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Characterization of the recombination enhancer, rec-1, in Caenorhabditis elegans Rattray, Bruce 1986

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CHARACTERIZATION OF THE RECOMBINATION ENHANCER, REC-IN CAENORHABDITIS ELEGANS by Bruce R a t t r a y B . S c , Queens U n i v e s i t y , Kingston, O n t a r i o . 1981. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES GENETICS We accept t h i s t h e s i s as conforming to the r e q u i r e d standard Dr. A.M. Rqsfi^, co-^Ajzlvis^r Dr. C. F. Wehrhahn, co-Advisor Dr. R. Ward A d v i s o r y Committee Dr. D. Juri.lof.fV Examiner Dr. F. D i T l , Chairman THE UNIVERSITY OF BRITISH COLUMBIA JANUARY 1986 BRUCE RATTRAY, 1986 7% In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of r^XeA^ c^  ( r e ^ t - ^ ^ 5 The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date T0—,. ~~2- °[ j *5 £ / R I ,\ A b s t r a c t The hyper-rec mutation r e c - 1 , i n C a e n o r h a b d i t i s elegans, has p r e v i o u s l y been shown to i n c r e a s e i n t e r g e n i c recombination frequency by a f a c t o r of three (Rose and B a i l l i e , 1979a). The f u r t h e r c h a r a c t e r i z a t i o n of the e f f e c t of rec-1 on recombination and other m e i o t i c processes has been the focus of t h i s t h e s i s . The e f f e c t of rec-1 on i n t r a g e n i c recombination, gene c o n v e r s i o n , f e c u n d i t y , c o m p e t i t i v e a b i l i t y , n o n d i s j u n c t i o n and mutation r a t e were examined. The rec-1 mutation produced a two to t h r e e - f o l d i n c r e a s e i n i n t r a g e n i c recombination and gene c o n v e r s i o n . P l e i o t r o p i c e f f e c t s are t y p i c a l of recombination mutants, however, o n l y the r a t e of X-chromosome n o n d i s j u n c t i o n and/or chromosome l o s s was a f f e c t e d by r e c - 1 , e x h i b i t i n g a t w o - f o l d i n c r e a s e r e l a t i v e t o the N2 (wild-type) s t r a i n . In a d d i t i o n to the above, a s t r a i n was c o n s t r u c t e d to determine i f the rec-1 mutation was an amber, nonsense mutation ( i e . a premature stop codon, p r e v e n t i n g t r a n s l a t i o n ) . The r e s u l t s showed t h a t the e x p r e s s i o n of the Rec-1 phenotype was not a f f e c t e d by a tRNA amber suppressor ( i e . sup-7). T h i s i n d i c a t e d t h a t rec-1 i s not an amber mutation. The a f f e c t of rec-1 as a recombination enhancer on the m o b i l i t y of the t r a n s p o s a b l e element, Tc1, was a l s o examined. E l e c t r o p h o r e t i c banding p a t t e r n s produced by DNA r e s t r i c t i o n fragments probed with Tc1 were compared f o r the N2 and Rec-1 s t r a i n s . An a l t e r e d banding p a t t e r n was found, s u g g e s t i n g t h a t rec-1 a c t i v a t e s Tc1 m o b i l i t y . However, p r e d i c t e d d i f f e r e n c e s between two Rec-1 DNA samples (one prepared two years a f t e r the other) were not found. S e v e r a l a l t e r n a t i v e e x p l a n a t i o n s are c o n s i d e r e d . High recombination mutations r e p o r t e d i n the l i t e r a t u r e are reviewed and s e v e r a l hypotheses c o n s i s t e n t with the above r e s u l t s are c o n s i d e r e d as p o s s i b l e e x p l a n a t i o n s of the rec-1 enhancer. i i i TABLE OF CONTENTS A b s t r a c t i i L i s t of Tables v i L i s t of F i g u r e s v i i Ackowledgements i x I. I n t r o d u c t i o n : an overview 1 I I . Background Inf o r m a t i o n A) L i f e H i s t o r y 8 B) S t r a i n s and C u l t u r e C o n d i t i o n s 9 C) S t r a i n C o n s t r u c t i o n s 10 I I I . Recombination E f f e c t s I n t r o d u c t i o n 20 Methods 23 R e s u l t s 31 D i s c u s s i o n 35 IV. P l e i o t r o p i c E f f e c t s I n t r o d u c t i o n 38 Methods 41 R e s u l t s 45 D i s c u s s i o n 51 V. The E f f e c t of an Amber Suppressor on rec-1 I n t r o d u c t i o n 55 Methods 56 R e s u l t s 57 D i s c u s s u i o n 60 i v VI. The E f f e c t of rec-1 on Tc1 M o b i l i t y I n t r o d u c t i o n 63 M a t e r i a l s and Methods 65 R e s u l t s 66 D i s c u s s i o n 68 V I I . D i s c u s s i o n : an overview 72 L i t e r a t u r e C i t e d 95 v L i s t of Tables Table 1: S t r a i n s used i n t h i s study 11 Table 2: Mutations used i n t h i s study 12 Table 3: V e r i f i c a t i o n of s t r a i n c o n s t r u c t i o n 18 Table 4: The e f f e c t of rec-1 on i n t r a g e n i c recombination and gene c o n v e r s i o n 32 Table 5: P u t a t i v e Rec-1, d u p l i c a t i o n b e a r i n g s t r a i n s 34 Table 6: The e f f e c t of rec-1 on f e c u n d i t y and c o m p e t i t i v e a b i l i t y 46 Table 7: The e f f e c t of rec-1 on n o n d i s j u n c t i o n and/or chromosome l o s s 48 Table 8: The e f f e c t of rec-1 on the frequency of r e c e s s i v e , l e t h a l mutations 50 Table 9: Summary of the r e s u l t s c h a r a c t e r i z i n g the rec-1 enhancer 73 Table 10: A review of l o c a l recombination enhancers 76 Table 11: A review of g e n e r a l enhancers of recombination 78 v i L i s t of F i g u r e s F i g u r e 1: Genetic map of C. elegans 13 F i g u r e 2: Determination of p, the recombination frequency 14 F i g u r e 3: C o n s t r u c t i o n of rec-1 homozygous s t r a i n s 16 F i g u r e 4: C o n s t r u c t i o n of a heterozygous unc-13 s t r a i n and i t s use i n measuring i n t r a g e n i c recombination 24 F i g u r e 5: Sample s i z e estimate f o r the number of chromosomes screened i n the i n t r a g e n i c expt 26 F i g u r e 6: C a l c u l a t i o n of i n t r a g e n i c recombination frequency 27 F i g u r e 7: C a l c u l a t i o n of gene c o n v e r s i o n frequency 28 F i g u r e 8 S t r a i n c o n s t r u c t i o n p r o t o c o l to determine the e f f e c t of rec-1 on l a r g e g e n e t i c d i s t a n c e s 30 F i g u r e 9: Assay f o r dete r m i n i n g the genotypes of competitors 43 F i g u r e 10: S t r a i n c o n s t r u c t i o n and procedure to determine the e f f e c t of rec-1 on mutation r a t e 44 F i g u r e 11: S t r a i n c o n s t r u c t i o n and procedure to determine the e f f e c t of sup-7 on the e x p r e s s i o n of rec-1 ....58 F i g u r e 12: The e f f e c t of sup-7 on the e x p r e s s i o n of the Rec-1 phenotype .....59 v i i F i g u r e 13: T c 1 - h y b r i d i z e d r e s t r i c t i o n fragment p a t t e r n s from DNA samples of Rec-1 and N2 67 F i g u r e 14: Screen f o r the i s o l a t i o n of a l l e l e s of rec-1 89 v i i i Acknowledgements I would l i k e to thank Ree, Kelly and Linda for t h e i r unflagging support; Terry, Linda and Kelly for t h e i r patience and help i n the lab; Dr. Dave Holm for his c r i t i c a l review of my work and my supervisor, Dr. Ann Rose, for her dedication to the rec-1 story ... II I t was a dark and stormy night II ix I. I n t r o d u c t i o n : An Overview Recombination i s c e n t r a l t o the process of m e i o s i s . By g e n e r a t i n g and c h a r a c t e r i z i n g recombination mutants, some i n s i g h t i n t o normal gene f u n c t i o n and mechanism may be o b t a i n e d . Rec-1, i n C a e n o r h a b d i t i s elegans, was i d e n t i f i e d and c h a r a c t e r i z e d as a spontaneous mutation by Rose and B a i l l i e (1979a). I t i s the o n l y known m e i o t i c enhancer of recombination i n eukaryotes which i s both g e n e r a l and uniform i n i t s e f f e c t (Rose, 1980). In the homozygous s t a t e the rec-1 mutation i n c r e a s e s the recombination frequency three to f o u r - f o l d . The same p r o p o r t i o n a l i n c r e a s e was found to be independent of both the s p e c i f i c i n t e r v a l and l i n k a g e group examined. I t was e s t a b l i s h e d t h a t a r e c e s s i v e mutation was r e s p o n s i b l e f o r the observed e f f e c t on recombination f r e q u e n c i e s . Given t h a t hyper-rec mutants are unusual, the rec-1 mutation p r o v i d e s an unique o p p o r t u n i t y to i n v e s t i g a t e r e c o m b i n a t i o n a l processes i n m e i o s i s . T h i s study d e a l s with the c h a r a c t e r i z a t i o n of r e c - 1 , and w i l l serve as a framework f o r f u t u r e i n v e s t i g a t i o n i n t o the m o l e c u l a r nature and e v o l u t i o n a r y r o l e of recombination. Hyper-rec mutations form an uncommon s u b c l a s s of recombination mutants. A number of examples have been found i n E s c h e r i c h i a c o l i : p o l A, uvrD, dut, dam and l i g (Konrad and Lehman, 1974; A r t h u r and L l o y d , 1980; Tye et a l , 1977; Konrad and Lehman, 1975; Marinus and Konrad, 1976; Bale et a l , 1979; Konrad et a l . , 1973). These mutants have a g e n e r a l e f f e c t on recombination frequency ( i e . recombination i s a f f e c t e d through out the genome), but the extent of the i n c r e a s e v a r i e s f o r d i f f e r e n t p a i r s of markers. A l l are mutators. 1 In yeast (Saccharomyces c e r e v i s i a e ) , c u r r e n t evidence suggests t h a t many recombination processes and t h e i r c o n t r o l s are h e l d i n common between m i t o s i s and m e i o s i s (eg. spo-7: E s p o s i t o e t a l . , 1975; spo-8: E s p o s i t o and E s p o s i t o , 1969; and see Haynes and Kunz, 1981 f o r a r e v i e w ) . However, d e s p i t e t h i s , o n l y m i t o t i c enhancers have been i s o l a t e d . UV-induced i n t r a - and i n t e r g e n i c recombination i s i n c r e a s e d by the r e p a i r d e f i c i e n t mutants rad-1, rad-3 and rad-4 (Hunnable and Cor, 1971; Kowalski and Lasowski, 1975). S i m i l a r r e s u l t s have been found f o r rev-1, rev-2 and rev-3 (Lemontt, 1971 ). The r e c e s s i v e mutation c h l i n c r e a s e s spontaneous m i t o t i c c r o s s i n g over but causes a h i g h frequency of l o s s of chromosome th r e e d u r i n g m e i o s i s and decreases m e i o t i c recombination (Haber, 1974; Haber p e r s . comm., c i t e d i n Baker et a l . , 1976). F i n a l l y , rem-1 i n c r e a s e s spontaneous i n t e r - and i n t r a g e n i c m i t o t i c recombination and spontaneous m i t o t i c mutation, but has no a f f e c t on m e i o s i s ( G o l i n and E s p o s i t o , 1977; Malone and Hoekstra, 1984). M i t o t i c mutants s i m i l a r to rem-1 have been r e p o r t e d i n other s p e c i e s (eg. Neurospora: uvs-3, Schroeder, 1970; A s p e r g i l l u s : uvs-B, S h a n f i e l d and Kafer, 1969; U s t i l a g o : uvs-1, H o l l i d a y et a l . , 1976). None of these mutants a l t e r m e i o t i c exchange. In Neurospora, three m e i o t i c hyper-rec mutations have been c h a r a c t e r i z e d . However, none of them are g e n e r a l i n t h e i r e f f e c t on recombination. M u t a t i o n s . i n rec-1 produce a 10 to 25 f o l d i n c r e a s e i n i n t r a g e n i c recombination and gene c o n v e r s i o n but o n l y a t two l o c i ( C atcheside et a l . , 1964). Mutations i n rec-2 i n c r e a s e gene c o n v e r s i o n and i n t r a - and i n t e r g e n i c recombination 2 by a f a c t o r of ten, but again, o n l y over two i n t e r v a l s (Smith, 1966). Mutations i n rec-3 behave s i m i l a r i l y to rec-1 but a l s o a f f e c t i n t e r g e n i c exchange (Smythe, 1973). In D r o s o p h i l a , s e l e c t i o n f o r l o c a l i n c r e a s e s i n recombination frequency have been s u c c e s s f u l ( C h i n n i c c i , 1971a,b; Brooks, 1984). However, o n l y two g e n e r a l , m e i o t i c enhancers have been r e p o r t e d and n e i t h e r has a uniform e f f e c t . The interchromosomal e f f e c t produces a 200 to 300% i n c r e a s e i n recombination frequency on a l l chromosomes and can be induced by a v a r i e t y of chromosomal rearrangments ( S t u t e r v a n t , 1919; L u c c h e c i and Suzuki, 1968). For example, an i n v e r s i o n on the X chromosome w i l l r e s u l t i n an i n c r e a s e i n recombination frequency at the d i s t a l ends and i n the r e g i o n of the centromere of the autosomes, while having no e f f e c t on the medial r e g i o n s ( S c h u l t z and R e d f i e l d , 1951; Ramel, 1962). Hinton (1966) compared these r e s u l t s with the e f f e c t of the p o i n t mutation c(3)G which i n c r e a s e s recombination when heterozygous and found a s i m i l a r p a t t e r n of nonuniform i n c r e a s e . T h i s mutant i s p a r t i c u l a r i l y unusual i n t h a t i t a b o l i s h e s recombination when homozygous (Gowen and Gowen, 1922; H a l l , 1972; C a r l s o n , 1972). In Zea mays, the mutant as a l s o produces a p a t t e r n of i n c r e a s e s i m i l a r t o the interchromosomal e f f e c t i n D r o s o p h i l a . A l l of the above examples d i f f e r from rec-1 i n C. elegans: e i t h e r they produce a uniform i n c r e a s e i n recombination frequency, but only i n some l o c a l r e g i o n ; or the extent of the i n c r e a s e i s q u i t e v a r i a b l e and a f f e c t s the e n t i r e genome. In C. 3 elegans, the i n c r e a s e i n recombinatiom frequency i s both uniform and g e n e r a l (Rose and B a i l l i e , 1979a; Rose, 1980). The s p e c i f i c o b j e c t i v e s of t h i s work were t h r e e - f o l d : 1) t o adequately d e s c r i b e the extent of the e f f e c t of the rec-1 mutation on i n t r a g e n i c recombination and gene c o n v e r s i o n ; 2) to determine the p l e i o t r o p i c e f f e c t s of rec-1 ( i e . i t s i n d i r e c t e f f e c t s on other m e i o t i c p r o c e s s e s ) ; and 3) to i n v e s t i g a t e the nature of the rec-1 mutation by u s i n g the g e n e t i c t o o l s a v a i l a b l e i n C. elegans. The f i r s t and second p o i n t s are of c r i t i c a l importance to the use of the Rec-1 s t r a i n i n p o p u l a t i o n b i o l o g y experiments intended to i n v e s t i g a t e the r o l e of recombination i n e v o l u t i o n . S ince F i s h e r (1930) and M u l l e r (1932) f i r s t q u e stioned the nominal advantage of recombination, numerous authors have ent e r e d the debate. Nei (1967) and Lewontin (1971) amoung ot h e r s have argued t h a t n a t u r a l s e l e c t i o n should a c t to reduce (and perhaps e l i m i n a t e ) recombination i n order to p r e s e r v e the l i n k a g e of " w e l l adapted" combinations of a l l e l e s . Although the long term advantages of recombination have been i n v e s t i g a t e d (eg. M u l l e r s r a t c h e t ) , such arguments n e c e s s i t a t e a group s e l e c t i o n i s t stance. E s h e l (1972) has shown t h a t the c o n d i t i o n s r e q u i r e d f o r group s e l e c t i o n to operate are q u i t e s t r i n g e n t . T h e r e f o r e , group s e l e c t i o n most l i k e l y o n l y operates under s p e c i a l circumstances and i s a l e s s g e n e r a l f o r c e i n e v o l u t i o n than s e l e c t i o n a t the l e v e l of the i n d i v i d u a l . In order to account f o r the p e r s i s t e n c e of recombination, a number of mechanisms have been proposed which y i e l d a s h o r t term advantage to the i n d i v i d u a l (Maynard Smith, 1971; 4 W i l l i a m s , 1975; Strobeck e t a l . , 1975; F e l s e n s t e i n , 1974; F e l s e n s t e i n and Yokoyama, 1976). The rec-1 mutation i n C. elegans seems i d e a l l y s u i t e d t o p r o v i d e e m p i r i c a l t e s t s f o r s e v e r a l of these hypotheses. C. elegans i s w e l l c h a r a c t e r i z e d g e n e t i c a l l y , has a g e n e r a t i o n time which i s s h o r t e r than D r o s o p h i l a and i s e a s i l y c u l t u r e d i n the l a b o r a t o r y (Brenner, 1974). By u s i n g d i f f e r e n t a l l e l e s of r e c - 1 , experiments c o u l d be designed to compare the e f f e c t of d i f f e r e n t amounts of recombination on the s h o r t and even long term f i t n e s s of an i n d i v i d u a l . Before such s t u d i e s can be attempted, the f u l l extent of the e f f e c t s of rec-1 on recombination and on o t h e r processes must be documented and then taken i n t o account i n the experimental d e s i g n . Chao and Gibson (1983) p o i n t out t h a t any e x p e r i m ental system i n p o p u l a t i o n b i o l o g y must be a b l e to d i s c r i m i n a t e between an i n t r i n s i c , c o m p e t i t i v e advantage or disadvantage due to the s i d e e f f e c t s of the mutant versus an i n c r e a s e d or decreased f i t n e s s caused by the p r i n c i p a l f u n c t i o n of the gene. One must attempt to answer the q u e s t i o n : " why i s one genotype more f i t than the o t h e r ? " The above i s a l s o of c r i t i c a l importance i n a i d i n g our understanding of recombination a t the l e v e l of the c e l l . Sandler et a l . (1968) have suggested t h a t such s i d e e f f e c t s can be used to deduce the nature of p a r t i c u l a r mechanisms and/or stages of m e i o s i s . In a d d i t i o n , p l e i o t r o p i c e f f e c t s are o f t e n used i n both the i s o l a t i o n and c h a r a c t e r i z a t i o n of recombination mutants. In the lower eukaryotes, one of three c r i t e r i a are t y p i c a l l y used f o r s c r e e n i n g : i ) reduced f e r t i l i t y , i n which gamete i n v i a b i l i t y 5 i s due to a high l e v e l of aneuploidy induced by the r e d u c t i o n or absence of c r o s s - o v e r t e t r a d s (eg. Baker et a l . , 1976; P e r k i n s , 1974; E s p o s i t o and E s p o s i t o , 1969); i i ) enhanced or depressed p r o t o t r o p h i c p r o d u c t i o n , which serves as an i n d i c a t o r of changes i n the amount of i n t r a g e n i c recombination (eg. Catcheside et a l . , 1964); and i i i ) m i t o t i c d i s t u r b a n c e s , d e t e c t e d by the enhancement or d e p r e s s i o n of spontaneous and induced mutation r a t e s , mutagen s e n s i t i v i t y , and a l t e r e d m i t o t i c recombination f r e q u e n c i e s (eg. G o l i n and E s p o s i t o , 1977). Mutants i s o l a t e d by the above procedures are then examined to determine t h e i r e f f e c t , i f any, on m e i o t i c recombination (eg. Dowling et a l . , 1985; Malone and Hoekstra, 1984). In the h i g h e r eukaryotes, the p l e i o t r o p i c e f f e c t s of recombination mutants a l s o r e s u l t i n a number of t y p i c a l g e n e t i c o b s e r v a t i o n s . For example, i n D r o s o p h i l a a r e d u c t i o n i n the number of c r o s s - o v e r t e t r a d s causes reduced f e r t i l i t y and i n c r e a s e d n o n d i s j u n c t i o n (eg. c(3)G: H a l l , 1972; G r e l l , 1962; and see Baker and H a l l , 1976, f o r a r e v i e w ) . A l s o , r e p a i r system d e f e c t s cause an i n c r e a s e i n spontaneous and induced mutation A1 r a t e (eg. mu: Gold and Green, 1974; mei-41 : Smith, 1973) as w e l l as r a d i a t i o n and mutagen s e n s i t i v i y (eg. mei-9: Boyd et D5 a l . , 1976; mei-41 : Boyd and Setlow, 1976). In summary, the t y p i c a l p l e i o t r o p i c e f f e c t s a s s o c i a t e d with recombination mutants i n c l u d e : reduced f e r t i l i t y and v i a b i l i t y , mutagen s e n s i t i v i t y , i n c r e a s e d spontaneous and/or induced mutation r a t e , and changes i n the r a t e of n o n d i s j u n c t i o n and chromosome l o s s . The rec-1 mutation i n C. elegans was examined to determine which, i f any, of these e f f e c t s were p r e s e n t . 6 F i n a l l y , e l a b o r a t i n g on the t h i r d o b j e c t i v e of t h i s study, a number of g e n e t i c t o o l s are a v a i l a b l e i n C. elegans which have allowed s e v e r a l s p e c i f i c hypotheses on the molecular b a s i s of rec-1 to be t e s t e d : i ) C. elegans i s the onl y h i g h e r eukaryote i n which tRNA nonsense suppressor mutants have been i s o l a t e d (Waterston and Brenner, 1978; Waterston, 1981; Hodgkin, 1985). By c o n s t r u c t i n g the a p p r o p r i a t e s t r a i n , the e f f e c t of rec-1 on recombination i n the presence of a tRNA amber suppressor was i n v e s t i g a t e d ; i i ) A mutation assay has been developed by Rosenbluth and B a i l l i e (1981) u s i n g the t r a n s l o c a t i o n eTl as a b a l a n c e r . Using t h i s system, the mutation r a t e i n the Rec-1 s t r a i n was measured to determine i f t h i s hyper-rec mutant a l s o a c t s as a mutator; and i i i ) Tc1 i s a t r a n s p o s a b l e element i n C. elegans (Emmons e t a l . , 1 983). Both nonhomologous and homologous recombination have been i m p l i c a t e d i n t r a n s p o s i t i o n mechanisms (Kleckner, 1981; Zupancic e t a l . , 1983). Given t h a t rec-1 i s a recombination enhancer, the e f f e c t of the rec-1 on Tc1 m o b i l i t y was examined. The p r e s e n t a t i o n of t h i s work has been o r g a n i z e d i n to ch a p t e r s . Each d e a l s with a s p e c i f i c s et of experiments and i n c l u d e s the methods, r e s u l t s and some d i s c u s s i o n as i s r e l e v a n t to t h a t work. The f i n a l chapter summarizes the s i g n i f i c a n c e of these r e s u l t s , reviews the proposed mechanisms of other hyper-rec mutants and s p e c u l a t e s on the nature of the rec-1 mutation. S e v e r a l experiments are suggested f o r f u t u r e i n v e s t i g a t i o n . 7 I I . Background Information A) L i f e H i s t o r y and General Reproductive C h a r a c t e r i s t i c s C a e n o r h a b d i t i s elegans i s a f r e e l i v i n g s o i l nematode which feeds on b a c t e r i a . I t i s h e r m a p h r o d i t i c ; however, males do occur spontaneously a t a frequency of one i n seven hundred (Brenner, 1974). The hermaphrodite i s XX and the male i s XO (Nigon, 1949); the spontaneous p r o d u c t i o n of males a r i s e s from n o n d i s j u n c t i o n and chromosome l o s s (Hodgkin e t a l , 1979). Male c u l t u r e s ( i e . a 1:1 sex r a t i o ) can be maintained i n the l a b by s e t t i n g up new mating p l a t e s each week. Hermaphrodites p r e f e r e n t i a l l y use sperm from the male when out c r o s s e d (Ward and C a r r e l , 1979). Four l a r v a l stages (L1 to L4) are passed through over a p e r i o d of three and one h a l f days; speed of development i n c r e a s e s with i n c r e a s i n g temperature. In the L4 stage a t r a n s l u c e n t c r e s c e n t i s v i s i b l e on the hermaphrodite body w a l l . T h i s i s the undeveloped v u l v a . T h i s c h a r a c t e r i s t i c can be used as an i n d i c a t e r of v i r g i n i t y f o r o u t - c r o s s experiments. Under s t a r v a t i o n c o n d i t i o n s the L2 stage moults to form a dauer l a r v a (Daugherty et a l , 1959). This s t a t e i s r e s i s t a n t to drought, high temperatures, s t a r v a t i o n e t c . . With the r e t u r n of f a v o u r a b l e growth c o n d i t i o n s the dauer l a r v a r e t u r n s t o the normal developmental pathway. The mean number of progeny per hermaphrodite i s approximately 300 (Brenner, 1974). Recombination decreases with age and i n c r e a s e s with temperature (Rose and B a i l l i e , 1979b). T h e r e f o r e , i n order to a c c u r a t e l y 8 measure the recombination frequency a l l progeny from an i n d i v i d u a l must be scored and the temperature h e l d c o n s t a n t . The optimal growth temperature i s 20 2C. The study of t h i s organism i s f u r t h e r enhanced by i t s a b i l i t y t o s u r v i v e f r e e z i n g . S t r a i n s can be s t o r e d i n l i q u i d n i t r o g e n ( H o r v i t z e t a l , 1979). B) Nematode S t r a i n s and C u l t u r e C o n d i t i o n s The nomenclature used i n t h i s t h e s i s f o l l o w s the p u b l i s h e d g u i d e l i n e s of H o r v i t z et a l . (1979). A s t r a i n i s a group of i n d i v i d u a l s of some p a r t i c u l a r genotype which has the a b i l i t y to produce i n d i v i d u a l s of the same genotype. Genes are typed i n u n d e r l i n e d , lower case l e t t e r s , w h i le phenotypes have t h e i r f i r s t l e t t e r c a p i t a l i z e d and are not u n d e r l i n e d . The term Wild-type s h a l l be used to r e f e r t o i n d i v i d u a l s with a phenotype l i k e t h a t of N2 s t r a i n worms. Linkage i s denoted by a continuous l i n e underneath the genes (eg. dpy-5 dpy-14/+ +). D i f f e r e n t l i n k a g e groups are separated by a semicolon (eg. dpy-5/dpy-5; unc-46/unc-46). The B r i s t o l v a r i e t y of C. elegans was s t u d i e d . The N2 s t r a i n and most of the mutant s t r a i n s were o b t a i n e d from D. B a i l l i e (S.F.U., Vancouver, Canada). Others were p r o v i d e d by the C. elegans Genetic Stock Center. The recombination maps of these mutant s t r a i n s are i d e n t i c a l to t h a t of N2 as a l l these s t r a i n s were o r i g i n a l l y i s o l a t e d from N2. The rec-1 mutation has the same e f f e c t i n a l l s t r a i n s . Table 1 l i s t s the s t r a i n s used i n t h i s study and i d e n t i f i e s those c o n s t r u c t e d by me. Table 2 l i s t s 9 the genes used, t h e i r a l l e l e s and t h e i r phenotypes. F i g u r e 1 i l l u s t r a t e s the r e l a t i v e p o s i t i o n s of these genes on the g e n e t i c map of C. elegans. Nematodes were c u l t u r e d i n 60 mm. p e t r i p l a t e s f i l l e d with nematode growth media and streaked with E. c o l i OP50 (Brenner, 1974). Stocks were maintained at 16 C, while a l l matings were performed at 20 C. In a l l recombination experiments c r e s c e n t stage hermaphrodites were t r a n s f e r r e d to separate c u l t u r e p l a t e s , allowed to s e l f c r o s s and then t r a n s f e r r e d to f r e s h c u l t u r e p l a t e s every twenty fo u r hours f o r three days. These t r a n s f e r s ( r e f e r r e d to as brooding) l i m i t e d the t o t a l number of progeny on any one p l a t e . T h i s made s c o r i n g e a s i e r and more r e l i a b l e . G enetic d i s t a n c e s were measured u s i n g the markers i n a c i s c o n f i g u r a t i o n . Recombination frequency was c a l c u l a t e d by p = 1 -J l - 2R, where R i s the f r a c t i o n of v i s i b l y recombinant progeny (Brenner, 1974) ( F i g u r e 2 ) . C) S t r a i n C o n s t r u c t i o n s The c h a r a c t e r i z a t i o n of the rec-1 mutation and i t s e f f e c t s on v a r i o u s processes r e q u i r e d the a v a l i b i l i t y of s e v e r a l s p e c i f i c s t r a i n s . The rec-1 homozygous s t r a i n s d e s c r i b e d by Rose and B a i l l i e (1979a) were used i n t h i s study. However, c e r t a i n genotypes had to be c o n s t r u c t e d , e s p e c i a l l y f o r the mutation r a t e experiment. 1 0 TABLE 1: S t r a i n s Used i n t h i s Study S t r a i n KR # Genotype Rec-1 BC00196 36 dpy-5 dpy-14/dpy-5 dpy-14; rec-1/rec-1 KR00177 177 dpy-5 unc-13/dpy-5 unc-13; rec-1/rec-1 BC00313 25 rec-1/rec-1 KR00370 370 unc-46/unc-46; rec-1/rec-1 * KR00371 371 eT1/eTl; rec-1/rec-1 * KR00372 372 dpy-18/dpy-18; rec-1/rec-1 KR00373 373 dpy-18/dpy-18; unc-46/unc-46; r e c - 1 / r e c - 1 * KR00377 377 dpy-18 unc-46/eT1; rec-1/rec-1 * BC00341 386 dpy-5 unc-13 unc-29; rec-1 dpy-5 unc-13 unc-29 rec-1 BC00452 387 unc-13/unc-13; rec-1/rec-1 N2 De r i v e d BC00338 18 dpy-18/dpy-18 BC00356 50 unc-46/unc-46 BC00070 300 eT1/eT1 KR00353 353 sDp2; let-362 dpy-5 unc-13 let-362 dpy-5 unc-13 BC00189 40 unc-13/unc-13 BC00279 dpy-5 unc-13 unc-29/dpv-5 unc-13 unc-29 RW02070 653 sup-7/sup-7; dpy-18/dpy-18 * C o n s t r u c t e d by B. R a t t r a y 1 1 TABLE 2: Mutations used i n t h i s study. GENE LINKAGE ALLELE VISIBLE GROUP PHENOTYPE dpy-•5 I (e61 ) short and f a t ; moves wel! dpy-•14 I (e188) short and f a t *; temperature s e n s i t i v e . dpy- 18 I I I (e364) short and f a t ; eTl I I I , V Unc-36; t r a n s l o c a t i o n ; uncoordinated movement. l e t - 362 I (h086) l e t h a l . r e c - 1 ? (s180) i n c r e a s e s recombination; otherwise Wt phenotype unc- 1 3 I (e51 ) par a l y z e d ; (e450) p a r a l y z e d . unc- 29 I (e403) s l i g h t l y uncoordunated movement. unc- 46 V (e177) very uncoordinated. sup- 7 X (st05) amber supressor; temperature s e n s i t i v e ; otherwise Wt phenotype. * The Dpy and Unc phenotypes are unique f o r each gene ( i e . Dpy-5 can be d i s t i n g u i s h e d from Dpy-14). With the ex c e p t i o n of Sup-7, a l l the othe r phenotypes are r e c e s s i v e . L I N K A G E G R O U P d p y - 1 4 l e t - 3 6 2 I d p y - 5 u n c - 1 3 u n c - 2 9 _i u i_ u n c - 5 4 1 s D p 2 0 5 % R E C O M B I N A T I O N U III h eT1 breakpoint d p y - 1 8 i i LY u n c - 4 6 eT1 breakpoint • • s u p - 7 F I G U R E 1 '. G e n e t i c M a p of C . elegans a n d p o s i t i o n s of g e n e s s t u d i e d . The number of v i s i b l y recombinant progeny (eg. Dpy-5 and Unc-13) 2 [ § of Dpy-5 ] The f r a c t i o n of v i s i b l y recombinant = 2 [ ff of Dpy-5 ] progeny,(R). T o t a l # Progeny The frequency of recombination: p = 1 - J l - 2R ; Where: 2R = the f r a c t i o n of progeny c a r r y i n g a recombinant chromosome; 1 - 2R = f r a c t i o n of progeny not c a r r y i n g a recombinant chromosome; J l - 2R ~ frequency of a non-recombinant event i n e i t h e r gamete, (oocyte o r spermatocyte); 1 - J l - 2R = the frequency of a recombinant event i n e i t h e r gamete. FIGURE 2: Determination of p, the recombination frequency f o r smal l i n t e r g e n i c d i s t a n c e s . The c a l c u l a t i o n i s based on the number of progeny t h a t v i s i b l y c a r r y a recombinant chromosome. The p a r e n t a l genotype was i n a c i s c o n f i g u r a t i o n (eg. dpy-5 unc-13). + + (modi f i e d from Rose, 1980) The rec-1 gene has not been mapped p r e c i s e l y . The rec-1 l o c u s i s not l i n k e d t o any o f t h e major gene c l u s t e r s (Rose, u n p u b l . d a t a ) . However, Rose has r e c e n t l y d e t e c t e d l o o s e l i n k a g e by rec-1 t o unc-54 a t the end of l i n k a g e group I . Rec-1 does not have a v i s i b l e phenotype, t h e r e f o r e , d u r i n g s t r a i n c o n s t r u c t i o n s i t was n e c e s s a r y t o c a l c u l a t e r e c o m b i n a t i o n f r e q u e n c i e s t o i d e n t i f y t h e r e c - 1 / r e c - 1 s e g r e g a n t s . Hermaphrodites of t h e genotype r e q u i r e d i n a rec-1 background were o u t - c r o s s e d w i t h Rec-1 males. The W i l d - t y p e male progeny were th e n c r o s s e d w i t h a Rec-1 d o u b l e mutant. F2 i n d i v i d u a l s were s e l f - c r o s s e d and t h e i r r ecombinant progeny were c o u n t e d . One h e r m a p h r o d i t e h a v i n g the d e s i r e d phenotype was t h e n p i c k e d from a " h i g h r e c o m b i n a t i o n " p l a t e and used as t h e p r o g e n i t o r of t h e new s t r a i n . F o r example, t h e s t r a i n Unc-46 Rec-1 was c o n s t r u c t e d as i n F i g u r e 3. The dpy-5 t o dpy-14 i n t e r v a l on l i n k a g e group I was used d u r i n g c o n s t r u c t i o n s as a means of i d e n t i f i n g s e g r e g a t i n g rec-1 homozygotes. Rec-1 males were c r o s s e d w i t h Unc-46 h e r m a p h r o d i t e s and the r e s u l t a n t males were c r o s s e d t o Dpy-5 Dpy-14 Rec-1 h e r m a p h r o d i t e s . The F2 were h e t e r o z y g o u s f o r dpy-5 and dpy-14 i n a c i s c o n f i g u r a t i o n and were e i t h e r r e c - 1 / r e c - 1 or rec-1/+ ( F i g u r e 3 ) . One h a l f o f the F2 were a l s o h e t e r o z y g o u s f o r unc-46. The F2 h e r m a p h r o d i t e s were s e l f c r o s s e d and brooded. The r e c o m b i n a t i o n f r e q u e n c i e s were c a l c u l a t e d from the F3 by c o u n t i n g Dpy-5 and Dpy-14 worms. A d r a m a t i c i n c r e a s e i n r e c o m b i n a t i o n f r e q u e n c y ( i e . a p p r o x i m a t e l y t h r e e f o l d ) was c o n s i d e r e d t o i n d i c a t e a Rec-1 s t r a i n . Ten Unc-46 i n d i v i d u a l s were p i c k e d from s e v e r a l h i g h r e c o m b i n a t i o n l i n e s and progeny 1 5 Po: rec-1 rec-1 [males] a [herm.] F1 : F2: F3: F4: ; rec-1 [males] X dpy-5 dpy-14 ; rec-1 + dpy-5 dpy-14 rec-1 [herm.] dpy-5 dpy-14 ; rec-1 or + + rec-1 ( s e l f - c r o s s ) r e c - 1 ; + or _+_ + [herm.] V a) Score recombination f r e q u e n c i e s b) P i c k 10 a/a worms (phenotype = A) from a p l a t e with a high recombination frequency. e 9 * a i rec-1 ; dpy-5 dpy-14 or rec-1 ( s e l f - c r o s s ) + + + + V Progeny t e s t - p i c k the p l a t e which does not segregate Dpy-5 Dpy-14 worms. FIGURE 3: C o n s t r u c t i o n of rec-1 homozygous s t r a i n s . The procedure i n v o l v e d u s i n g the p r e v i o u s l y c o n s t r u c t e d double Dpy mutant, Dpy-5 Dpy-14 Rec-1. "a" i s any mutation which produces a d i s t i n g u i s h a b l e phenotype when homozygous eg. Unc-46. 16 t e s t e d . Of t h o s e i n d i v i d u a l s which d i d not s e g r e g a t e Dpy-5 Dpy-14 progeny, one was p i c k e d as the p r o g e n i t o r o f t h e s t r a i n ( i e . unc-46/unc-46; r e c - 1 / r e c - 1 ) . The same method was used f o r t h e subsequent v e r i f i c a t i o n o f the s t r a i n s . I f t h e new s t r a i n was a c t u a l l y homozygous f o r r e c - 1 , t h e n a l l brooded F2 h e r m a p h r o d i t e s would have a h i g h r e c o m b i n a t i o n f r e q u e n c y . T a b l e 3 p r e s e n t s t h e r e s u l t s from t e s t i n g t h e Unc-46 Rec-1 s t r a i n . R e c o m b i n a t i o n f r e q u e n c i e s f o r the dpy-5 t o dpy-14 i n t e r v a l a r e p r o v i d e d f o r known Rec-1 and N2 s t r a i n s t o a l l o w comparison w i t h the c o n s t r u c t i o n d a t a . D e s p i t e the e x p e c t e d l a r g e c o n f i d e n c e i n t e r v a l s on t h e e s t i m a t e s o b t a i n e d from i n d i v i d u a l h e r m a p h r o d i t e s , i t can be c o n c l u d e d t h a t t h i s s t r a i n i s homozygous f o r r e c - 1 . The s t r a i n Dpy-18 Unc-46 Rec-1 was c o n s t r u c t e d from t h e Dpy-18 Rec-1 and t h e Unc-46 Rec-1 s t r a i n s . No r e c o m b i n a t i o n measures were r e q u i r e d as b o t h s t r a i n s were a l r e a d y homozygous f o r r e c - 1 . Unc-46 Rec-1 h e r m a p h r o d i t e s were c r o s s e d w i t h Rec-1 males. The h e t e r o z y g o u s male progeny were t h e n c r o s s e d w i t h Dpy-18 Rec-1 h e r m a p h r o d i t e s . Ten F2 h e r m a p h r o d i t e s were s e l f c r o s s e d and brooded. One h a l f of t h e s e worms were e x p e c t e d t o be h e t e r o z y g o u s f o r b o t h unc-46 and dpy-18. F i n a l l y , a s i n g l e s e g r e g a t i n g d o u b l e mutant ( i e . Dpy-18 Unc-46) was p i c k e d from the F3 t o e s t a b l i s h t h e d e s i r e d s t r a i n ( i e . Dpy-18 Unc-46 Re c - 1 ) . The r e c i p r o c a l t r a n s l o c a t i o n eT1 ( i e . t h e r i g h t h a l f o f l i n k a g e group I I I and t h e l e f t h a l f of V) produces an u n c o - o r d i n a t e d phenotype (Unc-36) when i n the homozygous s t a t e ( R o s e n b l u t h and B a i l l i e , 1981). An eTl Rec-1 s t r a i n was 17 Table 3: V e r i f i c a t i o n of s t r a i n c o n s t r u c t i o n ** S t r a i n N # Proqeny # Recombinants Recomb . F r e q . N2 9 2,907 40 1 .4 % _+ 0.4 Rec-1 10 3,104 1 65 5.3 % +_ 0.6 Unc-36; * 1 331 17 5.3 % +_ 2.5 Rec-1 1 335 17 5.2 % +_ 2.4 1 307 17 5.7 % +_ 2.7 1 359 15 4.3 % +_ 2.2 Unc-46; 1 267 17 6.6 % + 3.2 Rec-1 1 353 19 5.5 % 2.3 1 1 96 1 4 7.4 % + 3.8 1 245 13 5.5 % +_ 3.0 1 73 8 4.7 % + 2.0 1 247 1 6 6.7 % + 3.2 * Unc-36 i s the phenotype of eT1 homozygotes. * Recombination frequency f o r the dpv-5 dpy-14 i n t e r v a l . 18 c o n s t r u c t e d as d e s c r i b e d f o r the Unc-46 Rec-1 s t r a i n (Table 3 ) . T h i s was then used to c o n s t r u c t the s t r a i n dpy-18 unc-46/eT1; r e c - 1 / r e c - 1 . T h i s was obtained by c r o s s i n g Rec-1 males with eTl Rec-1 hermaphrodites. The F1 males were then c r o s s e d with Dpy-18 Unc-46 Rec-1 hermaphrodites. The F2 Wildtype progeny were dpy-18 unc-46/eT1; rec-1/rec-1 and dpy-18/+; unc-46/+; r e c - 1 / r e c - 1 . The eTl balanced s t r a i n was i d e n t i f i e d by the s e g r e g a t i o n of Unc-36 worms and the f a i l u r e of brooded F2 worms to segregate progeny of e i t h e r Dpy-18 or Unc-46 phenotypes ( i e . eT1 produces pseudolinkage between dpy-18 and unc-46; the genotypes of both Dpy-18 and Unc-46 would be a n e u p l o i d and hence l e t h a l ) . Progeny from dpy-18 unc-46/eT1 i n d i v i d u a l s occur i n the phenotypic r a t i o of 4 Wt : 1 Unc-36 : 1 Dpy-18 Unc-46. The s t r a i n i s maintained by always t r a n s f e r r i n g Wild-type i n d i v i d u a l s ( i e . dpy-18 unc-46/eTl; r e c - 1 / r e c - 1 ) . To date, 15 Rec-1 s t r a i n s have been c o n s t r u c t e d by e i t h e r Dr. Rose or myself. The a n a l y s i s and c h a r a c t e r i z a t i o n of the rec-1 mutation i s becoming more t r a c t a b l e as a d d i t i o n a l markers become a v a i l a b l e i n a rec-1 background. 19 I I I . Recombination E f f e c t s I n t r o d u c t i o n : I s o l a t e d i n C. elegans, rec-1 i s the onl y mutation i n eukaryotes which i s a g e n e r a l and uniform enhancer of m e i o t i c recombination (Rose and B a i l l i e , 1979a). Rose (unpubl. data) has been working on mapping the rec-1 l o c u s . At the same time she has o b t a i n e d data on the e f f e c t of rec-1 on one to two percent i n t e r v a l s a s s o c i a t e d with each of the major gene c l u s t e r s on a l l of the l i n k a g e groups. T h i s data supports the c o n c l u s i o n of Rose and B a i l l i e (1979a) t h a t rec-1 i s a g e n e r a l enhancer of recombination. In a d d i t i o n , i t demonstrates t h a t the i n c r e a s e i s f a i r l y uniform compared to other hyper-rec mutants i e . the extent of the i n c r e a s e ranges from two to s i x - f o l d , with a predominant e f f e c t of three to f o u r - f o l d . Very few hyper-recombination mutants have been r e p o r t e d i n the l i t e r a t u r e . S e v e r a l have been i s o l a t e d i n E s c h e r i c h i a c o l i and these have had d e f e c t i v e r e p a i r processes (eg. Glickman and Radman, 1980; P u k k i l a et a l . , 1983). Examples i n eukaryotes are much more d i f f i c u l t to come by. In yeast, rem-1 i n c r e a s e s the spontaneous m i t o t i c recombination frequency, but has no e f f e c t upon meiosis other than reducing f e r t i l i t y ( G o l i n and E s p o s i t o , 1977; Malone and Hoekstra, 1984). S i m i l a r i l y , uvs-3 i n Neurospora, uvs-B i n A s p e r g i l l u s and uvs-1 i n U s t i l a g o o n l y i n c r e a s e m i t o t i c recombination (Schroeder, 1970; S h a n f i e l d and Kafer, 1969; H o l l i d a y et a l . , 1976). C(3)G, a r e c e s s i v e mutation i n D r o s o p h i l a melanogaster, suppresses i n t r a - and i n t e r g e n i c m e i o t i c recombination almost completely when homozygous (Gowen and Gowen, 1922;), but produces a nonuniform i n c r e a s e throughout the genome when heterozygous (Gowen, 1933; Hinton, 1966; and C a r l s o n , 1972). The interchromosomal e f f e c t r e p o r t e d i n D. melanogaster produces l a r g e , nonuniform i n c r e a s e s i n m e i o t i c recombination f r e q u e n c i e s ( L u c c h e s i and Suzuki, 1968). The e f f e c t can be induced by a v a r i e t y of chromosomal rearrangements i n c l u d i n g i n v e r s i o n s , t r a n s l o c a t i o n s , and compound chromosomes (Ramel, 1962; Hinton, 1966; P r o c u n i e r and Suzuki, 1967, r e s p e c t i v e l y and f o r a review see L u c c h e s i , 1976). F i n a l l y , the as mutant i n Zea  mays i n c r e a s e s m e i o t i c recombination i n a nonuniform p a t t e r n ( M i l l e r , 1963). Rose and B a i l l i e (1979a) have shown t h a t rec-1 d i f f e r s from each of the above examples i n t h a t : 1) i t s e f f e c t on recombination i s uniform throughout the genome. The same t h r e e - f o l d i n c r e a s e i n recombination frequency i s found r e g a r d l e s s of the i n t e r v a l or l i n k a g e group examined. T h i s c o n t r a s t s with the D r o s o p h i l a mutants; and 2) i t a c t s m e i o t i c a l l y ( u n l i k e the m i t o t i c e f f e c t of rem-1 and other such mutations) (Rose, 1980). The purpose of the experiments r e p o r t e d here was to extend the c h a r a c t e r i z a t i o n of the rec-1 mutant i n C. elegans. I n t r a g e n i c recombination, gene c o n v e r s i o n and recombination f r e q u e n c i e s over l a r g e d i s t a n c e s were examined, with the i n t e n t i o n of determining the e f f e c t of rec-1 on recombination frequency over a broad range of g e n e t i c d i s t a n c e s . 21 The gene unc-13 was chosen t o study i n t r a g e n i c recombination and gene c o n v e r s i o n . T h i s gene was p a r t i c u l a r i l y s u i t e d f o r such an a n a l y s i s f o r s e v e r a l reasons: f i r s t , Moerman and B a i l l i e (1979) have developed a procedure f o r i n t r a g e n i c s t u d i e s i n C. elegans; second, the recombination frequency between the (e51) and (e450) a l l e l e s of unc-13 has been p r e v i o u s l y r e p o r t e d (Rose and B a i l l i e , 1980); and t h i r d , Rec-1 s t r a i n s c o n t a i n i n g both unc-13 a l l e l e s were a v a i l a b l e . I a l s o attempted t o examine the e f f e c t of rec-1 on recombination over l a r g e g e n e t i c d i s t a n c e s . The p r o t o c o l i n v o l v e d c o n s t r u c t i n g a homozygous rec-1 s t r a i n c o n t a i n i n g t h r e e markers and the d u p l i c a t i o n sDp2. The d e s i g n was such t h a t two markers (dpy-5 and unc-13) were c l o s e together while let-362 was f i f t e e n map u n i t s to the l e f t of dpy-5. The d u p l i c a t i o n was r e q u i r e d t o balance the l e t h a l mutation and suppress recombination over the marker i n t e r v a l s to m a i n t a i n the c i s c o n f i g u r a t i o n d u r i n g c o n s t r u c t i o n . In t h i s manner the l a r g e g e n e t i c d i s t a n c e was scored at the same time as the s m a l l e r i n t e r v a l . Hence, the s m a l l e r i n t e r v a l served as an " i n t e r n a l c o n t r o l " to i n d i c a t e the presence of a homozygous rec-1 i n d i v i d u a l d u r i n g s t r a i n c o n s t r u c t i o n . B r i e f l y , i t was found t h a t the Rec-1 s t r a i n e x h i b i t e d an i n c r e a s e i n both i n t r a g e n i c recombination and gene c o n v e r s i o n f r e q u e n c i e s to a l e v e l approximately three times g r e a t e r than the heterozygotes c o n s t r u c t e d from the KR386 and KR387 s t r a i n s . Secondly, the c o n s t r u c t i o n p r o t o c o l f o l l o w e d d i d not produce a homozygous rec-1 d u p l i c a t i o n b e a r i n g s t r a i n . P o s s i b l e e x p l a n a t i o n s of t h i s r e s u l t are examined. Methods: a ) I n t r a g e n i c Recombination and Gene Conversion - The e f f e c t of rec-1 on i n t r a g e n i c events was determined by u s i n g the f o l l o w i n g s t r a i n s : 1) dpy-5 unc-13(e51) unc-29; r e c - 1 ; and dpy-5 unc-13(e51) unc-29 rec-1 2) unc-13(e450 ); rec-1 . unc-13(e450) rec-1 The s c r e e n i n g method was m o d i f i e d from Rose and B a i l l i e (1980). Unc-13 (e450) Rec-1 hermaphrodites were c r o s s e d w i t h Rec-1 males. Heterozygous male progeny ( i e . unc-13/+; rec- 1 / r e c - 1 ) were mated to Dpy-5 Unc-13 Unc-29 Rec-1 hermaphrodites to produce i n d i v i d u a l s heterozygous f o r the two f l a n k i n g l o c i , with both a l l e l e s of unc-13 pres e n t ( F i g u r e 4). P a r a l y z e d hermaphrodites (Unc-13 phenotype) were p l a c e d i n d i v i d u a l l y on p l a t e s and allowed to s e l f - c r o s s . A l l p l a t e s were screened f o r two g e n e r a t i o n s . The normal segregants i n c l u d e d Dpy-5 Unc-13 and Unc-13 i n d i v i d u a l s (unc-13 i s e p i s t a t i c to unc-29). Dpy-5 Unc-13 worms were removed at r e g u l a r i n t e r v a l s to a v o i d overcrowding. Unc-13 (e450) homozygous i n d i v i d u a l s c o u l d not be d i s t i n g u i s h e d from the h e t e r o z y g o t e s ; t h i s d i d not a f f e c t the r e s u l t s of the screen as n e i t h e r c r o s s - o v e r or c o n v e r s i o n events would be d e t e c t e d i n t h i s case. E x c e p t i o n a l progeny c o n s i s t i n g of Wild-type (eg. normal movement) and Unc-29 (eg. s l i g h t l y unco-ordinated) worms were p i c k e d from the background of Unc-13 worms. Progeny t e s t i n g was performed to d i s t i n g u i s h between s i n g l e c r o s s - o v e r and apparent 23 CONSTRUCTION: rec-1 [males] X unc-13 (e450 ); rec-1 [herm.] rec-1 unc-13 (e450) rec-1 unc-13; rec-1 X dpy-5 unc-13 (e51) unc-29; rec-1 + rec-1 dpy-5 unc-13 (e51 ) unc-29 rec-1 [males] dpy-5 unc-13 (e51) + (e450) unc-13 [herm.] unc-29; rec-1 + rec-1 SCREEN: Po: 1 heterozygous Unc-13 hermaphrodite per p l a t e ; [ S e l f - c r o s s ] ; F1 : 1/2 of progeny (approx. 65 per p l a t e ) have the same genotype as the Po. These are s e l f - c r o s s e d ; Dpy Unc i n d i v i d u a l s are removed to a v o i d overcrowding and a s s i s t s c o r i n g i n the F2. F2: scre e n p l a t e s f o r e x c e p t i o n a l progeny eg. Wt (normal movement) i n an Unc (para l y z e d ) background. E x c e p t i o n a l Progeny from above a r e : Wt or Unc-29 eg. S i n g l e Cross-over Apparent eg. Double Cross--over + ++ unc-29 ; dpy-5 ++ unc-29 + unc-13 + or + unc-1 3 or + + ++ unc-29 . + ++ + dpy -5 unc-13 unc-29 + unc-13 or + + + + + dpy-5 unc-13 unc-29 FIGURE 4: C o n s t r u c t i o n of a heterozygous Unc-13 Rec-1 s t r a i n and i t s use i n measuring i n t r a g e n i c r e c o m b i n a t i o n . 24 double c r o s s - o v e r events ( i e . gene conversion) by l o o k i n g f o r the exchange of f l a n k i n g markers. Wild-type and Unc-29 e x c e p t i o n a l progeny c a r r y i n g a chromosome produced by a s i n g l e i n t r a g e n i c c r o s s - o v e r event segregated Unc-29 progeny, u n l i k e e i t h e r p a r e n t a l or c o n v e r s i o n genotypes. Depending on the genotype of the homologous chromosome, e x c e p t i o n a l progeny recovered due to a p u t a t i v e gene c o n v e r s i o n event segregated progeny i n one of the f o l l o w i n g phenotypic r a t i o s : 1) + unc-13 + > 3 Wt : 1 Unc-13; + + + 2) dpy-5 unc-13 unc-29 > 3 Wt : 1 Dpy-5 Unc-13 ; + + + 3) + unc-13 + > 1 Unc-13 : 2 Wt : 1 Dpy-5 Unc-29; dpy-5 + unc-29 4) dpy-5 unc-13 unc-29 > 1 Dpy-5 Unc-29 : 2 Wt : 1 Dpy-5 , dpy-5 + unc-29 Unc-13 The t o t a l number of chromosomes screened was e s t i m a t e d on the b a s i s of the mean number of progeny per hermaphrodite per g e n e r a t i o n (Rose and B a i l l i e , 1980) ( F i g u r e 5 ) . Apparent double c r o s s - o v e r s w i l l be r e f e r r e d to as gene c o n v e r s i o n s throughout the remainder of the t e x t . J u s t i f i c a t i o n f o r t h i s i n t e r p r e t a t i o n w i l l be p r o v i d e d l a t e r . C a l c u l a t i o n s f o r recombination and co n v e r s i o n f r e q u e n c i e s are presented i n F i g u r e s 6 and 7 r e s p e c t i v e l y . b) Recombination over l a r g e d i s t a n c e s - The p r o t o c o l f o r examining the e f f e c t of rec-1 on l a r g e g e n e t i c d i s t a n c e s was as f o l l o w s : Rec-1 males were c r o s s e d with a d u p l i c a t i o n - b e a r i n g s t r a i n ( i e . sDp2/let-362 dpy-5 unc-13/let-362 dpy-5 unc-13). 25 GIVEN: 1) dpy-5 (e51)(+) unc-29 ; + (+)(e450) + ( s e l f - c r o s s e d ) 2) Mean # progeny per hermaphrodite: X = 127 ; THEN: 3) T o t a l # F1 = X [ § C u l t u r e P l a t e s ]; = (127)(210) = 27,000 ; 4) # F1 Unc-13 Hets = 27,000 ]; 2 = 13,500 ; 5) T o t a l § F2 = (127)[ 13,500 ] ; = 1.7 m i l l i o n ; 6) T o t a l # screened = # F1 + # F2 ; = 1.7 m i l l i o n . THEREFORE: Approximately 1.7 m i l l i o n i n d i v i d u a l s or > 3 m i l l i o n chromosomes were screened. Sample counts were made and a s i m i l a r v a l u e was ob t a i n e d . FIGURE 5: Sample s i z e estimate f o r the number of chromosomes screened i n the i n t r a g e n i c experiment. GIVEN: 1) dpy-5 (e51) (+) unc-29 + (+)(e450) + ( s e l f - c r o s s ) V Recombinants a) + ++ unc-29; c) dpy-5 (e51)(e450) + ; + unc-13 + + + (e450) + b) + ++ unc-29; d) dpy-5 (e51)(e450) + . dpy-5 unc-13 unc-29 dpy-5 (e51) + unc-29 2) Recombination i s r e c i p r o c a l , i e . #(a) + #(b) = #(c) + #(d); 3) Recombinant chromosomes i n (a) and (b) are always " v i s i b l e " as Wt and Unc-29, r e s p e c t i v e l y ; however, (c) and (d) cannot be d i s t i n g u i s h e d from the segregants (Unc-13, Dpy-5 Unc-13), THEN: 4) T o t a l # Recombinant Chromosomes = 2[ # Unc-29 + # Wt ]. GIVEN: 5) Unc-29 i s under r e p r e s e n t e d due to the d i f f i c u l t y of s c o r i n g t h i s phenotype on a background of p a r a l y z e d worms (eg. Unc-13); 6) Based on the Punnett Square: # Wt = # Unc-29; 7) Recomb. F r e q . : p = T o t a l § Recombinant Chromosomes; T o t a l # Chromosomes THEN: 9) p = 4[ # Wt ] ; 2[ T o t a l # I n d i v i d u a l s ] THEREFORE: p = 2[ # Wt ] [ T o t a l # I n d i v i d u a l s Screened ] FIGURE 6: C a l c u l a t i o n of i n t r a g e n i c recombination frequency. I n t r a g e n i c recombinants are d i s t i n g u i s h e d from c o n v e r t a n t s (apparent double c r o s s - o v e r events) by examining t h e i r progeny and l o o k i n g f o r the exchange of f l a n k i n g markers i n the segregants. Note t h a t unc-13 i s e p i s t a t i c t o unc-29. 27 GIVEN: 1) dpy-5 (e51)(+) unc-29 + (+)(e450) + ( s e l f - c r o s s ) i Convertants Genotype Phenotype a) dpy-5 (+)(+) unc-29/dpy-5 unc-13 unc-29; Dpy-5 Unc-29; b) dpy-5 ( + )( + ) unc-29/+ unc-13 +; Wt; c) dpy-5 (e51)(e450) unc^29/dpy-5" unc-1 3 unc-29; * Dpy-5 Unc-13; d) dpy-5 (e51)(e45Q) unc-29/+ unc-13 + ; Unc-13; e) + (+)(+) +/dpy-5 unc-13 unc-29; Wt; f ) + (+)(+) + / + unc-13 +; Wt; g) + (e51)(e450) +/dpy-5 unc-13 unc-29; Unc-13; h) + (e51)(e450) + / + unc-13 +; Unc-13; 2) H a l f of the c o n v e r t a n t s can be d i s t i n g u i s h e d from the segregants: a) Dpy-5 Unc-29, b) Wt, e) Wt, and f ) Wt i n d i v i d u a l s can be p i c k e d from a background of Dpy-5 Unc-13 and Unc-13 worms; i e . # Convertant Chromosomes = 2 ( # wt + # Dpy-5 Unc-29 ) ; 3) The Dpy-5 Unc-29 worms are under r e p r e s e n t e d due to the d i f f i c u l t i e s i n s c o r i n g t h i s phenotype i n the above background; THEN: 4) # Convertant Chromosomes = 2 (4/3 [§ Wt c o n v e r t a n t s ] ); GIVEN: 5) Conversion F r e q . = # con v e r t a n t chromosomes T o t a l # chromosomes screened THEN: 6) Conversion F r e q . = 4/3 [ # Wt ] [ # i n d i v i d u a l s ] THEREFORE: Conversion Frequency = 4/3 [ # Wt ]  [ T o t a l # I n d i v i d u a l s ] FIGURE 7: C a l c u l a t i o n of c o n v e r s i o n frequency. The c o n v e r t a n t s are d i s t i n g u i s h e d from i n t r a g e n i c recombinants by examining t h e i r progeny and l o o k i n g f o r the absence of f l a n k i n g marker exchange i n the segregants. * unc-13 i s e p i s t a t i c t o unc-29. 28 The F1 heterozygotes c o n s i s t e d of d u p l i c a t i o n - b e a r i n g i n d i v i d u a l s and those without sDp2 (F i g u r e 8 ) . Four i n d i v i d u a l s " c a r r y i n g sDp2 were s e l e c t e d on the b a s i s of slower developement ( i e . younger apparent age on brooded p l a t e s ) and s l i g h t l y u nco-ordinated movement. These hermaphrodites were s e l f - c r o s s e d and brooded. The progeny segregated i n a 6 Wt : 1 Unc-13 phenotypic r a t i o . T h e o r e t i c a l l y , one h a l f of the Wildtype worms were d u p l i c a t i o n b e a r e r s . A l l of the Unc-13 worms c a r r i e d sDp2 ( F i g u r e 8 ) . One q u a r t e r of the Unc-13 worms were expected t o be homozygous f o r rec-1 (based on independent assortment). F i v e Unc-13 hermaphrodites were p i c k e d per p l a t e and c u l t u r e d i n d i v i d u a l l y as p o s s i b l e s t r a i n p r o g e n i t o r s . These independent l i n e s were t e s t e d to i d e n t i f y a rec-1 homozygote. For any giv e n l i n e , Rec-1 males were c r o s s e d with Unc-13 hermaphrodites. T h i s time the hermaphrodite progeny without the d u p l i c a t i o n were p i c k e d , s e l f c r o s s e d , brooded and recombinants scored ( F i g u r e 8). The e f f e c t of rec-1 on the smal l i n t e r v a l dpy-5 to unc-13 has been p r e v i o u s l y examined by Rose and B a i l l i e (1979a) and was found to e x h i b i t at l e a s t a t h r e e f o l d i n c r e a s e . T h e r e f o r e , i f the Unc-13 s t r a i n hermaphrodites being t e s t e d were r e c - 1 / r e c - 1 , the recombination f r e q u e n c i e s c a l c u l a t e d over the smal l i n t e r v a l were expected t o i n c r e a s e a c c o r d i n g l y . In e f f e c t , the dpy-5 unc-13 i n t e r v a l acted as an i n t e r n a l c o n t r o l t o i d e n t i f y a homozygous rec-1 background. The f i f t e e n map u n i t i n t e r v a l let-362 t o dpy-5 was scored a t the same time as the two map u n i t d i s t a n c e . Hence, give n a s t r a i n e x h i b i t i n g an i n c r e a s e i n recombination frequency over the dpy-5 unc-13 i n t e r v a l , the e f f e c t of rec-1 on 29 CONSTRUCTION: Po: [males] rec-1 X let-362 rec-1 let-362 dpy-5 unc-13 dpy-5 unc-13 [herm] F1 : V let-362 dpy-5 unc-13; + + + ( s e l f - c r o s s ) and l e t rec-1 dpy unc; rec-1 V F2: Pick, f i v e Unc-13 hermaphrodites from f o u r p l a t e s ; Set up i n d i v i d u a l l y , as p o s s i b l e s t r a i n p r o g e n i t o r s eg. + + let-362 dpy-5 unc-13; r e c - 1 ; or r e c - 1 ; or + ; let-362 dpy-5 unc-13 rec-1 + + STRAIN TEST: Based on random s e g r e g a t i o n , 1/4 of these p o s s i b l e p r o g e n i t o r s were expected to be rec-1 homozygotes. T h i s was t e s t e d by c r o s s i n g them to Rec-1 males eg. [males] rec-1 rec-1 let-362 X V let-362 let-362 dpy-5 dpy-5 unc-13 [herm.] unc-13; (rec-1 ? ) ; (rec-1 ?) d p y - s + + ( s e l f - c r o s s ) unc-13; (rec-1 ? ) ; [herm.] rec-1 SCORE: A recombination frequency over the dpy-5 to unc-13 i n t e r v a l of about 6 % i n d i c a t e s t h a t the s t r a i n was homozygous rec-1 . * D u p l i c a t i o n b e a r i n g i n d i v i d u a l s i n the F1 were i d e n t i f i e d by t h e i r slower developmental time and s l i g h t l y u n c o o r d i n a t e d movement. FIGURE 8: S t r a i n c o n s t r u c t i o n p r o t o c o l to determine the e f f e c t of rec-1 on l a r g e g e n e t i c d i s t a n c e s . 30 recombination frequency over the l a r g e i n t e r v a l c o u l d be examined s i m u l t a n e o u s l y . R e s u l t s : a) I n t r a g e n i c Recombination - The unc-13 gene was s t u d i e d to determine the e f f e c t of rec-1 on i n t r a g e n i c recombination and gene c o n v e r s i o n . A s t r a i n heterozygous f o r the a l l e l e s (e51) and (e450) was allowed t o s e l f - c r o s s f o r two g e n e r a t i o n s . E x c e p t i o n a l progeny were examined. Table 4 shows the r e s u l t s of these experiments. Two t r i a l s were performed; the r e s u l t s of the r e p l i c a t e s were c o n s i s t e n t and were t h e r e f o r e combined. Only the Wild-type recombinants are r e p o r t e d i n Table 4, and onl y Wild-type worms were used i n c a l c u l a t i n g the recombination f r e q u e n c i e s . T h i s was due to d i f f i c u l t i e s s c o r i n g Unc-29 worms on a background of Unc-13 worms. The f r e q u e n c i e s of both s i n g l e c r o s s - o v e r and gene co n v e r s i o n events i n c r e a s e d two to t h r e e f o l d i n the Rec-1 s t r a i n . Confidence i n t e r v a l s do not o v e r l a p i n the case of i n t r a g e n i c recombination, but j u s t o v e r l a p f o r the gene c o n v e r s i o n f r e q u e n c i e s . The c o e f f i c i e n t of c o i n c i d e n c e (c.c.) f o r N-2 was c a l c u l a t e d as: c.c. = observed frequency of apparent double c r o s s over events; expected frequency of double c r o s s over events 31 TABLE 4: The e f f e c t of rec-1 on i n t r a g e n i c recombination, and gene c o n v e r s i o n . Number of Number of # Chromosomes Recomb. Conversion Recombinants Convertants Screened Freq. Freq. * -6 -6 Rec-1 22 12 3.4 m i l l i o n 26 x 10 9.3 x 10 -6 -6 N2 5 3 2.2 m i l l i o n 8.7 x 10 3.5 x 10 * Apparent double c r o s s o v e r s . The p a r e n t a l genotype and mating was: dpy-5 (e51 ) ( + ) unc-29; rec-1 or +_ + (+) (e450) + rec-1 + ( s e l f - c r o s s ) CONFIDENCE INTERVALS: Recombination Conversion  Lower l i m i t Upper l i m i t Lower l i m i t Upper l i m i t -6 -6 -6 -6 Rec-1 19 x 10 32 x 10 7.0 x 10 12.2 x 10 -6 -6 -6 -6 N2 4.2 x 10 16 x 10 1.5 x 10 7.0 x 10 Confidence i n t e r v a l s are c a l c u l a t e d as i n Stevens (1942) where the observed frequency was c a l c u l a t e d as i n F i g u r e 7 and the expected v a l u e was equal to the recombination frequency -5 between the unc-13 a l l e l e s (e51) and (e450) ( i e . 10 %) m u l t i p l i e d by the recombination frequency between unc-13 and unc-29 ( i e . 2%). -6 i e . c.c. = 3.5 X 10 = 17.5; (0.02)(10"*) b) Recombination Over Large D i s t a n c e s - In order to examine the e f f e c t of rec-1 on recombination over l a r g e d i s t a n c e s I attempted to c o n s t r u c t a s t r a i n t h a t would measure recombination frequency over a two percent and a f i f t e e n p ercent i n t e r v a l a t the same time. As o u t l i n e d i n the Methods s e c t i o n the intended s t r a i n t o be c o n s t r u c t e d was: sDp2/let-362 dpy-5 unc-13/let-362 dpy-5 unc-13; r e c - 1 / r e c - 1 . E x p r e s s i o n of let-362 was suppressed by the d u p l i c a t i o n . Unc-13 hermaphrodites o b t a i n e d from the c o n s t r u c t i o n and hence p o s s i b l y of the d e s i r e d genotype were s e l f c r o s s e d to e s t a b l i s h independent l i n e s . During'subsequent out c r o s s i n g and progeny t e s t i n g one q u a r t e r of the twenty one l i n e s were expected t o be homozygous f o r r e c - 1 . However, none of these l i n e s showed a s i g n i f i c a n t i n c r e a s e i n recombination frequency f o r e i t h e r the c o n t r o l i n t e r v a l dpy-5 to unc-13 (2%) or the l a r g e g e n e t i c d i s t a n c e let-362 to dpy-5 (15%) (Table 5 ). D i s c u s s i o n : To c o n f i r m the occurrence of gene c o n v e r s i o n one must recover both products of a g i v e n exchange and show the n o n r e c i p r o c a l nature of the event. In C. elegans the g e n e t i c 33 Table 5: P u t a t i v e Rec-1 d u p l i c a t i o n - b e a r i n g s t r a i n s . These were i s o l a t e d from c o n s t r u c t i o n s intended to determine the e f f e c t of rec-1 on l a r g e g e n e t i c d i s t a n c e s . P a r e n t a l Recombinants Recombination Frequency Wt Dpy Unc Unc-13 Dpy-5 unc-13 to dpy-5 dpy-5 to let-362 482 56 5 0 1 .4% (0.7 to 2.5%)* 16.9% 1 .0 405 37 10 1 3.4% + 1 .4 13.1% + 1 .2 309 29 8 1 3.5% + 1 .5 13.4% + 1 .5 450 54 6 0 1 .8% (0.9 to 3.1%) 17.4% +_ 1 .0 244 32 3 0 1 .6% (0.6 to 3.5%) 19.0% _+ 1 .2 491 53 9 0 2.4% + 1.0 14.4% +_ 1 .1 395 32 5 1 1 .7% (0.8 to 3.2%) 12.1% + 1 .2 506 49 7 0 1 .9% (1 .0 to 3.1 %) 14.1% +_ 1 .0 407 44 3 1 1 .0% (0.4 to 2.2%) 15.6% _+ 1 .1 488 48 5 1 1 .4% (0.7 to 2.5%) 14.3% +_ 1 .1 280 30 6 2.8% (1 .5 to 4.9%) 14.2% + 1 .4 376 33 4 1 1 .4% (0.6 to 2.9%) 13.0% 1 .2 358 30 . 3 1 1.1% (0.4 to 2.5%) 12.7% + 1 .2 470 48 5 1 1 .5% (0.7 to 2.6%) 14.8% + 1 .0 51 4 41 6 1 1 .6% (0.8 to 2.8%) 12.1% + 1 .0 339 39 3 1 1 .2% (0.1 to 2.6%) 16.0% 1 .3 364 42 7 1 2.5% (1.4 to 4.3%) 16.0% + 1 .2 511 47 7 1 1 .9% (1 .0 to 3.1 %) 13.4% +_ 1 .0 423 44 8 1 2.5% + 1 .2 15.0% + 1 .1 41 9 30 4 0 1 .3% (0.6 to 2.6%) 11.0% 1 .2 351 35 6 1 2.3% (1 .2 to 4.0%) 14.4% + 1 .3 To score the recombination frequency when one of the markers i s a l e t h a l mutation use: R = 2 [ Recombinant c l a s s ] ; 4/3 [ Wt + Dpy Unc + Unc + 2 Dpy ] then, the recombination frequency i s c a l c u l a t e d as i n F i g u r e 2. * Confidence i n t e r v a l c a l c u l a t e d as i n Stevens, 1942 34 t o o l s necessary to perform a t e t r a d a n a l y s i s are not a v a i l a b l e . However, a l i n e of argument can be developed which s t r o n g l y suggests t h a t the experiments r e p o r t e d here demonstrate gene c o n v e r s i o n i n the unc-13 gene. F i r s t , as i n D. melanogaster, t h e r e i s reasonably high i n t e r f e r e n c e i n elegans. The frequency of double c r o s s i n g over has been examined on the X l i n k a g e group i n hermaphrodites and on l i n k a g e group IV i n males. In both cases the number of recovered double c r o s s - o v e r progeny was much l e s s than expected (Hodgkin e t a l . , 1979). Secondly, d e s p i t e such high i n t e r f e r e n c e , the data on apparent double c r o s s - o v e r events from my experiments with unc-13 r e s u l t e d i n a c o e f f i c i e n t of c o i n c i d e n c e of 17.5 (see R e s u l t s s e c t i o n ) . T h i s i n d i c a t e s t h a t f a r more double c r o s s - o v e r events o c c u r r e d than expected. The c l a s s i c a l e x p l a n a t i o n of a l o c a l i n c r e a s e i n exchange events ( i e . high n e g a t i v e i n t e r f e r e n c e ; P r i t c h a r d , 1960) i s p o s s i b l e but u n l i k e l y g i v e n t h a t i t has not been found p r e v i o u s l y i n C. elegans. The b e s t e x p l a n a t i o n of these r e s u l t s i s the d e t e c t i o n of gene c o n v e r s i o n by the system used ( F i n n e r t y , 1976). In D r o s o p h i l a , a t t a c h e d X chromosomes (Smith e t a l , 1970; and C a r l s o n , 1971) and compound autosomes (Chovnick et a l , 1970; and B a l l a n t y n e and Chovnick, 1971) are used to p r o v i d e a h a l f t e t r a d a n a l y s i s . In both the rosy l o c u s (Chovnick et a l , 1970) and the rudimentary l o c u s ( C a r l s o n , 1971) the absence of o u t s i d e marker exchange i n recovered w i l d - t y p e f l i e s was found to be completely a c c u r a t e i n i d e n t i f y i n g gene c o n v e r s i o n events. Rose and B a i l l i e (1980) c a l c u l a t e d the d i s t a n c e between the unc-13 a l l e l e s (e51 ) and (e450) i n t h N2 s t r a i n to be 8.0 X 35 -6 10 map u n i t s . T h i s i s i n agreement with the r e s u l t s of the -6 c o n t r o l s f o r the experiments performed here (p = 8.7 X 10 map u n i t s ; Table 4). In the Rec-1 s t r a i n the i n t r a g e n i c recombination frequency i n c r e a s e s t h r e e - f o l d (Table 4). T h i s i s c o n s i s t e n t with the recombination frequency i n c r e a s e s seen between i n t e r g e n i c markers (compare Table 3 and Rose and B a i l l i e , 1979a to Table 4). The e f f e c t of rec-1 on c o n v e r s i o n frequency i s l e s s c l e a r as the confidence i n t e r v a l s o v e r l a p s l i g h t l y . However, gi v e n t h a t c r o s s - o v e r events and gene c o n v e r s i o n are b e l i e v e d to be d i f f e r e n t end products of the same process, i t would seem most l i k e l y t h a t the l a c k of s t a t i s t i c a l s i g n i f i c a n c e i s due to sample s i z e l i m i t a t i o n s r a t h e r than a l a c k of e f f i c a c y by r e c - 1 . Rose and B a i l l i e (1979a) have demonstrated the e f f e c t of rec-1 on g e n e t i c d i s t a n c e s i n the order of one to two p e r c e n t . Since t h a t time, Rose has attempted to c o n s t r u c t s e v e r a l homozygous rec-1 s t r a i n s c o n t a i n i n g markers separated by f i v e to ten map u n i t s (Rose p e r s . comm.). These c o n s t r u c t i o n attempts were not s u c c e s s f u l and i t was not c l e a r whether rec-1 a f f e c t e d recombination f r e q u e n c i e s over l a r g e r d i s t a n c e s . The work performed i n t h i s study was designed to answer t h i s q u e s t i o n by examining a marker i n t e r v a l t h a t was known t o be a f f e c t e d by rec-1 and at the same time, w i t h i n the same c o n s t r u c t i o n , determine the e f f e c t of rec-1 on a l a r g e r i n t e r v a l . The f a i l u r e to r e c o v e r the d e s i r e d homozygous rec-1 s t r a i n i n the d u p l i c a t i o n c o n s t r u c t i o n i s p u z z l i n g (Table 5). Assuming independent assortment, the p r o b a b i l i t y of not r e c o v e r i n g a Rec-1 s t r a i n from the 21 l i n e s t e s t e d i s 0.24%. However, 36 Rose (unpubl. data) has r e c e n t l y o b t a i n e d data s u g g e s t i v e of l o o s e l i n k a g e of rec-1 to unc-54 at the r i g h t end of l i n k a g e group I (p = 0.27 _+ 0.16). T h i s makes the p o s s i b i l i t y of l i n k a g e by rec-1 to the markers of i n t e r e s t i n the c o n s t r u c t i o n a p o s s i b l e e x p l a n a t i o n f o r the r e s u l t s i n Table 5. However, e a r l i e r work by Rose (unpubl. data) has not shown any l i n k a g e by rec-1 to the unc-13 gene c l u s t e r on l i n k a g e group I (24 map u n i t s to the l e f t of unc-54). T h e r e f o r e , i f rec-1 i s l o o s e l y l i n k e d t o unc-54 i t must be p o s i t i o n e d to the r i g h t . T h i s would p l a c e rec-1 between 35 and g r e a t e r than 50 map u n i t s to the r i g h t of unc-13. Using t h i s range of p o s s i b l e l i n k a g e d i s t a n c e s between rec-1 and unc-13 the number of Rec-1 s t r a i n s expected to be recovered from the attempted c o n s t r u c t i o n i s between 3 and 5. Given t h a t no Rec-1 s t r a i n s were recovered, I f i n d an e x p l a n a t i o n of l i n k a g e to be p o s s i b l e , but u n s a t i s f a c t o r y . A c o n c l u s i v e answer to t h i s i s s u e r e q u i r e s a more p r e c i s e mapping of the rec-1 l o c u s . An a l t e r n a t i v e but s p e c u l a t i v e e x p l a n a t i o n f o r the f a i l u r e of the c o n s t r u c t i o n to produce a Rec-1 s t r a i n i s t h a t i n the presence of the d u p l i c a t i o n , sDp2, homozygosity f o r rec-1 i s l e t h a l to the i n d i v i d u a l . The only evidence i n support of t h i s p o s s i b i l i t y i s the i n a b i l i t y to i s o l a t e a sDp2 Rec-1 s t r a i n . T h i s hypothesis c o u l d be t e s t e d by r e p e a t i n g the experiment u s i n g markers on a d i f f e r e n t l i n k a g e group (eg. s e v e r a l d u p l i c a t i o n s comparable to sDp2 are a v a i l a b l e on the X chromosome). D u p l i c a t i o n a s s o c i a t e d l e t h a l i t y becomes a l i k e l y e x p l a n a t i o n i f a Rec-1 s t r a i n cannot be i s o l a t e d under these circumstances. IV. P l e i o t r o p i c E f f e c t s I n t r o d u c t i o n : I t i s important to know the nature and extent of the p l e i o t r o p i c e f f e c t s of the rec-1 enhancer. Such i n f o r m a t i o n may pr o v i d e some i n s i g h t i n t o the b a s i s of the mutation and the mechanism of i t s i n f l u e n c e on recombination. Moreover, i f the Rec-1 s t r a i n i s to be used i n p o p u l a t i o n experiments f o r st u d y i n g the e v o l u t i o n a r y importance of recombination (as d i s c u s s e d i n the f i r s t c h a p t e r ) , the a d d i t i o n a l e f f e c t s rec-1 might have on processes other than recombination must be documented. S e v e r a l types of " s i d e e f f e c t s " are a s s o c i a t e d with both hyper-rec and recombination d e f e c t i v e mutants. A number of s t r a i n s have a dramatic a f f e c t on f e r t i l i t y . T h i s i s due to i n c r e a s e d aneuploidy caused by s e g r e g a t i o n d i s o r d e r s d u r i n g m e i o s i s . Examples of such mutations i n c l u d e : the s p o r u l a t i o n d e f e c t i v e mutants i n y e a s t , spo-1 to spo-11, uvs-1 i n U s t i l a g o  maydis and c(3)G i n D. melanogaster ( E s p o s i t o and E s p o s i t o , 1969; K l a p o l t z et a l . , 1985; H o l l i d a y et a l . , 1976; Gowen and Gowen, 1922). R a d i a t i o n s e n s i t i v i t y i s a l s o t y p i c a l of a number of recombination mutants. In the Rad-3 and Rad-6 e p i s t a t i c groups of y e a s t , c e r t a i n mutations i n c r e a s e r a d i a t i o n induced m i t o t i c recombination. S i m i l a r i l y , both of the spontaneous, m i t o t i c hyper-rec mutants, uvs-1 i n U. maydis and rem-1 i n y e a s t , are 38 r a d i a t i o n s e n s i t i v e (Haynes and Kunz, 1981). A s c o s p o r e v i a b i l i t y i s a l s o a f f e c t e d by t h e s e mutants, s u g g e s t i n g some r o l e f o r t h e w i l d - t y p e gene p r o d u c t i n normal m e i o t i c development. I n D. m e l a n o g a s t e r , m e i - 9 d r a m a t i c a l l y r educes r e c o m b i n a t i o n f r e q u e n c y and i s d e f e c t i v e i n e x c i s i o n r e p a i r (Boyd e t a l . , 1976). S e v e r a l D5 (1) o t h e r r e c o m b i n a t i o n mutants (eg. mei-41 , mus , e t c . ) a r e d e f e c t i v e i n p o s t r e p l i c a t i v e r e p a i r (Boyd and S e t l o w , 1976). R e c o m b i n a t i o n mutants a r e a l s o o f t e n found t o a f f e c t t h e m u t a t i o n r a t e . The h y p e r - r e c mutants i n E. c o l i , f o r example, produce a d r a m a t i c i n c r e a s e i n t h e m u t a t i o n r a t e (eg. M a r i n u s and Konrad, 1976; B a l e e t a l . , 1979; A r t h u r and L l o y d , 1980). A l l o f t h e s e mutants i n v o l v e d e f e c t s i n a r e p a i r system, r e s u l t i n g i n n i c k s and b r e a k s i n t h e DNA. For example, t h e dam mutants a r e due t o the l o s s o f m e t h y l d i r e c t e d mismatch r e p a i r , p r o d u c i n g a h y p e r - r e c , hyper-mutable phenotype ( G l i c k m a n and Radman, 1980; P u k k i l a e t a l , 1983). R e c o m b i n a t i o n a s s o c i a t e d , spontaneous m u t a t i o n r a t e changes have a l s o been r e p o r t e d i n y e a s t . Spo-7, a r e c o m b i n a t i o n d e f e c t i v e mutant, has a reduced m i t o t i c m u t a t i o n r a t e ( E s p o s i t o e t a l . , 1975). C o n v e r s e l y , G o l i n and E s p o s i t o (1977) have shown t h a t rem-1 i n c r e a s e s b o t h m i t o t i c m u t a t i o n and r e c o m b i n a t i o n . I t has been s u g g e s t e d t h a t rem-1 causes i t s m i t o t i c h y p e r - r e c phenotype by p r o d u c i n g DNA l e s i o n s which a r e t h e n c o r r e c t e d by an e r r o r prone r e c o m b i n a t i o n - r e p a i r system (Malone and H o e k s t r a , 1984). The m e i o t i c m u t a t i o n r a t e , as w e l l as t h e r e c o m b i n a t i o n f r e q u e n c y , a r e a f f e c t e d by mu i n D. m e l a n o g a s t e r (Green, 1970; Green, 1976). The e f f e c t on r e c o m b i n a t i o n i s n o n u n i f o r m ; 39 c r o s s i n g over on the t i p of the X chromosome i s decreased, while a l l o ther r e g i o n s are u n a f f e c t e d . F i n a l l y , Moerman and Waterston (1984) have d e s c r i b e d the o n l y known mutator i n C. elegans. I t s e f f e c t on recombination i s not known. However, they have evidence t h a t suggests t h a t the mutator phenotype i s caused by the t r a n s p o s a b l e element, Tc1. T h i s would make any e f f e c t on recombination u n l i k e l y . F i n a l l y , s e g r e g a t i o n d i s o r d e r s r e s u l t i n g i n n o n d i s j u n c t i o n and chromosome l o s s have been found to be a s s o c i a t e d with recombination mutants. Studying D. melanogaster, S t u r t e v a n t and Beadle (1936) were the f i r s t t o d e s c r i b e a dramatic i n c r e a s e i n X chromosome n o n d i s j u n c t i o n a s s o c i a t e d with a decrease i n c r o s s - o v e r t e t r a d s . Baker and H a l l (1976) have reviewed the recombination d e f e c t i v e mutants i n D r o s o p h i l a and have concluded t h a t normal recombination i s necessary f o r r e g u l a r s e g r e g a t i o n . Crossover events appear to ensure t h a t a chromosome p a i r s p r o p e r l y with i t s homologue. Given a r e d u c t i o n i n recombination frequency, chromosomes tend to p a i r by s i z e ( i e . d i s t r i b u t i v e p a i r i n g , G r e l l , 1962; 1976). T h i s i n c r e a s e s the chance of i n c o r r e c t p a i r i n g ; i f recombination decreases, n o n d i s j u n c t i o n i n c r e a s e s (eg. f l i e s homozygous f o r c(3)G, Baker and H a l l , 1976). However, the converse may not h o l d t r u e as both a l l e l e s of c(3)G i n c r e a s e recombination when heterozygous, but do not decrease n o n d i s j u n c t i o n ( H a l l , 1972). In Neurospora, mei-1 and Mei-2 both decrease recombination and i n c r e a s e n o n d i s j u n c t i o n (Smith, 1975); w h i l e a s y n d e t i c mutants, i n higher p l a n t s , have a high frequency of u n i v a l e n t s at metaphase I with recombination reduced on the b i v a l e n t s (eg. 40 Beadle, 1933; Thomas and Rajhathy, 1966; S j o d i n , 1970; Baker et a l . , 1976). To date, i n v e s t i g a t i o n s s t u d y i n g the rec-1 enhancer i n C. elegans have focused on i t s e f f e c t on recombination, although p r e v i o u s work by Hartman and Herman (1982) has shown t h a t rec-1 i s not r a d i a t i o n s e n s i t i v e . To complete the c h a r a c t e r i z a t i o n of r e c - 1 , I have examined the p o s s i b l e e f f e c t s of t h i s mutation on f e c u n d i t y , v i a b i l i t y , s e g r e g a t i o n and mutation r a t e . In summary, i t was found t h a t rec-1 had no d e t e c t a b l e a f f e c t on f e c u n d i t y , v i a b i l i t y and mutation r a t e . However, n o n d i s j u n c t i o n and/or chromosome l o s s of the X l i n k a g e group appeared t o be i n c r e a s e d t w o - f o l d . Compared to other recombination mutants, rec-1 has s u r p r i s i n g l y few p l e i o t r o p i c e f f e c t s . Methods: a) F e c u n d i t y - Crescent stage hermaphrodites of both genotypes (rec-1 /rec-1 and +_/+) were t r a n s f e r r e d to separate c u l t u r e p l a t e s , s e l f - c r o s s e d and brooded. C u l t u r e s were maintained a t 20 2C and the number of progeny were counted when mature. b) Competitive A b i l i t y - Three c r e s c e n t stage hermaphrodites of both genotypes (rec-1 /rec-1 and +_/+_) were p l a c e d on a 100 mm p e t r i p l a t e c o n t a i n i n g nematode growth medium covered with a lawn of w i l d - t y p e E. c o l i . The p o p u l a t i o n was allowed to grow u n t i l s t a r v a t i o n symptoms appeared i n the most r e c e n t progeny 41 ( i e . dauer l a r v a e were produced). The p o p u l a t i o n was then t r a n s f e r r e d weekly to f r e s h c u l t u r e p l a t e s . A two by two cm. agar cube c o n t a i n i n g about 100 worms was cut from the o l d c u l t u r e and p l a c e d onto a new p l a t e . As the worms r a p i d l y over grew the p l a t e , they were r e g u l a r i l y exposed t o a s t a r v a t i o n s t r e s s . At the end of two months (approximately s i x t e e n g e n e r a t i o n s ) the genotypes of ten hermaphrodites were determined by o u t - c r o s s i n g to dpy-5 dpy-14/+ +; rec-1/rec-1 males and then s c o r i n g the recombinants i n the F2 g e n e r a t i o n ( F i g u r e 9 ) . c) N o n d i s j u n c t i o n - Twenty to t h i r t y c r e s c e n t stage hermaphrodites were i n d i v i d u a l l y p l a c e d on c u l t u r e p l a t e s f o r each experimental c o n d i t i o n (eg. temperature: 20 C and 26 C; genotype: rec-1 /rec-1 and +_/+) . Each hermaphrodite was brooded. The amount of n o n d i s j u n c t i o n (or chromosome l o s s ) was assayed by the number of males ( i e . XO i n d i v i d u a l s ) found i n the F1. d) Mutation Rate i n the Rec-1 S t r a i n - Mutation r a t e was estimated i n the Rec-1 s t r a i n u s i n g a m o d i f i c a t i o n of the methods o u t l i n e d i n Rosenbluth and B a i l l i e , (1981). Dpy-18 Unc-46 Rec-1 hermaphrodites were i n d i v i d u a l l y brooded. The progeny were counted to ensure t h a t the P o 1 s used d i d not c a r r y a spontaneous l e t h a l mutation. A l l of the progeny of an a c c e p t a b l e Po were then c r o s s e d to eT1/+; rec-1/rec-1 males. The F2 was comprised of two genotypes: dpy-18 unc-46/eTl; rec-1/rec-1 and dpy-18/+; unc-46/+; rec-1/rec-1 ( F i g u r e 10). The t r a n s l o c a t i o n eT1 produces pseudo-linkage between dpy-18 and unc-46 and suppresses recombination w i t h i n the t r a n s l o c a t e d 42 1) [males] r e c - 1 ; X dpy-5 dpy-14; r e c - 1 ; [herm.] rec-1 dpy-5 dpy-14 rec-1 V 2) [males] dpy-5 dpy-14; r e c - 1 ; X + + rec-1 _+_', or rec-1 ; [herm. ] + rec-1 3) V [herm.] dpy-5 dpy-14 or + + + + rec-1 or r e c - 1 ; + rec-1 SCORE: ( s e l f - c r o s s ) V a) I f the recombination frequency i s high (eg about 5.0%) then the Po was homozygous r e c - 1 . b) I f the recombination frequency was a t a normal l e v e l (eg. 1.5%) then the Po was N2. FIGURE 9: Assay f o r d e t e r m i n i n g the genotypes of competitors i n the p o p u l a t i o n experiment. 43 CONSTRUCTION: Po: 1 hermaphrodite ( s e l f - c r o s s e d ) F1 dpy-18 dpy-18 unc-46 unc-46 rec-1 rec-1 V Determine t h a t the Po i s not c a r r y i n g a l e t h a l by counting the number of progeny - a l e t h a l would reduce the f e c u n d i t y . P i c k 100 F1 [herm.] (genotype as above) X eT1 ; rec-1 [males] + rec-1 V F2: Set up 2,000 p l a t e s with 1 Wt hermaphrodite each and a l l o w to s e l f - c r o s s i e . dpy-18 unc-46; rec-1 eT1 + and SCREEN: F3: screen f o r absence of Dpy Unc i n d i v i d u a l s dpy-18 ; unc-46 ; rec-1 + + + V screen f o r : absence of Dpy worms; absence of Unc worms; reduced # of progeny. FIGURE 10: S t r a i n c o n s t r u c t i o n and procedure to determine the e f f e c t of rec-1 on the spontaneous mutation r a t e . The mutation r a t e was measured by s c r e e n i n g f o r l i n k e d l e t h a l s i n the r e g i o n balanced by e T l . Dpy-18 and unc-46 are pseudo-linked when balanced by e T l . 44 r e g i o n s (approximately 40 map u n i t s ) . In the absence of a l i n k e d l e t h a l mutation, the p r o p o r t i o n of Dpy-18 Unc-46 i n d i v i d u a l s i n the F3 would be 1/6th. The occurrence of a l e t h a l mutation i n the area balanced by eT1 would r e s u l t i n the absence of Dpy-18 Unc-46 worms i n the F3. Any p l a t e with two or l e s s Dpy Unc worms was progeny t e s t e d f o r a p u t a t i v e l e t h a l mutation ( i e . dpy-18  unc-46 l e t h a l / e T l ; s e l f - c r o s s e d ) . The progeny of hermaphrodites c a r r y i n g the w i l d - t y p e chromosome r a t h e r than eT1 were screened f o r the presence of a l i n k e d l e t h a l by n o t i n g : the absence or reduced number of Dpy-18 worms; the absence or reduced numbers of Unc-46 worms; and the reduced number of progeny. At the completion of the scre e n , w i l d - t y p e hermaphrodites were allowed to s e l f - c r o s s f o r s e v e r a l g e n e r a t i o n s ( i e . dpy-18  unc-46/eTl; rec-1/rec-1) . T h i s was to determine i f the s t r a i n was s t a b l e i n the presence of rec-1 ( i e . does eT1 suppress c r o s s i n g over i n the rec-1 homozygote). R e s u l t s : a) F e c u n d i t y - The mean number of progeny per hermaphrodite f o r the N2 and Rec-1 s t r a i n s were compared by brooding i n d i v i d u a l s and counting a l l progeny. Table 6a shows the e f f e c t of rec-1 on progeny number. No d i f f e r e n c e was found between the means of the f e c u n d i t y estimates of Rec-1 and N2 s t r a i n s . 45 TABLE 6: The e f f e c t of rec-1 on f e c u n d i t y and c o m p e t i t i v e a b i l i t y a) F e c u n d i t y : Mean Standard T o t a l # Progeny* D e v i a t i o n Progeny P - Rec-1 281 39.9 4,774 >0.50 N2 276 38.5 1,657 * S e l f c r o s s e d . b) Competition: Time Generation Genotype (weeks) Rec-1 N2 0 0 3 3 8 16 4 6 46 b) Competitve A b i l i t y - In order to assess the v i a b i l i t y of the Rec-1 s t r a i n a p o p u l a t i o n experiment was performed i n which Rec-1 hermaphrodites competed with N2 worms i n a food l i m i t e d environment. Genotypes of ten i n d i v i d u a l s were determined a f t e r 16 g e n e r a t i o n s . The hermaphrodites t e s t e d were randomly s e l e c t e d . The r e s u l t s of the genotype assay are presented i n Table 6b. Both Rec-1 and N2 i n d i v i d u a l s were p r e s e n t . There was no s u b s t a n t i a l d i f f e r e n c e i n the c o m p e t i t i v e a b i l i t y of the two s t r a i n s . Moreover, no d i f f e r e n c e was noted between the developmental times of the Rec-1 and N2 s t r a i n s d u r i n g the maintenance of c u l t u r e s . F r e s h s t o c k p l a t e s were e s t a b l i s h e d weekly and worms from both s t r a i n s were found to mature a t the same r a t e . c) Nondisj u n c t i o n - The frequency of males o c c u r r i n g on brooded p l a t e s was used as an i n d i c a t o r of the amount of n o n d i s j u n c t i o n and/or chromosome l o s s t a k i n g p l a c e i n the Rec-1 s t r a i n . D espite an apparent d o u b l i n g i n the frequency of males i n the Rec-1 s t r a i n c u l t u r e d a t 20 C, no s i g n i f i c a n t s t a t i s t i c a l d i f f e r e n c e was found r e l a t i v e to N2 (Table 7). Rose and B a i l l i e (1979b) have shown t h a t n o n d i s j u n c t i o n i n c r e a s e s with temperature and t h e r e f o r e , so does the frequency of males. Given t h a t the absence of a s i g n i f i c a n t r e s u l t might be a consequence of sample s i z e , the experiment was repeated a t a h i g h e r temperature so t h a t the number of males would be g r e a t e r . U n f o r t u n e t l y , the c o n t r o l ( i e . N2) hermaphrodites d i d not develope p r o p e r l y ; phenocopies with m u l t i p l e v u l v a and o t h e r a b n o r m a l i t i e s predominated to such an extent t h a t the worms 47 TABLE 7: The e f f e c t of rec-1 on n o n d i s j u n c t i o n and/or chromosome l o s s . Temperature # Males Sample S i z e # Males per 1,000 Rec-1 20°C 19 4,896 3.9* N2 20°C 3 1,657 1.8 Rec-1 26°C 32 1,825 17.5 N2** 26°C 17 1,933 8.8 * Not s i g n i f i c a n t at the 5% l e v e l (P=0.15). **From Rose and B a i l l i e , 1979b. 48 c o u l d not be r e l i a b l y s c o r e d . The N2 hermaphrodites used i n t h i s experiment were s l i g h t l y younger than t h e i r Rec-1 c o u n t e r p a r t s . They may have been a t a temperature s e n s i t i v e stage i n development when exposed to the temperature i n c r e a s e . The experiment was repeated, but was u n s u c c e s s f u l - i t may be t h a t the temperature used ( i e . 26 2C) was too c l o s e t o the upper l i m i t of the temperature t o l e r a n c e of C. elegans. Note however, t h a t Rose and B a i l l i e (1979b) have examined the e f f e c t of temperature on n o n d i s j u n c t i o n i n C. elegans. Although i t cannot be c o n s i d e r e d to be a proper c o n t r o l , a t w o - f o l d i n c r e a s e i s e v i d e n t i f one compares the frequency of males found i n the 26 aC Rec-1 experiment with the N2 r e s u l t s of Rose and B a i l l i e (1979b). d) Mutation Rate i n the Rec-1 S t r a i n - The e f f e c t of rec-1 on the mutation r a t e was examined by s c o r i n g f o r spontaneous, r e c e s s i v e , l e t h a l mutations i n the 40 map u n i t r e g i o n balanced by e T l . No such mutations were i s o l a t e d from the 1,877 screened chromosomes of the Rec-1 s t r a i n (Table 8). C a l c u l a t i n g the confidence i n t e r v a l as i n Stevens (1942), the upper l i m i t i s 1.6 -3 x 10 spontaneous, r e c e s s i v e , l e t h a l mutations per f o r t y map u n i t s . T h i s o v e r l a p s e x t e n s i v e l y with the c o n f i d e n c e i n t e r v a l of the N2 s t r a i n r e p o r t e d by Rosenbluth et a l . (1983). The rec-1 mutation does not appear to induce r e c e s s i v e l e t h a l s . Wild-type heterozygotes which were allowed to s e l f c r o s s f o r s e v e r a l g e n e r a t i o n s proved to be s t a b l y balanced by eT1 ( i e . dpy-18 unc-46/eTl; r e c - 1 / r e c - 1 ) . No Dpy-18 or Unc-46 49 TABLE 8 : The e f f e c t of r e c - 1 on the frequency of r e c e s s i v e , l e t h a l mutations. # L e t h a l s Sample S i z e Frequency* Conf. I n t e r v a l Rec - 1 0 1 , 8 7 1 0 0 to 1 . 6 x 1 0 " 3 N 2 * * 2 3 , 1 9 8 6 . 2 x 1 0 - 4 1 . 0 x 10 " J to 2 . 3 x 1 0 " 3 * Per 4 0 map u n i t s . ** From Rosenbluth e t a l . , 1 9 8 3 . recombinants were reco v e r e d . C r o s s i n g over d i d not occur w i t h i n the t r a n s l o c a t e d r e g i o n i n the Rec-1 s t r a i n . D i s c u s s i o n : Rec-1 appears to have i n c r e a s e d the r a t e of p r o d u c t i o n of males by a f a c t o r of two (Table 7 ) . T h i s i s i n agreement with the p r e l i m i n a r y work of Rose (1980). S t a t i s t i c a l l y s i g n i f i c a n t r e s u l t s were not o b t a i n e d ; however, t h i s was most l i k e l y due to sample s i z e r e s t r i c t i o n s . Males are XO; the p u t a t i v e r e c i p r o c a l event ( i e . XXX hermaphrodites) were not recovered i n t h i s experiment. T h e r e f o r e , e i t h e r X-chromosome n o n d i s j u n c t i o n and/or chromosome l o s s were i n c r e a s e d i n the Rec-1 s t r a i n . In o t h e r s e g r e g a t i o n mutants i n C. elegans both n o n d i s j u n c t i o n and chromosome l o s s are i n c r e a s e d (eg. the Him mutants: a s e r i e s of mutants which segregate an i n c r e a s e d p r o p o r t i o n of males, ranging from 2 to 35%). There are only two mutations i n C. elegans comparable to r e c - 1 : The f i r s t i s rad-4 which i n c r e a s e s recombination on the X chromosome by 24% and decreases X n o n d i s j u n c t i o n to one-tenth of the N2 v a l u e , without a l t e r i n g autosome s e g r e g a t i o n (Hartman and Herman, 1982). The authors suggest t h a t the a f f e c t on recombination i s due to d i f f e r e n c e s i n g e n e t i c background between the mutant and c o n t r o l s t r a i n s , r a t h e r than any r e a l e f f e c t by rad-4. The second i s him-8 (el 489) which decreases X recombination to one-eighth of the N2 v a l u e and i n c r e a s e s X-chromosome 51 n o n d i s j u n c t i o n ( i e . s e g r e g a t i n g 35% males). As with rad-4, autosomes are not a f f e c t e d (Hodgkins e t a l , 1979). Moreover, G o l d s t e i n (1 982 ) has shown t h a t him-8 (el 489 ) has s t r u c t u r a l l y complete synaptonemal complexes; t h e r e f o r e , i t appears t h a t homologue p a i r i n g i s normal. Rec e n t l y , P r a t t and Herman (pers. comm.) have i s o l a t e d a new a l l e l e of him-8 t h a t decreases X-chromosome recombination to 1/80th of i t s normal l e v e l and d r a m a t i c a l l y i n c r e a s e s X n o n d i s j u n c t i o n . The i n v e r s e r e l a t i o n s h i p between recombination and n o n d i s j u n c t i o n i l l u s t r a t e d by rad-4 and him-8 i s t y p i c a l of D r o s o p h i l a m e i o t i c mutants (Baker and H a l l , 1976; G r e l l , 1976). Rec-1, however, does not f i t t h i s p a t t e r n as i t i n c r e a s e s both recombination and a p p a r e n t l y n o n d i s j u n c t i o n . The f e c u n d i t y and v i a b i l i t y of the Rec-1 s t r a i n were not s i g n i f i c a n t l y d i f f e r e n t than the N2 s t r a i n (Table 6a and b ) . T h i s i s i n c o n t r a s t with most of the Him mutants i s o l a t e d by Hodgkins et a l . (1979). In these mutants l e t h a l i t y i s h i g h , r e f l e c t i n g the l a r g e amount of autosomal n o n d i s j u n c t i o n and hence aneuploidy t a k i n g p l a c e . S i m i l a r r e s u l t s are found i n yeast ( E s p o s i t o and E s p o s i t o , 1969). T h i s suggests t h a t rec-1 may a f f e c t o n l y X-chromosome s e g r e g a t i o n , s i m i l a r t o the n o n d i s j u n c t i o n e f f e c t of him-8 ( d e s c r i b e d above). However, even i f rec-1 caused autosomal n o n d i s j u n c t i o n a t the same r a t e as the X l i n k a g e group (Table 6b), i t would be d i f f i c u l t to d e t e c t such a small e f f e c t on f e c u n d i t y . A very l a r g e sample s i z e would be needed to determine i f autosomal d i s j u n c t i o n i s a c t u a l l y a l t e r e d . F i n a l l y , the above r e s u l t s are c o n s i s t e n t with the f i n d i n g s of Hartman and Herman (1982) which showed t h a t Rec-1 i s not r a d i a t i o n 52 s e n s i t i v e . Most r a d i a t i o n s e n s i t i v e mutants are not as robust as w i l d - t y p e and o f t e n have reduced f e c u n d i t y (eg. D r o s o p h i l a -Baker and H a l l , 1976; Yeast - Kowalski and Laskowski, 1975). Rosenbluth and B a i l l i e (1981) c h a r a c t e r i z e d the C. elegans mutation unc-72 (e_873) and determined t h a t i t was, i n f a c t , a t r a n s l o c a t i o n . Renaming i t eTl they proceeded to develope an e l e g a n t mutagenisis s c r e e n i n g technique p r e v i o u s l y not a v a i l a b l e i n t h i s s p e c i e s (Rosenbluth et a l . , 1983; 1985). In performing the screen u s i n g a Rec-1 s t r a i n , any e f f e c t rec-1 might have on the mutation r a t e might be r e l a t e d to the recombination process i t s e l f . Using a b a l a n c e r i n the screen to e l i m i n a t e c r o s s i n g over would then a l s o e l i m i n a t e any i n c r e a s e i n mutation r a t e . To a v o i d t h i s p o s s i b i l i t y the s c r e e n was prepared i n such a manner t h a t rec-1 c o u l d p u t a t i v e l y induce mutations i n a dpy-18; unc-46 background. The r e s u l t a n t l e t h a l mutation would then be "captured" over the b a l a n c e r eT1 i n the next g e n e r a t i o n . The Rec-1 s t r a i n d i d not induce r e c e s s i v e , l e t h a l mutations (Table 8). T h i s r e s u l t , i n c o n j u n c t i o n with the f a c t t h a t rec-1 i s not r a d i a t i o n s e n s i t i v e , makes i t u n l i k e l y t h a t the Rec-1 s t r a i n i s due to a r e p a i r system d e f e c t . Hyper-rec mutants i n o t h e r organisms may or may not have an i n c r e a s e d spontaneous mutation r a t e ; however, a l l those examined have shown some s i g n of r e p a i r system d e f i c i e n c y . In E. c o l i , the hyper-rec mutants are a l l hyper-mutable, with 10 to 1 0 0 - f o l d i n c r e a s e s i n mutation r a t e (eg. A r t h u r and L l o y d , 1980). Rem-1, i n y e a s t , i n c r e a s e s m i t o t i c i n t e r g e n i c recombination by a f a c t o r of t en and i n c r e a s e s the spontaneous mutation r a t e f i v e - f o l d . In D r o s o p h i l a , c(3)G has no a f f e c t on the spontaneous mutation r a t e i n me i o s i s ( H a l l , 1971; Green, 1970). However, i t does i n c r e a s e the frequency of X-ray induced, r e c e s s i v e , l e t h a l mutations, i m p l y i n g some d e f e c t i n a r e p a i r process (Watson, 1969, 1972). The e f f e c t of the interchromosomal e f f e c t on mutation r a t e has not been examined. C o n s i d e r i n g the above, i t i s p o s s i b l e t h a t rec-1 may have an e f f e c t on r e p a i r processes even though i t had no e v i d e n t e f f e c t on the spontaneous mutation r a t e . To r e s o l v e t h i s i s s u e the X-ray induced mutation r a t e should be measured i n the Rec-1 s t r a i n . 54 V. The E f f e c t of an Amber Suppressor on rec-1 I n t r o d u c t i o n ; A suppressor mutation i s a second a l t e r a t i o n i n the DNA t h a t r e v e r s e s the phenotypic e x p r e s s i o n of an e a r l i e r mutation (Smith, 1979). Reversions are not suppressors; the new mutation must occur a t a new s i t e . Three g e n e r a l c l a s s e s of suppressor mutations can be i d e n t i f i e d : i n t r a g e n i c , i n f o r m a t i o n a l and i n d i r e c t (Sherman, 1982). I n f o r m a t i o n a l suppressors a l t e r the f i d e l i t y of i n f o r m a t i o n flow from gene to p r o t e i n (eg. tRNA nonsense s u p r e s s o r s ) . Base p a i r s u b s t i t u t i o n mutations may convert c e r t a i n amino a c i d codons to c h a i n t e r m i n a t i o n codons ( i e . the stop codons: amber - UAG, ochre - UAA, o p a l - UGA). Suppression occurs by amino a c i d i n s e r t i o n a t the stop codon owing to an a l t e r e d a n t i c o d o n i n the tRNA s p e c i e s such t h a t i t now e r r o n e o u s l y r e c o g n i z e s and binds to the nonsense mutation. tRNA suppressors have been used e x t e n s i v e l y i n E. c o l i and y e a s t ( f o r reviews see: C e l i s and Smith, 1979, and Sherman, 1982). In h i g h e r eukaryotes the s t o r y i s q u i t e d i f f e r e n t . Amber suppressors have been s y n t h e s i z e d i n v i t r o u s i n g Xenopus l a e v i s c l o n e d DNA ( L a s k i et a l , 1 982)'. Through m i c r o i n j e c t i o n t e c h n i q u e s , t h i s suppressor has been s u c c e s s f u l l y expressed i n mammalian t i s s u e c u l t u r e s (Hudziak e t a l , 1982). However, the i s o l a t i o n of induced tRNA suppressor mutations has o n l y o c c u r r e d i n C. elegans. F i v e such mutants have been c h a r a c t e r i z e d and a l l are amber suppressors ( f o r a review see Hodgkin, 1985). 55 Sup-7 i s the most powerful suppressor found i n C. elegans and has a dominant e f f e c t (Waterston, 1981). Chemical a n a l y s i s of Unc-15 (e1214) worms ( i e . paramyosin d e f e c t i v e ) has shown t h a t sup-7 r e s t o r e s paramyosin l e v e l s to roughly 40% of the N2 l e v e l . Kimble et a l . (1982) and W i l l s et a l . (1983) demonstrated t h a t sup-7 i s an amber suppressor. More r e c e n t l y , B o l t e n e t a l . (1984) have c l o n e d and sequenced sup-7 (st5) and shown i t to be a tryptophan tRNA gene c o n t a i n i n g an a l t e r a t i o n a t the a n t i c o d o n which r e s u l t s i n i t r e a d i n g UAG codons. In the experiment r e p o r t e d here, I determined i f the rec-1 mutation c o u l d be suppressed by sup-7. A s t r a i n was c o n s t r u c t e d which segregated homozygous and heterozygous rec-1 i n d i v i d u a l s . The progeny were a l s o heterozygous f o r sup-7 and two c i s - l i n k e d markers. The recombination frequency of rec-1 homozygotes and heterozygotes were then compared i n a sup-7/+ background. These experiments c l e a r l y demonstrated t h a t the e f f e c t of rec-1 on recombination frequency i s not suppressed by sup-7. Methods: Sup-7 i s a dominant, amber suppressor mutation. I t was used to determine i f the e f f e c t s of rec-1 on recombination were due to an e a r l y stop s i g n a l p r e v e n t i n g the p r o d u c t i o n of a gene product. Sup-7 i s X - l i n k e d . 56 Rec-1 males were c r o s s e d with Sup-7 Dpy-18 hermaphrodites ( F i g u r e 11). Dpy-18 was present from the o r i g i n a l s t r a i n c o n s t r u c t i o n s (Waterston, 1981) and served as an i n d i c a t e r f o r the presence and absence of sup-7. The hermaphrodites were then t r a n s f e r r e d t o f r e s h p l a t e s a f t e r twenty f o u r hours. The F1 males, a l l of which were sup-7/0, were c r o s s e d with Dpy-5 Unc-13 Rec-1 hermaphrodites. The F2 hermaphrodites were heterozygous f o r sup-7, dpy-18, dpy-5 and unc-13, and were e i t h e r heterozygous or homozygous f o r rec-1 ( F i g u r e 11). Crescent stage F2 Wild-type hermaphrodites were brooded. C u l t u r e s were kept a t 22 2C as t h i s was the o p t i m a l growth temperature f o r Sup-7 worms. The F3 was screened f o r recombinants and recombination f r e q u e n c i e s were c a l c u l a t e d f o r each brooded l i n e . R e s u l t s : Based on random s e g r e g a t i o n , one h a l f of the hermaphrodites brooded should have been rec-1/+, while the other h a l f should have been r e c - 1 / r e c - 1 ; both were sup-7/+. I f sup-7 suppresses the e f f e c t of r e c - 1 , then a l l the recombination f r e q u e n c i e s should c l u s t e r around the w i l d t y p e v a l u e of about two p e r c e n t . A l t e r n a t i v e l y , i f rec-1 i s not suppressed, two d i f f e r e n t c l a s s e s of recombination f r e q u e n c i e s should be d i s c e r n a b l e : c a l c u l a t i o n s based on those brooded hermaphrodites t h a t were rec-1/rec-1 and those t h a t were rec-1/+. 57 CONSTRUCTION: Po: rec-1 [males] rec-1 sup-7; dpy-18 sup-7 dpy-18 [herm.] F1 : dpy-18; + sup-7; 0 rec-1 X dpy-5 unc-13 ; rec-1 [males] F2: dpy-1 8 or _+; + + SCORE RECOMBINANTS: dpy-5 unc-13 [herm.] rec-1 V sup-7; dpy-5 unc-13; + + + rec-1 or rec-1 [herm.] rec-1 ( s e l f - c r o s s ) F3: V a) I f sup-7 (which i s dominant) suppresses rec-1 then a l l the scored recombination f r e q u e n c i e s w i l l f a l l i n the range of t y p i c a l N2 v a l u e s . b) High recombination f r e q u e n c i e s t y p i c a l of Rec-1 i n d i c a t e s an absence of s u p p r e s s i o n . Given a l a r g e enough sample s i z e , a bimodal d i s t r i b u t i o n would be expected. FIGURE 11: S t r a i n c o n s t r u c t i o n and procedure to determine the e f f e c t of sup-7 on the e x p r e s s i o n of the Rec-1 phenotype 58 E x p e r i m e n t a l : N 7 * U ( s e l f - c r o s s ) M 6 * dpy-5 unc-13; rec-1 o r r e c - 1 ; B + + + rec-1 E 5 * * sup-7 R + 4 * * 0 F 3 * * * * JJ 2 * * * * * • * * E R " | * * * * * * * * * M A O 1 2 3 4 5 6 7 8 9 1 0 1 1 12 13 P H 5 C o n t r o l : R ( s e l f - c r o s s ) 0 4 dpy-5 unc-13; rec-1 D + + rec-1 1 3 * T E 2 * * * * S 1 * * * * * * * 0 1 2 3 4 5 6 7 £3 9 10 T\ 12 TT RECOMBINATION FREQUENCY (%) FIGURE 12: The e f f e c t of sup-7, an amber s u p p r e s s o r , on t h e e x p r e s s i o n o f the Rec-1 phenotype. I f s u p p r e s s e d , t h e r e c o m b i n a t i o n f r e q u e n c i e s s h o u l d c l u s t e r around t h e N2 v a l u e o f 2%. I f , on t h e o t h e r hand, rec-1 has i t s t y p i c a l e f f e c t on r e c o m b i n a t i o n f r e q u e n c y , two c l u s t e r s s h o u l d be e v i d e n t : rec-1/+ ( a p p r o x i m a t e l y 2%) and rec-1/rec-1 ( a p p r o x i m a t e l y 7%). 59 F i g u r e 12 c l e a r l y shows t h a t two c l a s s e s of recombination frequency are p r e s e n t . Confidence i n t e r v a l s are approximately +_1 m.u. f o r any p a r t i c u l a r i n d i v i d u a l . Although the p a t t e r n of recombination f r e q u e n c i e s does not p r o v i d e a good f i t f o r a bimodal d i s t r i b u t i o n , t h i s i s most l i k e l y a consequence of a l i m i t e d sample s i z e . C e r t a i n l y , the c l u s t e r of hermaphrodites with v a l u e s i n excess of 8% recombination i s due to the e f f e c t of r e c - 1 . The recombination frequency i s s l i g h t l y h i g h e r than the p r e v i o u s l y r e p o r t e d v a l u e ( F i g u r e 1). T h i s i s probably due to the i n c r e a s e d i n c u b a t i o n temperature of 22 2C. D i s c u s s i o n : Two q u e s t i o n s are of p a r t i c u l a r r e l e v a n c e a t t h i s p o i n t . F i r s t , how reasonable i s i t to expect rec-1 to be an amber mutant? The frequency of amber a l l e l e s i n other mutant genes i n C. elegans i s q u i t e v a r i a b l e . At t r a - 3 , t h r e e out of f o u r a l l e l e s are amber (Hodgkin, 1985), while s i x of 22 a l l e l e s are amber at unc-54 (Waterston, 1981). T h i s c o n t r a s t s w i t h fem-1 and t r a - 2 , where one of seven and one of 16 a l l e l e s , r e s p e c t i v e l y , are amber mutations (Doniach and Hodgkin, 1984; Hodgkin, 1985). C e r t a i n l y , amber mutations i n C. elegans are not r a r e events, making i t p o s s i b l e f o r rec-1 to be i n c l u d e d i n t h i s c l a s s of mutants. I t i s i n t e r e s t i n g to note t h a t the uvrD (210) hyper-rec mutation i n E. c o l i i s an amber mutant (Arthur and L l o y d , 1980). Secondly, what i n f o r m a t i o n would be gained i f sup-7 s u p p r e s s i o n of rec-1 was demonstrated? T h i s q u e s t i o n concerns i t s e l f w ith the u t i l i t y of nonsense mutations as a g e n e t i c t o o l ( f o r comments on t h i s t o p i c see: Waterston, 1981; Ferguson and 60 H o r v i t z , 1985; Hodgkin, 1985). In the case of r e c - 1 , p o s i t i v e r e s u l t s would have i n d i c a t e d t h a t the gene has a p r o t e i n product - a n o n t r i v i a l c o n c l u s i o n when the b i o c h e m i c a l f u n c t i o n of the gene product i s unknown. Moreover, the extent of s u p p r e s s i o n and any dosage e f f e c t ( i e . reduced s u p p r e s s i o n of the mutant phenotype when heterozygous f o r sup-7) would demonstrate whether the rec-1 gene product was r e q u i r e d i n c a t a l y t i c or s t o i c h i o m e t r i c q u a n t i t i e s . The former would suggest some enzymatic f u n c t i o n , whereas the l a t t e r would imply a s t r u c t u r a l component. For example, Unc-15 worms are paramyosin d e f e c t i v e and the p a r a l y z e d phenotype i s onl y p a r t i a l l y suppressed by one copy of sup-7 (Waterston, 1981). F i n a l l y , g i v e n t h a t most amber mutations are n u l l , the stop codon t y p i c a l l y o ccurs e a r l y i n the p o l y p e p t i d e s y n t h e s i s such t h a t no f u n c t i o n a l gene product i s produced. I f t h i s were t r u e f o r rec-1 i t would suggest t h a t the gene product may f u n c t i o n as a r e p r e s s o r ; i n i t s absence recombination i n c r e a s e s u n i f o r m l y throughout the genome. The r e s u l t s of t h i s study c l e a r l y show t h a t there i s no suppr e s s i o n of the e f f e c t of rec-1 on recombination ( F i g u r e 15). I am assuming t h a t at l e a s t p a r t i a l s u p p r e s s i o n of the mutant phenotype would be d e t e c t a b l e when sup-7 i s heterozygous. Even i f the gene product i s r e q u i r e d i n l a r g e amounts, amber mutants show some su p p r e s s i o n when heterozygous f o r sup-7 (Waterston, 1981). For example, Unc-54 worms have d e f e c t i v e myosin heavy cha i n molecules and are p a r a l y z e d ( E p s t e i n et a l . , 1974; MacLeod et a l . , 1977). The w i l d - t y p e gene product i s a major s t r u c t u r a l element and hence i s r e q u i r e d i n l a r g e amounts. De s p i t e t h i s , 61 p a r t i a l s u p p r e s s i o n o c c u r s i n the sup-7 h e t e r o z y g o t e , r e s u l t i n g i n an i n d i v i d u a l t h a t can move t o some e x t e n t . A n o t a b l e e x c e p t i o n has been r e p o r t e d ; l i n - 2 4 i s an amber m u t a t i o n w h i c h a f f e c t s v u l v a development and r e q u i r e s two c o p i e s of sup-7 t o show any s i g n s o f s u p p r e s s i o n (Ferguson and H o r v i t z , 1985). However, l i n - 2 4 has s e v e r a l u n u s u a l t r a i t s w hich may e x p l a i n t h i s r e s u l t . Only worms h e t e r o z y g o u s f o r l i n - 2 4 e x h i b i t a mutant phenotype, i m p l y i n g t h a t the phenotype i s due t o an i n t e r a c t i o n o f t h e w i l d - t y p e and mutant gene p r o d u c t s . A s i n g l e copy o f sup-7 p r o v i d e s f o r about a 15% i n c r e a s e i n t h e w i l d - t y p e amount of gene p r o d u c t ( W a t e r s t o n , 1981). I n t h e case of l i n - 2 4 t h i s a p p a r e n t l y s t i l l l e a v e s t o o much of the mutant p r o t e i n t o r e s u l t i n any improvement i n t h e phenotype. Only when t h e amount of a b n o r m a l l y f u n c t i o n i n g gene p r o d u c t i s d r a s t i c a l l y r e d u c e d ( i e . homozygous f o r sup-7 and h e t e r o z y g o u s f o r l i n - 2 4 ) i s s u p p r e s s i o n o f t h e mutant phenotype e v i d e n t . There i s , however, no r e a s o n t o b e l i e v e t h a t t h e above a p p l i e s t o r e c - 1 . The Rec-1 s t r a i n s e g r e g a t e s as an autosomal r e c e s s i v e m u t a t i o n and does not e x h i b i t any u n u s u a l f e a t u r e s (eg. semidominance e t c . ) (Rose and B a i l l i e , 1979a). T h e r e f o r e , g i v e n t h e above and t h e r e s u l t of t h i s e x p e r i m e n t ( F i g u r e 1 5 ) , I c o n c l u d e t h a t rec-1 i s not an amber mutant. 62 VI. The E f f e c t of rec-1 on Tel M o b i l i t y I n t r o d u c t i o n : Transposable elements are mobile DNA sequences whose p o s i t i o n i n the genome can be observed to change over r e l a t i v e l y s h o r t p e r i o d s of time ( f o r reviews see: K l e c k n e r , 1981,-pr o k a r y o t e s ; Cameron e t a l . , 1979, - Yeast; S p r a d l i n g and Rubin, 1981,- D r o s o p h i l a ) . Tc1 i s a t r a n s p o s a b l e element i n C. elegans. I t was d i s c o v e r e d as the cause of r e s t r i c t i o n fragment l e n g t h d i f f e r e n c e s (RFLDs) between the Bergerac v a r i e t y (BO) and the B r i s t o l (N2) s t r a i n (Emmons et a l . , 1979; Emmons et a l . , 1983). A 1.6 kb DNA sequence ( i e . Tc1) has a p p a r e n t l y undergone e x t e n s i v e t r a n s p o s i t i o n i n the BO s t r a i n . Tc1 has been sequenced and the s t r u c t u r e i s s i m i l a r to t r a n s p o s a b l e elements i n other s p e c i e s : i t c o n t a i n s t e r m i n a l r e p e a t s , t a r g e t s i t e d u p l i c a t i o n s and open r e a d i n g frames (Rosenweig et a l . , 1983). A l l proposed mechanisms of t r a n s p o s i t i o n i n v o l v e recombination i n one form or another (Kleckner, 1981). For example, the a n a l y s i s of mutants of IS50 sequences t h a t f l a n k Tn5 i n d i c a t e s t h a t a p o l y p e p t i d e r e q u i r e d f o r t r a n s p o s i t i o n i s a l s o necessary f o r recombination between IS50 sequences (Zupancic et a l . , 1983). The mechanism bears a c l o s e r resemblence to homologous recombination r a t h e r than s i t e s p e c i f i c recombination. The authors suggest t h a t the 63 IS50 encoded p o l y p e p t i d e f u n c t i o n s as both a r e s o l v a s e ( r e q u i r e d i n the t r a n s p o s i t i o n process) and a recombinase. The rec-1 mutation i n C. elegans i s a g e n e r a l enhancer of m e i o t i c recombination (Rose and B a i l l i e , 1979a; Rose, 1980; and see chapter I I I ) . The molecular b a s i s of t h i s e f f e c t i s not known; both homologous and/or nonhomologous recombination may be i n v o l v e d . Given t h a t rec-1 i n c r e a s e s recombination frequency d r a m a t i c a l l y , the q u e s t i o n addressed i n t h i s work was whether the Rec-1 s t r a i n showed any s i g n s of i n c r e a s e d t r a n s p o s i t i o n by Tc1 . The approach taken to answer t h i s q u e s t i o n i n v o l v e d examining the e l e c t r o p h o r e t i c banding p a t t e r n s produced by DNA r e s t r i c t i o n fragments probed with Tc1. Each band r e p r e s e n t s a d i f f e r e n t i n s e r t i o n s i t e by Tc1 i n the C. elegans genome. Tc1 m o b i l i t y i n N2 i s very low. L i a o et a l . (1983) found no d i f f e r e n c e s amoung the T c 1 - h y b r i d i z i n g r e s t r i c t i o n fragment banding p a t t e r n s of l a b o r a t o r y stocks of N2 t h a t had been separated f o r over 500 g e n e r a t i o n s (roughly e i g h t y e a r s ) . The Rec-1 and N2 s t r a i n s have been separated f o r i n excess of s i x y e a r s . The rec-1 mutation was i s o l a t e d from a heterozygote of two N2 d e r i v e d s t r a i n s : BC187 (dpy-5 (e61) unc-15 (e73) ) and CB51 (unc-13 (e51 ) ). I f rec-1 s i g n i f i c a n t l y i n c r e a s e s the m o b i l i t y of Tc1, then changes i n i n s e r t i o n s i t e s and hence the banding p a t t e r n should be observed. Such an a l t e r e d banding p a t t e r n was found. However, a l t e r n a t i v e i n t e r p r e t a t i o n s of t h i s r e s u l t are c o n s i d e r e d . S e v e r a l experiments are suggested f o r f u t u r e i n v e s t i g a t i o n . 64 M a t e r i a l s and Methods: 1) DNA P r e p a r a t i o n - C. elegans DNA samples were prepared by methods m o d i f i e d from Emmons et a l . (1979) ( B a i l l i e and Curran, p e r s . comm.). Worms were washed o f f of approximately 12 l a r g e c u l t u r e p l a t e s with 0.09 M NaCl and then c e n t r i f u g e d t o form a p e l l e t . The p e l l e t was resuspended i n 15 ml of I x P r o t e i n a s e K b u f f e r (1% SDS, 0.05 M EDTA, 0.1 M T r i s b u f f e r (pH 8.5), and 0.2 M NaCl); 100 micrograms/ml of p r o t e i n a s e K (EM Bi o c h e m i c a l s ) were added and the s o l u t i o n was incubated at 65 C f o r 30 minutes. The s o l u t i o n was then e x t r a c t e d t h r e e times w i t h equal volumes of w a t e r - s a t u r a t e d phenol, r e c o v e r i n g the aqueous phase each time. A f i n a l e x t r a c t i o n was performed u s i n g an equal volume of chloroform/isoamyl a l c o h o l (24:1 by v o l . ) . C o l d 95% ethanol was added and allowed to stand a t l e a s t one hour a t -20 C. The s o l u t i o n was c e n t r i f u g e d at 10,000 rpm f o r t en minutes. The r e s u l t a n t p e l l e t was a i r - d r i e d . The DNA was then resuspended i n one ml. of 1xTE (10 mM T r i s , 1 mM EDTA, pH 7.4). One drop of chloroform was added to prevent b a c t e r i a l contamination. Rec-1 DNA prepared i n 1983 and N2 DNA prepared i n 1985 were k i n d l y p r ovided by N. Mawji. 2) DNA H y b r i d i z a t i o n - Eco R l - r e s t r i c t e d genomic DNA d i g e s t s were separated by s i z e u s i n g h o r i z o n t a l 0.7% agarose e l e c t r o p h o r e t i c g e l s . The DNA was t r a n s f e r r e d t o n i t r o c e l l u l o s e f i l t e r s ( u n i d i r e c t i o n a l t r a n s f e r : Smith and Summers, 1980) and 32 h y b r i d i z e d with a P l a b e l l e d Tc1 probe p r o v i d e d by L-. H a r r i s . The Tc1 probe c o n s i s t e d of the c e n t r a l 1540 base p a i r s 65 of the Tc1 element; no f l a n k i n g sequence was pr e s e n t . The fragment used as the probe was cut out of the pUC-19 d e r i v e d plasmid (pCeh14) u s i n g Eco RV and i s o l a t e d by agarose g e l e l e c t r o p h o r e s i s and e l e c t r o e l u t i o n (prepared by L. H a r r i s ) . F i l t e r s were h y b r i d i z e d o v e r n i g h t a t 62 9C i n 5xSSPE, 0.3% SDS and then washed i n 2xSSPE, 0.2% SDS f o r a t l e a s t one hour a t 62 aC. They were then autoradiographed f o r three days u s i n g Kodak XRP-1 f i l m . R e s u l t s : Genomic b l o t h y b r i d i z a t i o n s were performed to determine i f rec-1 has any e f f e c t on Tc1 m o b i l i t y . T h i s was accomplished by examining the T c 1 - h y b r i d i z i n g Eco RI banding p a t t e r n i n the Rec-1 s t r a i n and comparing i t to t h a t of N2. Examination of N2 and Rec-1 DNA u s i n g Tc1 as a probe r e v e a l e d t h a t the Rec-1 DNA had an a l t e r e d p a t t e r n of Tc1 i n s e r t i o n s ( F i g u r e 13). At l e a s t f i v e new bands (lower arrows i n F i g u r e 13) were prese n t and one band (top arrow) was m i s s i n g i n the Rec-1 DNA. T h i s estimate i n c l u d e s one band which was present i n both Rec-1 and N2, but had double the i n t e n s i t y of h y b r i d i z a t i o n i n Rec-1 (center arrow). F i g u r e 13 a l s o shows a comparison of two samples of Rec-1 DNA: one was prepared i n 1983 while the other was prepared i n August, 1985. The two p r e p a r a t i o n s are separated by at l e a s t two hundred g e n e r a t i o n s . No d i f f e r e n c e s i n the T c 1 - h y b r i d i z a t i o n p a t t e r n s were v i s i b l e . 66 1 2 3 FIGURE 13: T c l - h y b r i d i z e d r e s t r i c t i o n fragment p a t t e r n s f r o m DNA samples o f t h e 1) Rec-1 ( 1 9 8 3 ) , 2) N2 and 3) Rec-1 (1985) s t r a i n s . Lambda s i z e m a r k e r s a r e shown on t h e r i g h t , w h i l e on t h e l e f t , a r r o w s i n d i c a t e t h e band d i f f e r e n c e s between t h e N2 and Rec-1 s t r a i n s . 67 D i s c u s s i o n : The N2 s t r a i n i n C. elegans has approximately 30 c o p i e s of Tc1 whereas BO has over 300 ( L i a o et a l . , 1983; Emmons et a l . , 1983). T h i s l a r g e d i f f e r e n c e has been accounted f o r by p o s t u l a t i n g e x t e n s i v e t r a n s p o s i t i o n i n BO and l i m i t e d Tc1 m o b i l i t y i n N2. T h i s s u g g e s t i o n of an a c t i v e and r e l a t i v e l y i n a c t i v e s t r a i n has r e c e i v e d support from a v a r i e t y of s o u r c e s : M o l e c u l a r i n v e s t i g a t i o n s have shown t h a t Tc1 r e a d i l y e x c i s e s i n BO and' t h a t t h i s e x c i s i o n i s p r i n c i p a l l y somatic (Emmons and Yesner, 1984). Tc1 somatic e x c i s i o n a l s o occurs i n N2, but a t a much lower r a t e than BO ( H a r r i s , p e r s . comm.). Extrachromosomal cop i e s of Tc1 have been i s o l a t e d i n BO, but not i n N2 (Rose and Snutch, 1984; Ruan and Emmons, 1984). F i n a l l y , no N2 l a b o r a t o r y stock d i f f e r e n c e s i n T c 1 - h y b r i d i z i n g r e s t r i c t i o n fragment banding p a t t e r n have been found, d e s p i t e the s e p a r a t i o n of these stocks f o r roughly 500 g e n e r a t i o n s ( L i a o e t a l . , 1983). Genetic s t u d i e s support the above r e s u l t . E i d e and Anderson (1985a) examined 65 independent spontaneous mutations i n N2 a t the unc-54 gene and found t h a t none were due to Tc1 t r a n s p o s i t i o n . T h i s c o n t r a s t s with the BO s t r a i n i n which 10 out of 18 spontaneous Unc-54 mutants arose from Tc1 i n s e r t i o n s (Eide and Anderson, 1985b). From the same work, the mutation r a t e i n BO was estimated to be a t l e a s t three times g r e a t e r than t h a t i n N2. A l l evidence to date argues f o r the s t a b i l i t y of Tc1 i n the N2 s t r a i n . Moreover, independently i s o l a t e d C. elegans s t r a i n s from g e o g r a p h i c a l l y d i s t a n t r e g i o n s have been shown to have 68 few, i f any, d i f f e r e n c e s i n Tc1 p a t t e r n s ( L i a o e t a l . , 1983). Thus i t appears t h a t Tc1 has been s t a b l e i n the genome of most s t r a i n s s i n c e t h e i r s e p a r a t i o n , p o t e n t i a l l y hundreds of years ago. Examination of N2 and Rec-1 DNA u s i n g Tc1 as a probe r e v e a l e d a number of banding d i f f e r e n c e s ( F i g u r e 1 3 ) . As expected, the N2 p a t t e r n observed was i d e n t i c a l t o p r e v i o u s l y r e p o r t e d r e s u l t s . T h e r e f o r e , the d i f f e r e n c e s between the N2 and Rec-1 s t r a i n s were not due to Tc1 a c t i v i t y i n N2. Any changes i n banding p a t t e r n must have o c c u r r e d i n Rec-1 or i t s p r o g e n i t o r s t r a i n . F i v e band a d d i t i o n s and one l o s s were found i n the Rec-1 banding p a t t e r n . T h i s r e s u l t suggested t h a t rec-1 was c a u s i n g a s i g n i f i c a n t i n c r e a s e i n Tc1 m o b i l i t y . To t e s t t h i s p o s s i b i l i t y I compared the banding p a t t e r n of Rec-1 DNA prepared two years ago (1983) with DNA prepared i n August, 1985. I f rec-1 was a c t i v a t i n g Tc1 m o b i l i t y , d i f f e r e n c e s i n the T c 1 - h y b r i d i z e d banding p a t t e r n would be expected between these p r e p a r a t i o n s . T h i s was not the case as the banding p a t t e r n s were i d e n t i c a l , r u l i n g out rec-1 as an a c t i v a t o r of Tc1 m o b i l i t y ( F i g u r e 13). A l t e r n a t i v e l y , the T c 1 - h y b r i d i z e d band a d d i t i o n and l o s s may be due to i n c r e a s e d gene c o n v e r s i o n o c c u r r i n g i n the Rec-1 s t r a i n . A Tc1 v a r i a n t e x i s t s with an Eco RI cut s i t e i n the transposon. The cut s i t e has y e t to be p r e c i s e l y l o c a t e d , however, the most l i k e l y l o c a t i o n where a s i n g l e base p a i r change c o u l d r e s u l t i n an Eco RI cut s i t e i s approximately 1,400 base p a i r s i n t o the element ( H a r r i s , pers. comm.). Increased gene c o n v e r s i o n c o u l d r e s u l t i n an i n c r e a s e i n the number of 69 c o p i e s of the Eco RI v a r i a n t . As Eco RI d i g e s t s were used i n t h i s study, both band a d d i t i o n and band l o s s would be expected due to the above; what appeared as a s i n g l e , l a r g e T c 1 - h y b r i d i z i n g band would now show up as two s m a l l e r bands. However, t h i s e x p l a n a t i o n r e q u i r e s t h a t the i n c r e a s e i n gene c o n v e r s i o n d i s p r o p o r t i o n a t e l y favours the c o r r e c t i o n of heteroduplex DNA to the Eco R1 v a r i a n t . Moreover, to account f o r the absence of T c 1 - h y b r i d i z e d banding d i f f e r e n c e s between the new and o l d Rec-1 DNA p r e p a r a t i o n s , one would have to assume t h a t the gene c o n v e r s i o n r a t e s between the d i f f e r e n t Tc1 v a r i a n t s have reached a new e q u i l i b r i u m . Although p o s s i b l e , t h i s seems u n l i k e l y . A t h i r d e x p l a n a t i o n , which i s c o n s i s t e n t with the r e s u l t s i n F i g u r e 13, i s t h a t the d i f f e r e n t Tc1 i n s e r t i o n s i t e s found i n Rec-1 are due to c e r t a i n c o n d i t i o n s t h a t produced a s h o r t b u r s t of Tc1 a c t i v i t y . T h i s event c o u l d have o c c u r r e d i n the Rec-1 s t r a i n or one of i t s p r o g e n i t o r s t r a i n s . I f so, attempts to reproduce these c o n d i t i o n s may p r o v i d e some i n s i g h t i n t o the c o n t r o l of Tc1 m o b i l i t y . M cClintock (1978) suggested t h a t environmental s t r e s s might induce t r a n s p o s i t i o n . Experimental evidence i n support of t h i s p r o p o s a l i n c l u d e s the d i s c o v e r y of a heat shock promoter 5' to the open r e a d i n g frame i n the D r o s o p h i l a c o p i a element (Strand and McDonald, 1985). Moreover, i n D i c t y o s t e l i u m discoideum, the RNA t r a n s c r i p t s of a 2.5 kb repeated DNA sequence l o c a t e d i n a transposon i n c r e a s e d r a m a t i c a l l y as a r e s u l t of heat shocking the c e l l s (Zuker et a l . , 1983). S e v e r a l p u t a t i v e heat shock promoters have been found i n the 5' r e g i o n of Tc1 i n C. elegans (Rose and Snutch, per s . comm.). 70 Two experiments can be suggested f o r f u t u r e i n v e s t i g a t i o n : 1) the b u r s t of Tc1 movement may have taken p l a c e much e a r l i e r , b e f o r e the i s o l a t i o n of Rec-1. Stocks of the p r o g e n i t o r s t r a i n s are a v a i l a b l e , having been s t o r e d i n l i q u i d n i t r o g e n . Hence, the T c 1 - h y b r i d i z i n g r e s t r i c t i o n fragment banding p a t t e r n s of these s t r a i n s c o u l d be compared t o those of N2 and Rec-1; and 2) the Rec-1 phenotype, i t s e l f , may be a consequence of the changes i n i n s e r t i o n s i t e s r a t h e r than a cause. One of the new T c 1 - h y b r i d i z i n g bands i n the Rec-1 DNA may r e p r e s e n t the i n s e r t i o n of a copy of Tc1 i n t o the rec-1 gene, i m p l y i n g t h a t the b u r s t of Tc1 m o b i l i t y o c c u r r e d c o n c u r r e n t l y w i t h the appearance of the Rec-1 phenotype. T h i s c o u l d be t e s t e d by determ i n i n g i f any of the new bands segregate w i t h the Rec-1 phenotype d u r i n g o u t c r o s s experiments. P o s i t i v e r e s u l t s c o u l d then l e a d t o the c l o n i n g of the rec-1 gene. The i n s e r t e d transposon c o u l d be used to i d e n t i f y the gene sequences of i n t e r e s t i n a c l o n e d DNA l i b r a r y c o n s t r u c t e d from the Rec-1 s t r a i n (Bingham et a l . , 1981). 71 V I I . D i s c u s s i o n : Overview The work presented i n t h i s study c h a r a c t e r i z e d the recombination enhancer r e c - 1 , i n C. elegans. The r e s u l t s of the v a r i o u s experiments r e p o r t e d i n chapters I I I to VI are summarized i n Table 9. The rec-1 mutant produced a two to t h r e e - f o l d i n c r e a s e i n i n t r a g e n i c c r o s s i n g over and gene c o n v e r s i o n . An attempt was made to examine l a r g e g e n e t i c i n t e r v a l s , but problems i n the s t r a i n c o n s t r u c t i o n prevented t h i s p r o j e c t from being completed (see chapter I I I ) . The e f f e c t s of rec-1 on other m e i o t i c processes were a l s o s t u d i e d . Mutation r a t e , f e c u n d i t y and competitve a b i l i t y showed no s u b s t a n t i a l a f f e c t by r e c - 1 ; however, X-chromosome n o n d i s j u n c t i o n and/or chromosome l o s s appeared to be i n c r e a s e d by a f a c t o r of two. I f r e a l , t h i s i n c r e a s e concomitant with an i n c r e a s e i n recombination frequency i s very unusual. For example, Baker and H a l l (1976) surveyed m e i o t i c mutants i n D r o s o p h i l a and found t h a t the amount of n o n d i s j u n c t i o n was d i r e c t l y p r o p o r t i o n a l to the extent of the decrease i n recombination (see chapter I V ) . A s t r a i n was c o n s t r u c t e d to determine i f the rec-1 enhancer was due to the presence of a premature stop codon, p r e v e n t i n g t r a n s l a t i o n . The hyper-rec mutant i n E. c o l i , uvrD(210), has been shown to be a consequence of an amber, nonsense mutation (Arthur and L l o y d , 1980). The experiment performed i n chapter V showed t h a t the e x p r e s s i o n of rec-1 was not a f f e c t e d by a tRNA amber suppressor ( i e . sup-7), i n d i c a t i n g t h a t rec-1 i s not an amber mutation. TABLE 9: Summary of r e s u l t s c h a r a c t e r i z i n g the rec-1 enhancer Character E f f e c t Source I. Recombination: I I . F e c u n d i t y : I I I . Competitive A b i l i t y : IV. Mutation Rate: V. R a d i a t i o n S e n s i t i v i t y : VI. N o n d i s j u n c t i o n : V I I . Amber Suppression: V I I I . Tc1 m o b i l i t y : i n c r e a s e s gene c o n v e r s i o n , chapter I I I ; i n t r a - and i n t e r g e n i c Rose and B a i l l i e , exchange 3 t o 4 - f o l d no e f f e c t no e f f e c t no e f f e c t no e f f e c t 2 - f o l d i n c r e a s e no e f f e c t 1 979a chapter IV. chapter IV. chapter IV. Hartman and Herman, 1 982 chapter IV. Rose, 1980 a l t e r e d T c 1 - h y b r i d i z e d r e s t r i c t i o n fragment banding p a t t e r n i n N2 vs Rec-1; i n t e r p r e t a t i o n i s u n c l e a r chapter V. chapter VI. 73 F i n a l l y , the e f f e c t of rec-1 as a recombination enhancer on the m o b i l i t y of the t r a n s p o s a b l e element Tc1 was examined (see chapter V I ) . E l e c t r o p h o r e t i c banding p a t t e r n s produced by DNA r e s t r i c t i o n fragments probed with Tc1 were compared f o r the N2 and Rec-1 s t r a i n s . An a l t e r e d p a t t e r n was found, suggesting t h a t rec-1 a c t i v a t e s Tc1 m o b i l i t y . However, p r e d i c t e d p a t t e r n d i f f e r e n c e s were not found between two Rec-1 DNA samples (one prepared two years a f t e r the o t h e r ) . S e v e r a l a l t e r n a t i v e e x p l a n a t i o n s were c o n s i d e r e d (chapter V I ) . A d i s t i n c t i o n can be made between f i n e and coarse c o n t r o l s of recombination (Simchen and Stamberg, 1969). Genes of coarse c o n t r o l presumably a f f e c t the e s s e n t i a l , s e q u e n t i a l steps necessary f o r m e i o s i s (eg. s y n a p s i s , breakage, r e p a i r , s e g r e g a t i o n e t c . ) and hence have a g e n e r a l and u s u a l l y extreme e f f e c t on recombination. Examples are found i n many g e n e t i c a l l y w e l l c h a r a c t e r i z e d s p e c i e s (eg. E. c o l i : recA - W i l l e t s e t a l . , 1969; D r o s o p h i l a ; mei-9 - Baker and Carpenter, 1972; c(3)G -Gowen and Gowen, 1922; Yeast: spo-8, spo-11 - E s p o s i t o and E s p o s i t o , 1969; K l a p h o l z e t a l . , 1985; Neurospora: mei-1 -Smith, 1975; Zea mays: a m e i o t i c - Palmer, 1971; B r a s s i c a  campestris: as3 - Stringham, 1970). Genes of f i n e c o n t r o l have a more l i m i t e d and l o c a l e f f e c t on recombination. R e g u l a t i o n of exchange i n s p e c i f i c r e g i o n s of the genome i s a p p a r e n t l y achieved by c o n t r o l l i n g access to r e c o g n i t i o n s i t e s which i d e n t i f y the l o c a t i o n t o the recombinase enzymes (Smichen and Stamberg, 1969; Stamberg and K o l t i n , 1973). Li n d s e y and Sandler (1977) have extended t h i s analogy by proposing t h a t r e g i o n a l r e g u l a t i o n of exchange i n v o l v e s two components: 1) a c o n t r o l l i n g system, which s p e c i f i e s the d i s t r i b u t i o n of exchange; and 2) a responding system, which i s rec o g n i z e d by the c o n t r o l l i n g elements. Examples i n c l u d e cog ( i n t e r a c t i n g with rec-2) i n Neurospora, mei-1 i n D r o s o p h i l a and e l i n Zea mays (Angel e t a l . , 1970; V a l e n t i n , 1973; N e l , 1975). C o n s i d e r i n g hyper-rec mutants as a s u b c l a s s of the above, the same c l a s s i f i c a t i o n of l o c a l and g l o b a l e f f e c t s can be a p p l i e d . Recombination hotspots are DNA sequences t h a t s t i m u l a t e recombination i n t h e i r immediate v i c i n i t y ( f o r a review see Smith, 1983). They have been found i n both p r o k a r y o t e s and eukaryotes; the known mutants are summarized i n Table 10. Chi s i t e s i n E. c o l i s t i m u l a t e recombination by the recA-recBC-dependent pathway (McMilin et a l . , 1974). The maximum e f f e c t on exchange occurs near the Chi s i t e , but extends a t a reduced l e v e l up t o 10 kb away ( S t a h l e t a l . , 1980). In eukaryotes the HOT1 mutation i n ye a s t , the cog mutation i n Neurospora c r a s s a , the ade6 mutation i n Schizosaccharomyces  pombe and the b u f f (YS17) mutation i n S o r d a r i a b r e v i c o l l i s a l l s t i m u l a t e m e i o t i c recombination i n adjacent sequences ( K e i l and Roeder, 1984; Angel et a l . , 1970; Gutz, 1971; and MacDonald and Whitehouse, 1979). A l l of the above are presumed to i n v o l v e changes i n DNA sequences t h a t c r e a t e r e c o g n i t i o n s i t e s f o r recombinase enzymes. D e t e c t i o n of such a s i t e r e s u l t s i n the i n i t i a t i o n or r e s o l u t i o n of a H o l l i d a y j u n c t i o n . A number of l o c a l , hyper-rec mutants have a l e s s r e g u l a r e f f e c t on recombination and are not i n c l u d e d i n Table 10. These mutants produce a l o c a l i n c r e a s e over perhaps one or two i n t e r v a l s , but decrease the recombination frequency elsewhere. TABLE 10: A review of l o c a l recombination enhancers Extent of Increase Species/gene M i t o t i c M e i o t i c Reference E. c o l i : Chi s i t e s S. c e r e v i s i a e : Hot 1 S. pombe: ade 6 (M26) N. c r a s s a : rec-1  rec-2/cog rec-3/con S_. b r e v i c o l l u s : b u f f (YS17) 1 5 - f o l d i n c r e a s e 7 - f o l d * * * * none Smith, 1983 K e i l and Roeder, 1984 2 0 - f o l d i n t r a g e n i c only; 10 to 2 5 - f o l d i n t r a g e n i c ; 1 0 - f o l d i n t r a - and i n t e r g e n i c a l l y ; 10 to 2 5 - f o l d i n t r a -and i n t e r g e n i c a l l y 1 0 - f o l d i n t r a g e n i c only; Gutz, 1971 Catcheside, 1964 Smith, 1966 Angel et a l . , 1970 Catcheside, 1966 MacDonald and Whitehouse, 1979 * I n d i c a t e s t h a t the e f f e c t s on m i t o t i c recombination were not r e p o r t e d . No p l e i o t r o p i c e f f e c t s were r e p o r t e d f o r any of the l o c a l enhancers. Parry (1973) and Carpenter and Sandler (1974) have shown t h a t mei-S282 and mei-218 i n D r o s o p h i l a produce i n c r e a s e s i n recombination i n the centromeric r e g i o n s of both the X and second chromosomes, a p p a r e n t l y compensating f o r the g e n e r a l but nonuniform decrease caused by both mutations. The c h l mutant i n y e a s t i n c r e a s e s m i t o t i c exchange, but decreases m e i o t i c recombination on chromosome three (Haber, 1974). In h i g h e r p l a n t s , m e i o t i c d e f e c t s i n synapsis are t y p i c a l l y b e l i e v e d t o be i n v o l v e d (Baker et a l . , 1976). Moens (1969) r e p o r t e d a 2 and 2 . 5 - f o l d l o c a l i n c r e a s e i n as(4) and as(b) homozygotes i n Lycopersicum esculentum a s s o c i a t e d with a g e n e r a l decrease i n chiasma frequency and an i n c r e a s e d p r o p o r t i o n of u n i v a l e n t s a t metaphase I. The e l mutation i n Zea mays causes a two f o l d i n c r e a s e i n recombination on chromosome 5, but decreases recombination to two t h i r d s the w i l d - t y p e v a l u e on chromosome 9 (Nel, 1975). F i n a l l y , the e x i s t e n c e of l o c a l m o d i f i e r s of recombination have been demonstrated through s e l e c t i o n experiments and i n t e r s t r a i n c r o s s e s (Brooks, 1984; C h i n n i c c i , 1971a,b; Simchin et a l . , 1971). General enhancers of recombination do not form as c o h e s i v e a group. T h e i r p l e i o t r o p i c e f f e c t s are more e x t e n s i v e and v a r i a b l e , s u g g e s t i n g t h a t no s i n g l e h y p o t h e s i s can account f o r t h e i r e f f e c t on recombination (Table 11). Based on Simchen and Stambergs' p r o p o s a l , i t appears l i k e l y t h a t these mutants a f f e c t d i f f e r e n t stages i n the s e q u e n t i a l c o n t r o l of m e i o s i s . Sandler et a l . (1968) p r o v i d e d a schematic r e p r e s e n t a t i o n of the c o n t r o l of chromosome behavior i n D r o s o p h i l a . They suggested t h a t "landmark" events ( i e . processes with g e n e t i c a l l y d e t e c t a b l e consequences) can be used to determine the mode and time of 77 TABLE 1 1; A review of general enhancers of recombination Extent of Increase Soecies/cene M i t o t i c Meiotic P l e i o t r o o v Reference E. c o l i : uvr D 12-fold hyper-mutable Arthur and Llo y d , rad. s e n s i t i v e 1980. dam 3 to 17 f o l d hyper-mutable Marinus and Konrad rad. s e n s i t i v e 1976. l i e hyper-rec hyper-mutable Konrad et al.,1972 (extent not reported) t . s . l e t h a l dut hyper-rec hyper-mutable Tye e t a l . , 1977. pol A hyper-rec t . s . l e t h a l Konrad and Lehman, 1974. Saccharomvces ; rem-1 TO-fold i n t r a - none hyper-mutable G o l i n and E s n c s i t c and i n t e r g e n i c rad. s e n s i t i v e 1977." U s t i l l a g c : uvs-1 A s p e r g i l l u s : uvs-3 Neurosoora: uvs-3 D r o s o c h i l a : Ir.-er-chrcmcsomaJ e f f e c t 1 0 - f o l d i n t r a - none and i n t e r g e n i c 1-0-fold i n t r a - none and i n t e r g e n i c 2 - f o l d i n t r a - none and i n t e r g e n i c U.V. s e n s i t i v e H c l l i d a y et a l . , reduced f e c u n d i t v 1976. reduced f e c u n d i t y S h a n f i e l d and Kafer, 1.969 . reduced f e c u n d i t y Schroeder, 1970, Both mi t o s i s and meiosis; reduced 2 to 3 - f o l d ; nonuniform; f e c u n d i t y i n t e r g e n i c oniv. Sturtevant, 191 Lu c c h e s i , 1976. c ( 3 ) G C. elecans: rec-1 Z. mays: as 2 to 3-fol d ; low f e c u n d i t y Hinton, V962 ; i n t r a and i n t e r g e n i c ; Watson, 1972. nonuniform 3 to 4-fold; increases Rose and B a i l l i e , i n t r a and i n t e r g e n i c ; n o n d i s j u n c t i o n 1979a; general and uniform; Th i s work. 1 to 10-fold; reduced , , = i i - w , f e c u n d i t y v a r i a b l e penetrance M i l l e r , 1963 i n t e r g e n c ; i n d i c a t e s that the e f f e c t s on m i t o t i c recombination were not reported. 78 a c t i o n of m e i o t i c mutants. The p l e i o t r o p i c e f f e c t s of g e n e r a l , hyper-rec mutants may r e p r e s e n t such landmarks. The E. c o l i mutants are a l l hyper-mutable and are radiation/mutagen s e n s i t i v e , i m p l i c a t i n g d e f e c t s i n t h e i r r e p a i r systems (Table 12; Glickman and Radman, 1980; P u k k i l a e t a l . , 1983). S i m i l a r i l y , the m i t o t i c enhancers, rem-1 i n y e ast and uvs-1 i n U s t i l a g o maydis are mutagen s e n s i t i v e , have reduced f e r t i l i t y and are hyper-mutable (rem-1 only) (Malone and Hoekstra, 1984; H o l l i d a y e t a l . , 1976). Double mutants of rem-1 and rad-6 are v i a b l e , however, rem-1, rad-50 i n d i v i d u a l s are not. A l l of the above suggests t h a t rem-1 induces DNA l e s i o n s of a k i n d t y p i c a l l y c o r r e c t e d by the Rad-50 r e p a i r pathway (Malone and Hoekstra, 1984). The induced DNA breaks are presumed to be r e p a i r e d by a r e c o m b i n a t i o n - r e p a i r mechanism. The p l e i o t r o p i c e f f e c t s a s s o c i a t e d with the f o u r m e i o t i c mutants are more d i f f i c u l t to i n t e r p r e t . The interchromosomal e f f e c t i n D r o s o p h i l a i s t y p i c a l l y found i n f l i e s heterozygous f o r a chromosomal rearrangement (eg. i n v e r s i o n , d u p l i c a t i o n e t c . ) . However, V a l e n t i n (1972) and Suzuki (1963) have r e p o r t e d e x c e p t i o n a l i n v e r s i o n s which enhance recombination even when homozygous. The interchromosomal e f f e c t i n c r e a s e s both m i t o t i c and m e i o t i c , i n t e r g e n i c recombination two to t h r e e - f o l d (Ronen, 1964; and see L u c c h e s i , 1976 f o r a r e v i e w ) . However, i n t r a g e n i c recombination appears to be only s l i g h t l y a f f e c t e d ( C a r l s o n , 1972). The p a t t e r n of i n c r e a s e i s nonuniform, with the g r e a t e s t e f f e c t s o c c u r r i n g d i s t a l l y on the X-chromosome and both d i s t a l l y and c e n t r a l l y on the autosomes. Exchange i n heterochromatic r e g i o n s i s not a f f e c t e d . P r o c u n i e r and Suzuki (1967) noted t h a t the interchromosomal e f f e c t decreases i n t e r f e r e n c e ( i e . the r a t i o of m u l t i p l e to s i n g l e c r o s s - o v e r t e t r a d s i n c r e a s e s ) . T h i s suggests t h a t the interchromosomal e f f e c t a l t e r s the p r e c o n d i t i o n s f o r recombination r a t h e r than the p r o b a b i l i t y per se of an exchange t a k i n g p l a c e , as the l a t t e r does not a f f e c t i n t e r f e r e n c e (Carpenter and Sandler, 1974; but see G r e l l , 1978, 1984; she c h a l l e n g e s the assumption t h a t the p r o b a b i l i t y of exchange i s constant throughout the genome). F i n a l l y , Harger and Holm (1980) have shown t h a t when recombination i s i n c r e a s e d by an i n v e r s i o n on a compound autosome, n o n d i s j u n c t i o n of the X-chromosome i s reduced r e l a t i v e t o the c o n t r o l ( i e . a compound autosome without the i n v e r s i o n ) . T h i s i m p l i e s t h a t n o n d i s j u n c t i o n i s g e n e r a l l y decreased due to the enhancement of recombination by the interchromosomal e f f e c t , c o n s i s t e n t with the o b s e r v a t i o n s of Baker and H a l l (1976). Two models have been suggested to e x p l a i n the e f f e c t of s t r u c t u r a l rearrangements on recombination. Mather (1936) and Roberts (1969) proposed t h a t b i v a l e n t s compete f o r some l i m i t e d m a t e r i a l which i s r e q u i r e d f o r exchange (eg. a recombinase). S t r u c t u r a l h e t e r o z y g o s i t y t h a t reduces the amount of c r o s s i n g over i n one p a r t i c u l a r r e g i o n of the genome would leave a l a r g e r amount of the r e q u i r e d enzyme f o r exchange i n other competing i n t e r v a l s . As an a l t e r n a t i v e , L u c c h e s i and Suzuki (1968) have developed a time-delay model i n which the time r e q u i r e d f o r s y n a p s i s i s i n c r e a s e d due to the p h y s i c a l and s p a t i a l c o n s t r a i n t s imposed by the rearrangement. They p o s t u l a t e t h a t the i n c r e a s e d time spent i n s y n a p s i s may a l s o i n c r e a s e the 80 p r o b a b i l i t y of exchange. T e s t i n g these hypotheses, as w e l l as i n v e s t i g a t i n g other p o s s i b i l i t i e s , has proven to be r a t h e r d i f f i c u l t . R e c e n t l y , Szauter (1984) has shown t h a t the nonuniform i n c r e a s e induced by the interchromosomal e f f e c t i s not due to a d e f e c t i n the c o n t r o l l i n g system r e g u l a t i n g r e g i o n a l exchange. The rec-1 mutant i n C. elegans d i f f e r s from the interchromosmal e f f e c t i n s e v e r a l ways. F i r s t , rec-1 i s r e c e s s i v e ; an i n c r e a s e i n recombination i s o n l y found i n the homozygote. T h i s makes i t u n l i k e l y f o r the induced i n c r e a s e to be a consequence of a s m a l l rearrangment. Secondly, u n l i k e the interchromosomal e f f e c t , rec-1 i n c r e a s e s i n t r a g e n i c recombination and a p p a r e n t l y gene c o n v e r s i o n to the same extent as i t does i n t e r g e n i c exchange. F i n a l l y , rec-1 has a uniform e f f e c t on recombination, i n c r e a s i n g the r a t e of exchange by a f a c t o r of t h r e e or f o u r . The a§_ mutant i n Zea mays i s unusual i n t h a t i t has v a r i a b l e penetrance; any g i v e n p l a n t may e x h i b i t a dramatic i n c r e a s e i n recombination frequency (eg. up to a 1 0 - f o l d i n c r e a s e ) or almost none. T h i s c o n t r a s t s with rec-1 i n C. elegans, which i s 100% p e n e t r a n t . M i l l e r (1963) has observed p a i r i n g a t pachytene i n as p l a n t s and found i r r e g u l a r i t i e s i n s y n a p s i s . However, p a i r i n g appeared normal around the centromere and d i s t a l ends of the chromosomes. These were the same r e g i o n s r e p o r t e d to have enhanced recombination f r e q u e n c i e s . M i l l e r suggested t h a t a compensatory mechanism s i m i l a r t o the interchromosomal e f f e c t i n D r o s o p h i l a may be r e s p o n s i b l e f o r the observed i n c r e a s e s and 81 p r e d i c t e d t h a t i f medial r e g i o n s were examined, a decreased recombination frequency would be found. N o n d i s j u n c t i o n and chromosome l o s s are a l s o i n c r e a s e d by the as mutant, as expected i f s y n a p s i s i s d e f e c t i v e . As a consequence f e r t i l i t y i s a l s o reduced. The mutant c(3)G i n D r o s o p h i l a i s a r e c e s s i v e c r o s s - o v e r suppressor (Gowen and Gowen, 1922). Remarkably, i n t r a - and i n t e r g e n i c recombination i n c r e a s e two to t h r e e - f o l d i n females heterozygous f o r c(3)G. The p a t t e r n of i n c r e a s e i s q u i t e s i m i l a r to t h a t of the interchromosomal e f f e c t ( C a r l s o n , 1972; Hinton, 1966). N o n d i s j u n c t i o n i s not decreased i n the het e r o z y g o t e , d e s p i t e the i n c r e a s e i n recombination frequency ( H a l l , 1972). In terms of i t s p l e i o t r o p i c e f f e c t s , c(3)G does not i n c r e a s e the spontaneous mutation r a t e ( H a l l , 1971). However, Watson (1972) has shown an i n c r e a s e i n the frequency of X-ray induced, r e c e s s i v e l e t h a l s from c(3)G f l i e s , s u g g e s t i n g some d e f e c t i n a r e p a i r p r o c e s s . The homozygote does not have a synaptonemal complex, e x h i b i t s e x t e n s i v e n o n d i s j u n c t i o n and has reduced f e c u n d i t y (Smith and King, 1968; Gowen, 1933; H a l l , 1972). Hinton (1966), found t h a t a f l y heterozygous f o r a d e f i c i e n c y c o n t a i n i n g the c(3)G l o c u s e x h i b i t e d a recombination frequency i n t e r m e d i a t e between those of mutant homozygote and w i l d - t y p e f l i e s . T h i s prompted him to suggest t h a t : i ) c(3)G i s hypomorphic f o r some s y n a p t i c process such t h a t s ynapsis i n c r e a s e s over the genotypic s e r i e s -def i c i e n c y / c ( 3 )G, c ( 3 )G/c ( 3 )G, deficiency/-!-, c ( 3 )G/+ , +/+_ and; 82 i i ) the frequency of exchange i s i n c r e a s e d by the amount of d i s t u r b a n c e encountered by a b i v a l e n t d u r i n g s y n a p s i s ( S c h u l t z and R e d f i e l d , 1951; c i t e d i n Hinton, 1966). Presumably, s y n a p s i s i n c(3)G/+ f l i e s i s a l t e r e d s l i g h t l y and t h e r e f o r e , causes an i n c r e a s e i n recombination frequency r e l a t i v e to the w i l d - t y p e s t r a i n . T h i s s y n a p t i c d i s t u r b a n c e h y p o t h e s i s has some i n t e r e s t i n g s i m i l a r i t i e s with the time-delay model of the interchromosomal e f f e c t . Based on the above examples and the r e s u l t s o b t a i n e d i n t h i s study a number of hypotheses about the nature of the rec-1 mutation and hence the f u n c t i o n of the w i l d - t y p e gene product can be proposed. In chapter IV i t was suggested t h a t rec-1 may r e s u l t i n a d e f e c t i n some r e p a i r p r o c e s s , l i k e the E. c o l i mutants. A l t e r n a t i v e l y , l i k e rem-1, i t might induce DNA l e s i o n s . In e i t h e r case, an i n c r e a s e d number of breaks i n the DNA would p r o v i d e an e l e v a t e d s u b s t r a t e supply f o r i n i t i a t i n g c r o s s - o v e r events. I t f o l l o w s from the above t h a t the spontaneous mutation r a t e would a l s o be i n c r e a s e d (as found i n both the E. c o l i and ye a s t mutants). Using the b a l a n c e r eTl and the mutation r a t e assay developed by Rosenbluth et a l . (1983), I have shown t h a t rec-1 does not induce l e t h a l mutations (Table 8: 0 l e t h a l mutations recovered from 1,871 chromosomes examined). Moreover, Hartman and Herman (1982) r e p o r t e d t h a t the Rec-1 s t r a i n i s not r a d i a t i o n s e n s i t i v e - a phenotype t h a t would have been expected i f a r e p a i r system was e i t h e r d e f e c t i v e or s t r e s s e d by an e x t r a source of DNA damage. This evidence does not c o n c l u s i v e l y prove t h a t rec-1 i s not due to a r e p a i r system r e l a t e d d i s o r d e r ( r e c a l l the example of c(3)G as d i s c u s s e d i n chapter I V ) , but i t does make i t an 83 u n l i k e l y e x p l a n a t i o n . A l t e r n a t i v e l y , the rec-1 w i l d - t y p e gene product may be a recombinase enzyme. T h i s enzyme c o u l d be i n v o l v e d i n the f o r m a t i o n of the H o l l i d a y j u n c t i o n , as i s the case w i t h recA and rec-1 p r o t e i n s i n E. c o l i and U s t i l a g o maydis, r e s p e c t i v e l y (Tsang e t a l . , 1985; Tsang e t a l . , 1985; Kmiec and Holloman, 1982, 1984). On the other hand, the p u t a t i v e recombinase c o u l d a s s i s t i n the r e s o l u t i o n of the H o l l i d a y j u n c t i o n . Symington and Kolodner (1985) have r e c e n t l y p u r i f i e d an enzyme i n y e a s t which c l e a v e s H o l l i d a y j u n c t i o n s . In e i t h e r case, an i n c r e a s e i n the amount of enzyme ( i e . a hypermorph) or an i n c r e a s e i n the e f f i c i e n c y of the enzyme c o u l d r e s u l t i n an i n c r e a s e i n recombination frequency. However, such mutations would have a dominant e f f e c t on recombination; rec-1 behaves as a r e c e s s i v e mutation (Rose and B a i l l i e , 1979a). T h i s makes i t u n l i k e l y t h a t rec-1 codes f o r such an enzyme. T h i r d l y , rec-1 may be a r e p r e s s o r i n v o l v e d i n r e g u l a t i n g m e i o t i c recombination. A precedent has been r e p o r t e d i n Neurospora c r a s s a , although the genes i n v o l v e d have only a l o c a l e f f e c t on recombination. The r e c - 1 , rec-2 and rec-3 genes r e g u l a t e exchange i n s e v e r a l s p e c i f i c r e g i o n s . Mutations a t these l o c i r e s u l t i n a dramatic i n c r e a s e i n recombination frequency i n the a s s o c i a t e d i n t e r v a l s (Catcheside et a l . , 1964; C a t c h e s i d e 1966; Smith, 1966). Two elements have been i d e n t i f i e d + t h a t modify the e x p r e s s i o n of the rec mutants: the cog f a c t o r i n c r e a s e s recombination i n the r e c mutants (Angel e t a l . , 1970; Catcheside and Angel, 1974), whereas the con element m o d i f i e s the extent of s u p p r e s s i o n of recombination by the 84 r e c a l l e l e s ( C atcheside, 1975). Angel et a l . (1970) and C a t c h e s i d e (1975) have suggested t h a t the r e c gene products b i n d with an o p e r a t o r - l i k e s i t e (the con element), thereby b l o c k i n g t r a n s l a t i o n of an adjacent "recombinase" coding sequence. T h i s would prevent or l i m i t the p r o d u c t i o n of the enzymes necessary f o r exchange. In the derepressed s t a t e ( i e . r e c ) these enzymes are a v a i l a b l e t o i n t e r a c t with the cog f a c t o r , a r e c o g n i t i o n sequence p r o v i d i n g s p e c i f i c i t y f o r the s i t e of enhanced exchange. In C. elegans, the rec-1 enhancer a f f e c t s the e n t i r e genome, not j u s t one l o c a l i t y . T h e r e f o r e , i f the w i l d - t y p e gene product of rec-1 i s a r e p r e s s o r , the enzyme under i t s c o n t r o l must be a g e n e r a l recombinase with no s p e c i f i c i t y f o r p a r t i c u l a r r e c o g n i t i o n s i t e s . T h i s hypothesis would r e q u i r e the absence of a f u n c t i o n a l gene product i n the rec-1 homozygote. A nonsense mutation i s one of s e v e r a l p o s s i b l e c l a s s e s of mutations which meet t h i s requirement. As noted e a r l i e r the amber tRNA suppressor, sup-7, does not have an a f f e c t on the Rec-1 s t r a i n . Ochre and o p a l mutations remain as p o s s i b i l i t i e s , but cannot be t e s t e d u n t i l the a p p r o p r i a t e tRNA suppressors are i s o l a t e d i n C. elegans. Other mutation events such as a s m a l l d e l e t i o n , a p o i n t mutation i n a c r i t i c a l r e g i o n of the p o l y p e p t i d e , or a f r a m e s h i f t mutation are p l a u s a b l e means of p r e v e n t i n g the mutant gene from producing a f u n c t i o n a l gene product. T h i s h y p o t h e s i s can account f o r the g e n e r a l absence of p l e i o t r o p i c e f f e c t s , as the o n l y aspect of m e i o s i s a f f e c t e d would be the p r o b a b i l i t y of exchange. However, n o n d i s j u n c t i o n 85 and/or chromosome l o s s appear to be i n c r e a s e d i n Rec-1. There i s no mechanism i n the r e p r e s s o r model t h a t c o u l d produce such an i n c r e a s e . F i n a l l y , the p o s s i b i l i t y of a synaptonemal d i s t u r b a n c e , as proposed f o r c(3)G and the interchromosomal e f f e c t must be c o n s i d e r e d . Both of these D r o s o p h i l a mutants i n c r e a s e recombination i n the h eterozygote (Hinton, 1966; L u c c h e s i , 1976; but see V a l e n t i n , 1972), whereas the e f f e c t s of rec-1 are only expressed i n the homozygote. T h e r e f o r e , i f rec-1 a f f e c t s s y n a p s i s i t i s not a consequence of a p h y s i c a l c o n s t r a i n t ( i e . a rearrangement) or some hypomorph induced d i s t u r b a n c e such as t h a t i n c(3)G. Instead, i t i s more l i k e l y to i n v o l v e some d i s c r e t e f u n c t i o n which u n i f o r m l y and c o n s i s t e n t l y i n c r e a s e s the time spent i n s y n a p s i s and hence the p r o b a b i l i t y of exchange. Th i s would suggest some r e g u l a t o r y f u n c t i o n f o r the w i l d - t y p e gene product. I t i s i n t e r e s t i n g to note t h a t the o n l y s i d e e f f e c t the rec-1 mutation appears to pocess i s a s e g r e g a t i o n a l d i s o r d e r , perhaps r e f l e c t i n g an a l t e r a t i o n i n the c o n t r o l of s y n a p s i s . G o l d s t e i n (pers. comm.) i s i n the process of examining synaptonemal complexes i n the Rec-1 s t r a i n . To d i s c r i m i n a t e i n any c o n c l u s i v e f a s h i o n between these v a r i o u s hypotheses w i l l r e q u i r e f u r t h e r c h a r a c t e r i z a t i o n of the Rec-1 s t r a i n . At the end of each chapter a number of experiments have been proposed f o r f u t u r e i n v e s t i g a t o r s . I would a s s i g n the f o l l o w i n g order or p r i o r i t y t o these p r o j e c t s . F i r s t , the rec-1 locus must be mapped. Any other experiments designed to g e n e t i c a l l y c h a r a c t e r i z e rec-1 c o u l d be accomplished more 86 e f f i c i e n t l y i f the l o c a t i o n of the recombination enhancer was known. Next, I would i s o l a t e d e f i c i e n c i e s c o v e r i n g the immediate r e g i o n about the rec-1 l o c u s . By v a r y i n g the gene dosage such d e f i c i e n c i e s would al l o w the i n v e s t i g a t o r to determine the l e v e l of p r o d u c t i o n of the gene product i n the mutant. For example, a hypomorph would e x h i b i t a more extreme phenotype as a hemizygote ( i e . d e f i c i e n c y / r e c - 1 ) than as a homozygote ( M u l l e r , 1 933). However, an amorph would not produce a f u n c t i o n a l gene product and, t h e r e f o r e , i t s mutant phenotype would be independent of gene dosage. The d e f i c i e n c i e s can a l s o be u s e f u l i n i s o l a t i n g other a l l e l e s of r e c - 1 . I t should be emphasized t h a t t h e r e i s no easy way of s c r e e n i n g f o r recombination mutants i n high e r eukaryotes, e s p e c i a l l y when the mutant i s s e l e c t e d on the b a s i s of i t s e f f e c t on recombination r a t h e r than some a s s o c i a t e d p l e i o t r o p i c e f f e c t . In ye a s t , i n which m e i o t i c s i d e e f f e c t s are o f t e n used as the b a s i s of a s c r e e n i n g procedure, one runs the r i s k of i s o l a t i n g a mutant t h a t i s f u n c t i o n a l l y i n c i d e n t a l t o the recombination process ( E s p o s i t o and E s p o s i t o , 1969). G r e l l (1984) has made use of a d e f i c i e n c y to p r e f e r e n t i a l l y recover induced recombination d e f e c t i v e mutants from a s p e c i f i c r e g i o n of the genome. Her approach r e q u i r e s only three g e n e r a t i o n s to i d e n t i f y the mutant. The i s o l a t i o n of s e v e r a l rec-1 a l l e l e s would al l o w a comparison of the extent of t h e i r e f f e c t s on recombination and other processes (eg. p l e i o t r o p i c e f f e c t s ) . The procedure i s o u t l i n e d i n F i g u r e 14. Rec-1 males homozygous f o r some v i s i b l e marker, B, are mutagenized. These 87 are mated to hermaphrodites c a r r y i n g a second marker, C, a d e f i c i e n c y (Def.) c o v e r i n g the rec-1 lo c u s and a dominant marker, A, on the homologue ( F i g u r e 14). In the F1, w i l d - t y p e males c a r r y i n g the t r e a t e d chromosome ( i e . B +/+ C; d e f i c i e n c y / r e c - 1 ) are s e l e c t e d and c r o s s e d with homozygous double mutant hermaphrodites. Any induced, r e c e s s i v e mutation i n the r e g i o n exposed by the d e f i c i e n c y (eg. the rec-1 l o c u s ) w i l l be expressed i n the hemizygous c o n d i t i o n i n the F1. T h e r e f o r e , r e v e r t a n t s and pseudorevertants of rec-1 w i l l be d e t e c t e d by s c o r i n g the recombinants i n the F2. Any s i g n i f i c a n t r e d u c t i o n i n the frequency of the recombinant phenotypes ( i e . Wild-type worms) should be r e a d i l y observed. The screen can be adapted to the i s o l a t i o n of hyper-rec a l l e l e s of rec-1 by d e c r e a s i n g the d i s t a n c e between the t r a n s markers B and C such t h a t recombinants i n the N2 s t r a i n become q u i t e r a r e (eg. 0.3 map u n i t s - most broods of about 100 progeny would not have a recombinant). A t h r e e - f o l d or g r e a t e r i n c r e a s e i n recombination frequency would make the presence of at l e a s t one Wt recombinant per brood a much more common event, and hence i d e n t i f y the recombination mutant. The o n l y c o n s t r a i n t on the above i s t h a t the marker, B, must not s i g n i f i c a n t l y reduce the mating e f f i c i e n c y of the mutagenized males. Hodgkin (1983) has r e p o r t e d on the mating e f f i c i e n c y of a number of mutants i n C. elegans. His r e s u l t s c o u l d be used to s e l e c t an a p p r o p r i a t e marker. F i n a l l y , g i v e n a set of a l l e l e s with some v a r i a t i o n i n the extent of t h e i r e f f e c t on c r o s s i n g over, long term p o p u l a t i o n experiments c o u l d be performed to i n v e s t i g a t e the e v o l u t i o n a r y 88 Po: EMS [males] r e c - 1 ; B rec-1 B X Def. _C_ C [herm.] V F i : P i c k Wt males ( r e c a l l A i s dominant) and c r o s s t o double mutant hermaphrodites. Eg. [1 male] (Wt) Def . rec-1 B X [ 1 herm.] B V F_2: Score f o r the absence of recombinants. For example, few or no Wt progeny i n the F2 i d e n t i f i e s a r e v e r t a n t or p u t a t i v e recombination d e f i c i e n t a l l e l e of r e c - 1 . F i g u r e 14: Screen f o r the i s o l a t i o n of a l l e l e s of r e c - 1 . M o d i f i e d from G r e l l (1984). The use of a d e f i c i e n c y t a r g e t s the rec-1 l o c u s f o r mutant i n d u c t i o n . 89 r o l e of recombination. Given the i n t u i t i v e need to p r e s e r v e the l i n k a g e of w e l l adapted combinations of a l l e l e s , one would expect recombination to be s e l e c t e d a g a i n s t (Nei, 1967; Turner, 1967; E s h e l , 1972). A number of models have attempted to account f o r the p e r s i s t e n c e of recombination. These hypotheses can be d i v i d e d i n t o f i v e c a t e g o r i e s . F i r s t , g e n e t i c a l l y v a r i a b l e ( i e . s e x u a l l y produced) o f f s p r i n g may be advantageous when the environment v a r i e s e i t h e r s p a t i a l l y or t e m p o r a l l y . The d i v e r s i t y of the a v a i l a b l e n i c h e s w i l l be l a r g e and the o p t i m a l genotype should, t h e r e f o r e , out compete o t h e r s i n the m i c r o h a b i t a t (Maynard Smith, 1971; W i l l i a m s , 1975; Hamilton, 1975; Bulmer, 1980). Secondly, frequency dependent s e l e c t i o n may operate i n favour of g e n e t i c v a r i a t i o n produced by recombination. T h i s model i s based on the b i o t i c environment. The s e l e c t i v e advantage possessed by the m i n o r i t y genotype i s due to the a c t i o n of pathogens and p r e d a t o r s , b i o t i c f a c t o r s t h a t a d j u s t t h e i r own s t r a t e g y to e x p l o i t the most r e a d i l y a v a i l a b l e r e s o u r c e . J a e n i k e (1978) and R i c e (1983a,b) have suggested t h a t the e f f e c t s of p e s t s and pathogens on the o f f s p r i n g are reduced by sexual r e p r o d u c t i o n i n t r o d u c i n g d i s i m i l a r i t i e s i n phenotype ( i e . defences) between the progeny and t h e i r p a r e n t s . T h i r d l y , d e n s i t y dependent s e l e c t i o n may a l s o p r o v i d e an advantage f o r s e x u a l l y r e p r o d u c i n g i n d i v i d u a l s . Maynard Smith (1978) and P r i c e and Waser (1982) proposed t h a t g e n e t i c a l l y v a r i a b l e s i b l i n g s have d i f f e r e n t r e s o u r c e requirements and hence, have a g r e a t e r f i t n e s s than a s e x u a l l y produced progeny when competing i n a uniform, r e s o u r c e l i m i t e d environment. 90 Fourth, the persistence of recombination promoting a l l e l e s may be determined by t h e i r association with favourable mutations. The recombination gene may hitch-hike with favourable recombinants that i t creates (Felsenstein and Yokoyama, 1976; Strobeck et a l . , 1975). F i n a l l y , a l l of the above hypotheses are conceptually si m i l a r i n that they assume that the s e l e c t i v e advantage recombination provides to an i n d i v i d u a l stems from i t s a b i l i t y to increase the genotypic v a r i a t i o n of the progeny. Bernstein et a l . (1985) have proposed a d i f f e r e n t basis for t h i s nominal sel e c t i v e advantage - the recombinational repair of damaged DNA. Drawing from natural history data, they suggest that t h i s more basic role for recombination explains why many species have abandoned out-crossing but have retained recombination. In t h e i r view, any advantage produced by increased genetic v a r i a t i o n i s lim i t e d to spe c i a l i z e d s i t u a t i o n s . As an aside, Shields (1982) has taken a novel approach by suggesting that the e f f e c t s of recombination (whatever advantages or disadvantages these might e n t a i l ) may be regulated by behavioural modification. The amount of inbreeding, and hence the reduced e f f i c a c y of recombination i n producing v a r i a t i o n i n the progeny, i s controlled by the organism's dispersal patterns. Selection may act on the distance t r a v e l l e d before choosing a breeding s i t e and a mate, such that an optimal l e v e l of genetic v a r i a b i l i t y i s maintained - not too much and not too l i t t l e . A major l i m i t a t i o n of these various models has been the absence of an appropriate system to test t h e i r predictions. Very few attempts have been made to experimentally investigate the 91 r o l e of recombination i n the d e t e r m i n a t i o n of i n d i v i d u a l f i t n e s s . Malmberg (1977) used T4 bacteriophage t o determine i f recombination i n c r e a s e s the r a t e of response to s e l e c t i o n f o r a t r a i t under p o l y g e n i c c o n t r o l . Although the f r e q u e n c i e s of recombination were not w e l l q u a n t i f i e d , i t appears from h i s r e s u l t s t h a t the h i g h recombination l i n e " e v o l v e d " more r a p i d l y than the o t h e r s . Contrary to these r e s u l t s , Thompson (1977) found t h a t recombination had no a f f e c t . He made use of complex, chromosomal rearrangements a v a i l a b l e i n D r o s o p h i l a t o suppress c r o s s i n g over i n a c o n s t r u c t e d s t r a i n . He then compared the r a t e of response to s e l e c t i o n i n the suppressed f l i e s t o t h a t found i n a w i l d - t y p e s t r a i n . To date, o n l y one p a i r of i n v e s t i g a t o r s have attempted to t e s t any of the hypotheses concerning the s e l e c t i v e f o r c e s f a v o u r i n g recombination. Antonovics and E l l s t r a n d (1984) and E l l s t r a n d and Antonovics (1985) have shown t h a t i n the grass Anthoxanthum odoratum s e x u a l l y reproduced p l a n t s have a f i t n e s s advantage over asexual p l a n t s i n environments s u b j e c t to frequency dependent s e l e c t i o n . No such advantage was observed when p l a n t s were exposed to d e n s i t y dependent s e l e c t i o n . U n f o r t u n a t e l y , these s t u d i e s cannot make any c o n c l u s i o n s about the o p t i m a l amount of recombination, or the f a c t o r s which determine t h a t optimum. The rec-1 l o c u s i n C. elegans may p r o v i d e at l e a s t a p a r t i a l s o l u t i o n t o t h i s problem, e s p e c i a l l y i f s e v e r a l a l l e l e s of t h i s gene can be i s o l a t e d . As an example c o n s i d e r the " h i t c h - h i k i n g " h y p o thesis of Strobeck et a l . (1975). They suggested a mechanism which c o u l d i n c r e a s e the frequency of a recombination enhancing a l l e l e . J u s t as a mutator gene can i n c r e a s e i n frequency by h i t c h - h i k i n g with a f a v o u r a b l e mutation i t has induced (Cox and Gibson, 1974; Chao and Cox, 1983), computer s i m u l a t i o n s by the authors demonstrated t h a t a recombination enhancer can i n c r e a s e i n frequency because of i t s a s s o c i a t i o n with a f a v o u r a b l e recombinant. They made the f o l l o w i n g assumptions i n t h e i r model: i ) two a l l e l e s A and a are maintained by h e t e r o s i s ; i i ) the p o p u l a t i o n i s f i x e d f o r the a l l e l e b; i i i ) the a l l e l e , D, i s a dominant suppressor of recombination, where as d u n i f o r m l y i n c r e a s e recombination t o some set l e v e l ; and i v ) a t the s t a r t of the experiment a f a v o u r a b l e mutation, B (codominant with b ) , i s i n t r o d u c e d i n c o u p l i n g phase with d a t a frequency of one i n a thousand i n d i v i d u a l s . By choosing the a p p r o p r i a t e g e n e t i c markers each of these assumptions c o u l d be s a t i s f i e d i n an experimental system u s i n g C. elegans. I n i t i a l a l l e l e f r e q u e n c i e s and g e n e t i c d i s t a n c e s between the markers c o u l d be v a r i e d . S e l e c t i o n c o e f f i c i e n t s c o u l d be estimated from f e c u n d i t y and developmental d i f f e r e n c e s between mutants. The a f f e c t of v a r y i n g the amount of recombination c o u l d be determined by u s i n g d i f f e r e n t p a i r s of rec-1 a l l e l e s i n d i f f e r e n t experimental t r i a l s . The computer s i m u l a t i o n s of Strobeck e t a l . (1975) i n d i c a t e t h a t roughly 70 ge n e r a t i o n s are r e q u i r e d f o r a l l e l e f r e q u e n c i e s to reach e q u i l i b r i u m i n such a p o p u l a t i o n . Given C. elegans' r a p i d g e n e r a t i o n time, over 100 generations i n a year i s p o s s i b l e . Such a long term study does not seem i m p r a c t i c a l when compared 93 to the one year D r o s o p h i l a p o p u l a t i o n experiment performed by E n d l e r (1973). Taking the above as a s t a r t i n g p o i n t , a number of l i n e s of i n v e s t i g a t i o n c o u l d be developed. 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