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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=GANSbyMONIQUE-CLAIRE ZETKAB.Sc., The University of British Columbia, 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESGENETICS PROGRAMMEWe accept this thesis as conforming to the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust 1993©Monique-Claire Zetka, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  Medical GeneticsThe University of British ColumbiaVancouver, CanadaDate 10 September 1993DE-6 (2/88)IIABSTRACTThe process of meiosis ensures heritable genetic material is passed faithfully from onegeneration to the next. To identify the mechanisms involved in this process, the effects ofsex, mutation, and rearrangement on meiotic recombination in Caenorhabditis elegans wereinvestigated. The short life cycle and existence of meiotic mutants make this organism anideal system in which to study meiosis. To determine the effect of sex on meioticrecombination, crossing over was characterized in male gametes and then compared to thefrequencies observed in hermaphrodite gametes. Male recombination across chromosome /was approximately one-third less than that observed in the hermaphrodite. This decreasevaried with the interval being measured and in one interval, no difference was observedbetween the sexes. The frequency of recombination in hermaphrodite spermatocytes wastwo-fold higher than that observed in oocytes and male spermatocytes . Thus,recombination frequencies appear to be a function of gonad physiology rather than sexualphenotype. To test this further, recombination was measured in males sexually transformedby the her-1 mutation. The results indicated that the sexual phenotype, rather thankaryotype, determined the recombination frequency characteristic of a certain sex. Likerecombination in the hermaphrodite, male recombination was also found to increase withtemperature and decrease with age. Therefore, recombination frequency in C. elegans isinfluenced by physiological factors such as sexual phenotype and age, and environmentalfactors such as temperature.Mutations in genes that regulate meiosis can affect the frequency of recombinationand the distribution of exchange events. A recessive mutation in the gene rec-1 wasmapped, and its effects on the distribution of crossing over on LG / were determined. Thismutation was mapped to the right end of chromosome /using the duplications sDp1 (whichcarries a wild-type allele of the gene) and sDp2 (which does not). A high resolution mapposition was determined using several deficiencies of the right end of the chromosome tomap the mutation. The ribosomal deficiency eDf24 failed to complement rec-1, indicatingthe locus was located within its boundaries. Crossing over in five intervals on chromosome Iwas measured in rec-1 homozygotes. The frequency of recombination in one interval locatedin the, centre of the chromosome showed a ten-fold increase, whereas an interval located onthe right end showed a three-fold decrease. Despite the changes to the frequencies ofrecombination in these intervals, the total genetic length of chromosome /remainedunchanged, indicating that the rec-1 mutation affected the distribution of a wild-typenumber of exchange events. This implies that the rec-1(+) gene product is necessary inestablishing the distribution of crossovers along the chromosome.Chromosome rearrangements can reduce or eliminate crossing over by physicallydisrupting the normal organization of the chromosome. In this study, a crossover suppressorfor the right end of LG /was isolated and characterized. By inducing markers on therearrangement and establishing the gene order in the homozygote, hInl(I) was demonstratedto be the first inversion isolated in C. elegans. Crossing over in the heterozygote wascharacterized, and intrachromosomal (but not interchromosomal) effects were observed. Theinteraction of hInl(I) with two translocations demonstrated that small homologous regionscan pair and recombine efficiently, and that the formation of a chiasma between twohomologues is necessary for their proper segregation. Rare recombinants bearingduplications and deficiencies were isolated from inversion heterozygotes, leading to theproposal that hIn1(I) is paracentric with the meiotic centromere to its left. The meioticbehaviour of the inversion was found to be consistent with the proposal that the meioticchromosomes of C. elegans are monocentric.TABLE OF CONTENTSABSTRACT^TABLE OF CONTENTS^ ivLIST OF TABLES viiiLIST OF FIGURESACKNOWLEDGEMENTS^ xiGENERAL INTRODUCTION 1Chapter 1: Sex-Related Differences in Crossing OverINTRODUCTION^ 4MATERIALS and METHODSStrain maintenance and culture conditions^ 7Recombination mapping^ 11Recombination in hermaphrodite germlines^ 12Recombination in Her-1 hermaphrodites 13Measuring the effect of age on recombination^ 13Measuring the effect of temperature on recombination^13RESULTSComparison of male and hermaphrodite recombinationfrequencies^ 15ivComparison of recombination frequency inhermaphrodite gametes^ 15Variation of recombination frequency with age^ 20Variation of recombination frequency with temperature^24Effect of Rec-1 on male recombination^ 24DISCUSSION^ 27Chapter 2: The Characterization and Mapping of rec - 1INTRODUCTION^ 31MATERIALS and METHODSDuplication mapping^ 33Deficiency mapping 34RESULTSEffect of rec - 1 on the genetic map of LG /^ 36Duplication mapping of rec - 1^ 36Deficiency mapping of rec - 1 40DISCUSSION^ 44Chapter 3: The Meiotic Behaviour of an InversionINTRODUCTION^ 48VMATERIALS and METHODSIsolation of hIn1(I)^ 50Measuring egg-hatching frequencies^ 50Induction of genetic markers on hIn1(I) 50Measuring recombination in hIn1(I) homozygotes^ 51Analysis of recombinants from hIn1(I) heterozygotes^52DAPI-staining of hDp132 meiotic cells^ 53Interaction of hIn1(I) with szT1(I;X)^ 53Interaction of hInl (I) with hT2(I;III) 54hIn1(I) efficacy as a balancer^ 54RESULTSIsolation of a crossover suppressor forthe right end of LG /^ 55Zygotic viability of hInl (I) heterozygotes^ 55Gene order in hIn1(I)^ 58Crossing over in inversion homozygotes^ 58Effect of hIn1(I) on crossing over on LG /^ 59Structure of recombinants derived fromhIn1 (I) heterozygotes^ 62Effect of hIn1(I) on crossing over on other chromosomes^64viviiInteraction of hIn1(1) with szT1(1,../Y)^ 64Interaction of hInl(D with hT2(I;IID:(a) Crossing over^ 67(b)Segregation 67hInl(D as a balancer^ 70DISCUSSION^ 72CONCLUSIONS 82BIBLIOGRAPHY^ 86APPENDIXI. Distributive pairing of sDp1^ 103II. Cosmid mapping of rec-1^ 104III. PCR mapping of eDf24^ 108IV. List of strains used in this thesis^ 112LIST OF TABLESTable 1^Abbreviations^ 9Table 2^Male brood analysis 16Table 3^Hermaphrodite brood analysis^ 17Table 4^Male recombination on Linkage Group /^ 19Table 5^Recombination in hermaphrodite germlines 21Table 6^Recombination in Her-1 (X0) hermaphrodites^ 22Table 7^Effect of temperature on male recombination 25Table 8^Effect of rec-1 on male recombination^ 26Table 9^Effect of rec-1 on crossing over on LG I^ 38Table 10^Duplication mapping of rec-1^ 39Table 11^Deficiency mapping of rec-1^ 42Table 12^Effect of hIn1(I) on crossing over on linkage group / ^56Table 13^Egg-hatching frequencies of hIn1(1) heterozygotes and recombinants^57Table 14^Recombination in hIn1(I) homozygotes^ 60Table 15^Effect of hInl(I) on crossing over on other chromosomes^66Table 16^Effect of hIn1(I) on crossing over with LG / translocations^68Table 17^Cosmid mapping of rec-1^ 106viiiixTable 18^List of strains^ 112LIST OF FIGURESFigure 1^Partial genetic map of Linkage Group I^ 8Figure 2^Male and hermaphrodite meiotic maps of LG I ^18Figure 3^Variation of recombination frequency with parental age^23Figure 4^Meiotic map of LG /in the presence of Rec-1^ 37Figure 5^Deficiency map of the unc-54 region^ 43Figure 6^Meiotic map of LG /in hIn1(I) heterozygotes^ • 61Figure 7^Structure of h/n/(/)-derived recombinants 63Figure 8^DAP I-stainingof hDp132 meiotic cells^ 65Figure 9^Punnett diagramming recombination inhIn1(I)/szT1(I;X) heterozygotes^ 69Figure 10^Punnett diagramming segregation inhInl(I)/hT2(I;III) heterozygotes^ 71Figure 11^Pairing of inverted sequences in hIn1(I) heterozygote^74Figure 12^Effect of single crossover in hInl(I) heterozygote 79Figure 13^Cosmid map of the unc-54 region^ 105Figure 14^Sequences of primers used in PCR analysis^ 109Figure 15^PCR analysis of eDf24^ 110xxiACKNOWLEDGEMENTSI would like to thank my supervisor Ann Rose for the patience, guidance, andinterest shown to me during the course of this thesis. I would also like to thank mysupervisory committee Drs. Fred Dill, Don Moerman, and in particular David Baillie andDavid Holm for their helpful advice. I would also like to thank Raja Rosenbluth for herconstructive criticism of my manuscripts and this thesis. The cytogenetic techniques used inthis thesis were taught to me by Dr. Peter Moens and I would like to thank him for his timeand effort. The years I have spent in the Rose lab have priviledged me with the friendshipof many people and I would like to thank Ken Peters, Jennifer McDowall, Colin Thackerand especially Kim McKim for their help and discussion. I would especially like to thankmy family for their stoic support of all my endeavours and my friend Ralph Allan for yearsof silly fun.This work was supported in part by a University Graduate Fellowship.GENERAL INTRODUCTIONMeiosis is the process by which sexually reproducing organisms produce haploidgametes. This process consists of one round of DNA replication followed by a reductionaldivision at meiosis I and an equational division, resembling mitosis, at meiosis II. Prophaseof meiosis I is marked by two distinct processes that culminate in the segregation ofreplicated homologous chromosomes: pairing and recombination (reviewed by HAWLEY1988; HAWLEY and ARBEL 1993).Pairing between homologues is achieved by several temporally distinct events.During the first phase, called homologue recognition, homologous chromosomes are thoughtto find one another and align themselves at a distance in the diffuse nucleus (MAGUIRE1984). In C. elegans, homologue recognition regions have been identified on everychromosome (ROSE, BAILLIE and CURRAN 1984; McKIM, HOWELL and ROSE 1988)and are absolutely required for pairing and recombination. Once aligned, the chromosomesare brought into tighter association through a homology search that may be mediated byRecA-type proteins (CONLEY and WEST 1989), which locate and homologously pairdiscrete sites on the chromosome (CAO, ALANI and KLECKNER 1990; KLECKNER,PADMORE and BISHOP 1991). The existence of such sites has been documented in avariety of organisms including Drosophila raelanogaster, where pairing sites have beenmapped along the X chromosome of the female (HAWLEY 1980) and in the ribosomalcluster 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 andSIMCHEN 1993; GOLD WAY et al. 1993). During the second phase of pairing, this earlyalignment is locked in place by recombinational intermediates which result from the repair ofdouble-strand breaks that appear early in meiosis (SUN et al. 1989; PADMORE, CAO andKLECKNER 1991). The chromosomes then begin to condense, a process thought to be12crucial to the next stage of pairing, which results in intimate synapsis and the formation of atripartite laminar structure called the synaptonemal complex (SC), between the homologues(KLECKNER, PADMORE and BISHOP 1991). The ZIP1 locus of yeast encodes acomponent of the central region of the SC, indicating that specific proteins are required forthe formation of the structure (SYM, ENGEBRECHT and ROEDER 1993). MAGUIRE(1978) proposed that only those recombination intermediates which occur in the context ofthe SC have the potential to form chiasma. This is supported by the fact that double-strand breaks, thought to be the substrate for recombination, appear before and at the sametime as the first appearance of the SC (PADMORE, CAO and KLECKNER 1991). Inaddition, mutants in RED1, MER1, and HOPI are defective in SC formation but stillcompetent in meiotic exchange, indicating recombination can be initiated in the absence ofthe synaptonemal complex (ROCKMILL and ROEDER 1990; ENGEBRECHT, HIRSCHand ROEDER 1988; HOLLINGSWORTH, GOETSCH and BYERS 1990). In menlmutants, however, the exchange events that occur do not ensure faithful disjunction of thehomologues (ENGEBRECHT, HIRSCH and ROEDER 1990). This may be explained ifthe role of the SC during meiosis is the conversion of a number of sites of alignment andrecombination into a bivalent united by a chiasmata.An essential feature of meiosis is recombination between homologues, which serves toreassort genetic information and promote proper segregation of the chromosomes. Crossingover refers to a reciprocal event resulting in an exchange of flanking markers. The frequencyand distribution of crossing over are regulated, and a number of mutations which disruptthis pattern have been identified (reviewed by BAKER et al. 1976).The nematode C. elegans is an ideal system for the study of meiosis. Populationsconsist mostly of self-fertilizing hermaphrodites that are capable of producing about 300progeny each and that have a short generation time (3.5 days at 200). Males can be usedfor the introduction of genetic markers and the genetic maps of the five autosomes and theX chromosome are well marked with visible mutations. Recessive mutations have beenisolated that reduce crossing over (HODGKIN, HORVITZ and BRENNER 1979), conferradiation sensitivity (HARTMAN and HERMAN 1982), and increase both crossing overand conversion (ROSE and BAILLIE 1979b; RATTRAY and ROSE 1988). Thus, in C.elegans, gene products important in meiosis can be identified by mutations which producephenotypes that have also been described in other systems (BAKER et al. 1976). In thisstudy, meiosis in C. elegans has been investigated by examining the effect of sex, mutation,and rearrangement on recombination.34Chapter 1: Sex-Related Differences in Crossing OverINTRODUCTIONThe biology of C. elegans provides a unique opportunity to examine the effect of sexon recombination. Laboratory populations consist largely of self-fertilizing hermaphrodites(5AA;XX). Males (5AA;X0), arise spontaneously as a result of X-chromosomenondisjunction (HODGKIN, HORVITZ and BRENNER 1979) and are maintained bycross-fertilization with hermaphrodites. The standard genetic map of C. elegans (EDGLEYand RIDDLE 1990) is based upon hermaphrodite recombination frequencies that are theproduct of crossover events in two germlines: oocyte and hermaphrodite spermatocyte. Thefrequency of recombination in these two germlines has been shown to be different (ROSEand 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 relationshipbetween sex and recombination frequency. The first is the absence of recombination in onesex, a characteristic of D. melanogaster males (MORGAN 1912) and Bombyx moni females(TANAKA 1913). The second, more common situation, is one where recombination existsin both sexes, but with a reduced frequency in one (reviewed by DUNN and BENNETT1967). 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 generallyhigher in maize (RHOADES 1941; ROBERTSON 1984), and in Tribolium cast aneum(SOKOLOFF 1964). However, sex-related differences in recombination frequency are notuniform for all regions of the genome. In maize, some intervals have been reported to belonger in the female meiosis (ROBERTSON 1984). In mice, significant sex differences inrecombination frequency went in opposite directions on different chromosomes (DAVISSONand RODERICK 1981) and in humans, some regions were the same genetic size in both5sexes (DONIS-KELLER et al. 1987). This suggests local differences in recombinationbetween 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 hasbeen investigated. Each of the autosomes in C. elegans is marked by a region where genescluster on the meiotic map as a result of less recombination per base pair than the genomeaverage (BRENNER 1974; GREENWALD et al. 1987; KIM and ROSE 1987; PRASADand 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 hasbeen determined.One approach in studying the relationship between sex and recombination frequencyis measuring recombination in sexually transformed individuals. Hormone treatments havebeen used in the Medaka, Oryzias latipes, to transform XY fish, normally male, intofunctional females. Crossing over in these transformed males was found to occur at a higherfrequency than in normal males (YAMAMOTO 1961). This suggests that differences inrecombination between the sexes are not completely the result of the sex chromosomeconstitution, but also depend on the physiological differences associated with sex. In C.elegans, mutations exist which result in the complete transformation of the sexualphenotype. One such mutation, her-1, transforms fertile XO males into self-fertilehermaphrodites (HODGKIN 1980), and has been used in this study to examine the effect ofkaryotype on recombination frequency in the nematode.Meiotic recombination frequency in higher eukaryotes is affected by several knownparameters. Recombination frequency increases at temperature extremes in D. melanogaster(PLOUGH 1917, 1921), Neurospora crassa (McNELLY-INGLES, LAMB, and FROST1966) and Coprinus lagopus (LU 1969, 1974). A decrease in meiotic recombinationfrequency with maternal age has been observed in D. melanogaster (BRIDGES 1927; NEEL1941), in C. elegans (ROSE and BAILLIE 1979a), and on some chromosomes in the mouse,Mus musculus (FISHER 1949; BODMER 1961; REID and PARSONS 1963). Existinghuman data is not conclusive about maternal age effects although some evidence suggests apaternal age effect may exist (LANGE, PAGE and ELSTON 1975; ELSTON, LANGE andNAMBOODIRI 1976). In C. elegans, hermaphrodite recombination frequency decreaseswith maternal age and increases with temperature (ROSE and BAILLIE 1979a) and in thepresence of the rec-1 mutation (ROSE and BAILLIE 1979b). In this study, the effect oftemperature, age, and rec-1 on recombination in C. elegans males has been investigated.67Chapter 1: MATERIALS AND METHODSGeneral Methods: C. elegans population consist largely of self-fertilizing hermaphrodites(5AA;XX). Males (5AA;X0) arise spontaneously as a result of X-chromosomenondisjunction (HODGKIN, HORVITZ and BRENNER 1979) and were maintained bymating to hermaphrodites. Wild-type and mutant strains were maintained and mated onpetri plates containing nematode growth medium (NGM) and streaked with Escherichia(BRENNER 1974). All experiments were carried out at 200 unless otherwise noted. Thewild-type strain N2 and most mutant strains of C. elegans var. Bristol used in this studywere obtained from D.L. Baillie at Simon Fraser University, British Columbia or from theCaenorhabditis Genetics Centre at the University of Missouri, Columbia, Missouri. RW3072was 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 usedin the course of this work:LG^bli-3(e579); unc-11(e47); dpy-5(e61); bli-4(e937); dpy-14(e188); unc-13(e450); uric-29(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. elegansnomenclature 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 anumber 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, uric-101 (ml) is a mutation in a gene which results in an uncoordinated, or unc, phenotype. Itwas the one hundred and first unc gene identified, and the first mutation isolated in theFIGURE 1.-A partial genetic map of Linkage Group /showing the major markers used inthis study.8aunc- 13dpy- 14 unc-29^bli-4^lin-11dpy- 5 unc- 75 unc- 59let-50unc-54let-49bli - 3^unc- 11 unc-101Ilev-115 m.u. Linkage Group ITable 1AbbreviationsAbbreviation^ Phenotypebli^ blistered cuticledpy dumpyher^ helmaphrodization of XO animalshim high incidence of maleslet^ lethallev levamisole resistantlin^ abnormal cell lineageion longrec^ abnormal recombinationunc uncoordinated910laboratory with the m allele designation. The allele designation for the Rose lab is h and thestrain designation is KR.The following translocations were used in this study: szT/(/;X) (FODOR andDEAK 1985; McKIM, HOWELL and ROSE 1988), hT2(/;///) (McKIM, PETERS andROSE in press), hT1(I;17) (McKIM, HOWELL and ROSE 1988), and hT3(I;X) (McKIM1990). 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 ahomozygote and is marked with b/i-4 on /NHL, where L denotes the left arms ofchromosomes 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 componentsof the translocation (i.e. szT1(I;X)). When discussing the individual componentchromosomes of a translocation, the nomenclature describes the segregational properties ofthe 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 rightof 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), whichsegregates 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(/;///)/// (ofstructure /NHL), which segregates from chromosome III. hT1(I;V) consists of hT1(LV)I (ofstructure 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 (ofstructure /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 oninversion chromosomes are shown in square brackets (e.g. hInl(I)klpy-5 unc-41), similar tothe system in use for translocations. The nomenclature does not necessarily provideinformation 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 freeduplications 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 thelaboratory designation) and followed in parentheses by their chromosome of origin and thedesignation 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 homologueswhereas sDp2(1,1) duplicates the left end of the chromosome and does not pair andrecombine (ROSE, CURRAN and BAILLIE 1984). Deficiencies are abbreviated Df andare followed in parentheses by their chromosome of origin. eDf2.4 complements unc-54 andpartially deletes the ribosomal cluster, the most distal genetic marker on LG I. Theremaining eDf deficiencies fail to complement tine-54 and were isolated using eDf24 as abalancer (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 measuredby scoring the number of recombinant progeny of a cis-heterozygote, under the conditionsdescribed by ROSE and BAILLIE (1979a). The recombination frequency (p) between twomarkers was calculated using the formula p = 1 - (1 - 2R)172, where R is the number ofvisible 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 Wtsone recombinant class) where Wts is the number of wild-type progeny. Map distances inthe male were determined by scoring the progeny resulting from mass mating seven malesheterozygous for a pair of cis-linked markers to five homozygous hermaphrodites (newhermaphrodites each day) every 24 hours for four days. On the fourth day the males wereleft on plates with the same hermaphrodites for a fifth day, after which the hermaphrodites1112were 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 ofthe hermaphrodite for the following reasons. In both male and hermaphroditerecombination experiments, the double homozygote class is not scored because of its reducedviability, and the total progeny number is calculated from the wild-type class. Mapping inthe hermaphrodite involves crossing two heterozygous germlines, whereas mapping in themale involves crossing one germline heterozygous for a pair of markers to one which ishomozygous. For this reason, the ratio of wild-type progeny to progeny homozygous for themarkers differs in hermaphrodite and male recombination experiments. Thus, the number ofwild-type progeny must be multiplied by 4/3 and 2 respectively to correct for the inviableclass. Both classes of recombinants were used in the calculations unless otherwise noted. Incases 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 forthe presence of both male and hermaphrodite individuals of the recombinant phenotype. Inthe case of the bli- 3 unc - 11 interval, bli- 3 penetrance is low and Bli-3 recombinants werescored as wild-type and later subtracted. The unc - 75 unc - 101 and unc - 101 unc - 54 mapdistances 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 wereapproximated 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 oocyteswas 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. Thetranslocation 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)[++;dpy-18Jor unc-11 dpy-5; + /hT2(1;III)[+ +;dpy-18] were mated to heterozygoushermaphrodites every 24 hours and the male progeny were scored. The oocyterecombination frequency (a), is 2 X the number of recombinant individuals divided by thetotal progeny. The total number of progeny is 4/3 X (number of Wts + one recombinantclass). Knowing the value of R for the hermaphrodite and a, the recombination frequencyin the oocytes, the following equation was solved for b, the recombination frequency inhermaphrodite spermatocytes.R = 1/2b(1 - a) + 1/2a(1 - b)^1/2abRecombination 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-1homozygous 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 thegenotype + ; her-//hT4/;V)func-29; + J. These males were then crossed to hT3(I;X)[dpy-5unc-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 wild-type 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-75recombinants.Variation With Age: The variation of recombination with parental age was examined intwo intervals; dpy-5 unc-75 and dpy-5 unc-13. Young heterozygous males were individuallymated to 5 new homozygous hermaphrodites every 12 hours for 4 days. Heterozygous L4hermaphrodite 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 wasexamined in the dpy-5 anc-75 and dpy-5 unc-13 intervals. Seven heterozygous males weremass mated to five homozygous hermaphrodites and transferred to new hermaphroditesevery 24 hours at temperatures of 150 or 250. Hermaphrodite controls were picked from the13same plates as experimental males and were transferred every day. All progeny werepermitted to develop at 200 to avoid any inviablity produced by temperature extremes.14Chapter 1: RESULTSMale recombination frequency is lower than hermaphrodite: Differences inrecombination frequencies between the sexes were initially studied in two intervals; dpy- 5unc - 75 and dpy- 5 unc - 13. The latter interval is located within the chromosome /geneticcluster and the former includes the cluster and a genetically large interval to the right. Inboth 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 inthe male was general across the length of chromosome /, other intervals inside and outsidethe cluster were investigated. The results for six intervals spanning LG /is shown in Figure2 (data shown in Table 4). In the dpy- 5 unc -29 unc - 75 interval, only hermaphrodites werescored because the phenotypes of male recombinants were subtle and progeny testing wasnot possible. Male recombination frequency was lower in five of the intervals tested whencompared to hermaphrodite controls. The differences in recombination frequencies betweenthe hermaphrodite and the male in these intervals were not uniform; they varied from 1.3-fold in unc - 11 dpy- 5 to 2 -fold in dpy- 5 unc - 13 and unc - 75 unc - 101. In the unc - 101 unc - 54region, 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-relateddifferences are interval-dependent and not size-dependent. Thus, the greatest differences incrossover frequency were observed near the gene cluster, and no difference was observed atthe right end of the chromosome. The total genetic length of the meiotic map of LG /is31.7 m.u. in the male, compared to 44.1 m.u. in the hermaphrodite (data from Table 4). Asis the case with the hermaphrodite meiotic map, the male map is also marked by a centrallylocated cluster.Recombination in hermaphrodite spermatocytes is higher than in oocytes: Therecombination formula normally used in measuring map distances in the hermaphrodite isbased on the assumption that the frequency of recombination is equal in both germlinesalthough this has been shown not to be the case (ROSE and BAILLIE 1979a). To measure15Table 2Male brood analysisGenotype^Wts^Recombinants^pX100(C I )a9^d^Dpy^Unc9 d 9 ddpy-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)Totalsdpy-5 unc-75/ + +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)16a16ba C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).17Table 3Hermaphrodite brood analysisGenotype^ Wts^Recombinants^pX100(C.I.)aDpy^Uncdpy-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)Totalsdpy-5 unc-75I + +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)a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).FIGURE 2.-Male and hermaphrodite meiotic maps of LG I. Three factor experimentspositioned unc - 75 between dpy- 5 and uric - 101. The LG /cluster extends from unc - 11 tounc -29 (EDGLEY and RIDDLE 1990).18abli- 3^unc- I 1unc- 13dpy- 5 unc-29unc-54unc- 751 unc- 101I^IMaleunc- 13unc-29bli- 3dpy- 5unc- 11 unc-75 unc- 101^ unc- 545 m. u.^ Hermaphrodite19aTable 4Male recombination on Linkage Group IGenotype^Wts^Recombinants^pX100(C I.)a9^ 'blz-3 unc-11I +male b^1392^1206^135 Unc^109 Unc^9.4C(8.2-10.6)hermaphrodite^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 Unchermaphrodite^3786^58 Dpy 2.3(2.0-2.8)61 Uncdpy-5 unc-29/male^2536^2451^29 Dpy^35 Dpy^1.2(1.0-1.5)44 Unc^61 Unchermaphrodite^1822^30 Dpy 2.8(2.2-3.5)39 Uncdpy-5 unc-29 unc-75/ +male^581^ 11 Dpy-5d6 Unc-29 Unc-75d^1.4(0.8-2.2)17 Unc-75e 2.9(1.6-4.3)hermaphrodite^1598^34 Dpy-5d36 Unc-29 Unc-75d^3.4(2.6-4.2)2 UflC2gd,eunc-29 unc-75/ + +maleunc-75 unc-101/ + +malehermaphroditeunc-101 unc-54I + +malehermaphrodite63 Unc-75e 6.0(4.7-7.6)3374 3568 95 Unc-75 80 Unc-75 2.7(2.6-2.8)126 Unc-29 90 Unc-292634 2553 45 Unc 42 Unc 1.6c(1.3-2.0)3192 68 Unc 3.2c(2.7-3.8)392 362 71 Unc 62 Unc 15.0c(12.7-17.2)1187 116 Unc 14.4c(11.8-17.1)19ba 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.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 ofLC /markers, and the male progeny scored (see MATERIALS and METHODS). Theresults are shown in Table 5. In measuring the unc-11 dpy-5 interval, an unusually smallnumber of Dpy-5 recombinants were recovered. The most conservative approach was to useonly the Unc-11 recombinants in the calculations, since this would give the minimumestimate of differences in recombination between the two germlines. In both intervalsstudied, the frequency of recombination in hermaphrodite spermatocytes was higher thanthat observed in oocytes; 2-fold in dpy-5 unc-75 and 1.5-fold in unc-11 dpy-5. To furtherexamine the effect of sexual phenotype on recombination frequency, crossing over wasmeasured in males transformed into fertile hermaphrodites by the her-I mutation. Theresults 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 thosethat were fertile produced few progeny. For this reason, recombinants that proved to besterile upon progeny testing were also included in the calculations. The crossover frequencyin these transformed males was significantly higher than that observed in normal males. Anattempt was made to examine recombination in transformed hermaphrodites using the tra-1(e1099) mutation but these males mated poorly and rarely produced progeny.Male recombination varies with age: ROSE and BAILLIE (1979a) foundhermaphrodite recombination frequency to decrease with age. The effect of parental age onrecombination in the dpy-5 unc-75 and dpy-5 unc-13 intervals is shown in Figure 3 (datashown in Tables 2 and 3 respectively). In both intervals, male recombination frequencyshows a general decrease with age. Consistent with the previous results, the recombinationfrequencies of hermaphrodite controls also decreased with age. The variation in malerecombination with age shows some periodicity in both intervals tested. The statistical ofthis 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 greatestnumber of self-fertilization progeny were also produced in this period20Genotype^Wts^ Recombinants^pX100(C.I.) aDpy^Uncdpy-5 unc-75/ + +oocytebspermcunc-11 dpy-5/ + +oocytebspermc4290 83 92 6.0(5.2-6.9)12.43707 7 24 1.9(1.2-2.8)2.721Table 5Recombination in hermaphrodite germlinesa 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).22Table 6Recombination in Her-1(X0) hermaphroditesRecombinantsWild typesDpy^UncGenotype 9^9^9^pX100(C I )adpy-5 unc-75/ + +; her-I/her-1(X°)dpy-5 unc-75/ + +bmale53108032503 24574623148 132707122 13512.5(6.2-23.1)9.1(8.9-9.2)5.1(5.0-5.3)a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).b data from Table 3.FIGURE 3.-The variation of recombination frequency with parental age in the (a) dpy- 5unc - 75 interval and (b) dpy- 5 unc - 13 interval. Brood analysis for male heterozygotes isrepresented by the dashed line. Hermaphrodite controls are represented by the solid line.Vertical bars represent 95% confidence intervals.23a0(b)14o 1211=C l 10▪ 8O 6scp^4CC2o-(a)Time (hr)0^20^40^60^80^100Time (hr)—•—• Hermaphrodite^'4 Male23balthough the variation between individual males was high. In one experiment examining thedpy - 5 unc - 75 interval in the male, a small number of progeny were recovered in the 49-60 hrperiod and this was likely the result of the poor physical condition of the hermaphroditesused in the matings since it was not reproduced in later experiments.Male recombination frequency increases with temperature: Crossing over in thehermaphrodite has been found to vary with temperature (ROSE and BAILLIE 1979a). Todetermine if temperature has a similar effect in the male, recombination was measured incis-heterozygous males at experimental temperatures of 150 and 250. The results are shownin Table 7 with 200 controls for comparison. Recombination frequency in the male and inthe hermaphrodite decreased at 15° and increased at 25° in both intervals tested. In thedpy-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 leasttwo-fold greater in males when compared to that of hermaphrodite controls. Male crossoverfrequency remained lower than that observed in the hermaphrodite at all temperatures andin both intervals tested.Male recombination frequency increases with Rec-1: The rec - 1 mutation increasedmeiotic recombination three-fold in the hermaphrodite (ROSE and BAILLIE 1979b). Thisincrease retained the meiotic distribution of crossover events. To determine if this mutationhad 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 areshown in Table 8. Recombination frequency in the male increased three-fold in the unc - 11dpy-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-1hermaphrodite crossover frequencies remained higher than those observed in the male.2425aTable 7Effect of temperature on male recombinationGenotype^Wts^ Recombinants^pX100(C I )a9^d Dpy^Unc9^d^9^d15°Cdpy-5 unc-13I + +malebhermaphroditedpy-5 unc-751 + +malehermaphrodite20°Cdpy-5 unc-13/ + + cmalehermaphroditedpy-5 unc-75/ + + cmalehermaphrodite25°Cdpy-5 unc-13I^+malehermaphroditedpy-5 unc-75I +1870 1992 9 14 6 12 0.5(0.4-0.7)1218 9 8 1.0(0.6.-1.6)2345 2528 93 103 111 89 3.9(3.8-4.0)2206 121 130 8.4(7.4-9.5)4242 4140 36 28 34 40 0.8(0.7-1.0)3119 34 32 1.6(1.2-2.0)4231 4308 234 258 226 229 5.3(5.2-5.4)10803 623 707 9.1(8.9-9.2)968 1077 15 10 10 11 1.1(0.8-1.5)3024 52 42 2.3(1.9-2.8)25bmale^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.dpy-5 unc-13 rec-11 + + rec-1male 922hermaphrodite 2111dpy-5 unc-13/ + +maled 4241hermaphroditee 311926Table 8The effect of Rec-1 on male recombinationGenotype^Wts^ Recombinants^pX100(C.I.) a9^o'' Dpy^Unc9 d 9 d755 40 43 57 30 5.0(4.3-5.7)91 91 6.7(5.7-7.6)962 19 12 15 25 1.8(1.4-2.2)58 61 2.3(2.0-2.8)908 46 41 36 43 4.3(3.7-5.0)103 86 6.6(5.7-7.7)4140 36 28 34 40 0.8(0.7-1.0)34 32 1.6(1.2-2.0)unc-11 dpy-5/ + +male c^983hermaphrodite c^3786unc-11 dpy-5 rec-11 + + rec-1maleb^866hermaphrodite^2033C.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.Chapter 1: DISCUSSIONBRENNER (1974) first observed that each C. elegans autosome is marked by acluster 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 bythe enhancement observed in the clusters when treated with gamma radiation (KIM andROSE 1987) and elevated temperatures (ROSE and BAILLIE 1979a; this study). Theresults presented in this thesis show that the frequency of recombination is generally higherin the C. elegans hermaphrodite than in the male, although the increases are not uniformalong the length of the chromosome and one interval showed no sex-related difference. Inaddition, the gene cluster of LG I appeared to be larger in the male than in thehermaphrodite. That the genetic map of the male is smaller than that of thehermaphrodite, while the gene cluster is larger, may be explained if the recombinationsuppression observed in the hermaphrodite is more pronounced in the male, or if interferencevalues in the male (leading to double-crossing over) are low. HODGKIN, HORVITZ, andBRENNER (1979) found complete interference on the X chromosome of the hermaphroditebut 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 Xchromosome but neither possibility has been confirmed. It is unlikely that low interferencein the male is the basis of sex-related differences in recombination frequency for severalreasons. Firstly, large decreases in the male meiotic map were observed in small intervals inthe cluster, a region in which double-crossing over would be extremely rare. Secondly, in alarge interval like blz- 3 unc - 11, the male meiotic map showed a 36% decrease inrecombination when compared to the hermaphrodite. The number of double-crossovers onewould expect in this interval (approximately 2), cannot account for the magnitude of thisdecrease. Thus, while it is possible that interference values differ between the27hermaphrodite and the male, it is unlikely to be the sole explanation of differential rates ofcrossing over between the sexes.Elevated temperatures produce increases in recombination values in a number oforganisms including Drosophila (PLOUGH 1917), Coprinus (LU 1969, 1974), andNeurospora (McNELLY-INGLES, LAMB and FROST 1966). In Drosophila, the greatesttemperature related changes in crossover frequency occur in centromeric regions, whererecombination is normally suppressed (PLOUGH 1917; BRIDGES 1915, 1927; STERN1926; 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 atelevated temperatures. In this study, similar increases of recombination values have beenobserved in the male in two intervals. The dpy- 5 unc - 13 interval has been wellcharacterized and includes a portion of the chromosome which is the most recombinationallysuppressed (KIM and ROSE 1988; PRASAD et al. 1993). The adjacent unc - 13 unc - 75interval 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 genecluster on the genetic map extends further in the male than in the hermaphrodite and canbe expanded by temperature over a larger interval.The results of experiments using the rec - 1 mutation can also be interpreted in lightof sex-related differences in cluster size. This mutation increased the frequency of malerecombination in both intervals tested. In both sexes, a greater enhancement effect (6-8-foldincrease) was observed in the dpy- 5 unc - 13 interval, located within the cluster, whencompared to the uric - 11 dpy- 5 interval (3-fold increase), a larger region at the left end of thecluster. 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 tothe effects of rec - 1.BRENNER (1974) measured recombination frequency in oocytes on the Xchromosome and found this frequency to be the same as the hermaphrodite frequency. Inthis study, oocyte recombination frequency was measured in two intervals on LG / and28found to be lower than both the total hermaphrodite frequency and the crossover frequencyin hermaphrodite spermatocytes. These results may be explained if differences inrecombination frequency between the hermaphrodite germlines are genetic interval-dependent or limited to the autosomes. Although recombination was found to vary with agein male spermatocytes, it is unlikely hermaphrodite spermatocytes contribute to thevariation of recombination frequency with age in the hermaphrodite since spermatogenesis inhermaphrodites is restricted to the fourth larval stage, at which time about 300 sperm areproduced (HIRSH, OPPENHEIM and KLASS 1976; WARD and CARREL 1979). Thishas previously been pointed out in studies examining the variation of recombination withhermaphrodite age (ROSE and BAILLIE 1979a). If the recombination frequency inhermaphrodite spermatocytes (b) is constant, it follows that as the oocyte recombinationfrequency approaches zero with increasing age, the value of R in the hermaphrodite shouldnever fall below 1/2b. For example, in the dpy- 5 unc - 75 interval the value of R in the finalbrood (0.08) is still higher than 1/2b (0.06). Of further interest is the possibility that thevariation of recombination frequency with age is a continuum of the two germlines. Sincethe first brood measures the earliest oocyte recombination frequency (those events occurringright after the switch from spermatogenesis), one would expect the two germlines to havesimilar frequencies in this brood. In the dpy- 5 unc - 75 interval for example, knowing thevalue of R in the first brood (0.10) and the value of b (0.12), the value of a (0.09), thefrequency of recombination in the oocyte, can be calculated. As predicted, the oocyterecombination frequency in this brood is close to, but not higher, than the spermatocytefrequency.YAMAMOTO (1961) measured recombination in hormonally transformed XY malesof the Medaka and found the recombination frequency to be much higher than thatobserved in normal males. In this study, recombination was measured in males sexuallytransformed by the her- 1 mutation. Similar to the previous results, the recombinationfrequency was significantly higher in the transformed males when compared to normal29males. This result can be interpreted as evidence that it is the sexual phenotype and notgenotype that determines the frequency of recombination during gametogenesis.Meiotic recombination frequency in both sexes of C. elegans is affected by age andtemperature. Recombination frequency decreases with maternal age in Drosophila(BRIDGES 1927; NEEL 1941), in mice (FISHER 1949), and in C. elegans (ROSE andBAILLIE 1979a). A fall in crossover frequency with paternal age was observed in twointervals. This variation of recombination frequency with parental age does not affect theresults of other experiments. As described in MATERIALS and METHODS, only L4hermaphrodites, which are easily identifiable at that stage, and young males were used inlater experiments further characterizing recombination. The population of males used inthese experiments was considered to be synchronous since all male recombinationexperiments were replicated and reproducible results were obtained. For example, thecurves derived from four separate experiments examining the variation of recombinationfrequency 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 beapproximately one-third less than that observed in the hermaphrodite. This decrease,however, was not uniform and one interval showed no sex-related difference in crossoverfrequency. By measuring recombination in the two germlines of the hermaphrodite and intransformed males, it has been concluded it is the physiology of the gonad, rather than thesexual karyotype of the germline, that determines the recombination frequency characteristicof a specific sex. It was also observed that male recombination in C. elegans varies with ageand temperature, suggesting recombination is quantitatively rather than qualitativelydifferent between the sexes. For this reason, it is recommended that the standard practicessuggested by ROSE and BAILLIE (1979a) for hermaphrodite recombination experiments bealso applied to male recombination studies (i.e. that studies measuring recombinationfrequency be carried out at 200 and all progeny from the male should counted).30Chapter 2: Characterization and Mapping of rec - 1INTRODUCTIONMutations which disrupt the normal frequency and distribution of crossing over canidentify genes important in the control of meiosis. Study of these mutations has revealedthat in organisms in which recombination normally occurs, one crossover between thehomologues is necessary for their proper disjunction (reviewed by JONES 1984, 1987). Oneclass of genes is defined by mutants which are defective in the recombinational machineryand 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 recombinationdefective and result in reduced levels of recombination. In D. melanogaster, recombinationdefective mutants have been divided into three groups based on the distribution of theirexchange events (reviewed by BAKER et al. 1976). One group is represented by the genemei-9, whose mutations decrease the frequency of crossing over but maintain the wild-typepattern of events (BAKER and CARPENTER 1972; CARPENTER and SANDLER1974). The second group includes the genes mei-218, mei-41, and mei-251, whose mutationsreduce the frequency of crossing over by differing amounts in different intervals, therebydisrupting both the frequency and pattern of exchange events (BAKER and CARPENTER1972; CARPENTER and SANDLER 1974; SANDLER and SZAUTER 1978). The thirdgroup is represented by mutations in mei-352 which alter the distribution, but not theoverall frequency of crossing over (BAKER and CARPENTER 1972).In C. elegans, the majority of meiotic mutants have been isolated as recessivemutations that increased the nondisjunction frequency of the X, resulting in a Him (highincidence of males) phenotype (HODGKIN, HORVITZ and BRENNER 1979). Mutationsin the genes him-6 and him-14 are recombination defective and produce nondisjunction ofthe autosomes as well as of the X chromosome, presumably as a result of the reduction incrossing over (HODGKIN, HORVITZ and BRENNER 1979; KEMPHUES, KUSCH andWOLF 1988). A class of meiotic mutant not previously described is represented by the31recessive rec - 1 mutation which increases both crossing over (ROSE and BAILLIE 1979b)and conversion (RATTRAY and ROSE 1988) on all chromosomes. Since rec - 1 mutantsare not radiation sensitive (HARTMAN and HERMAN 1982), the function of this geneappears to be specific to meiosis rather than to general DNA metabolism. In this thesis, theeffect of rec - 1 on the distribution of crossing over has been characterized and a map positionhas been determined.32Chapter 2: MATERIALS AND METHODSThe 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 locuswas examined by measuring recombination in the dpy-5 dpy-14 interval in the presence ofthe duplication (sDp1 carries wild-type alleles of both of these markers). Rec-1 or N2 maleswere mated to dpy-5 dpy-1.4 rec-1/dpy-5 dpy-1.4 rec-1/sDpl(I,f) hermaphrodites and dpy-5dpy-14/dpy-5 dpy-14/sDpl(I,f) controls respectively. Wild-type hermaphrodite progenyresulting from this cross were individually plated and their progeny scored. sDpi-bearinghermaphrodites have lower brood sizes (duplication homozygotes are inviable) and increasednondisjunction 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 forsDp1(I,I) were examined for the presence of males, and the frequency of the doublehomozygote class was determined. This class was expected to approach a frequency of 0.125in the presence of the duplication and and a frequency of 0.25 in its absence. Therecombination frequency in individuals lacking the duplication was calculated as describedin Chapter 1: MATERIALS and METHODS. A gametic frequency of 0.43 forsDp1(I,f)(ROSE, BAILLIE and CURRAN 1984) was used to calculate the frequency ofcrossing over in individuals determined to be of the genotype dpy-5 dpy-14 rec-1/ + + rec-1/sDp1(I,l) and dpy-5 dpy-14 + / + + rec-1/sDp1(1j) using the formula:p = 1 - [1 - 148D/17(D + W)]1/2where 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 thatrecombination 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-1/3334+ + 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 thefrequency of segregation of the double mutant as described for sDpl. A gametic frequency of0.38 for sDp2(I;f) (ROSE, BAILLIE and CURRAN 1984) was used to calculate thefrequency of crossing over in the presence of the duplication using the formula:p = 1 - [1 - 75D/19(D + W)]112where D is the number of Dpy-5 progeny and W is the number of wild types. This formulaassumes that the sDp2 homozygote is not viable. Recombination was also measured in dpy-5 unc-13 rec-1/ + + rec-1jsDp2 hermaphrodites and in dpy-5 unc-13 rec-1/ + + + /sDp2controls. 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-13segregants amongst their progeny. Recombination in the dpy-5 unc-13 interval wascalculated using the formula:p =1 - [1 - 19D/(D w.)]1/216where 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 therec-1 locus, dpy-11 unc-42/ + + ; rec-1/rec-1 or dpy-11 unc-42/ + + males were crossed tounc-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 progenyand failed to segregate Unc-54 individuals (indicating the presence of the deficiency) werescored. Recombination was measured in the dpy-11 unc-42 interval using the generalrecombination formula discussed in Chapter 1: MATERIALS and METHODS. Thedeficiencies eDf4, eDf9, eDf10, and eDf13 were isolated using eDf24 as a balancer and allcomplement 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 matedto unc-54/ + males and the resulting Unc-54 hermaphrodites were then mated to males ofthe genotype dpy-11 unc-42/ + + ; rec-1/rec-1 or dpy-11 unc-42/ + +. Wild-typehermaphrodite progeny were individually plated and their progeny screened for the presenceof Dpy-11 Unc-42 segregants and the absence of Unc-54 segregants. The recombinationfrequency in the dpy-11 unc-42 interval was then measured and calculated as describedabove for eDf24.35Chapter 2: RESULTSrec - 1 alters the distribution of crossing over: ROSE and BAILLIE (1979b) showedthat rec - 1 greatly enhanced the frequency of crossing over in small intervals. To determinethe effect of rec - 1 on recombination along the whole chromosome, four intervals spanningLG /were examined and the results are shown in Figure 4 (Data shown in Table 9). Thebli- 3 unc - 11 interval, located on the left arm of LG I, was 12.8 in rec - 1 homozygotes and14.8 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 - 1homozygotes (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 extensiveenhancement in the presence of rec - 1. The unc - 101 unc - 54 interval, located on the rightarm of the chromosome, was severely reduced from 14.4 m.u. in controls to 4.6 m.u. in rec - 1homozygotes. The dpy- 5 unc- 54 interval, however, was 31.6 m.u. in controls and 30.6 rec - 1 homozygotes, indicating that total recombination on the right arm of LG /in rec - 1homozygotes did not change when compared to controls. The total genetic length of LG Iwas 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 nolinkage between rec - 1 and any markers located in the gene clusters of the autosomes. Whenmarkers located at the ends of the chromosomes were tested, rec - 1 showed loose linkage tothe gene unc - 54, located on the right end of LG I (ROSE unpublished results). Since rec - 1is completely recessive (ROSE and BAILLIE 1979b), a strategy using two largeduplications of LG I, sDp1 and sDp2, to map the gene was developed (data shown in Table10; extent of duplications shown in Figure 4). Although sDp1 does pair and recombine withLG 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 withthe duplication. In the absence of the duplication, the frequency of crossing over in dpy- 536FIGURE 4.-Meiotic maps of LG /in the presence of the rec - 1 mutation and in controls.37aunc- 13bli- 3^ unc- 11dpy- 5 unc- 5unc- 101Rec-1unc- 13unc- 101unc- 54dpy- 5bli- 3^ unc- 11I1^isDp2sDp 1Linkage Group I5 m.u.Table 9The effect of rec - 1 on crossing over on LG /38Wts Recombinants pX100(C.I.)a1686 170 Unc 14.8(12.4-17.4)990 79 Unc 12.8(10.0-16.1)3786 58 Dpy 2.3(2.0-2.8)2033 91 Dpy 91 Unc 6.7(5.7-7.6)3119 34 Dpy 32 Unc 1.6(1.2-2.0)3706 156 Dpy 6.3(5.3-7.3)889 79 Dpy 66 Unc 12.0(10.0-14.0)1369 183 Dpy 213 Unc 21.2(20.1-22.2)1187 116 Unc-101 14.4(11.8-17.1)1973 61 Unc-101 4.6(3.6-5.8)1620 349 Dpy 31.6(30.3-32.9)1698 355 Dpy 267 Unc 30.6(29.2-32.0)Genotypebli-3 unc-11/ + + bbli-3 unc-11 rec-1/ + + rec-1unc-11 dpy-5/ + + bunc-11 dpy-5 rec-1/ + + rec-1dpy-5 unc-13/ + +edpy-5 unc-13 rec-1/ + + rec-1ddpy-5 unc-101/ + + edpy-5 unc-101 rec-1/ + + rec-1unc-101 unc-54/ + +bunc-101 unc-54 rec-1/ + + rec-1dpy-5 unc-54/ + +dpy-5 unc-54 rec-1/ + + rec-1C.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.Table 10Duplication mapping of rec-13 9Wts Recombinants pX100(C.I.) a3238 28 Dpy-5 1.3(0.88-1.8)1614 6 Dpy-5 1.6(0.7-3.5)8659 321 Dpy-5 5.5(5.4-5.6)1201 5 Dpy-5 1.8(0.7-4.1)2213 25 Dpy-5 1.7(1.1-2.4)1976 7 Dpy-5 0.7(0.3-1.4)1729 67 Dpy-5 5.8(4.4-7.2)1002 13 Dpy-5 2.6(1.3-4.2)3119 34 Dpy 32 Unc 1.6(1.2-2.0)2133 23 Dpy 1.6(1.0-2.4)3706 156 Dpy 6.25(5.3-7.3)1057 4 Dpy 0.2(0.08-0.6)1977 41 Dpy 1.4(0.9-1.9)Genotypedpy-5 dpy-14 + / + + rec-1dpy-5 dpy-14 + / + + rec-1/sDp1dpy-5 dpy-14 rec-1/ + + rec-1dpi-5 dpy-14 rec-1/ + + rec-1/sDp1dpy-5 dpy-14 rec-1/ + + +dpy-5 dpy-14 rec-1/ + + +/sDp2dpy-5 dpy-14 rec-1/ + + rec-1dpy-5 dpy-14 rec-1/ + + rec-1/sDp2dpy-5 unc-13/ + + bdpy-5 unc-13 + / + + rec-1dpy-5 unc-13 rec-1/ + + rec-1dpy-5 unc-13 + / + + rec-1/sDp2dpy-5 unc-13 rec-1/ + + rec-1/sDp2C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).b Data from Table 3.40dpy-14 rec-1/ + + + heterozygotes was 0.013. In rec-1 homozygotes, the frequency ofrecombination increased to 0.055 in this interval. In the presence of sDp1, the frequency ofrecombination 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. Theseresults are also consistent with the finding that rec-1 is completely recessive to its wild-typeallele (ROSE and BAILLIE 1979b). To ensure that the suppression observed was not ageneral feature of LG /duplications, recombination was also measured in the presence ofsDp2, a large duplication of the left half of the chromosome. The frequency of crossing overbetween dpy-5 and dpy-1.4 in the presence of sDp2 and Rec-1 (0.026) was 3-fold higher thanin the absence of Rec-1 (0.007), indicating that the Rec-1 phenotype was expressed despitethe presence of the duplication. Although the frequency of crossing over in the presence ofsDp2 and Rec-1 was significantly higher than in the absence of Rec-1, the frequencies weremuch lower than those obtained in the absence of the duplication (0.06 in rec-1homozygotes and 0.02 in heterozygotes). To confirm the possibility that the overall decreasein recombination frequencies could be attributed to a reduced recovery of recombinants inthe presence of sDp2, recombination was examined in another interval. The frequency ofcrossing 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 inthe presence of sDp2 and Rec-1 (0.014) was 7-fold higher than that observed in the absenceof Rec-1 (0.002). Since the recombination formula used to calculate the frequencies assumesthat both sDp1 and sDp2 are inviable as a homozygotes, these results may be explained ifsDp2 homozygotes can be recovered and are affecting the recovery of recombinants. Analternative explanation may be that sDp2 suppresses recombination between the twohomologues, however, recombination between the duplication and the chromosomes /hasnot been observed (ROSE, BAILLIE and CURRAN 1984).eDf24 (I) fails to complement rec-1: The duplication sDp1 covers the right arm of LGI, including most of the centrally located cluster. Since rec-1 was suppressed by sDpl,deficiencies of the right end were tested for failure to complement the mutation (data shownin Table 11). The deficiencies used in this study and their known breakpoints are shown inFigure 5. The dpy-11 unc-42 interval, normally 2.7 m.u., increases to 6.4 in rec- 1homozygotes. In eDf24/rec-1; dpy- 11 unc-42/ + + heterozygotes, this interval showed a 2-fold enhancement in recombination (5.6 m.u.) when compared to eDf24/ + ; dpy-11 unc-42controls (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-54locus (including eDf4, eDf9, eDf10, and eDf13) (ANDERSON and BRENNER 1984) whichhad undefined right breakpoints. Although these deficiencies genetically complementedeDf24, the possibility remained they physically overlapped eDf24 in a region that did notinclude any essential genes. All of the deficiencies tested complemented rec-1, indicatingthat if the deletions did overlap with eDf24, rec-1 was not included in the region of overlap.41Table 11Deficiency mapping of rec-142Wts Recombinants pX100(C.I.) a1250 26 Dpy 20 Unc 2.7(2.0-3.6)1219 66 Dpy 59 Unc 7.6(6.4-9.0)999 19 Dpy 23 Unc 3.1(2.2-4.1)693 8 Dpy 11 Unc 2.0(1.2-3.1)1127 29 Dpy 3.8(2.5-5.4)1558 46 Dpy 49 Unc 4.5(3.6-5.5)1668 35 Dpy 33 Unc 3.0(2.3-3.8)2119 88 Dpy 72 Unc 5.6(4.8-6.5)Genotypeunc-42 dpy-11/ +unc-42 dpy-11/^; rec-1/rec-1unc-42 dpy-11/ + + ; rec-1/eDf4unc-42 dpy-11/ + + ; rec-1/eDf9unc-42 dpy-11/ + + ; rec-1/eDf10unc-42 dpy-11/ + + ; rec-1/eDf13unc-42 dpy-11/^; /eDf24unc-42 dpy-11/^; rec-1/eDf24a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).43aFIGURE 5.-Deficiency map of the unc-54 region of LG I. The right breakpoint of eDf24 isknown to map within the ribosomal cluster whose genetic locus is rrn-1 (see APPENDIXIII). The left breakpoint maps within nonribosomal sequences to the right of unc-54 (seeChapter 2: RESULTS). It is not known how far eDf9 and eDf4 extend to the right, onlythat they complement eDf24. Both deficiencies may delete common sequences with eDf24, ifno essential genes are included.unc— 54gus— 1let-20643blet-50let-208unc— 59^lev— 111 lev— 10I^Ices-2 rrn— 1eDf24 1 HeDf 1 0eDf 13 IeDf4 ^eDf9 ^LG I (right)144Chapter 2: DISCUSSIONThe rec - 1 mutation, initially described as a general recombination enhancer, increasesmeiotic crossing over (ROSE and BAILLIE 1979b) and conversion (RATTRAY and ROSE1988), without disrupting the normal pattern of meiotic exchange. The intervals tested inthese studies, however, were located in the central gene clusters, or at the distal tips of thechromosome arms, where a small clustering effect also exists. BRENNER (1974) firstproposed that the gene clusters observed on the meiotic map were the result ofrecombination suppression in the region. This has been supported by several studies thathave shown that the cluster is a result of less recombination per base pair than the genomicaverage (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 enhancedin rec - 1 homozygotes, consistent with previous results (ROSE and BAILLIE 1979b). Thelevel of enhancement, however, was dependent on the interval tested within the cluster; thefrequency of recombination increased 3-fold in the uric - 11 dpy-5 interval and 5-fold in thedpy- 5 unc - 13 interval. The differential level of enhancement may be explained if rec- 1alleviates the recombination suppression normally present in the cluster and if thissuppression is more extreme in some regions. This interpretation is supported by the factsthat the dpy- 5 unc - 13 interval contains the most recombinationally suppressed region of thecluster (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 twolarge 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 - 101anc - 54, a comparably-sized interval on the right end, showed a 4-fold reduction. Theapparent suppression of crossing over in this interval may be explained if rec - 1 reducedinterference values on the right arm, or if the recombination frequency normally observed inthat region is enhanced per base pair, compared to the genome average, however, neitherpossibility has been confirmed.Darlington (1937) proposed that the formation of a chiasmata between homologueswas necessary for their proper disjunction. In C. elegans, the suppression of crossing overbetween homologues results in their random segregation (ZETKA and ROSE 1992; thisstudy). 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 inversionheterozygotes (ZETKA and ROSE 1992; this study). The conservation of a 50 m.u. geneticlength can be explained by some cytological evidence suggesting that the meioticchromosomes are held together by a terminalized chiasma at metaphase of meiosis I(ALBERTSON and THOMSON 1993). The genetic size of chromosome /in rec - 1homozygotes also approached 50 m.u., compatible with an average of one crossover everymeiosis. Although rec - 1 does not affect the total number of crossovers, its effect ofexpanding some regions and contracting others, disrupted the normal distribution ofexchanges. This suggests the mechanism responsible for ensuring that one crossover occursbetween the homologues every meiosis is epistatic to rec - 1(+), the role of which appears tobe in determining preferred sites of exchange. The existence of such sites is supported bystudies 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 andin its absence, the distribution of crossover events on the chromosome becomes related tophysical size. On chromosome /, a preferential site of exchange may be located on the rightarm and would explain the high frequency of recombination in the unc - 101 unc - 54 region inspite 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 alsoreduce the frequency of crossing over. Mutations in the exceptional gene, mei- 352, disruptthe distribution of exchanges along the chromosome, but do not alter their frequency45(BAKER and CARPENTER 1972), similar to rec - 1. In both mutants, the frequency ofrecombination is increased in regions which are normally recombinationally suppressed.Recombination in mei- 352 mutants is enhanced in the centric heterochromatin and on thefourth chromosome of mei- 352 mutants (BAKER and CARPENTER 1972; SANDLER andSZAUTER 1978). In rec - 1 mutants, crossing over is enhanced in the gene clusters, and inthe case of chromosome /, also in a region adjacent to the cluster (the unc -29 unc - 101interval). This region has not been cloned (COULSON et al. 1986, 1988), presumablybecause of the presence of repetitive sequences. The meiotic centromere of chromosome Ihas been mapped to the dpy- 5 unc - 75 interval and is thought to be tightly linked to the leftbreakpoint of the inversion hIn1(I), which lies between unc -29 and unc - 75 (ZETKA andROSE 1992; this study). This is supported by the structure of two recombinantchromosomes, derived from inversion heterozygotes, which proved to be deficiencies withindistinguishable left breakpoints. These deficiencies are thought to have arisen as a resultof the breakage of a dicentric bridge at anaphase II. That two independent events couldresult 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 theunc -29 unc - 75 interval) and as a result, the region pulled apart at anaphase is small, andthe probability of two independent breaks in the region is high. In addition to the presenceof potential centromeric sequences, a family of repetitive sequences (La VOLPE,CIARAMELLA, BAllICALUPO 1988) have also been mapped to this region. For thesereasons, it is possible that rec - 1 disrupts two distinct forms of crossover suppression; oneform responsible for establishing the gene cluster and the other responsible for the crossoversuppression associated with repetitive sequences. mei- 352 mutants also exhibit a decreasedfrequency 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 suppressionobserved on the right arm of LG /in rec - 1 mutants can be explained by lower interferencevalues, the number of double crossovers expected in the unc - 101 unc - 54 interval46(approximately two) cannot explain the 3-fold reduction in genetic length. Furthermore, thegenetic length of chromosome / in rec - 1 mutants approached the 50 m.u. observed incontrols, 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 forexchange necessary for determining its probability or distribution on the chromosome, theydiffer in two important respects. Firstly, mei- 352 specifically increases recombination inrecombinationally-suppressed regions and does not decrease recombination in any interval,whereas rec - 1 increased recombination in the cluster and also decreased the probability ofexchange in the unc - 101 unc - 54 region. Secondly, mei- 352 females are partially sterile andthis phenotype cannot be explained by nondisjunction of the autosomes as a result ofreduced recombination, since mutants have normal levels of exchange (BAKER andCARPENTER 1972). Paradoxically, mei- 352 mutants exhibit increased nondisjunctionand 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 geneproduct is actually involved in the sex-determination pathway and that its effect on meiosisis indirect (K. McKIM pers. comm.); the mutation may disrupt sex-determination geneswhich regulate meiosis-specific genes. These observations suggest that while mei-352 andrec - 1 clearly disrupt a meiotic process necessary for establishing spatial limits on thedistribution of exchange, rec - 1 specifically disrupts this process and as a result, remains thelone representative of an unusual class of meiotic mutants.47Chapter 3: The Meiotic Behaviour of an InversionINTRODUCTIONChromosome rearrangements that result in crossover suppression are useful for awide range of genetic experiments, including the dissection of chromosomal featuresresponsible for meiotic behaviour. An understanding of the mechanisms responsible for theelimination of meiotic events in the presence of the rearrangement can lead to the discoveryand description of sites necessary for the recognition and synapsis of homologues, meioticexchange, and subsequent disjunction. For example, studies of translocations in C. eleganshave led to the proposal that each chromosome contains a single region necessary forhomologue recognition and pairing (ROSENBLUTH and BAILLIE 1981; McKIM,HOWELL and ROSE 1988; reviewed by ROSE and McKIM 1992). Translocations are themajor 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 ROSE1988), although intrachromosomal crossover suppressors have also been described(HERMAN 1978; ANDERSON and BRENNER 1984; ROSENBLUTH, JOHNSEN andBAILLIE 1990). For example, deletions of the chromosome V end that does not containthe region necessary for homologue recognition were found to suppress recombination forseveral map units beyond the breakpoint of the deletion (ROSENBLUTH, JOHNSEN andBAILLIE 1990). The authors proposed that the deletions eliminated sites required formeiotic synapsis which occurs after homologue recognition has taken place. Insertionalduplications have a polar effect on recombination (HERMAN, ALBERTSON andBRENNER 1976; McKIM 1990; reviewed by ROSE and McKIM 1992). Theintrachromosomal suppressor mnCl(II) has been used to balance a large region ofchromosome // (HERMAN 1978) and although mnC1(H) is widely believed to be aninversion, no reversal of gene order has been demonstrated.48In this thesis, the first genetic inversion in C. elegans is described. Since thisinversion, hIn1(I), suppresses crossing over in a region not previously balanced bytranslocations, 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 centromericbehavior of chromosome I has been characterized.49Chapter 3: MATERIALS AND METHODSThe 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 matingwere individually plated and their progeny screened for the absence of Unc-101recombinants. In total, 900 chromosomes were screened and one isolate recovered thatsuppressed 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 platedand allowed to lay eggs for two 10-12 hour periods. The hermaphrodites were thentransferred and the eggs remaining on the plate counted. All resulting progeny werecounted three days later.Induction of genetic markers on hInl (I): hIn1(1) homozygous males were treatedwith 25 mM EMS (ROSENBLUTH, CUDDEFORD and BAILLIE 1983) using theprocedure described by BRENNER (1974). The mutagenized males were then mated tounc - 75(e950) unc - 101(m1) homozygotes for 24 hours. These hermaphrodites wereindividually plated and their progeny screened for the presence of Unc-75 individuals. Sincethe 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 aninduction of a new mutation on the hIn1(I) chromosome. 18,700 chromosomes werescreened and two mutations were recovered; unc - 75(h1041), and unc - 75(h1042). Both newunc - 75 alleles were lethal as homozygotes and were maintained as heterozygotes. Both newalleles produced the Unc-75 visible phenotype when crossed to males heterozygous for unc-75(e950). To induce an unc - 54 mutation on the hInl(I) chromosome, hIn1(1) malesmutagenized in the procedure described above were mated to lev- 11 let -49 + + I + + unc-5054 let-50 (RW3072) hermaphrodites for 24 hours. These hermaphrodites were thenindividually plated, and their progeny screened for Unc-54 individuals. A total of 5200chromosomes were screened, of which one-half are heterozygous with the unc-54 let-50chromosome. Some Unc-54 isolates could arise from recombination between unc-54 and let-50 in the parental strain. To test if the new unc-54 mutations were linked to hIn1(I), theprogeny of putative hIn1(1)t+ + unc-541/unc-101 1ev-11 + hermaphrodites were screened forthe presence of Unc-101 and Lev-11 recombinants. One strain, KR2151, exhibited completerecombination 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 examinecrossing over in dpy-5 unc-75, Dpy-5 recombinants were picked from amongst the progeny ofhInl(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 fromthis cross were then mated to dpy-5 unc-75(e950)/+ + males to ensure the dpy-5 mutationwas still present. The resulting hIni(I)Icipy-5 unc-75,Vdpy-5 unc-75(e950) progeny werecrossed to hIn1(1)1-unc-54Y + males and a fraction of the wild-type individuals resultingfrom this cross were of the desired genotype hInl(Opy-5 + unc-75j/hIn1(1)1+ unc-54This experiment also confirmed the gene order in hInl (I) was dpy-5 unc-54 unc-75 (seeChapter 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 inthe same heterozygotes used in the three-factor experiment using the formula:p = 9 - [81 - 20(2D W)(9D - W)/(D W)2]1/210(2D W)/(D W)51where 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, themutations can be crossed onto the inversion chromosome. Since hIn1(I) is viable as ahomozygote, recombination was measured in hInl(I)[+ +]/hInl(Didpy-5 unc-29J andhIn1(01-+ +J/hIn1(Dldpy-5 unc-54] heterozygotes using the general mapping methodsdescribed in Chapter 1: MATERIALS and METHODS.Analysis of recombinants from hIn1 (I) heterozygotes: Four rare recombinantsfalling 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-101unc-54 and hIn1(k+ +J/unc-75 unc-101 heterozygotes respectively. The duplicationshDp131 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 tovisible markers. For example, markers inside the region of hIn1(I) crossover suppressionwere tested by mating males of the genotype unc-75 unc-101/hInl(Di + + Jto hDp(D/unc-101 1ev-11 hermaphrodites. A fraction of the wild-type progeny from the cross were of thedesired genotype hDp(I)/unc-75 unc-101 + / + unc-101 1ev-11. Upon examining theprogeny of such individuals, Unc-75 individuals were observed if the duplication did notcarry unc-75(+). In the event the duplication did carry unc-75(+), no Unc-75 individualswere observed. A similar procedure was followed for markers outside the region of crossoversuppression with the exception that males heterozygous for a wild-type, rather than ahIn1(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-75u,nc-101 +/unc-75 + unc-54. Since Unc-75 Unc-54 individuals are similar in phenotype toUnc-54 individuals, several wild type progeny from the latter heterozygote were plated andtheir progeny examined. If the duplication carried unc-54(+), a fraction of these individualswould be of the genotype hDp(I)/unc-75 unc-54.52The deficiencies hDfll and hDf12 were complementation tested with several visiblemarkers by mating hDfll/unc-101 unc-54 and hDf12/unc-75 unc-101 hermaphrodites toeither unc-x/ + or lev-11/ + males. The F1 progeny resulting from this mating werescreened for both males and hermaphrodites Unc-x or Lev-11 in phenotype, the presence ofwhich 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 meioticchromosomes of an hDp132/unc-29 unc-75 hermaphrodite were stained with DAPI asdescribed by MOENS and PEARLMAN (1991). Hermaphrodites were placed in a solutionof 0.03% TWEEN and the gonads were removed. The tissue and cells were then fixed in4% formaldehyde and allowed to dry at room temperature overnight. The slides were thentreated with 5-10 ul/ml of DAPI solution (0.1 mg/ml DAPI in PBS) in 1 ml of moui:i.tingsolution 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 ofhIn1(1) crossover suppression and the szT/(/;X)/breakpoint was measured by scoring theUnc-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-101hermaphrodite progeny were scored from + ; /szT1(I;X)1-unc-1O1; lon-21hermaphrodites. 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 thefollowing formula:p = 4 - [16 - 60 1_7(U + W)]1/2653where U is the number of Unc-101 recombinants and W is the number of wild-typeprogeny.54Interaction of hIn I (I) with hT2(/;///): To examine the interaction of hIn1(1) with thetranslocation hT2(1;III), recombination was measured in +^+ /hT2(1,117)[bli-.4 dpy-5 unc-541 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. Thesegregation of hIn 1 (I) and hT2(/;///)/ was examined by scoring the Dpy-5 Unc-29 progenyof 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 genotypehIn1(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 progenyscreened for the absence of Unc-101 Lev-11 individuals.55Chapter 3: RESULTSIsolation of a crossover suppressor for the right end of LG^hIn1(I) was identifiedin 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 0.04m.u. in hIn1(1)1+ +Yunc-101 unc-54 heterozygotes (see Table 12). Since recombination inthis interval was measured using the Unc-101 recombinant class (Unc-54 recombinants areindistinguishable from the double mutant), the possibility that hIn1(I) was a suppressor ofthe Unc-101 phenotype remained. For this reason, crossing over was examined in unc-1011ev-11 heterozygotes from which both recombinant classes were recovered. This interval was9.0 m.u. in unc-101 lev-11/ + + heterozygotes and 0.07 m.u. in hInl(I) f+ +ilunc-101 1ev-11heterozygotes, demonstrating extensive crossover suppression of the right arm of LG /inhInl (I) heterozygotes. Individuals homozygous for hInl (I) were fertile and wild type inappearance. Since most crossover suppressors identified in C. elegans are tranlocations, thesegregation of hIn1(I) from a normal homologue marked with an unc-101 mutation wasexamined. The predicted segregation pattern of wild-type and Unc progeny for atranslocation 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 intrachromosomalrearrangement.hIril (I) heterozygotes have wild-type zygote viability: To further confirm thathIn1(I) was an intrachromosomal rearrangement, the egg-hatching frequency of individualshomozygous and heterozygous for the mutation was determined and is shown in Table 13.The egg-hatching frequencies for heterozygotes is not statistically different than forhomozygotes, both of which are high, suggesting that few or no aneuploid gametes are beingproduced in the former. The egg-hatching frequencies of two recombinants derived fromhIn1(I) heterozygotes is also shown for comparison. The egg-hatching frequency ofTable 12Effects of hIn1(I) on crossing over on Linkage Group IGenotype^ Wild types^Recombinants^pX100(C.I.)aunc-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 Dpyc36 Unc-29 Unc-75c^3.4(2.6-4.2)2 Unc-29c,d63 Unc-75d^6.0(4.7-7.6)dpy-5 unc-29 unc-75IhIn1^1669^68 Dpyc52 Unc-29 Unc-75c^5.5(4.6-6.5)4 Unc-29c,d81 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)56aa 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.56bTable 13Egg-hatching frequencies of hInl (I) heterozygotes and recombinantsGenotype^ Egg-hatchingFrequencyh/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)5758hDfll/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 lociin D. melanogaster and D. simulans were not in the same sequence on the genetic maps ofthe 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 rearrangedchromosome: unc-54(h1040), unc-75(h104 1), and unc-75(h10.42). The unc-75 mutationswere recessive lethals that produced an Unc-75 phenotype when heterozygous with talc-75(e950), and both were used in the following experiments. Dpy-5 and Unc-54 recombinantprogeny from a hIn1(1)[dpy-5 unc-54 + j/hIn1(1)[ + + unc-75I hermaphrodite wereindividually mated to unc-75(e950)/ + males to determine if the recombinant chromosomecarried one of the lethal unc-75 mutations. The normal order of these genes is dpy-5 unc-75unc-54 (EDGLEY and RIDDLE 1990). If the order of unc-75 and unc-54 were reversed, allDpy-5 recombinants should fail to complement unc-75(e950), whereas the Unc-54recombinants should complement unc-75. Of 17 Dpy progeny examined with h1042 and 10with WV, all 27 failed to complement unc-75. Of 12 Unc-54 progeny examined with h/042and 6 with h1041, all 18 complemented unc-75. This demonstrated that either the geneorder in hIn1(1.) is dpy-5 unc-54 unc-75, or that the order is unchanged but unc-54 is nowtightly linked to unc-75. To distinguish between these two possibilities, Dpy-5 progeny froma hIn1(1)1-clpy-5 + unc-75(h1042)1/hIn1A+ unc-54 +1 hermaphrodite were individuallyplated and their progeny examined for Dpy-5 Unc-54 segregants. If the gene order inhIn1(1) were dpy-5 unc-54 unc-75, only some of the Dpy-5 recombinants were expected tosegregate Dpy Uncs. Of 243 Dpy progeny examined, 120 segregated the double mutant and123 did not, indicating that gene order of unc-75 and unc-54 is reversed in hIn1(1) withrespect to wild type and establishing that hIn1(1) is an inversion.Recombination frequency in hInl(I) homozygotes is normal: Inversionhomozygotes do not experience the pairing problems inherent in heterozygotes. Todetermine if crossing over occurred in hIn1(1) homozygotes, and at what frequency,recombination was measured in three intervals: one interval outside the boundary ofcrossover suppression in heterozygotes and two spanning the boundary. The results areshown in Table 14. The dpy-5 unc-75(h1042) distance in hinl(I) homozygotes was obtainedfrom the same experiment as the gene order. The dpy-5 unc-54 and dpy-5 unc-75 distancesin hInl (I) homozygotes were 9.8 and 18.8 respectively. The dpy-5 unc-75 distance isprobably an underestimate since it was measured in trans and relied upon the recovery of aless viable double homozygote class. The map distances between dpy-5 and unc-54 and dpy-5 and unc-75 in controls were 26.4 and 9.4 (data from Table 12) respectively. This confirmsthe gene order indicated by the three-factor experiment and suggests that recombinationfrequency in homozygotes is wild type. Recombination was also measured in the dpy-5 unc-29 interval, a region located outside the inversion in the LG /cluster. The map distance inthis interval in homozygotes was found to be 3.7 m.u., not significantly different thanobserved in wild types (data in Table 12).hInl (I) crossover suppression is associated with recombination enhancement onLG I: To determine the extent of him' (I) mediated crossover suppression, intervals to theleft of unc-101 were examined and the results are shown in Figure 6 (data shown in Table12). 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 inhinl (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 theinterval contained a region of recombination enhancement with an associated region ofrecombination suppression. To distinguish between these alternatives, recombination wasexamined in dpy-5 unc-29 unc-75 heterozygotes. Recombination in the dpy-5 unc-75 intervalwas 13.0 m.u. in hInl(I) heterozygotes and 9.4 m.u. in the control. Crossing over in theunc-29 unc-75 region was not significantly affected by the presence of hInl (I), whereas thedpy-5 unc-29 interval showed a 1.6-fold increase in recombination in hIn1(1)[+ 4-J/dpy-5unc-29 heterozygotes. To further map the boundary of crossover suppression, recombinationwas measured between unc-29 and 12n-11. This interval was 4.7 mu. in heterozygotes and59Table 14Recombination in hInl (I) homozygotesGenotype Wild types Recombinants pX100(C.I.) ahInlidpy-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)hInlidpy-5 + unc-75(h1042)7/hIn1f + unc-54 + J 1135 243 Dpyb 9.0(6.6-11.4)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).60FIGURE 6.-Meiotic maps of LG /in hIn1(1) heterozygotes and controls. The LG /clusterextends from unc-11 to unc-29 (EDGLEY and RIDDLE 1990).61aunc -29dPY-5 lin-11^unc- 101bli- 3^unc- 11^unc- 75unc- 54lev- 11LG Iunc-29lin- 11unc -54lev- 11unc- 101unc- 75dpy- 5bli- 3^ unc- 11hin 1 602 m.0 .1.4 m.u. in controls (3-fold enhancement), thus localizing the hIn1(I) boundary of crossoversuppression between lin-11 and unc-75. To determine if the recombination enhancementobserved in the dpy-5 unc-29 interval extended to the left arm of LG /, recombination wasexamined in the bli-3 unc-11 and unc-11 dpy-5 regions. In hIn1(1) heterozygotes, a 1.5-foldincrease in recombination was observed in the bli-3 unc-11, and a 2-fold increase wasobserved in the unc-11 dpy-5 interval when compared to controls. The total genetic lengthof 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 anddeficiencies: Single crossovers within a classical inversion heterozygote producechromosomes that contain duplications and deficiencies. Four rare (— 1/2500) recombinantswere recovered from hIn1(1) heterozygotes. Three of these originated from mappingexperiments (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 determineif the individuals homozygous for the chromosome of interest were viable, all fourrecombinants were crossed to N2 males. The progeny of wild-type hermaphrodites resultingfrom this cross were screened for the presence of individuals with the original recombinantphenotype. Two of the four recombinants proved to be homozygous lethal, and both failedto complement unc-59 and 1ev-11 establishing them as deficiencies, later designated as hDfl 1and hDf12. hDf11 was known to complement unc-54 because of the original phenotype ofthe recombinant (Unc-101 when heterozygous with an unc-101 unc-54 chromosome). hDfllcomplemented 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 unc-101, suggesting the deficiency bearing chromosome carries the original unc-101 mutation (J.-Y. HO unpublished results). hDf12 also complemented unc-54 and was known tocomplement unc-101 based on the original recombinant phenotype (Unc-75 whenheterozygous with an unc-75 unc-101 chromosome). Thus the left breakpoint of hDf12 is tothe 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 phenotype62FIGURE 7.-Position of breakpoints of recombinant chromosomes derived from hIn1(1)heterozygotes. hDfll complements unc - 75 and unc - 101. The right breakpoints of hDp131and hDp132 are not known but both duplications cover unc- 54.63aunc-54bli-3Iunc-11 dpy-5 unc-29 unc-75 unc-101^unc-59 1ev-11II^I^I^I i^I IhDf11hDf12I^hDp131hDp132 I^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, suggestingthe recombinants were diploid for the unc - 101 1ev - 11 chromosome and carried a duplicationof unc - 101. These two duplications were mapped to visible markers and segregated fromchromosome / as though unlinked. The meiotic chromosomes of an hDp132/unc -29 unc - 75hermaphrodite 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 freeduplication. Both hDp131 and hDp132 have breakpoints between unc -29 and unc - 75, andcarry unc - 75(+), unc - 101(+), unc - 59(+), and unc - 54(+). That the duplications are Lev-11in phenotype when heterozygous with unc - 101 1ev - 11 chromosomes suggests that they arelinked to the original 1ev- 11 mutation. The extent of the duplications and their knownbreakpoints 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 inregions surrounding the centric heterochromatin and the distal tips of chromosome arms onthe 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. Theresults are shown in Table 15. Two regions located on autosomes and one located on the Xchromosome were examined. In all three cases, the presence of hIn1(0 did not significantlyaffect recombination in heterozygotes.hInl (I) recombines with szT1(I;X): The meiotic behaviour of the translocationszT1(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 theleft of unc -29, and translocation homozygotes are inviable. The extent of crossoversuppression was determined; recombination was suppressed to the left of the breakpoint andenhanced to the right (McKIM, HOWELL and ROSE 1988). Since crossing over issuppressed in the unc - 75 unc - 54 interval in hIn1(.1) heterozygotes, it was of interest to6465aFIGURE 8.-DAPI staining of chromosomes from the meiotic cells of an hDp132/unc-29 laic-75 hermaphrodite showing a) an oocyte bearing a seventh chromosome, indicating theduplication is unlinked and b) an oocyte lacking the duplication.a)65bTable 15Effect of hInl (I) on crossing over on other chromosomesGenotype^ Wild types^Recombinants^pX100(C.I.) adpy-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).66determine if pairing was possible between the two rearrangements. Crossing over betweenthe szT 1 (I ;X ) breakpoint and the hInl (I) boundary of crossover suppression was measuredin hermaphrodites of the genotype hIn1(1)1 + + /szT1(I;X)[unc-101;lon-21 The mapdistance between the breakpoint of szT/(/;X) and the boundary of hIn1(1) crossoversuppression (between lin-11 and unc-75) was 45 m.u. The recombination frequency betweenthe szT1 breakpoint and unc-101 was measured in + ; + /szT/(1;X)/unc-/0/;/on-2/ controlsand was 25 m.u., approximately 2-fold lower. The data for these experiments are shown inTable 16 and the Punnett square diagramming recombination between the tworearrangements is shown in Figure 9.hIn1 (I)/ hT2(I;III) heterozygotes suppress crossing over on LG I: Thetranslocation hT2(I;III) is comprised of two chromosomes; hT2(I;III)I segregates fromchromosome 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 thismarker. Since hInl(I) suppresses recombination from unc-75 to unc-54, it was of interest todetermine whether recombination could be completely suppressed on LG I. Crossing overbetween dpy-5 and unc-54 was measured in hIn1(I)[+ + +_1/hT2(I;III)I-dpy-5 bli-4 unc-54,1heterozygotes and + + + /hT2(I;III)idpy-5 bli-4 unc -54J controls (results shown in Table16). 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 effectivelysuppressed along the entire length of the chromosome.hIn1 (I) and hT2(I;III)I segregate randomly: While examining recombination inhIn1(1)1+ + +_1/hT2(I;III)[dpy-5^unc-54_1heterozygotes, an unusually small number ofDpy-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-29Jheterozygotesand + + + /hT2(I;III)[dpy-5 bli-4 unc-29Jcontrols. Since both dpy-5 and unc-29 map inthe crossover-suppressed arm of hT2(I;III)III, the recovery of the double mutant,representing the viable translocation homozygote class, is dependent upon the proper6768Table 16Effect of hInl (I) on crossing over with LG I translocationsGenotype 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 ..0.8(0.15-2.8)a C.I. = 95% confidence interval (see Chapter 1: MATERIALS and METHODS).b Both Dpy individuals were fertile and gave Dpy progeny.69aFIGURE 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 presumedlethal zygotes resulting from severe aneuploidy. Viable classes are indicated by phenotypeand any aneuploidy they may carry. szDp1 progeny are duplicated for AXII and are viable(McKIM, HOWELL and ROSE 1988).IRxR[u/0112];II-XLIRxR[u101/2];XNIN;IIALIN;XNu/O/IN;II-XLu/WIN;XNiRxR[+121;ILXI-iltxR[4_121;XNIRXREu/0//2];ILxL WT Unc-101IRXR[u/0//2];XNWT Unc-101IN;IIAL WT WTINT;XN WT WT WTu10/IN;ILL Unc-101 WT/WIN;XN Unc-101 Unc-101 WTiltxR[ +12];IIALWT WTittxR[+/2];XNWT WTsegregation of the translocation from the normal homologues. In the control, the predictedratio (5:1) of wild types to Dpy-5 Unc-29 progeny was observed (771 Wild types: 164 DpyUnc). The frequency observed in hIn1(I) heterozygotes, however, was 13.7:1 (411 Wildtypes: 30 Dpy Unc), close to the predicted ratio of 11:1 if hT2(I;III)I and hIn1(I) weresegregating randomly, resulting in aneuploid gametes (shown in Figure 10). Both therecovery of rare recombinants in the previous experiment and the difference between thepredicted and observed segregation ratios may be explained by a low frequency of pairingbetween the two rearrangements.hIni (I) effectively balances lethal mutations: One objective in isolating a crossoversuppressor for a region associated with the homologue recognition region was thedemonstration that such rearrangements, presumably intrachromosomal, would be effectivebalancers. The efficiency of hIn1(I) was tested by screening for recessive lethal mutations inthe region of crossover suppression. In total, 1412 mutagenized chromosomes were screenedand 54 mutations, including those resulting in adult sterility, were recovered. Strainsrepresenting the recovered mutations were effectively balanced in hIn1(I) heterozygotes forat least 20 generations (before being frozen) without breakdown of the balancer beingobserved.7071aFIGURE 10.-Punnett square diagramming the predicted segregation of a hInl(I)[+ ++YhT2(I;III)idpy-5 bli-4 unc-29Jheterozygote (see RESULTS). Empty boxes representpresumed lethal zygotes resulting from severe aneuploidy. Viable classes are indicated byphenotype and any aneuploidy they may carry. hDp13 progeny are duplicated for /R/HRand are viable (McKIM, PETERS and ROSE in press). Progeny duplicated for /L/HL areviable (K. McKIM pers. comm.).'NHL[d5u29);hInl'NHL[d5u291;IRIIIR'NHL[d5 u2.91'NHL[d5u29];IRIHR;hInlHO;hInlIIIN;IRHIRIIIN IIIN;IRHIR;hInlILIIIL[d5u29];hInlWT'NHL[d5u29];/R/HRDpy-5Unc-29WT/NHL[d5 u29] WT/NHL[d5u29];IRIHR;hInlWTHIN;hInl WT WT WThDp134IIIN;IRIIIR WT/MNVVT/MN./R///k;hInlWT WThDp134Chapter 3: DISCUSSIONIn 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 meioticproperties of hInI(I) were similar to those observed for inversions in Drosophila, includingcrossover suppression within the inverted region and intrachromosomal effects. hIn1(I) iscapable of recombining efficiently with the translocation szT/(/;X)/, indicating that the tworearrangements also synapse efficiently, a prerequisite to chaisma formation. For thisreason, it has been concluded that hInl(I) and chromosome /are capable of homologuerecognition and synapsis, but that physical constraints inside the inversion loop limitchiasma 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, hasbeen well characterized. NOVITSKI and BRAVER (1954) designed a system to recover theproducts of single exchanges inside In(1)d1-49 using a compound chromosome. Theyobserved a 75% reduction in crossing over in heterozygotes despite cytological evidence thatthis inversion was capable of pairing by forming loops in mitotic cells (PAINTER 1933).This suggests that topological constraints exist that reduce the frequency of chiasmataformation inside such inversions when heterozygous. By analogy, hIn1 (I), which is alsolocated at the end of a chromosome and is even smaller than In(1)d1-49, should experienceconstraints in pairing for recombination in heterozygotes. Secondly, compensatory increasesin recombination are large in hIn1(I) heterozygotes, as would be expected if exchangeswithin the inversion were rare (i.e. in hIn1(I) heterozygotes, the map distance from bli-3 tolin-11, normally 22 m.u., approaches 50 m.u.). The fact that hInl(I) heterozygotesefficiently recombine in other regions of the chromosome indicates that the ability of thehomologues to recognize one another is intact, and that recombination suppression on theright arm is limited to the inverted segment. Thirdly, reciprocal recombination events wereisolated from hInl(I) heterozygotes, suggesting that all meiotic products can be recovered,72but that their frequency is low. Figure 11 shows the possible pairing conformations betweenthe inversion and the normal chromosome. The first shows a pairing loop resulting fromhomologous 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) andhT2(I.III). The pairing portion of szT./(/;X)/ and hIni(I) share sequences not included ineither rearrangement, whereas the pairing portion of hT2(I;III)Iand hIn1(I) have nocommon unrearranged sequences. The crossover frequency between hIni(I) and szT.I(I.X)Iwas 0.45, demonstrating that synapsis and recombination were efficiently conducted betweenthe 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. .Theseresults agree with the conclusion that exchanges within the inversion are rare, since the onlyDNA available for pairing is within the inverted segment.DARLINGTON (1937) suggested that the formation of a chiasma betweenhomologues during meiosis facilitates their proper disjunction (reviewed by HAWLEY1988). 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 inC. elegans, the formation of a chiasma between two homologues is necessary to ensure theirproper disjunction at meiosis I. This interpretation is supported by cytogenetic studieswhich have documented that at metaphase I the bivalents orient axially and may be heldtogether at the metaphase plate by a terminalized chiasma (ALBERTSON and THOMSON1993).In Drosophila, it has been observed that inversions can effect increases inrecombination frequency on the rearranged chromosome and on the other majorchromosomes (STURTEVANT 1919; STURTEVANT 1931; DOBZHANSKY 1933;reviewed by LUCHESSI 1976). The analysis presented here showed that the total geneticlength of chromosome I was 41 m.u. in hInl(I) heterozygotes and 44 mu. in controls. Thesevalues are similar to the recombination frequency reported for the pairing portion of7374aFIGURE 11.-Possible pairing conformations within a chromosome arm heterozygous forhIn1T. The hInl (I) chromosome is represented by gray lines and the normal homologue byblack. a) Synapsis is shown for both the inversion and the normal homologue, resulting in aconventional pairing loop. b) Synapsis is shown only for the uninverted regions. Theinversion does not pair with the normal homologue.b)74bchromosome 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 onboth arms of LG /, the amount of exchange is limited to approximately one crossover eventper meiosis. Unlike inversions in Drosophila, hIn41)-mediated recombination enhancementin heterozygotes did not extend to other linkage groups. No increase in crossing over wasobserved in the three intervals examined in the presence of hIn1(.1.), regardless of theirlocation on the autosomes (small interval inside the cluster or large interval spanning thecluster) or on the X chromosome. Interchromosomal effects have been observed in C.elegans with mutations that result in X-chromosome nondisjunction (HODGKIN, HORVITZand BRENNER 1979; HERMAN and KARI 1989). Thus in C. elegans, as in D.melanogaster, the mechanism that regulates the number of crossovers per meiosis mayinvolve compensatory increases of events on other chromosomes in the event crossing over issuppressed or reduced along an entire chromosome. The failure to observeinterchromosomal effects in hIn1 (I) heterozygotes may have been expected sincerecombination was not reduced on chromosome /as a whole.Exchange events resulting from an intrachromosomal effect are not distributedrandomly along the chromosome. In Drosophila for example, such increases occur in regionssufficiently removed from the inversion breakpoint (GRELL 1962), and near the centricheterochromatin and distal tips of other chromosomes, regions of low intrinsic exchange(SCHULTZ and REDFIELD 1951; RAMEL 1962). Each of the autosomes in C. elegansare marked by a region where genes cluster on the meiotic map resulting from a reduction inrecombination (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 inintervals both inside (1.5 fold in dpy-5 unc-29) and outside (1.5 fold in bli-3 unc-11) thechromosome Igene cluster. This suggests that the regulatory mechanism responsible forestablishing the distribution of crossing over is independent of the mechanism determining75the number of exchanges. The meiotic pattern specific to chromosome I is retained; theenhancement observed is not greater in the cluster than it is at the left end. The frequencyand distribution of exchange events was found to be normal in hIni(I) inversionhomozygotes. This suggested that when the pairing difficulties experienced in heterozygoteswere removed in homozygotes, exchange within the inversion maintained the distributionobserved in wild types. Chromosomal sites that are necessary for normal levels of meioticexchange have been mapped in Drosophila (HAWLEY 1980; SZAUTER 1984). A similarmechanism may exist in C. elegans since recombination frequency is enhanced in the regionadjacent to the szT.1(IX) breakpoint on LC I, suggesting the break may have disrupted themechanism responsible for the regional distribution of exchange (McKIM, HOWELL andROSE 1988). If this mechanism is mediated by chromosomal elements, the level of crossingover in hIn1(I) homozygotes suggests that such elements can operate normally in eitherorientation.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 thatinverted 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 anddicentric fragments. The formation of these structures had been observed cytogeneticallyduring meiosis in Zea mays (McCLINTOCK 1933). Single exchanges within paracentricinversions were not observed in Drosophila until single crossover products were recoveredfrom individuals heterozygous for a long paracentric inversion on the X using an attachedchromosome (SIDOROV et al. 1935). These results demonstrated that single crossovers dooccur but that single crossover recombinants are not recovered. Nevertheless, informationtransfer in the form of gene conversion occurred in undiminished frequency in inversionheterozygotes, except near the breakpoints where effective homologous pairing may not bepossible (CHOVNICK 1973). Unexpectedly, no concomitant loss of zygote viability wasobserved in heterozygotes despite the formation of aberrant chromosomes (STURTEVANT76and BEADLE 1936; NOVITSKI 1952). To explain this, STURTEVANT and BEADLE(1939) proposed that chromatids involved in single exchanges were excluded from afunctional 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 chromosomeswith terminal duplications and deficiencies that were segregated into gametes and resultedin reduced fertility (ROBERTS 1967). The frequency at which single exchanges occurredwas dependent on the size and location of the inversion; a reduced frequency of such eventswas observed with both small inversions, and inversions located at the ends of chromosomearms (STURTEVANT and BEADLE 1936; NOVITSKI and BRAVER 1954). Individualsheterozygous for hIn1(I) showed no reduction in egg-hatching frequencies, compatible withthe behaviour of a paracentric inversion for which the products of single exchanges are eitherexcluded from functional nucleii or for which single exchanges in the inverted segment arerare.The recovery of a chromosomal rearrangement in C. elegans that behaves as aparacentric inversion may seem suprising given that the mitotic chromosomes are holokinetic(ALBERTSON and THOMSON 1982) and evidence for holocentric meiotic chromosomeshas been reviewed (HERMAN 1988). Recent cytological studies, however, suggest that theends of C. elegans chromosomes adopt centromeric functions for meiotic disjunction; one endholds the bivalent together and the other probably provides a site for the attachment ofmicrotubules. These roles do not appear to be specific to one end of the chromosome andeither end can be the inner or outer end of the bivalent (ALBERTSON and THOMSON1993). This meiotic behaviour is similar to that observed in other mitotically holokineticspecies; the nematode Parascaris univalens (GODAY, CIOFI LUZZATTO andPIMPINELLI 1985; PIMPINELLI and GODAY 1989; GODAY and PIMPINELLI 1989),the insects Euchistus servas (HUGHES-SCHRADER and SCHRADER 1961) and Myrmusmirzformis (NOKKALA 1985), where the mitotic chromosomes are holocentric but during77meiosis centromeric activity is restricted to a limited chromosome region, often atchromosome ends.The results of genetic analyses in C. elegans have consistently been compatible withthe predicted behaviour of monocentric chromosomes. For example, the segregation ratios ofaneuploid and viable progeny observed from translocation heterozyotes were compatiblewith the presence of a single centromere (HERMAN 1978; ROSENBLUTH and BAILLIE1981; McKIM, HOWELL and ROSE 1988; this study). In the case of the translocationeT1(III;V), recombination is suppressed to one side of the translocation breakpoint while theother recombines and segregates from the chromosome with which it had paired(ROSENBLUTH and BAILLIE 1981). Thus, in any one meiosis, only one meioticsegregator (centromere) was functional. The data presented in this thesis strongly suggestthat the meiotic chromosomes are monocentric, a suggestion compatible with both geneticand cytogenetic observations.The isolation of four recombinants from hIn1(1) heterozygotes raised the possibilitythat their genotypes would provide information on the location of the meiotic centromere.Two free duplications, hDp131 and hDp132 were recovered following a single exchange eventinside the inversion loop. The structure of these duplications is consistent with the productsformed by the events illustrated in in Figure 12. In this model, hDp131 and hDp132 arerepresented by the acentric fragment that results from a single exchange within aparacentric inversion where the centromere is to the left of unc - 75. The facts that theduplications were isolated independently, and that the left endpoints were nonrandom andcoincided 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 singleexchange within a paracentric inversion loop is not recovered under ordinary circumstances.In C. elegans, however, free duplications are readily recovered (HERMAN, ALBERTSONand BRENNER 1976). According to the model shown in Figure 12, the reciprocal productis a duplication of the sequences to the left of the inversion including the centromericsequences. This structure is analogous to the dicentric chromosomes generated by single7879aFIGURE 12.-Effects of single crossing over within a chromosome arm heterozygous forhInl(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. Ldenotes the left end and R denotes the right end of LG I. C represents the gene cluster, aand 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 thecase of hDf12, a = unc-75 and b = unc-101. Wild-type alleles of these markers are shownon the inverted chromosome. b) Meiotic products resulting from the exchange event (seeChapter 3: DISCUSSION). The chromosome duplicated for the cluster is proposed to bedicentric and the origin of hDf11 and hDf12. The small chromosome duplicated for the rightends has the same structure as hDp131 and hDp132.ab+aa^b +aa79bb)exchanges within paracentric inversions in Drosophila. Although this reciprocal product wasnot recovered intact in our experiments, hDfl 1 and hDf12 may have resulted from itsbreakage. Dicentric chromosomes in other organisms have been observed to form chromatidbridges at anaphase I, and as a result are meiotically unstable and subject to chromosomebreakage (McCLINTOCK 1933, 1941; CARSON 1946; HABER, THORBURN andROGERS 1984). The two deficiencies recovered, hDfl 1 and hDf12, could have resultedfrom a similar event followed by the broken end of one product being capped by sequenceson the right end (including uric- 54 (+)), presumably derived from its normal homologue towhich it is still attached. To stabilize broken ends of chromosomes, double-stranded breakscan be repaired by fusing with other chromosomes (McCLINTOCK 1941; 1942) or byrecombining with homologous sequences (HABER and THORBURN 1984). KADYK andHART WELL (1992) have shown that sister chromatids are preferred over homologues assubstrates for recombinational repair. Both deficiencies recovered have breakpointsindependent of the site of the original exchange event. Thus, the structures of therecombinant chromosomes recovered are compatible with the interpretation that h/n/(/) is aparacentric inversion with the centromere to its left. That cytogenetic analysis hasdemonstrated that both ends of LC / can adopt centromeric function (ALBERTSON andTHOMSON 1993) is not inconsistent with the genetic data. Firstly, in any one meiosisonly one end of the chromosome adopts centromeric function (i.e. spindles do not attach toboth ends). Secondly, the right end of chromosome /was observed to take on centromericfunction in the majority of meioses (ALBERTSON and THOMSON 1993), therebyexplaining the preferential recovery of recombinant products whose derivation is most easilyexplained by centromeric activity of the right end of LC /rather than the left.The recombinant products recovered are not consistent with the predicted behaviourof a paracentric inversion with centromeric sequences to the right of the inversion. Therecovery of hDp 131 and hDp132 is not compatible with dicentric products (see below), andno 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 the80following reasons. No significant reduction in egg-hatching frequency was observed inheterozygotes as would have been expected if the inversion were pericentric,however, it is possible that the frequency of recombination within the inversion was so lowthat no reduction was observed. Most importantly, however, if hin1(I) included thecentromere, hDp131 and hDp132 would be centric and would have segregated from theirhomologue at meiosis II. To recover these duplications, a nondisjunction event would beneeded to generate a viable zygote, and the probability of recovering two such rare events isvery low. The reciprocal recombinant product would also have possessed one centromereand 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, KARIand HARTMAN 1982; SIGURDS ON et al. 1986; McKIM 1990; ALBERTSONunpublished results).In conclusion, the meiotic behaviour of a C. elegans inversion hIn1(1), that suppressescrossing over in a previously unbalanced region, has been characterized. The simplestinterpretation of the data presented is that hIn.1(I) is a paracentric inversion with themeiotic centromere to its left. hIn1(1) was used to successfully balance lethal mutations in aregion previously impenetrable to extensive essential gene analysis, demonstrating its valueas a new class of balancer for the genome of C. elegans.81CONCLUSIONSThe intent of this thesis has been the description of conventional meiotic phenomenain C. elegans. Meiosis in C. elegans is marked by the classical features that distinguish itfrom mitosis: pairing, recombination, and segregation of homologous chromosomes. Someof the functional elements responsible for these processes have now been described in thenematode. The term "pairing" has been used to describe several chromosomal behavioursthat are now understood to be temporally and functionally distinct. Homologue pairing isthe alignment of chromosomes at a distance, perhaps as early as interphase, and is thoughtto 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 fibrullarproteins that anchor the two homologues together during interphase and early prophase. InC. elegans, the characterization of two free chromosome / duplications demonstrated thatone covering the right arm of the chromosome was capable of pairing for recombination withthe normal chromosomes, while the duplication covering the left arm was not. This led tothe proposal that the right end of chromosome /contained sequences necessary forrecombination and pairing between the homologues (ROSE, BAILLIE and CURRAN 1984;McKIM, HOWELL and ROSE 1988). The characterization of translocations andduplications has led to the identification of a single site on each chromosome that isnecessary for pairing and recombination, discussed by McKIM, HOWELL and ROSE(1988). This site, called the homologue recognition region, may correspond to the regiondiscussed by MAGUIRE (1984), described as the site of first contact between homologuesduring meiosis. The genetic behaviour of rearrangements lacking this region supports thisinterpretation since such rearrangements fail to pair for recombination with theirhomologues. This demonstrates that the function associated with this site temporallyprecedes any later meiotic event in the pathway, consistent with the predicted behaviour ofa specialized site required for initial homologue recognition early in meiosis. Such behaviour,however, would also be predicted for rearrangements that delete the site of telomere82attachment to the nuclear membrane. The failure of the chromosomes to pair andrecombine could be attributed to the failure of the homologue lacking the telomereattachment site to become properly oriented and anchored in the spatial organization of thenucleus. As a result, it would float free in the nucleus, unable to participate in later meioticevents. In C. elegans, electron microscopy of pachtene nucleii has demonstrated that whileonly one telomere of each chromosome is attached to the nuclear membrane, both telomereshave the ability to do so (GOLDSTEIN 1982, 1984a, 1984b, 1985, 1986; GOLDSTEIN andSLATON 1982), indicating the telomere attachment site does not correspond to thehomologue recognition region.The second form of meiotic pairing is thought to bring the homologues into a tighterassociation as a result of numerous recombination events which occur at certain sites. at ahigher 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 shownto be a recombination hotspot (GOLD WAY, ARBEL and SIMCHEN 1993; GOLD WAY etal. 1993). In C. elegans, these secondary pairing sites are not sufficient to ensurerecombination between homologues in the absence of the homologue recognition region(ROSENBLUTH, JOHNSEN and BAILLIE 1990; McKIM, PETERS and ROSE 1993). Inthe presence of the HRR, however, a small set of these sites may be preferentially used toensure secondary pairing between rearranged chromosomes. Intrachromosomal effects intranslocation (McKIM, HOWELL and ROSE 1988; McKIM, PETERS and ROSE 1993)and inversion heterozygotes (ZETKA and ROSE 1992) enhance crossing over in the regionscapable 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 crossingover had to occur in a small region flanked on one side by nonhomologous translocatedsequences, and on the other by the inversion. This supports the interpretation that pairingbetween such sites is independent of the pairing of neighbouring sites, and does not supporta model whereby pairing for recombination is initiated only at the end(s) of a chromosome83and progresses from this site. Rather, it suggests that recombination events can be initiatedinternally.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 onboth the X chromosome and the autosome (HODGKIN, HORVITZ and BRENNER 1979).The second class is represented by one mutation, rec-1, which disrupts the normaldistribution 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 unitsobserved in rearrangement heterozygotes (McKIM, HOWELL and ROSE 1988; ZETKAand ROSE 1992; McKIM, PETERS and ROSE 1993) and wild-type individuals (ZETKAand ROSE 1990), suggesting a flexible mechanism exists to ensure the formation of onecrossover between the homologues. In this study, crossing over was eliminated inheterozygotes resulting in the random segregation of the twochromosomes. This demonstrates that in C. elegans, one chiasma per bivalent is necessaryfor the proper disjunction of homologous chromosomes.Recombination in C. elegans is also regulated by a mechanism based on the sexualphenotype of the individual. Male recombination frequency on chromosome I is reduced byone third when compared to the hermaphrodite frequency, suggesting the formation of achiasma is not guaranteed in every male meiosis. Three possibilities may explain the orderlydisjunction 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 wherecrossing over is difficult to measure. Secondly, male meiosis may make more use ofsecondary pairing sites to ensure proper segregation in the absence of chiasma formation. InDrosophila, deletion mapping of the ribosomal cluster has demonstrated that sequencesbetween the rRNA genes function as X- Y pairing sites in male meioses (McKEE, HABERAand VRANA 1992). Thirdly, studies have documented that free duplications segregatefrom the X chromosome in the male and it is possible that nonrecombinant chromosomesmay pair distributively (McKIM, PETERS and ROSE 1993; this study APPENDIX I).84Recombination-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 thehomologues that is mediated by the formation of the synaptonemal complex. Normalsynaptonemal complexes, consisting of two lateral elements and a central element form inboth 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 thisis not true for meiosis. Analysis of recombinants derived from inversion heterozygotes hassuggested 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 haveconcluded that while centromeric activity is localized to one end of the chromosome duringmeiosis, 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 tospindles (monocentric), rather than both ends (dicentric), or the whole chromosome(holocentric). On chromosome /, the right end of LG /adopts centromere function in themajority of meioses (ALBERTSON and THOMSON 1993). This is consistent with thestructure of hIn1 (0-derived recombinants since each recombinant chromosome isrepresentitive of one meiosis, and their derivation has been explained by the presence of ameiotic centromere on the right arm (ZETKA and ROSE 1992). This proposal was basedupon the genetic definition of the centromere, defined as the last point of attachmentbetween sister chromatids at anaphase II. By this definition, C. elegans chromosomes havetwo potential spindle attachment sites at Meiosis I (of which only one is used) and atMeiosis II they possess one genetic (and cytological) centromere. 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Distributive pairing of the X and sDp1 (I;1) in males: To test if the freeduplication sDp1 distributively paired with the single X chromosome in males, dpy-5 uric-13;0 /szT1(I;_,X)[ + + ;lon-2] males were mated to dpy-5 dpy-14/sDp1 hermaphrodites. Allwild-type males resulting from this cross were of the genotype dpy-5 unc-13/dpy-5 dpy-14/sDp1 and were mated to dpy-5; unc-36 homozygous hermaphrodites and the wild-typeand Dpy-5 progeny were scored. If the duplication and the X chromosome segregated fromone another, the resulting male progeny were predicted be wild-type as consequence ofinheriting sDp1 and the hermaphrodite progeny were predicted to Dpy-5 as a consequence ofinheriting the paternal X. If the duplication and the X segregated randomly, one half of thewild-type and Dpy-5 progeny were expected to be male. 269/293 wild-type progeny weremale 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 theproposal that distributive pairing occurs in males.103Cosmid mapping of rec -1: A Rec-1 transgenic strain (KR2357), constructed bycoinjecting 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 thesecosmids carried a wild-type allele of rec-1, Rol-6 transformants (rol-6 is dominant andestablishes the presence of the extrachromasomal array containing the cosmids discussedabove) 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 screenedfor the presence of Dpy-11 Unc-42 segregants. Recombination in these individuals wascalculated 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 isthe number of wild types and R is the number of roller progeny. Rol-6 animals will not rollin an Unc-42, Dpy-11 or Dpy-11 Unc-42 background.The region of chromosome /located between unc-54 and the ribosomal cluster (rrn-1) at the right distal tip is spanned by six overlapping cosmids shown in Figure 13. In orderto map rec-1 to one of these cosmids, the six cosmids were coinjected as sets of three (onegroup containing the cosmids ZK219, B0195, CO1F5 and the other ZK340, B0467, ZC556)into rec-1 homozygotes (J. McDOWALL unpublished results) using the rol-6 transformationsystem (MELLO et al. 1991). Attempts to construct a transgenic line containing thesecond group of cosmids (ZK340, B0467, ZC556) were unsuccessful and no stable lines wereisolated (J. McDowall pers. comm.). Transgenic Rol-6 hermaphrodites bearing anextrachromosomal array containing the cosmids ZK219, B0195 and CO1F5 were tested forthe presence of Rec-1 by measuring recombination in dpy-11 unc-42/ + + ; rec-1/rec-1individuals (data shown in Table 17). Since the cosmids were injected into a Rec-1individual, the presence of a wild-type allele of rec-1 on one of the cosmids would result in anormal map distance for the dpy-11 unc-42 interval. The dpy-11 unc-42 interval was 8.8104105aFIGURE 13.-Physical map of cosmids in the region of unc - 54.B0195 1 ^, ,^B0467^i1 ^iCO1F5^ZC556ZK219 ZK340i^1unc-54^ kin-1^ rrn-1tr.106Table 17Cosmid mapping of rec-1Wts Rol-6 Dpy Unc pX100(C.I.)a1219 66 59 7.6(6.4-9.0)1250 17 12 2.7(2.0-3.6)637 271 60 49 8.9(7.3-10.6)494 403 19 11 2.5(1.7-3.5)Genotypedpy-11 unc-42/ + + ;rec-1/rec-1bdpy-11 unc-4.2/ + +bdpy-11 unc-42/ + + ;rec-1/rec-1;hEx12cdpy-11 unc-42/ + + ;rec-1/ + ;hEx12cC.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 andCO1F5 in unknown copy number (J. McDOWALL unpublished results).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. Inthe 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 theextrachromasomal array. The frequency of recombination was 2.5 m.u. in transgenicindividuals heterozygous for rec-1 and 2.7 m.u. in dpy-11 unc-42/ + + controls, indicatingthe presence of the array does not affect recombination in this interval. Because thepresence of the array was not confirmed by PCR, however, the negative results of theseexperiments are not conclusive.107PCR 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. Eggswhich remained unhatched after 20 hours were removed to agar plates lacking 0P50 andtreated according to the protocol of BARSTEAD, KLEIMAN and WATERSTON (1991)with the exception that the eggs were transferred using cut fishing line which then remainedin 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 ofTaq 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 at55°C. After these cycles, the samples were incubated for 7 min at 72°C and cooled to 4°Cusing 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/ ofeach 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 clusterlocated at the right end of LG I, but could not determine whether the deficiency containedsequences to the left of the cluster or lay completely within it. To distinguish between thesetwo possibilities, primers flanking the left ribosomal junction (one specific for nonribosomalsequences adjacent to the cluster and the other specific for 26S ribosomal RNA) were usedto determine if the junction was present in eDf24 homozygotes. The sequences of theseprimers and control primers derived from the adenosyl homocysteine hydrolase gene(AHH)(PRASAD, STARR and ROSE 1993) are shown in Figure 14 and the PCR productsare shown in Figure 15. The primer set specific for the ribosomal junction produces a 517b.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,108109aFIGURE 14.-Sequences of PCR primers used in this study. a) RL30 anneals tononribosomal DNA adjacent to the ribosomal cluster and RL31 anneals to 26S ribosomalgenes. b) RL12 and RL14 anneal to sequences within the AHH locus, located in the centralcluster of chromosome I.RL30 5' - TGG GAA TTT TCT GTT CAG GT - 3'RL31 5' - CGC AAT AAC AAG TCA ACA GT - 3''12 5' - CGT CCG TTC TTG AGG GTG - 3'a)b)RL14 51 - CTA AGA TGC TCG CCA AGG - 3'110aFIGURE 15.-PCR analysis of e Df24(I). PCR products obtained from lane 2) N2 L4hermaphrodite, 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) L4hermaphrodite 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 the517 b.p. product, indicating that the deficiency spans the junction (THACKER and ZETKAunpublished results).110b1 2 3 4 5 6 7 8 9 10 11 12 -or 577-6( 517indicating the deficiency spans the ribosomal junction and includes nonribosomal DNAadjacent to the cluster. Identical primer sets were also used to determine which of the threeremaining cosmids contained the ribosomal junction. The cosmids ZC556 and B0467 (notZK340) produced a 517 b.p. product, indicating the ribosomal junction maps to thesecosmids (data not shown).111112aIII. Table 18Strains used in this studyStrain Genotype^ Strain GenotypeBC62 dpy-5(e61)unc-75(e950)BC64 unc-35 (e259)dpy-5 (e61)BC89 dpy-5(e61)unc-54(e190)BC196 dpy-5(e61)dpy-14(e88)rec-1(s180)BC207 dpy-5 (e61)unc-29(e403)BC251 unc-42(e270)dpy-11(e224)BC260 unc-11(e47)dpy-5(e61)rec-1(s180)BC415 dpy-5(e61)unc-13(e450)BC563 dpy-180364)unc-36(e.261)BC 1195 sDpVdpy-17(e251)unc-36(e261)CB51 dpy-5 (e51)CB88 dpy-7(e88)CB151 unc-3 (e151)CB190 unc-54(e190)CB261 unc-59(e261)CB450 unc-13(e450)CB719 unc-1(e719)CB1479 him-S (e1423)DR1^unc-101(m1)KK596 him-/4(i/44ts)KR181 unc-42(e270)dpy-11(e224)rec-1(sl 80)KR236 dpy-5061)unc-13('e450)/sDp2KR307 dpy-5('e61)unc-101(m1)KR309 bli-3(e579)unc-11(e47)rec-1(s180)KR387 unc-13('e450)rec-1(s180)KR642 dpy-5(e61)rec-1(s180)KR900 dpy-5(e61)unc-13('e450)/szTi(I;X)[+ +;lon-2JKR1004 dpy-5(e61)dpy-14(e88)KR1005 sDp2/dpy-5061Ppy-14(e88)rec-1(s180)KR1012 dpy-5(e61)unc-29(e403)unc-75(e950)KR1064 bli-3(e579)unc-11(e47)KR1071 dpy-5(e61)unc-11(e47)112bStrain Genotype^ Strain GenotypeKR1301 rec-1(s180) malesKR1473 unc-101(ml)unc-54(e190)KR1546 sDp1/dpy-5(e61)dpy-14(e88)rec-1(s180)KR1701 dpy-7(e88)unc-3(e151)KR1714 unc-29(e403)unc-75(e950)KR1734 unc-75(e950) unc-101(m1)KR1735 sDp1/dpy-5(e61)dpy-14(e88)KR1898 unc-101(m1)ley-11('x12)KR1906 unc-11(e47)rec-1(s180)KR1949 hIn1(1) malesKR1953 unc-54(e190)/eDf24KR1954 dpy-5(e61) unc-75(e950); her-1(e1520)KR1955 dpy-5(e61)unc-101/szTliUnc-101;lon-21KR1956 dpy-5(e61) unc-13(e450) rec-1(s180)KR2151^func-54('h10401KR2152 hDp131/unc-75(e950)unc-101(m1)KR2153^fdpy-5(e61)unc-54(h1040)JKR2156 unc-29(e403)1zn-11(n389)KR2158 unc-75(e950)unc-59(e261)KR2159 unc-75(h1042)/unc-75(e950)unc-101(m1)KR2226 hIn11-unc-54(h1040)J/unc-101(m1)1ey-11(x12)KR2228 ley-11(x12) malesKR2387 unc-1(e719)dpy-7(e88)KR2390 hDp132/unc-75(e950)unc-54(e190)KR2391 hDp131/unc-75(e950)unc-59(e261)KR2392 hDfil/unc-101(ml)unc-54(e190)KR2394 hDp2/dpy-5(e61)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^RW3072 1ey-11(x12)10-49(st44)/unc-54(st60)let-50(st33)KR2020 hDf12/unc-75(e950)unc-101(m1)^SP580 mri164(/;X)KR2025 hDp131/unc-101(ml)ley-11(x12)^ZZ3003 ley-11(x12)


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