t e r i a 32 12 S t r i p p i n g Nytran F i l t e r s 33 13. O l i g o L a b e l l i n g of Probes 34 14 Screening Phage L i b r a r i e s 35 15. DNA Sequencing 37 I I I . R e s u l t s 1. I s o l a t i n g DNA from the LGI Gene C l u s t e r 40 1.1 Polymorphisms w i t h i n LGI 40 1.2 L o c a l i z a t i o n of the Polymorphic S i t e s on LGI 40 1.3 T h r e e - f a c t o r Mapping of the Polymorphic S i t e s 42 1.4 O r i e n t a t i o n of the Polymorphic S i t e s 45 1.5 I d e n t i f i c a t i o n of the Polymorphic S i t e hP9 50 1.6 I s o l a t i o n of DNA Surrounding sPl 52 1.7 P o s i t i o n i n g sPl and hP9 R e l a t i v e to the D u p l i c a t i o n s hDp58 and hDp62 55 1.8 R e s t r i c t i o n Enzyme Map of Cosmids Surrounding dpy-14 56 1.9 Amount of DNA Within Cosmids 59 1.10 Amount of DNA Surrounding Polymorphic S i t e s 59 2. Coding Regions Near the dpy-14 Gene 2.1 I d e n t i f i c a t i o n of Conserved Regions Between C. elegans and C. briggsae 64 2.2 I d e n t i f i c a t i o n of Coding Elements Using Conserved Regions 68 2.3 P o s i t i o n i n g Conserved Regions on the P h y s i c a l Map 70 V l l l 2.4 R e l a t i v e Abundance of I s o l a t e d cDNAs 70 2.5 C h a r a c t e r i s t i c s of Conserved Regions 73 3. Genetic Analyses i n the dpy-14 Region 94 3.1 Screening f o r Tc1 Induced Mutations of dpy-14 94 3.2 Screening f o r Formaldehyde Induced Mutations of dpy-14 94 3.3 Complementation T e s t i n g with h572 95 3.4 Southern A n a l y s i s of h572 97 IV D i s c u s s i o n 1. P h y s i c a l Map of the LGI Gene C l u s t e r 104 2. The Genetic L o c i W i thin the dpy-5 unc-13 Region of LGI 1 1 1 3. C o r r e l a t i n g the Genetic and P h y s i c a l Maps i n the dpy-14 Region of LGI ^ 113 4. I d e n t i f i c a t i o n and C h a r a c t e r i z a t i o n of Coding Elements Near dpy-14 115 5. Candidate Sequences f o r the dpy-14 Gene 117 6. DNA Sequence A n a l y s i s of Coding Elements Conserved Between C. elegans and Mammals 118 7. Homologies between C. elegans and Mammals 122 8. Summary and C o n c l u s i o n s 125 9. L i s t of F i n d i n g s 129 10. Pr o p o s a l s f o r Future Research 131 References 133 Appendix 1. S t r a i n s Used 144 Appendix 2. T h r e e - f a c t o r Mapping S t r a i n s 146 ix LIST OF TABLES Table 1 Probes f o r polymorphic s i t e s 11 Table 2 Regional l o c a l i z a t i o n of polymorphic s i t e s 43 Table 3 T h r e e - f a c t o r mapping of polymorphic s i t e s 47 Table 4 Mapping of polymorphisms 48 Table 5 Cosmids used 53 Table 6 C a l c u l a t i o n of the average amount of DNA per cosmid 60 Table 7 I s o l a t i o n of phage surrounding polymorphic s i t e s 61 Table 8 Conserved r e g i o n s between C. el egans and C. b r i g g s a e i n the r e g i o n near dpy-14 67 Table 9 C h a r a c t e r i s t i c s of conserved regions 69 Table 10 Complementation t e s t i n g with H57 2 96 Table 11 Dosage a n a l y s i s of the H57 2 mutation 101 Table 12 Amount of DNA i n the c o n t i g s from the LGI c l u s t e r 109 X LIST OF FIGURES F i g . 1 A p a r t i a l g e n e t i c map of the unc-11 unc-29 i n t e r v a l of l i n k a g e group I 15 F i g . 2 C o n s t r u c t i o n of three f a c t o r mapping s t r a i n s 18 F i g . 3 L o c a l i z a t i o n of the polymorphic s i t e s to the sDp2 region of LGI 41 F i g . 4 Three f a c t o r mapping of polymorphic s i t e s 46 F i g . 5 O r i e n t i n g the polymorphic s i t e s sPl and hP9 49 F i g . 6 I d e n t i f i c a t i o n of the polymorphic s i t e hP9 51 F i g . 7 A g e n e t i c and cosmid map of the hP5 unc-13 i n t e r v a l 54 F i g . 8 P o s i t i o n i n g hDp58 and hDp62 r e l a t i v e to sPl and hP9 57 F i g . 9 Merged r e s t r i c t i o n enzyme map of the cosmids near dpy-14 58 F i g . 10 Cosmids between hP9 and unc-13 63 F i g . 11 Screening f o r conserved r e g i o n s between C. elegans- and C. briggsae 66 F i g . 12 Screening f o r conserved regions between C. elegans and C. briggsae using fragments from cosmids 71 F i g . 13 P l a c i n g the coding elements on the cosmid map 72 F i g . 14 I d e n t i f i c a t i o n of a cDNA belonging to a gene f a m i l y 74 F i g . 15 H y b r i d i z a t i o n of sPl-I to Mammalian DNA 78 F i g . 16 Sequencing s t r a t e g y of sPl-1 79 xi F i g . 17 P a r t i a l sequence of sPl-1 80 F i g . 18 D i r e c t i o n of t r a n s c r i p t i o n of sPl-1 82 F i g . 19 I d e n t i f i c a t i o n of a fragment c o n t a i n i n g U1 83 F i g . 20 Northern a n a l y s i s of conserved regions 85 F i g . 21 H y b r i d i z a t i o n of sPl-3 to mammalian DNA 86 F i g . 22 Sequencing s t r a t e g y of sPl-3 87 F i g . 23 P a r t i a l sequence of sPl-3 88 F i g . 24 A comparison of the deduced amino a c i d sequence of sPl-3 and S-adenosyl-L-homocysteine h y d r o l a s e from r a t and D. discoideum. 91 F i g . 25 Dosage a n a l y s i s of the h57 2 mutation 98 F i g . 26 Scanning densitometer t r a c i n g s of the h572 99 mutation F i g . 27 Southern a n a l y s i s of the h57 2 mutation 102 F i g . 28 Genetic map of the dpy-14 r e g i o n * 112 1 I. I n t r o d u c t i o n One approach to the study of genome o r g a n i z a t i o n has been through the study of gene f a m i l i e s . In D r o s o p h i l a melanogaster gene f a m i l i e s have been d e f i n e d i n d i f f e r e n t c a t e g o r i e s (reviewed by S p r a d l i n g and Rubin, 1981; Wright, 1987): 1) r e i t e r a t e d genes; f o r example ribosomal RNA genes (Long and Dawid, 1980) or h i s t o n e genes ( L i f t o n et al ., 1977); 2) gene f a m i l i e s that arose from tandem gene d u p l i c a t i o n s and subsequent divergence; eg. c u t i c l e p r o t e i n s (Snyder and Davidson, 1983) and the dopa decarboxylase gene c l u s t e r (Ddc) ( G i l b e r t et a l . , 1984; Wright 1987); 3) f u n c t i o n a l l y but not s t r u c t u r a l l y r e l a t e d gene c l u s t e r s ; f o r example, two tropomyosin isoforms are l o c a t e d w i t h i n 140 Kb of the i n d i r e c t f l i g h t muscle s p e c i f i c a c t i n gene (act88F) ( K a r l i k et a l . , 1984); and 4) gene complexes such as the b i t h o r a x complex (Lewis, 1978). S i m i l a r c a t e g o r i e s have been observed i n other organisms. Some examples a r e : 1) tandem repeats of ribosomal RNA genes i n C. elegans ( S u l s t o n and Brenner,1974; Nelson and Honda, 1985); 2) genes f a m i l i e s t h a t r e s u l t e d from tandem d u p l i c a t i o n s such as the g l o b i n gene c l u s t e r s on human chromosomes 11 and 16 (reviewed K a r l s s o n and N i e n h u i s , 1985); and 3) randomly d i s p e r s e d gene f a m i l i e s such as tRNA genes (Cortese et a l . , 1978) and c o l l a g e n genes (Cox et a l . , 1985). Another approach to the study of genome o r g a n i z a t i o n has been by means of the d e s c r i p t i o n , by mu t a t i o n a l a n a l y s i s , of a l l the genes i n a genomic r e g i o n . Many re g i o n s of the 2 D. melanogaster genome have been c h a r a c t e r i z e d g e n e t i c a l l y . For example, i n the zest e-whi t e r e g i o n on the X chromosome most of the e s s e n t i a l genes were i d e n t i f i e d (Judd, Shen, and Kaufman, 1972). In the region adjacent t o the rosy gene on chromosome t h r e e , 20 complementation groups were i d e n t i f i e d ( H i l l i k e r et a l . , 1980). Many r e g i o n s of the C. elegans genome have a l s o been c h a r a c t e r i z e d g e n e t i c a l l y . For example, some r e g i o n s that have undergone e x t e n s i v e g e n e t i c a n a l y s i s with regard to the d i s t r i b u t i o n of e s s e n t i a l genes a r e : the reg i o n near the gene dpy-5 on chromosome I (Rose and B a i l l i e , 1980; Howell et al . , 1987; Howell, 1989); the re g i o n around the c r o s s - o v e r supressor, mnCl, on chromosome II (Sigurdson et a l . , 1984); two l a r g e autosomal r e g i o n s balanced by the r e c i p r o c a l t r a n s l o c a t i o n eTl (Rosenbluth and B a i l l i e , 1981) on chromosomes I I I and V (Rosenbluth, Cuddeford and B a i l l i e , 1983, 1985; Rosenbluth et a l . , 1988); the region near the unc-22 gene on chromosome IV ( R o g a l s k i , Moerman, and B a i l l i e , 1982; R o g a l s k i and B a i l l i e , 1985, C l a r k et a l . , 1988); the reg i o n near the ama-1 gene on chromosome IV (Rogalski and R i d d l e , 1988); and a re g i o n of the X chromosome (Meneely and Herman, 1979, 1981). An elegant example of a combined g e n e t i c and molecular a n a l y s i s of a re g i o n was the p r e v i o u s l y mentioned dopa decarboxylase region i n D. melanogaster. Using d e f i c i e n c i e s found i n the r e g i o n , 16 of the 18 genes i n the Ddc c l u s t e r were shown by g e n e t i c a n a l y s i s to have separate f u n c t i o n s . Mutations i n any of the genes were l e t h a l . A molecular 3 a n a l y s i s showed that some of the genes shared DNA homologies. Thus a combined g e n e t i c and molecular a n a l y s i s of a region can pr o v i d e i n f o r m a t i o n about the o r g a n i z a t i o n of a re g i o n that c o u l d not be obtained by j u s t one method. I t f o l l o w s that a combined g e n e t i c and molecular a n a l y s i s of an e n t i r e genome would a l s o y i e l d more i n f o r m a t i o n about a re g i o n than e i t h e r ' analyses would on i t s own. The molecular analyses of l a r g e regions of the genome of higher eucaryotes have lagged behind the g e n e t i c a n a l y s i s . However, i n t e r e s t i n understanding the g e n e t i c d e f e c t s of human d i s e a s e s has l e d to the p r o p o s a l to sequence the e n t i r e human genome. The human genome i s 3 X 10^ bp and w i l l generate an enormous amount of sequence d a t a . The r e a l c h a l l e n g e of t h i s p r o j e c t w i l l be to e x t r a c t the b i o l o g i c a l l y important i n f o r m a t i o n from the DNA sequence. One method to do t h i s would be to f i r s t t r y a v a r i e t y of approaches, i n p a r a l l e l with the human genome p r o j e c t , using a smal l e r model g e n e t i c system. One model g e n e t i c system c o n s i d e r e d to be s u i t a b l e f o r a complete g e n e t i c and molecular a n a l y s i s i s the nematode C. elegans (Lewin, 1989). The s o i l nematode C. el egans i s w e l l s u i t e d f o r the study of genomic o r g a n i z a t i o n . Large r e g i o n s of the genome have-been c h a r a c t e r i z e d g e n e t i c a l l y (Rosenbluth et al ., 1983, 1985, 1988; Sigurdson et a l . , 1984; Howell et a l . , 1987) with g r e a t e r than 800 genes i d e n t i f i e d m u t a t i o n a l l y ( i n Edgley and R i d d l e , 1987). In a d d i t i o n , 95% of the genome has been c l o n e d i n t o cosmids and a p h y s i c a l map of the e n t i r e genome i s near 4 completion (Coulson et al. , 1986, 1 988). Sequence data, from regions where the ge n e t i c and p h y s i c a l maps have been a l i g n e d , c o u l d be used to e x t r a c t b i o l o g i c a l i n f o r m a t i o n by c o r r e l a t i n g regions that c o n t a i n e d open read i n g frames with m u t a t i o n a l l y d e f i n e d genes. T h i s type of a n a l y s i s can p r e s e n t l y be a p p l i e d to r e g i o n s that are a l r e a d y w e l l c h a r a c t e r i z e d both g e n e t i c a l l y and m o l e c u l a r l y . Thus a p r e c i s e alignment of the ge n e t i c and p h y s i c a l maps i s one p r e r e q u i s i t e f o r s t u d y i n g genome o r g a n i z a t i o n . C. el egans are found throughout the world. They are approximately 1mm i n l e n g t h , feed on b a c t e r i a , have a gen e r a t i o n time of 3.5 days at 20°C and produce an average of 300 progeny by s e l f - f e r t i l i z a t i o n (Brenner 1974; R i d d l e 1978). A f t e r h a t c h i n g , the young worms develop through four l a r v a l stages shedding i t s c u t i c l e at each stage. The mature a d u l t has approximately 1000 somatic n u c l e i (959 i n the hermaphrodite and 1031 i n males ( S u l s t o n and H o r v i t z , 1977; Su l s t o n et al., 1983) and i s composed of h i g h l y d i f f e r e n t i a t e d t i s s u e types (eg. muscle, c u t i c l e , i n t e s t i n e , nervous system). They are tran s p a r e n t throughout t h e i r l i f e t i m e a l l o w i n g v i s u a l i z a t i o n of i n t e r n a l s t r u c t u r e s and the o b s e r v a t i o n of c e l l d i v i s i o n s i n l i v e animals. The e n t i r e embryonic and post-embryonic c e l l l i n e a g e s have been determined ( S u l s t o n and H o r v i t z , 1977; Su l s t o n et al., 1983). They are easy to manipulate and maintain i n the l a b o r a t o r y and s t r a i n s can be kept f r o z e n i n l i q u i d n i t r o g e n . These a t t r i b u t e s make 5 C. elegans a u s e f u l experimental organism f o r the study of metazoan development and genome o r g a n i z a t i o n . The C. el egans genome has f i v e p a i r s of autosomes and a sex chromosome (X). Hermaphrodites have two X chromosomes and males have one. Males a r i s e spontaneously at a frequency of 0.1% (Rose and B a i l l i e , 1979) by X chromosome n o n d i s j u n c t i o n (Hodgkin et a l . , 1979). There are approximately 2000 to 4000 genes i n the genome (Brenner, 1974; Moerman and B a i l l i e , 1979; C l a r k et a l . , 1988; Howell 1989) of which n e a r l y 800 have been m u t a t i o n a l l y i d e n t i f i e d (Edgley and R i d d l e , 1987). The h a p l o i d genome s i z e i s 1 X 10^ n u c l e o t i d e p a i r s ( J . S u l s t o n p e r s o n a l communication) and c o n s i s t s of 83% s i n g l e copy and 17% r e p e t i t i v e DNA ( S u l s t o n and Brenner, 1974). The C. el egans P h y s i c a l Map A. Coulson and J . Su l s t o n at the Medical Research C o u n c i l , Cambridge, England have undertaken to c o n s t r u c t a complete p h y s i c a l map of the C. elegans genome. The map c o n s i s t s of a c o l l e c t i o n of o v e r l a p p i n g fragments of DNA cl o n e d i n t o cosmid v e c t o r s (Coulson et al ., 1986). R e s t r i c t i o n enzyme d i g e s t e d ^ ^ P - l a b e l e d cosmid DNA and DNA from known standard markers was separated by e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e g e l s and analyzed by autoradiography (DNA f i n g e r p r i n t i n g ) . Data from the f i n g e r p r i n t e d DNA i s s t o r e d i n a computer database where i n d i v i d u a l cosmid f i n g e r p r i n t s are compared to one another. Cosmids with o v e r l a p p i n g f i n g e r p r i n t s " are j o i n e d i n t o a cosmid " c o n t i g " . Using t h i s 6 method alone approximately 90% of the genome had been cl o n e d i n t o c o n t i g s . To f a c i l i t a t e the l i n k i n g of c o n t i g s , C. elegans DNA was c l o n e d i n t o yeast a r t i f i c i a l chromosome v e c t o r s (Burke et al ., 1987) and probed with cosmids to d e t e c t any o v e r l a p . Using t h i s method the number of cosmid c o n t i g s was reduced from 700 to 346, with some c o n t i g s extending g r e a t e r than 2000 Kb (Coulson et al ., 1988). One b e n e f i t of t h i s p h y s i c a l map i s that i t w i l l allow access t o l a r g e segments of DNA u s i n g s i n g l e phage c l o n e s . I f a phage c l o n e i s known to be p h y s i c a l l y l i n k e d to a gene of i n t e r e s t then i d e n t i f y i n g a c o n t i g with a phage c l o n e w i l l a i d i n the i d e n t i f i c a t i o n of the gene. For example, a phage clone c l o s e l y l i n k e d to the gene s q t - J (Kramer et a l . , 1988) was i s o l a t e d and used to i d e n t i f y a c o n t i g of 250 k i l o b a s e p a i r s (Kb). W i t h i n t h i s c o n t i g Kramer and co-workers were ab l e to i d e n t i f y the sqt-1 gene. Another b e n e f i t of having a p h y s i c a l map i s the p o t e n t i a l to o v e r l a y the p h y s i c a l map on the g e n e t i c map. T h i s would allow access to a gene without f i r s t having to i d e n t i f y a p h y s i c a l l y l i n k e d phage, p r o v i d e d the g e n e t i c l o c a t i o n of the gene was known. There f o r e a u s e f u l undertaking would be the c o n s t r u c t i o n of a u n i f i e d g e n e t i c and p h y s i c a l map of the genome. The j o i n i n g of the g e n e t i c and p h y s i c a l maps r e q u i r e s the i d e n t i f i c a t i o n of s i t e s common to both maps. Since the g e n e t i c l o c a t i o n of a gene i s u s u a l l y determined p r i o r to i t s c l o n i n g , a match of the c l o n e d gene to a c o n t i g forms an 7 anchor p o i n t between the two maps {eg. the unc-22 gene, Moerman et al., 1986). T h i s approach would e v e n t u a l l y l e n d i t s e l f to the complete j o i n i n g of the maps pr o v i d e d genes from a l l r e g i o n s of the genome are c l o n e d . To a c c e l e r a t e the development of the map, non-coding DNA sequences from a de f i n e d i n t e r v a l of the genome c o u l d be i d e n t i f i e d , mapped, and then used to i d e n t i f y a c o n t i g . The two commonly used l a b o r a t o r y s t r a i n s of C. elegans, v a r i e t y B r i s t o l (N2) and v a r . Bergerac (BO), d i f f e r i n the r e l a t i v e number of Tc1 tr a n s p o s a b l e elements present i n t h e i r genomes (Emmons et al., 1983). N2 s t r a i n s possess approximately 30 co p i e s of Tc1, while BO s t r a i n s possess approximately 300 copies.. T h i s d i f f e r e n c e p r o v i d e s a means by which DNA sequences d e t e c t i n g r e s t r i c t i o n fragment l e n g t h polymorphisms (RFLP) between the two s t r a i n s can be r e a d i l y i s o l a t e d (Rose et al. , 1982). B a i l l i e et al. (1985) d e s c r i b e d the method of Tc1- l i n k a g e s e l e c t i o n as a gen e r a l method f o r the t a r g e t e d i s o l a t i o n of cl o n e d probes. By s e l e c t i v e l y m a i n t a i n i n g a region of the genome with DNA d e r i v e d from BO worms Tc1 elements l i n k e d to the reg i o n can be i s o l a t e d and mapped. B a i l l i e et al. (1985) i d e n t i f i e d and mapped s i x polymorphic probes from the unc-43 unc-31 i n t e r v a l of chromosome IV and used these probes to i n i t i a t e an intens e i n v e s t i g a t i o n i n t o the molecular o r g a n i z a t i o n of the unc-22 r e g i o n . A l t e r n a t i v e l y , s t r a i n polymorphisms can be sought by usin g e x i s t i n g c l o n e d DNA t o screen Southern b l o t s c o n t a i n i n g N2 and BO DNA. Since the BO genome c o n t a i n s approximately 300 8 c o p i e s of Tc1 and the genome s i z e i s 1 X IO** base p a i r s then, on average, the d i s t a n c e between T d s i s about 330 Kb. Hence an N2/B0 s t r a i n polymorphism, due to the presence of a Tc1 element i n the BO genome, should be observed approximately every 300 Kb. These polymorphisms can be q u i c k l y p o s i t i o n e d on the g e n e t i c map and the clone i d e n t i f y i n g the s i t e can be matched to a c o n t i g . T h e r e f o r e , each polymorphic s i t e p r o v i d e s an anchor p o i n t between the p h y s i c a l and g e n e t i c maps. In t h i s way l a r g e segments of c l o n e d DNA can be j o i n e d to the g e n e t i c map. Once j o i n e d any r e s e a r c h e r i n t e r e s t e d i n a gene from the j o i n e d r e g i o n can e a s i l y o b t a i n the DNA i n the form of cosmid c l o n e s . The Linkage Group I Gene C l u s t e r Each autosome of C. elegans c o n t a i n s a s i n g l e r e g i o n of the m e i o t i c map that encompasses a h i g h d e n s i t y of genes. These r e g i o n s are commonly r e f e r r e d to as gene c l u s t e r s and were f i r s t observed by Brenner (1974). The exact boundaries of the c l u s t e r s are not known, however the endpoints are d e f i n e d on m e i o t i c maps by v i s i b l e mutations c o n s i d e r e d to be near the boundaries (Edgley and R i d d l e 1987). The chromosome I gene c l u s t e r spans the i n t e r v a l from unc-11 to unc-29, a g e n e t i c d i s t a n c e of 5.0 map u n i t s (mu) (Howell et a l . , 1987; McKim et al ., 1988). T h i s gene c l u s t e r has been s t u d i e d with regard to recombination frequency between genes (Rose and B a i l l i e 1979a, 1979b; Kim and Rose, 1987), m e i o t i c behavior of rearrangements (Rose et al ., 1984; 9 McKim et a l . , 1988; McKim and Rose, 1989) and i d e n t i f i c a t i o n of e s s e n t i a l genes (Rose and B a i l l i e 1980; Howell et a l . , 1987; Howell 1989). These and other s t u d i e s have r e s u l t e d i n the c o n s t r u c t i o n of an e x t e n s i v e g e n e t i c map of t h i s c l u s t e r (Edgley and R i d d l e , 1987). The f r e e d u p l i c a t i o n sDp2 covers the l e f t t h i r d of chromosomel, approximately 17 map u n i t s , and breaks w i t h i n the chromosome I gene c l u s t e r (Rose et a l . , 1984). Using t h i s d u p l i c a t i o n a method f o r r e c o v e r i n g and a n a l y z i n g a l a r g e number of e s s e n t i a l genes was developed (Howell et a l . , 1987; Howell, 1989). So f a r , 495 l e t h a l mutations have been recovered a f t e r mutagenizing 31,000 chromosomes with e t h y l methane s u l f o n a t e . From t h i s study Howell (1989) est i m a t e s that 70% of the e s s e n t i a l genes i n the sDp2 r e g i o n have been mutated. Most of these mutations have been two-factor mapped, complementation t e s t e d , and mapped i n t o zones (Howell et al .,1987; McKim et al ., 1988; P i l g r i m and Rose, 1988; Howell, 1989; McDowall and Rose, 1989; McKim and Rose, 1989). Thus, t h i s r e g i o n i s a r i c h source of m u t a t i o n a l l y d e f i n e d genes. An important e x t e n s i o n of these s a t u r a t i o n s t u d i e s would be to c o r r e l a t e t h i s g e n e t i c a n a l y s i s with c l o n e d DNA to form a combined g e n e t i c and p h y s i c a l map of the r e g i o n . At the s t a r t .of t h i s t h e s i s no cosmid DNA from the chromosome I gene c l u s t e r had yet been i d e n t i f i e d . However f i v e DNA sequences d e t e c t i n g polymorphic s i t e s from the dpy-5 to unc-29 i n t e r v a l had been i d e n t i f i e d (Table 1) and were a v a i l a b l e f o r use to i d e n t i f y cosmid c o n t i g s . One of these 10 s i t e s , sPl, had been mapped near to the gene dpy-14 (Rose et al . , 1982; H a r r i s and Rose, 1986) and the phage s1 ( i s o l a t e d by A.M. Rose) c o n t a i n i n g the sPl l o c u s had been sent to the MRC Cambridge, England to be compared to the cosmid c o n t i g s . D e t e c t i o n of coding elements T. Snutch (1984) demonstrated that w i t h i n the r e g i o n around the hsplO gene of C. elegans the o n l y DNA that c r o s s -h y b r i d i z e d to C. briggsae DNA con t a i n e d the coding element f o r the hsplO gene. Using t h i s method of c r o s s - h y b r i d i z a t i o n between C. elegans and C. briggsae DNA Heine and Blumenthal (1986) were able to det e c t 12 conserved r e g i o n s and showed that ten of these regions produced a t r a n s c r i p t . Again u s i n g the method of c r o s s - h y b r i d i z a t i o n Prasad (1988) and Prasad and B a i l l i e (1989) were able to de t e c t nine conserved DNA fragments c o n t a i n e d w i t h i n a 150 Kb region of DNA near the unc-22 gene on chromosome IV of C. elegans. These authors demonstrated that seven of the c r o s s - h y b r i d i z i n g fragments c o n t a i n e d genes. In a d d i t i o n , Heschl (1988) used t h i s method to a i d i n the i d e n t i f i c a t i o n of the 5' r e g u l a t o r y r e g i o n of the hsp70 gene. Hence, the method of c r o s s s p e c i e s h y b r i d i z a t i o n • b e t w e e n C. elegans and C. briggsae DNA has proven to be a f a s t and e f f i c i e n t means to i d e n t i f y coding elements. The method of c r o s s - s p e c i e s h y b r i d i z a t i o n as a means to i d e n t i f y coding elements can a l s o be a p p l i e d to d i s t a n t l y r e l a t e d organisms. Thus, one of the goals of t h i s t h e s i s was Table 1 PROBES FOR POLYMORPHIC SITES Plasmid N2 s i z e (Kb) 1 BO s i z e (Kb) 1 S i t e pCeh51 2 2.4 4.0 hP5 p C e s l 8 3 6.0 7.6 sPl pCeh52 2 3.2 4.8 hP7 pCeh50 2 2.0 3.6 hP4 pCeh48 2 0.8 2.4 hP6 S i z e of EcoRI fragment on genomic Southern b l o t s . I d e n t i f i e d by J . McDowall i n S t a r r et al. ,( 1989). I d e n t i f i e d by K. Beckenbach i n Rose et al . , (1982). 12 to undertake a comprehensive survey u s i n g a set of contiguous coding elements from C. el egans to search f o r conserved re g i o n s i n mammalian DNA. The dpy-14 Region of chromosome I The gene dpy-14 maps near the middle of the chromosome I gene c l u s t e r and f a l l s w i t h i n the region spanned by sDp2 (Rose et al ., 1984). T h i s gene i s a member of a major c l a s s of mo r p h o l o g i c a l mutants, the Dpy mutants. The Dpy mutants are c h a r a c t e r i z e d by a s h o r t , f a t phenotype. To date 28 d i f f e r e n t Dpy genes have been i d e n t i f i e d (Edgley and R i d d l e , 1987). Two separate c l a s s e s of Dpy mutants have been i d e n t i f i e d . The genes dpy-21, dpy-26, dpy-27, and dpy-28 are i n v o l v e d i n c o n t r o l l i n g X chromosome e x p r e s s i o n (Hodgkin, 1983; Meneely and Wood, 1984; Meyer and Casson, 1986; Hodgkin, 1987). The second c l a s s of Dpy mutations i n c l u d e the c o l l a g e n genes dpy-13 (von Mende et al . , 1988) and dpy-10 (A. Levy and J . Kramer, p e r s o n a l communication). The chromosome I gene c l u s t e r c o n t a i n s the two Dpy mutants dpy-5 and dpy-14, which are separated by 1.5 map u n i t s (mu) (Rose and B a i l l i e , 1979). These mutants can be e a s i l y d i s t i n g u i s h e d by o b s e r v a t i o n with a d i s e c t i n g microscope. U n l i k e the c o l l a g e n coding genes dpy-10 and dpy-13 the cl o n e d dpy-5 gene ( J . B a b i t y , p e r s o n a l communication) and dpy-20 gene (D. C l a r k , p e r s o n a l communication) do not appear to be members of the c o l l a g e n gene f a m i l y . I t w i l l be i n t e r e s t i n g to determine i f the f u n c t i o n of dpy-14 i s s i m i l a r to the f u n c t i o n of dpy-5 or i f 13 i t i s another member of the c o l l a g e n gene f a m i l y a f f e c t i n g c u t i c l e s t r u c t u r e . The dpy-14 region c o n t a i n s a number of genes t h a t a f f e c t muscle s t r u c t u r e . The unc-15 gene has been cloned and shown to code f o r paramyosin (H. Kagawa, p e r s o n a l communication) and mutants i n unc-87 show an abnormal d i s t r i b u t i o n of t h i c k and t h i n f i l a m e n t s (Waterston et al ., 1980). Mutants i n let-75 show abnormal pharyngeal muscle s t r u c t u r e (R. Waterston, p e r s o n a l communication) and t h i s gene maps c o i n c i d e n t with myo-1 (A. Rose; J . S u l s t o n and A. Coulson, p e r s o n a l communication). In a d d i t i o n , dpy-14 mutants are f i r s t - s t a g e l a r v a l l e t h a l s when homozygous f o r the n o n - l e t h a l muscle mutations unc-15 or unc-54 (Rose and B a i l l i e , 1980). As p r e v i o u s l y mentioned, the dpy-14 region i s a p a r t of chromosome I that has undergone e x t e n s i v e g e n e t i c a n a l y s i s . Many e s s e n t i a l genes from the r e g i o n have been i d e n t i f i e d and mapped. Many i n t e r e s t i n g genes a f f e c t i n g muscle s t r u c t u r e and perhaps c u t i c l e formation are i n the r e g i o n . Furthermore, a polymorphic s i t e , sPl, s u i t a b l e f o r i d e n t i f y i n g DNA i n the re g i o n was a v a i l a b l e f o r use. T h i s made the dpy-14 region an i d e a l l o c a t i o n to s t a r t a molecular a n a l y s i s of the chromosome I gene c l u s t e r . The r e s e a r c h d e s c r i b e d i n t h i s t h e s i s was designed to address s e v e r a l d i f f e r e n t q u e s t i o n s p e r t a i n i n g to genes and genomic o r g a n i z a t i o n of a region from chromosome I of C. elegans. The i n i t i a l o b j e c t i v e of t h i s t h e s i s was to use mapped polymorphisms to i d e n t i f y cosmid c o n t i g s from a 3.0 map 14 u n i t i n t e r v a l between the genes dpy-5 and unc-29 on chromosome. I ( F i g . 1). The cosmid DNA obtained from A. Coulson and J . Su l s t o n would be used to e s t a b l i s h a rough alignment between the g e n e t i c and p h y s i c a l maps of the region and to examine the DNA with r e s p e c t t o the amount of DNA per map u n i t i n s e v e r a l d i f f e r e n t r e g i o n s . A second o b j e c t i v e was to l o c a t e and c h a r a c t e r i z e coding elements near the dpy-14 gene u s i n g the method of c r o s s - s p e c i e s h y b r i d i z a t i o n t o C. briggsae (Prasad, 1988; Prasad and B a i l l i e , 1989). Any coding elements i d e n t i f i e d by t h i s method would be used to produce a d e t a i l e d p h y s i c a l and g e n e t i c map of the r e g i o n . The t h i r d o b j e c t i v e was to c r o s s - h y b r i d i z e the i d e n t i f i e d C. elegans coding elements to mammalian DNA i n an attempt to i d e n t i f y e v o l u t i o n a r i l y conserved genes. Any C. elegans gene i d e n t i f i e d i n t h i s manner would be sequenced and compared t o the sequences of other known genes to t r y and e s t a b l i s h i t s f u n c t i o n or c e l l u l a r l o c a t i o n . 15 F i g u r e 1 A PARTIAL GENETIC MAP OF THE UNC-11 UNC-29 INTERVAL OF CHROMOSOME I unc-87 i i dpy-14 let-75 unc-11 dpy-5 unc-37\\ \unc~15 unc-29 i i i I I I I i • • _ ! I I l_J L_J I • • . . L sDp2 ! 1.0 mu ! Based on p u b l i s h e d map d i s t a n c e s : unc-11 dpy-5 (Howell, 1989); dpy-5 unc-29 (McKim et al., 1988^; dpy-14 unc-15 (Rose and B a i l l i e , 1980). The map p o s i t i o n of let-75 (Rose and B a i l l i e , 1980) and unc-14 i s based on data from McKim and Rose, (1989). The f r e e d u p l i c a t i o n sDp2 was d e s c r i b e d by Rose et al ., ( 1984) . 16 I I . MATERIALS AND METHODS 1. Nematode c u l t u r i n q c o n d i t i o n s Nematode s t r a i n s were grown and maintained at 16^C or 20^C on P e t r i p l a t e s c o n t a i n i n g nematode growth media (NGM) s t r e a k e d with E. c o l i OP50 (Brenner 1974). Two s p e c i e s of Ceanorhabdi t is were used i n t h i s study, C. elegans and C. b r i g g s a e . Two w i l d - t y p e C. elegans s t r a i n s were used: var. B r i s t o l s t r a i n N2, and v a r . Bergerac s t r a i n BO. The w i l d - t y p e C. b r i g g s a e s t r a i n used was G16 (Fodor et al . , 1983). The nomenclature used i n t h i s t h e s i s f o l l o w s the recommended g u i d e l i n e s of H o r v i t z et a l . , (1979). S t r a i n s used, along with the names of the i n d i v i d u a l s who c o n s t r u c t e d the s t r a i n s , are l i s t e d i n Appendix 1. The l a b o r a t o r y of o r i g i n i s d e s i g n a t e d by the upper case l e t t e r ( s ) p receding the s t r a i n number. The lower case l e t t e r ( s ) p receding the a l l e l e number desi g n a t e s the l a b o r a t o r y i n which the mutant was i s o l a t e d . For example, BC,s: Dr. D.L. B a i l l i e , Simon F r a s e r U n i v e r s i t y ; KR,h: Dr. A.M. Rose, U n i v e r s i t y of B r i t i s h Columbia; CB,e: Dr. S. Brenner, Cambridge U n i v e r s i t y . 2. C o n s t r u c t i o n of t h r e e - f a c t o r mapping s t r a i n s T h r e e - f a c t o r a n a l y s i s of the polymorphisms was done by c o n s t r u c t i n g recombinant chromosome I c o n t a i n i n g both N2 and BO DNA. In most cases, N2 c / s - l i n k e d double mutant (mm) hermaphrodites were mated with w i l d - t y p e N2 males. The 17 heterozygous males were then mated to wil d - t y p e BO hermaphrodites. Recombinant ( s i n g l y mutant, m) progeny were i s o l a t e d from N2/B0 h y b r i d s s e g r e g a t i n g the doubly marked chromosome. These recombinants were m+/mm. From t h e i r progeny, i n d i v i d u a l s which were homozygous f o r the recombinant chromosome (m+) were i s o l a t e d to generate recombinant s t r a i n s . A flow diagram of t h i s method i s shown i n F i g . 2. The t h r e e - f a c t o r mapping s t r a i n s used i n t h i s t h e s i s are l i s t e d i n Appendix 2. 3. Screening f o r Dpy-14 mutants Two s c r e e n i n g procedures were used to look f o r new a l l e l e s of dpy-14. The f i r s t method was to look f o r Tc1 i n s e r t i o n s i n t o dpy-14. Males of the genotype dpy-14 unc-13 / + + were c r o s s e d to mut-6 hermaphrodites (Mori et al. , 1988). A f t e r mating ov e r n i g h t at 20^C, hermaphrodites were t r a n s f e r r e d to i n d i v i d u a l p l a t e s and the F1 progeny screened fo r Dpy-14 i n d i v i d u a l s i n a background of wi l d - t y p e worms. The F1 progeny from 236 mated hermaphrodites were screened and no Dpy-14 animals were observed. The second s c r e e n i n g method used formaldehyde as the mutagen as d e s c r i b e d by Moerman and B a i l l i e (1980) at a formaldehyde c o n c e n t r a t i o n of 0.1% as recommended by Johnsen and B a i l l i e (1988). A 1% stock s o l u t i o n of formaldehyde was prepared as f o l l o w s : F i v e grams of paraformaldehyde i n 50 mL of d i s t i l l e d water was warmed to 65^C. One molar NaOH was added dropwise u n t i l the s o l u t i o n c l e a r e d and the pH was 18 F i g u r e 2 CONSTRUCTION OF THREE FACTOR-MAPPING STRAINS BO X ml m2 N2 i + V + ! i i + i ml i m2 V s e l f - f e r t i l i z e p i c k recombinants + i m2 ml I ml i m2 s e l f - f e r t i l i z e V i ml j m2 s e l f - f e r t i l i z e V + m2 i i i i i i i i + m2 ml + i i i i — — — — • N f ^ ^ V ^ V / V ' W — — — — — — Aw<VSf<Nf *V I I I I ml + 19 a d j u s t e d to 7.2. The s o l u t i o n was d i l u t e d to 150 mL with d i s t i l l e d water and to 500 mL with M9 b u f f e r . A 0.1% s o l u t i o n was prepared by d i l u t i n g the 1% stock s o l u t i o n with M9 b u f f e r . One hundred and f i f t y w i l d - t y p e N2 males were suspended i n a 0.1% s o l u t i o n f o r four hours at 20^C. The worms were removed from the formaldehyde, t r a n s f e r r e d to P e t r i p l a t e s and allowed to recover f o r two hours at 20^C. The males were t r a n s f e r r e d to a mating p l a t e c o n t a i n i n g 9J0 dpy-5 dpy-14 unc-13 hermaphrodites. A f t e r mating o v e r n i g h t the hermaphrodites were t r a n s f e r r e d to i n d i v i d u a l P e t r i p l a t e s . The F1 progeny were screened f o r Dpy-14 animals i n a background of w i l d - t y p e and Dpy-5 Dpy-14 Unc-13 worms. One dpy-14 (h572) / dpy-5 dpy-14 unc-13 i n d i v i d u a l was observed from 1,154 o u t c r o s s e d progeny. From t h i s i n d i v i d u a l the s t r a i n KR1000 (+ (h572) + / dpy-5 dpy-14^unc-13) was e s t a b l i s h e d . 4. Complementation t e s t i n g using hDf8 The mutation h572 has been shown to be a d e l e t i o n which i s c a l l e d hDf8. Mutations were t e s t e d f o r complementation with hD/8. Heterozygous males c o n t a i n i n g the mutation to be t e s t e d were mated to hermaphrodites of the genotype dpy-5 + unc-13 / + hDf8 + or dpy-5 + unc-75 / + hDf8 unc-75. Mated hermaphrodites were brooded and the p l a t e s screened f o r a p a r t i c u l a r phenotype i f a v i s i b l e mutation was being t e s t e d , or f o r the presence or absence of dpy-14 progeny i f a l e t h a l mutation was being t e s t e d . 20 5. Cosmids used A l l cosmids used i n t h i s study were p r o v i d e d by A. Coulson and J . S u l s t o n at the MRC, Cambridge, England. Kanamycin r e s i s t a n t cosmids were l o r i s t 2 cosmids (Cross and L i t t l e , 1986; Gibson et a l . , 1987). A m p i c i l l i n r e s i s t a n t cosmids were pJB8 cosmids (Ish-Horowicz and Burke, 1981). 6. DNA i s o l a t i o n 6.1 Nematode genomic DNA i s o l a t i o n C. el egans genomic DNA was prepared by a method m o d i f i e d by Curran and B a i l l i e ( p e rsonal communication) from that of Emmons et al . ( 1979). C. elegans s t r a i n s were grown on 60 mm P e t r i p l a t e s u n t i l c u l t u r e s were crowded. F i v e mm square pads were cut and t r a n s f e r r e d to 100 mm P e t r i p l a t e s c o n t a i n i n g NGM, 0.7% agarose, and s t r e a k e d with w i l d - t y p e E. c o l i . C u l t u r e s were incubated at 20^C f o r f i v e t o ten days u n t i l the p l a t e s were crowded with a d u l t worms. The worms were washed from the p l a t e s with 0.5% NaCl and t r a n s f e r r e d t o 50 mL Fal c o n tubes. The worms were p e l l e t e d at 2,000 rpm f o r 10 minutes, r i n s e d once with 10 mL of 0.5% NaCl, and p e l l e t e d a t 2,000 rpm f o r 10 minutes. The worm p e l l e t was resuspended i n 5 mL of p r o t e i n a s e K b u f f e r , incubated with 5 mg p r o t e i n a s e K at 65^C f o r 10 minutes or u n t i l the worms were d i s s o l v e d . The volume was i n c r e a s e d to 15 mL with p r o t e i n a s e K b u f f e r and g e n t l y mixed f o r one hour with 15 mL w a t e r - s a t u r a t e d phenol. The mixture was spun at 2,000 rpm f o r 10 minutes and the aqueous 21 l a y e r removed l e a v i n g the i n t e r f a c e behind. The phenol e x t r a c t i o n was repeated one to two more times u n t i l the i n t e r f a c e was c l e a r of c e l l u l a r d e b r i s . The aqueous l a y e r was g e n t l y mixed f o r one minute with 15 mL of Sevag ( c h l o r o f o r m 24:1 isoamyl a l c o h o l ) , c e n t r i f u g e d a t 2,000 rpm f o r 5 minutes and t r a n s f e r r e d to a new tube. The DNA was p r e c i p i t a t e d by adding 1/10 volume of 4 M NH^OAc, one volume of i s o p r o p a n o l and g e n t l y i n v e r t i n g . The DNA was p u l l e d out of the s o l u t i o n with a s e a l e d 1 mL p i p e t t e , r i n s e d with 70% e t h a n o l , d i s s o l v e d i n 1 mL of 1X TE c o n t a i n i n g 20 ug RNase A, g e n t l y i n v e r t e d o v e r n i g h t at room temperature, and s t o r e d at 4^C. P r o t e i n a s e K b u f f e r NGM medium 0.1 M T r i s (pH 8.0) 17 g agar 0.05 M EDTA (pH 8.0) 2.5 g bacto peptone 0.2 M NaCl 3.0 g NaCl 1% SDS 1 mL c h o l e s t e r o l 0.001 M C a C l 2 0.001 M MgS0 4 0.025 M KH 2P0 4 (pH 6.0) Water to 500 mL 6.2 Yeast genomic DNA i s o l a t i o n ( J . S t i l e s , 1983) The yeast c l o n e s (YAC 11, YAC 58) c o n t a i n i n g N2 genomic DNA i n s e r t e d i n t o yeast a r t i f i c i a l chromosomes were pr o v i d e d by R. Waterston. S i n g l e c o l o n i e s were p i c k e d from YPD p l a t e s 22 and t r a n s f e r r e d to 50 mL of YPD media. The c e l l s were incubated f o r 18 hours at 30^C with shaking. The c e l l s were p e l l e t e d at 2,500 rpm f o r 10 minutes and resuspended i n 3.5 mL of s o l u t i o n 1. To t h i s 0.1 mL of l y t i c a s e (2.5 mg/mL) was added and incubated at 37^C f o r one hour. The c e l l s were p e l l e t e d at 2,500 rpm f o r 10 minutes, resuspended i n s o l u t i o n 2, mixed, and incubated f o r 30 minutes at 65^C. To t h i s 0.8 mL of 5 M KOAc was added and incubated on i c e f o r one hour. The c e l l u l a r d e b r i s was p e l l e t e d at 2500 rpm f o r 10 minutes and the supernatant was removed. The supernatant was e x t r a c t e d twice with an equal volume of w a t e r - s a t u r a t e d phenol and once with sevag. The DNA was p r e c i p i t a t e d by adding 1/10 volume of 4 M NH4OAc and one volume of i s o p r o p a n o l and l e f t at -20^C f o r 30 minutes. The DNA was p u l l e d out of s o l u t i o n w i t h a s e a l e d 1 mL p i p e t t e , r i n s e d with 70% e t h a n o l , and d i s s o l v e d i n 500 uL of 1X TE c o n t a i n i n g 20 ug/mL RNase A. Samples were s t o r e d at 4°C. YPD medium 1% Bacto yeast e x t r a c t 2% Bacto peptone 2% Bacto dextrose YPD p l a t e s 1% Bacto yeast e x t r a c t 2% Bacto peptone 2% Bacto dextrose 2% Bacto agar 23 s o l u t i o n 1 s o l u t i o n 2 1 M s o r b i t o l 3.2 mL 1X TE (pH 7.5) 0.1 M EDTA 320 uL 0.5 M EDTA (pH 8.0) 14 mM beta-mercaptoethanol 160 uL 2 M T r i s (pH 8.0) 160 uL 10% SDS 10 uL d i e t h l y pyrocarbonate 6.3 plasmid and cosmid DNA i s o l a t i o n 6.3.1 m i n i - p r e p a r a t i o n (Davis et al . , 1980) S i n g l e c o l o n i e s were p i c k e d from L b r o t h p l a t e s and t r a n s f e r r e d to 10 mL of L b r o t h c o n t a i n i n g 50 ug/mL of the a p p r o p r i a t e a n t i b i o t i c . The c e l l s were incubated at 37^C f o r 18 hours. C e l l s were p e l l e t e d at 2000 rpm f o r 10 minutes, resuspended i n 300 uL of 50 mM sucrose; 10 mM EDTA; 25 mM T r i s (pH 8.0) c o n t a i n i n g 5 mg/mL lysozyme (Boehringer Mannheim), and t r a n s f e r r e d to a 1.9 mL Eppendorf tube. A f t e r a f i v e minute i n c u b a t i o n at room temperature 400 uL of 0.2 M NaOH; 1% SDS was added, v i g o r o u s l y i n v e r t e d , and p l a c e d on i c e f o r f i v e minutes. To t h i s mixture 300 uL of c o l d KOAc s o l u t i o n (see below) was added, v i g o r o u s l y i n v e r t e d , and p l a c e d on i c e f o r f i v e minutes. The c e l l u l a r d e b r i s was p e l l e t e d at 12,000 rpm i n a m i c r o c e n t i f u g e (Eppendorf 5415) f o r f i v e minutes. The aqueous l a y e r was removed and e x t r a c t e d twice with 850 uL w a t e r - s a t u r a t e d phenol and once with Sevag. The DNA was p r e c i p i t a t e d by adding 850 uL i s o p r o p a n o l and p e l l e t e d at 12,000 rpm f o r 10 minutes. The DNA p e l l e t was washed twice 24 with 70% e t h a n o l , a i r d r i e d , and d i s s o l v e d i n 100 uL 1X TE c o n t a i n i n g 20 ug RNase A (BRL). Samples were s t o r e d at -20°C. KOAc s o l u t i o n 3 M potassium a c e t a t e 11.6% g l a c i a l a c e t i c a c i d (58 mL/500 mL) Water to 500 mL 6.3.2 Large s c a l e plasmid and cosmid DNA i s o l a t i o n (Davis et al . , 1980) S i n g l e c o l o n i e s were p i c k e d from L broth p l a t e s and t r a n s f e r r e d to 10 mL of L broth c o n t a i n i n g 50 ug/mL of the a p p r o p r i a t e a n t i b i o t i c . The c e l l s were incubated at 37^C f o r 18 hours. The c u l t u r e was t r a n s f e r r e d to 500 mL of f r e s h L broth c o n t a i n i n g 50 ug/mL a m p i c i l l i n or kanamycin and incubated o v e r n i g h t at 37^C with shaking. The c e l l s were t r a n s f e r r e d to 250 mL Beckman tubes, c e n t r i f u g e d at 7,000 rpm fo r 10 minutes, the supernatant d i s c a r d e d , the p e l l e t resuspended i n 5 mL of l y s i s b u f f e r , and t r a n s f e r r e d to a 45 mL Beckman tube. The mixture was incubated at room temperature f o r f i v e minutes, then 20 mL of 0.2 M NaOH; 1% SDS was added, i n v e r t e d v i g o r o u s l y , and c o o l e d i n an ice-water bath f o r 10 minutes. To t h i s 10 mL of c o l d s o l u t i o n I was added, i n v e r t e d v i g o r o u s l y , and incubated on i c e f o r 10 minutes. A f t e r c e n t r i f u g i n g at 16,000 rpm f o r 20 minutes at 4^C, 36 mL of the supernatant was t r a n s f e r r e d to a c l e a n tube. 25 The DNA was p r e c i p i t a t e d and p e l l e t e d by adding 24 mL of i s o p r o p a n o l , g e n t l y i n v e r t i n g , s t o r i n g at -20^C f o r 30 minutes, and p e l l e t i n g at 2000 rpm f o r 10 minutes. The p r e c i p i t a t e was washed twice with 70% e t h a n o l . The p e l l e t was d i s s o l v e d i n 5 mL of 1X TE; 7.5 mL water s a t u r a t e d C s C l ; 0.65 mL ethidium bromide (10 mg/mL), t r a n s f e r r e d to a 12 mL heat s e a l a b l e tube, and c e n t r i f u g e d at 60,000 rpm f o r 18 hours at 15^C. The DNA band was removed, t r a n s f e r r e d t o a c l e a n tube and r e p e a t e d l y e x t r a c t e d with an equal volume of water-s a t u r a t e d butanol u n t i l the aqueous l a y e r was f r e e of ethidium bromide. The aqueous l a y e r was mixed with two volumes of water, s i x volumes of 95% e t h a n o l , and l e f t at -20^C f o r one hour. The DNA was c e n t r i f u g e d at 2,000 rpm f o r 10 minutes, the p e l l e t was r i n s e d with 70% e t h a n o l , d i s s o l v e d i n 1 mL of 1X TE, and s t o r e d at 4°C. L y s i s B u f f e r S o l u t i o n 1 25 mM T r i s (pH 8) 3 M potassium a c e t a t e 10 mM EDTA (pH 8) 11.6 % g l a c i a l a c e t i c a c i d 50 mM sucrose 5 mg/mL lysozyme (Boehringer Mannheim) added j u s t p r i o r to use. 6.4 Phage DNA i s o l a t i o n ( M a n i a t i s et al. , 1982) 6.4.1 Bacteriophage lambda g t l O and Charon 4A F i v e mL of L broth was i n o c u l a t e d with E. col i , s t r a i n C600. The c e l l s were incubated at 37°C f o r 18 hours. The 26 c e l l s were p e l l e t e d at 2,000 rpm f o r f i v e minutes, the supernatant d i s c a r d e d , and the c e l l s resuspended i n 2.5 mL of 10 mM MgCl2. The resuspended c e l l s (1.1 mL) were i n f e c t e d with 110 uL of phage ( d i l u t e d to 1x10** plaque forming u n i t s {pfu} with X d i l u t i o n b u f f e r {10 mM T r i s [pH 7.5]; 10 mM MgCl 2})f and incubated at 37°C f o r 10 minutes. The i n f e c t e d c e l l s were mixed with 3 mL of top agarose, poured onto L b r o t h p l a t e s , and incubated o v e r n i g h t at 37^C. F i v e mL of X d i l was poured on top of each c o n f l u e n t l y s e d p l a t e , and incubated at 4^C o v e r n i g h t . The X d i l o v e r l a y was c o l l e c t e d , c e n t r i f u g e d at 10,000 rpm f o r 10 minutes, and the supernatant (phage stock) s t o r e d at 4°C. To 80 mL of phage stock 8.0 g of p o l y e t h y l e n e g l y c o l (PEG) and 4.5 g NaCl was added. The mixture was g e n t l y i n v e r t e d at room temperature u n t i l a l l the s a l t d i s s o l v e d , then incubated at 4^C f o r three hours. The r e s u l t i n g white p r e c i p i t a t e was c e n t r i f u g e d at 10,000 rpm f o r 10 minutes and resuspended i n 9.0 mL of X d i l . To t h i s 6.75 g of C s C l was added, mixed, t r a n s f e r r e d t o a 12 mL heat s e a l a b l e tube, and c e n t r i f u g e d at 60,000 rpm f o r 18 hours i n a Beckman Ti70 r o t o r . The phage band was removed with a 1 mL s y r i n g e , t r a n s f e r r e d to a 1.5 mL microfuge tube, and s t o r e d at 4^C. A 50 uL a l i q u o t of the phage i n C s C l was mixed with 5 uL of 2 M T r i s ; 0.2 M EDTA (pH 8.0), 50 uL formamide, and l e f t f o r two hours at room temperature. The DNA was p r e c i p i t a t e d and p e l l e t e d by adding 50 uL water and 300 uL 95% e t h a n o l , mixing, then c e n t r i f u g i n g at 12,000 rpm f o r 5 seconds. The 27 p r e c i p i t a t e d DNA was r i n s e d twice with 70% e t h a n o l , a i r d r i e d , and d i s s o l v e d i n 50 uL 1X TE. 6.4.2 Lambda ZAP DNA i s o l a t i o n DNA from lambda ZAP phage was i s o l a t e d as plasmid DNA by the p r o t o c o l recommended by the lambda ZAP manufacturer, Stratagene. Two hundred uL of E. c o l i , s t r a i n BB4, was mixed with 1 X 10 5 lambda ZAP phage and 1 X 10 7 R408 h e l p e r phage. The c e l l s were incubated f o r 15 minutes at 37^C. A f t e r i n i t i a l i n f e c t i o n 5 mL of 2X YT medium was added and incubated with shaking f o r four hours at 37^C. The c e l l s were heated f o r 20 minutes at 70^C, then spun f i v e minutes at 2,000 rpm. The supernatant was decanted and s t o r e d (as a plasmid packaged i n the F1 phage p a r t i c l e ) at 4^C. To rescue the packaged plasmid 200 uL of BB4 c e l l s were mixed with 200 uL of supernatant packaged plasmid and incubated f o r 15 minutes at 37^C. A f t e r i n c u b a t i o n 10 uL was p l a t e d on L b r o t h p l a t e s c o n t a i n i n g 50 ug/mL a m p i c i l l i n and incubated o v e r n i g h t at 37^C. S i n g l e c o l o n i e s from these p l a t e s were used to i s o l a t e p lasmid DNA as d e s c r i b e d i n s e c t i o n II—6.3.1 and II-6.3.2. 7. Aqarose-qel e l e c t r o p h o r e s i s R e s t r i c t i o n enzyme d i g e s t e d genomic, plasmid, and cosmid DNA was s i z e f r a c t i o n a t e d by means of agarose g e l e l e c t r o p h o r e s i s . Agarose, d i s s o l v e d i n 1X TBE b u f f e r (0.5% to 1.2% W:V) c o n t a i n i n g 1 ug e t h i d i u m bromide per mL of g e l , was poured i n t o a h o r i z o n t a l mold and allowed to s o l i d i f y at room 28 temperature. The s o l i d i f i e d g e l s were submerged i n 1X TBE b u f f e r . The DNA was e l e c t r o p h o r e s e d at e i t h e r 30 v o l t s f o r 18 hours f o r l a r g e g e l s (15cm x 20cm) or at 90 v o l t s f o r one to two hours f o r small g e l s (6cm x 10cm). The DNA was v i s u a l i z e d and photographed using a 300 nm t r a n s i l l u m i n a t o r . IX TBE b u f f e r 0.89 M T r i s 0.89 M b o r i c a c i d 0.00.1 M EDTA (pH 8.0) 8. Subcloning of DNA from cosmids (Davis et al. , 1980) One ug of cosmid DNA and 0.2 ug of plasmid DNA was added to a s o l u t i o n c o n t a i n i n g 4.0 uL of r e s t r i c t i o n enzyme b u f f e r , 4.0 uL 10X BSA, 2 u n i t s of r e s t r i c t i o n enzyme, and d i s t i l l e d water (to b r i n g the t o t a l r e a c t i o n volume to 40 uL). The mixture was incubated f o r two hours at 37^C, then f o r 10 minutes at 65^C. A 10 uL a l i q u o t was removed f o r e l e c t r o p h o r e s i s as d e s c r i b e d ( s e c t i o n I I - 7 ) , to check that the d i g e s t i o n was complete. E i t h e r pUCl9 (Yanish- Perron et al. , 1985) or b l u e s c r i p t (Stratagene) were the plasmids used f o r s u b c l o n i n g . A 10 uL a l i q u o t of the d i g e s t e d DNA was added to a s o l u t i o n c o n t a i n i n g 4.0 uL of 10X l i g a t i o n b u f f e r (500 mM T r i s , 100 mM M g C l 2 ) , 4.0 uL of 10X BSA (1 mg/mL), 4.0 uL of 10 mM ATP, 4.0 uL of 0.1 M d i t h i o t h r e i t o l , 1 u n i t T4 DNA l i g a s e 29 (BRL), 4.0 uL water. T h i s l i g a t i o n mixture was incubated f o r 18 hours at 16°C. Ten uL of the above l i g a t i o n mixture was added to 100 uL of competent E. c o l i c e l l s , s t r a i n JM83 or D5a and incubated f o r 60 minutes at O^C. A f t e r i n c u b a t i n g f o r 10 minutes at 42^C, 1 mL of s t e r i l e L broth was added and incubated f o r 45 minutes at 37^C. A 50 uL a l i q u o t was p l a t e d onto X-gal p l a t e s and incubated f o r 18 hours at 37^C. S i n g l e white c o l o n i e s were p i c k e d and streaked on L broth p l a t e s c o n t a i n i n g 50 ug/mL a m p i c i l l i n . S i n g l e c o l o n i e s from these p l a t e s were used to prepare DNA ( s e c t i o n II-6.3.1; I I - 6 . 3 . 2 ) . 9. Southern t r a n s f e r and h y b r i d i z a t i o n (Southern, 1975) Approximately 3 to 5 ug of C. el egans genomic DNA was d i g e s t e d u s i n g c o n d i t i o n s as recommended by the enzyme manufacturer i n a volume of 80 uL. A f t e r d i g e s t i o n f o r 18 hours at 37^C the samples were heated f o r 10 minutes at 65^C. Si x t e e n uL of l o a d i n g b u f f e r (30% g l y c e r o l i n water; 0.25% bromophenol blue) was added and thoroughly mixed. A f t e r e l e c t r o p h o r e s i s ( s e c t i o n II-7) the g e l was submerged at room temperature i n 0.25 M HCI f o r 15 minutes, r i n s e d i n d i s t i l l e d water, submerged i n 0.5 M NaOH; 1.5 M NaCl f o r 45 minutes, r i n s e d with d i s t i l l e d water, and submerged i n 1 M NH^OAc f o r 30 minutes. The t r e a t e d g e l was p l a c e d on a 3MM Whatman paper wick that was soaked i n 10X SSC. A Nytran f i l t e r cut to the s i z e of the g e l and which had been soaked i n 1 M NH^OAc f o r 15 30 minutes was l a y e r e d on top of the g e l . Any a i r bubbles were c a r e f u l l y removed. On top of the Nytran were p l a c e d two 3MM Whatman f i l t e r s wetted with 1 M NH^OAc and then a two inch stack of paper towels. The DNA was allowed to t r a n s f e r to the Nytran . f i l t e r by c a p i l l a r y a c t i o n f o r two hours. The f i l t e r was removed, a i r d r i e d , and baked at 80^C f o r one hour. The baked f i l t e r was t r a n s f e r r e d to a h e a t - s e a l a b l e bag and approximately 1 mL of 5X SSPE; 0.3% SDS (prewarmed to h y b r i d i z a t i o n temperature) was added f o r each 20 cm 2 of Nytran ( i e . 10 mL f o r a f i l t e r 15 cm X 13 cm). To t h i s approximately 10 ng of ^ 2 P - l a b e l l e d probe ( s e c t i o n 11-13) was added. The h y b r i d i z a t i o n and wash c o n d i t i o n s v a r i e d and the d i f f e r e n t c o n d i t i o n s used are d e s c r i b e d i n s e c t i o n 11-11. The f i l t e r s were washed twice f o r f i v e minutes i n prewarmed wash s o l u t i o n (2X SSPE, 0.3% SDS or 0.5X SSC, 0.3% SDS) then once f o r 60 minutes i n prewarmed wash s o l u t i o n . The f i l t e r s were removed, a i r d r i e d f o r at l e a s t one hour, s e a l e d i n p l a s t i c wrap, and exposed to Kodak RP or AR f i l m u s ing DuPont L i g h t n i n g Plus i n t e n s i f y i n g screens f o r 2 to 18 hours at -70^C. 10. Northern t r a n s f e r and h y b r i d i z a t i o n ( M a n i a t i s et al . , 1982) RNA i s o l a t e d from the d i f f e r e n t l a r v a l stages of C. elegans was provided by T. Snutch and D.L. B a i l l i e . T o t a l RNA was p r o v i d e d by S. Prasad and D.L. B a i l l i e . Northern f i l t e r s c o n t a i n i n g RNA from the d i f f e r e n t l a r v a l stages were prepared by S. Prasad. 31 F i f t e e n ug of C. elegans RNA was denatured i n 2.2 M formaldehyde; 50% d e - i o n i z e d formamide; 1X MOPS f o r 15 minutes at 60^C. The RNA was e l e c t r o p h o r e s e d i n a 1.1% agarose g e l (6 cm X 10 cm) c o n t a i n i n g 1X MOPS; 2.2 M formaldehyde (pH 7.0) at 80 v o l t s f o r two to three hours. A f t e r e l e c t r o p h o r e s i s the ge l was soaked twice f o r 15 minutes i n d i s t i l l e d water. The t r e a t e d g e l was p l a c e d on a 3MM Whatman paper wick which was soaked i n 10X SSC. A Nytran f i l t e r cut to the s i z e of the g e l which had been wetted i n 10X SSC was l a y e r e d on top of the g e l . Any a i r bubbles were c a r e f u l l y removed. On top of the Nytran was p l a c e d two 3MM Whatman f i l t e r s wetted with 10X SSC and then a two inch stack of paper towels. The RNA was allowed to t r a n s f e r to the Nytran f i l t e r by c a p i l l a r y a c t i o n o v e r n i g h t . The f i l t e r was removed, a i r d r i e d , and baked at 80^C f o r two hours. The baked f i l t e r was t r a n s f e r r e d to a heat s e a l a b l e bag and p r e h y b r i d i z e d i n 20 mL of 5X SSPE; 0.3% SDS f o r two hours at 68^C. The p r e h y b r i d i z a t i o n s o l u t i o n was d i s c a r d e d and 1 mL of f r e s h h y b r i d i z a t i o n s o l u t i o n (prewarmed to 68^C) was added f o r each 10 cm 2 of Nytran ( i e . 6 mL f o r a f i l t e r 6 cm X 10 cm). Approximately 10 ng of 3 2 P - l a b e l e d probe ( s e c t i o n 11-13) was added. The f i l t e r s were washed twice f o r 5 minutes i n prewarmed 2X SSPE; 0.5 % SDS wash s o l u t i o n then once f o r 60 minutes i n 2X SSPE; 0.5 % SDS at 68^C. The f i l t e r s were removed, a i r d r i e d f o r at l e a s t one hour, s e a l e d i n p l a s t i c wrap, and exposed to Kodak RP or AR f i l m u s i n g DuPont L i g h t n i n g Plus i n t e n s i f y i n g screens f o r 1 to 3 days at -70°C. 32 1X MOPS s o l u t i o n 0.2 M morpholinopropanesulfonic a c i d (pH 7.0) 50 mM sodium a c e t a t e 1 mM EDTA (pH 8.0) 11. DNA h y b r i d i z a t i o n and wash c r i t e r i a D i f f e r e n t h y b r i d i z a t i o n and wash c r i t e r i a were .employed depending on the source of DNA being probed (eg. C. elegans genomic, genomic DNA from other s p e c i e s , and cosmid DNA). A l l h y b r i d i z a t i o n s except h y b r i d i z a t i o n to mammalian DNA were done at 65°C i n 5X SSPE; 0.3% SDS f o r 18 hours. H y b r i d i z a t i o n to f i l t e r s c o n t a i n i n g mammalian DNA was i n the same s o l u t i o n but at 62^C. The f i l t e r s were not p r e h y b r i d i z e d but were wetted with prewarmed h y b r i d i z a t i o n s o l u t i o n added immediately p r i o r to adding the ^ ^ P - l a b e l l e d probe. 11.1 Low s t r i n g e n c y c o n d i t i o n s F i l t e r s were washed twice with shaking f o r f i v e minutes in approximately 200 mL of 2X SSPE; 0.2% SDS prewarmed to 65^C. The f i l t e r s were then washed f o r one hour at 65^C using f r e s h prewarmed wash s o l u t i o n . The f i l t e r s were removed from the wash s o l u t i o n and a i r d r i e d f o r an hour. 11.2 Moderate s t r i n g e n c y F i l t e r s were washed twice with shaking f o r f i v e minutes i n approximately 200 mL of 0.5X SSC; 0.2% SDS prewarmed to 33 65^C. The f i l t e r s were then washed f o r one hour at 65^C using f r e s h prewarmed wash s o l u t i o n . The f i l t e r s were removed from the wash s o l u t i o n and a i r d r i e d f o r an hour. 11.3 High s t r i n g e n c y F i l t e r s were washed twice with shaking f o r f i v e minutes i n approximately 200 mL of 0.2X SSC; 0.2% SDS prewarmed to 65^C. The f i l t e r s were then washed f o r one hour at 65^C using f r e s h prewarmed wash s o l u t i o n . The f i l t e r s were removed from the wash s o l u t i o n and a i r d r i e d f o r an hour. 12. S t r i p p i n g n u c l e i c a c i d s from f i l t e r s 12.1 S t r i p p i n g DNA from Southern b l o t s 3 2 p - l a b e l l e d probe was more e a s i l y removed from f i l t e r s which had not been allowed t o dry out completely. A l l washes were done at room temperature with shaking f o r 10 minutes. The f i l t e r s were f i r s t washed i n approximately 200 mL of 0.5 M NaOH; 1.5 M NaCl, then i n 0.5 M T r i s (pH 7.5); 1.5M NaCl, and f i n a l l y i n 1X SSPE. The f i l t e r s were removed, a i r d r i e d , and s t o r e d at room temperature i n the dark u n t i l r e q u i r e d . F i l t e r s c o u l d r o u t i n e l y be used f i v e times with no n o t i c e a b l e d e p r e c i a t i o n of s i g n a l i n t e n s i t y although o l d e r f i l t e r s tended to show an i n c r e a s e d background l e v e l . 12.2 S t r i p p i n g RNA from Northern b l o t s The Northern f i l t e r s were washed i n approximately 200 mL of 70% d e - i o n i z e d formamide; 0.1X SSPE at 70°C f o r two hours. 34 The f i l t e r s were removed, a i r d r i e d , and s t o r e d at room temperature i n the dark u n t i l r e q u i r e d . 13. O l i q o l a b e l l i n g (Feinberg and V o g e l s t e i n , 1984) T h i r t y uL of g e l p u r i f i e d DNA d i l u t e d to 1 ng/uL with d i s t i l l e d water was b o i l e d i n an 1.5 mL Eppendorf tube f o r 10 minutes. The DNA was removed and immediately c o o l e d i n an ice-water bath f o r 5 minutes. To t h i s was added 10 uL of OLB-A, 5 uL of VOX BSA, one u n i t Klenow (Pharmacia), and 5 uL 32 p_ ATP (3,000 Ci/mmol; 10.0 mCi/mL; New England N u c l e a r ) . The mixture was incubated at 16^C f o r 18 hours and the DNA p u r i f i e d i n a Sephadex G25 (Pharmacia) s p i n column. Immediately p r i o r to use the 3 2 P - l a b e l l e d probe was b o i l e d f o r 10 minutes then c o o l e d i n an ice-water bath f o r f i v e minutes. OLB-A s o l u t i o n A:B:C (1:2.5:1.5) s o l u t i o n A 1.0 mL 1.25 M T r i s (pH 8.0); 0.125 M M g C l 2 18 uL 2-mercaptoethanol 5 uL 0.1 M dTTP (Pharmacia) 5 uL 0.1 M dCTP (Pharmacia) 5 uL 0.1 M dGTP (Pharmacia) 35 s o l u t i o n B 2 M Hepes (pH 6.6) s o l u t i o n C h e x a n u c l e o t i d e s (Pharmacia) suspended i n 1X TE at 90 OD/mL. 14. Screening phage l i b r a r i e s (Davis et a l . , 1980) Four d i f f e r e n t phage l i b r a r i e s were screened by the method of Benton and Davis (1977). A N2 genomic l i b r a r y c o n s t r u c t e d by T. Snutch i n the v e c t o r Charon 4A was p r o v i d e d by T. Snutch and D.L. B a i l l i e . A N2 cDNA l i b r a r y c o n s t r u c t e d i n the v e c t o r lambda gtlO was p r o v i d e d by B. Meyer. An N2 cDNA (24 hour stage) l i b r a r y c o n s t r u c t e d i n the v e c t o r lambda gtlO was p r o v i d e d by J . Ahringer and J . Kimble. An N2 cDNA l i b r a r y c o n s t r u c t e d i n the v e c t o r Lambda ZAP (Stratagene) was p r o v i d e d by R. Barstead and R. Waterston. A l l the l i b r a r i e s were screened i n the same manner, however the method of DNA i s o l a t i o n f o r the l i b r a r i e s was d i f f e r e n t ( s e c t i o n I I - 6 . 4 ) . Phage from stock tubes having a t i t r e of 10^ to 1 0 1 1 plaque forming u n i t s (pfu) per mL were s e r i a l l y d i l u t e d with lambda d i l (10 mM T r i s ; 10 mM MgCl 2) to a t i t r e of 2X10 5 pfu/mL. E. c o l i c e l l s ( s t r a i n C600 or BB4) grown ove r n i g h t at 37°C i n 10 mL of L broth were p e l l e t e d at 2,000 rpm and resuspended i n 5 mL of 10 mM M g C l 2 . Approximately 2,000 p f u were mixed with 100UL of resuspended C600 or BB4 c e l l s and incubated at room temperature f o r 15 minutes. A f t e r 36 i n c u b a t i o n the phage and c e l l s were mixed with 3 mL of melted top agarose (L broth c o n t a i n i n g 0.75 agarose), vortexed, and poured on L broth p l a t e s (100 mm X 15 mm). A f t e r the top agarose had s o l i d i f i e d the p l a t e s were incubated at 37^C ov e r n i g h t . The p l a t e s were then c o o l e d at 4^C f o r at l e a s t one hour. Dry n i t r o c e l l u l o s e f i l t e r s ( S c h l e i c h e r and S c h u e l l ) were l a y e r e d on top of the p l a t e s f o r 5 minutes at room temperature. The f i l t e r s were removed and t r e a t e d with 0.5 M NaOH; 1.5 M NaCl f o r 5 minutes, then with 1 M NH 4OAc f o r 5 minutes. The t r e a t e d f i l t e r s were a i r d r i e d and then baked at 80^C f o r one hour. The baked f i l t e r s were h y b r i d i z e d as d e s c r i b e d i n s e c t i o n II-9 and washed at high s t r i n g e n c y ( s e c t i o n 11-11). Any p o s i t i v e plaques a f t e r autoradiography were p i c k e d from p l a t e s by removing a small plug and t r a n s f e r r i n g the plug to a 1.5 mL tube c o n t a i n i n g 1 mL of SM b u f f e r . The t i t r e of phage i n t h i s tube ranged from 1X10^ to 1X10 7 pfu/mL. The above procedure was repeated with the exc e p t i o n that only 50 to 100 p f u were p l a t e d i n subsequent rounds of s c r e e n i n g . The sc r e e n i n g was repeated u n t i l a l l plaques p l a t e d h y b r i d i z e d to the probe. SM b u f f e r 0.1 M NaCl 0.01 M M g C l 2 0.05 M T r i s {pH 7.5} 0.01% g e l a t i n 37 15. DNA sequencing (Sanger et al., 1977; H a t t o r i and Sakaki, 1986) Plasmid DNA was prepared as d e s c r i b e d i n s e c t i o n I I -6.3.1. C l o s e d c i r c u l a r plasmid DNA was separated by e l e c t r o p h o r e s i s i n low m e l t i n g p o i n t agarose and removed by c u t t i n g a block of agarose from the g e l . To the block of agarose was added 500 uL of 20 mM T r i s ; 1 mM EDTA then heated to 70°C to melt the g e l . The melted agarose was e x t r a c t e d twice with 70°C phenol f o l l o w e d by one e x t r a c t i o n with Sevag. To the aqueous s o l u t i o n was added 1/10 volume of 8 M NH4OAc and one volume of i s o p r o p a n o l to p r e c i p i t a t e the DNA. The p e l l e t was r i n s e d with 70% ethanol and vacuum d r i e d . The p r e c i p i t a t e d DNA was resuspended i n 16 uL of 1 X TE. Approximately 1.0 ug of DNA i n a volume of 16 uL (1X TE) was mixed with 4 uL of 1 M NaOH; 1 mM EDTA and l e f t at room temperature f o r f i v e minutes. To t h i s 2 uL of 2 M NH4OAc (pH 5.4) was added and mixed. The DNA was p r e c i p i t a t e d by adding 150 uL of 95% ethanol and p e l l e t e d f o r 10 minutes i n a m i c r o c e n t r i f u g e . The p e l l e t was r i n s e d twice with 70% e t h a n o l , d r i e d f o r .10 minutes i n a vacuum d e s s i c a t o r , and d i s s o l v e d i n 10 uL d i s t i l l e d water. The denatured plasmid DNA was mixed with 1 uL M13 o l i g o n u c l e o t i d e primer (5 ng/uL), 1.5 uL Klenow b u f f e r and incubated f o r 15 minutes at 60^C. The M13 forward primer was the 17-mer 5'-TCACGACGTTGTAAAAC-3'and the reverse primer was 5'-CAGGAAACAGCTATGAC-3'. A f t e r c o o l i n g to room temperature f o r 15 minutes 1 u n i t of DNA polymerase I (Klenow fragment; Pharmacia; 1 u n i t / uL) and 2.0 uL of -^S-ATP (NEN; 1350 Ci/mM) were added. The mixture was dispensed i n t o four tubes, each c o n t a i n i n g 1.5 uL of the d i f f e r e n t d i d e o x y n u c l e o t i d e mixes (Pharmacia) and incubated f o r 15 minutes at 45^C. One uL of chase s o l u t i o n (0.125 mM f o r each dNTP) was added and incubated f o r 15 minutes at 45^C. To each sample 6.0 uL of l o a d i n g b u f f e r was added, and the samples s t o r e d on i c e u n t i l r e q u i r e d . Immediately p r i o r to l o a d i n g , the samples were heated to 95^C f o r three minutes. Two uL of sample were loaded on a 6% acrylamide-7 M urea g e l and e l e c t r o p h o r e s e d a t 50 watts using 0.5X T r i s - b o r a t e b u f f e r f o r two to four hours. An aluminum p l a t e was clamped onto the sequencing apparatus to maintain a constant heat d i s t r i b u t i o n . A f t e r e l e c t r o p h o r e s i s the g e l was soaked f o r 15 minutes i n 5% a c e t i c a c i d ; 6% methanol, t r a n s f e r r e d to Whatman 3MM paper and vacuum d r i e d f o r 30 minutes at 80^C. The f i l t e r was wrapped i n p l a s t i c and exposed to Kodak RP f i l m f o r one to three days. 10X Klenow b u f f e r 70 mM T r i s (pH 7.5) 0.2 M NaCl 70 mM M g C l 2 1 mM EDTA l o a d i n g b u f f e r 95% formamide 12.5 mM EDTA 0.1% bromophenol blue 0.1% xylene c y a n o l IX TBE b u f f e r 0.89 M T r i s 0.89 M b o r i c a c i d 0.001 M EDTA (pH 8.0) acrylamide-urea g e l 0.15 g b i s - a c r y l a m i d e 2.85 g acrylamide 25.0 g urea 26.7 mL H 20 5 mL 5X TBE \ \ 40 I I I . R e s u l t s 1. ISOLATING DNA FROM THE LGI GENE CLUSTER 1.1 Polymorphisms w i t h i n l i n k a g e group I The probe pCesl8 i d e n t i f i e d the s t r a i n polymorphism sPl and was mapped to LGI (Rose et al. , 1982) near to dpy-14 ( H a r r i s and Rose, 1986). The probes pCeh50, pCeh51, pCeh48, and pCeh52 i d e n t i f i e d the s t r a i n polymorphisms hP4, hP5, hP6, and hP7 r e s p e c t i v e l y and were i s o l a t e d by J . McDowall using the method of Tc1 - l i n k a g e s e l e c t i o n ( B a i l l i e et al. , 1985). A d e s c r i p t i o n of each probe and r e s p e c t i v e polymorphic s i t e are l i s t e d i n Table 1 (page 11). These probes were used to o b t a i n lambda phage, each c o n t a i n i n g approximately 15 Kb of N2 genomic DNA, that were sent to A. Coulson and J . S u l s t o n at the MRC Cambridge, England. 1.2 L o c a l i z a t i o n of the polymorphic s i t e s on LGI To map a polymorphic s i t e w i t h i n LGI, the s t r a i n KR800 (let-(h539) dpy-5 (hl4) unc-29 (h2)/I et - (h5 39) dpy-5 (hl4) unc-29 (H2); sDp2 (I:f)) was used. In t h i s s t r a i n the dpy-5 unc-29 i n t e r v a l c o n s i s t s of BO DNA and sDp2 c o n s i s t s of N2 DNA. If the polymorphic s i t e i s w i t h i n the reg i o n spanned by sDp2 the probe that d e t e c t e d the s i t e would h y b r i d i z e to both N2 and BO DNA; i f the s i t e l i e s o u t s i d e sDp2 the probe would h y b r i d i z e to only BO DNA. An example of t h i s a n a l y s i s i s demonstrated f o r the hP5 s i t e and i s shown i n F i g . 3. The plasmid probe pCeh51 i d e n t i f i e d the s t r a i n polymorphism hP5. T h i s plasmid c o n t a i n e d a 2.4 Kb £coRI fragment that d e t e c t e d bands on Southern b l o t s at 2.4 Kb i n N2 DNA and at 4.0 Kb i n BO DNA. I f hP5 i s w i t h i n the region 41 F i g u r e 3 LOCALIZATION OF THE POLYMORPHIC SITES  TO THE sDp2 REGION OF LGI KR800 m 1 2 3 a) A cartoon of the DNA makeup f o r the s t r a i n KR800. S o l i d bar de r i v e d from N2 DNA. Hatched bar d e r i v e d from BO DNA. b) Southern b l o t s were h y b r i d i z e d with the probe pCeh51 (hP5): lane 1 ) N2; 2) BO; 3 ) KR800. pCeh5l h y b r i d i z e s to both the 2.4 and 4.0 Kb bands in the KR800 lane i n d i c a t i n g t h at i t l i e s w i t h i n the region spanned by sDp2. 42 spanned by sDp2 then pCeh51 would h y b r i d i z e to both the 2.4 Kb N2 and 4.0 Kb BO bands; a s i n g l e 4.0 Kb BO band would be seen i f hP5 l i e s o u t s i d e sDp2. F i g . 3 shows pCeh5l d e t e c t e d both the 2.4 Kb N2 and the 4.0 Kb BO bands, i n d i c a t i n g that hP5 l i e s w i t h i n the region spanned by sDp2. The r e s u l t s f o r the remaining polymorphic probes are l i s t e d i n Table 2. 1.3 Three f a c t o r mapping of the polymorphic s i t e s The polymorphisms were p o s i t i o n e d on the g e n e t i c map using N2/BO recombinant s t r a i n s f o r t h r e e - f a c t o r mapping. For each set of markers chosen many independent recombinant s t r a i n s were e s t a b l i s h e d (Appendix 2 ) . Each of these s t r a i n s has been pr e s e r v e d as a frozen c u l t u r e that can be r e v i v e d and used i n fu t u r e mapping experiments as a d d i t i o n a l polymorphic s i t e s are i d e n t i f i e d . If a polymorphic s i t e i s between the two phenotypic markers, the f r a c t i o n of recombinants showing an N2 or BO band i s an estimate of the g e n e t i c d i s t a n c e between the v i s i b l e mutant markers and the DNA s i t e . I f the s i t e i s o u t s i d e the chosen markers, the type of recombinant determines the r i g h t / l e f t p o s i t i o n of the s i t e r e l a t i v e to one of the two markers. An example of t h i s a n a l y s i s i s demonstrated f o r the s P l s i t e (Rose et al . , 1982). The plasmid probe pCes18 i d e n t i f i e s the s t r a i n polymorphism s P l . T h i s plasmid c o n t a i n s a 1.6 Kb EcoRI H i n d l l l fragment that d e t e c t s bands on Southern b l o t s i n EcoRI d i g e s t e d N2 DNA at 6.0 Kb and at 7.6 Kb in the BO genome due to the i n s e r t i o n of a T d ( H i n ) element i n the BO genome at t h i s s i t e 43 Table 2 REGIONAL LOCALIZATION OF POLYMORPHIC SITES S i t e probe sDp2 hP5 pCeh51 i n sPl pCesl8 i n . hP9 pCehl65 i n hP7 pCeh52 out hP4 pCeh50 out hP6 pCeh48 out Probes f o r the polymorphic s i t e s were h y b r i d i z e d t o the s t r a i n KR800 that c o n t a i n sDp2 (N2 DNA) and two BO chromosome I ' s . Probes that d e t e c t e d both an N2 and BO bands were i n s i d e the i n t e r v a l spanned by sDp2. Probes that d e t e c t e d only a BO band were o u t s i d e the sDp2 i n t e r v a l . 44 (Rose et al. , 1985). Southern b l o t s c o n t a i n i n g DNA from ten Dpy-14 or Unc-13 recombinant s t r a i n s generated from the heterozygous parent dpy-14 unc-13 (N2) / + + (BO) were h y b r i d i z e d with the probe f o r sPl. A l l s i x Unc-13 recombinants {+ (BO) unc-13 (N2)} had the 7.6 Kb BO band; a l l four' Dpy-14 recombinants {dpy-14 (N2) + (BO)} had the 6.0 Kb N2 band. These r e s u l t s p l a c e sPl to the l e f t of unc-13. Southern b l o t s c o n t a i n i n g DNA from three Unc-37 or Dpy-14 recombinant s t r a i n s generated from the heterozygous parent unc-37 dpy-14 (N2) were h y b r i d i z e d with the probe f o r sPl. Both Dpy-14 recombinants {+ (BO) dpy-14 (N2)} had the 6.0 Kb N2 band; the Unc-37 recombinant {unc-37 (N2) + (BO)} had the 7.6 Kb BO band. These r e s u l t s p l a c e sPl to the r i g h t of unc-37. Using 43 recombinant s t r a i n s generated i n the 1 ..8 mu i n t e r v a l between dpy-5 and unc-13, no recombination event has been d e t e c t e d between sPl and dpy-14 (Appendix 2 ) . The r e s u l t s o b t a i n e d with pCesl8 f o r two of these s t r a i n s are shown i n F i g . 4. Table 3 summarizes the three f a c t o r mapping data f o r a l l the polymorphisms. F i g . 4 a l s o shows a common f e a t u r e found on autoradiographs of Southern b l o t s h y b r i d i z e d with Tc1; that of somatic e x c i s i o n . Tc1 e x h i b i t s a high l e v e l of t r a n s p o s i t i o n i n BO s t r a i n s (Moerman and Waterston, 1984; Eide and Anderson, 1985) and i n N2 s t r a i n s ( H a r r i s and Rose, 1986). Since the BO genome c o n t a i n s many more Tc1 elements, an e x c i s i o n band o f t e n appears on autoradiographs c o n t a i n i n g BO DNA that i s 1600 bp s m a l l e r than the main h y b r i d i z a t i o n band. Such an e x c i s i o n band can be seen i n F i g . 4 lane 3. 45 1.4 O r i e n t i n g the polymorphic s i t e s The polymorphic s i t e s d e s c r i b e d above have been mapped with resp e c t t o each other by h y b r i d i z i n g each t o the same set of recombinant s t r a i n s . I f the recombination event that gave r i s e to a s t r a i n f a l l s between a p a i r of polymorphic markers then t h i s event o r d e r s the polymorphic s i t e s on the g e n e t i c map. The polymorphic s i t e s sPl and hP9 were mapped r e l a t i v e to each other u s i n g the recombinant s t r a i n s l i s t e d i n Table 4. Southern b l o t s c o n t a i n i n g EcoRI d i g e s t e d DNA from 12 recombinant s t r a i n s generated from the heterozygous parent dpy-5 bli-4 unc-13 (N2) / + + + (BO) were h y b r i d i z e d with the probes f o r sPl and hP9. Two Unc-13 s t r a i n s were i n f o r m a t i v e f o r o r i e n t i n g sPl and hP9. The genotype of these s t r a i n s i s + + (BO) unc-13 (N2). The probe f o r sPl d e t e c t e d a 7.6 Kb BO band whereas the probe f o r hP9 d e t e c t e d a 5.5 Kb N2 band i n these recombinant s t r a i n s . These r e s u l t s i n d i c a t e t hat the recombination events g i v i n g r i s e t o these Unc-13 s t r a i n s f a l l between sPl and hP9. From t h i s data the g e n e t i c order was determined to be dpy-5 sPl hP9 unc-13. A cartoon of the DNA makeup of the recombinant s t r a i n s i s shown i n F i g . 5. The probe which i d e n t i f i e s hP5 was a l s o h y b r i d i z e d to the recombinant s t r a i n s l i s t e d i n Table 4 ( S t a r r et al., 1989). Using t h i s i n f o r m a t i o n the gen e t i c order was determined to be dpy-5 hP5 bli-4 sPl hP9 unc-13. The polymorphic s i t e s hP4, hP6, and hP7 l i e o u t s i d e of the reg i o n spanned by sDp2, p l a c i n g these s i t e s to the r i g h t of the hP9 s i t e . Probes f o r each of these polymorphic s i t e s were 46 F i g u r e 4 THREE FACTOR MAPPING OF POLYMORPHIC SITES unc-37 dpy-14 N2 BO Dpy-14(D) 1 0 Unc-37(U) 0 1 a) Genomic Southern b l o t s h y b r i d i z e d with the probe pCesl8 ( s P l ) : lane 1) BO; 2) N2; 3) N2/BO unc-37 recombinant; 4) N2/BO dpy-14 recombinant, b) A cartoon of the DNA makeup f o r the N2/BO dpy-14 and unc-37 recombinants. S o l i d bar d e r i v e d from N2 DNA. Hatched bar d e r i v e d from BO DNA. The lower band i n lane 3 i s a Tel e x c i s i o n band. 47 Table 3 THREE-FACTOR MAPPING OF POLYMORPHIC SITES S i t e R e s u l t Genomic l o c a t i o n hP5 dpy-5 (8/15) hP5 (7/15) dpy-14 1.0 mu l e f t unc- 13 sPl dpy-5 (15/15) (sPl, dpy-14) i n s e p a r a b l e f rom (sPl, dpy-14) 25/25 unc-13 dpy-14 unc-37 (3/3) (sPl, dpy-14) hP9 dpy-5 (10/10) (dpy-14, hP9) 0.2 mu l e f t unc- 13 unc-37 (3/3) (dpy-14, hP9) dpy-5 bli-4 (6/14) hP9 (8/14) unc -13 dpy-14 (4/10) hP9 (6/10) unc- 13 hP7 dpy-5 (12/14) hP7 (2/14) unc- 29 0.4 mu l e f t unc -29 hP4 dpy-5 (7/8) hP4 (1/8) unc-29 0.4 mu l e f t unc -29 hP6 dpy-5 (21/22) hP6 (1/22) unc- 29 0.1 mu l e f t unc -29 C a l c u l a t i o n s based on map d i s t a n c e s of dpy-5 unc-13 = 1.8; dpy-14 unc-13 =0.3 (Rose and B a i l l i e , 1979); and dpy-5 unc-29 = 3.0 (McKim et al. , 1988). 48 TABLE 4 MAPPING OF POLYMORPHISMS S t r a i n Phenotype dpy-5 hP5 P a t t e r n bl i-4 sPl hP9 unc- 13 KR1206 Unc-13 BO BO BO X N2 N2 N2 KR1196 Unc-13 BO BO BO x N2 N2 N2 KR1199 Unc-13 BO BO BO X N2 N2 N2 KR1219 Dpy-5 B l i - 4 N2 N2 N2 X BO BO BO KR1207 Unc-13 BO BO BO BO x N2 N2 KR1208 Unc-13 BO BO BO BO x N2 N2 KR1197 Unc-13 BO BO BO BO BO x N2 KR1198 Unc-13 BO BO BO BO BO X N2 KR1203 Unc-13 BO BO BO BO BO X N2 KR1205 Unc-13 BO BO BO BO BO X N2 KR1209 Unc-13 BO BO BO BO BO X N2 KR1220 Dpy-5 B l i - 4 N2 N2 N2 N2 N2 X BO Dpy-5 B l i - 4 or Unc-13 recombinant s t r a i n s were generated from the heterozygous parent dpy-5 bli-4 unc-13 (N2) / + + + (BO). Each x denotes the s i t e of a s i n g l e c r o s s o v e r event. 49 F i g u r e 5 ORIENTING THE POLYMORPHIC SITES sPl AND hP9 dpy-5 bli-4 sPl hP9 unc-13 i i i BO DNA : N2 DNA £coRI d i g e s t e d DNA from the N2/BO recombinants s t r a i n s KR1207 and KR1208 generated from the heterozygous parent dpy-5 bli-4 unc-13 (N2) / + + + (BO) were h y b r i d i z e d with the probes that d e t e c t the sPl and hP9 s i t e s . The probe f o r sPl d e t e c t e d a 7.6 Kb BO band and the probe f o r hP9 d e t e c t e d a 5.5 Kb N2 band i n both s t r a i n s . These r e s u l t s p l a c e d hP9 c l o s e r to unc-13 than sPl. 50 h y b r i d i z e d to e i g h t Dpy-5 and Unc-29 recombinants generated from the heterozygous parent dpy-5 unc-29 (BO) / + + (N2). The recombination event i n one s t r a i n separated a l l three s i t e s from unc-29. Another recombinant s t r a i n separated hP7 from hP4, hP6 and unc-29. The r e s u l t s from t h i s a n a l y s i s demonstrates the g e n e t i c order t o be dpy-5 hP7 (hP4, hP6) unc-29. Combining a l l the mapping data i n t h i s work the genomic order was found to be dpy-5 hP5 bli-4 unc-37 (sPl dpy-14) hP9 unc-13 hP7 (hP4 hP6) unc-29. 1.5 I d e n t i f i c a t i o n of the polymorphic s i t e hP9. Twelve cosmids, r e p r e s e n t i n g over 400 Kb of genomic DNA, were h y b r i d i z e d to Southern b l o t s c o n t a i n i n g EcoRI d i g e s t e d N2 and BO DNA. The cosmid F15C11 d e t e c t e d a RFLP between the N2 and BO s t r a i n s . T h i s s i t e was named hP9. The i n d i v i d u a l EcoRI r e s t r i c t i o n fragment from F15C11 that d e t e c t e d hP9 i s 5.5 Kb i n s i z e and d e t e c t s a 5.5 Kb band i n EcoRI d i g e s t e d N2 DNA and 3.3 and 3.7 Kb bands i n BO DNA ( F i g . 6a). A 0.8 Kb p o r t i o n of the 5.5 Kb fragment was subcloned as plasmid pCeh165. When b l o t s c o n t a i n i n g N2 and BO genomic DNA d i g e s t e d with the enzyme B g l l l were h y b r i d i z e d with pCeh165, bands at 3.8 Kb and 3.2 Kb i n N2 DNA and at 5.5 Kb and 3.2 Kb i n BO DNA were d e t e c t e d ( F i g . 6b). These r e s u l t s are c o n s i s t e n t with the i n t e r p r e t a t i o n t h a t the BO genome c o n t a i n s an i n s e r t i o n at the hP9 s i t e . However, f u r t h e r v e r i f i c a t i o n i s r e q u i r e d . F i g u r e 6 IDENTIFICATION OF THE POLYMORPHIC SITE hP9 51 Genomic Southern b l o t s c o n t a i n i n g DNA from N2 and BO worms were h y b r i d i z e d with the probe pCeh165. a) EcoRI d i g e s t ; lane 1) N2; 2) BO. b) BglII d i g e s t ; lane 1) N2; 2) BO. 52 1.6 I s o l a t i o n of DNA surrounding sPl Table 5 l i s t s a l l the cosmids used i n t h i s work. F i g . 7 shows the r e s u l t s presented i n t h i s s e c t i o n . The lambda phage s1 i d e n t i f i e d a cosmid c o n t i g c o n t a i n i n g the polymorphic s i t e sPl. I n i t i a l l y t h i s c o n t i g c o n s i s t e d of the three cosmids T21G5, C14A12, and TlOBI 1. An attempt was made to extend the sPl c o n t i g s i n c e sPl had not been separated from dpy-14. The l e f t end fragment from the cosmid T21G5 was i d e n t i f i e d and used to probe a yeast a r t i f i c i a l chromosome (YAC) l i b r a r y (Burke et al.., 1987) of N2 genomic DNA c o n s t r u c t e d by R. Waterston and A. Coulson (Coulson et al., 1988). T h i s l i b r a r y was s u p p l i e d by A. Coulson as a set of approximately 2100 yeast c o l o n i e s c o n t a i n e d on three h y b r i d i z a t i o n f i l t e r s . H y b r i d i z a t i o n with the l e f t end fragment of T21G5 d e t e c t e d two p o s i t i v e YAC c l o n e s Y11G8 and Y17H5. These YAC c l o n e s c o u l d be used to l i n k w ith other cosmid c o n t i g s . F o r t u i t o u s l y the YAC c l o n e s Y58 and Y22F2 were si m u l t a n e o u s l y i d e n t i f i e d by A. Coulson and J . S u l s t o n and these two YAC c l o n e s were used to f u r t h e r c h a r a c t e r i z e the cosmids surrounding the sPl s i t e . The YAC c l o n e Y58 l i n k e d the cosmid T10B11 t o the cosmid F15C11. Southern b l o t s c o n t a i n i n g EcoRI d i g e s t s of T10B11 and F15C11 were h y b r i d i z e d with 3 2 P - l a b e l e d T10B11 and F15C11. No o v e r l a p between these two cosmids was d e t e c t e d . Southern b l o t s c o n t a i n i n g yeast DNA from Y58 were h y b r i d i z e d with 3 2 P - l a b e l e d T10B11 and F15C11. Both cosmids h y b r i d i z e d to more than one band c o n f i r m i n g the Y58 l i n k a g e . Table 5 COSMIDS USED A m p i c i l l i n r e s i s t a n t Kanamycin r e s i s t a n t E02D9 C1 4A1 2 E02H2 F1 4G7 KO1B11 T1 3C1 T14D10 T21G5 T10B11 F15C11 T06D11 T04E7 T07C11 F52A8 A l l cosmids were obtained from A. Coulson and J . S u l s t o n , MRC Cambridge, England. 54 F i g u r e 7 A GENETIC AND COSMID MAP OF THE hP5 unc-13 INTERVAL a) (let-83, let-86) dpy-14 unc-87 j Iet-75 dpy-5 hP5 bli-4 unc~37 \ sPl hP9 unc-13 i i i i i i i i • • i i i i i i i I I I i I I I ==== Cloned DNA co n t a i n e d i n mapped cosmids ! Anchor p o i n t s between the g e n e t i c and p h y s i c a l maps b) sPl hP9 T13C1 T21G5 T10B11 E02H2 T14D10 C14A12 F15C11 _____ _Y58~ cosmid DNA 25 Kb ====___ YAC DNA An expanded view of the cosmid and YAC clo n e s from the sPl and hP9 r e g i o n . The amount of DNA i n the YAC clon e Y58 i s not known. 55 The s t r a i n polymorphism hP9 ( s e c t i o n III—1.5) was d e t e c t e d with the cosmid F15C11. The hP9 s i t e maps to the r i g h t of sPJ ( s e c t i o n 111-1.4). Since T10B11 was l i n k e d to F15C11 by Y58 and F15C11 was to the r i g h t of T10B11, t h i s o r i e n t e d the sPl c o n t i g with r e s p e c t to the nearby g e n e t i c markers. From these r e s u l t s T21G5 i s c l o s e r to dpy-14 and F15C11 i s c l o s e r t o unc-13. Subsequent to t h i s A. Coulson and J . S u l s t o n found the cosmid F15C11 overlapped with the cosmid E02H2. E02H2 i s the l e f t - m o s t cosmid i n the c o n t i g c o n t a i n i n g unc-13. Hence, a l l the N2 genomic DNA from sPl to unc-13 has been i d e n t i f i e d and i s o l a t e d i n o v e r l a p p i n g cosmid or YAC c l o n e s . The YAC clone Y22F2 l i n k e d the cosmid T21G5 to the cosmid T14D10. The cosmid T14D10 i s the right-most cosmid from a l a r g e c o n t i g that extends to hP5. Southern b l o t s c o n t a i n i n g EcoRI d i g e s t s of T21G5 and T14D10 were h y b r i d i z e d with 32P-labeled T14D10. Overlap between T14D10 and the two outermost fragments from T21G5 was d e t e c t e d c o n f i r m i n g the Y22F2 l i n k a g e . Hence, a l l the N2 genomic DNA from hP5 to sP1 has been i d e n t i f i e d and i s o l a t e d i n o v e r l a p p i n g cosmid or YAC c l o n e s . 1.7 P o s i t i o n i n g sPl and hP9 r e l a t i v e to the d u p l i c a t i o n s  hDp58 and hDp62 The f r e e d u p l i c a t i o n s hPp58 and hPp62 cover the l e f t r e g i on of LGI and break between dpy-14 and unc-13 (McKim and Rose, 1989). Southern b l o t s c o n t a i n i n g EcoRI d i g e s t e d DNA of s t r a i n s c o n t a i n i n g hDp58 and hPp62 over two BO chromosome I were h y b r i d i z e d with the probes f o r sPl and hP9. As shown i n F i g . 8a 56 the probe f o r sPl h y b r i d i z e d to a 6.0 Kb band i n the N2 lane, a 7.6 Kb band i n the BO lane and both the 6.0 and 7.6 Kb bands i n the hDp62 l a n e . In F i g . 8b the probe f o r hP9 d e t e c t s only the BO bands (3.7 and 3.3 Kb) i n the hDp62 l a n e . These r e s u l t s p l a c e the breakpoint of hDp62 between sPl and hP9. F i g . 8c and 8d show that i n the hDp58 lane both the N2 and BO bands were d e t e c t e d when probed with sPl and hP9 r e s p e c t i v e l y . These r e s u l t s p l a c e the b reakpoint of hDp58 to the r i g h t of hP9. 1.8 R e s t r i c t i o n enzyme map of the cosmids surrounding dpy-14 The seven cosmids, T13C1, T14D10, T21G5, C14A12, T10B11, F15C11, and E02H2 surrounding dpy-14 were r e s t r i c t i o n mapped. These cosmids c a r r y a t o t a l of 208 Kb of DNA and represent a 173 Kb continuous segment of N2 genomic DNA. F i g . 9 shows the merged r e s t r i c t i o n enzyme map of these seven cosmids. The three cosmids T21G5, C14A12, and T10B11 were r e s t r i c t i o n mapped with the enzyme EcoRI. The remaining four cosmids T13C1, T14D10, F15C11, and E02H2 were r e s t r i c t i o n mapped with the enzyme Pstl. The merged r e s t r i c t i o n map was generated as f o l l o w s : 1) The r e g i o n of o v e r l a p between cosmids was determined by h y b r i d i z i n g cosmids to Southern b l o t s c o n t a i n i n g s e t s of cosmids; 2) the r e g i o n of o v e r l a p between cosmids and phage, obtained from phage walks i n the r e g i o n by N. Mawji and myself, was determined by h y b r i d i z i n g 3 2 p - l a b e l l e d phage to Southern b l o t s c o n t a i n i n g s e t s of cosmids; and 3) the order of fragments w i t h i n a cosmid was determined by i s o l a t i n g i n d i v i d u a l BamHI, EcoRI, Pstl, SacI, and Sail F i g u r e 8 POSITIONING hDP58 AND hDp62 RELATIVE TO sPl AND hP9 57 Genomic Southern b l o t s of EcoRI d i g e s t e d DNA from N2, BO, hDp58 (KR1775) and hDp62 (KR1758) were h y b r i d i z e d with the probes f o r sPl and hP9. The genotype of the two d u p l i c a t i o n s t r a i n s are l i s t e d i n appendix 1. a) probed with sPl ; lane 1) N2; 2) hPp62; 3)B0. b) probed with hP9; lane 1) N2; 2) hPP62; 3)B0: c) probed with sPl ; lane 1) N2; 2) hPp58; 3)B0. d) probed with hP9; lane 1) N2; 2) hPP58; 3)B0. 58 Figure 9 MERGED RESTRICTION ENZYME WAP OF  THE COSMIDS NEAR DPY-14 1 1 10 Kb A B C D E F G B I J K L M N O P Q R S T U (V XY)Z a b c d(efg)h i j k l m PPP P P P P P E E EE E E E EE E E E * EEE E E E E E EEE E E EE EE E m 1 1 1 1 1 1 1 111 1 1 111 11 i n 1 1 111 i n 111111 1 PPP p i n 1 T13C1 E E EE E E 1 1 11 1 1 E EE E 1 111 T21G5 E E E EEE E E EE EE E 111 i n 111111 1 TlOBll P P 1 1 P E 1 1 T14D10 E E EEE E E E E E EEE E 11 i n 11 111 i n 1 C14A12 Above the five cosmids T13C1, T14D10, T21G5, C14A12, and TlOBll is the merged restriction map of the contiguous region. For convenience each fragment has been given a single letter label. In some instances the odering of fragments was not uniquely determined and this is denoted by brackets ( ) around the fragment letters. P: Pstl, E: EcoRI A merged restriction map joining the cosmid TlOBll to the cosmid F15C11 was not possible because there was a YAC clone joining them. The merged restriction map for the cosmids F15C11 and E02B2 is shown below. o p q P P P P 1 1 1 1 PP 11 P P P P 1 1 1 1 F15C11 E02H2 PP 11 10 Kb 59 fragments, 3 2 P - l a b e l l i n g , and h y b r i d i z i n g to Southern b l o t s c o n t a i n i n g s e t s of cosmids. In some i n s t a n c e s the o r d e r i n g of fragments c o u l d not be u n i q u e l y determined. These fragments are i n d i c a t e d i n parentheses i n F i g . 9. 1.9 Amount of DNA w i t h i n cosmids The cosmid maps generated by A. Coulson and J . S u l s t o n are based on shared H i n d l l l s i t e s and are not a measure of the amount of DNA c o n t a i n e d w i t h i n the cosmids. However, Coulson et al., (1986) d i d estimate that an average cosmid c o n t a i n s 34 Kb of genomic DNA. Table 6 l i s t s 12 cosmids from the dpy-14 unc-13 r e g i o n of LGI. Each cosmid was d i g e s t e d with the enzyme EcoRI, agarose g e l e l e c t r o p h o r e s e d , and the amount of DNA contained t a l l i e d . The 12 cosmids c o n t a i n e d 406 Kb of DNA g i v i n g an average of 33.8 Kb per cosmid. Prasad (1988) estimated that 11 cosmids around the unc-22 gene on LG IV c o n t a i n an average of 35 Kb. Thus the average amount of DNA per cosmid i n the dpy-14 unc-13 i n t e r v a l i s i n good agreement with the average value found f o r cosmids from other r e g i o n s i n the genome. 1.10 Amount of DNA surrounding the polymorphic s i t e s . Phage c o n t a i n i n g N2 genomic i n s e r t s surrounding each polymorphic s i t e were i s o l a t e d (Table 7 ) . Matches to cosmid c o n t i g s were o b t a i n e d with the phage s1, KR#3.2, and KR#51 . I n i t i a l l y the c o n t i g s i d e n t i f i e d by these phage, and consequently anchored to the g e n e t i c map, were not connected to each o t h e r . As the cosmid c l o n i n g p r o j e c t proceeded, a d d i t i o n a l unmapped c o n t i g s were l i n k e d to the hP5 and sPl c o n t i g s and these 60 Table 6 CALCULATION OF THE AVERAGE AMOUNT OF DNA PER COSMID Cosmid Hindlll s i t e s DNA content (Kb) K01B11 10 38.4 E02D9 10 31.4 T1 4D1 0 1 1 33.5 T21G5 10 35.5 C14A12 13 33 . 8 T10B11 15 27.7 F1 5C1 1 13 34.0 E02H2 12 30.7 T04E7 1 1 34.5 T06D11 8 35.1 F52A8 10 36.4 T07C11 12 35.3 T o t a l average 135 12.1 406.3 33.8 Table 7 ISOLATION OF PHAGE SURROUNDING POLYMORPHIC SITES s i t e probe phage o r i g i n hP4 pCeh50 KR#84 T. S t a r r hP5 pCeh5l KR#32 J . Babity hP6 pCeh48 KR#51 T. S t a r r hP7 pCeh52 KR#88 T. S t a r r sPl pCesl8 Si A.M. Rose The o r i g i n of the plasmid probes used to i d e n t i f y the phage l i s t e d i n t a b l e 7 are l i s t e d i n t a b l e 1 (page 11). 62 two c o n t i g s were e v e n t u a l l y l i n k e d t o g e t h e r . The e n t i r e i n t e r v a l from hP5 to unc-13 i s now connected by cosmids. The amount of DNA between polymorphic s i t e s was estimated u s i n g an average value of 34 Kb per cosmid. There are approximately 30 cosmid e q u i v a l e n t s of DNA i n the hP5 to unc-13 i n t e r v a l . T h e r e f o r e the p h y s i c a l d i s t a n c e i s estimated to be 1000 Kb. Between hP5 and sPl t h e r e are 18 cosmid e q u i v a l e n t s of DNA r e p r e s e n t i n g approximately 600 Kb of genomic DNA. The p h y s i c a l d i s t a n c e between sPl and hP9 was not based on the number of cosmids but r e l i e d on a r e s t r i c t i o n enzyme map of t h i s i n t e r v a l and was found to be 110 Kb. Between hP9 and unc-13 there are 9 cosmid e q u i v a l e n t s of DNA r e p r e s e n t i n g approximately 300 Kb of genomic DNA ( F i g . 10). Surrounding the polymorphic s i t e hP6 there are f i v e cosmid e q u i v a l e n t s of DNA r e p r e s e n t i n g 170 Kb of genomic DNA. 63 F i g u r e 10 COSMIDS BETWEEN hP9 AND unc-13^ hP9 unc-13 — I : 1 — E02H2 T07G11 T04E7 T06D11 C44E7 C03C3 C09G6 F_TC3 T07C11 1 D e r i v e d from the cosmid maps of A. Coulson and J . S u l s t o n . 64 2. CODING REGIONS NEAR THE DPY-14 GENE 2.1 I d e n t i f i c a t i o n of conserved regions between C. elegans  and C. briggsae A search f o r p o t e n t i a l coding elements c o n t a i n e d w i t h i n the seven cosmids surrounding the dpy-14 gene was i n i t i a t e d . T h i s search was based on the o b s e r v a t i o n that around the hsp70 gene (heat shock 70 kdal) of C. elegans, the only DNA that c r o s s h y b r i d i z e s with C. briggsae c o n t a i n s the coding element f o r the hsp70 gene (Snutch 1984). Prasad and B a i l l i e (1989) e l e g a n t l y demonstrated i n the unc-22 region of chromosome IV that the DNA fragments from C. elegans cosmids that c r o s s e d h y b r i d i z e d t o C. briggsae DNA con t a i n e d coding elements. Furthermore, these authors r e p o r t that none of the DNA fragments from C. elegans cosmids t h a t d i d not c o n t a i n conserved r e g i o n s were a b l e to d e t e c t . t r a n s c r i p t s on northern b l o t s . Since no a d d i t i o n a l t r a n s c r i p t s were i d e n t i f i e d on northen b l o t s then the method of i d e n t i f y i n g p o t e n t i a l C. elegans coding sequences by c r o s s homology to C. briggsae i s as s e n s i t i v e as t r a n s c r i p t mapping. The b i o l o g i c a l and bio c h e m i c a l nature of most of the genes on LGI i s unknown. Cross s p e c i e s h y b r i d i z a t i o n p r o v i d e s a powerful method f o r i d e n t i f y i n g coding elements of unknown f u n c t i o n s i n c e i t does not r e l y on the r e l a t i v e abundance of mRNA or knowing the time of e x p r e s s i o n . Prasad (1988) showed t h a t DNA sequences between C. elegans and C. briggsae s h a r i n g 80% i d e n t i t y c o u l d be de t e c t e d on Southern b l o t s using low s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s 65 ( s e c t i o n 11-11). Each of the seven cosmids were ^ ^ P - l a b e l l e d and used to probe Southern b l o t s c o n t a i n i n g EcoRI d i g e s t e d DNA from C. elegans, s t r a i n N2, and C. briggsae, s t r a i n G16 under low s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s . From these seven cosmids, r e p r e s e n t i n g 173 Kb of N2 genomic DNA, ten h y b r i d i z a t i o n bands were observed i n C. briggsae. F i g . 11 shows the r e s u l t s with the cosmid T21G5. T h i s cosmid c o n t a i n s 35 Kb of DNA and d e t e c t s 2 bands i n C. briggsae. The r e s u l t s with the remaining cosmids are summarized i n Table 8. When compared to.the N2 bands the s i g n a l i n t e n s i t y of most G16 bands from autoradiographs of N2/G16 Southerns were r e l a t i v e l y weak. Th e r e f o r e each cosmid was l a b e l l e d and h y b r i d i z e d t o N2/G16 f i l t e r s more than once to ensure that the bands were not a r t i f a c t u a l . O c c a s i o n a l l y a band i n the G16 lane was not v i s u a l i z e d i n one autor a d i o g r a p h compared to another. T h i s was not observed when using i n d i v i d u a l g e l - p u r i f i e d 32 p_ l a b e l l e d fragments from the cosmids. To be reasonably c o n f i d e n t t h a t most of the conserved fragments were d e t e c t e d , a l l the EcoRI fragments from the cosmids T13C1 and E02H2 were agarose g e l p u r i f i e d and used as DNA probes. Using the cosmids T13C1 and E02H2 as probes each cosmid h y b r i d i z e d to two bands on Southern b l o t s c o n t a i n i n g C. briggsae DNA. Using the i n d i v i d u a l g e l - p u r i f i e d EcoRI fragments from these cosmids the same bands were d e t e c t e d i n C. briggsae DNA. A f t e r d etermining the number of bands i n G16 each cosmid d e t e c t e d , the s p e c i f i c EcoRI fragment from the cosmid c o n t a i n i n g the conserved r e g i o n was i d e n t i f i e d . T h i s was done because whole 66 F i g u r e 1 1 SCREENING FOR CONSERVED REGIONS BETWEEN  C. elegans AND C. briggsae Genomic Southern b l o t s were h y b r i d i z e d with the cosmid T21G5: lane 1) C. elegans', 2) C. briggsae. Two conserved regions were detected with t h i s cosmid. Table 8 CONSERVED REGIONS BETWEEN C. elegans AND C. b r i z g s a e IN THE REGION NEAR dpy-14 Cosmid l o c u s cosmid cDNA s i z e G16 (Kb) subclone subclone T1 3C1 S P 1 -1 pCehl76 pCeh168 m u l t i p l e T1 3C1 S P 1 -2 pCehl77 NF 1 .9 T14D10 S P 1 -3 pCehl24 pCeh127 4.0 T1 4D1 0 S P 1 -4 pCehl23 pCehl66 6.5 T21G5 S P 1 -5 pCes200 1 pCeh16 2 4.3 T21G5 S P 1 -6 pCeh80 pCehl13 4.8 C1 4A1 2 S P 1 -7 pCeh89 pCeh167 2.3 T1 OB 1 1 S P 1 -8 pCeh8l NF 2.8 + 3.1 F1 5C1 1 S P 1 -9 pCeh114 NF 2.0 E02H2 S P 1 -10 ND pCehl64 4.3 1 i s o l a t e d by K. Beckenbach 2 i s o l a t e d by N. Mawji ND: not done ; NF: not found 68 cosmids c o u l d not be used to screen cDNA l i b r a r i e s due to the shared DNA sequences between the cosmid v e c t o r and lambda phage. F i g . 12 shows the autoradiographs f o r a l l ten conserved fragments. One fragment from the cosmid T13C1 d e t e c t e d m u l t i p l e bands i n both the N2 and G16 lanes under low s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s . The s i z e of the d e t e c t e d G16 bands and the subclone d e t e c t i n g them are l i s t e d i n Table 8 (page 67). 2.2 I d e n t i f i c a t i o n of coding elements using conserved  regions Each conserved fragment was ^ P - l a b e l l e d and used to screen cDNA l i b r a r i e s . Three d i f f e r e n t cDNA l i b r a r i e s were used i n t h i s study ( s e c t i o n 11-14). These l i b r a r i e s were c o n s t r u c t e d by: 1) B. Meyer, MIT; 2) J . A h r i n g e r and J . Kimble, Univ. of Wisconsin; and 3) R. Waterston and R. Barstead, Washington Univ., S t . L o u i s . One conserved fragment, S P 1 - 5 , had been p r e v i o u s l y used t o i d e n t i f y a 0.9 Kb cDNA by N. Mawji (unpublished d a t a ) . From the remaining nine conserved fragments s i x cDNAs were d e t e c t e d from the l i b r a r y of R. Waterston and R. Barstead. The d e t a i l s of each cDNA are l i s t e d i n Table 9. The three conserved fragments not d e t e c t i n g cDNAs i n t h i s l i b r a r y were used to e x t e n s i v e l y screen the 24 hour stage s p e c i f i c l i b r a r y of J . Ahringer and J . Kimble. No cDNAs were d e t e c t e d . Furthermore, these same three conserved fragments were a l s o used to probe northern b l o t s and no RNAs were d e t e c t e d . These r e s u l t s suggest that these three conserved fragments c o n t a i n coding elements that are not h i g h l y expressed, or are stage s p e c i f i c , or are not coding elements. Table 9 CHARACTERISTICS OF CONSERVED REGIONS lo c u s i s o l a t e d RNA1 s i z e (Kb) phage percent cDNA s i z e (Kb) screened sP1-1 0 . 9 UD 1 / 6 0 , 0 0 0 0.002 SP1-2 UD UD 0 / 9 2 , 0 0 0 -SP1-3 1 .1 1 .8 65/16 , 000 0.4 SP1-4 1 . 5 1 .7 1 / 8 , 0 0 0 0.01 SP1 -5 0.9 2 2.0 2 NR -SP1 -6 0.7 2 . 5 1 /8,000 0.01 SP1-7 1.0 1.6 1 /8,000 0.01 SP1 -8 UD UD 0 / 9 2 , 0 0 0 sP1 - 9 UD UD 0/92,000 -sPI - 1 0 1 . 6 •1.7 4 /8,000 0 . 0 5 1 Estimated from northern b l o t s c o n t a i n i n g t o t a l RNA i s o l a t e d from a l l growth stages. 2 N. Mawgi unpublished data UD: undetected NR: not recorded 70 2.3 P o s i t i o n i n g conserved regions on the p h y s i c a l map F i g . 13 shows the p o s i t i o n of the coding elements, conserved fragments which d i d not d e t e c t coding elements and the two polymorphic s i t e s sPl and hP9 on a p h y s i c a l map of the dpy-14 r e g i o n . To s i m p l i f y the nomenclature each conserved fragment was given the name sP1 f o l l o w e d by a number to i d e n t i f y the l o c u s . The conserved r e g i o n f a r t h e s t to the l e f t r e l a t i v e to the g e n e t i c map was give n the number 1. The genomic and cDNA c l o n e s r e p r e s e n t i n g these l o c i are l i s t e d i n Table 8 (page 67). As expected a l l the cDNAs h y b r i d i z e d to the cosmid fragments that were used to i d e n t i f y the cDNAs. However, the three cDNA cl o n e s sPl-5, sPl-6 and sPl-10 h y b r i d i z e d to more than one EcoRI cosmid fragment (see s e c t i o n I I I - 2 . 5 ) . 2.4 R e l a t i v e abundance of i s o l a t e d cDNAs A comparison of the r e l a t i v e abundance of the cDNA c l o n e s i s o l a t e d i n t h i s study shows a 250 f o l d d i f f e r e n c e between the l e a s t abundant (sPl-1) to the most abundant (sPl-3). The cDNA clone i d e n t i f i e d as sPl-1 was i s o l a t e d only once i n 60,000 phage screened. T h i s r e p r e s e n t s approximately 0.002% of the mRNAs present when t h i s l i b r a r y was c o n s t r u c t e d . In c o n t r a s t , the cDNA clo n e i d e n t i f i e d as sPl-3 was i s o l a t e d 65 times i n 16,000 phage screened i n the primary screen. Four of these phage were p u r i f i e d and a l l four h y b r i d i z e d to a s i n g l e 1.8 Kb EcoRI fragment i n N2 DNA. Th e r e f o r e sPl-3 r e p r e s e n t s 71 F i g u r e 12 SCREENING FOR CONSERVED REGIONS BETWEEN C. elegans AND  C. b r i g g s a e USING FRAGMENTS FROM COSMIDS Genomic Southern b l o t s c o n t a i n i n g EcoRI d i g e s t e d DNA from C. elegans ( l e f t lane) and C. b r i g g s a e ( r i g h t lane) were h y b r i d i z e d (low s t r i n g e n c y c o n d i t i o n s ) with agarose g e l p u r i f i e d fragments from the cosmids. Each fragment i s i d e n t i f i e d by the cosmid name fol l o w e d by the EcoRI s i z e (Kb). Panel a) T13C1-1.6; b) T13C1-2.8; c) T14D10-1.8; d) T14D10-3.6; e) T21G5-2.7; f ) T21G5-4.8; g) C14A12-2.2; h) T10B11-3.0; i ) F15C11-2.8; j ) E02H2-5.5 72 F i g u r e 13 PLACING THE CODING ELEMENTS ON THE COSMID MAP. sPl hP9 • n • • • | • • n p - j 1 2 3 4 5 6 7 8 9 10 T13C1 T21G5 TIOBI 1 E02H2 T14D10 C14A12 F15C11 25 Kb • Coding elements n Conserved regions Each i d e n t i f i e d conserved region was h y b r i d i z e d to Southern b l o t s of the cosmids from the dpy-14 r e g i o n . From the r e s u l t i n g data the conserved r e g i o n s were p l a c e d on a p h y s i c a l map of the r e g i o n . To s i m p l i f y the nomenclature each conserved r e g i o n was gi v e the name s P l f o l l o w e d by a number. The conserved region f a r t h e s t to the l e f t was given the number 1. 73 approximately 0.4% (65/16,000) of the mRNAs present at the time of c o n s t r u c t i o n of t h i s l i b r a r y . For comparison the C. elegans a c t i n (provided by M. Krause) and c o l l a g e n probes (provided by J . Kramer) were h y b r i d i z e d to t h i s cDNA l i b r a r y . The four a c t i n genes were found to be present approximately 0.6 % (96/16000) and the approximate 100 c o l l a g e n genes were found to represent 5% (361/8000). By n o r m a l i z i n g the abundance found f o r a s i n g l e gene the r e l a t i v e abundance of sPl-3 i s t h r e e - f o l d g r e a t e r than that of a s i n g l e a c t i n gene and approximately 10 f o l d g r e a t e r than that of a s i n g l e c o l l a g e n gene. 2.5. CHARACTERISTICS OF CONSERVED REGIONS 2.5.1 sPl-1 The cDNA clo n e sPl-1 c o n t a i n s a 0.9 Kb i n s e r t . T h i s cDNA h y b r i d i z e s to a s i n g l e 1.6 Kb band i n the cosmid T13C1 but to approximately 15 bands i n N2 DNA (low s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s : s e c t i o n 11-11). The genomic clone corresponding to sPl-\ a l s o d e t e c t e d m u l t i p l e bands when h y b r i d i z e d to Southern b l o t s c o n t a i n i n g C. briggsae DNA ( F i g . 14a). However at moderate h y b r i d i z a t i o n s t r i n g e n c y only bands at 1.6 Kb and 3.0 Kb are det e c t e d ( F i g . 14b). T h i s may i n d i c a t e that sPl-r i s a member of a two-gene f a m i l y not a multi-gene f a m i l y . Supporting t h i s idea was the o b s e r v a t i o n that sPl-1 c r o s s - h y b r i d i z e d with the probes col-4 and col-12 under low s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s . Under moderate s t r i n g e n c y c o n d i t i o n s sPl-1 d i d not h y b r i d i z e to col-12, but d i d h y b r i d i z e to col-4. I t has been shown that the 74 F i g u r e 14 IDENTIFICATION OF A cDNA BELONGING TO A GENE FAMILY a) Genomic S o u t h e r n s c o n t a i n i n g DNA fr o m C. elegans ( l a n e 1) and C. briggsae ( l a n e 2) ( F o d o r et al., 1983) were h y b r i d i z e d w i t h t h e genomic c l o n e r e p r e s e n t i n g t h e sPl-1 l o c u s under low s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s . Under t h e s e c o n d i t i o n s a p p r o x i m a t e l y 15 d i f f e r e n t bands were o b s e r v e d i n b o t h t h e N2 and G16 l a n e s , b) Genomic S o u t h e r n s c o n t a i n i n g DNA from C. el egans was h y b r i d i z e d w i t h t h e cDNA c l o n e r e p r e s e n t i n g t h e sPl-1 l o c u s under m o d e r a t e s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s . Under t h e s e c o n d i t i o n s o n l y two bands were o b s e r v e d i n t h e N2 l a n e a t 1.6 and 3.0 Kb. 75 col-4 gene i s not a member of the c o l l a g e n gene f a m i l y yet i t c r o s s - h y b r i d i z e s with c o l l a g e n probes ( J . Kramer, p e r s o n a l communication). Perhaps the sPl-1 probe i s behaving i n a s i m i l a r manner, under low s t r i n g e n c y c o n d i t i o n s the sPl-1 probe i s d e t e c t i n g members of the c o l l a g e n gene f a m i l y but l i k e col-4 i s not a member of t h i s f a m i l y . The col-4 probe was h y b r i d i z e d t o a N2 genomic Southern f i l t e r and d e t e c t e d a s i n g l e band at 2.0 Kb (moderate h y b r i d i z a t i o n c o n d i t i o n s ) . Since the sPl-1 probe d e t e c t e d bands at 1.6 and 3.0 Kb t h i s i n d i c a t e d that the e x t r a band at 3.0 Kb det e c t e d by sPl-1 i s not col-4. Northern A n a l y s i s When probed with the cDNA sPl-1 no h y b r i d i z a t i o n bands were observed on northern b l o t s c o n t a i n i n g e i t h e r mixed stage t o t a l RNA or pol y A + RNA. The f a c t t h at the abundance of sPl-1 was one in 60,000 phage screened may account f o r the northern r e s u l t . I f the gene rep r e s e n t e d by sPl-1 i s r e q u i r e d at a c o n c e n t r a t i o n of only a few molecules per c e l l or i s expressed f o r a b r i e f time d u r i n g development i t would be d i f f i c u l t to de t e c t on northern b l o t s . Northern b l o t s c o n t a i n i n g RNA from the d i f f e r e n t l a r v a l stages of C. elegans were probed with sPl-1. Again no bands were observed. These r e s u l t s emphasize a major advantage of us i n g c r o s s - s p e c i e s h y b r i d i z a t i o n to de t e c t coding elements s i n c e t h i s p a r t i c u l a r gene would have been overlooked i f northern a n a l y s i s was the onl y means used. 76 H y b r i d i z a t i o n to mammalian DNA Normally a l l c r o s s - s p e c i e s h y b r i d i z a t i o n s to C. briggsae or mammalian DNA were c a r r i e d out under low s t r i n g e n c y c o n d i t i o n s . However, to a v o i d h y b r i d i z a t i o n to members of the c o l l a g e n f a m i l y the probe f o r sPl-1 was h y b r i d i z e d under moderate s t r i n g e n c y c o n d i t i o n s . As shown i n F i g . 15, sPl-1 d e t e c t e d a major band at 1.6 Kb and a minor band at 3.0 Kb i n N2 DNA (lane 1). Six bands can be d e t e c t e d i n the mouse lane (two of these bands are i n the high molecular weight smear and r e q u i r e v i s u a l i z a t i o n on a l i g h t box to be d e t e c t e d ) . The s i g n a l i n t e n s i t y of the bands i n the mouse lane are of g r e a t e r i n t e n s i t y than the minor band a t 3.0 Kb i n the N2 l a n e . Perhaps t h i s i n d i c a t e s that sPl-1 shares more sequence i d e n t i t y with i t s mouse c o u n t e r p a r t than with the c l o s e s t r e l a t e d sequence i n C. elegans. Np attempt was made to e q u a l i z e the amount of DNA loaded i n t o each l a n e . The sPl-1 cDNA was sequenced s i n c e t h i s cDNA c r o s s - h y b r i d i z e d to mammalian DNA. F i g . 16 shows the sequencing s t r a t e g y used and the sPl-1 sequence i s shown i n F i g . 17. An i n t e r e s t i n g f e a t u r e found w i t h i n the sequence of sPl-1 i s a 100 bp segment c o n t a i n i n g a r e p e t i t i v e m o t i f . The deduced amino a c i d sequence from t h i s segment c o n t a i n s an abundance of the amino a c i d glutamine. Using the r e p e t i t i v e segment from sPl-1 to search the NIBR p r o t e i n data base d e t e c t e d matches with the OPA r e p e t i t i v e sequence found w i t h i n the D. melanogaster genes Notch, Antennapedia and other D r o s o p h i l a genes i n v o l v e d with neurogenesis (Wharton et a l . , 1985; Stroeher et al ., 1986; 77 Laughon et al . , 1986). The remaining sequence of sPl-1 d i d not d e t e c t any other matches. I t i s i n t e r e s t i n g t o s p e c u l a t e that perhaps the sPl-1 gene i s i n v o l v e d with neurogenesis of C. el egans . D i r e c t i o n of T r a n s c r i p t i o n F i g . 18 shows an expanded view of the Pstl r e s t r i c t i o n enzyme map of the cosmid T13C1 that c o n t a i n s the sPl-1 c l o n e . The c l o n e s pCehl72 and pCehl73 (subclones of the sPl-1 cDNA) were sequenced and from that sequence data the pCehl73 c l o n e was shown to c o n t a i n the 3' end of sPl-1. Southern a n a l y s i s of Pstl d i g e s t s of the cosmid T13C1 i n d i c a t e d that pCeh172 h y b r i d i z e d to fragment "B" whereas pCeh173 h y b r i d i z e d to fragments "B" and "C". Thus, the d i r e c t i o n of t r a n s c r i p t i o n i s from l e f t to r i g h t when compared to the g e n e t i c map. 2.5.2 sPl-2 The sPl-2 s i t e was i d e n t i f i e d by the c l o n e pCehl77 from the cosmid T14D10. The probe f o r sPl-2 d i d not d e t e c t a cDNA nor d i d i t d e t e c t an RNA molecule on a northern f i l t e r . W i t h in the cosmid T14D10 the snRNA U1 gene had been i d e n t i f i e d (T. Blumenthal and J . Thomas, p e r s o n a l communication). As shown i n F i g . 19 a Southern b l o t of a Pstl d i g e s t of the cosmid T14D10 was h y b r i d i z e d with the probe pGem R13 that c o n t a i n e d the gene f o r U1(Russnak and Candido, 1985). T h i s probe h y b r i d i z e d to a 6.0 Kb £coRI fragment, the same fragment used to i d e n t i f y sPl-2. Thus, the sPl-2 probe d e t e c t e d the gene f o r the snRNA U1. F i g u r e 15 HYBRIDIZATION OF s P l - 1 TO MAMMALIAN DNA 78 a r 2 » • Genomic Southern b l o t s were h y b r i d i z e d under moderate s t r i n g e n c y c o n d i t i o n s with the probe f o r s P l - 1 : Lane 1) C. elegans (N2); lane 2) mouse. 79 F i g u r e 16 SEQUENCING STRATEGY FOR RI RV P RI RI: EcoRI; P: P s t I ; RV: EcoRV | | 1 OObp Arrows i n d i c a t e the d i r e c t i o n and extent of sequencing s t a r t i n g from the i n d i c a t e d r e s t r i c t i o n enzyme s i t e s . The EcoRI s i t e s are w i t h i n the c l o n i n g v e c t o r and are not pa r t of the C. elegans genomic DNA. 80 gure 17 PARTIAL SEQUENCE OF s P l - 1 SP1-1 TATCCTGGTAATGCACAGCAGTTTCGTCCGCCTCCACAGCAGTCA 45 p r o t e i n Y P G N A Q Q F R P P P Q Q S 15 SP1-1 CACACCGATACACGCAGCAGCAGCAGCAACACTGCGCTCTACAAA 90 p r o t e i n H T D T R S S S S N T A L Y K 3 0 SP1 -1 CAACCACTGTACAGGATACTCTGCCGTTGCGGACGCACCAAAAAA 13 5 p r o t e i n Q P L Y R I L C R C G R T K K 4 5 SP1 -1 GAAGAAGCGACCCACGAAAAAGCAGAAGGAAGCAAGCTGCCGCTG 18 0 p r o t e i n E E A T H E K A E G S K L P L 6 0 SP1-1 CATGAGCAACAGCAACAACAGCAGCAGCAACAAATGCAAGCATAT 225 p r o t e i n H E Q Q Q Q Q Q Q Q Q M Q A Y 7 5 S P 1 - 1 TATGGACAGCAGCAACAACAGCAAGAAAGAATGCAAATGATGCAG 270 p r o t e i n Y G Q Q Q Q Q Q E R M Q M M Q 9 0 SP1-1 CAACAGCAACAACAAGGCAAATGTGCAGCAACCGGGATATCCAGG 315 p r o t e i n Q Q Q Q Q G K C A A T G I S R 105 SP1 -1 ACTAGGTTATCCTGGAATCCCCCTCCACAAGGAGCTTTTTCCCCC 360 p r o t e i n T R L S W N P P P Q G A F S P 120 SP1 -1 AGGCTATCTTCGGGGAGGAGCTCAGCCAACACACACATATGGCAC 405 p r o t e i n R L S S G R S S A N T H I W H 135 SP1-1 CACACACAGTTACACACACGATGCATGTTCACAGCAGCAGCAGAG 450 p r o t e i n H T Q L H T R C M F T A A A E 150 S P 1-1 GTCAGCACCCGGTGGTCACACACATGGGGCATGGCACACACGCAG 495 p r o t e i n V S T R W S H T W G M A H T Q 165 SP1 -1 CTTCTGTTCATACCGTTCGTGTTCCAGTCGTTGGGCCACCAGGTG 540 p r o t e i n L L F I P F V F Q S . L G H Q V 180 SP1-1 TGTGGTGACCCTGTACCGGTGTTCGTGTTCCAGTCGTTGGGCCAC 585 p r o t e i n C G D P V P V F V F Q S L G H 195 81 PARTIAL SEQUENCE OF 5 P 1 - 1 SP1 -1 CAGGTGTGTGGTGACCCTGTACCGGTTGGAAACAATTATCGCCGG 630 p r o t e i n Q V C G D P V P V G N N Y R R 210 sP1 -1 ATCGTGTATACAGCAGCAGAATTCCGATTCCAGAGCAACCATCGA 675 p r o t e i n I V Y T A A E F R F Q S N H R 225 sP1 -1 GCACCGATGTCTGGAGAGTTCGACCACCACCTGTTTCGTCTGGAA 720 p r o t e i n A P M S G E F D H H L F R L E 240 SP1 -1 AAGTCATCCACAGCAACAGCAGCAAGGATCTTTTCATCGAAGTTA 765 p r o t e i n K S S T A T A A R I F S S K L 255 SP1-1 GTCGTCGGCAACGTCTACTCTGGTAGTCACACACCATTCGGACAA 810 p r o t e i n V V G N V Y S G S H T P F G Q 270 S P 1 - 1 CAAGGTCCATCTCAATCTGAACCCACTGCAGTCATAGCAGACACA 855 p r o t e i n Q G P S Q S E P T A V I A D T 285 SP1-1 ACAAACCGAATTATGAATCAACTATTAAACTCTTCCCGAACCTAC 900 p r o t e i n T N R I M N Q L L N S S R T Y 300 SP1 -1 TTACTAATCTTCCCAACTTAATGTGTTCTGGTAGGTCTCGCGCCT 945 p r o t e i n L L I F P T * 306 SP1 -1 GGTGTTTGGGTTCCCCTGTGTCGGCTGAAAATCGCCGAGTCTTCA 990 SP1-1 AAAAAAAAAAAAAAAA 1006 The sPl-1 cDNA clone c o n t a i n e d an open reading frame of 918 bases. T r a n s l a t i o n of sPl-1 terminated at a UAA (ochre) codon and 68 n u c l e o t i d e s downstream a poly-A t a i l was observed. The i n f e r r e d amino a c i d s are presented below each codon. U n d e r l i n e d i s a r e g i o n c o n t a i n i n g a r e p e t i t i v e m o t i f . 82 F i g u r e 18 DIRECTION OF TRANSCRIPTION OF sPl-1 T1 3C1 5' > 3' P P P P P A B C D 2 Kb PstI r e s t r i c t i o n enzyme map of the cosmid T13C1. Southern b l o t s c o n t a i n i n g PstI d i g e s t s of T13C1 were h y b r i d i z e d with the sPl-1 cDNA subclones pCeh172 and pCehl73. The c l o n e s pCehl72 and pCeh173 were sequenced and from the sequence data the pCeh173 cl o n e was shown to c o n t a i n the 3' end of sPl-1. Southern a n a l y s i s i n d i c a t e d that the pCeh172 subclone h y b r i d i z e d to fragment "B" whereas the pCeh173 subclone h y b r i d i z e d to fragments "B" and "C". The arrow ( > ) above the "B" and "C" fragments i n d i c a t e s the d i r e c t i o n of t r a n s c r i p t i o n . 83 F i g u r e 19 IDENTIFICATION OF A FRAGMENT CONTAINING A TJ1 GENE 6.0-1 Southern b l o t of an EcoRI d i g e s t of the cosmid T14D10 was h y b r i d i z e d with the probe pGem R13 (provided by R. Russnak and P. M. Candido). A s i n g l e 6.0 Kb band was d e t e c t e d . The 6.0 Kb fragment from the cosmid T14D10 was the same fragment used to i n i t i a l l y i d e n t i f y the conserved s i t e sPl-2. 84 2.5.3 sP1-3 The cDNA clo n e sPl-3 c o n t a i n s a 0.8 Kb i n s e r t . T h i s cDNA h y b r i d i z e d to a s i n g l e genomic and cosmid EcoRI fragment of 1.8 Kb. Northern a n a l y s i s ( F i g . 20) showed that the sPl-3 cDNA h y b r i d i z e d t o a 1.8 Kb message. H y b r i d i z a t i o n to Mammalian DNA As shown i n F i g . 21 sPl-Z h y b r i d i z e d to a s i n g l e 1.8 Kb band i n N2 DNA. C a r e f u l i n s p e c t i o n of the human DNA lane r e v e a l e d s i x bands h y b r i d i z e d with sPl-3. These bands are of r e l a t i v e l y weak s i g n a l i n t e n s i t y however t h i s a u t o r a d i o g r a p h appears very s i m i l a r to many of the C. briggsae autoradiographs that a l s o show weak h y b r i d i z a t i o n s i g n a l s when probed with C. elegans sequences. The s i x bands i n the human lane may r e f l e c t the f a c t that the human co u n t e r p a r t to sPl-3 i s composed of a number of exons spread over a l a r g e i n t e r v a l . A l t e r n a t i v e l y the s i x bands may represent independent fragments that share sequence i d e n t i t y with sPl-3 and each o t h e r . The sPl-3 cDNA was sequenced s i n c e t h i s cDNA c r o s s - h y b r i d i z e d to mammalian DNA. F i g . 22 shows the sequencing s t r a t e g y used and the sPl-3 sequence i s shown i n F i g . 23. The p a r t i a l sequence of the sPl-3 cDNA clo n e d e t e c t e d sequence s i m i l a r i t i e s with the gene S-adenosyl-L-homocysteine h y d r o l a s e (AHH) (D. B a i l l i e , p e r s o n a l communication) from r a t (Ogawa et al . , 1987) and Di ct yos t el i um discoideum (Kaser et al . , 1988). Over a s t r e t c h of 800 bases the sequence of sPl-3 was 85 F i g u r e 20 NORTHERN ANALYSIS OF CONSERVED REGIONS Northern b l o t s c o n t a i n i n g t o t a l RNA were h y b r i d i z e d with the probes d e t e c t i n g the conserved r e g i o n s . The s i z e of the message det e c t e d i s l i s t e d a f t e r the probe: panel 1) sPI-1; 2) sPl-3 (1.8 Kb); 3) sPl-4 (1.7 Kb); 4) sP\-6 (2.5 Kb); 5) sPl-7 (1.6 Kb); 6) sPl-10 (1.7 Kb). 86 F i g u r e 21 HYBRIDIZATION OF s P l - 3 TO MAMMALIAN DNA Genomic Southern b l o t s were h y b r i d i z e d under low s t r i n g e n c y c o n d i t i o n s with the probe f o r s P l - 3 : Lane 1 ) C. elegans ( N 2 ) ; lane 2 ) human. 87 FIGURE 22 SEQUENCING STRATEGY FOR sP\~3 Rl H Rl I 1 1 I > < - 1 < 1 > R l : EcoRI; H: H i n d l l l | 1 lOObp Arrows i n d i c a t e the d i r e c t i o n and extent of sequencing s t a r t i n g from the i n d i c a t e d r e s t r i c t i o n enzyme s i t e s . The EcoRI s i t e s are w i t h i n the c l o n i n g v e c t o r and are not p a r t of the C. elegans genomic DNA. FIGURE 23 PARTIAL SEQUENCE OF sPl~3 SP1-3 CGCACGGGAACCGAAAGCCTTGAGGGAAGCAGCAGATCCCTTTCC 45 p r o t e i n R T G T E S L E G S S R S L S 15 SP1-3 GACATCGATCCAATCAATGCTCTTCAAGCCGCTATGGAAGGATAC 90 p r o t e i n D l D P I N A L Q A A M E G Y 30 S P 1 - 3 'GAAGTGACCACCCTCGAGGAGGCTGCACCAAAGGCTAACATCATT 135 p r o t e i n E V T T L E E A A P K A N I I 45 S P 1 - 3 GTCACCACCACTGGATGCAAGGACATCGTTACCGGAAAGCACTTC 180 p r o t e i n V T T T G C K D I V T G K H F 6 0 SP1-3 GAGCTTCTTCCAAATGACGCTATCGTCTGCAACGTTGGTCACTTC 225 p r o t e i n E L L P N D . A I V C N V G H F 75 S P1-3 GATTGCGAGATTGACGTCAAATGGCTCACACAATGCACAAGAAGG 270 p r o t e i n D C E I D V K W L T Q C T R R 9 0 SP1 -3 ACACATCAAGCACAGGTTGACCGTTACACCCTCAAGAACGGACGT 315 p r o t e i n T H Q A Q V D R Y T L K N G R 105 SP1-3 CACGTCATCCTCCTTGCCGAAGGACGTCTTGTCAACCTTGGATGT 360 p r o t e i n H V I L L A E G R L V N ' L G C 120 SP1-3 GCCACCGGACATCCATCTTTCGTCATGTCCAACTCCTTCACCAAC 405 p r o t e i n A T G H P S F V M S N S F T N 135 SP1 -3 CAAGTTCTCGCTCAAGTTGAGCTCTGGACCAAGTTCGGAACTCCA 450 p r o t e i n Q V L A Q V E L W T K F G T P 150 89 PARTIAL SEQUENCE OF s P l ~ 3 SP1-3 CAAGAATACAAGCTCGGACTCAAAAACCCTGACAGAGTCGCTACT 495 p r o t e i n Q E Y K L G L K N P D R V A T 165 S P 1-3 CACTTGGCTCAATGGAGTCAAGCTTCAAAGCTTTCCGACGAGCAA 540 p r o t e i n H L A Q W S Q A S K L S D E Q 180 SP1-3 GCTTCATACCTTGGAGTTCAAGTTGCCGGACCATACAAGCCAGAC 585 p r o t e i n A S Y L G V Q V A G P Y K P D 195 S P 1-3 CACTACAGATATTAATTTTGTTGGTTCTACCCCATACGGATATTT 630 p r o t e i n H Y R Y * 2 1 0 S P 1-3 TTTTAACTGTTTTAAATTGTATTGCCCTCAAACTTCATATGATCT 675 p r o t e i n 225 S P 1-3 GACTGGTTGTATTGTTCCCATCTACCCGCTTTTCCCCCAATACAT 720 p r o t e i n 240 SP1 -3 AATCGCCTGATTTAGTCGTTTTTCTTCAACTCGTTGATTTGGTTC 765 p r o t e i n ^ 242 S P1 -3 TGTTGTTTGTCTTTTGGACATAAATGTTTTATGGAAAAAAAAAAA 810 S P 1-3 AAAAAAAAAAA 820 The s P l - 3 cDNA c l o n e c o n t a i n e d an open reading frame of 597 bases. T r a n s l a t i o n of s P l - 1 terminated at a TAA codon and 204 n u c l e o t i d e s downstream a poly-A t a i l was observed. The i n f e r r e d amino a c i d s are presented below each codon. The p u t a t i v e poly-A a d d i t i o n s i g n a l i s u n d e r l i n e d . 90 approximately 58% i d e n t i c a l to that of AHH. A comparison of the deduced amino a c i d sequence of sPI-3 with S-adenosyl-L-homocysteine hydrolase i s shown i n F i g . 24. Approximately 60 % of the amino a c i d s between sPl-3 and AHH are i d e n t i c a l ; and, i f c o n s e r v a t i v e changes are c o n s i d e r e d the homology between the .C. el egans sequence and the AHH sequence from r a t i s 82% while between C. elegans and D. discoideum the homology i s 84%. Thus the high degree of s i m i l a r i t y between sPl-3 and AHH suggests that the sPl-3 l o c u s encodes the gene S-adenosyl-L-homocysteine h y d r o l a s e . 2.5.4 sPl-4 The cDNA clo n e sPl-4 c o n t a i n s a 1.5 Kb i n s e r t . T h i s cDNA h y b r i d i z e d to a s i n g l e genomic and cosmid EcoRI fragment of 3.8 Kb. Northern a n a l y s i s ( F i g . 20, page 84) showed that the sPl-4 cDNA h y b r i d i z e d to a 1.7 Kb message. H y b r i d i z a t i o n of sPl-4 to Southern b l o t s c o n t a i n i n g mammalian DNA d i d not d e t e c t any homologies. 2.5.5 sPl-5 The cDNA c l o n e sPl-5 c o n t a i n s a 0.9 Kb i n s e r t . T h i s cDNA h y b r i d i z e s to 4.8 and 6.5 Kb EcoRI fragments i n N2 DNA but to only a 4.8 Kb fragment i n cosmid DNA. T h i s suggests that other r e l a t e d sequences e x i s t elsewhere i n the genome. I used the genomic plasmid c l o n e c o r r e s p o n d i n g to sPl-5 ( i s o l a t e d by K. Beckenbach) to i d e n t i f y a phage c l o n e . T h i s phage was matched to a cosmid c o n t i g by A. Coulson and J . S u l s t o n t h a t was not from 91 F i g u r e 24. A COMPARISON OF THE DEDUCED AMINO ACID SEQUENCE OF sPl~3 AND  S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE FROM RAT AND D. DISCOIDEUM Rat CAQALRGFGARVIITEIDPINALQAAMEGYEVTTMDEACKEGNIF • • • • • • • « • • • • • • « • • • • « • • • • • • • • • • • • • • • • • • • « • • • • • • • • • • • • • • C. elegans RTGTESLEGSSRSLSDIDPINALQAAMEGYEVTTLEEAAPKANII • • • • • • • • • • • • • • • • • • • » • D. di scoi deum CAQSLSKMGARVLVTEIDPINALQACMDGYQIVTMETAAPLSNIF Rat VTTTGCVTJILLGRHFEQMKDDAIVCNIGHFDVEIDVKWLNENAVE • ••••• •• • ••• •••••• •••• ••••••• • ••••• ••• ••••• • •••••••••••• ••••••••• • C. elegans VTTTGCKDIVTGKHFELLPNDAIVCNVGHFDCEIDVKWLTQ--CT D. discoideum VTTTGCRDIVRGEHFAVMKEDAIYCNIGHFDCEIDVAWLNA--NA Rat KVNIKPQVDRYLLKNGHRIILLAEGRLVNLGCAMGHPSFVMSNSF C. elegans RRTHQAQVDRYTLKNGRHVILLAEGRLVNLGCATGHPSFVMSNSF • • • • • • • • • • • • • * • • * * • • • • • • • • • • • • • • • • D. di scoi deum KKTVKPQVDRYTLANGVH11LLAEGRLVNLGCGTGHPSFVMSNSF Rat TNQVMAQIELWTHPDKYPVGVHF-LPKKLDEAVAEAHLGKLNVKL • • • * • • • « • • • • » • • • • • • • • • C. elegans TNQVLAQVELWT KFGTPQEYKLGLKNPDRVA-THLAQWS-QA • • • • • • • • •' • • * • • • • • • • • • • • • ••• • • • • • • • » » D. di s c o i deum CNQTLAQIALWT KTEEPLGVHLLPKDEEVAR-LHLDQLA-KL Rat TKLTEKQAQYLGMPINGPFKPDHYRY* C. elegans SKLSDEQASYLGVQVAGPYKPDHYRY* • a o a • • • • • • • • • • • • • D. di scoi deum TTLTEKQSEYLSVPVAGPYKVDHYRY* A comparison of the deduced amino a c i d sequence of sPl-3, AHH from r a t (Ogawa et al ., 1987), and from D. discoideum ( K a s i r et a l . , 1988). I d e n t i c a l amino a c i d s are i n d i c a t e d by (:). Conserved amino a c i d changes are i n d i c a t e d by (.). Gaps to a l i g n the sequences are i n d i c a t e d by (-). 92 the dpy-14 r e g i o n of LGI (A. Coulson and J . S u l s t o n , p e r s o n a l communication). A p o s s i b l e e x p l a n a t i o n f o r these f i n d i n g s i s that the gene corresponding to sPl-5 i s p a r t of a two member gene f a m i l y and th a t the second member i s from o u t s i d e the seven cosmids an a l y z e d i n t h i s study. Northern a n a l y s i s showed that the sPl-5 cDNA h y b r i d i z e d to a 2.0 Kb message (N. Mawji, unpublished r e s u l t s ) . H y b r i d i z a t i o n of sPl-5 to Southern b l o t s c o n t a i n i n g mammalian DNA f a i l e d to d e t e c t any homologies. 2.5.6 sPl-6 The cDNA clo n e sPl-6 c o n t a i n s a 0.7 Kb i n s e r t . T h i s cDNA h y b r i d i z e d t o genomic and cosmid EcoRI fragments of 4.8 and 3.3 Kb. From the r e s t r i c t i o n enzyme map of the cosmid T21G5 the 4.8 and 3.3 Kb fragments* are found to be adjacent t o each o t h e r . Since the sPl-6 cDNA clo n e does not c o n t a i n an EcoRI s i t e then sPl-6 can not span the EcoRI s i t e between these fragments. T h i s i n d i c a t e s t h a t the genomic r e g i o n c o r r e s p o n d i n g to sPl-6 c o n t a i n s an i n t r o n t h a t possesses an EcoRI s i t e . Northern a n a l y s i s ( F i g . 20, page 84) showed that the sPl-6 cDNA h y b r i d i z e d to a 2.5 Kb message. H y b r i d i z a t i o n of sPl-6 to Southern b l o t s c o n t a i n i n g mammalian DNA d i d not de t e c t any homologies. 2.5.7 sPl-7 The cDNA c l o n e sPl-7 c o n t a i n s a 1.0 Kb i n s e r t . T h i s cDNA h y b r i d i z e d t o a s i n g l e genomic and cosmid EcoRI fragment of 2.2 Kb. Northern a n a l y s i s ( F i g . 20, page 84) showed that the sPl-7 93 cDNA h y b r i d i z e d to a 1.6 Kb message. H y b r i d i z a t i o n of sPl-7 to Southern b l o t s c o n t a i n i n g mammalian DNA f a i l e d to d e t e c t any homologies. 2.5.8 sPl-8 and sPl-9 The conserved fragments d e t e c t i n g the sPl-8 and sPl-9 s i t e s f a i l e d to d e t e c t any cDNAs. Furthermore, these conserved fragments d i d not d e t e c t an RNA molecule on northern b l o t s c o n t a i n i n g e i t h e r mixed-stage t o t a l RNA or poly A + RNA. One e x p l a n a t i o n f o r these o b s e r v a t i o n s i s that the genes represented by the sPl-8 and sPl-9 conserved regions are p o o r l y expressed. Another e x p l a n a t i o n i s that they are expressed f o r a short time i n t e r v a l and t h e r e f o r e would be under represented i n mixed stage RNA or cDNA l i b r a r i e s . 2.5.9 sPl-10 The cDNA c l o n e sPl-10 c o n t a i n s a 1.6 Kb i n s e r t . T h i s cDNA h y b r i d i z e d to EcoRI genomic and cosmid fragments of 5.5, 2.0, and 0.8 Kb. These fragments were found to be adjacent to each other w i t h i n the cosmid F15C11. An EcoRI d i g e s t of the 1.6 Kb cDNA l i b e r a t e d fragments of 0.8, 0.5 and 0.3 Kb. The 0.8 cDNA fragment was ^ 2 P - l a b e l l e d a n d found to h y b r i d i z e to the 5.5 Kb N2 genomic fragment and not to the 0.8 Kb N2 genomic fragment. Since n e i t h e r of the remaining two EcoRI cDNA fragments are l a r g e enough to span the remaining genomic fragments, the gene cor r e s p o n d i n g to sPI-10 c o n t a i n s at l e a s t one i n t r o n . 94 Northern a n a l y s i s ( F i g . 20, page 84) showed that the sPl-10 cDNA h y b r i d i z e d to a 1.7 Kb message. H y b r i d i z a t i o n of sPl-10 to Southern b l o t s c o n t a i n i n g mammalian DNA f a i l e d to de t e c t any homologies. 3. GENETIC ANALYSIS IN THE DPY-14 REGION 3.1 Screening f o r Tc1-induced mutations of dpy-14 Males of the genotype dpy-14 unc-13/ + + were c r o s s e d to mut-6 hermaphrodites (Mori et al., 1988a). A f t e r mating, 236 hermaphrodites were t r a n s f e r r e d to i n d i v i d u a l p l a t e s and t h e i r progeny were screened f o r Dpy-14 i n d i v i d u a l s i n a background of wild- t y p e animals. Approximately 40,000 F1 progeny were screened and no Dpy-14 i n d i v i d u a l was observed. The f a c t that no Tc1-induced Dpy-14 animals were found was not s u r p r i s i n g s i n c e the Tc1 mutation r a t e f o r the unc-22 gene which c o n t a i n s 10 Tc1 consensus s i t e s was 1 x 10~ 4 ( Moerman et al., 1986) and f o r I in-12 was 5 x 10" 5 (Greenwald, 1985). 3.. 2 Screening f o r formaldehyde-induced mutations of dpy-14 Wild-type males were t r e a t e d with 0.1 % formaldehyde (Johnsen and B a i l l i e , 1988) and then mated to dpy-5 dpy-14 unc-13 hermaphrodites. A f t e r mating, the hermaphrodites were t r a n s f e r r e d to i n d i v i d u a l p l a t e s and the F1 progeny screened f o r Dpy-14 i n d i v i d u a l s i n a background of wi l d - t y p e s and Dpy-5 Dpy-14 Unc-13 animals. From 1,154 c r o s s progeny one Dpy-14 animal was i s o l a t e d . From the s e l f - f e r t i l i z e d progeny of t h i s worm the 95 s t r a i n KR1000 was e s t a b l i s h e d . Twenty i n d i v i d u a l Dpy-14 animals were t r a n s f e r r e d to f r e s h p l a t e s and allowed t o s e l f - f e r t i l i z e . A l l 20 segregated Dpy-14 and Dpy-5 Dpy-14 Unc-13 progeny suggesting that the formaldehyde induced a l l e l e of dpy-14 (hDf8) was homozygous i n v i a b l e . T h i s i n t e r p r e t a t i o n was l a t e r s u b s t a n t i a t e d by A.M. Rose (unpublished d a t a ) . 3.3 Complementation t e s t i n g with hDf8 The l e t h a l mutation hDf8 was balanced over dpy-5 dpy-14 unc-13. hDf8 complements both dpy-5 and unc-13 but f a i l e d to complement unc-37 and unc-87. Males of the genotype dpy-5 unc-87 / + + or dpy-5 unc-37 / + + were mated to dpy-5 + unc-13 / + hDf8 + hermaphrodites. Ten Unc-37 animals from t h i s c r o s s were progeny-tested and a l l 10 segregated Unc-37 and Dpy-5 Unc-37. In a d d i t i o n the Unc-37 hermaphrodites (genotype dpy-5 + unc-37 / + hDf8 + ) were c r o s s e d to dpy-14 unc-29 / + + males. From t h i s c r o s s Dpy-14 animals were observed c o n f i r m i n g that the hDf8 chromosome i s present i n the Unc-37 hermaphrodites. S i m i l a r r e s u l t s were ob t a i n e d f o r unc-87. Table 10 shows a l l the complementation r e s u l t s o b t ained with hDf8. One i n t e r p r e t a t i o n as to the nature of the hDf8 mutation i s that the hDf8 bearing chromosome c o n t a i n s a d e l e t i o n t h a t i n c l u d e s the i n t e r v a l from unc-37 t o dpy-14. The d e l e t i o n was t e n t a t i v e l y r e f e r r e d to as hDf8 (see s e c t i o n IV-3 f o r a d i s c u s s i o n of these f i n d i n g s ) . Table 10 COMPLEMENTATION TESTING WITH h57£ Cross Wt ml m2 dpy-5 + unc-37 X dpy-5 + unc-75 91 Dpy-5 (37) Unc-37 (42) + + + + h572 + dpy-5 + unc-13 X dpy-5 + unc-87 . 175 Dpy-5 (73) Unc-87 (71) + h572 + + + + dpy-5 + unc-13 X dpy-5 + unc-14 103 Dpy-5 (49) Unc-14 (0) + h572 + + + dpy-5 4 unc-75 X dpy-14 let-83 unc-13 92 Dpy -14 (0) + h572 + + + 4 dpy-5 + unc-13 X dpy-14 let-86 unc-13 81 Unc-13 (37) Dpy-14 (30) 1 4 h572 + + + + dpy-5 4 unc-13 X dpy-14 let-75 unc-13 63 Unc-13 (30) Dpy-14 (24) + h572 4 4 4 1 These Dpy-14 animals were arrested at the f i r s t larval stage 97 3.4 Southern analysis of hDf8 Southern blots containing EcoRI digested genomic DNA from N2, and KR1000 worms were hybridized with probes spanning a 300 Kb i n t e r v a l . As an internal control these blots were simultaneously hybridized with the probe for sP4 which has been mapped to LGIV ( B a i l l i e et al., 1985). Autoradiographs of these blots were scanned using a LKB scanning densitometer. If a probe originated from a deleted region (1 copy) then the re l a t i v e signal i n t e n s i t y in the hDf8 lane should be 50% of that in the N2 lane (2 copies). If the test probe originated from outside the deletion (2 copies), then the r e l a t i v e signal i n t e n s i t y should be similar to N2. The r a t i o between the test probe and the probe for sP4 was calculated in each lane and this r a t i o was compared between lanes. F i g . 25 shows the results obtained with the probes sPl-1, pCeh167 (from the cosmid T10B11) and sPl-10. F i g . 26a shows the tracing obtained from t h i s autoradiograph. The sPl-1 clone comes from the cosmid T13C1 and i s 43 Kb to the l e f t of the mapped polymorphism sPl. The ra t i o of the area under these tracings was calculated and i s presented in the form of a bar graph in F i g . 26b. From th i s graph i t i s apparent that the signal i n t e n s i t y from the sPl-1 probe i s less than 50% compared to the N2 lane indicating that sPl-1 i s within a deleted region. In contrast the probe for sPl-10 shows similar signal intensity in both the hDf8 and N2 lanes. To i d e n t i f y the fragment that detects the breakpoint, additional probes were hybridized to the above Southern blot s . The order of the probes l i s t e d from top 98 F i g u r e 25 DOSAGE ANALYSIS OF THE hDf8 MUTATION pCeh167 sP4 Genomic Southern b l o t s c o n t a i n i n g EcoRI d i g e s t e d DNA from wild-type N2 and hDf8 d e l e t i o n i n s t r a i n KR1000 (dpy-5 dpy-14 unc-13 / + hDf8 + ) were h y b r i d i z e d with the probes pCes233 (sP4), pCehl76 (sPl-1), pCehl67 (from the cosmid T10B11), and pCeh165 (sPl-10). lane 1) N2; 2) KR1000. 99 F i g u r e 26 SCANNING DENSITOMETER TRACINGS OF THE hDf8 MUTATION a) panel 1 2 pCeh165 : 7 panel 2 DCehl76 2 n f l I / b) 1 00% 80% 40% pCeh176 isPl-1) pCehl67 pCeh165 {sPl-10) a) Scanning densitometer t r a c i n g s o b t a i n e d from the autoradiograph shown i n F i g u r e 26 of N2 and KR1000 a f t e r h y b r i d i z a t i o n with the probes pCes233 (sP4), pCeh176 (sPI-1), pCeh167, and pCehl65 (sPl-10). panel 1) N2; panel 2) KR1000: b) A bar graph p l o t of the c a l c u l a t e d area under t r a c i n g s (above) with the r a t i o of the t e s t probe to pCes233 (sP4) normalized to 100% f o r the N2 lane. lane 1) N2; lane 2) KR1000. 100 to bottom i n Table 11 corresponds with the g e n e t i c map going from l e f t to r i g h t . R e l a t i v e to the g e n e t i c map, DNA sequences from the l e f t end of the cosmid C14A12 are w i t h i n a reg i o n that i s d e l e t e d i n the hDf8 mutation. DNA sequences from the r i g h t end of t h i s cosmid are o u t s i d e of the d e l e t i o n . T h i s suggested that one of the HD/8 b r e a k p o i n t s f a l l s w i t h i n C14A12. F i g . 26a shows the r e s u l t s u s i n g a 3.7 Kb EcoRI fragment from C14A12 as a probe. T h i s probe d e t e c t e d a s i n g l e 3.7 Kb EcoRI band i n the N2 lane but det e c t e d two a d d i t i o n a l bands of 2.8 and 10 Kb i n the KR1000 lane, s u g g e s t i n g that t h i s fragments spans a br e a k p o i n t . Supporting evidence comes from a 2.3 Kb fragment -. T h i s fragment has been p o s i t i o n e d on a r e s t r i c t i o n enzyme map of C14A12 to be 1.5 Kb to the r i g h t of the 3.7 Kb fragment and was shown to be o u t s i d e of the d e l e t i o n by scanning densitometer t r a c i n g s . In a d d i t i o n , Southern b l o t s c o n t a i n i n g EcoRI, Bglll, PstI, and. Hindlll d i g e s t e d DNA from N2 and a d i f f e r e n t AD/S-containing s t r a i n (KR1793) were h y b r i d i z e d with the C14A12-3.7 Kb fragment. The KR1793 s t r a i n was used to ensure that the a d d i t i o n a l bands observed were r e p r o d u c i b l y observed i n another AZ)/5-containing s t r a i n . F i g . 27b shows t h a t the same two e x t r a bands of 2.8 and 10 Kb i n the EcoRI d i g e s t (lane 2) were observed. In a d d i t i o n e x t r a bands were observed i n both the Bglll, (lane 4), and Hindlll (lane 8) d i g e s t s although no e x t r a bands were seen in the PstI d i g e s t (lane 6). The presence of d e t e c t a b l e band s h i f t s in both hDf8 s t r a i n s and the f a c t t h at DNA sequences only 1.5 Kb 101 Table 11 DOSAGE ANALYSIS OF THE hDf8 MUTATION t e s t probe cosmid r e l . area (N2:hDf8) hDf8 F14G7-3.0 1 F14G7 1 : 0.41 in pCeh168 T1 3C1 1 : 0.40 in pCes200 T21G5 1 : 0.47 in C14A12-2.3 2 C14A12 1 : 0.94 out pCehl67 T10B11 1 : 0.82 out pCehl65 F15C11 1 : 0.95 out 1 The 3.0 Kb EcoRI fragment from the cosmid F14G7 was cut from an agarose g e l , e l e c t r o e l u t e d , and used as a h y b r i d i z a t i o n probe. 2 The 2.3 Kb EcoRI fragment from the cosmid C14A12 was cut from an agarose g e l , e l e c t r o e l u t e d , and used as a h y b r i d i z a t i o n probe. 102 F i g u r e 27 SOUTHERN ANALYSIS OF THE hDf 8 MUTATION J * i 1 2 3 4 1 2 3 4 5 6 7 * 8 a) Genomic Southern b l o t s c o n t a i n i n g DNA from N2 and KR1000 (dpy-5 dpy-14 unc-13 / + hDf8 + ) were probed w i t h a 3.7 Kb EcoRI fragment from the cosmid C14A12. EcoRI d i g e s t : l a n e 1) N2; 2) KR1000. Bglll d i g e s t : l a n e 3) N2; 4) KR1000. b) Genomic Southern b l o t s c o n t a i n i n g DNA from N2 and KR1793 ( l e t - 3 5 2 dpy-5 + unc-13 / + + hDf8 + ) were probed w i t h a 3.7 Kb EcoRI fragment from the cosmid C14A12. EcoRI d i g e s t : l a n e 1) N2; 2) KR1793. B g l l l d i g e s t : l a n e 3) N2; 4) KR1793. P s t I d i g e s t : l a n e 5) N2; 6) KR1793. H i n d l l l d i g e s t : l a n e 7) N2; 8) KR1793. 103 away are o u t s i d e of the d e l e t i o n i n d i c a t e that one breakpoint of hDf8 f a l l s w i t h i n the 3.7 Kb EcoRI fragment of C14A12. The o b s e r v a t i o n that there are always two e x t r a bands i n d i c a t e s t h a t hDf8 r e s u l t e d from a complex rearrangement. One p o s s i b l e mechanism to e x p l a i n the data i s a d e l e t i o n / i n s e r t i o n event. The i n t e r p r e t a t i o n of these data i s d i s c u s s e d i n s e c t i o n IV-3. 104 IV. DISCUSSION 1. PHYSICAL MAP FROM THE LGI GENE CLUSTER The s i x polymorphic s i t e s d e s c r i b e d here have been i n s t r u m e n t a l i n the i d e n t i f i c a t i o n of more than 1100 Kb of DNA wi t h i n the dpy-5 unc-29 i n t e r v a l of chromosome I. The four polymorphic s i t e s hP4, hP5, hP6, and hP7 from the dpy-5 unc-29 i n t e r v a l of LG I were i d e n t i f i e d by the method of Tc 1 - l i n k a g e s e l e c t i o n ( B a i l l i e et al., 1985). A f i f t h s i t e (hP9) was i d e n t i f i e d i n cosmid DNA obtained from A. Coulson and J . S u l s t o n , Cambridge, England. The s i x t h s i t e (sPJ) a n a l y z e d . i n t h i s t h e s i s came from a pr e v i o u s study (Rose et al., 1982). The ge n e t i c order of these s i x polymorphic DNA markers with res p e c t to v i s i b l e g e n e t i c markers was found to be dpy-5 hP5 bli-4 unc-37 (sPl, dpy-14) hP9 unc-13 hP7 {hP4, hP6) unc-29. Except f o r the hP9 s i t e , phage DNA surrounding each polymorphic s i t e was i s o l a t e d and used to screen a cosmid DNA l i b r a r y . From these phage t h r e e cosmid c o n t i g s were i d e n t i f i e d . A f o u r t h c o n t i g c o n t a i n i n g the hP9 s i t e had been p r e v i o u s l y mapped to LGI and shown to c o n t a i n the gene unc-15 (A. Coulson and J . S u l s t o n , p e r s o n a l communication). I n i t i a l l y , these four c o n t i g s were not p h y s i c a l l y l i n k e d to each other, however as the genome p h y s i c a l map p r o j e c t p r o gressed a s i n g l e c o n t i g c o n t a i n i n g hP5, sPl, and hP9 was formed. The amount of DNA i n a cosmid c o n t i g i s best determined by c o n s t r u c t i n g cosmid o v e r l a p maps based on h y b r i d i z a t i o n data. Each cosmid i s ^ 2 P - l a b e l l e d and used to probe Southern b l o t s c o n t a i n i n g cosmid DNA from i t s neighbors. From the r e s u l t i n g 105 autoradiographs the amount of DNA shared by a p a i r of cosmids can be determined and s u b t r a c t e d from the t o t a l amount of DNA c o n t a i n e d i n the two cosmids. T h i s was repeated f o r a l l p a i r s of cosmids i n the c o n t i g . For small c o n t i g s t h i s method i s f e a s i b l e . However, f o r very l a r g e c o n t i g s i t i s time consuming and c o s t l y . Two simple methods to estimate the amount of DNA co n t a i n e d i n a cosmid c o n t i g were used. The f i r s t method r e l i e d on e s t i m a t i n g both the number of cosmids i n the c o n t i g and the average amount of DNA per cosmid. By choosing cosmids from the maps of A. Coulson and J . S u l s t o n that do not share any common Hindlll s i t e s the minimum number of cosmids i n a c o n t i g can be determined. The amount of DNA per cosmid can be estimated by r e s t r i c t i o n enzyme d i g e s t i o n and agarose g e l e l e c t r o p h o r e s i s . The second method estimates the average s i z e of a Hindlll fragment and measures the number of Hindlll fragments i n a c o n t i g from the c o n t i g maps of A. Coulson and J . S u l s t o n . The 3.0 map u n i t i n t e r v a l between dpy-5 and unc-29 i s repr e s e n t e d by four cosmid c o n t i g s . Two of these c o n t i g s were i d e n t i f i e d i n t h i s t h e s i s . The remaining two c o n t i g s were anchored to t h i s r e gion by c l o n i n g the genes dpy-5 ( J . B a b i t y , p e r s o n a l communication) and I in-10 (S. Kim, p e r s o n a l communication). Table 12 g i v e s an estimate of the amount of DNA c o n t a i n e d i n each c o n t i g to date. The DNA v a l u e s l i s t e d i n t h i s t a b l e are based on an estimate of 34 Kb per cosmid and 106 Table 12 AMOUNT OF DNA IN THE CONTIGS FROM THE LGI CLUSTER Co n t i g DNA Content (Kb) method 1 method 2 dpy-5 510 660 hP5-sP1-hP9-unc-13 1400 1740 l i n - 1 0 270 260 hP6 170 160 T o t a l 2350 2820 Method 1: c a l c u l a t e d by counting the number of cosmids with no common Hindlll s i t e s m u l t i p l i e d by 34 Kb per cosmid. Method 2: c a l c u l a t e d by counting the number of Hindlll s i t e s and m u l t i p l y i n g by 3.0 Kb per Hindlll s i t e . As more cosmids are i d e n t i f i e d and l i n k e d to the e x i s t i n g LGI c o n t i g s , as p a r t of the on-going genomic c l o n i n g p r o j e c t , the amount of DNA i n the c l u s t e r i n c r e a s e s . The Dpy-5 c o n t i g was anchored t o the g e n e t i c map by J . Ba b i t y (personal communication). The Lin-10 c o n t i g was anchored to the ge n e t i c map by S. Kim (per s o n a l communication). The hP5-sP1-hP9-unc-13 and hP6 c o n t i g s were anchored to the gen e t i c map by S t a r r et al. , 1989. 107 3.0 Kb per Hindlll fragment. The c o n t i g s are d e s c r i b e d by the known g e n e t i c markers c o n t a i n e d w i t h i n them. The four c o n t i g s mentioned above represent g r e a t e r than 2300 Kb of c l o n e d N2 DNA (A. Coulson and J . S u l s t o n p e r s o n a l communication). Since these four c o n t i g s are not yet j o i n e d t h i s i s a minimum esti m a t e . The amount of DNA between dpy-14 and unc-13 was compared to the d i s t a n c e i n map u n i t s . The 95% c o n f i d e n c e l i m i t s f o r the g e n e t i c d i s t a n c e between dpy-14 and unc-13 i s 0.16 - 0.49 map u n i t s (Rose and B a i l l i e , 1979). Using an average value of 333 Kb the DNA/mu value between dpy-14 to unc-13 would range between 53 to 163 Kb i f t h i s r e g i o n was an average r e g i o n . Making the assumption that sPl i s s u f f i c i e n t l y c l o s e to dpy-14 the amount of DNA from the dpy-14 unc-13 i n t e r v a l was estimated to be 400 Kb u s i n g a value of 34 Kb per cosmid and 500 Kb using a value of 3.0 Kb per Hindlll s i t e . T h e r e f o r e the amount of DNA per map u n i t i n the dpy-14 unc-13 i n t e r v a l ranges between a minimum of 2.5 (400/163) to a maximum of 9.5 (500/53) times the average v a l u e . The f i r s t evidence that recombination frequency c o u l d be i n c r e a s e d a c r o s s the LGI gene c l u s t e r was presented by Kim and Rose ( 1987).. These authors demonstrated that the recombination frequency i n the dpy-5 to unc-13 i n t e r v a l i n c r e a s e d f o l l o w i n g treatment with gamma r a d i a t i o n . Moreover, they showed that the i n c r e a s e was not constant throughout the c l u s t e r and p o s t u l a t e d that t h e i r r a d i a t i o n map r e p r e s e n t s the p h y s i c a l map more a c c u r a t e l y than does the m e i o t i c map. With the c o n s t r u c t i o n of the p h y s i c a l map p a r t i a l l y completed (Coulson et al. , 1986; Coulson et al., 1988), and with 108 of the g e n e t i c mapping of the DNA polymorphic s i t e s from w i t h i n the hP5-unc-13 i n t e r v a l presented i n t h i s t h e s i s , a comparison to the gamma map of Kim and Rose (1987) can be made. Kim and Rose found i n the dpy-14 to unc-13 i n t e r v a l the i n c r e a s e i n recombination frequency was found to i n c r e a s e 4.7 times a f t e r i r r a d i a t i o n which compares w e l l with the DNA per map u n i t value found to range between 2.5 to 9.5 times the genomic average. Increases i n the DNA per map u n i t value have a l s o been found i n the gene c l u s t e r s on other chromosomes. Near the gene I in-12 i n the LGIII c l u s t e r Greenwald et al ., (1987) d e s c r i b e d a three to f o u r - f o l d i n c r e a s e , and i n the LGIV c l u s t e r between the genes dpy-20 to unc-22 Prasad (1988) found a f o u r - f o l d i n c r e a s e . A l l these r e s u l t s are c o n s i s t e n t with the hypothesis (Brenner 1974) that the gene c l u s t e r s of C. elegans r e s u l t from a r e d u c t i o n i n recombination frequency compared to the genome average. Non-uniform recombination has been w e l l documented i n many organisms (reviewed Baker et al., 1976). Both r e g i o n s of i n c r e a s e d and reduced l e v e l s of m e i o t i c exchange have been observed. Increased recombination (sometimes r e f e r r e d to as rec o m b i n a t i o n a l hotspots) can be promoted at s p e c i f i c DNA sequences. W i t h i n the genome of E. coli recombination i s f r e q u e n t l y i n i t i a t e d by the RecBC enzyme at a s p e c i f i c o c t a n u c l e o t i d e sequence c a l l e d Chi ( P o n t i c e l l i et al ., 1985). These s i t e s are present approximately 1000 times throughout the genome. S i m i l a r l y i n humans a reco m b i n a t i o n a l hot spot has been observed w i t h i n the 0-globin gene c l u s t e r (reviewed O r k i n and Kazazian 1984). C h a k r a v a r t i et al., (1984) estimated that w i t h i n 109 a 9 Kb i n t e r v a l 5' to the /3-globin gene recombination was i n c r e a s e d approximately 30 f o l d . Reduced r a t e s of m e i o t i c exchange have been observed i n the c e n t r i c heterochromatin of D r o s o p h i l a melanogaster (Roberts 1969; Carpenter and Baker 1972). Lower r a t e s of recombination are a l s o found i n euchromatic regions adjacent to the centromere (centromere e f f e c t , Beadle 1932). S t u d i e s of D r o s o p h i l a have shown that i n the presence of s p e c i f i c m e i o t i c mutants the frequency of recombination per chromosomal arm i s the same as i n w i l d type but the d i s t r i b u t i o n of c r o s s o v e r s has been a l t e r e d (reviewed Baker et al ., 1976). The above r e s u l t s i n d i c a t e that the non-uniform d i s t r i b u t i o n of c r o s s o v e r events i s r e g u l a t e d by a g e n e t i c a l l y c o n t r o l l e d system. In C. elegans recombination frequency i s a f f e c t e d by temperature and p a r e n t a l age (Rose and B a i l l i e 1979a), the gene rec-1 (Rose and B a i l l i e 1979b; R a t t r a y and Rose 1988), r a d i a t i o n (Kim and Rose 1987; McKim, Howell, and Rose, 1988), and t r a n s l o c a t i o n s (Rosenbluth and B a i l l i e 1981; Herman et a l . , 1982; McKim, Howell, and Rose, 1988). These s t u d i e s have not s p e c i f i c a l l y addressed the mechanism f o r the gene c l u s t e r s . A f e a s i b l e argument put f o r t h by Szauter (1984) f o r the reason r e g i o n a l d i f f e r e n c e s i n recombination e x i s t i s t h a t they r e s u l t from a secondary consequence of the p h y s i c a l s t r u c t u r e of chromosomes d u r i n g m e i o s i s . F a c t o r s such as the degree of chromosomal condensation, d i f f e r e n t i a l a s s o c i a t i o n of the chromosome with the synaptonemal complex, the nature of chromosomal p r o t e i n attachment, or some other f e a t u r e of 110 chromosome s t r u c t u r e may have s i g n i f i c a n t e f f e c t s on r a t e s of recombination. Within the LGI gene c l u s t e r of C. elegans McKim, Howell, and Rose (1988) observed that the r e g i o n adjacent to the breakpoint of the t r a n s l o c a t i o n szTl(I;X), underwent an enhancement i n recombination frequency. These authors proposed that the t r a n s l o c a t i o n d i s r u p t s r e g i o n a l c o n s t r a i n t s on recombination frequency due e i t h e r to the removal of chromosome I sequences r e s p o n s i b l e f o r the suppression of recombination or the i n t r o d u c t i o n of X chromosome sequences that do not c o n t a i n the s u p p r e s s i o n s i g n a l s . In t h i s t h e s i s the dpy-14 unc-13 i n t e r v a l showed an i n c r e a s e d amount of DNA per map u n i t v a l u e . Kim and Rose (1987) showed t h i s r e g i o n a l s o had the h i g h e s t l e v e l of i n c r e a s e d recombination frequency f o l l o w i n g gamma i r r a d i a t i o n . Regions on e i t h e r s i d e of t h i s i n t e r v a l showed l e s s than h a l f the r e l a t i v e i n c r e a s e i n recombination frequency (Kim and Rose 1987; McKim, Howell, and Rose, 1988). Perhaps sequences r e s p o n s i b l e f o r these o b s e r v a t i o n s r e s i d e i n the dpy-14 unc-13 i n t e r v a l . Depending on the nature of t h i s phenomenon i t may be p o s s i b l e to map the sequences r e s p o n s i b l e to w i t h i n a couple of cosmid e q u i v i l e n t s of DNA. I f so, then a d e t a i l e d examination of t h i s r e g i o n may r e v e a l sequences that are r e q u i r e d f o r the normal r e g u l a t i o n of recombination i n C. elegans. I l l 2. The Genetic L o c i W ithin the dpy-5 unc-13 r e g i o n of LGI The dpy-5 unc-13 i n t e r v a l of LGI has been e x t e n s i v e l y analyzed at the ge n e t i c l e v e l with approximately 80 genes m u t a t i o n a l l y i d e n t i f i e d (Brenner 1974; Rose and B a i l l i e , 1980; Edgley and R i d d l e , 1987; Howell et al., 1987; P i l g r i m and Rose 1988; Howell 1989; J . McDowall, p e r s o n a l communication). Many of these genes have been mapped i n t o zones using o v e r l a p p i n g s e t s of fr e e d u p l i c a t i o n s (McKim and Rose, 1989; J . McDowall and A.M. Rose, unpublished r e s u l t s ) . In a d d i t i o n , three f a c t o r mapping data (Appendix 3) pl a c e d the genes / e t - 3 8 1 , I et-382, let-383, let-385, let-386, lel-391, and let-392 to the l e f t of dpy-14 and the gene let-400 to the r i g h t . The gene let-389 was not separated from dpy-14 i n t h i s t h e s i s , but other work has p o s i t i o n e d let-389 to the r i g h t of dpy-14 (K. McKim, p e r s o n a l communication). F i g . 28 shows the g e n e t i c map of t h i s r e g i o n c o n s t r u c t e d from p u b l i s h e d data of oth e r s (Brenner 1974; Rose and B a i l l i e 1980; Edgley and R i d d l e , 1 987; Howell et al . , 1987; P i l g r i m and Rose 1988; Howell 1989; J . McDowall, p e r s o n a l communication) and from mapping data i n t h i s t h e s i s . 112 F i g u r e 28 GENETIC MAP OF THE dpy-14 REGION (I et-381) (/ et-382 I et-390) I et-75 (/ et-383 I et-391) | (let-386 let-392) Uet-86) unc-14 unc-15 (let-385 let-83) \ {I et-389,I et-400) \\ unc-37 (dpy-14, sPl) hP9 unc-13 I I J I I I IJ J hDf 8 hDP62 hDp58 sDp2 0.1 mu The f r e e d u p l i c a t i o n sDp2 was d e s c r i b e d by Rose et al, . (1984). The f r e e d u p l i c a t i o n s hDp58, and hDp62 were d e s c r i b e d by McKim and Rose, (1989). The map p o s i t i o n of let-75 (Rose and B a i l l i e , 1980) and unc-14 were based on data from McKim and Rose (1989). The p o s i t i o n i n g of let-86 and let-389 to the r i g h t of dpy-14 was based on data by K. McKim (p e r s o n a l communication). 113 3. C o r r e l a t i o n of the g e n e t i c and p h y s i c a l maps i n the  dpy-14 region of LGI Using N2/B0 mapping s t r a i n s f o r t h r e e - f a c t o r mapping, the sPl polymorphic s i t e was mapped between the genes unc-37 and unc-13, i n s e p a r a b l e from dpy-14. The hP9 polymorphic s i t e was mapped between dpy-14 and unc-13 and was p h y s i c a l l y l i n k e d 110 Kb to the r i g h t of sPl. T h i s r e s u l t o r i e n t e d the cosmids with r e s p e c t to the g e n e t i c map. The breakpoint of hDp62 was found to be between sPl and hP9. The breakpoint of hDp58 was found to be to the r i g h t of hP9. The gene unc-14 was found to be i n s i d e the breakpoint of sDp2 but o u t s i d e the breakpoint of hDp58 (McKim and Rose, 1989). hDf8 f a i l e d to complement the genes unc-37, unc-87, dpy-14, let-83, and let-86 but not the gene lei-75. The r i g h t breakpoint of hDf8 was mapped between sPl and hP9 i n the cosmid C14A12. Adjacent DNA sequences to the l e f t of the breakpoint were found by DNA dosage s t u d i e s to l i e w i t h i n the d e l e t i o n . Adjacent DNA sequences to the r i g h t of the breakpoint were found by DNA dosage s t u d i e s to l i e o u t s i d e of the d e l e t i o n . T h i s p l a c e s the genes unc-37, unc-87, dpy-14, let-83, and let-86 to the l e f t of the hDf8 b r e a k p o i n t . Although I have i n t e r p r e t e d the hDf8 chromosome to c o n t a i n a d e l e t i o n of the unc-37 to dpy-14 r e g i o n , t h i s mutation does not behave as expected f o r a simple d e l e t i o n . F i r s t , the C14A12-3.7 fragment d e t e c t s two e x t r a bands i n the hDf8 DNA. A simple d e l e t i o n would be expected to d e t e c t only one j u n c t i o n fragment with the C14A12-3.7 probe. A p o s s i b l e mechanism to e x p l a i n the two e x t r a fragments observed would be a d e l e t i o n event a s s o c i a t e d 114 with a d u p l i c a t i o n of the region c o n t a i n i n g the C14A12-3.7 r e g i o n . I f such an event had occured on the hDf8 chromosome then the C14A12-3.7 probe c o u l d h y b r i d i z e t o the j u n c t i o n fragment of the d e l e t i o n and to the j u n c t i o n fragment of the d u p l i c a t i o n . Such a d e l e t i o n / d u p l i c a t i o n event has been p r e v i o u s l y observed with the d e l e t i o n eDf 1 ( R i d d l e and Brenner, 1978; R. Waterston, 1988). Second, the hDf8 mutation completely suppresses recombination from the l e f t end of chromosome I to near the middle of chromosome I (K. McKim and A.M. Rose, p e r s o n a l communication). These r e s u l t s suggest that the hDf8 mutation i s a complex rearrangement. T h i s must be kept i n mind when i n t e r p r e t i n g r e s u l t s d e r i v e d from i n f o r m a t i o n o b t a i n e d with hDf8. One goal towards understanding the c o m p l e x i t i e s of how the genome of a higher eukaryote i s o r g a n i z e d i s to c h a r a c t e r i z e both m u t a t i o n a l l y and p h y s i c a l l y a l a r g e p o r t i o n of the genome. The dpy-5 unc-13 i n t e r v a l of LGI has been e x t e n s i v e l y analyzed at the g e n e t i c l e v e l . Most of the DNA i n t h i s i n t e r v a l i s now mapped, so an attempt to i d e n t i f y a l l the coding elements can be i n i t i a t e d . As the coding elements are i d e n t i f i e d a one-to-one c o r r e l a t i o n with the mutated genes can be made. The recent advances i n DNA m i c r o - i n j e c t i o n (Kimble et al., 1982) and i n the c o n s t r u c t i o n of t r a n s g e n i c s t r a i n s ( F i r e , 1986) i n C. elegans w i l l g r e a t l y a i d i n the d e t e r m i n a t i o n of these c o r r e l a t i o n s . One method would be to i n j e c t a mutant worm with c l o n e d DNA and observe the progeny f o r the rescue of the mutant phenotype. Another approach to rescue mutations i n e s s e n t i a l genes, would be to use a s t r a i n t h a t c a r r i e d a p i e c e of i n j e c t e d 115 DNA ( i n t e g r a t e d i n t o the genome or a c t i n g l i k e a f r e e d u p l i c a t i o n ) and t e s t f o r complementation to l e t h a l b e a r i n g s t r a i n s . However, rescue by i n j e c t i o n r e q u i r e s the f o l l o w i n g c o n d i t i o n s : 1) mutations i n genes from the re g i o n of i n t e r e s t have been i d e n t i f i e d and are a v a i l a b l e f o r use; 2) the DNA from the r e g i o n has been c l o n e d and i s a v a i l a b l e f o r i n j e c t i o n ; and 3) a c o r r e l a t i o n between the g e n e t i c and p h y s i c a l map of the re g i o n has been made. These c r i t e r i a have been met f o r the re g i o n immediately surrounding the dpy-14 gene. 4. I d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of coding elements  near dpy-14 The method f o r i s o l a t i n g coding elements r e l i e d on f i r s t i d e n t i f y i n g DNA sequences that were conserved between C. elegans and C. b r i g g s a e . T h i s method was based on the o b s e r v a t i o n s of T. Snutch that around the hsp70 gene of C. elegans, the only DNA that c r o s s h y b r i d i z e d with C. b r i g g s a e c o n t a i n e d the coding element f o r the hsp 70 gene (Snutch 1984). Prasad (1988) i d e n t i f i e d and i s o l a t e d nine DNA fragments from C. elegans near the unc-22 gene on LGIV that c r o s s - h y b r i d i z e d with C. b r i g g s a e . He i s o l a t e d the genes corre s p o n d i n g to two of the fragments from both C. elegans and C. b r i g g s a e . A comparison of the DNA sequences f o r these elements showed t h a t they were 80% conserved at the n u c l e o t i d e l e v e l w i t h i n the coding p o r t i o n of these genes. T h e r e f o r e , under the h y b r i d i z a t i o n c o n d i t i o n s used by Prasad (1988) DNA sequences that are 80% i d e n t i c a l can be d e t e c t e d by 116 Southern a n a l y s i s . I d e n t i c a l c o n d i t i o n s were used i n t h i s study to d e t e c t conserved fragments i n the dpy-14 r e g i o n of LGI. Within 173 Kb of N2 genomic DNA near dpy-14 ten conserved fragments were i d e n t i f i e d . These ten fragments d e t e c t e d seven cDNAs. Of the seven cDNA c l o n e s only s i x d e t e c t e d RNAs on northern b l o t s . The one cDNA c l o n e , sPl-1, that f a i l e d to d e t e c t a RNA molecule was the l e a s t abundant cDNA i s o l a t e d i n t h i s study. T h i s has i m p l i c a t i o n s f o r the conserved fragments sPI-8 and sPl-9 that d i d not d e t e c t cDNAs. If these two conserved fragments c o n t a i n genes that were expressed at lower l e v e l s than sPl-1 then i t i s not s u r p r i s i n g t h at they f a i l e d to i d e n t i f y any cDNAs or mRNAs. In the re g i o n around dpy-14 the average s p a c i n g between conserved fragments was found to be approximately 17 Kb (173 Kb per 10 fragments). In the re g i o n around unc-22 Prasad (1988) found the average spacing between genes to be 19 Kb (150 Kb per 8 genes). During a search f o r v i t e l l o g e n i n genes Heine and Blumenthal (1986) found the spacing between genes to be 20 Kb. Thus, the gene d e n s i t y i n the dpy-14 region i s comparable to other r e g i o n s of C. elegans. 5. Candidate sequences f o r the dpy-14 gene The Dpy mutants are a c l a s s of mutants showing the same s h o r t , f a t phenotype and have proven to be e x c e l l e n t g e n e t i c markers. As mentioned i n the I n t r o d u c t i o n , 28 d i f f e r e n t Dpy genes have been i d e n t i f i e d to date (Edgley and R i d d l e , 1987) and 117 some of the Dpy genes have been c l a s s i f i e d . The genes dpy-21, dpy-26, dpy-27, and dpy-28 are i n v o l v e d i n c o n t r o l l i n g X chromosome e x p r e s s i o n (Hodgkin, 1983; Meneely and Wood, 1984; Meyer and Casson, 1986; Hodgkin, 1987) and the genes dpy-13 (von Mende et al . , 1988) and dpy-10 (A. Levy and J . Kramer, p e r s o n a l communication) have been shown to code f o r c o l l a g e n s . Mutations i n the dpy-14 gene a l s o r e s u l t i n a worm with a sh o r t , f a t body shape. The only a l l e l e of dpy-I4(e188) that e x i s t s was o r i g i n a l l y i s o l a t e d by Brenner (1974). T h i s a l l e l e produces abnormal l a r v a e at ha t c h i n g t h a t remain Dpy throughout t h e i r a d u l t l i f e , and i t d i s p l a y s a t e m p e r a t u r e - s e n s i t i v e phenotype. Worms maintained at 16^C resemble the wi l d - t y p e phenotype whereas worms maintained at 20^C are Dpy. The dpy-14 gene has been shown t o i n t e r a c t with the gene sqt-1, which i s one of a group of genes thought to p l a y a r o l e i n the formation of the c u t i c l e (Kusch and Edgar, 1986; Kramer et al., 1988). The sqt-l(scl3) a l l e l e i s a r e c e s s i v e mutation that " r e s u l t s i n a l e f t r o l l e r phenotype. However, l i k e other r e c e s s i v e a l l e l e s of sqt-1 i t behaves as a " c r y p t i c dominant" l e f t r o l l e r i n c e r t a i n g e n e t i c backgrounds. Kusch and Edgar (1986) proposed that t h i s may be the r e s u l t of an i n t e r a c t i o n of genes that code f o r s t r u c t u r a l p r o t e i n s that form the c u t i c l e . A dpy-14 g e n e t i c background p r o v i d e s a phenotypic context i n which sqt-1(sc13) behaves dominantly. The gene f o r sqt-1 has been c l o n e d and shown to be a member of the c o l l a g e n gene f a m i l y (Kramer et al ., 1988). However, no c o l l a g e n genes were found i n 118 the cosmids surrounding the sPl s i t e t h e r e f o r e i t i s u n l i k e l y that the dpy-14 gene encodes a c o l l a g e n gene. 6. DNA sequence a n a l y s i s of coding elements conserved  between C. el egans and mammals With the advent of l a r g e shared computer databases, one p o s s i b l e method tp i d e n t i f y the f u n c t i o n of a gene product i s to sequence the gene and compare i t s sequence to other sequenced genes. As many more genes of known f u n c t i o n are sequenced and entered i n t o the database t h i s method becomes more u s e f u l . Since t h i s method r e l i e s on the p r i o r sequencing of an analogous gene, most l i k e l y from an organism other than C. elegans, candidate genes f o r t h i s a n a l y s i s should demonstrate c r o s s s p e c i e s h y b r i d i z a t i o n between d i s t a n t s p e c i e s (eg. nematodes and mammals). Both sPl-1 and sPl-3 meet t h i s c r i t e r i a . The sequences from these two cDNAs were compared to the sequence database of C. elegans and EMBL R18 (D. B a i l l i e , p e r s o n a l communication). Included i n the C. el egans sequence database are the sequences f o r col-1 and col-2. These two c o l l a g e n s show the t y p i c a l gly-X-Y repeat p a t t e r n found among members of the C. elegans c o l l a g e n gene f a m i l y (Kramer, et al . , 1982). Since t h i s gly-X-Y p a t t e r n was not observed i n the sequence of the sPl-1 cDNA, sPl-1 can not be co n s i d e r e d a member of the c o l l a g e n f a m i l y u n l e s s the repeat p a t t e r n i s found i n a f u l l l e n g t h cDNA not yet sequenced. The sPl-1 genomic clone was de t e c t e d by 119 c r o s s - h y b r i d i z a t i o n at low s t r i n g e n c y with the probe col-12 and at moderate s t r i n g e n c y with col-4. I t f o l l o w s that the sPl-1 c l o n e i s more c l o s e l y r e l a t e d to col-4 (at the l e v e l of DNA h y b r i d i z a t i o n ) than to col-12. The col-4 probe has been sequenced and found not to c o n t a i n any gly-X-Y repeats ( J . Kramer p e r s o n a l communication). In a d d i t i o n , and comparable to sPl-1, no RNA was de t e c t e d with the col-4 probe ( J . Kramer p e r s o n a l communication). An i n t e r e s t i n g r e s u l t found w i t h i n the sequence of sPl-1 i s a small repeat motif of approximately 30 amino a c i d s . The deduced amino a c i d sequence from t h i s r e g ion shows a h i g h l e v e l of the amino a c i d glutamine. A comparison of the deduced amino a c i d sequence to the NIBR sequence database d e t e c t e d matches with the neurogenic repeat OPA (Wharton et al., 1985; Stroeher et al., 1986; Laughon et al., 1986). The OPA repeat was estimated to be present at over 300 s i t e s i n the D. melanogaster genome (Wharton et a l . , 1985). Under low h y b r i d i z a t i o n c o n d i t i o n the sPl-1 cDNA h y b r i d i z e d to many C. elegans genomic fragments. Perhaps, l i k e OPA, i t i s the glutamine r i c h r e g i o n found w i t h i n sPl-1 that i s r e s p o n s i b l e f o r d e t e c t i n g these c r o s s homologies. I f so, t h i s would i n d i c a t e that the sPl-1 gene may be a member of a group of genes that are i n v o l v e d with neurogenesis i n C. elegans. F u r t h e r c h a r a c t e r i z a t i o n of sPl-1 w i l l be r e q u i r e d i n order to determine i t s f u n c t i o n . The p a r t i a l sequence of the sPl-3 cDNA c l o n e d e t e c t e d sequence s i m i l a r i t i e s with the gene S-adenosyl-L-homocysteine h y d r o l a s e (AHH) (D. B a i l l i e , p e r s o n a l communication) from r a t 120 (Ogawa et al . , 1987) and Di ct yost el i um di scoi deum (Kaser et al., 1988). Over a s t r e t c h of 800 bases the DNA sequence of sPl-3 was approximately 58% i d e n t i c a l to that of AHH from both r a t and D. dis coedeum. A comparison of the deduced amino a c i d sequence of sPl-3 with S-adenosyl-L-homocysteine h y d r o l a s e i s shown i n F i g . 24. Approximately 60 % of the amino a c i d s between sPl-3 and AHH from both r a t and D. discoideum are i d e n t i c a l ; and, i f c o n s e r v a t i v e changes are c o n s i d e r e d the homology between the C. el egans sequence and the AHH sequence from r a t i s 82% while between C. elegans and D. discoideum the homology i s 84%. The high degree o f s i m i l a r i t y between sPl-3 and AHH suggests that the sPl-3 l o c u s encodes the gene S-adenosyl-L-homocysteine h y d r o l a s e . AHH c a t a l y s e s the h y d r o l y s i s of S-adenosyl-L-homocysteine (SAH) to S-adenosine and L-homocysteine d u r i n g the b i o s y n t h e s i s of c y s t e i n e from methionine (de l a Haba and Cantoni, 1959). AHH has been found i n a l a r g e v a r i e t y of organisms (Shimizu et al., 1984), c o n s i s t s of a number of su b u n i t s , and r e q u i r e s NAD+ f o r a c t i v i t y (Richards et al., 1978). A c h a r a c t e r i s t i c NAD +-binding s i t e was observed i n both the ra t AHH sequence (Ogawa et al., 1987) and i n the D. discoideum AHH sequence ( K a s i r et al., 1988). T h i s f e a t u r e was not observed i n the sequence of sPl-3 because the sPl-3 cDNA clone d i d not extend f a r enough to in c l u d e t h i s s i t e . The sequencing of a f u l l l e n g t h cDNA clone or of the sPl-3 genomic c l o n e would be r e q u i r e d to c o n f i r m that sPl-3 c o n t a i n s a NAD + b i n d i n g s i t e . 121 AHH re p r e s e n t s approximately 2% of the s o l u b l e p r o t e i n s i n D. discoideum (Hohman et a l . , 1984) and approximately 0.2% of the s o l u b l e p r o t e i n s i n c a l f l i v e r (Richards et a l . , 1978). Although no i n f o r m a t i o n i s a v a i l a b l e r e g a r d i n g the AHH p r o t e i n i n C. elegans, i t i s f e a s i b l e t h a t the amount of AHH i n C. elegans i s s i m i l a r to that found i n other organisms ( i . e . , very abundant). The o b s e r v a t i o n that the r e l a t i v e abundance of the s P l - 3 cDNA cl o n e was found to be 0.4% i s c o n s i s t e n t with the hyp o t h e s i s t h a t the s P l - 3 l o c i encodes AHH. Since AHH i s a very abundant p r o t e i n found w i t h i n the c y t o p l a s i m of a l l c e l l s , i t i s i n t e r e s t i n g to s p e c u l a t e why high l e v e l s are r e q u i r e d . Hohman et a l . , (1984) p o s t u l a t e that the high abundancy of AHH found i n e u k a r y o t i c c e l l s was to r e g u l a t e the l e v e l s of S-adenosyl-L-homocysteine (SAH). Since SAH i s a potent i n h i b i t o r of S-adenosyl-L-methionine dependent transmethylases, c o n t r o l of the c e l l u l a r SAH l e v e l s v i a AHH may be a key r e g u l a t i o n step i n many c e l l u l a r m e t h y l a t i o n r e a c t i o n s . The a c t i v a t e d form of S-adenosylmethionine (SAM) i s the primary methyl group donor f o r most common c e l l u l a r m e t h l y l a t i o n r e a c t i o n s . The a c t i v a t e d form of SAM i s regenerated from m e t h y l a t i o n of homocysteine. AHH c a t a l i z e s the p r o d u c t i o n of homocysteine i n the me t h l a t i o n c y c l e , t h e r e f o r e AHH i s i n v o l v e d i n most c e l l u l a r m e t h y l a t i o n r e a c t i o n s . Another enzyme i n v o l v e d i n the methylation c y c l e i s methionine a d e n o s y l t r a n s f e r a s e (MAT). The c e l l u l a r l e v e l s of MAT were found to be 0.3% of the s o l u b l e p r o t e i n s i n r a t l i v e r (Tabor and Tabor, 1984) which i s s i m i l a r to the AHH l e v e l s . 122 A l t e r n a t i v e l y , AHH may have an a d d i t i o n a l f u n c t i o n i n the c e l l not yet determined. I f the sPl-3 l o c u s does encode AHH, i t w i l l be i n t e r e s t i n g to determine which mutant from the dpy-14 r e g i o n corresponds to AHH. 7. Homologies between C. el egans and mammals An i n t e r e s t i n understanding the ge n e t i c d e f e c t s of human dis e a s e s . h a s l e d to the p r o p o s a l to sequence the human genome. The sequence data would be used to i d e n t i f y open reading frames (ORF) and some b i o l o g i c a l i n f o r m a t i o n about the ORF's c o u l d be determined by d e t e c t i n g sequence s i m i l a r i t i e s with a gene of known f u n c t i o n . Hennikoff and Wallace (1988) demonstrated that t h i s approach i s f e a s i b l e by s u c c e s s f u l l y matching ten sequences, from a v a r i e t y of d i f f e r e n t organisms, with known genes. Since a l a r g e amount of sequence data i s generated from organisms that are d i s t a n t l y r e l a t e d to humans i t would be of i n t e r e s t to know what p r o p o r t i o n of genes have an analogous human c o u n t e r p a r t . To a f i r s t approximation t h i s c o u l d be determined by a syste m a t i c survey of a contiguous set of coding elements f o r t h e i r a b i l i t y to c r o s s - h y b r i d i z e to human DNA. In t h i s study, ten sequences conserved between C. elegans and C. b r i g g s a e were used t o survey f o r sequence s i m i l a r i t i e s between C. elegans and humans. Three of the ten fragments c o n t a i n e d sequences that would c r o s s - h y b r i d i z e to human DNA. One fragment, r e p r e s e n t i n g the sPl-1 l o c u s , was shown under moderate s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s to c r o s s - h y b r i d i z e to mammalian DNA. Since no s i g n i f i c a n t sequence s i m i l a r i t i e s 123 were found with sPl-J, the amount of c r o s s homology cannot be determined. A second fragment, r e p r e s e n t i n g the sPl-2 l o c u s , was shown to c o n t a i n the h i g h l y conserved small n u c l e a r RNA gene U1. T h i s gene has been i s o l a t e d from a v a r i e t y of organisms (Ro-Choi and Busch, 1974). A comparison of the sequence data r e v e a l e d that the U1 gene has r e t a i n e d approximately 75% i d e n t i t y between organisms as d i v e r s e as D. melanogast er and D. discoideum (Reddy et al., 1974; Wise and Weiner, 1981). A t h i r d fragment, r e p r e s e n t i n g the sPl-3 l o c u s , was shown under low s t r i n g e n c y h y b r i d i z a t i o n c o n d i t i o n s to c r o s s - h y b r i d i z e to human DNA. As d i s c u s s e d p r e v i o u s l y the sPl-3 gene probably encodes the gene AHH. A comparison of the sequence data r e v e a l e d that the sPl-3 gene shared approximately 60% DNA i d e n t i t y over and 800 base p a i r r e g i o n with the AHH cou n t e r p a r t from r a t and D. discoideum. However, the DNA s i m i l a r i t y was not uniform over the e n t i r e r e g i o n but reached a maximum value of approximately 75% i d e n t i t y over a 120 base p a i r s t r e t c h . T h e r e f o r e , i t appears that C. el egans sequences s h a r i n g 75% DNA i d e n t i t y can be used t o det e c t analogous human sequences. The C. elegans sequences from the dpy-14 r e g i o n that c r o s s - h y b r i d i z e d to mammalian DNA represent 30% of the genes d e t e c t e d i n t h i s study. The gene d e n s i t y i n the dpy-14 region was found to be s i m i l a r to the gene d e n s i t y i n the regions i n v e s t i g a t e by Prasad (1988) and Heine and Blumenthal (1986). T h e r e f o r e , there i s no reason to b e l i e v e that the dpy-14 region i s v a s t l y d i f f e r e n t from other r e g i o n s i n C. elegans. T h i s means 124 that p o s s i b l y as many as one t h i r d of the genes from C. elegans would be u s e f u l i n i d e n t i f y i n g human genes. A d d i t i o n a l i n f o r m a t i o n other than sequence data i s r e q u i r e d to i n i t i a l l y i d e n t i f y the f u n c t i o n of a gene. T h e r e f o r e , e x t r a c t i n g the b i o l o g i c a l i n f o r m a t i o n from the sequenced human genome w i l l r e q u i r e a comparison to p r e v i o u s l y i d e n t i f i e d sequences. Since C. elegans w i l l have many gene i n common to humans then perhaps the sequence data from C. elegans w i l l p l a y a v i t a l r o l e i n the i d e n t i f i c a t i o n of many human genes. 125 SUMMARY AND CONCLUSIONS The o b j e c t i v e s of t h i s t h e s i s were: 1. to map N 2 / B 0 s t r a i n polymorphisms from the dpy-5 unc-29 i n t e r v a l of the C. elegans LGI gene c l u s t e r with r e s p e c t to each other and v i s i b l e g e n e t i c markers from the r e g i o n ; 2 . to use the mapped s t r a i n polymorphisms to o b t a i n cosmid DNA from A. Coulson and J . S u l s t o n ; 3. to use the cosmid DNA to e s t a b l i s h a rough alignment between the g e n e t i c and p h y s i c a l maps of the r e g i o n ; 4. to examine the DNA with r e s p e c t to the amount of DNA per map u n i t i n s e v e r a l d i f f e r e n t r e g i o n s ; 5 . to l o c a t e and c h a r a c t e r i z e c o d i n g elements near the gene dpy-14; 6. to a l i g n the g e n e t i c and p h y s i c a l maps i n the dpy-14 r e g i o n ; 7 . to survey a contiguous r e g i o n i n C. elegans f o r DNA homologies with mammals. 126 8. to i d e n t i f y and sequence C. elegans coding elements conserved between C. el egans and mammals i n an e f f o r t to e s t a b l i s h t h e i r f u n c t i o n . The s i x polymorphisms used i n t h i s study were mapped using the N2/B0 recombinant s t r a i n s l i s t e d i n appendix 2. From t h i s a n a l y s i s the g e n e t i c order of these s i x polymorphic DNA markers with respect to v i s i b l e g e n e t i c markers was found to be dpy-5, hP5, bli-4, unc-37, {dpy-14, sPl), hP9, unc-13, hP7, (hP4, hP6), unc-29. Subsequent to t h i s S. Prasad p o s i t i o n e d dpy-14 to the l e f t of sPl a f t e r r e s c u i n g Dpy-14 worms by DNA m i c r o - i n j e c t i o n with the cosmid T14D10 (S. Prasad p e r s o n a l communication). Thus, the g e n e t i c order of the markers i s now e s t a b l i s h e d to be dpy-5, hP5, bli-4, unc-37, dpy-14, sPl, hP9, unc-13, hP7, (hP4, hP6), unc-29. N2 phage c o n t a i n i n g the hP5, sPl, and hP6 s i t e s were used to i d e n t i f y cosmid c o n t i g s from the LGI gene c l u s t e r . In a d d i t i o n , the cosmid c o n t a i n i n g the hP9 s i t e was used to l i n k the sPl and unc-13 cosmid c o n t i g s t o g e t h e r . T h e r e f o r e , the polymorphisms mapped in t h i s study were i n s t r u m e n t a l i n l i n k i n g together a s i n g l e c o n t i g that spans the 1.2 mu between hP5 and unc-13. The hP5, sPl, hP9, and unc-13 l o c i p r o v i d e anchor p o i n t s e s t a b l i s h i n g a rough alignment between the g e n e t i c and p h y s i c a l maps i n t h i s r e g i on of LGI. The amount of DNA per cosmid, based on an estimate d e r i v e d from 12 cosmids i n the hP5 unc-13 i n t e r v a l , was found to be 34 Kb per cosmid. There are approximately 30 cosmid e q u i v i l e n t s of DNA 127 i n the hP5 unc-13 i n t e r v a l . T h e r e f o r e , the p h y s i c a l d i s t a n c e between these markers i s estimated to be 1000 Kb. The amount of DNA between hP5 and unc-13 (1000 Kb) was compared to the d i s t a n c e i n map u n i t s (1.2 mu). Based on the expected average DNA content of 333 Kb/mu i t can be concluded that m e i o t i c recombination i s suppressed i n t h i s r e g i o n . A c l o s e r i n s p e c t i o n of d i f f e r e n t r e g i o ns i n the hP5 unc-13 i n t e r v a l r e v e a l e d t h a t the g r e a t e s t s uppression oc c u r r e d between hP9 and unc-13. T h i s i s i n agreement with the gamma-ray expansion data of Kim and Rose (1987) that showed the g r e a t e s t r e g i o n of expansion o c c u r r e d between dpy-14 and unc-13. The region around dpy-14 has undergone an e x t e n s i v e g e n e t i c a n a l y s i s (Rose and B a i l l i e 1980; Howell et al., 1987; P i l g r i m and Rose 1988; Howell 1989; J . McDowall, unpublished r e s u l t s ) and many e s s e n t i a l genes have been i d e n t i f i e d and mapped (Rose and B a i l l i e 1980; Howell et al., 1987; McKim, Howell and Rose, 1988; McKim and Rose, 1989; J . McDowall and A.M. Rose, unpublished r e s u l t s ; K. McKim, unpublished r e s u l t s ) . Furthermore, the polymorphic s i t e , sPl (Rose et al., 1982), s u i t a b l e f o r i d e n t i f y i n g DNA i n the r e g i o n was a v a i l a b l e f o r use. T h i s made the dpy-14 region an i d e a l l o c a t i o n to s t a r t a search f o r coding elements c o n t a i n e d w i t h i n the cosmids. The technique of h y b r i d i z i n g C. el egans DNA probes to the c l o s e l y r e l a t e d s p e c i e s C. briggsae had been proven to be a r a p i d and s e n s i t i v e method f o r the i d e n t i f i c a t i o n of coding elements (Snutch, 1984; Prasad, 1988). W i t h i n 173 Kb of DNA (contained w i t h i n seven cosmids near to dpy-14) 10 conserved regions were 128 i d e n t i f i e d . Of these ten conserved r e g i o n s only seven d e t e c t e d cDNAs. One of the conserved regions not d e t e c t i n g a cDNA, sPl-2, c r o s s - h y b r i d i z e d with a probe c o n t a i n i n g a coding element f o r the gene U1. Most l i k e l y the i n a b i l i t y of the remaining two conserved regions to d e t e c t a cDNA i s because of t h e i r low r e p r e s e n t a t i o n due to e i t h e r poor or stage s p e c i f i c e x p r e s s i o n . A d e t a i l e d p h y s i c a l map of the dpy-14 r e g i o n was e s t a b l i s h e d by p o s i t i o n i n g the sPl and hP9 s i t e s r e l a t i v e to the rearrangements hDf8, hDp58, hDp62, and sDp2. The p h y s i c a l map was a l i g n e d with the e x i s t i n g g e n e t i c map which makes the dpy-14 r e g i o n one of the few regions i n C. elegans to be c h a r a c t e r i z e d at both the g e n e t i c and molecular l e v e l s . A systematic survey f o r homology to mammalian DNA us i n g the set of conserved regions i d e n t i f i e d i n t h i s t h e s i s found that three coding elements were conserved. The two C. elegans coding elements sPl-1 and sPl-3 c r o s s - h y b r i d i z e d to mammalian DNA. Both of the c o r r e s p o n d i n g cDNAs were p a r t i a l l y sequenced and compared to the EMBL sequence data base. No s i m i l a r i t i e s were found with the sPl-1 cDNA. The sPl-3 cDNA c o n t a i n e d approximately 60% i d e n t i t y with the gene AHH from r a t and D. discoideum. LIST OF FINDINGS The f o l l o w i n g i s a l i s t of the f i n d i n g of t h i s t h e s i s . 1. The polymorphisms hP5, sPl, and hP9 were mapped to the r e g i o n spanned by the f r e e d u p l i c a t i o n sDp2 and the polymorphisms hP4, hP6, and hP7 were mapped o u t s i d e the sDp2 r e g i o n . 129 2. The polymorphism hP9 was i d e n t i f i e d , mapped between dpy-14 and unc-13, and mapped to the r i g h t of the sPl s i t e . 3. The g e n e t i c order of the polymorphic s i t e s with r e s p e c t to the g e n e t i c map was found to be dpy-5, hP5, unc-37, {dpy-14, sPl) hP9, unc-13, hP7, (hP4, hP6) , unc-29. 4. Phage c o n t a i n i n g the hP5, sPl, and hP6 s i t e s i d e n t i f i e d and anchored cosmid c o n t i g s to the g e n e t i c map. The i n t e r v a l from the l e f t of hP5 to the r i g h t of unc-13 i s now c o n t a i n e d i n a s i n g l e c o n t i g of approximately 1400 Kb. 5. The DNA per map u n i t value was found to vary i n the hP5 to unc-13 i n t e r v a l with the g r e a t e s t value found between hP9 and unc-13. 6. Seven cosmids r e p r e s e n t i n g 173 Kb of N2 genomic DNA near the gene dpy-14 were i d e n t i f i e d . Using c r o s s - s p e c i e s h y b r i d i z a t i o n to C. briggsae ten conserved r e g i o n s were i d e n t i f i e d w i t h i n these seven cosmids. The ten conserved fragments were used to i d e n t i f y seven cDNAs, s i x of which i d e n t i f i e d RNAs on Northern b l o t s . 7. The r e l a t i v e abundance of the i s o l a t e d cDNAs v a r i e d 250 f o l d w ith the most abundant s i m i l a r to that found f o r a c t i n . 130 8. A comprehensive survey f o r mammalian homologies i n a contiguous set of ten C. elegans coding regions found three coding elements to be conserved. 9. The sPl-2 conserved r e g i o n c o n t a i n e d the small n u c l e a r RNA gene U1. 10. The sPl-3 cDNA was p a r t i a l l y sequenced and found to share sequence s i m i l a r i t i e s with the gene S-adenosyl-L-homocysteine h y d r o l a s e . 11. The sPl-1 cDNA was p a r t i a l l y sequenced and the sequence used to search the EMBL sequence data base. No homologies were i d e n t i f i e d . 12. A formaldehyde-induced mutation t h a t f a i l e d to complement the genes unc-37, unc-87, dpy-14, let-83 and let-86 was i s o l a t e d . T h i s mutation appeared to be the r e s u l t of a DNA rearrangement with one breakpoint f a l l i n g w i t h i n the cosmid C14A12. 13. Using the conserved elements i d e n t i f i e d i n t h i s t h e s i s and the rearrangements and mapped genes from the r e g i o n , a d e t a i l e d p h y s i c a l and g e n e t i c map i n the v i c i n i t y of dpy-14 was c o n s t r u c t e d . 131 PROPOSALS FOR FUTURE RESEARCH The f o l l o w i n g i s a l i s t of experiments that c o u l d be done to f u r t h e r t h i s work. 1.) Try to narrow down the r e g i o n between hP9 and unc-13 that has the h i g h e s t DNA per map u n i t v a l u e . T h i s c o u l d be accomplished by g e n e r a t i n g many (ie. 100) N2/BO recombinant s t r a i n s and i d e n t i f y i n g a d d i t i o n a l polymorphisms between dpy-14 and unc-13. Once the region has been narrowed down to a few cosmids ( i f p o s s i b l e ) then examine the DNA f o r unusual f e a t u r e s , such as r e p e t i t i v e DNA or f o r coding elements l i k e SIR ( G o t t l i e b and E s p o s i t o , 1989) which i s known to a f f e c t recombination r a t e s . 2) By DNA rescue t r y to e s t a b l i s h i f any of the coding elements, sPl-3, sPl-4, or sPl-5 correspond to the dpy-14 gene. 3) By DNA rescue t r y to e s t a b l i s h which of the coding elements i d e n t i f i e d i n t h i s work correspond to the e s s e n t i a l genes mapped in the r e g i o n . 4) With the technique of in situ h y b r i d i z a t i o n , determine i f any of the coding elements i d e n t i f i e d i n t h i s work are expressed i n a t i s s u e s p e c i f i c manner. 132 5) H y b r i d i z e the coding elements i d e n t i f i e d i n t h i s work to Northern b l o t s c o n t a i n i n g RNA from the d i f f e r e n t stages of development to determine when the genes are maximally expressed. 6) I f the f u n c t i o n of s P l - 1 i s determined, then i s o l a t e and map to a chromosome the human co u n t e r p a r t gene. 7) Determine the d i r e c t i o n of t r a n s c r i p t i o n f o r each of the i d e n t i f i e d coding elements. 8) Use the cosmids as probes to screen northern b l o t s i n a t r a n s c r i p t i o n mapping experiment to determine i f a d d i t i o n a l coding elements from the region can be d e t e c t e d . 9) I s o l a t e the C. b r i g g s a e genomic r e g i o n using the C. elegans coding elements i d e n t i f i e d near dpy-14 to determine whether the gene order i s maintained. 133 REFERENCES BAILLIE, D. 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MESSING, 1985. Improved M13 phage c l o n i n g v e c t o r s and host s t r a i n s : N u c l e o t i d e sequences of the M13mpl8 and pUCl9 v e c t o r s . Gene 33:103-119. Appendix 1 STRAINS USED S t r a i n Genotype O r i g i n N2 v i l d - t y p e Brenner S, B a i l l i e D.L. BO wild-type Hirsh D G16 wild-type Fodor A BC69 • dpy-14(el88) unc-13(e51) Rose A BC105 unc-S7(e262) dpy-14(el8S) Rose A KR1124 dpy-14(elS8) Ut-75(sl0l) unc-lS(c51)/ szTl{l;I) Rose A KR1073 dpy-14(el88) Iet-B6(sl4l) unc-13(e51)/ szTl(l;l) Rose A KR1036 dpy-H(tlSS) Ut-83 (s97) unc-13(eSl)/ szft(I;X) Rose A BC62 dpy-5(e61) %nc-75(e950) Rose A KR173 dpy-5(e6l) v.nc-lS(t450) Rose A KR446 dpy-5(e€l) let-S76(hlS0) unc-13(e450) Rose A KR281 dpy-5(e6l) Ict-S90(h44) unc-13(e450) Rose A KR425 dpy-5(e61) lei-392(hl22) unc-13(e450) Rose A KR352 dpy-5(e6l) Iet-S89(k89) unc-13(t450) Rose A KR429 dpy-5(e61) let-381(hl07) unc-13(e450) Rose A KR432 dpy-5(t6l) Ut-S86(hll7) unc-13(e450) Rose A KR424 dpy-S(eBl) let-38S(hll5) unc-13(e450) Rose A KR357 dpy-5(e6l) Ut-S82(h82) unc-13(e450) Rose A KR354 dpy-5(e61) let-3S5(h85) unc-13(e450) Rose A KR350 dpy-5(e6l) Ut-S9l(h91) unc-13(e450) Rose A KR610 dpy-5(eei) let-400(h269) unc-lS(e450) Rose A Strain Genotype Origin KR598 dpy-5(e6l) Iet-S98(h257) unc-13(e450) Rose A KR542' dpy-5(e6l) let-S97(h£28) unc-lS(e450) Rose A KR800 Ut-(h5S9) dpy-5(hl4) unc-£9(h2) ; sDp2 (I:f) Rose A KR1000 dpy-5(e6l) dpy-14(el88) unc-13(e4S0) / h572 Starr T KR1758 dpy-S(eei) dpy-149(el8S) ; hDp62 McKim K KR1775 dpy-S(t6l) ipy-U9(el88) ; hDpSS McKim K KR1793 lei-35£(h45) dpy-5 (t6l) unc-13 (e450) / h572 McKim R Appendix 2. THREE-FACTOR MAPPING STRAINS strain heterozygous parent genotype recombinant genotype origin KR1561 dpy-14 unc-13 (N2) / + + (BO) KR1562 dpy-14 unc-13 (N2) / + + (BO) KR1564 dpy-14 unc-13 (N2) / + + (BO) RR1565 dpy-14 unc-13 (N2) / + + (BO) KR1676 dpy-14 unc-lS (N2) / + + (BO) KR1677 dpy-14 unc-lS (N2) / + + (BO) KR1678 dpy-14 unc-13 (N2) / + + (BO) KR1679 dpy-14 unc-13 (N2) / + + (BO) KR409 + + (N2) / dpy-5 unc-29 (BO) KR410 + + (N2) / dpy-5 unc-29 (BO) KR411 + + (N2) / dpy-5 unc-29 (BO) KR412 + + (N2) / dpy-5 unc-29 (BO) KR413 + + (N2) / dpy-5 unc-29 (BO) KR414 + + (N2) / dpy-5 unc-29 (BO) KR984 + + (N2) / dpy-5 unc-29 (BO) KR985 + + (N2) / dpy-5 unc-29 (BO) + (BO) unc-13 (N2) + (BO) unc-13 (N2) + (BO) unc-13 (N2) ' + (BO) unc-13 (N2) dpy-14 (N2) + (BO) dpy-14 (N2) + (BO) dpy-14 (N2) + (BO) dpy-14 (N2) + (BO) + (N2) unc-29 (BO) + (N2) unc-29 (BO) + (N2) unc-29 (BO) + (N2) unc-29 (BO) + (N2) unc-29 (BO) 4 (N2) unc-29 (BO) + (N2) unc-29 (BO) + (N2) unc-29 (BO) Starr T. Starr T. Starr T. Starr T. Starr T. Starr T. Starr T. Starr T. Howell A.M. Howell A.M. Howell A.M. Howell A.M. Howell A.M. Howell A.M. Howell A.M. Howell A.M. Appendix 2. THREE-FACTOR HAPPING STRAINS strain heterozygous parent genotype recombinant genotype origin KR986 + + (H2) / dpy-5 unc -ts (BO) + (N2) unc-29 (BO) Howell A.M. KR987 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR988 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR989 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR990 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR991 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR992 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. RR993 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR994 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR995 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. RR996 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR997 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR998 + + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. KR999 4 + (N2) / dpy-5 unc -29 (BO) dpy-5 (BO) + (N2) Howell A.M. RR976 unc-37 dpy-14 (N2) / + + (BO) unc-37 (N2) 4 (BO) Starr T. KR977 UnC- 57 dpy-14 (N2) / + + (BO) + (BO) dpy-14 (N2) Starr T. Appendix 2. THREE-FACTOR MAPPING STRAINS strain heterozygous parent genotype recombinant genotype origin KR1402 unc-37 dpy-14 (N2) / + + (BO) KR1196 dpy-5 bli-4 unc-13 / + + + (BO) KR1197 dpy-5 bli-4 unc-13 / + + + (BO) KR1198 dpy-5 bli-4 unc-13 / + + + (BO) KR1199 dpy-5 bli-4 unc-13 / + + + (BO) KR1203 dpy-5 bli-4 unc-lS / 4 + + (BO) KR1205 dpy-5 bli-4 unc-13 / + + + (BO) KR1206 dpy-5 bli-4 unc-lS / + + + (BO) KR1207 dpy-5 bli-4 unc-13 / + + + (BO) KR1208 dpy-5 bli-4 unc-lS / + + + (BO) KR1209 dpy-5 bli-4 unc-13 / + + + (BO) KR1219 dpy-5 bli-4 unc-13 / + + + (BO) KR1220 dpy-5 bli-4 unc-13 / + + + (BO) + (BO) dpy-14 (N2) Starr T. + + (BO) unc-13 (N2) Peters R. + + (BO) unc-lS (N2) Peters K. + + (BO) unc-13 (N2) Peters K. + + (BO) unc-13 (N2) Peters K. + + (BO) unc-13 (N2) Peters K. + + (BO) unc-13 (N2) Peters K. + + (BO) unc-13 (N2) Peters K. + + (BO) unc-13 (N2) Peters K. + + (BO) unc-13 (N2) Peters K. + + (BO) unc-13 (N2) Peters K. dpy-5 bli-4 (N2) + (BO) Peters K. dpy-5 bU-4 (N2) + (BO) Peters K. 

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