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Distortions of the genetic map of chromosome I in Caenorhabditis elegans Zetka, Monique-Claire 1993

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DISTORTIONS OF THE GENETIC MAP OF CHROMOSOME /IN CAENORHABDITIS =GANS by MONIQUE-CLAIRE ZETKA B.Sc., The University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES GENETICS PROGRAMME We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1993 ©Monique-Claire Zetka, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of Medical Genetics The University of British Columbia Vancouver, Canada  Date 10  DE-6 (2/88)  September 1993  II  ABSTRACT The process of meiosis ensures heritable genetic material is passed faithfully from one generation to the next. To identify the mechanisms involved in this process, the effects of sex, mutation, and rearrangement on meiotic recombination in Caenorhabditis elegans were investigated. The short life cycle and existence of meiotic mutants make this organism an ideal system in which to study meiosis. To determine the effect of sex on meiotic recombination, crossing over was characterized in male gametes and then compared to the frequencies observed in hermaphrodite gametes. Male recombination across chromosome / was approximately one-third less than that observed in the hermaphrodite. This decrease varied with the interval being measured and in one interval, no difference was observed between the sexes. The frequency of recombination in hermaphrodite spermatocytes was two-fold higher than that observed in oocytes and male spermatocytes . Thus, recombination frequencies appear to be a function of gonad physiology rather than sexual phenotype. To test this further, recombination was measured in males sexually transformed by the her-1 mutation. The results indicated that the sexual phenotype, rather than karyotype, determined the recombination frequency characteristic of a certain sex. Like recombination in the hermaphrodite, male recombination was also found to increase with temperature and decrease with age. Therefore, recombination frequency in C. elegans is influenced by physiological factors such as sexual phenotype and age, and environmental factors such as temperature. Mutations in genes that regulate meiosis can affect the frequency of recombination and the distribution of exchange events. A recessive mutation in the gene rec-1 was mapped, and its effects on the distribution of crossing over on LG / were determined. This mutation was mapped to the right end of chromosome /using the duplications sDp1 (which carries a wild-type allele of the gene) and sDp2 (which does not). A high resolution map position was determined using several deficiencies of the right end of the chromosome to map the mutation. The ribosomal deficiency eDf24 failed to complement rec-1, indicating  the locus was located within its boundaries. Crossing over in five intervals on chromosome I was measured in rec-1 homozygotes. The frequency of recombination in one interval located in the, centre of the chromosome showed a ten-fold increase, whereas an interval located on the right end showed a three-fold decrease. Despite the changes to the frequencies of recombination in these intervals, the total genetic length of chromosome /remained unchanged, indicating that the rec-1 mutation affected the distribution of a wild-type number of exchange events. This implies that the rec-1(+) gene product is necessary in establishing the distribution of crossovers along the chromosome. Chromosome rearrangements can reduce or eliminate crossing over by physically disrupting the normal organization of the chromosome. In this study, a crossover suppressor for the right end of LG /was isolated and characterized. By inducing markers on the rearrangement and establishing the gene order in the homozygote, hInl(I) was demonstrated to be the first inversion isolated in C. elegans. Crossing over in the heterozygote was characterized, and intrachromosomal (but not interchromosomal) effects were observed. The interaction of hInl(I) with two translocations demonstrated that small homologous regions can pair and recombine efficiently, and that the formation of a chiasma between two homologues is necessary for their proper segregation. Rare recombinants bearing duplications and deficiencies were isolated from inversion heterozygotes, leading to the proposal that hIn1(I) is paracentric with the meiotic centromere to its left. The meiotic behaviour of the inversion was found to be consistent with the proposal that the meiotic chromosomes of C. elegans are monocentric.  iv  TABLE OF CONTENTS  ABSTRACT^ TABLE OF CONTENTS^  iv  LIST OF TABLES ^  viii  LIST OF FIGURES ACKNOWLEDGEMENTS^  xi  GENERAL INTRODUCTION^  1  Chapter 1: Sex-Related Differences in Crossing Over INTRODUCTION^  4  MATERIALS and METHODS Strain maintenance and culture conditions^  7  Recombination mapping^  11  Recombination in hermaphrodite germlines ^  12  Recombination in Her-1 hermaphrodites^  13  Measuring the effect of age on recombination ^  13  Measuring the effect of temperature on recombination^13 RESULTS Comparison of male and hermaphrodite recombination frequencies^  15  V  Comparison of recombination frequency in hermaphrodite gametes^  15  Variation of recombination frequency with age ^  20  Variation of recombination frequency with temperature ^24 Effect of Rec-1 on male recombination ^  24 27  DISCUSSION^ Chapter 2: The Characterization and Mapping of rec 1 -  31  INTRODUCTION^ MATERIALS and METHODS Duplication mapping^  33  Deficiency mapping^  34  RESULTS Effect of rec 1 on the genetic map of LG -  /^  36  Duplication mapping of rec 1^  36  Deficiency mapping of rec 1^  40  -  -  DISCUSSION^  44  Chapter 3: The Meiotic Behaviour of an Inversion INTRODUCTION^  48  vi  MATERIALS and METHODS Isolation of hIn1(I)^  50  Measuring egg-hatching frequencies ^  50  Induction of genetic markers on hIn1(I)^  50  Measuring recombination in hIn1(I) homozygotes^ 51 Analysis of recombinants from hIn1(I) heterozygotes^52 DAPI-staining of hDp132 meiotic cells^  53  Interaction of hIn1(I) with szT1(I;X)^  53  Interaction of hInl (I) with hT2(I;III)^  54  hIn1(I) efficacy as a balancer^  54  RESULTS Isolation of a crossover suppressor for the right end of LG /^  55  Zygotic viability of hInl (I) heterozygotes^  55  Gene order in hIn1(I)^  58  Crossing over in inversion homozygotes ^  58  Effect of hIn1(I) on crossing over on LG /^  59  Structure of recombinants derived from hIn1 (I) heterozygotes^  62  Effect of hIn1(I) on crossing over on other chromosomes ^64  vii  Interaction of hIn1(1) with szT1(1,../Y)^  64  Interaction of hInl(D with hT2(I;IID: (a) Crossing over^  67  (b)Segregation^  67  hInl(D as a balancer^  DISCUSSION^  70 72  CONCLUSIONS^  82  BIBLIOGRAPHY^  86  APPENDIX I. Distributive pairing of sDp1^  103  II. Cosmid mapping of rec-1^  104  III. PCR mapping of eDf24^  108  IV. List of strains used in this thesis^  112  viii  LIST OF TABLES  Table 1^Abbreviations^  9  Table 2^Male brood analysis^  16  Table 3^Hermaphrodite brood analysis^  17  Table 4^Male recombination on Linkage Group /^  19  Table 5^Recombination in hermaphrodite germlines ^  21  Table 6^Recombination in Her-1 (X0) hermaphrodites ^  22  Table 7^Effect of temperature on male recombination^  25  Table 8^Effect of rec-1 on male recombination^  26  Table 9^Effect of rec-1 on crossing over on LG I^  38  Table 10^Duplication mapping of rec-1^  39  Table 11^Deficiency mapping of rec-1^  42  Table 12^Effect of hIn1(I) on crossing over on linkage group /  ^56  Table 13^Egg-hatching frequencies of hIn1(1) heterozygotes and recombinants^57 Table 14^Recombination in hIn1(I) homozygotes^  60  Table 15^Effect of hInl(I) on crossing over on other chromosomes^ 66 Table 16^Effect of hIn1(I) on crossing over with LG / translocations ^68 Table 17^Cosmid mapping of rec-1^  106  ix  Table 18^List of strains^  112  x  LIST OF FIGURES  Figure 1^Partial genetic map of Linkage Group I^  8  Figure 2^Male and hermaphrodite meiotic maps of LG I  ^18  Figure 3^Variation of recombination frequency with parental age ^23 Figure 4^Meiotic map of LG /in the presence of Rec-1 ^  37  Figure 5^Deficiency map of the unc-54 region^  43  Figure 6^Meiotic map of LG /in hIn1(I) heterozygotes^  •  61  Figure 7^Structure of h/n/(/)-derived recombinants ^  63  Figure 8^DAP I-stainingof hDp132 meiotic cells^  65  Figure 9^Punnett diagramming recombination in hIn1(I)/szT1(I;X) heterozygotes^  69  Figure 10^Punnett diagramming segregation in hInl(I)/hT2(I;III) heterozygotes^  71  Figure 11^Pairing of inverted sequences in hIn1(I) heterozygote^74 Figure 12^Effect of single crossover in hInl(I) heterozygote^ 79 Figure 13^Cosmid map of the unc-54 region^  105  Figure 14^Sequences of primers used in PCR analysis ^  109  Figure 15^PCR analysis of eDf24^  110  xi  ACKNOWLEDGEMENTS  I would like to thank my supervisor Ann Rose for the patience, guidance, and interest shown to me during the course of this thesis. I would also like to thank my supervisory committee Drs. Fred Dill, Don Moerman, and in particular David Baillie and David Holm for their helpful advice. I would also like to thank Raja Rosenbluth for her constructive criticism of my manuscripts and this thesis. The cytogenetic techniques used in this thesis were taught to me by Dr. Peter Moens and I would like to thank him for his time and effort. The years I have spent in the Rose lab have priviledged me with the friendship of many people and I would like to thank Ken Peters, Jennifer McDowall, Colin Thacker and especially Kim McKim for their help and discussion. I would especially like to thank my family for their stoic support of all my endeavours and my friend Ralph Allan for years of silly fun.  This work was supported in part by a University Graduate Fellowship.  1  GENERAL INTRODUCTION  Meiosis is the process by which sexually reproducing organisms produce haploid gametes. This process consists of one round of DNA replication followed by a reductional division at meiosis I and an equational division, resembling mitosis, at meiosis II. Prophase of meiosis I is marked by two distinct processes that culminate in the segregation of replicated homologous chromosomes: pairing and recombination (reviewed by HAWLEY 1988; HAWLEY and ARBEL 1993). Pairing between homologues is achieved by several temporally distinct events. During the first phase, called homologue recognition, homologous chromosomes are thought to find one another and align themselves at a distance in the diffuse nucleus (MAGUIRE 1984). In C. elegans, homologue recognition regions have been identified on every chromosome (ROSE, BAILLIE and CURRAN 1984; McKIM, HOWELL and ROSE 1988) and are absolutely required for pairing and recombination. Once aligned, the chromosomes are brought into tighter association through a homology search that may be mediated by RecA-type proteins (CONLEY and WEST 1989), which locate and homologously pair discrete sites on the chromosome (CAO, ALANI and KLECKNER 1990; KLECKNER, PADMORE and BISHOP 1991). The existence of such sites has been documented in a variety of organisms including Drosophila raelanogaster, where pairing sites have been mapped along the X chromosome of the female (HAWLEY 1980) and in the ribosomal cluster of the male (McKEE and KARPEN 1990). Recently in Saccharomyces cerevisiae, such a site has been identified on the left arm of chromosome 11/(GOLDWAY, ARBEL and SIMCHEN 1993; GOLD WAY et al. 1993). During the second phase of pairing, this early alignment is locked in place by recombinational intermediates which result from the repair of double-strand breaks that appear early in meiosis (SUN et al. 1989; PADMORE, CAO and KLECKNER 1991). The chromosomes then begin to condense, a process thought to be  2  crucial to the next stage of pairing, which results in intimate synapsis and the formation of a tripartite laminar structure called the synaptonemal complex (SC), between the homologues (KLECKNER, PADMORE and BISHOP 1991). The ZIP1 locus of yeast encodes a component of the central region of the SC, indicating that specific proteins are required for the formation of the structure (SYM, ENGEBRECHT and ROEDER 1993). MAGUIRE (1978) proposed that only those recombination intermediates which occur in the context of the SC have the potential to form chiasma. This is supported by the fact that doublestrand breaks, thought to be the substrate for recombination, appear before and at the same time as the first appearance of the SC (PADMORE, CAO and KLECKNER 1991). In addition, mutants in RED1, MER1, and HOPI are defective in SC formation but still competent in meiotic exchange, indicating recombination can be initiated in the absence of the synaptonemal complex (ROCKMILL and ROEDER 1990; ENGEBRECHT, HIRSCH and ROEDER 1988; HOLLINGSWORTH, GOETSCH and BYERS 1990). In menl mutants, however, the exchange events that occur do not ensure faithful disjunction of the homologues (ENGEBRECHT, HIRSCH and ROEDER 1990). This may be explained if the role of the SC during meiosis is the conversion of a number of sites of alignment and recombination into a bivalent united by a chiasmata. An essential feature of meiosis is recombination between homologues, which serves to reassort genetic information and promote proper segregation of the chromosomes. Crossing over refers to a reciprocal event resulting in an exchange of flanking markers. The frequency and distribution of crossing over are regulated, and a number of mutations which disrupt this pattern have been identified (reviewed by BAKER et al. 1976). The nematode C. elegans is an ideal system for the study of meiosis. Populations consist mostly of self-fertilizing hermaphrodites that are capable of producing about 300 progeny each and that have a short generation time (3.5 days at 200). Males can be used for the introduction of genetic markers and the genetic maps of the five autosomes and the X chromosome are well marked with visible mutations. Recessive mutations have been  3  isolated that reduce crossing over (HODGKIN, HORVITZ and BRENNER 1979), confer radiation sensitivity (HARTMAN and HERMAN 1982), and increase both crossing over and conversion (ROSE and BAILLIE 1979b; RATTRAY and ROSE 1988). Thus, in C. elegans,  gene products important in meiosis can be identified by mutations which produce  phenotypes that have also been described in other systems (BAKER et al. 1976). In this study, meiosis in C. elegans has been investigated by examining the effect of sex, mutation, and rearrangement on recombination.  4  Chapter 1: Sex-Related Differences in Crossing Over INTRODUCTION  The biology of C. elegans provides a unique opportunity to examine the effect of sex on recombination. Laboratory populations consist largely of self-fertilizing hermaphrodites (5AA;XX). Males (5AA;X0), arise spontaneously as a result of X-chromosome nondisjunction (HODGKIN, HORVITZ and BRENNER 1979) and are maintained by cross-fertilization with hermaphrodites. The standard genetic map of C. elegans (EDGLEY and RIDDLE 1990) is based upon hermaphrodite recombination frequencies that are the product of crossover events in two germlines: oocyte and hermaphrodite spermatocyte. The frequency of recombination in these two germlines has been shown to be different (ROSE and BAILLIE 1979a). Sexual differences in crossing over are known to occur in a number of organisms. There may exist two qualitatively different situations when examining the relationship between sex and recombination frequency. The first is the absence of recombination in one sex, a characteristic of D. melanogaster males (MORGAN 1912) and Bombyx moni females (TANAKA 1913). The second, more common situation, is one where recombination exists in both sexes, but with a reduced frequency in one (reviewed by DUNN and BENNETT 1967). Recombination frequency in the female is generally higher in D. ananassae (MORIWAKI 1937), in mice (SLIZYNSKI 1960), and in humans (WHITE et al. 1985a; DONIS-KELLER et al. 1987). Alternatively, male recombination frequency is generally higher in maize (RHOADES 1941; ROBERTSON 1984), and in Tribolium cast aneum (SOKOLOFF 1964). However, sex-related differences in recombination frequency are not uniform for all regions of the genome. In maize, some intervals have been reported to be longer in the female meiosis (ROBERTSON 1984). In mice, significant sex differences in recombination frequency went in opposite directions on different chromosomes (DAVISSON and RODERICK 1981) and in humans, some regions were the same genetic size in both  5  sexes (DONIS-KELLER et al. 1987). This suggests local differences in recombination between the sexes are not representative of the chromosome, nor of the genome as a whole. In this study, the effect of sex on recombination in the nematode Caenorhabditis elegans has been investigated. Each of the autosomes in C. elegans is marked by a region where genes cluster on the meiotic map as a result of less recombination per base pair than the genome average (BRENNER 1974; GREENWALD et al. 1987; KIM and ROSE 1987; PRASAD and BAILLIE 1989; STARR et al. 1989). By examining intervals spanning linkage group (LG) I, the effect of sex on recombination in intervals inside and outside such a region has been determined. One approach in studying the relationship between sex and recombination frequency is measuring recombination in sexually transformed individuals. Hormone treatments have been used in the Medaka, Oryzias latipes, to transform XY fish, normally male, into functional females. Crossing over in these transformed males was found to occur at a higher frequency than in normal males (YAMAMOTO 1961). This suggests that differences in recombination between the sexes are not completely the result of the sex chromosome constitution, but also depend on the physiological differences associated with sex. In C. elegans,  mutations exist which result in the complete transformation of the sexual  phenotype. One such mutation, her-1, transforms fertile XO males into self-fertile hermaphrodites (HODGKIN 1980), and has been used in this study to examine the effect of karyotype on recombination frequency in the nematode. Meiotic recombination frequency in higher eukaryotes is affected by several known parameters. Recombination frequency increases at temperature extremes in D. melanogaster (PLOUGH 1917, 1921), Neurospora crassa (McNELLY-INGLES, LAMB, and FROST 1966) and Coprinus lagopus (LU 1969, 1974). A decrease in meiotic recombination frequency with maternal age has been observed in D. melanogaster (BRIDGES 1927; NEEL 1941), in C. elegans (ROSE and BAILLIE 1979a), and on some chromosomes in the mouse, Mus musculus (FISHER 1949; BODMER 1961; REID and PARSONS 1963). Existing  human data is not conclusive about maternal age effects although some evidence suggests a  6  paternal age effect may exist (LANGE, PAGE and ELSTON 1975; ELSTON, LANGE and NAMBOODIRI 1976). In C. elegans, hermaphrodite recombination frequency decreases with maternal age and increases with temperature (ROSE and BAILLIE 1979a) and in the presence of the rec-1 mutation (ROSE and BAILLIE 1979b). In this study, the effect of temperature, age, and rec-1 on recombination in C. elegans males has been investigated.  7  Chapter 1: MATERIALS AND METHODS General Methods: C. elegans population consist largely of self-fertilizing hermaphrodites (5AA;XX). Males (5AA;X0) arise spontaneously as a result of X-chromosome nondisjunction (HODGKIN, HORVITZ and BRENNER 1979) and were maintained by mating to hermaphrodites. Wild-type and mutant strains were maintained and mated on petri plates containing nematode growth medium (NGM) and streaked with Escherichia (BRENNER 1974). All experiments were carried out at 200 unless otherwise noted. The wild-type strain N2 and most mutant strains of C. elegans var. Bristol used in this study were obtained from D.L. Baillie at Simon Fraser University, British Columbia or from the Caenorhabditis Genetics Centre at the University of Missouri, Columbia, Missouri. RW3072 was supplied by R.W. Waterston at Washington University School of Medicine, St. Louis, Missouri. The following genetic markers (for list of strains, see APPENDIX IV) were used in the course of this work: LG^bli-3(e579); unc-11(e47); dpy-5(e61); bli-4(e937); dpy-14(e188); unc-13(e450); uric29(e403); unc-29(e193); lin-11(n389); uric-75(e950); unc-75(h1041); unc-75(h1042); uric-101(ml); unc-59(e261); 1ev-11(x12); let-49(st44);  ?MC-  54 (e190); uric-54(h1040); unc-  54(st40); let-50(st33); rec-1(s180)  LG /1/: dpy-18(e364); unc-36(e251) LG V: unc-42(e270); her-1(e1520); dpy-11(e224); him-5(e1467) LG X: lon-2(e678); unc-1(e719); dpy-3(e27) The locations of some genes on chromosome / are shown in Figure 1. C. elegans nomenclature of genes and alleles conforms to the system outlined by HORVITZ et al. (1979). The names of genetic loci are abbreviated using a three letter code followed by a number and then by an allele designation defining the laboratory of origin in parentheses. The most commonly used abbreviations are described in Table 1. For example, uric101 (ml) is a mutation in a gene which results in an uncoordinated, or unc, phenotype. It  was the one hundred and first unc gene identified, and the first mutation isolated in the  8a  FIGURE 1.-A partial genetic map of Linkage Group /showing the major markers used in this study.  ^ ^  unc- 13 let-50 dpy- 14 unc-29 unc-54 bli-4^lin-11 let-49 unc- 59 dpy- 5^unc- 75 bli - 3^unc- 11 lev-11 unc-101 I  5 m.u.  Linkage Group I  9  Table 1 Abbreviations  Abbreviation^  Phenotype  bli^  blistered cuticle  dpy^  dumpy  her^  helmaphrodization of XO animals  him^  high incidence of males  let^  lethal  lev^  levamisole resistant  lin^  abnormal cell lineage  ion^  long  rec^  abnormal recombination  unc^  uncoordinated  10  laboratory with the m allele designation. The allele designation for the Rose lab is h and the strain designation is KR. The following translocations were used in this study: szT/(/;X) (FODOR and DEAK 1985; McKIM, HOWELL and ROSE 1988), hT2(/;///) (McKIM, PETERS and ROSE in press), hT1(I;17) (McKIM, HOWELL and ROSE 1988), and hT3(I;X) (McKIM 1990). szT1(I;X) is inviable as a homozyote and is marked with the lon-2 mutation on /RR, where R denotes the right arms of chromosomes l and X. hT2(I;III) is viable as a  homozygote and is marked with b/i-4 on /NHL, where L denotes the left arms of chromosomes l and Hi Both hT1(1,T) and 1)113(I;X) are inviable as homozygotes. Mutations on translocation chromosomes (T) are shown in square brackets (McKIM, HOWELL and ROSE 1988) and the formal name is used when discussing both components of the translocation (i.e. szT1(I;X)). When discussing the individual component chromosomes of a translocation, the nomenclature describes the segregational properties of the new chromosomes. The translocation szT/(/;X) is comprised of two chromosomes; szT1(I;X)I (of structure /RXR, where R denotes the portion of the chromosome to the right  of the breakpoint), which segregates from chromosome I, and szT1(I;X)X (of structure /LX-L, where L denotes the portion of the chromosome to the left of the breakpoint), which  segregates from the X chromosome. Similarly, hT2(/;///) is comprised of two chromosomes; hT2(I;III)I (of structure /R///R), which segregates from chromosome I, and hT2(/;///)/// (of  structure /NHL), which segregates from chromosome III. hT1(I;V) consists of hT1(LV)I (of structure IR V1) which segregates from chromosome /, and hTl(I;v)v- (of structure /L VR), which segregates from chromosome V. hT3(I;X) consists of two chromosomes; hT3(I;X)I (of structure /RXR) which segregates from chromosome I, and hT3(I;X)X (of structure ILXL), which segregates from the X chromosome. Inversions in C.elegans are written In (HORVITZ et al. 1979). Mutations on inversion chromosomes are shown in square brackets (e.g. hInl(I)klpy-5 unc-41), similar to the system in use for translocations. The nomenclature does not necessarily provide  11  information on gene order and does not implicate the marker in the rearrangement, indicating only that the mutations are linked to the inversion. The following duplications and deficiencies were used in this study: the free duplications sDp1(1,1), sDp2(If) (ROSE, BAILLIE and CURRAN 1984), hDp131(1,1), hDp132(If) (ZETKA and ROSE 1991; this study), the deficiencies eDf4(I), eDf9(I), eDflO(I), eDf13(I), eDf24(I) (ANDERSON and BRENNER 1984), hDf11(I), and hDf12(I)  (ZETKA and ROSE 1992). Duplications in C. elegans are written as Dp (preceeded by the laboratory designation) and followed in parentheses by their chromosome of origin and the designation f if they are free duplications (unlinked to an intact chromosome). sDpl(If) duplicates the right end of LG / and pairs and recombines with the normal homologues whereas sDp2(1,1) duplicates the left end of the chromosome and does not pair and recombine (ROSE, CURRAN and BAILLIE 1984). Deficiencies are abbreviated Df and are followed in parentheses by their chromosome of origin. eDf2.4 complements unc-54 and partially deletes the ribosomal cluster, the most distal genetic marker on LG I. The remaining eDf deficiencies fail to complement tine-54 and were isolated using eDf24 as a balancer (ANDERSON and BRENNER 1984). The origin and structure of hDp131, hDp132, hDf11, and hDf12 are discussed in Chapter 3. Recombination Mapping: Recombination frequency in the hermaphrodite was measured  by scoring the number of recombinant progeny of a cis-heterozygote, under the conditions described by ROSE and BAILLIE (1979a). The recombination frequency (p) between two markers was calculated using the formula p = 1 - (1 - 2R)172, where R is the number of visible recombinant individuals divided by the number of total progeny (BRENNER 1974). The total progeny number of the hermaphrodite is estimated as 4/3 X (number of Wts one recombinant class) where Wts is the number of wild-type progeny. Map distances in the male were determined by scoring the progeny resulting from mass mating seven males heterozygous for a pair of cis-linked markers to five homozygous hermaphrodites (new hermaphrodites each day) every 24 hours for four days. On the fourth day the males were left on plates with the same hermaphrodites for a fifth day, after which the hermaphrodites  12  were transferred. Since mapping in the male involves recombination in only one germline, the recombination frequency (p) is equal to R. The total progeny number of the male is 2 X (number of Wts + one recombinant class). This differs from the total progeny number of the hermaphrodite for the following reasons. In both male and hermaphrodite recombination experiments, the double homozygote class is not scored because of its reduced viability, and the total progeny number is calculated from the wild-type class. Mapping in the hermaphrodite involves crossing two heterozygous germlines, whereas mapping in the male involves crossing one germline heterozygous for a pair of markers to one which is homozygous. For this reason, the ratio of wild-type progeny to progeny homozygous for the markers differs in hermaphrodite and male recombination experiments. Thus, the number of wild-type progeny must be multiplied by 4/3 and 2 respectively to correct for the inviable class. Both classes of recombinants were used in the calculations unless otherwise noted. In cases where only one class of recombinants was used, R = 2 X (one recombinant class) divided by the total progeny number. All hermaphrodite recombinants were progeny tested. The progeny of putative recombinants that had mated before being picked were screened for the presence of both male and hermaphrodite individuals of the recombinant phenotype. In the case of the bli 3 unc 11 interval, bli 3 penetrance is low and Bli-3 recombinants were -  -  -  scored as wild-type and later subtracted. The unc 75 unc 101 and unc 101 unc 54 map -  -  -  -  distances were based on the Unc-75 and Unc-101 recombinant classes respectively. 95% confidence intervals were calculated using the statistics of CROW and GARDNER (1959). In the event the number of recombinants exceeded 300, confidence intervals were approximated using the equation 1.96(nxy)1/2 where x is the number of recombinants (n), divided by the number of wild-types plus recombinants, and y is equal to 1 - x. Recombination in Hermaphrodite Germlines: Recombination frequency in oocytes was measured by scoring the male progeny of dpy 5 unc 75/ + + or unc 11 dpy 5/ + + -  -  -  -  hermaphrodites mated to a male carrying an appropriate crossover suppressor. The translocation hT2(/;///) was chosen because it suppresses crossing over in both these regions (McKIM, PETERS and ROSE 1993). Males of the genotype dpy 5 unc 75; + /hT2(I;III)[+ -  -  13  +;dpy-18Jor unc-11 dpy-5; + /hT2(1;III)[+ +;dpy-18] were mated to heterozygous  hermaphrodites every 24 hours and the male progeny were scored. The oocyte recombination frequency (a), is 2 X the number of recombinant individuals divided by the total progeny. The total number of progeny is 4/3 X (number of Wts + one recombinant class). Knowing the value of R for the hermaphrodite and a, the recombination frequency in the oocytes, the following equation was solved for b, the recombination frequency in hermaphrodite spermatocytes.  R = 1/2b(1 - a) + 1/2a(1 - b)^1/2ab Recombination in Her-1 Hermaphrodites : To measure recombination in Her-1(X0) individuals, hT1(1-;V)[unc-29; + ; + j/szT1(I;X)[ + ; + ; lon-2Jmales were crossed to her-1 homozygous hermaphrodites. Because of the segregational properties of sz T./(/;X) (McKIM, HOWELL and ROSE 1988), all wild-type males resulting from this cross were of the genotype + ; her-//hT4/;V)func-29; + J. These males were then crossed to hT3(I;X)[dpy-5 unc-29; + Jhomozygotes to produce + + ;0/hT3(I;X)idpy-5 unc-29; + her-1/ + males.  When the latter males were mated to dpy-5 unc-75; her-1 hermaphrodites, the only wildtype hermaphrodites that resulted were of the genotype + + /dpy-5 unc-75; her-l/her-1; + /0. Recombination was measured in these individuals by scoring Dpy-5 and Unc-75  recombinants. Variation With Age: The variation of recombination with parental age was examined in two intervals; dpy-5 unc-75 and dpy-5 unc-13. Young heterozygous males were individually mated to 5 new homozygous hermaphrodites every 12 hours for 4 days. Heterozygous L4 hermaphrodite controls were brooded every 12 hours for 3 days under the same conditions. The recombination frequency in every 12 hour period was calculated as described above. Variation with Temperature: The effect of temperature on male recombination was examined in the dpy-5 anc-75 and dpy-5 unc-13 intervals. Seven heterozygous males were mass mated to five homozygous hermaphrodites and transferred to new hermaphrodites every 24 hours at temperatures of 150 or 250. Hermaphrodite controls were picked from the  14 same plates as experimental males and were transferred every day. All progeny were permitted to develop at 200 to avoid any inviablity produced by temperature extremes.  15  Chapter 1: RESULTS Male recombination frequency is lower than hermaphrodite: Differences in  recombination frequencies between the sexes were initially studied in two intervals; dpy 5 -  unc 75 and dpy 5 unc 13. The latter interval is located within the chromosome /genetic -  -  -  cluster and the former includes the cluster and a genetically large interval to the right. In both intervals, the frequency of recombination was approximately two-fold lower in the male (data shown in Tables 2 and 3). To determine if the reduced recombination frequency in the male was general across the length of chromosome /, other intervals inside and outside the cluster were investigated. The results for six intervals spanning LG /is shown in Figure 2 (data shown in Table 4). In the dpy 5 unc 29 unc 75 interval, only hermaphrodites were -  -  -  scored because the phenotypes of male recombinants were subtle and progeny testing was not possible. Male recombination frequency was lower in five of the intervals tested when compared to hermaphrodite controls. The differences in recombination frequencies between the hermaphrodite and the male in these intervals were not uniform; they varied from 1.3fold in unc 11 dpy 5 to 2 fold in dpy 5 unc 13 and unc 75 unc 101. In the unc 101 unc 54 -  -  -  -  -  -  -  -  -  region, the male meiotic distance was not different from that observed in the hermaphrodite. The difference for a comparably sized interval, bli 3 unc 11 was 1.6, suggesting sex-related -  -  differences are interval-dependent and not size-dependent. Thus, the greatest differences in crossover frequency were observed near the gene cluster, and no difference was observed at the right end of the chromosome. The total genetic length of the meiotic map of LG /is 31.7 m.u. in the male, compared to 44.1 m.u. in the hermaphrodite (data from Table 4). As is the case with the hermaphrodite meiotic map, the male map is also marked by a centrally located cluster. Recombination in hermaphrodite spermatocytes is higher than in oocytes: The  recombination formula normally used in measuring map distances in the hermaphrodite is based on the assumption that the frequency of recombination is equal in both germlines although this has been shown not to be the case (ROSE and BAILLIE 1979a). To measure  16a Table 2 Male brood analysis  Genotype^Wts^Recombinants ^pX100(C I )a  9^d^Dpy^Unc 9  d  9  d  dpy-5 unc-13/ + +  0-12 hr  594  535  10  8  6  14  1.7(1.2-2.2)  13-24 hr  789  761  11  5  9  10  1.1(0.8-1.5)  25-36 hr  751  703  2  2  4  5  0.4(0.2-0.7)  37-48 hr  849  890  6  8  7  3  0.7(0.4-1.0)  49-60 hr  453  503  5  3  2  3  0.7(0.3-1.1)  61-72 hr  442  382  2  1  0  3  0.4(0.2-0.8)  73-84 hr  233  238  0  1  5  1  0.7(0.3-1.4)  85-96 hr  109  113  0  0  1  1  0.4(0.1-1.5)  Totals  4241  4140  36  28  34  40  0.8(0.7-1.0)  0-12 hr  667  693  46  49  58  58  '7.2(6.3-8.1)  13-24 hr  556  568  28  41  29  41  6.8(4.9-6.8)  25-36 hr  712  782  48  61  44  52  6.4(5.6-7.3)  37-48 hr  879  794  45  42  34  34  4.4(3.8-5.1)  49-60 hr  378  389  21  15  18  7  3.8(3.0-4.8)  61-72 hr  611  655  27  31  27  19  3.9(3.2-4.7)  73-84 hr  352  346  16  17  16  12  4.2(3.3-5.2)  85-96 hr  76  81  3  2  0  6  3.4(1.7-5.7)  Totals  4231  4308  234  258  226  229  5.3(5.2-5.4)  dpy-5 unc-75/ + +  16b  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).  17 Table 3 Hermaphrodite brood analysis  Genotype  ^  Wts^Recombinants ^pX100(C.I.)a Dpy^Unc  dpy-5 unc-131 + +  0-12 hrs  334  6  7  2.8(1.5-4.6)  13-24 hrs  647  11  9  2.3(1.5-3.5)  25-36 hrs  786  5  7  1.1(0.6-1.9)  37-48 hrs  718  5  6  1.1(0.5-2.0)  49-60 hrs  383  5  1  1.2(0.5-2.5)  61-72 hrs  251  2  2  1.1(0.4-2.9)  3119  34  32  1.6(1.2-2.0)  0-12 hrs  1120  73  88  10.6(9.1-12.3)  13-24 hrs  2583  171  179  10.0(9.7-10.4)  25-36 hrs  2893  175  197  9.5(9.2-9.8)  37-48 hrs  2560  114  154  7.8(6.8-8.7)  49-60 hrs  1051  51  64  8.1(6.7-9.7)  61-72 hrs  596  39  25  8.0(6.1-9.9)  Totals  10803  623  707  9.1(8.9-9.2)  Totals  dpy-5 unc-75I + +  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).  18a  FIGURE 2.-Male and hermaphrodite meiotic maps of LG I. Three factor experiments positioned unc 75 between dpy 5 and uric 101. The LG /cluster extends from unc 11 to -  -  unc 29 (EDGLEY and RIDDLE 1990). -  -  -  unc- 13 dpy- 5 bli- 3^unc- I 1  unc-29 unc- 75 1 unc- 101  unc-54  I^I  Male unc- 13 dpy- 5 bli- 3  unc- 11  5 m. u.^  unc-29 unc-75 unc- 101^  unc- 54  Hermaphrodite  19a Table 4 Male recombination on Linkage Group I  Genotype  ^  Wts^Recombinants^pX100(C I.)a  9^ '  blz-3 unc-11I +  male b  ^  hermaphrodite  ^  1392^1206^135 Unc  ^  109 Unc^9.4C(8.2-10.6)  1686^170 Unc^  14.8c(12.4-17.4)  uric-11 dpy-5I + +  male^983^962^19 Dpy^12 Dpy^1.8(1.4-2.2) 15 Unc^25 Unc hermaphrodite^3786^58 Dpy^  2.3(2.0-2.8)  61 Unc  dpy-5 unc-29/  male^2536^2451^29 Dpy^35 Dpy^1.2(1.0-1.5) 44 Unc^61 Unc hermaphrodite^1822^30 Dpy^  2.8(2.2-3.5)  39 Unc dpy-5 unc-29 unc-75/ +  male^581^ 11 Dpy-5d 6 Unc-29 Unc-75d^1.4(0.8-2.2) 17 Unc-75e^ 2.9(1.6-4.3) hermaphrodite^1598^34 Dpy-5d 36 Unc-29 Unc-75d^3.4(2.6-4.2) 2 UflC2gd,e  19b 63 Unc-75e  6.0(4.7-7.6)  unc-29 unc-75/ + +  male  3374  3568  95 Unc-75  80 Unc-75  126 Unc-29  90 Unc-29  45 Unc  42 Unc  2.7(2.6-2.8)  unc-75 unc-101/ + +  male  2634  hermaphrodite  3192  2553  68 Unc  1.6c(1.3-2.0) 3.2c(2.7-3.8)  unc-101 unc-54I + +  male  392  hermaphrodite  1187  362  71 Unc  62 Unc  116 Unc  15.0c(12.7-17.2) 14.4c(11.8-17.1)  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Recombination measured in individuals of indicated sex.  c Calculated from one recombinant class (see Chapter 1: MATERIALS and METHODS). d dpy-5 unc-29; e unc-29 unc-75.  20  the difference in recombination frequency between the germlines, dpy-5 unc-x; + /hT2(I;III)[+ +; dpy-167 males were crossed to hermaphrodites cis-heterozygous for a pair of  LC /markers, and the male progeny scored (see MATERIALS and METHODS). The results are shown in Table 5. In measuring the unc-11 dpy-5 interval, an unusually small number of Dpy-5 recombinants were recovered. The most conservative approach was to use only the Unc-11 recombinants in the calculations, since this would give the minimum estimate of differences in recombination between the two germlines. In both intervals studied, the frequency of recombination in hermaphrodite spermatocytes was higher than that observed in oocytes; 2-fold in dpy-5 unc-75 and 1.5-fold in unc-11 dpy-5. To further examine the effect of sexual phenotype on recombination frequency, crossing over was measured in males transformed into fertile hermaphrodites by the her-I mutation. The results of experiments measuring recombination in the dpy-5 unc-75 interval in Her-1 (XO) hermaphrodites is shown in Table 6. Most of these hermaphrodites were sterile and those that were fertile produced few progeny. For this reason, recombinants that proved to be sterile upon progeny testing were also included in the calculations. The crossover frequency in these transformed males was significantly higher than that observed in normal males. An attempt was made to examine recombination in transformed hermaphrodites using the tra1(e1099) mutation but these males mated poorly and rarely produced progeny. Male recombination varies with age: ROSE and BAILLIE (1979a) found  hermaphrodite recombination frequency to decrease with age. The effect of parental age on recombination in the dpy-5 unc-75 and dpy-5 unc-13 intervals is shown in Figure 3 (data shown in Tables 2 and 3 respectively). In both intervals, male recombination frequency shows a general decrease with age. Consistent with the previous results, the recombination frequencies of hermaphrodite controls also decreased with age. The variation in male recombination with age shows some periodicity in both intervals tested. The statistical of this fluctuation is difficult to assess due to the low recovery of recombinants in later broods. In the male, the most reproducible results were obtained in the first 36 hours. The greatest number of self-fertilization progeny were also produced in this period  21 Table 5 Recombination in hermaphrodite germlines  Genotype^Wts^  Recombinants^pX100(C.I.) a Dpy^Unc  dpy-5 unc-75/ + +  oocyteb  4290  83  92  spermc  6.0(5.2-6.9) 12.4  unc-11 dpy-5/ + +  oocyteb  3707  7  24  spermc  1.9(1.2-2.8) 2.7  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Only male progeny scored. C Recombination frequency in hermaphrodite sperm (see Chapter 1: MATERIALS and METHODS).  22 Table 6 Recombination in Her-1(X0) hermaphrodites  Recombinants Wild types Dpy^Unc Genotype  9^9^9^pX100(C I )a  dpy-5 unc-75/ + +; her-I/her-1(X°)  53  4  dpy-5 unc-75/ + +b  10803  623  male  2503  2457  148  12.5(6.2-23.1) 707 132  122  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b data from Table 3.  9.1(8.9-9.2) 135  5.1(5.0-5.3)  23a  FIGURE 3.-The variation of recombination frequency with parental age in the (a) dpy 5 -  unc 75 interval and (b) dpy 5 unc 13 interval. Brood analysis for male heterozygotes is -  -  -  represented by the dashed line. Hermaphrodite controls are represented by the solid line. Vertical bars represent 95% confidence intervals.  23b  (a) 14  o 12 11= C l 10 ▪  8  O  6  scp^4 CC 2 o-  Time (hr) (b)  0  0^20^40^  60  ^  80  ^  Time (hr) —•—• Hermaphrodite ^ Male '4  100  24  although the variation between individual males was high. In one experiment examining the dpy 5 unc 75 interval in the male, a small number of progeny were recovered in the 49-60 hr -  -  period and this was likely the result of the poor physical condition of the hermaphrodites used in the matings since it was not reproduced in later experiments. Male recombination frequency increases with temperature: Crossing over in the hermaphrodite has been found to vary with temperature (ROSE and BAILLIE 1979a). To determine if temperature has a similar effect in the male, recombination was measured in cis-heterozygous males at experimental temperatures of 150 and 250. The results are shown in Table 7 with 200 controls for comparison. Recombination frequency in the male and in the hermaphrodite decreased at 15° and increased at 25° in both intervals tested. In the dpy 5 unc 13 interval, the magnitude of the temperature effects was the same in both sexes; -  -  at 250 recombination frequency increased approximately 40% and at 150, it decreased 40%. In the dpy 5 unc 75 interval, however, the magnitude of the temperature effect was at least -  -  two-fold greater in males when compared to that of hermaphrodite controls. Male crossover frequency remained lower than that observed in the hermaphrodite at all temperatures and in both intervals tested. Male recombination frequency increases with Rec-1: The rec 1 mutation increased -  meiotic recombination three-fold in the hermaphrodite (ROSE and BAILLIE 1979b). This increase retained the meiotic distribution of crossover events. To determine if this mutation had the same effect in the male, recombination was measured in unc 11 dpy 5 rec 11 + + -  -  -  rec 1 and dpy 5 unc 13 rec 11 + + rec 1 individuals. The results of these experiments are -  -  -  -  -  shown in Table 8. Recombination frequency in the male increased three-fold in the unc 11 -  dpy 5 interval (from 1.8 to 5.0, data in Tables 4 and 8 respectively) and five-fold in the dpy-  5 unc 13 interval (from 0.8 to 4.3, data in Tables 7 and 8 respectively). Rec-1 -  hermaphrodite crossover frequencies remained higher than those observed in the male.  25a Table 7 Effect of temperature on male recombination  Genotype^Wts^  Recombinants^pX100(C I )a  9^d^Dpy^Unc 9^d^9^d  15°C dpy-5 unc-13I + +  maleb  1870  hermaphrodite  1218  1992  9  14  9  6  12  0.5(0.4-0.7) 1.0(0.6.-1.6)  8  dpy-5 unc-751 + +  male  2345  hermaphrodite  2206  2528  93  103  121  111  89  3.9(3.8-4.0) 8.4(7.4-9.5)  130  20°C dpy-5 unc-13/ + + c  male  4242  hermaphrodite  3119  4140  36  28  34  34  40  0.8(0.7-1.0) 1.6(1.2-2.0)  32  dpy-5 unc-75/ + + c  male  4231  hermaphrodite  10803  4308  234  258  226  229  707  623  5.3(5.2-5.4) 9.1(8.9-9.2)  25°C dpy-5 unc-13I^+  male  968  hermaphrodite  3024  dpy-5 unc-75I +  1077  15 52  10  10 42  11  1.1(0.8-1.5) 2.3(1.9-2.8)  25b male^1105^1142^139^122^114^107^9.7(94-10.0) hermaphrodite^1574  ^  125^140^12.4(11.0-13.9)  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Recombination measured in individuals of indicated sex.  c Data from brooding experiments.  26  Table 8 The effect of Rec-1 on male recombination  Genotype^Wts^  Recombinants ^pX100(C.I.) a  9^o''^ Dpy^Unc 9  d  9  d  40  43  57  30  unc-11 dpy-5 rec-11 + + rec-1  maleb^866  755  hermaphrodite^2033  91  5.0(4.3-5.7) 6.7(5.7-7.6)  91  unc-11 dpy-5/ + +  male c  ^  hermaphrodite c  ^  983  962  3786  19  12  58  15  25  61  1.8(1.4-2.2) 2.3(2.0-2.8)  dpy-5 unc-13 rec-11 + + rec-1  male  922  hermaphrodite  2111  908  46  41  103  36  43  4.3(3.7-5.0) 6.6(5.7-7.7)  86  dpy-5 unc-13/ + +  maled  4241  hermaphroditee  3119  4140  36 34  28  34 32  C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Recombination measured in individuals of indicated sex.  C Data from Table 4. d Data from Table 2.  C Data from Table 3.  40  0.8(0.7-1.0) 1.6(1.2-2.0)  27  Chapter 1: DISCUSSION BRENNER (1974) first observed that each C. elegans autosome is marked by a cluster of genes and proposed that this clustering was a result of recombination suppression. This has been supported by studies which have compared the genetic and physical maps (GREENWALD et al. 1987; PRASAD and BAILLIE 1989; STARR et al. 1989) and by the enhancement observed in the clusters when treated with gamma radiation (KIM and ROSE 1987) and elevated temperatures (ROSE and BAILLIE 1979a; this study). The results presented in this thesis show that the frequency of recombination is generally higher in the C. elegans hermaphrodite than in the male, although the increases are not uniform along the length of the chromosome and one interval showed no sex-related difference. In addition, the gene cluster of LG I appeared to be larger in the male than in the hermaphrodite. That the genetic map of the male is smaller than that of the hermaphrodite, while the gene cluster is larger, may be explained if the recombination suppression observed in the hermaphrodite is more pronounced in the male, or if interference values in the male (leading to double-crossing over) are low. HODGKIN, HORVITZ, and BRENNER (1979) found complete interference on the X chromosome of the hermaphrodite but measured a moderate C value (coefficient of coincidence) on an autosome in the male. This may be explained if high interference is limited either to the hermaphrodite or to the X chromosome but neither possibility has been confirmed. It is unlikely that low interference in the male is the basis of sex-related differences in recombination frequency for several reasons. Firstly, large decreases in the male meiotic map were observed in small intervals in the cluster, a region in which double-crossing over would be extremely rare. Secondly, in a large interval like blz 3 unc 11, the male meiotic map showed a 36% decrease in -  -  recombination when compared to the hermaphrodite. The number of double-crossovers one would expect in this interval (approximately 2), cannot account for the magnitude of this decrease. Thus, while it is possible that interference values differ between the  28  hermaphrodite and the male, it is unlikely to be the sole explanation of differential rates of crossing over between the sexes. Elevated temperatures produce increases in recombination values in a number of organisms including Drosophila (PLOUGH 1917), Coprinus (LU 1969, 1974), and Neurospora (McNELLY-INGLES, LAMB and FROST 1966). In Drosophila, the greatest temperature related changes in crossover frequency occur in centromeric regions, where recombination is normally suppressed (PLOUGH 1917; BRIDGES 1915, 1927; STERN 1926; MATHER 1939). ROSE and BAILLIE (1979a) examined two intervals in the LG / cluster of the hermaphrodite and found 2-3 fold increases in recombination frequency at elevated temperatures. In this study, similar increases of recombination values have been observed in the male in two intervals. The dpy 5 unc 13 interval has been well -  -  characterized and includes a portion of the chromosome which is the most recombinationally suppressed (KIM and ROSE 1988; PRASAD et al. 1993). The adjacent unc 13 unc 75 -  -  interval showed a two-fold map expansion in the male compared to the hermaphrodite. This suggests that the recombination suppression responsible for the appearance of the gene cluster on the genetic map extends further in the male than in the hermaphrodite and can be expanded by temperature over a larger interval. The results of experiments using the rec 1 mutation can also be interpreted in light -  of sex-related differences in cluster size. This mutation increased the frequency of male recombination in both intervals tested. In both sexes, a greater enhancement effect (6-8-fold increase) was observed in the dpy 5 unc 13 interval, located within the cluster, when -  -  compared to the uric 11 dpy 5 interval (3-fold increase), a larger region at the left end of the -  -  cluster. The dpy 5 unc 13 region is more recombinationally suppressed (discussed above) -  -  than flanking regions, suggesting that the most suppressed regions may be more sensitive to the effects of rec 1. -  BRENNER (1974) measured recombination frequency in oocytes on the X chromosome and found this frequency to be the same as the hermaphrodite frequency. In this study, oocyte recombination frequency was measured in two intervals on LG / and  29  found to be lower than both the total hermaphrodite frequency and the crossover frequency in hermaphrodite spermatocytes. These results may be explained if differences in recombination frequency between the hermaphrodite germlines are genetic intervaldependent or limited to the autosomes. Although recombination was found to vary with age in male spermatocytes, it is unlikely hermaphrodite spermatocytes contribute to the variation of recombination frequency with age in the hermaphrodite since spermatogenesis in hermaphrodites is restricted to the fourth larval stage, at which time about 300 sperm are produced (HIRSH, OPPENHEIM and KLASS 1976; WARD and CARREL 1979). This has previously been pointed out in studies examining the variation of recombination with hermaphrodite age (ROSE and BAILLIE 1979a). If the recombination frequency in hermaphrodite spermatocytes (b) is constant, it follows that as the oocyte recombination frequency approaches zero with increasing age, the value of R in the hermaphrodite should never fall below 1/2b. For example, in the dpy 5 unc 75 interval the value of R in the final -  -  brood (0.08) is still higher than 1/2b (0.06). Of further interest is the possibility that the variation of recombination frequency with age is a continuum of the two germlines. Since the first brood measures the earliest oocyte recombination frequency (those events occurring right after the switch from spermatogenesis), one would expect the two germlines to have similar frequencies in this brood. In the dpy 5 unc 75 interval for example, knowing the -  -  value of R in the first brood (0.10) and the value of b (0.12), the value of a (0.09), the frequency of recombination in the oocyte, can be calculated. As predicted, the oocyte recombination frequency in this brood is close to, but not higher, than the spermatocyte frequency. YAMAMOTO (1961) measured recombination in hormonally transformed XY males of the Medaka and found the recombination frequency to be much higher than that observed in normal males. In this study, recombination was measured in males sexually transformed by the her 1 mutation. Similar to the previous results, the recombination -  frequency was significantly higher in the transformed males when compared to normal  30  males. This result can be interpreted as evidence that it is the sexual phenotype and not genotype that determines the frequency of recombination during gametogenesis. Meiotic recombination frequency in both sexes of C. elegans is affected by age and temperature. Recombination frequency decreases with maternal age in Drosophila (BRIDGES 1927; NEEL 1941), in mice (FISHER 1949), and in C. elegans (ROSE and BAILLIE 1979a). A fall in crossover frequency with paternal age was observed in two intervals. This variation of recombination frequency with parental age does not affect the results of other experiments. As described in MATERIALS and METHODS, only L4 hermaphrodites, which are easily identifiable at that stage, and young males were used in later experiments further characterizing recombination. The population of males used in these experiments was considered to be synchronous since all male recombination experiments were replicated and reproducible results were obtained. For example, the curves derived from four separate experiments examining the variation of recombination frequency with paternal age in the dpy-5 unc-13 interval could be superimposed. In conclusion, male recombination across the length of LG / was found to be approximately one-third less than that observed in the hermaphrodite. This decrease, however, was not uniform and one interval showed no sex-related difference in crossover frequency. By measuring recombination in the two germlines of the hermaphrodite and in transformed males, it has been concluded it is the physiology of the gonad, rather than the sexual karyotype of the germline, that determines the recombination frequency characteristic of a specific sex. It was also observed that male recombination in C. elegans varies with age and temperature, suggesting recombination is quantitatively rather than qualitatively different between the sexes. For this reason, it is recommended that the standard practices suggested by ROSE and BAILLIE (1979a) for hermaphrodite recombination experiments be also applied to male recombination studies (i.e. that studies measuring recombination frequency be carried out at 200 and all progeny from the male should counted).  31  Chapter 2: Characterization and Mapping of rec 1 -  INTRODUCTION  Mutations which disrupt the normal frequency and distribution of crossing over can identify genes important in the control of meiosis. Study of these mutations has revealed that in organisms in which recombination normally occurs, one crossover between the homologues is necessary for their proper disjunction (reviewed by JONES 1984, 1987). One class of genes is defined by mutants which are defective in the recombinational machinery and another is defined by mutants that alter the normal patterns of exchange during meiosis (reviewed by BAKER et al. 1976). The majority of mutations identified are recombination defective and result in reduced levels of recombination. In  D. melanogaster, recombination  defective mutants have been divided into three groups based on the distribution of their exchange events (reviewed by BAKER et al. 1976). One group is represented by the gene  mei-9, whose mutations decrease the frequency of crossing over but maintain the wild-type pattern of events (BAKER and CARPENTER 1972; CARPENTER and SANDLER 1974). The second group includes the genes mei-218, mei-41, and mei-251, whose mutations reduce the frequency of crossing over by differing amounts in different intervals, thereby disrupting both the frequency and pattern of exchange events (BAKER and CARPENTER 1972; CARPENTER and SANDLER 1974; SANDLER and SZAUTER 1978). The third group is represented by mutations in mei-352 which alter the distribution, but not the overall frequency of crossing over (BAKER and CARPENTER 1972). In C. elegans, the majority of meiotic mutants have been isolated as recessive mutations that increased the nondisjunction frequency of the X, resulting in a Him (high incidence of males) phenotype (HODGKIN, HORVITZ and BRENNER 1979). Mutations in the genes him-6 and him-14 are recombination defective and produce nondisjunction of the autosomes as well as of the X chromosome, presumably as a result of the reduction in crossing over (HODGKIN, HORVITZ and BRENNER 1979; KEMPHUES, KUSCH and WOLF 1988). A class of meiotic mutant not previously described is represented by the  32  recessive rec 1 mutation which increases both crossing over (ROSE and BAILLIE 1979b) -  and conversion (RATTRAY and ROSE 1988) on all chromosomes. Since rec 1 mutants -  are not radiation sensitive (HARTMAN and HERMAN 1982), the function of this gene appears to be specific to meiosis rather than to general DNA metabolism. In this thesis, the effect of rec 1 on the distribution of crossing over has been characterized and a map position -  has been determined.  33  Chapter 2: MATERIALS AND METHODS The source of mutations and chromosomal rearrangements is given in Chapter 1: MATERIALS and METHODS. Duplication mapping of rec - I: The possibility that sDp1(1,7) included the rec-1 locus was examined by measuring recombination in the dpy-5 dpy-14 interval in the presence of the duplication (sDp1 carries wild-type alleles of both of these markers). Rec-1 or N2 males were mated to dpy-5 dpy-1.4 rec-1/dpy-5 dpy-1.4 rec-1/sDpl(I,f) hermaphrodites and dpy-5 dpy-14/dpy-5 dpy-14/sDpl(I,f) controls respectively. Wild-type hermaphrodite progeny  resulting from this cross were individually plated and their progeny scored. sDpi-bearing hermaphrodites have lower brood sizes (duplication homozygotes are inviable) and increased nondisjunction of the X resulting in male progeny (ROSE, BAILLIE and CURRAN 1984). To identify the individuals that carried the duplication, broods of the size characteristic for sDp1(I,I) were examined for the presence of males, and the frequency of the double  homozygote class was determined. This class was expected to approach a frequency of 0.125 in the presence of the duplication and and a frequency of 0.25 in its absence. The recombination frequency in individuals lacking the duplication was calculated as described in Chapter 1: MATERIALS and METHODS. A gametic frequency of 0.43 for sDp1(I,f)(ROSE, BAILLIE and CURRAN 1984) was used to calculate the frequency of  crossing over in individuals determined to be of the genotype dpy-5 dpy-14 rec-1/ + + rec1/sDp1(I,l) and dpy-5 dpy-14 + / + + rec-1/sDp1(1j) using the formula:  p = 1 - [1 - 148D/17(D + W)]1/2  where D is the number of Dpy-5 recombinants and W is the number of wild-type progeny. This formula is based upon the assumptions that the sDp1 homozygote is inviable and that recombination between sDp1 and LC /does not occur in the dpy-5 dpy-14 interval. Similarly, sDp2 was used to map rec-1 by measuring recombination in dpy-5 dpy-14 rec-1/  34  + + rec-1/sDp2(I;f) hermaphrodites and in dpy-5 dpy-14 rec-1/ + + + /sDp2 controls.  This duplication covers both markers and sDp2-bearing worms were identified by the frequency of segregation of the double mutant as described for sDpl. A gametic frequency of 0.38 for sDp2(I;f) (ROSE, BAILLIE and CURRAN 1984) was used to calculate the frequency of crossing over in the presence of the duplication using the formula:  p = 1 - [1 - 75D/19(D + W)]112  where D is the number of Dpy-5 progeny and W is the number of wild types. This formula assumes that the sDp2 homozygote is not viable. Recombination was also measured in dpy5 unc-13 rec-1/ + + rec-1jsDp2 hermaphrodites and in dpy-5 unc-13 rec-1/ + + + /sDp2  controls. In this case, however, the duplication does not extend to unc-13 and as a result, sDp2-bearing hermaphrodites were identified by the presence of a large number of Unc-13 segregants amongst their progeny. Recombination in the dpy-5 unc-13 interval was calculated using the formula:  p =1 - [1 - 19D/(D w.)]1/2  16  where D is the number of Dpy-5 recombinants and W is the number of wild-type progeny. Deficiency mapping of rec - 1: To test if the ribosomal deficiency eDf2.4(I;f) deleted the rec-1 locus, dpy-11 unc-42/ + + ; rec-1/rec-1 or dpy-11 unc-42/ + + males were crossed to unc-54/eDf24 hermaphrodites and the resulting wild-type progeny individually plated.  Since eDf24 does not include zinc-54, only plates that segregated Dpy-11 Unc-42 progeny and failed to segregate Unc-54 individuals (indicating the presence of the deficiency) were scored. Recombination was measured in the dpy-11 unc-42 interval using the general recombination formula discussed in Chapter 1: MATERIALS and METHODS. The  35  deficiencies eDf4, eDf9, eDf10, and eDf13 were isolated using eDf24 as a balancer and all complement eDf24 and fail to complement unc-54 (ANDERSON and BRENNER 1984). To test if any of these deficiencies included rec-1, eDfX/eDf24 hermaphrodites were mated to unc-54/ + males and the resulting Unc-54 hermaphrodites were then mated to males of the genotype dpy-11 unc-42/ + + ; rec-1/rec-1 or dpy-11 unc-42/ + +. Wild-type hermaphrodite progeny were individually plated and their progeny screened for the presence of Dpy-11 Unc-42 segregants and the absence of Unc-54 segregants. The recombination frequency in the dpy-11 unc-42 interval was then measured and calculated as described above for eDf24.  36  Chapter 2: RESULTS rec 1 alters the distribution of crossing over: ROSE and BAILLIE (1979b) showed -  that rec 1 greatly enhanced the frequency of crossing over in small intervals. To determine -  the effect of rec 1 on recombination along the whole chromosome, four intervals spanning -  LG /were examined and the results are shown in Figure 4 (Data shown in Table 9). The bli 3 unc 11 interval, located on the left arm of LG I, was 12.8 in rec 1 homozygotes and -  -  -  14.8 m.u. in controls, showing no recombination enhancement in the presence of rec 1. -  Recombination in the unc 11 dpy 5 interval, however, showed a 3-fold enhancement in rec 1 -  -  -  homozygotes (6.7 m.u. compared to 2.3 m.u. in controls). The dpy 5 unc 101 interval, -  -  normally 12.0 m.u., was 21.2 mu. in rec 1 homozygotes, demonstrating extensive -  enhancement in the presence of rec 1. The unc 101 unc 54 interval, located on the right -  -  -  arm of the chromosome, was severely reduced from 14.4 m.u. in controls to 4.6 m.u. in rec 1 -  homozygotes. The dpy 5 unc 54 interval, however, was 31.6 m.u. in controls and 30.6 m.u. -  -  in rec 1 homozygotes, indicating that total recombination on the right arm of LG /in rec 1 -  -  homozygotes did not change when compared to controls. The total genetic length of LG I was 45.3 m.u. in rec 1 homozygotes and 43.5 m.u. in controls. -  sDp1 (I,T) suppresses the Rec-1 phenotype: ROSE and BAILLIE (1979b) found no  linkage between rec 1 and any markers located in the gene clusters of the autosomes. When -  markers located at the ends of the chromosomes were tested, rec 1 showed loose linkage to -  the gene unc 54, located on the right end of LG I (ROSE unpublished results). Since rec 1 -  -  is completely recessive (ROSE and BAILLIE 1979b), a strategy using two large duplications of LG I sDp1 and sDp2, to map the gene was developed (data shown in Table ,  10; extent of duplications shown in Figure 4). Although sDp1 does pair and recombine with LG I, it does so rarely in the dpy 5 dpy 1.4 region (ROSE, BAILLIE and C URRAN 1984; -  -  McKIM, PETERS and ROSE 1993) and in conjunction with the small size of this interval, it is unlikely that any recombinants recovered were the result of a recombination event with the duplication. In the absence of the duplication, the frequency of crossing over in dpy 5 -  37a  FIGURE 4.-Meiotic maps of LG /in the presence of the rec 1 mutation and in controls. -  unc- 13 bli- 3^  unc- 11  dpy- 5  unc- 101  unc- 5  Rec-1 unc- 13 dpy- 5 ^ bli- 3 unc- 11  unc- 101 unc- 54  I 1^i  sDp2 sDp 1 5 m.u.  Linkage Group I  38 Table 9 The effect of rec - 1 on crossing over on LG /  Genotype  Wts  Recombinants  pX100(C.I.)a  bli-3 unc-11/ + + b  1686  170 Unc  14.8(12.4-17.4)  bli-3 unc-11 rec-1/ + + rec-1  990  79 Unc  12.8(10.0-16.1)  unc-11 dpy-5/ + + b  3786  58 Dpy  2.3(2.0-2.8)  unc-11 dpy-5 rec-1/ + + rec-1  2033  91 Dpy 91 Unc  6.7(5.7-7.6)  dpy-5 unc-13/ + +e  3119  34 Dpy 32 Unc  1.6(1.2-2.0)  dpy-5 unc-13 rec-1/ + + rec-1d  3706  156 Dpy  6.3(5.3-7.3)  dpy-5 unc-101/ + + e  889  79 Dpy 66 Unc  12.0(10.0-14.0)  dpy-5 unc-101 rec-1/ + + rec-1  1369  183 Dpy 213 Unc  21.2(20.1-22.2)  unc-101 unc-54/ + +b  1187  116 Unc-101  14.4(11.8-17.1)  unc-101 unc-54 rec-1/ + + rec-1  1973  61 Unc-101  4.6(3.6-5.8)  dpy-5 unc-54/ + +  1620  349 Dpy  31.6(30.3-32.9)  dpy-5 unc-54 rec-1/ + + rec-1  1698  355 Dpy 267 Unc  30.6(29.2-32.0)  C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Data from Table 4. C Data from Table 3.  d Data from Table 10. e Data from Table 12.  39  Table 10 Duplication mapping of rec-1  Genotype  Wts  Recombinants  pX100(C.I.) a  dpy-5 dpy-14 + / + + rec-1  3238  28 Dpy-5  1.3(0.88-1.8)  dpy-5 dpy-14 + / + + rec-1/sDp1  1614  6 Dpy-5  1.6(0.7-3.5)  dpy-5 dpy-14 rec-1/ + + rec-1  8659  321 Dpy-5  5.5(5.4-5.6)  dpi-5 dpy-14 rec-1/ + + rec-1/sDp1  1201  5 Dpy-5  1.8(0.7-4.1)  dpy-5 dpy-14 rec-1/ + + +  2213  25 Dpy-5  1.7(1.1-2.4)  dpy-5 dpy-14 rec-1/ + + +/sDp2  1976  7 Dpy-5  0.7(0.3-1.4)  dpy-5 dpy-14 rec-1/ + + rec-1  1729  67 Dpy-5  5.8(4.4-7.2)  dpy-5 dpy-14 rec-1/ + + rec-1/sDp2  1002  13 Dpy-5  2.6(1.3-4.2)  dpy-5 unc-13/ + + b  3119  34 Dpy 32 Unc  1.6(1.2-2.0)  dpy-5 unc-13 + / + + rec-1  2133  23 Dpy  1.6(1.0-2.4)  dpy-5 unc-13 rec-1/ + + rec-1  3706  156 Dpy  6.25(5.3-7.3)  dpy-5 unc-13 + / + + rec-1/sDp2  1057  4 Dpy  0.2(0.08-0.6)  dpy-5 unc-13 rec-1/ + + rec-1/sDp2  1977  41 Dpy  1.4(0.9-1.9)  C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Data from Table 3.  40  dpy-14 rec-1/ + + + heterozygotes was 0.013. In rec-1 homozygotes, the frequency of recombination increased to 0.055 in this interval. In the presence of sDp1, the frequency of recombination in the dpy-5 dpy-14 interval was reduced to 0.018 in sDp1/dpy-5 dpy-1.4 rec-  1/ + + rec-1 heterozygotes, similar to the value of 0.016 observed in sDpl/dpy-5 dpy-14 + / + + rec-1 heterozygotes, indicating that sDp1 carried a wild-type allele of rec-1. These results are also consistent with the finding that rec-1 is completely recessive to its wild-type allele (ROSE and BAILLIE 1979b). To ensure that the suppression observed was not a general feature of LG /duplications, recombination was also measured in the presence of  sDp2, a large duplication of the left half of the chromosome. The frequency of crossing over between dpy-5 and dpy-1.4 in the presence of sDp2 and Rec-1 (0.026) was 3-fold higher than in the absence of Rec-1 (0.007), indicating that the Rec-1 phenotype was expressed despite the presence of the duplication. Although the frequency of crossing over in the presence of  sDp2 and Rec-1 was significantly higher than in the absence of Rec-1, the frequencies were much lower than those obtained in the absence of the duplication (0.06 in rec-1 homozygotes and 0.02 in heterozygotes). To confirm the possibility that the overall decrease in recombination frequencies could be attributed to a reduced recovery of recombinants in the presence of sDp2, recombination was examined in another interval. The frequency of crossing over was examined in sDp2/dpy-5 unc-13 rec-1/ + + rec-1 and sDp2/dpy-5 unc-  13/ + + rec-1 heterozygotes. The frequency of recombination between dpy-5 and unc-13 in the presence of sDp2 and Rec-1 (0.014) was 7-fold higher than that observed in the absence of Rec-1 (0.002). Since the recombination formula used to calculate the frequencies assumes that both sDp1 and sDp2 are inviable as a homozygotes, these results may be explained if  sDp2 homozygotes can be recovered and are affecting the recovery of recombinants. An alternative explanation may be that sDp2 suppresses recombination between the two homologues, however, recombination between the duplication and the chromosomes /has not been observed (ROSE, BAILLIE and CURRAN 1984).  eDf24 (I) fails to complement rec-1: The duplication sDp1 covers the right arm of LG I, including most of the centrally located cluster. Since rec-1 was suppressed by sDpl,  41  deficiencies of the right end were tested for failure to complement the mutation (data shown in Table 11). The deficiencies used in this study and their known breakpoints are shown in Figure 5. The dpy-11 unc-42 interval, normally 2.7 m.u., increases to 6.4 in rec- 1 homozygotes. In eDf24/rec-1; dpy- 11 unc-42/ + + heterozygotes, this interval showed a 2fold enhancement in recombination (5.6 m.u.) when compared to eDf24/ + ; dpy-11 unc-42 controls (3.0 m.u.), indicating that the deletion failed to complement the rec-1 mutation. eDf24 had previously been used as a balancer to isolate a number of deletions of the unc-54  locus (including eDf4, eDf9, eDf10, and eDf13) (ANDERSON and BRENNER 1984) which had undefined right breakpoints. Although these deficiencies genetically complemented eDf24, the possibility remained they physically overlapped eDf24 in a region that did not include any essential genes. All of the deficiencies tested complemented rec-1, indicating that if the deletions did overlap with eDf24, rec-1 was not included in the region of overlap.  42 Table 11 Deficiency mapping of rec-1  Genotype  Wts  Recombinants  pX100(C.I.) a  unc-42 dpy-11/ +  1250  26 Dpy 20 Unc  2.7(2.0-3.6)  unc-42 dpy-11/^; rec-1/rec-1  1219  66 Dpy 59 Unc  7.6(6.4-9.0)  unc-42 dpy-11/ + + ; rec-1/eDf4  999  19 Dpy 23 Unc  3.1(2.2-4.1)  unc-42 dpy-11/ + + ; rec-1/eDf9  693  8 Dpy 11 Unc  2.0(1.2-3.1)  unc-42 dpy-11/ + + ; rec-1/eDf10  1127  29 Dpy  3.8(2.5-5.4)  unc-42 dpy-11/ + + ; rec-1/eDf13  1558  46 Dpy 49 Unc  4.5(3.6-5.5)  unc-42 dpy-11/^; /eDf24  1668  35 Dpy 33 Unc  3.0(2.3-3.8)  unc-42 dpy-11/^; rec-1/eDf24  2119  88 Dpy 72 Unc  5.6(4.8-6.5)  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).  43a  FIGURE 5.-Deficiency map of the unc-54 region of LG I. The right breakpoint of eDf24 is known to map within the ribosomal cluster whose genetic locus is rrn-1 (see APPENDIX III). The left breakpoint maps within nonribosomal sequences to the right of unc-54 (see Chapter 2: RESULTS). It is not known how far eDf9 and eDf4 extend to the right, only that they complement eDf24. Both deficiencies may delete common sequences with eDf24, if no essential genes are included.  43b  unc— 54 gus— 1 let-206 unc— 59^lev— 11 1 lev— 10  let-50 let-208 ces-2  rrn— 1  I^I  eDf24  1  H  eDf 1 0 eDf 13 I eDf4 ^ eDf9 ^  1 LG I (right)  44  Chapter 2: DISCUSSION The rec 1 mutation, initially described as a general recombination enhancer, increases -  meiotic crossing over (ROSE and BAILLIE 1979b) and conversion (RATTRAY and ROSE 1988), without disrupting the normal pattern of meiotic exchange. The intervals tested in these studies, however, were located in the central gene clusters, or at the distal tips of the chromosome arms, where a small clustering effect also exists. BRENNER (1974) first proposed that the gene clusters observed on the meiotic map were the result of recombination suppression in the region. This has been supported by several studies that have shown that the cluster is a result of less recombination per base pair than the genomic average (GREENWALD et al. 1987; KIM and ROSE 1987; PRASAD and BAILLIE 1989; STARR et al. 1989). In this thesis, crossing over in the gene cluster of LG / was enhanced in rec 1 homozygotes, consistent with previous results (ROSE and BAILLIE 1979b). The -  level of enhancement, however, was dependent on the interval tested within the cluster; the frequency of recombination increased 3-fold in the uric 11 dpy 5 interval and 5-fold in the -  -  dpy 5 unc 13 interval. The differential level of enhancement may be explained if rec 1 -  -  -  alleviates the recombination suppression normally present in the cluster and if this suppression is more extreme in some regions. This interpretation is supported by the facts that the dpy 5 unc 13 interval contains the most recombinationally suppressed region of the -  -  cluster (STARR et al. 1989), and shows the most enhancement in the presence of Rec-1. This enhancement, however, is not a general feature of the rec 1 phenotype since in two -  large intervals flanking the cluster the frequency of crossing over was unaffected or reduced. Recombination in the bli 3 unc 11 interval was not different in controls, whereas unc 101 -  -  -  anc 54, a comparably-sized interval on the right end, showed a 4-fold reduction. The -  apparent suppression of crossing over in this interval may be explained if rec 1 reduced -  interference values on the right arm, or if the recombination frequency normally observed in that region is enhanced per base pair, compared to the genome average, however, neither possibility has been confirmed.  45  Darlington (1937) proposed that the formation of a chiasmata between homologues was necessary for their proper disjunction. In C. elegans, the suppression of crossing over between homologues results in their random segregation (ZETKA and ROSE 1992; this study). Consistent with this result, the genetic length of chromosome / approaches 50 m.u. (corresponding to an average of one crossover per meiosis) in wild-type hermaphrodites, males (ZETKA and ROSE 1990; this study), translocation heterozygotes (McKIM, HOWELL and ROSE 1988; McKIM, PETERS and ROSE 1993) and inversion heterozygotes (ZETKA and ROSE 1992; this study). The conservation of a 50 m.u. genetic length can be explained by some cytological evidence suggesting that the meiotic chromosomes are held together by a terminalized chiasma at metaphase of meiosis I (ALBERTSON and THOMSON 1993). The genetic size of chromosome /in rec 1 -  homozygotes also approached 50 m.u., compatible with an average of one crossover every meiosis. Although rec 1 does not affect the total number of crossovers, its effect of -  expanding some regions and contracting others, disrupted the normal distribution of exchanges. This suggests the mechanism responsible for ensuring that one crossover occurs between the homologues every meiosis is epistatic to rec 1(+), the role of which appears to -  be in determining preferred sites of exchange. The existence of such sites is supported by studies in S. cerivisiae where a secondary pairing site on chromosome /// (GOLD WAY, ARBEL and SIMCHEN 1993) was found to be a recombination hotspot (GOLDWAY et al. 1993). The role of rec 1(+) may be in the identification of such sites during pachytene and -  in its absence, the distribution of crossover events on the chromosome becomes related to physical size. On chromosome /, a preferential site of exchange may be located on the right arm and would explain the high frequency of recombination in the unc 101 unc 54 region in -  -  spite of its small physical size (COULSON et al. 1986, 1988). In Drosophila, mutations that alter the distribution of exchanges have been isolated (reviewed by Baker et al. 1976). Of these, however, mutations in all but one gene also reduce the frequency of crossing over. Mutations in the exceptional gene, mei 352, disrupt -  the distribution of exchanges along the chromosome, but do not alter their frequency  46  (BAKER and CARPENTER 1972), similar to rec 1. In both mutants, the frequency of -  recombination is increased in regions which are normally recombinationally suppressed. Recombination in mei 352 mutants is enhanced in the centric heterochromatin and on the -  fourth chromosome of mei 352 mutants (BAKER and CARPENTER 1972; SANDLER and -  SZAUTER 1978). In rec 1 mutants, crossing over is enhanced in the gene clusters, and in -  the case of chromosome /, also in a region adjacent to the cluster (the unc 29 unc 101 -  -  interval). This region has not been cloned (COULSON et al. 1986, 1988), presumably because of the presence of repetitive sequences. The meiotic centromere of chromosome I has been mapped to the dpy 5 unc 75 interval and is thought to be tightly linked to the left -  -  breakpoint of the inversion hIn1(I), which lies between unc 29 and unc 75 (ZETKA and -  -  ROSE 1992; this study). This is supported by the structure of two recombinant chromosomes, derived from inversion heterozygotes, which proved to be deficiencies with indistinguishable left breakpoints. These deficiencies are thought to have arisen as a result of the breakage of a dicentric bridge at anaphase II. That two independent events could result in identical breakpoints can be explained by the presence of a fragile site in the region, or if the meiotic centromere is tightly linked to the breakpoint of hInl(I) (mapping to the unc 29 unc 75 interval) and as a result, the region pulled apart at anaphase is small, and -  -  the probability of two independent breaks in the region is high. In addition to the presence of potential centromeric sequences, a family of repetitive sequences (La VOLPE, CIARAMELLA, BAllICALUPO 1988) have also been mapped to this region. For these reasons, it is possible that rec 1 disrupts two distinct forms of crossover suppression; one -  form responsible for establishing the gene cluster and the other responsible for the crossover suppression associated with repetitive sequences. mei 352 mutants also exhibit a decreased -  frequency of single-exchange tetrads and an increased frequency of double-exchange tetrads, suggesting the defect lies in the establishment of spatial restrictions on chiasma formation (BAKER and CARPENTER 1972). While it is possible that the crossover suppression observed on the right arm of LG /in rec 1 mutants can be explained by lower interference -  values, the number of double crossovers expected in the unc 101 unc 54 interval -  -  47  (approximately two) cannot explain the 3-fold reduction in genetic length. Furthermore, the genetic length of chromosome / in rec 1 mutants approached the 50 m.u. observed in -  controls, suggesting that if double-crossing over occurred, it did so at a low frequency. While rec 1 and mei 352 may be defective in genes that specify a precondition for -  -  exchange necessary for determining its probability or distribution on the chromosome, they differ in two important respects. Firstly, mei 352 specifically increases recombination in -  recombinationally-suppressed regions and does not decrease recombination in any interval, whereas rec 1 increased recombination in the cluster and also decreased the probability of -  exchange in the unc 101 unc 54 region. Secondly, mei 352 females are partially sterile and -  -  -  this phenotype cannot be explained by nondisjunction of the autosomes as a result of reduced recombination, since mutants have normal levels of exchange (BAKER and CARPENTER 1972). Paradoxically, mei 352 mutants exhibit increased nondisjunction -  and chromosome loss which occurs at a frequency too low to explain the sterile phenotype (BAKER and CARPENTER 1972). Recent evidence suggests that the mei 352 gene -  product is actually involved in the sex-determination pathway and that its effect on meiosis is indirect (K. McKIM pers. comm.); the mutation may disrupt sex-determination genes which regulate meiosis-specific genes. These observations suggest that while mei 352 and -  rec 1 clearly disrupt a meiotic process necessary for establishing spatial limits on the -  distribution of exchange, rec 1 specifically disrupts this process and as a result, remains the -  lone representative of an unusual class of meiotic mutants.  48  Chapter 3: The Meiotic Behaviour of an Inversion INTRODUCTION  Chromosome rearrangements that result in crossover suppression are useful for a wide range of genetic experiments, including the dissection of chromosomal features responsible for meiotic behaviour. An understanding of the mechanisms responsible for the elimination of meiotic events in the presence of the rearrangement can lead to the discovery and description of sites necessary for the recognition and synapsis of homologues, meiotic exchange, and subsequent disjunction. For example, studies of translocations in C. elegans have led to the proposal that each chromosome contains a single region necessary for homologue recognition and pairing (ROSENBLUTH and BAILLIE 1981; McKIM, HOWELL and ROSE 1988; reviewed by ROSE and McKIM 1992). Translocations are the major class of dominant crossover suppressor in C. elegans (HERMAN 1978; ROSENBLUTH and BAILLIE 1981; HERMAN, KARI and HARTMAN 1982; FERGUSON and HORVITZ 1985; CLARK et al. 1988; McKIM, HOWELL and ROSE 1988), although intrachromosomal crossover suppressors have also been described (HERMAN 1978; ANDERSON and BRENNER 1984; ROSENBLUTH, JOHNSEN and BAILLIE 1990). For example, deletions of the chromosome V end that does not contain the region necessary for homologue recognition were found to suppress recombination for several map units beyond the breakpoint of the deletion (ROSENBLUTH, JOHNSEN and BAILLIE 1990). The authors proposed that the deletions eliminated sites required for meiotic synapsis which occurs after homologue recognition has taken place. Insertional duplications have a polar effect on recombination (HERMAN, ALBERTSON and BRENNER 1976; McKIM 1990; reviewed by ROSE and McKIM 1992). The intrachromosomal suppressor mnCl(II) has been used to balance a large region of chromosome // (HERMAN 1978) and although mnC1(H) is widely believed to be an inversion, no reversal of gene order has been demonstrated.  49 In this thesis, the first genetic inversion in C. elegans is described. Since this inversion, hIn1(I), suppresses crossing over in a region not previously balanced by translocations, it is representative of a new class of balancers for the genome. Furthermore, the meiotic behavior of hIn1(I) with respect to homologue recognition and the centromeric behavior of chromosome I has been characterized.  50  Chapter 3: MATERIALS AND METHODS The source of mutations and chromosomal rearrangements is given in Chapter 1: MATERIALS and METHODS. Isolation of hInl (I): N2 males were treated with 1500 rads of gamma radiation (ROSENBLUTH, CUDDEFORD and BAILLIE 1985) and mated to anc 101(m1) unc-  54(e190) homozygotes. unc 101 unc 541^+ hermaphrodites resulting from this mating -  -  were individually plated and their progeny screened for the absence of Unc-101 recombinants. In total, 900 chromosomes were screened and one isolate recovered that suppressed crossing over. Egg-hatching frequency: Hermaphrodites of the genotype unc 101 lev 11/hIn1(k+ -  -  hInl(I)/hIn1(1), hDp131/unc 101 1ev 11 or hDfll/unc 101 unc 54 were individually plated -  -  -  -  and allowed to lay eggs for two 10-12 hour periods. The hermaphrodites were then transferred and the eggs remaining on the plate counted. All resulting progeny were counted three days later. Induction of genetic markers on hInl (I): hIn1(1) homozygous males were treated with 25 mM EMS (ROSENBLUTH, CUDDEFORD and BAILLIE 1983) using the procedure described by BRENNER (1974). The mutagenized males were then mated to unc 75(e950) unc 101(m1) homozygotes for 24 hours. These hermaphrodites were -  -  individually plated and their progeny screened for the presence of Unc-75 individuals. Since the unc 75 unc 101 interval is located in the crossover suppressed region of hInl(I) -  -  heterozygotes, any Unc-75 individuals recovered were expected to be the result of an induction of a new mutation on the hIn1(I) chromosome. 18,700 chromosomes were screened and two mutations were recovered; unc 75(h1041), and unc 75(h1042). Both new -  -  unc 75 alleles were lethal as homozygotes and were maintained as heterozygotes. Both new -  alleles produced the Unc-75 visible phenotype when crossed to males heterozygous for unc75(e950). To induce an unc 54 mutation on the hInl(I) chromosome, hIn1(1) males -  mutagenized in the procedure described above were mated to lev 11 let 49 + + I + + unc-  -  51  54 let-50 (RW3072) hermaphrodites for 24 hours. These hermaphrodites were then  individually plated, and their progeny screened for Unc-54 individuals. A total of 5200 chromosomes were screened, of which one-half are heterozygous with the unc-54 let-50 chromosome. Some Unc-54 isolates could arise from recombination between unc-54 and let50 in the parental strain. To test if the new unc-54 mutations were linked to hIn1(I), the  progeny of putative hIn1(1)t+ + unc-541/unc-101 1ev-11 + hermaphrodites were screened for the presence of Unc-101 and Lev-11 recombinants. One strain, KR2151, exhibited complete recombination suppression in heterozygotes indicating the new mutation, unc-54(h1040), was linked to hInl(I). Recombination in hIn1 (I) homozygotes: Recombination was examined in hIn.1(I)  homozygotes in three intervals; dpy-5 unc-75, dpy-5 unc-29, and dpy-5 unc-54. To examine crossing over in dpy-5 unc-75, Dpy-5 recombinants were picked from amongst the progeny of hInl(I)[+ unc-75 +]/hIn1(1)[dpy-5 + unc-54] hermaphrodites and mated to unc-75(e950)/+  males to confirm the presence of unc-75(h1041 or h1042). Unc-75 progeny resulting from this cross were then mated to dpy-5 unc-75(e950)/+ + males to ensure the dpy-5 mutation was still present. The resulting hIni(I)Icipy-5 unc-75,Vdpy-5 unc-75(e950) progeny were crossed to hIn1(1)1-unc-54Y + males and a fraction of the wild-type individuals resulting from this cross were of the desired genotype hInl(Opy-5 + unc-75j/hIn1(1)1+ unc-54 This experiment also confirmed the gene order in hInl (I) was dpy-5 unc-54 unc-75 (see Chapter 3: RESULTS). Knowing the map distance between dpy-5 and unc-54 in hInl(I) homozygotes (see Table 13), recombination was measured in the unc-54 unc-75 interval in the same heterozygotes used in the three-factor experiment using the formula:  p = 9 - [81 - 20(2D W)(9D - W)/(D W)2]1/2  10(2D W)/(D W)  52  where D is the number of Dpy-5 recombinants and W is the number of wild-type progeny. Since dpy-5 and unc-29 are outside the boundary of hIn1(I) crossover suppression, the mutations can be crossed onto the inversion chromosome. Since hIn1(I) is viable as a homozygote, recombination was measured in hInl(I)[+ +]/hInl(Didpy-5 unc-29J and hIn1(01-+ +J/hIn1(Dldpy-5 unc-54] heterozygotes using the general mapping methods  described in Chapter 1: MATERIALS and METHODS. Analysis of recombinants from hIn1 (I) heterozygotes: Four rare recombinants  falling into two classes, duplications and deficiencies, were recovered from hIn 1 (I) heterozygotes. The deficiencies hDf11 and hDf12 were recovered from hIn1(1)1+ +]/unc-101 unc-54 and hIn1(k+ +J/unc-75 unc-101 heterozygotes respectively. The duplications hDp131 and hDp132 were both recovered from hInl(I)1+ qq/unc-101 1ev-11 heterozygotes,  based on the Lev-11 visible phenotype. These duplications were mapped with respect to visible markers. For example, markers inside the region of hIn1(I) crossover suppression were tested by mating males of the genotype unc-75 unc-101/hInl(Di + + Jto hDp(D/unc101 1ev-11 hermaphrodites. A fraction of the wild-type progeny from the cross were of the  desired genotype hDp(I)/unc-75 unc-101 + / + unc-101 1ev-11. Upon examining the progeny of such individuals, Unc-75 individuals were observed if the duplication did not carry unc-75(+). In the event the duplication did carry unc-75(+), no Unc-75 individuals were observed. A similar procedure was followed for markers outside the region of crossover suppression with the exception that males heterozygous for a wild-type, rather than a hIn1(I) chromosome, were used. To determine if the duplications also carried unc-54(+), hIn1(I)[+ +J/unc-75 unc-54 males were mated to hDp(D/unc-75 unc-101 hermaphrodites.  A number of the progeny resulting from this mating were of the genotype hDp(I)/unc-75 u,nc-101 +/unc-75 + unc-54. Since Unc-75 Unc-54 individuals are similar in phenotype to  Unc-54 individuals, several wild type progeny from the latter heterozygote were plated and their progeny examined. If the duplication carried unc-54(+), a fraction of these individuals would be of the genotype hDp(I)/unc-75 unc-54.  53  The deficiencies hDfll and hDf12 were complementation tested with several visible markers by mating hDfll/unc-101 unc-54 and hDf12/unc-75 unc-101 hermaphrodites to either unc-x/ + or lev-11/ + males. The F1 progeny resulting from this mating were screened for both males and hermaphrodites Unc-x or Lev-11 in phenotype, the presence of which indicated the deficiency did not carry either unc-x(+) or lev-11(+). DAPI staining of hDp132: To determine if hDp132 was a free duplication, the meiotic chromosomes of an hDp132/unc-29 unc-75 hermaphrodite were stained with DAPI as described by MOENS and PEARLMAN (1991). Hermaphrodites were placed in a solution of 0.03% TWEEN and the gonads were removed. The tissue and cells were then fixed in 4% formaldehyde and allowed to dry at room temperature overnight. The slides were then treated with 5-10 ul/ml of DAPI solution (0.1 mg/ml DAPI in PBS) in 1 ml of moui:i.ting solution and examined under a fluorescence microscope (330-380 nm, reflector 420 nm, barrier 420 nm). Interaction of hinl (I) with szT1(I;X): Recombination between the boundary of hIn1(1) crossover suppression and the szT/(/;X)/breakpoint was measured by scoring the  Unc-101 progeny from hermaphrodites of the genotype hIn(1)1 + + /szT1(1;X)I-unc-1O1; lon-21. To measure crossing over between szT/(I,'X)/ and chromosome /, Unc-101  hermaphrodite progeny were scored from + ; /szT1(I;X)1-unc-1O1; lon-21 hermaphrodites. In both cases, the crossover frequency (p) between the szT1(I;X) breakpoint and unc-101 (or the hIn1(I) boundary of crossover suppression) is defined by the following formula:  p = 4 - [16 - 60 1_7(U + W)]1/2  6  where U is the number of Unc-101 recombinants and W is the number of wild-type progeny.  54  Interaction of hIn I (I) with hT2(/;///): To examine the interaction of hIn1(1) with the translocation hT2(1;III), recombination was measured in +^+ /hT2(1,117)[bli-.4 dpy-5 unc541 and hInl(I)[+ +^dpy-5 une-5Jheterozygotes using the formula:  p = 1 - [1 - 20D(3D W)/(4D 2W)2]1/2  (3D + W)/(2D W)  where D is the number of Dpy-5 recombinants and W the number of wild types. The segregation of hIn 1 (I) and hT2(/;///)/ was examined by scoring the Dpy-5 Unc-29 progeny of a hIn1(I)1-+ +^dpy-5 unc-29] hermaphrodite and + + dpy-5 unc-29] control.  Lethal screen using hInl (I) as a balancer: Hermaphrodites of the genotype hIn1(1)[unc-54_1/unc-101 1ev-11 + were treated with 17 mM EMS (ROSENBLUTH,  CUDDEFORD and BAILLIE 1983) using the procedure described by BRENNER (1974). Wild-type F1 progeny from these hermaphrodites were individually plated and their progeny screened for the absence of Unc-101 Lev-11 individuals.  55  Chapter 3: RESULTS Isolation of a crossover suppressor for the right end of LG ^hIn1(I) was identified  in a screen for gamma mutations that suppressed crossing over between unc-101 and unc-54, a 14 m.u. interval located at the right end of LG I. This map distance was reduced to 0.04 m.u. in hIn1(1)1+ +Yunc-101 unc-54 heterozygotes (see Table 12). Since recombination in this interval was measured using the Unc-101 recombinant class (Unc-54 recombinants are indistinguishable from the double mutant), the possibility that hIn1(I) was a suppressor of the Unc-101 phenotype remained. For this reason, crossing over was examined in unc-101 1ev-11 heterozygotes from which both recombinant classes were recovered. This interval was 9.0 m.u. in unc-101 lev-11/ + + heterozygotes and 0.07 m.u. in hInl(I) f+ +ilunc-101 1ev-11  heterozygotes, demonstrating extensive crossover suppression of the right arm of LG /in hInl (I) heterozygotes. Individuals homozygous for hInl (I) were fertile and wild type in  appearance. Since most crossover suppressors identified in C. elegans are tranlocations, the segregation of hIn1(I) from a normal homologue marked with an unc-101 mutation was examined. The predicted segregation pattern of wild-type and Unc progeny for a translocation heterozygote is 5:1 (HERMAN 1978; ROSENBLUTH and BAILLIE 1981). heterozygotes segregated wild-type and Unc progeny in a 3:1 ratio (2060 wild types: 672 Unc-101 individuals); a segregation pattern characteristic of an intrachromosomal rearrangement. hIril (I) heterozygotes have wild-type zygote viability: To further confirm that hIn1(I) was an intrachromosomal rearrangement, the egg-hatching frequency of individuals  homozygous and heterozygous for the mutation was determined and is shown in Table 13. The egg-hatching frequencies for heterozygotes is not statistically different than for homozygotes, both of which are high, suggesting that few or no aneuploid gametes are being produced in the former. The egg-hatching frequencies of two recombinants derived from hIn1(I) heterozygotes is also shown for comparison. The egg-hatching frequency of  56a Table 12 Effects of hIn1(I) on crossing over on Linkage Group I  Genotype  ^  Wild types^Recombinants^pX100(C.I.)a  unc-101 unc-54I + + b^1187^116 Unc-101^14.4(11.8-17.1) unc-101 unc-54IhInl^1584^1 Unc-101^0.04(0.002-0.25) unc-101 ley-11I + +^1492^99 Unc 82 Lev^9.0(7.8-10.3) unc-101 ley-111 hInl^2062^1 Lev^0.07(0.004-0.39) unc-75 unc-101I + + b^3192^68 Unc-101^3.2(2.7-3.8) unc-75 unc-101IhInl^2211^1 Unc-75^0.07(0.003-0.30) dpy-5 unc-101I ± +^889^79 Dpy 66 Unc^12.0(10.1-14.0) dpy-5 unc-101IhInl^1975^165 Dpy 148 Unc^11.7(11.2-12.2) unc-29 lin-11/ + + ; hzm-5/+^1514^14 Unc-29^1.4(0.8-2.3) unc-29 lin-11/hInl; him-5/+ ^1381^44 Unc-29^4.7(3.4-6.3) dpy-5 unc-29 unc-75I + + +b 1598^34 Dpyc  36 Unc-29 Unc-75c^3.4(2.6-4.2) 2 Unc-29c,d 63 Unc-75d^6.0(4.7-7.6) dpy-5 unc-29 unc-75IhIn1 ^1669^68 Dpyc  52 Unc-29 Unc-75c^5.5(4.6-6.5) 4 Unc-29c,d 81 Unc-75d^7.5(5.9-9.5) unc-11 dpy-5/ + + b ^3786^58 Dpy 61 Unc^2.3(2.0-2.8) zinc-11 dpy-5/hInl^1345^46 Dpy 41 Unc^4.8(3.9-5.9) blz-3 unc-11I + +b ^1686^170 Unc^14.8(12.4-17.4) blz-3 unc-11IhIn1^1232^191 Unc^22.7(19.4-26.0)  56b a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Data from Table 4. C dpy-5 unc-29  interval.  d unc-29 unc-75 interval.  57 Table 13 Egg-hatching frequencies of hInl (I) heterozygotes and recombinants  Genotype^  Egg-hatching Frequency  h/n//h/n/^  0.985(465/472)  unc-101 lev-11/hInli + + J^  0.983(567/577)  unc-101 unc-54/hDf11^  0.356(73/205)  unc-101 lev-11/hDp131^  0.982(567/577)  58  hDfll/unc-101 unc-54 and hDp131/unc-101 1ev-11 heterozygotes was 36% and 98%  respectively. Gene order is inverted in hInl(I): STURTEVANT (1921) established that three loci  in D. melanogaster and D. simulans were not in the same sequence on the genetic maps of the two species, thus defining the first inversion. To determine if hInl(I) was an inversion, the order of genes was examined by the induction of three mutations on the rearranged chromosome: unc-54(h1040), unc-75(h104 1), and unc-75(h10.42). The unc-75 mutations were recessive lethals that produced an Unc-75 phenotype when heterozygous with talc75(e950), and both were used in the following experiments. Dpy-5 and Unc-54 recombinant  progeny from a hIn1(1)[dpy-5 unc-54 + j/hIn1(1)[ + + unc-75I hermaphrodite were individually mated to unc-75(e950)/ + males to determine if the recombinant chromosome carried one of the lethal unc-75 mutations. The normal order of these genes is dpy-5 unc-75 unc-54 (EDGLEY and RIDDLE 1990). If the order of unc-75 and unc-54 were reversed, all  Dpy-5 recombinants should fail to complement unc-75(e950), whereas the Unc-54 recombinants should complement unc-75. Of 17 Dpy progeny examined with h1042 and 10 with WV, all 27 failed to complement unc-75. Of 12 Unc-54 progeny examined with h/042 and 6 with h1041, all 18 complemented unc-75. This demonstrated that either the gene order in hIn1(1.) is dpy-5 unc-54 unc-75, or that the order is unchanged but unc-54 is now tightly linked to unc-75. To distinguish between these two possibilities, Dpy-5 progeny from a hIn1(1)1-clpy-5 + unc-75(h1042)1/hIn1A+ unc-54 +1 hermaphrodite were individually plated and their progeny examined for Dpy-5 Unc-54 segregants. If the gene order in hIn1(1) were dpy-5 unc-54 unc-75, only some of the Dpy-5 recombinants were expected to  segregate Dpy Uncs. Of 243 Dpy progeny examined, 120 segregated the double mutant and 123 did not, indicating that gene order of unc-75 and unc-54 is reversed in hIn1(1) with respect to wild type and establishing that hIn1(1) is an inversion. Recombination frequency in hInl(I) homozygotes is normal: Inversion  homozygotes do not experience the pairing problems inherent in heterozygotes. To determine if crossing over occurred in hIn1(1) homozygotes, and at what frequency,  59  recombination was measured in three intervals: one interval outside the boundary of crossover suppression in heterozygotes and two spanning the boundary. The results are shown in Table 14. The dpy-5 unc-75(h1042) distance in hinl(I) homozygotes was obtained from the same experiment as the gene order. The dpy-5 unc-54 and dpy-5 unc-75 distances in hInl (I) homozygotes were 9.8 and 18.8 respectively. The dpy-5 unc-75 distance is probably an underestimate since it was measured in trans and relied upon the recovery of a less viable double homozygote class. The map distances between dpy-5 and unc-54 and dpy5 and unc-75 in controls were 26.4 and 9.4 (data from Table 12) respectively. This confirms  the gene order indicated by the three-factor experiment and suggests that recombination frequency in homozygotes is wild type. Recombination was also measured in the dpy-5 unc29 interval, a region located outside the inversion in the LG /cluster. The map distance in  this interval in homozygotes was found to be 3.7 m.u., not significantly different than observed in wild types (data in Table 12). hInl (I) crossover suppression is associated with recombination enhancement on  LG I: To determine the extent of him' (I) mediated crossover suppression, intervals to the  left of unc-101 were examined and the results are shown in Figure 6 (data shown in Table 12). The unc-75 unc-101 interval, normally 3.2 m.u., was reduced to 0.07 m.u. in hInl (I) heterozygotes. The dpy-5 unc-101 interval, however, was not significantly different in hinl (I) heterozygotes when compared to the control (11.7 and 12.0 m.u. respectively),  thereby raising two possibilities; recombination to the left of unc-75 was normal or the interval contained a region of recombination enhancement with an associated region of recombination suppression. To distinguish between these alternatives, recombination was examined in dpy-5 unc-29 unc-75 heterozygotes. Recombination in the dpy-5 unc-75 interval was 13.0 m.u. in hInl(I) heterozygotes and 9.4 m.u. in the control. Crossing over in the unc-29 unc-75 region was not significantly affected by the presence of hInl (I), whereas the dpy-5 unc-29 interval showed a 1.6-fold increase in recombination in hIn1(1)[+ 4-J/dpy-5 unc-29 heterozygotes. To further map the boundary of crossover suppression, recombination  was measured between unc-29 and 12n-11. This interval was 4.7 mu. in heterozygotes and  60 Table 14 Recombination in hInl (I) homozygotes  Genotype  Wild types  Recombinants  hInlidpy-5 unc-29.1/hInlf + +1  1500  35 Dpy 46 Unc 3.7(2.9-4.6)  dpy-5 unc-54/hInli + + .7  1464  151 Dpy  15.2(12.7-17.7)  hinl[dpy-5 unc-54Y + +  1285  136 Dpy  15.6(13.2-18.2)  hInlidpy-5 unc-54j/hInlf + +1  1312  87 Dpy  9.8(7.9-11.9)  243 Dpyb  9.0(6.6-11.4)  hInlidpy-5 + unc-75(h1042)7/hIn1f + unc-54 + J 1135  pX100(C.I.) a  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b unc-54 unc-75 distance measured in trans (see Chapter 3: MATERIALS and METHODS).  61a  FIGURE 6.-Meiotic maps of LG /in hIn1(1) heterozygotes and controls. The LG /cluster extends from unc-11 to unc-29 (EDGLEY and RIDDLE 1990).  unc -29 dPY-5 lin-11^unc- 101 bli- 3^unc- 11^unc- 75  bli- 3  ^  unc- 54 lev- 11  unc-29 lin- 11 dpy- 5 unc- 11  LG I  unc -54 lev- 11 unc- 101 unc- 75  hin 1 60  2 m.0 .  62  1.4 m.u. in controls (3-fold enhancement), thus localizing the hIn1(I) boundary of crossover suppression between lin-11 and unc-75. To determine if the recombination enhancement observed in the dpy-5 unc-29 interval extended to the left arm of LG /, recombination was examined in the bli-3 unc-11 and unc-11 dpy-5 regions. In hIn1(1) heterozygotes, a 1.5-fold increase in recombination was observed in the bli-3 unc-11, and a 2-fold increase was observed in the unc-11 dpy-5 interval when compared to controls. The total genetic length of chromosome /was 44 m.u. in controls and 41 m.u. in hIn1(I) heterozygotes (see Figure 6). Rare recombinants from hInl (I) heterozygotes contain duplications and deficiencies: Single crossovers within a classical inversion heterozygote produce  chromosomes that contain duplications and deficiencies. Four rare (— 1/2500) recombinants were recovered from hIn1(1) heterozygotes. Three of these originated from mapping experiments (see Table 12), while the fourth was isolated independently from a hIn1(1)1-1-q/unc-101 1ev-11 hermaphrodite on the basis of its visible Lev-11 phenotype. To determine  if the individuals homozygous for the chromosome of interest were viable, all four recombinants were crossed to N2 males. The progeny of wild-type hermaphrodites resulting from this cross were screened for the presence of individuals with the original recombinant phenotype. Two of the four recombinants proved to be homozygous lethal, and both failed to complement unc-59 and 1ev-11 establishing them as deficiencies, later designated as hDfl 1 and hDf12. hDf11 was known to complement unc-54 because of the original phenotype of the recombinant (Unc-101 when heterozygous with an unc-101 unc-54 chromosome). hDfll complemented unc-75, indicating the left deficiency breakpoint is to the right of this gene. PCR analysis of the left breakpoint of hDf11 indicates the deficiency does not include unc101, suggesting the deficiency bearing chromosome carries the original unc-101 mutation (J.-  Y. HO unpublished results). hDf12 also complemented unc-54 and was known to complement unc-101 based on the original recombinant phenotype (Unc-75 when heterozygous with an unc-75 unc-101 chromosome). Thus the left breakpoint of hDf12 is to the right of unc-101. The extent of these deficiencies is shown diagramatically in Figure 7. The remaining two recombinants, hDp131 and hDp132, were both Lev-11 in phenotype  63a  FIGURE 7.-Position of breakpoints of recombinant chromosomes derived from hIn1(1) heterozygotes. hDfll complements unc 75 and unc 101. The right breakpoints of hDp131 -  -  and hDp132 are not known but both duplications cover unc 54. -  unc-54 bli-3  I  unc-11 dpy-5 unc-29 unc-75 unc-101^unc-59 1ev-11 II^I^I^I^i^I  hDf11 hDf12 hDp131 hDp132  I^ I^  I  64  when heterozygous with an unc - 101 1ev - 11 chromosome. When crossed to N2 males, however, all resulting wild-type progeny segregated Unc-101 Lev-11 individuals, suggesting the recombinants were diploid for the unc - 101 1ev - 11 chromosome and carried a duplication of unc - 101. These two duplications were mapped to visible markers and segregated from chromosome / as though unlinked. The meiotic chromosomes of an hDp132/unc - 29 unc - 75 hermaphrodite were stained with DAPI to determine if the duplication was unlinked. Figure 8 shows a cell carrying a seventh chromosome, indicating that hDp132 is a free duplication. Both hDp131 and hDp132 have breakpoints between unc - 29 and unc - 75, and carry unc - 75(+), unc - 101(+), unc - 59(+), and unc - 54(+). That the duplications are Lev-11 in phenotype when heterozygous with unc - 101 1ev - 11 chromosomes suggests that they are linked to the original 1ev - 11 mutation. The extent of the duplications and their known breakpoints is shown in Figure 7. hInl (I) has no effect on crossing over on other chromosomes: In D. melanogaster, inversion heterozygosity produces interchromosomal effects; an increase in crossing over in regions surrounding the centric heterochromatin and the distal tips of chromosome arms on the other pairs of chromosomes (SCHULTZ and REDFIELD 1951; RAMEL 1962; reviewed by L UCCHESI 1976). To determine if hIn 1 (I) produces a similar effect in C. elegans, recombination was measured on other chromosomes in hIn1(1) heterozygotes. The  results are shown in Table 15. Two regions located on autosomes and one located on the X chromosome were examined. In all three cases, the presence of hIn1(0 did not significantly affect recombination in heterozygotes. hInl (I) recombines with szT1(I;X): The meiotic behaviour of the translocation szT1(I;X) has been extensively characterized (FODOR and DEAK 1985; McKIM,  HOWELL and ROSE 1988). The breakpoint of the translocation on LG / is close to the left of unc - 29, and translocation homozygotes are inviable. The extent of crossover suppression was determined; recombination was suppressed to the left of the breakpoint and enhanced to the right (McKIM, HOWELL and ROSE 1988). Since crossing over is suppressed in the unc - 75 unc - 54 interval in hIn1(.1) heterozygotes, it was of interest to  65a  FIGURE 8.-DAPI staining of chromosomes from the meiotic cells of an hDp132/unc-29 laic75 hermaphrodite showing a) an oocyte bearing a seventh chromosome, indicating the duplication is unlinked and b) an oocyte lacking the duplication.  65b  a)  66 Table 15 Effect of hInl (I) on crossing over on other chromosomes  Genotype  ^  Wild types^Recombinants^pX100(C.I.) a  dpy-18 unc-36/ + +^1561^77 Dpy 94 Unc^8.1(6.9-9.3) dpy-18 unc-36I + +;hIn1/+^1881^98 Dpy 96 Unc^7.6(6.6-8.7) unc-1 dpy-3/ + +^1373^23 Dpy 27 Unc^2.7(2.1-3.5) zinc-1 dpy-3I + +;hIn1/+^2147^32 Dpy 36 Unc^2.4(1.8-2.9) unc-42 dpy-11/ + +^1357^22 Dpy 20 Unc^2.3(1.8-3.2) unc-42 dpy-11/ + +;hIn1/+^1324^19 Dpy 14 Unc^1.9(1.3-2.6)  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).  67  determine if pairing was possible between the two rearrangements. Crossing over between the szT 1 (I ;X ) breakpoint and the hInl (I) boundary of crossover suppression was measured in hermaphrodites of the genotype hIn1(1)1 + + /szT1(I;X)[unc-101;lon-21 The map distance between the breakpoint of szT/(/;X) and the boundary of hIn1(1) crossover suppression (between lin-11 and unc-75) was 45 m.u. The recombination frequency between the szT1 breakpoint and unc-101 was measured in + ; + /szT/(1;X)/unc-/0/;/on-2/ controls and was 25 m.u., approximately 2-fold lower. The data for these experiments are shown in Table 16 and the Punnett square diagramming recombination between the two rearrangements is shown in Figure 9. hIn1 (I)/ hT2(I;III) heterozygotes suppress crossing over on LG I: The  translocation hT2(I;III) is comprised of two chromosomes; hT2(I;III)I segregates from chromosome l and, hT2(I;III)III segregates from chromosome /IL In heterozygotes, recombination on LG /is suppressed to the left of unc-101 and enhanced to the right of this marker. Since hInl(I) suppresses recombination from unc-75 to unc-54, it was of interest to determine whether recombination could be completely suppressed on LG I. Crossing over between dpy-5 and unc-54 was measured in hIn1(I)[+ + +_1/hT2(I;III)I-dpy-5 bli-4 unc-54,1 heterozygotes and + + + /hT2(I;III)idpy-5 bli-4 unc 54J controls (results shown in Table -  16). The map distance of chromosome /was reduced to 0.8 mu. in hIn1(I) heterozygotes, compared to 32.1 m.u. in controls, thus demonstrating recombination could be effectively suppressed along the entire length of the chromosome. hIn1 (I) and hT2(I;III)I segregate randomly: While examining recombination in hIn1(1)1+ + +_1/hT2(I;III)[dpy-5^unc-54_1heterozygotes, an unusually small number of  Dpy-5 Unc-54 progeny, representing the viable translocation homozygote, were recovered. To investigate the possibility that hIn1(I) and hT2(/;///)/ were segregating abnormally, segregation was examined in hIn1(I)1+ + +J/hT2(I;III)idpy-5 bli-4 unc-29Jheterozygotes and + + + /hT2(I;III)[dpy-5 bli-4 unc-29Jcontrols. Since both dpy-5 and unc-29 map in the crossover-suppressed arm of hT2(I;III)III, the recovery of the double mutant, representing the viable translocation homozygote class, is dependent upon the proper  68 Table 16 Effect of hInl (I) on crossing over with LG I translocations  Genotype  Wild types  Recombinants  pX100(C.I.)a  +; + /szT 1 (I ;X)unc- 101;lon-21  1110  200 Unc  25.0(24.0-26.0)  hInl(I)1 +^+ /szT1a;Xffunc-101;lon-2]  1775  563 Unc  45.5(45.4-48.7)  + + + /hT2(I;III)[dpy-5 blz-4 unc-541  441  59 Dpy  32.1(24.1->50)  hIn1(1-)1 + + + .1/hT2(I;III)[dpy-5 bli-4 unc-54]  591  2 Dpyb  a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b Both Dpy individuals were fertile and gave Dpy progeny.  ..0.8(0.15-2.8)  69a  FIGURE 9.-Punnett square diagramming the predicted segregation of a hInl (I) I+ .1; + /szT1(I,X)tunc-101;lon-2] heterozygote (see RESULTS). Empty boxes represent presumed  lethal zygotes resulting from severe aneuploidy. Viable classes are indicated by phenotype and any aneuploidy they may carry. szDp1 progeny are duplicated for AXII and are viable (McKIM, HOWELL and ROSE 1988).  IRx R  IRx R  [u/0112];  [u101/2];  II-XL  XN  IN; IIAL  IN; XN  u/O/IN;  II-XL  u/WIN; XN  iRxR [+121; ILXI-  iltxR [4_121; XN  IRXR  Eu/0//2];  WT  ILxL  Unc-101  IRXR  [u/0//2];  WT  XN IN; IIAL INT; XN  Unc-101  WT  WT  WT  u10/IN; ILL  WT  WT  Unc-101  WT  /WIN;  XN  Unc-101  Unc-101  iltxR [ +12]; IIAL ittxR [+/2]; XN  WT  WT  WT  WT  WT  70  segregation of the translocation from the normal homologues. In the control, the predicted ratio (5:1) of wild types to Dpy-5 Unc-29 progeny was observed (771 Wild types: 164 Dpy Unc). The frequency observed in hIn1(I) heterozygotes, however, was 13.7:1 (411 Wild types: 30 Dpy Unc), close to the predicted ratio of 11:1 if hT2(I;III)I and hIn1(I) were segregating randomly, resulting in aneuploid gametes (shown in Figure 10). Both the recovery of rare recombinants in the previous experiment and the difference between the predicted and observed segregation ratios may be explained by a low frequency of pairing between the two rearrangements. hIni (I) effectively balances lethal mutations: One objective in isolating a crossover  suppressor for a region associated with the homologue recognition region was the demonstration that such rearrangements, presumably intrachromosomal, would be effective balancers. The efficiency of hIn1(I) was tested by screening for recessive lethal mutations in the region of crossover suppression. In total, 1412 mutagenized chromosomes were screened and 54 mutations, including those resulting in adult sterility, were recovered. Strains representing the recovered mutations were effectively balanced in hIn1(I) heterozygotes for at least 20 generations (before being frozen) without breakdown of the balancer being observed.  71a  FIGURE 10.-Punnett square diagramming the predicted segregation of a hInl(I)[+ + +YhT2(I;III)idpy-5 bli-4 unc-29Jheterozygote (see RESULTS). Empty boxes represent  presumed lethal zygotes resulting from severe aneuploidy. Viable classes are indicated by phenotype and any aneuploidy they may carry. hDp13 progeny are duplicated for /R/HR and are viable (McKIM, PETERS and ROSE in press). Progeny duplicated for /L/HL are viable (K. McKIM pers. comm.).  'NHL [d5u29); hInl  'NHL [d5u291; IRIIIR  'NHL [d5 u2.91  ILIIIL [d5u29]; hInl  'NHL [d5u29]; IRIHR; hInl  HO  ;  hInl  IIIN; IRHIR  IIIN  WT  'NHL [d5u29]; /R/HR  Dpy-5 Unc-29  WT  /NHL [d5 u29]  WT  /NHL [d5u29]; IRIHR; hInl HIN; hInl IIIN; IRIIIR  WT  WT  WT  WT  /MN VVT /MN. /R///k; hInl  IIIN; IRHIR; hInl  WT  WT hDp134  WT hDp134  72  Chapter 3: DISCUSSION In this thesis, evidence has been presented for an inversion in C. elegans, that inverts a region of chromosome I, including the genes unc-75 and unc-54. The meiotic properties of hInI(I) were similar to those observed for inversions in Drosophila, including crossover suppression within the inverted region and intrachromosomal effects. hIn1(I) is capable of recombining efficiently with the translocation szT/(/;X)/, indicating that the two rearrangements also synapse efficiently, a prerequisite to chaisma formation. For this reason, it has been concluded that hInl(I) and chromosome /are capable of homologue recognition and synapsis, but that physical constraints inside the inversion loop limit chiasma formation, resulting in the suppression of crossing over in the region. That exchange events are rare seems likely for three reasons. Firstly, in Drosophila, crossing over inside In(1)d1-49, an inversion located at the end of the X chromosome, has been well characterized. NOVITSKI and BRAVER (1954) designed a system to recover the products of single exchanges inside In(1)d1-49 using a compound chromosome. They observed a 75% reduction in crossing over in heterozygotes despite cytological evidence that this inversion was capable of pairing by forming loops in mitotic cells (PAINTER 1933). This suggests that topological constraints exist that reduce the frequency of chiasmata formation inside such inversions when heterozygous. By analogy, hIn1 (I), which is also located at the end of a chromosome and is even smaller than In(1)d1-49, should experience constraints in pairing for recombination in heterozygotes. Secondly, compensatory increases in recombination are large in hIn1(I) heterozygotes, as would be expected if exchanges within the inversion were rare (i.e. in hIn1(I) heterozygotes, the map distance from bli-3 to lin-11, normally 22 m.u., approaches 50 m.u.). The fact that hInl(I) heterozygotes  efficiently recombine in other regions of the chromosome indicates that the ability of the homologues to recognize one another is intact, and that recombination suppression on the right arm is limited to the inverted segment. Thirdly, reciprocal recombination events were isolated from hInl(I) heterozygotes, suggesting that all meiotic products can be recovered,  73  but that their frequency is low. Figure 11 shows the possible pairing conformations between the inversion and the normal chromosome. The first shows a pairing loop resulting from homologous pairing of all sequences and the second shows the inversion remaining unpaired. It is not possible to distinguish between these two configurations genetically. Recombination was examined between hInl(I) and two translocations; szT/(I,.X) and hT2(I.III). The pairing portion of szT./(/;X)/ and hIni(I) share sequences not included in  either rearrangement, whereas the pairing portion of hT2(I;III)Iand hIn1(I) have no common unrearranged sequences. The crossover frequency between hIni(I) and szT.I(I.X)I was 0.45, demonstrating that synapsis and recombination were efficiently conducted between the two in spite of the genetically small size of the homologously paired region. In contrast, the frequency of recombination between hIn1(1) and hT2(I;III)I was less than 0.01. .These results agree with the conclusion that exchanges within the inversion are rare, since the only DNA available for pairing is within the inverted segment. DARLINGTON (1937) suggested that the formation of a chiasma between homologues during meiosis facilitates their proper disjunction (reviewed by HAWLEY 1988). One consequence of the crossover suppression observed in hInl(I)/hT2(I;III) heterozygotes was the random segregation of hIn1(I) and hT2(I;III)I. This suggests that in C. elegans, the formation of a chiasma between two homologues is necessary to ensure their  proper disjunction at meiosis I. This interpretation is supported by cytogenetic studies which have documented that at metaphase I the bivalents orient axially and may be held together at the metaphase plate by a terminalized chiasma (ALBERTSON and THOMSON 1993). In Drosophila, it has been observed that inversions can effect increases in recombination frequency on the rearranged chromosome and on the other major chromosomes (STURTEVANT 1919; STURTEVANT 1931; DOBZHANSKY 1933; reviewed by LUCHESSI 1976). The analysis presented here showed that the total genetic length of chromosome I was 41 m.u. in hInl(I) heterozygotes and 44 mu. in controls. These values are similar to the recombination frequency reported for the pairing portion of  74a  FIGURE 11.-Possible pairing conformations within a chromosome arm heterozygous for hIn1T. The hInl (I) chromosome is represented by gray lines and the normal homologue by  black. a) Synapsis is shown for both the inversion and the normal homologue, resulting in a conventional pairing loop. b) Synapsis is shown only for the uninverted regions. The inversion does not pair with the normal homologue.  74b  b)  75  chromosome I in individuals heterozygous for four translocations involving chromosome I: hT1(I;V), szT1(I;X) (McKIM, HOWELL and ROSE 1988), hTS(I;X), and hT2(/;///)  (McKIM 1990). These results indicated that while compensatory increases can occur on both arms of LG /, the amount of exchange is limited to approximately one crossover event per meiosis. Unlike inversions in Drosophila, hIn41)-mediated recombination enhancement in heterozygotes did not extend to other linkage groups. No increase in crossing over was observed in the three intervals examined in the presence of hIn1(.1.), regardless of their location on the autosomes (small interval inside the cluster or large interval spanning the cluster) or on the X chromosome. Interchromosomal effects have been observed in C. elegans with mutations that result in X-chromosome nondisjunction (HODGKIN, HORVITZ  and BRENNER 1979; HERMAN and KARI 1989). Thus in C. elegans, as in D. melanogaster, the mechanism that regulates the number of crossovers per meiosis may  involve compensatory increases of events on other chromosomes in the event crossing over is suppressed or reduced along an entire chromosome. The failure to observe interchromosomal effects in hIn1 (I) heterozygotes may have been expected since recombination was not reduced on chromosome /as a whole. Exchange events resulting from an intrachromosomal effect are not distributed randomly along the chromosome. In Drosophila for example, such increases occur in regions sufficiently removed from the inversion breakpoint (GRELL 1962), and near the centric heterochromatin and distal tips of other chromosomes, regions of low intrinsic exchange (SCHULTZ and REDFIELD 1951; RAMEL 1962). Each of the autosomes in C. elegans are marked by a region where genes cluster on the meiotic map resulting from a reduction in recombination (BRENNER 1974) per base pair compared to the genomic average (GREENWALD et al. 1987; KIM and ROSE 1987; PRASAD and BAILLIE 1989; PRASAD et al. 1993). In hIn1(I) heterozygotes, recombination frequency was enhanced in intervals both inside (1.5 fold in dpy-5 unc-29) and outside (1.5 fold in bli-3 unc-11) the chromosome Igene cluster. This suggests that the regulatory mechanism responsible for establishing the distribution of crossing over is independent of the mechanism determining  76  the number of exchanges. The meiotic pattern specific to chromosome I is retained; the enhancement observed is not greater in the cluster than it is at the left end. The frequency and distribution of exchange events was found to be normal in hIni(I) inversion homozygotes. This suggested that when the pairing difficulties experienced in heterozygotes were removed in homozygotes, exchange within the inversion maintained the distribution observed in wild types. Chromosomal sites that are necessary for normal levels of meiotic exchange have been mapped in Drosophila (HAWLEY 1980; SZAUTER 1984). A similar mechanism may exist in C. elegans since recombination frequency is enhanced in the region adjacent to the szT.1(IX) breakpoint on LC I, suggesting the break may have disrupted the mechanism responsible for the regional distribution of exchange (McKIM, HOWELL and ROSE 1988). If this mechanism is mediated by chromosomal elements, the level of crossing over in hIn1(I) homozygotes suggests that such elements can operate normally in either orientation. Inversions are classically defined by their exclusion (paracentric) or inclusion (pericentric) of the centromere (MULLER 1938; reviewed by ROBERTS 1976). Cytogenetic analysis of the salivary glands of inversion heterozygotes demonstrated that inverted homologous segments were capable of pairing by forming a loop (PAINTER 1933). A single exchange in a paracentric inversion loop led to the formation of acentric and dicentric fragments. The formation of these structures had been observed cytogenetically during meiosis in Zea mays (McCLINTOCK 1933). Single exchanges within paracentric inversions were not observed in Drosophila until single crossover products were recovered from individuals heterozygous for a long paracentric inversion on the X using an attached chromosome (SIDOROV et al. 1935). These results demonstrated that single crossovers do occur but that single crossover recombinants are not recovered. Nevertheless, information transfer in the form of gene conversion occurred in undiminished frequency in inversion heterozygotes, except near the breakpoints where effective homologous pairing may not be possible (CHOVNICK 1973). Unexpectedly, no concomitant loss of zygote viability was observed in heterozygotes despite the formation of aberrant chromosomes (STURTEVANT  77  and BEADLE 1936; NOVITSKI 1952). To explain this, STURTEVANT and BEADLE (1939) proposed that chromatids involved in single exchanges were excluded from a functional nucleus, a theory later corroborated by genetic and cytological evidence (STURTEVANT and BEADLE 1939; CARSON 1946; HINTON and LUCHESSI 1960). In contrast, single crossovers in pericentric inversion heterozygotes produced chromosomes with terminal duplications and deficiencies that were segregated into gametes and resulted in reduced fertility (ROBERTS 1967). The frequency at which single exchanges occurred was dependent on the size and location of the inversion; a reduced frequency of such events was observed with both small inversions, and inversions located at the ends of chromosome arms (STURTEVANT and BEADLE 1936; NOVITSKI and BRAVER 1954). Individuals heterozygous for hIn1(I) showed no reduction in egg-hatching frequencies, compatible with the behaviour of a paracentric inversion for which the products of single exchanges are either excluded from functional nucleii or for which single exchanges in the inverted segment are rare. The recovery of a chromosomal rearrangement in C. elegans that behaves as a paracentric inversion may seem suprising given that the mitotic chromosomes are holokinetic (ALBERTSON and THOMSON 1982) and evidence for holocentric meiotic chromosomes has been reviewed (HERMAN 1988). Recent cytological studies, however, suggest that the ends of C. elegans chromosomes adopt centromeric functions for meiotic disjunction; one end holds the bivalent together and the other probably provides a site for the attachment of microtubules. These roles do not appear to be specific to one end of the chromosome and either end can be the inner or outer end of the bivalent (ALBERTSON and THOMSON 1993). This meiotic behaviour is similar to that observed in other mitotically holokinetic species; the nematode Parascaris univalens (GODAY, CIOFI LUZZATTO and PIMPINELLI 1985; PIMPINELLI and GODAY 1989; GODAY and PIMPINELLI 1989), the insects Euchistus servas (HUGHES-SCHRADER and SCHRADER 1961) and Myrmus mirzformis (NOKKALA 1985), where the mitotic chromosomes are holocentric but during  78  meiosis centromeric activity is restricted to a limited chromosome region, often at chromosome ends. The results of genetic analyses in C. elegans have consistently been compatible with the predicted behaviour of monocentric chromosomes. For example, the segregation ratios of aneuploid and viable progeny observed from translocation heterozyotes were compatible with the presence of a single centromere (HERMAN 1978; ROSENBLUTH and BAILLIE 1981; McKIM, HOWELL and ROSE 1988; this study). In the case of the translocation eT1(III;V), recombination is suppressed to one side of the translocation breakpoint while the  other recombines and segregates from the chromosome with which it had paired (ROSENBLUTH and BAILLIE 1981). Thus, in any one meiosis, only one meiotic segregator (centromere) was functional. The data presented in this thesis strongly suggest that the meiotic chromosomes are monocentric, a suggestion compatible with both genetic and cytogenetic observations. The isolation of four recombinants from hIn1(1) heterozygotes raised the possibility that their genotypes would provide information on the location of the meiotic centromere. Two free duplications, hDp131 and hDp132 were recovered following a single exchange event inside the inversion loop. The structure of these duplications is consistent with the products formed by the events illustrated in in Figure 12. In this model, hDp131 and hDp132 are represented by the acentric fragment that results from a single exchange within a paracentric inversion where the centromere is to the left of unc 75. The facts that the -  duplications were isolated independently, and that the left endpoints were nonrandom and coincided with the boundary of hIn1(1) crossover suppression (between unc 29 and unc 75) -  -  support this interpretation. In Drosophila, the acentric fragment generated by a single exchange within a paracentric inversion loop is not recovered under ordinary circumstances. In C. elegans, however, free duplications are readily recovered (HERMAN, ALBERTSON and BRENNER 1976). According to the model shown in Figure 12, the reciprocal product is a duplication of the sequences to the left of the inversion including the centromeric sequences. This structure is analogous to the dicentric chromosomes generated by single  79a  FIGURE 12.-Effects of single crossing over within a chromosome arm heterozygous for hInl(I). a) Synapsis is shown only for the inversion. hInl(I) is represented by gray lines  (inversion boundaries shown by parentheses) and the normal homologue by black lines. L denotes the left end and R denotes the right end of LG I. C represents the gene cluster, a and b represent markers on the normal chromosome; in the case of hDp131 and hDp132, unc-101 and 1ev-11 respectively. In the case of hDfll, a = unc-101 and b = unc-54. In the  case of hDf12, a = unc-75 and b = unc-101. Wild-type alleles of these markers are shown on the inverted chromosome. b) Meiotic products resulting from the exchange event (see Chapter 3: DISCUSSION). The chromosome duplicated for the cluster is proposed to be dicentric and the origin of hDf11 and hDf12. The small chromosome duplicated for the right ends has the same structure as hDp131 and hDp132.  79b  a a  b  +  a^b +  b)  a a  80  exchanges within paracentric inversions in Drosophila. Although this reciprocal product was not recovered intact in our experiments, hDfl 1 and hDf12 may have resulted from its breakage. Dicentric chromosomes in other organisms have been observed to form chromatid bridges at anaphase I, and as a result are meiotically unstable and subject to chromosome breakage (McCLINTOCK 1933, 1941; CARSON 1946; HABER, THORBURN and ROGERS 1984). The two deficiencies recovered, hDfl 1 and hDf12, could have resulted from a similar event followed by the broken end of one product being capped by sequences on the right end (including uric- 54 (+)), presumably derived from its normal homologue to which it is still attached. To stabilize broken ends of chromosomes, double-stranded breaks can be repaired by fusing with other chromosomes (McCLINTOCK 1941; 1942) or by recombining with homologous sequences (HABER and THORBURN 1984). KADYK and HART WELL (1992) have shown that sister chromatids are preferred over homologues as substrates for recombinational repair. Both deficiencies recovered have breakpoints independent of the site of the original exchange event. Thus, the structures of the recombinant chromosomes recovered are compatible with the interpretation that h/n/(/) is a paracentric inversion with the centromere to its left. That cytogenetic analysis has demonstrated that both ends of LC / can adopt centromeric function (ALBERTSON and THOMSON 1993) is not inconsistent with the genetic data. Firstly, in any one meiosis only one end of the chromosome adopts centromeric function (i.e. spindles do not attach to both ends). Secondly, the right end of chromosome /was observed to take on centromeric function in the majority of meioses (ALBERTSON and THOMSON 1993), thereby explaining the preferential recovery of recombinant products whose derivation is most easily explained by centromeric activity of the right end of LC /rather than the left. The recombinant products recovered are not consistent with the predicted behaviour of a paracentric inversion with centromeric sequences to the right of the inversion. The recovery of hDp 131 and hDp132 is not compatible with dicentric products (see below), and no acentric fragment of the predicted structure was recovered. The possibility that hIn 1 (I) is a pericentric inversion cannot formally be ruled out, however, it is unlikely for the  81  following reasons. No significant reduction in egg-hatching frequency was observed in heterozygotes as would have been expected if the inversion were pericentric, however, it is possible that the frequency of recombination within the inversion was so low that no reduction was observed. Most importantly, however, if hin1(I) included the centromere, hDp131 and hDp132 would be centric and would have segregated from their homologue at meiosis II. To recover these duplications, a nondisjunction event would be needed to generate a viable zygote, and the probability of recovering two such rare events is very low. The reciprocal recombinant product would also have possessed one centromere and would have been meiotically stable. Size is unlikely to be a consideration since large, rearranged chromosomes exist in C. elegans which are meiotically stable (HERMAN, KARI and HARTMAN 1982; SIGURDS ON et al. 1986; McKIM 1990; ALBERTSON unpublished results).  In conclusion, the meiotic behaviour of a C. elegans inversion hIn1(1), that suppresses crossing over in a previously unbalanced region, has been characterized. The simplest interpretation of the data presented is that hIn.1(I) is a paracentric inversion with the meiotic centromere to its left. hIn1(1) was used to successfully balance lethal mutations in a region previously impenetrable to extensive essential gene analysis, demonstrating its value as a new class of balancer for the genome of C. elegans.  82  CONCLUSIONS The intent of this thesis has been the description of conventional meiotic phenomena in C. elegans. Meiosis in C. elegans is marked by the classical features that distinguish it from mitosis: pairing, recombination, and segregation of homologous chromosomes. Some of the functional elements responsible for these processes have now been described in the nematode. The term "pairing" has been used to describe several chromosomal behaviours that are now understood to be temporally and functionally distinct. Homologue pairing is the alignment of chromosomes at a distance, perhaps as early as interphase, and is thought to be mediated by discrete sites whose number may be related to chromosome size (MAGUIRE 1984). These homologue pairing sites may be the attachment sites for fibrullar proteins that anchor the two homologues together during interphase and early prophase. In C. elegans,  the characterization of two free chromosome / duplications demonstrated that  one covering the right arm of the chromosome was capable of pairing for recombination with the normal chromosomes, while the duplication covering the left arm was not. This led to the proposal that the right end of chromosome /contained sequences necessary for recombination and pairing between the homologues (ROSE, BAILLIE and CURRAN 1984; McKIM, HOWELL and ROSE 1988). The characterization of translocations and duplications has led to the identification of a single site on each chromosome that is necessary for pairing and recombination, discussed by McKIM, HOWELL and ROSE (1988). This site, called the homologue recognition region, may correspond to the region discussed by MAGUIRE (1984), described as the site of first contact between homologues during meiosis. The genetic behaviour of rearrangements lacking this region supports this interpretation since such rearrangements fail to pair for recombination with their homologues. This demonstrates that the function associated with this site temporally precedes any later meiotic event in the pathway, consistent with the predicted behaviour of a specialized site required for initial homologue recognition early in meiosis. Such behaviour, however, would also be predicted for rearrangements that delete the site of telomere  83  attachment to the nuclear membrane. The failure of the chromosomes to pair and recombine could be attributed to the failure of the homologue lacking the telomere attachment site to become properly oriented and anchored in the spatial organization of the nucleus. As a result, it would float free in the nucleus, unable to participate in later meiotic events. In C. elegans, electron microscopy of pachtene nucleii has demonstrated that while only one telomere of each chromosome is attached to the nuclear membrane, both telomeres have the ability to do so (GOLDSTEIN 1982, 1984a, 1984b, 1985, 1986; GOLDSTEIN and SLATON 1982), indicating the telomere attachment site does not correspond to the homologue recognition region. The second form of meiotic pairing is thought to bring the homologues into a tighter association as a result of numerous recombination events which occur at certain sites. at a higher frequency. Such pairing sites have been mapped on the X chromosome of Drosophila (HAWLEY 1980) and in yeast a pairing site identified on chromosome ///has been shown to be a recombination hotspot (GOLD WAY, ARBEL and SIMCHEN 1993; GOLD WAY et al. 1993). In C. elegans, these secondary pairing sites are not sufficient to ensure  recombination between homologues in the absence of the homologue recognition region (ROSENBLUTH, JOHNSEN and BAILLIE 1990; McKIM, PETERS and ROSE 1993). In the presence of the HRR, however, a small set of these sites may be preferentially used to ensure secondary pairing between rearranged chromosomes. Intrachromosomal effects in translocation (McKIM, HOWELL and ROSE 1988; McKIM, PETERS and ROSE 1993) and inversion heterozygotes (ZETKA and ROSE 1992) enhance crossing over in the regions capable of homologous pairing to levels approaching 50 map units. In hIn1(1)/szT1(I,X) heterozygotes, the genetic size of chromosome /was 50 map units even though all crossing over had to occur in a small region flanked on one side by nonhomologous translocated sequences, and on the other by the inversion. This supports the interpretation that pairing between such sites is independent of the pairing of neighbouring sites, and does not support a model whereby pairing for recombination is initiated only at the end(s) of a chromosome  84  and progresses from this site. Rather, it suggests that recombination events can be initiated internally. Two major classes of mutations that disrupt meiotic exchange exist in C. elegans. The largest is represented by several him mutants which are recombination-defective on both the X chromosome and the autosome (HODGKIN, HORVITZ and BRENNER 1979). The second class is represented by one mutation, rec-1, which disrupts the normal distribution of crossing over and does not decrease the viability and brood sizes of mutants. The genetic size of chromosome /in rec-1 homozygotes approaches the 50 map units observed in rearrangement heterozygotes (McKIM, HOWELL and ROSE 1988; ZETKA and ROSE 1992; McKIM, PETERS and ROSE 1993) and wild-type individuals (ZETKA and ROSE 1990), suggesting a flexible mechanism exists to ensure the formation of one crossover between the homologues. In this study, crossing over was eliminated in heterozygotes resulting in the random segregation of the two chromosomes. This demonstrates that in C. elegans, one chiasma per bivalent is necessary for the proper disjunction of homologous chromosomes. Recombination in C. elegans is also regulated by a mechanism based on the sexual phenotype of the individual. Male recombination frequency on chromosome I is reduced by one third when compared to the hermaphrodite frequency, suggesting the formation of a chiasma is not guaranteed in every male meiosis. Three possibilities may explain the orderly disjunction of chromosomes in male gametes in the event a chiasma does not form. Firstly, substantial levels of recombination may occur at the distal tips of the chromosome where crossing over is difficult to measure. Secondly, male meiosis may make more use of secondary pairing sites to ensure proper segregation in the absence of chiasma formation. In Drosophila, deletion mapping of the ribosomal cluster has demonstrated that sequences between the rRNA genes function as X- Y pairing sites in male meioses (McKEE, HABERA and VRANA 1992). Thirdly, studies have documented that free duplications segregate from the X chromosome in the male and it is possible that nonrecombinant chromosomes may pair distributively (McKIM, PETERS and ROSE 1993; this study APPENDIX I).  85  Recombination-independent segregation systems have been documented in Drosophila (GRELL 1962) and in yeast (DAWSON, MURRAY and SZOSTAK 1986). The third form of meiotic pairing culminates in the intimate association of the homologues that is mediated by the formation of the synaptonemal complex. Normal synaptonemal complexes, consisting of two lateral elements and a central element form in both males and hermaphrodites (GOLDSTEIN and SLATON 1982). Although C. elegans chromosomes behave holokinetically during mitosis (ALBERTSON and THOMSON 1982), evidence has been presented in this thesis that this is not true for meiosis. Analysis of recombinants derived from inversion heterozygotes has suggested that hIn1(1) is a paracentric inversion with the meiotic centromere to its left (ZETKA and ROSE 1992). This is supported by recent cytological studies which have concluded that while centromeric activity is localized to one end of the chromosome during meiosis, either end can perform this function (ALBERTSON and THOMSON 1993). Thus, in any one meiosis, one end of the chromosome adopts centromeric activity by attaching to spindles (monocentric), rather than both ends (dicentric), or the whole chromosome (holocentric). On chromosome /, the right end of LG /adopts centromere function in the majority of meioses (ALBERTSON and THOMSON 1993). This is consistent with the structure of hIn1 (0-derived recombinants since each recombinant chromosome is representitive of one meiosis, and their derivation has been explained by the presence of a meiotic centromere on the right arm (ZETKA and ROSE 1992). 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All  wild-type males resulting from this cross were of the genotype dpy-5 unc-13/dpy-5 dpy14/sDp1 and were mated to dpy-5; unc-36 homozygous hermaphrodites and the wild-type  and Dpy-5 progeny were scored. If the duplication and the X chromosome segregated from one another, the resulting male progeny were predicted be wild-type as consequence of inheriting sDp1 and the hermaphrodite progeny were predicted to Dpy-5 as a consequence of inheriting the paternal X. If the duplication and the X segregated randomly, one half of the wild-type and Dpy-5 progeny were expected to be male. 269/293 wild-type progeny were male and 275/298 Dpy-5 progeny were hermaphrodite indicating that in approximately 87% of the male meioses, sDp1 and the single X segregated from one another, consistent with the proposal that distributive pairing occurs in males.  104  Cosmid mapping of rec - 1: A Rec-1 transgenic strain (KR2357), constructed by coinjecting rol-6 cosmid pRF4 and three overlapping cosmids (ZK219, B0195, CO1F5) (mapping to the right of unc-54), was obtained from J. McDowall. To determine if these cosmids carried a wild-type allele of rec-1, Rol-6 transformants (rol-6 is dominant and establishes the presence of the extrachromasomal array containing the cosmids discussed above) were mated to dpy-11 unc-.42/ + + ; rec-1/rec-1 or dpy-11 unc-42/ + + males. Rol-6 progeny resulting from this cross were individually plated and their progeny screened for the presence of Dpy-11 Unc-42 segregants. Recombination in these individuals was calculated using the formula:  p=1  -  [1  -  3(D + U)/2(W R D)  where D is the number of Dpy recombinants, U is the number of Unc recombinants, W is the number of wild types and R is the number of roller progeny. Rol-6 animals will not roll in an Unc-42, Dpy-11 or Dpy-11 Unc-42 background. The region of chromosome /located between unc-54 and the ribosomal cluster (rrn1) at the right distal tip is spanned by six overlapping cosmids shown in Figure 13. In order  to map rec-1 to one of these cosmids, the six cosmids were coinjected as sets of three (one group containing the cosmids ZK219, B0195, CO1F5 and the other ZK340, B0467, ZC556) into rec-1 homozygotes (J. McDOWALL unpublished results) using the rol-6 transformation system (MELLO et al. 1991). Attempts to construct a transgenic line containing the second group of cosmids (ZK340, B0467, ZC556) were unsuccessful and no stable lines were isolated (J. McDowall pers. comm.). Transgenic Rol-6 hermaphrodites bearing an extrachromosomal array containing the cosmids ZK219, B0195 and CO1F5 were tested for the presence of Rec-1 by measuring recombination in dpy-11 unc-42/ + + ; rec-1/rec-1 individuals (data shown in Table 17). Since the cosmids were injected into a Rec-1 individual, the presence of a wild-type allele of rec-1 on one of the cosmids would result in a normal map distance for the dpy-11 unc-42 interval. The dpy-11 unc-42 interval was 8.8  105a  FIGURE 13.-Physical map of cosmids in the region of unc 54. -  CO1F5 B0195  ^ 1  ^i  ZK219  ^ ,  ZC556  ,^  B0467 ^ 1  i  ZK340  i^  1  unc-54  ^  kin-1  ^  rrn-1  tr.  106 Table 17 Cosmid mapping of rec-1  Dpy  Unc  pX100(C.I.)a  1219  66  59  7.6(6.4-9.0)  dpy-11 unc-4.2/ + +b  1250  17  12  2.7(2.0-3.6)  dpy-11 unc-42/ + + ;rec-1/rec-1;hEx12c  637  271  60  49  8.9(7.3-10.6)  dpy-11 unc-42/ + + ;rec-1/ + ;hEx12c  494  403  19  11  2.5(1.7-3.5)  Genotype  Wts  dpy-11 unc-42/ + + ;rec-1/rec-1b  Rol-6  C.I. 95% confidence interval (see Chapter 1: MATERIALS and METHODS). b data from Table 10. c The extrachromosomal array hEx12 includes the rol-6 dominant gene, and the cosmids ZK219, B0195 and CO1F5 in unknown copy number (J. McDOWALL unpublished results).  107 m.u. in transgenic individuals homozygous for rec-1 and 6.4 m.u. in dpy-11 unc-42/ + + ;  rec-1/rec-1 controls, indicating that the cosmids did not carry a wild-type allele of rec-1. In the event normal meiotic recombination was affected by the presence of the cosmids, recombination was also measured in dpy-11 unc-42/ + + ; rec-1/ + individuals carrying the extrachromasomal array. The frequency of recombination was 2.5 m.u. in transgenic individuals heterozygous for rec-1 and 2.7 m.u. in dpy-11 unc-42/ + + controls, indicating the presence of the array does not affect recombination in this interval. Because the presence of the array was not confirmed by PCR, however, the negative results of these experiments are not conclusive.  108  PCR mapping of eDf24 (I): Young eDf24/hIn1[unc- 101] or h/n./(/)/h/n/(I) hermaphrodites were plated and allowed to lay eggs for 12 hours and then removed. Eggs which remained unhatched after 20 hours were removed to agar plates lacking 0P50 and treated according to the protocol of BARSTEAD, KLEIMAN and WATERSTON (1991) with the exception that the eggs were transferred using cut fishing line which then remained in the PCR tubes. The cosmids ZK340, B0467, and ZC556 were provided by J. McDowall. The reaction volume of 22.5 u/ included 2.5 u/ of DNA preparation, 0.125 units of Taq polymerase (Sigma), 4 u/ each of 2.5 mM dNTP (dATP, dCTP, dGTP, and dTTP)  and 1 u/ of each oligonucleotide in a PCR buffer (Sigma) containing 1.5 u/ of 25 mM MgC12. The reaction mixtures were overlaid with mineral oil (Fisher), incubated for 3 min at 95°C, 30 sec at 50°C, taken through 30 cycles of 1 min at 72°C, 45 sec at 94°C, and 20 sec at 55°C. After these cycles, the samples were incubated for 7 min at 72°C and cooled to 4°C using a brand name thermal cycler. After cycling, 5 u/ of 5 X DNA sample buffer (1 X = 0.25% bromophenol blue, 0.25% xylene cyanole, 15% Ficoll) was added the sample. 20 u/ of each sample was removed and analyzed on a 1.2% agarose gel containing ethidium bromide. After electrophoresis at 140 V for about 1 hour, the gels were removed and photographed. ALBERTSON (1984b) observed that eDf24 deletes a portion of the ribosomal cluster located at the right end of LG I, but could not determine whether the deficiency contained sequences to the left of the cluster or lay completely within it. To distinguish between these two possibilities, primers flanking the left ribosomal junction (one specific for nonribosomal sequences adjacent to the cluster and the other specific for 26S ribosomal RNA) were used to determine if the junction was present in eDf24 homozygotes. The sequences of these primers and control primers derived from the adenosyl homocysteine hydrolase gene (AHH)(PRASAD, STARR and ROSE 1993) are shown in Figure 14 and the PCR products are shown in Figure 15. The primer set specific for the ribosomal junction produces a 517 b.p. product and the All primers a 577 b.p. product. Both products are present in hInl (I) homozygote controls, however, only the 577 b.p. product is present in eDf24 homozygotes,  109a  FIGURE 14.-Sequences of PCR primers used in this study. a) RL30 anneals to nonribosomal DNA adjacent to the ribosomal cluster and RL31 anneals to 26S ribosomal genes. b) RL12 and RL14 anneal to sequences within the AHH locus, located in the central cluster of chromosome I.  a) RL30  5' - TGG GAA TTT TCT GTT CAG GT - 3'  RL31  5' - CGC AAT AAC AAG TCA ACA GT - 3'  b)  '12 5' - CGT CCG TTC TTG AGG GTG - 3' RL14  51 - CTA AGA TGC TCG CCA AGG - 3'  110a  FIGURE 15.-PCR analysis of e Df24(I). PCR products obtained from lane 2) N2 L4 hermaphrodite, lane 3) N2 egg, lanes 4-9) eDf2. 4 (I)/eDf24 (I) egg, lane 10) eDf2.4 (I)/hIn 1 ( kunc- 10.1j L4 hermaphrodite, and lane 11) hIn .1 (I)/hInl (I) L4  hermaphrodite using the left ribosomal junction primers (517 b.p. predicted product size) and All control primers (577 b.p. predicted product size). eDf24 (I) homozygotes lack the 517 b.p. product, indicating that the deficiency spans the junction (THACKER and ZETKA unpublished results).  110b  1 2 3 4 5 6 7 8 9 10 11 12  -or 577 -6( 517  111  indicating the deficiency spans the ribosomal junction and includes nonribosomal DNA adjacent to the cluster. Identical primer sets were also used to determine which of the three remaining cosmids contained the ribosomal junction. The cosmids ZC556 and B0467 (not ZK340) produced a 517 b.p. product, indicating the ribosomal junction maps to these cosmids (data not shown).  112a III. Table 18 Strains used in this study  Strain Genotype  ^  Strain Genotype  BC62 dpy-5(e61)unc-75(e950)  CB719 unc-1(e719)  BC64 unc-35 (e259)dpy-5 (e61)  CB1479 him-S (e1423)  BC89 dpy-5(e61)unc-54(e190)  DR1^unc-101(m1)  BC196 dpy-5(e61)dpy-14(e88)rec-1(s180)  KK596 him-/4(i/44ts)  BC207 dpy-5 (e61)unc-29(e403)  KR181 unc-42(e270)dpy-11(e224)rec-1(sl 80)  BC251 unc-42(e270)dpy-11(e224)  KR236 dpy-5061)unc-13('e450)/sDp2  BC260 unc-11(e47)dpy-5(e61)rec-1(s180)  KR307 dpy-5('e61)unc-101(m1)  BC415 dpy-5(e61)unc-13(e450)  KR309 bli-3(e579)unc-11(e47)rec-1(s180)  BC563 dpy-180364)unc-36(e.261)  KR387 unc-13('e450)rec-1(s180)  BC 1195 sDpVdpy-17(e251)unc-36(e261)  KR642 dpy-5(e61)rec-1(s180)  CB51 dpy-5 (e51)  KR900 dpy-5(e61)unc-13('e450)/szTi(I;X)[+ +;lon-2J  CB88 dpy-7(e88)  KR1004 dpy-5(e61)dpy-14(e88)  CB151 unc-3 (e151)  KR1005 sDp2/dpy-5061Ppy-14(e88)rec-1(s180)  CB190 unc-54(e190)  KR1012 dpy-5(e61)unc-29(e403)unc-75(e950)  CB261 unc-59(e261)  KR1064 bli-3(e579)unc-11(e47)  CB450 unc-13(e450)  KR1071 dpy-5(e61)unc-11(e47)  112b  Strain Genotype^  Strain Genotype  KR1301 rec-1(s180) males  KR2151^func-54('h10401  KR1473 unc-101(ml)unc-54(e190)  KR2152 hDp131/unc-75(e950)unc-101(m1)  KR1546 sDp1/dpy-5(e61)dpy-14(e88)rec-1(s180)  KR2153^fdpy-5(e61)unc-54(h1040)J  KR1701 dpy-7(e88)unc-3(e151)  KR2156 unc-29(e403)1zn-11(n389)  KR1714 unc-29(e403)unc-75(e950)  KR2158 unc-75(e950)unc-59(e261)  KR1734 unc-75(e950) unc-101(m1)  KR2159 unc-75(h1042)/unc-75(e950)unc-101(m1)  KR1735 sDp1/dpy-5(e61)dpy-14(e88)  KR2226 hIn11-unc-54(h1040)J/unc-101(m1)1ey-11(x12)  KR1898 unc-101(m1)ley-11('x12)  KR2228 ley-11(x12) males  KR1906 unc-11(e47)rec-1(s180)  KR2387 unc-1(e719)dpy-7(e88)  KR1949 hIn1(1) males  KR2390 hDp132/unc-75(e950)unc-54(e190)  KR1953 unc-54(e190)/eDf24  KR2391 hDp131/unc-75(e950)unc-59(e261)  KR1954 dpy-5(e61) unc-75(e950); her-1(e1520)  KR2392 hDfil/unc-101(ml)unc-54(e190)  KR1955 dpy-5(e61)unc-101/szTliUnc-101;lon-21  KR2394 hDp2/dpy-5(e61)  KR1956 dpy-5(e61) unc-13(e450) rec-1(s180)  KR2423 dpy-5(e61)unc-36(e261)  KR2017 unc-75(h1041)/unc-75(e950)unc-101(m1)MT633 u-11(n389);him-5(e1467) KR2019 hDp132/unc-75(e950)unc-101  ^  KR2020 hDf12/unc-75(e950)unc-101(m1) KR2025 hDp131/unc-101(ml)ley-11(x12)  ^  ^  RW3072 1ey-11(x12)10-49(st44)/unc-54(st60)let-50(st33) SP580 mri164(/;X)  ZZ3003 ley-11(x12)  


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