The Drosophila Mei-S332 Gene Promotes Sister-Chromatid Cohesionin Meiosis Following Kinetochore Differentiation

The Drosophila mei-S332 Gene Promotes Sister-Chromatid Cohesionin Meiosis Following Kinetochore Differentiation

Anne W. Kerrebrock,* Wesley Y. Miyazaki,*9t Deborah Birnbyt”and Terry L. Orr-Weaver*9t92 *Whitehead Institute, Cambridge, Massachusetts 02142, and +Department ofBiology,Massachusetts Institute of Technology, Cambridge, Massachusetts 02142 Manuscript received September 3, 1991 Accepted for publication December24, 1991

ABSTRACT The Drosophila meiS332 gene acts to maintain sister-chromatid cohesion before anaphase I1 of meiosis in both males and females. By isolating and analyzing seven new alleles and a deficiency uncovering the mei-S332 gene we have demonstrated that the onset of the requirement for mei-S332 is not until late anaphaseI. All of our alleles result primarilyin equational (meiosis11) nondisjunction with low amounts of reductional (meiosis I) nondisjunction. Cytological analysis revealed that sister chromatids frequently separate in late anaphaseI in these mutants. Sincethe sister chromatids remain associated until late in the first division, chromosomes segregate normallyduring meiosis I, and the genetic consequencesof premature sister-chromatid dissociationare seen as nondisjunction in meiosis 11. The late onsetof mei-S332 action demonstratedby the mutations was not a consequence of residual gene function because two strong, and possibly null, alleles give predominantly equational nondisjunction both as homozygotes and in trans to a deficiency. mei-S332 is not required until after metaphase I, when the kinetochore differentiates from a single hemispherical kinetochore jointly organized by the sister chromatids into two distinct sister kinetochores.Therefore, we propose that the mei-S322 product acts to hold the doubled kinetochore together until anaphase 11. All of the alleles are fully viable when in trans to a deficiency, thusmei-S332 is not essential for mitosis. Four of the alleles show an unexpected sex specificity.

N meiosis haploid gametes are produced by two meiosis I1 (MAGUIRE197813, 1979). Since these uni- I successive rounds of chromosome segregation that valents can still organize theaxial (or lateral) elements are notseparated by DNA replication. In thefirst, or of the SC between the two sister chromatids, it is likely reductional, meiotic division the homologs pair and that completeSC isrequired to prevent the premature segregate from each other.The second, or equational, separation of sister chromatids in meiosis I (MACUIRE meiotic division resembles mitosis in that the sister 1990). Another mechanism that could ensure sister- chromatidssegregate. Since the sisterchromatids chromatid cohesion would be the catenation of sister move as a unit to thepoles in the reductionaldivision, chromatids that arises as a consequence of DNA rep- functions promoting cohesionof the sister chromatids lication (MURRAYand SZOSTAK1985). However, must exist in meiosis I. Such functions might also act analysis of plasmids in yeast failed todemonstrate in meiosis I1 or mitosis to maintain the association of extensive interlocking prior to anaphase of mitosis the sister chromatids until anaphase. (KOSHLANDand HARTWELL1987). Candidate regu- The mechanisms thatpromote sister-chromatid latory or structural proteins that act to holdthe sister cohesion are currently not well understood. Sister- chromatids together have yet to be identified. chromatid cohesion is likely to require both structural Functions required for meiotic chromosome segre- proteinsthat hold the sisters togetherand timing gation can be identified by the isolation of mutants, mechanisms that delay separation until the appropri- an approachemployed inseveral organisms. However, ate anaphase. Cytological studies inmaize suggest that only a few mutations have been isolated that poten- sister-chromatid cohesion in early meiosis I may in- tially affect sister-chromatid cohesion.In thedesynap- volve the synaptonemal complex found between (SC) tic (dy) mutant of maize, homologs pair and undergo homologous chromosomesduring the pachytenestage recomRnation, but the chiasmata are IT& maimained (reviewed in MACUIRE1990). Unpaired homologs (MAGUIRE1978a). The resultingunivalents often (univalents) in trisomic strains ofmaize undergo sister- undergo sister-chromatid separation during meiosis I. chromatidseparation during meiosis I ratherthan The dy gene is proposed to have a role in promoting

I Presentaddress: Department of Genetics, University of Washington, sister-chromatid cohesion, which in turn is required Seattle, Washington 98195. ‘To whom correspondence should be addressed at The Whitehead Insti- for chiasma maintenance. Cytological analysis of the tute, Nine Cambridge Center, Cambridge, Massachusetts 02142. PC mutant in tomato reveals premature sister-chro- Genetics 130 827-841 (April, 1992) 828 A. W. Kerrebrock et al. matid separation in late anaphase I (CLAYBERG1959). mei-S332 and ord acted at different times in meiosis 1 In yeast, one interpretation of the mutations in the to promote sister cohesion. Previous data alsosug- REDZ (ROCKMILLand ROEDER1988) andDISZ (ROCK- gested that these genes might play a role in mitotic MILL and FOGEL1988) genes is that they result in chromosome segregation (BAKER,CARPENTER and Rr- defects in sister-chromatid cohesion during meiosis I. POLL 1978). The red1 mutant fails to assemble synaptonemal com- Only a single allele existed for mei-S332, and no plex (ROCKMILLand ROEDER1990); this could leadto deficiencies wereidentified that uncovered this locus. premature separation of the sister chromatids. Consequently, it was not known whether the observed TWOgenes have been identified in Drosophila mel- phenotype corresponded to loss of gene function, and anogaster,mei-S332 and ord, whichhave been pro- whether this phenotype accurately reflected the true posed to maintain sister-chromatid cohesion until an- biological role of the gene. Therefore it was essential aphase I1 of meiosis (DAVIS 1971; GOLDSTEIN1980; to isolate additional alleles to determine whether the MASON1976; SANDLERet al. 1968). Mutations in mei- wild-type mei-S332 function was to promote sister- S332 and ord are unusual in that they affect chromo- chromatid cohesion in meiosis.New alleles would also some segregation during meiosis I in both males and permit the role of mei-S332 in mitosis to be examined. females. The majority ofmutations that affect meiotic Moreover, the apparent difference in time of action chromosome segregation in Drosophila show sex spe- of mei-S332 and ord in meiosis I could be explained as cificdefects inmeiosis I (BAKERand HALL 1976), a result of the initial allele of meiS332 being leaky, indicating that meiosis I differs profoundly in Dro- and this hypothesis could be tested with additional sophila males and females. In females, the homologs alleles. We have isolated seven new alleles mei-S332of undergo recombination and formsynaptonemal com- aswell as deficiencies uncovering the locus. In this plex, and mutants defective in recombination show paper we present a genetic analysis of these new alleles high levels ofnondisjunction (BAKERand HALL1976). which demonstrates that the mei-S332 gene product Thus, as has beenobserved in a numberof organisms, promotes sister-chromatid cohesion in meiosis I. The recombination is linked to proper chromosome seg- onset of the requirement for meiS332 action is after regation in Drosophila females.In addition, a backup metaphase I, following differentiation of the kineto- system for the segregation of nonrecombinant chro- chore. mosomes (the distributive system) has been demonstrated genetically in females (GRELL1976). In males MATERIALS AND METHODS the synaptonemal complex is not formed and recombination does not occur. Homologs appear to pair via Stocks: All Drosophila stocks and crosses were raised at 25"on standard cornmeal-brewer's yeast-molasses-agar specialized pairing sites or collochores (MCKEE and food. Unless noted, all stockswere received from the Bloom- KARPEN1990). Despite the differences between males ington Stock Center atthe University of Indiana. The and females the phenotypes of mei-S332 and ord imply phenotypes of the original meiotic mutations mez-s332 and that some aspectsof meiosis I such assister-chromatid ord are fully described in (DAVIS1971; GOLDSTEIN 1980; MASON 1976; SANDLERet al. 1968). In our experiments, we cohesion are under common genetic control in both utilized a deficiency uncovering meiS332 named Df12RX58- sexes. 6. We isolated this deficiency in a screen for deficiencies in The previously characterized phenotype resulting cytological interval 58 by screening for X-ray-induced loss from a mutation in the meiS332 locus indicated that of a P element containing the white gene inserted into 58D (the P[(w)AR]4-043transformant, obtained from R. LEVIS this gene promotes sister-chromatid cohesion in at the Fred Hutchinson Cancer Research Center) (LEVIS, meiosis. Cytological analysis of male meioticsegrega- HAZELRIGCand RUBIN 1985). The breakpoints of tion in the mei-S332 mutant demonstrated that the DJT2R)X58-6 are approximately 58A3-B2; 58E3-10. Other sister chromatids prematurely dissociate in meiosis I deficiencies isolated in this screen will be described in a (DAVIS1971; GOLDSTEIN1980). In this mutant non- separate report. All other mutations used in these experiments are de- disjunction does not occur until meiosis I1 becausethe scribed in (LINDSLEYand GRELL1968). The cn bw sp chro- sister chromatids do not precociously disjoinuntil late mosome used for ethyl methane sulfonate (EMS) mutagen- anaphase I, thus meiosis I segregation is unaffected. esis was derived from a stock obtained from J. TAMKUN The onset of mei4332 activity during anaphase I was (University of California at Santa Cruz) via R. LEHMANN (Whitehead Institute). Stocksused in the recombination in contrast to observations obtained with the single mapping experiments included a1 dp b pr c px sp/CyO, a1 d allele of ord. ord, like mei-S332, appears to promote b pr BC c px sp/SMI and In(2R) mamNzG,S Sp Tft mamNZGPu B/ sister-chromatid cohesion. However, in flies mutant Cy0 (from R. LEHMANN) anda px sp (from the Mid-America for the ord locus mostly reductional (meiosis I) non- Stock Center atBowling Green State University). The latter disjunction occurs (MASON 1976), and cytologically, two stocks were crossed to obtain the recombinant In(2R) mamNzG,Tft mamNZGPaz a px sp chromosome used in the the sister chromatids were observed to be aberrantly second mapping experiment. The isogenized X and Y chro- associated as early as prophase I (GOLDSTEIN1980). mosomes,which were crossed intothe meiS332 mutant Thus the previous analysis raised the possibility that stocks (see below), came from a y/@Y stock and a y snfs/ The Drosophila mei-S332 Gene 829 FM7a/y+Y; spa*" stock, respectively. A y cu uf car stock (from 85; mei-S33Z4, 67; mei-S3325, 48; mei-S33Z6, 63; mei-S332' R. S. HAWLEY,University of California at Davis) was crossed 62; mei-S3328, 6 1. to Canton-S to isolate the cu ufcar recombinant chromo- Construction of isogenic stocks: In order to minimize some used in the female nondisjunction tests. Compound differences between the mei-S332 alleles due to genetic chromosomes usedin the nondisjunction tests included: background, and to remove lethals and steriles from the C(l)RM,y2 su(U")u";pX.r" y+, In(l)EN,y u f B; C(2)EN, b pr; EMS-mutagenized chromosomes, we constructed stocks C(3)EN; C(4)RM, ci ef. Forthe rest of this,report,the which were isogenic for the sex chromosomes and which c(I)mchromosome will be symbolized as "XX," andLhe had most of the original mutagenized second chromosome PX. yZ y+, In(1)EN chromosome will be symbolized as"XY." replaced. We first made a stock of the genotype y/y+Y; +/ EMS mutagenesis: Adult y/y+Y; cn bw SF males were SMl; spa*"' in which the X and Y chromosomes had been mutagenized with 0.035 M EMS as described (LEWISand isogenized; this stockwill be referred toas the iso-X,Y stock. BACHER1968), and mated to y'; cnmei-S332' ord'/SMI Recombinants for all eight mei-S332 alleles and the nonre- females (see Figure I). From the progeny of this cross, males combinant DJT2R)X58-6 chromosome were crossed into the with a mutagenized cn bw sp chromosome over the cn mei- iso-X,Y background. S332' ord' tester chromosome were selected and individually Recombinants of the eight mei-S332 alleles were isolated tested for nondisjunction in matings to yellow females. Ex- as follows. The left arm of chromosome 2 was replaced by ceptional nullo-XY sperm produced by these males resulted recombining a pr cn bw chromosome with either a al dp b in yellow (y/O) males in a background of yellow+ males (y/ pr c mei-S332 bw sp recombinant chromosome from the first y+Y) and females (y/y+). Vials with more than two yellow mapping experiment (mei-S3322*'~4~6),the original mutagen- males were scored as positive for nondisjunction; most vials ized cn bw sp chromosome (mei-S3325n7.8)or a recombinant had 20-30 progeny. Positives were retested over the single chromosome with the distal right arm of chromosome 2 ord and meiS332 mutations. replaced with px and sp (mei-S332'), and then selecting for Lacto-aceto orcein squashes of salivary gland chromo- recombinant chromosomes which were marked with either somes (ASHBURNER1989) from the eight mei-S332 noncom- pr cn bw sp (mei-S33Z2") or pr cn px sp (mei-S332'). Approx- plementers showed that only one of them contained a visible imately 10 recombinant chromosomes were stocked for each chromosomal rearrangement, a deficiency in region 58 that allele, and these stocks were scored for homozygote viability we have named Dx2R)Rl-8. and fertility and thenondisjunction phenotype. A single line Recombination mapping:We mapped the eight mei-S332 for each allele was selected, and crossed into the iso-X,Y alleles intwo separate experiments using standard tech- background (the "A" stock). niques. In the first experiment, six of the eight meiS332 The homozygous meiS332"' alleles were male sterile in noncomplementers from the EMS screen (mei-S3322r4,637*8, the iso-X,Y background, presumably due to mutations else- Dx2R)Rl-8) were mapped to thec (75.5)-px(100.5) interval where on the second chromosome that interacted with iso- on chromosome 2. Recombination took placein females X,Y. We therefore crossed recombinants from the second which had the second chromosome containing the noncom- mapping experiment, which had the distal right arm of the plementer over a a1 dp b pr c px sp chromosome. These chromosome 2 replaced by px and sp, into the iso-X,Y back- females were crossed to y/y+Y; cn mei-S332' px/SMl males, ground(the "B" stock). Fertile homozygous males were and their male progeny with recombinant or nonrecombi- obtained by crossing the A and B stocks together. For the nant second chromosomes over the original meiS332' allele mei-S33Z4, meiS332 , and mei-S3328 alleles, we also crossed were isolated. These males, whichwere also y/y+Y or y+/y+Y, the A and B stocks to obtain fertile homozygous females. were mated singly to y; a1 dp b pr BI c px sp/CyO,bw virgin Otherwise, we used homozygous females from the A stock females. The progeny ofthis cross were scored for the for nondisjunction tests. visible mutations and the presence of yellow (X/O) males To avoid the accumulation of modifiers which decrease arising from nondisjunction events in the male parent giving the frequencies of nondisjunction in meiS332 stocks (DAVIS rise to nullo-XY sperm. Only vials with at least 20 progeny 197 1; HALL 1972), we maintained the mei-S332' stock by were scored: those with three or more yellow males were crossing heterozygous males and virgin females each gen- scored as mei-S332-, those with one or no yellowmales eration. This was not necessary for the other alleles, since were scored as mei-S332', and those with two yellow males homozygotes in those stocks were either male sterile or were retested. The number of recombinants in the c-px inviable. interval scored for each allele was: mei-S33Z2, 42; mei-S3324, Nondisjunction tests: All experiments were performed 60;mei-S33Z6, 59; mei-S33Z7, 53; Dj(ZR)R1-8,58.In all cases, at 25", using the iso-X,Y stock (y/y+Y; +ISMI; spa*") as the the meiotic nondisjunction phenotype was found to map wild-type control. Haplo-4 Minute flies, triploids, triploid close topx (99.1-100.0 cM). In the mei-S3328 mapping cross, intersexes and metafemales appeared rarely amongthe we recovered one mei-S332-px recombinant, placing this progeny of the nondisjunction tests and were not included allele at 99.5 cM. in the final progeny totals. Tests to measure sex chromo- We performed a second experiment to map the original some nondisjunction were set up as described (ZITRONand allele and sevenof the EMS-induced noncomplementers HAWLEY1989): crosses were set up on day 0, parents were (excluding Dx2R)RI-8) more precisely. These mutations discarded on day 5, and progeny were scored until day 18. were mapped within the Pu (97 cM)-px (100.5) interval using Sex chromosome nondisjunctionin males: xondisjunction an In(2R)mamNZG,Tft mumN2' Puz a px sp chromosome. was measured in crosses of y/y+Y males to XX, y2 su(wa) w' Recombinants in this interval were selected overthe females. The frequencies of six out of the nine possible types DfT2R)X58-6 chromosome, which uncovers both mei-S332 of sperm could be deduced from the phenotypes of the and px. Single males that were recombinant in the Pu-px progeny of this cross (see Figure 2A). Nondisjunction at interval were mated to y; Sco/SMI females to score for either meiotic division or chromosome loss events resulted nondisjunction as described above. For the wsak mei-S3325 in nullo-XY sperm, nondisjunction at the first meiotic divi- allele, recombinant males were mated to XX, y2 su(wa)w. sion resulted in XY sperm, nondisjunction at the second females to score for nullo-XY and diplo-X exceptions. The meiotic division resulted in XX sperm, and nondisjunction number of fertile recombinants in the Pu-px interval scored at both divisions resulted in XXY sperm. for each allele was: mei-S332', 35; mei-S332', 49; mei-SjjP, Percentages for each exceptional class were calculated by 830 A. W. Kerrebrock et ai. dividing the number of progeny in that class by the total number of progeny; these percentages were summed to give the valueof "Total observed nondisjunction". However, exceptional diplo-Y sperm were not scored in this cross because they were phenotypically indistinguishable from regular mono-Y sperm. Moreover, since diplo-Y sperm are not efficiently recovered in XX females (GOLDSTEIN 1980; LINDSLEYand GRELL1968), the equational diplo-Y exceptional class was greatly underrepresented among the prog- "; cn bw so* X YisODrcn eny. Consequently, the"Total observed nondisjunction" y+Y cn mei-S332 ord y SMI values in these tests (see Tables 1 and 2) are underestimates of the actual levels of nondisjunction in males. Since we could not determine thefrequency of diplo-Y sperm in these tests, we were unable to calculate the frequencies of chromosome loss in males. Sex chromosome nondisjunction in females: Nondisjunctip Scorefor sex chromosomenondisjunction was measured in crossesof y/cu v f car or y/y females to XU, Regular progeny: I (yellow+ females) v f B males. All four types of ova gaverise to phenotypically y/y+ distinguishable progeny in this cross (see Figure 2B). In this y/y+Y (yellow+males) cross, only half ofthe total number of exceptions, but all of the regular X gametes, were recovered (Figure 2B). There- Exceptionalprogeny: yf0 (yellowmales) fore, percentages for the exceptional classes were deter- FIGURE1.-Screen for new alleles of mei4332 and ord. Males mined by doubling the number of exceptional progeny and with a y+Y and a second chromosome marked with c7t bw sp were dividing by the "Adjusted total," which is the number of mutagenized with EMS and crossed to a stock with a tester chro- progeny in the regular X classes plus twice the number of mosome mutated for both mei-S332 and ord. Single males were progeny in the exceptional classes. scored for mutations failing to complement either meiS332 or ord Females in the diplo-X exceptional class that arose from by crossing them to yellow mutant females and scoring for sex first (reductional) or second (equational) division nondis- chromosome nondisjunction. Progeny from vials in which excep- junction were distinguished using the centromere-linked tional X0 male progeny were observed were stocked over the mutation carnation. Carnation females were immediately balancer and retested. scored as pguational exceptions. Carnation+ females were mated to XY males, and their male progeny were scored for gamete class to the expected numbers, determined by mul- the presence of the car mutation to determine whether the tiplying the product of the frequencies of the individual mother was a reductional (car'lcar) or equational (car+/ classes of X and fourth chromosome exceptions by the total car+) exception. Recombination in four intervals spanning number of progeny. the X chromosome was also assayed in the female tests by Nondisjunction of the fourth chromosomes in males was scoring the regular X class (Bar+ males) for the recessive X- assayed by crossing y/y+Y; spaPo' males to y; C(4)RM ci ef linked mutations y, cu, u, f and car. females. The types of progeny resulting from regular and Autosomal nondisjunction: To test nondisjunction of chro- exceptional gametes for the fourth chromosomes are de- mosomes 2 and 3 in males, 10 males which were homozy- scribed above. Independence of the sex and fourth chro- gous, heterozygous or wild type for the eightmeiS332 alleles mosomes were not monitored in this cross, because dipio-X were mated to 15 C(2)EN or C(3)EN virgin females. Recip- exceptional sperm were not recovered. rocal crosses were performed to test autosomal nondisjunc- Cytology of meiosis in males: Testis squashes to analyze tion in females, using the same number of parents per vial. meiotic chromosome behavior were prepared, and staging For both sets of crosses, parents were discarded on day 7, was determined as described (GOLDSTEIN1980). Precocious and the total number of progeny were counted until day sister-chromatid segregation was scored only for the sex 18. When flies withcompound C(2)EN or C(3)EN autosomes chromosomes and the autosomes, not for the fourth chro- are crossed to flies with unattached second and third chromosome. Phase contrast microscopy was done using a Zeiss mosomes, virtually no progeny are obtained due to lethal Axioskop or Axiophot equipped with Plan Neofluar 40X zygotic aneuploidy (Figure 2C). The only surviving progeny and 1OOX and Plan Apochromat 63X objectives. in these crosses arise from nondisjunction events in the mei- Viabilitytests: Males that were cn mei-S332"* sp/SMI S332+ ormei-S332- parents. were crossed to DfT2R)X58-6, pr cn/SMI females in bottles. Nondisjunction of the X andfourth chromosgmes in The parents were discarded on day 5, and progeny were females was assayed by crossing y; spapo'females to XU, vfB; counted until day 18. The three surviving classes of progeny C(4)RM ci ef males. Regular mono-4 ova resulted in either could be distinguished phenotypically: mei-S332/+ flies were triplo-4 progeny, which were wild type for fourth chromo- Curly speck, Of/+ flies were Curly speck+,and meiS3321Df some markers, or haplo-4 sparkling-poliert Minute progeny flies were Curly+ speck+ (the mei-S332+ allele was on the (not included in the final totals). Nullo-4 exceptional ova balancer). Percentages for each class were determined by gave rise to cubitus interruptus eyeless-Russian progeny, dividing the number of flies in each class by the total. and diplo-4 exceptional ova gave rise to sparkling-poliert progeny that were not Minute. The types of regular and RESULTS exceptional gametes with respect to the X chromosomes in this test have already been described (Figure 2B). Frequen- Isolation of new alleles of mei-S332: We conducted cies of the exceptional nullo-X and diplo-X ova were cor- an EMS screen for mutations that failed to comple- rected as previously described; no such correction was necessary for chromosome 4 exceptional ova. Independent ment the initial meiS332 allele (Figure I). Noncom- segregation of the X and 4 chromosomes could be monitored plementers of bothord' and mei-S332' were recovered in this cross by comparing the observed numbers for each over a second chromosome carrying both mutations The Drosophila mei-S332 Gene 83 1 by screening for sex chromosome nondisjunction in A. Cross: y/y+~d X X"X. y2 SUW) Ira 9 males (MATERIALS AND METHODS). A totalof eight mei- Gametes Ova S332 noncomplementers and five ord noncomplemen- x"x, y2 au(w.) w 0 ters were found out of 9900 chromosomes screened. Regular Sperm All pairwise combinations of the 13 noncomplemen- X lethal yellow male Y or YV yellow+. suppressed lethal ters were tested in both males and females for nondis- white-apricot female junction of the sex chromosomes. The eight mei-S332 Exceptlonal noncomplementers and five ord noncomplementers Sperm 0 yellow*,suppressed lethal fell into two separate complementation groups (data white-aprlcot female XV or XVY lethal yellow' male not shown). The five noncomplementers of ord will xx lethal yellow female be described further in a separate report (W. MIYA- XXY or XXYY lethal yellow' female ZAKI and T. ORR-WEAVER, manuscript in prepara- tion). B. Cross: cv v f ceHy Q X X"V, v f B d We mappedthe mei-S332 noncomplementers to demonstrate that they were true alleles of mei-S332 Gametes Sperm (MATERIALS AND METHODS). The strongest noncomplementers were first mapped to the c-px interval on Regular Ova Bar female Bar+ male distal 2R. One of these noncomplementers was found to Exceptional Ova be adeficiency in cytological interval 58 (Dx2R)RI- q"Bar male lethal 8); unfortunately, high sterility associated with this xx lethal Bar+ female deficiency precluded its use in subsequentexperi- ments. To map the remaining seven noncomplementers and the original mei-Sj32' allele more precisely, c. Cross: +I+ X C(2)EN recombinants in the Pu-px interval were recovered Gametes C(2)EN parent overthe deficiency Dx2R)X58-6 (MATERIALS AND METHODS) and tested for nondisjunctionin males. mei- Regular S332' was found tomap to position 99.2 cM, a position lethal lethal which is more consistent with its cytological location Exceptlonal viable lemal of 58B-D (DAVIS1977; A. KERREBROCK,unpublished 7232 lethal viable data) than was the previously reported map position FIGURE2.-Crosses to test for nondisjunction. A, Sex chromo- of 95 cM (DAVIS1971). The map positions of the some nondisjunction inmales. Progeny arising from regular or seven noncomplementers in this experiment were: exceptional sperm are distinguishable by their sex and their eye- mei-S3322, 99.3 cM; mei-S332j, 99.5 cM; mei-S3324, color and body-color phenotypes. Unless otherwise noted, all progeny have wild type eye color. B, Sex chromosome nondisjunction 99.3 cM; mei-S3325, 99.1 cM; mei-S3326, 99.3 cM; mei- in females. Progeny arising from regular or exceptional ova are S3327, 99.5 cM; mei-S3328, 99.5 cM. Thus we con- distinguishable by their sex and their phenotype with respect to clude that all seven newly isolated noncomplementers Bar. C, Chromosome 2 nondisjunction. Only progeny arising from map to themei-S332 locus and are authenticalleles of exceptional gametes survive. Progeny arising from regular mono-2 mei4332. gametes die due to lethal zygotic aneuploidy. Similar results are obtained in crosses to C(3)EN stocks. Sex chromosome nondisjunction in males: We assayed nondisjunction of the sex chromosomes in alleles in males did not correlate with the strength of males in order to determine whether the new mei- the allele, and they may have been due to background S332 alleles affected chromosome segregation in the mutations in the stocks. Although the frequencies of first or second meiotic division (Figure 2A). In this exceptional sperm that we observed for the mei-S332' cross, XY sperm were diagnostic of nondisjunction in allele were slightly lower than previously reported the first meiotic division and XX sperm were diagnos- frequencies (GOLDSTEIN1980), the relative frequen- tic of meiosis I1nondisjunction. The othermajor class cies of exceptional gametes were essentially the same of exceptional gametes,nullo-XYsperm, was produced (nullo-XY > diplo-X >> XU). from nondisjunction at either meiotic division or by All eight mei-S332 alleles overa deficiency also chromosome loss. The six strongest mei-S332 alleles resulted in muchhigher frequencies of meiosisI1 resulted primarily in meiosis I1 nondisjunction when nondisjunction relative to meiosis I nondisjunction in homozygous in males (Table 1). For most of the males (Table 2). Most alleles gave from 10- to 20-fold alleles, the frequency of observed equational excep- higher frequencies of equational exceptions relative tions (XX sperm) ranged from five to 15-fold higher toreductional exceptions. If these values are cor- thanthe frequency of reductionalexceptions (XU rected to include diplo-Y equational exceptions (MA- sperm). The low levels of reductional exceptions that TERIALS AND METHODS), then most alleles over a defi- we observed for each of the homozygous mei-S332 ciency probably gave 20-30-fold more equational 832 A. W. Kerrebrock et al.

TABLE 1 Sex chromosome nondisjunctionin males homozygousfor the indicated allele

Regular sperm X 1357 1815 2990 1026 1934 1766 1947 2076 1915 Y(V) 1412 1630 2303 1081 1781 1490 1569 2300 1718 Exceptional sperm 0 3 1015 400 267 713 28 51 1332 895 XYlU) 0 30 18 3 31 23 10 37 74 xx 0 43 1 130 97 264 11 13 580 385 xxym 0 5 0 0 3 0 0 1 6 Total progeny 2772 4926 5841 2474 4726 3318 3590 6326 4993 % nullo-XY 20.6 0.1 6.8 10.8 15.1 0.8 1.4 21.1 17.9 % XYV) 0.0 0.6 0.3 0.1 0.7 0.7 0.3 0.6 1.5 % xx 0.0 8.7 2.2 3.9 0.45.6 0.3 9.2 7.7 % XXYp,) 0.0 0.1 0.0 0.0 0.06 0.0 0.0 0.02 0.1 Total observed non- 0.1 30.014.8 9.3 21.5 1.8 2.1 30.9 27.2 disjunction

TABLE 2

Sex chromosome nondisjunctionin males with the indicatedalleles over DfT2R)X58-6

SamplemeiS332'meiS332' mei-S3326 meiS332' mei4332' mei-S3323 mei-S332* meiS332' Regular sperm X 767 2069 899 792 1597 1901 1478 1470 Y(r) 709 1821 932 723 1539 1489 1753 1488 Exceptional sperm 0 609 849 303 446 103 31 1 1108 83 1 xym 12 38 8 25 12 28 41 37 xx 203 399 148 215 28 122 638 452 XXYP) 4 0 1 2 0 0 5 5 Total progeny 2304 5176 2291 2203 3279 3851 5023 4283 % nullo-XY 26.4 16.4 13.2 20.2 3.1 8.1 22.1 19.4 % XY(Y) 0.5 0.7 0.3 1.1 0.4 0.7 0.8 0.9 % xx 8.8 7.7 6.5 9.8 0.9 3.2 12.7 10.6 % XXY(U) 0.2 0.0 0.04 0.09 0.0 0.0 0.1 0.1 Total observed non- 35.9 24.8 20.0 31.2 4.4 12.0 35.7 31.0 disiunction

than reductional exceptions. This assumes similar fre- S??26 were weak alleles (2% totalnondisjunction) quencies of X and Y chromosome nondisjunction, as (Table 1). When placed over a deficiency, all alleles was observed by Goldstein (GOLDSTEIN1980) for the showed an increase in total nondisjunction that was homozygous mei-S??Z' allele. Therefore,the high due primarily to increases in the nullo-XY and diplo-X numbers of equational exceptions produced by males exceptional gamete classes. These increases were less homozygous or hemizygous for the eight mei-S??Z dramatic for the strong alleles mei-S??Z', mei-S??Z7 alleles indicate that most nondisjunction in these males and mei-S??2* (1.1-1.2-fold) than for the weak mei- occurredat the second meiotic division. Since the S??Z6 allele (6-fold). strong alleles did not show a shift to higher frequen- Two of the fourstrongest alleles (mei-S332' and mei- cies of reductionalexceptions when placed overa S3?27) are likely candidatesfor nulls, since they deficiency, the preponderance of meiosis I1 nondis- showed only a slight increase in total nondisjunction junction did not result from residual activity of hy- when placed over a deficiency in both males and in pomorphic alleles. females (see below). Forboth alleles, however,the The alleles can be placed into three classes, based increase observed in males was significant by the x* on the observed frequency of nondisjunction. mei- contingency test (LINDREN,MCELRATH and BERRY S332', mei-S3?Z4, mei-S??Z7 and mei-S?3Z8 were clas- 1978). If these alleles are indeed nulls, then this slight sified as strong alleles (20-30% total nondisjunction), increase in nondisjunction may reflect variability in mei-S??Z2 and mei-S??23 were moderate alleles (10- the strengthof the mutantphenotype due to different 15% total nondisjunction), and mei-S??2' and mei- geneticbackgrounds. It is alsopossible thatthe The Drosophila mei-S332 Gene 833

l)f(2R)X58-6 deficiency uncovers a second gene that 1980). Our results and those of GOLDSTEIN(1 980) enhances the mei-S332- phenotype. demonstratethat the earliest manifestation ofthe Cytology of malemeiosis: We performed testis requirementfor mei-S332 productthat can be ob- squashes in order to examine the behavior of chro- served cytologically is in mid-late anaphase I. mosomes during meiosis in males homozygous for the In meiosis -11 ali of the alleles showed precocious new mei-S332 alleles. Early meiosis I stages (prophase sister-chromatid separation and aberrant anaphase I1 I to early anaphase I) appeared to be normal for all figures (Table 3 and data not shown). For the strong of the alleles (Figure 3D and Table3). In later meiotic alleles (mei-S33Z3,mei-S33z4, mei-~332', mei-S33Zx), stages (mid anaphase I to prometaphase 11), the pri- the frequency of premature separation of sister chro- mary defect observed in mutant males was a failure matids (PSSC) observed in prophase I1 and prometa- to maintain sister-chromatid cohesion (Figure 3E and phase I1 cells was high, with mei-S33ZR showing the Table 3). Although it was difficult to obtain scorable highest levels of precocious sister-chromatid separa- cells for the early stages, premature sister-chromatid tion. The levels of aberrant segregation events quan- separation was readily seen in mid-late anaphase I in tified in Table 3 are an underestimate, because am- the alleles which were strong in males in the genetic biguous chromosomes were not scored. This is partic- tests (mei-S3323, mei-S33Z4, mei-S33Z7 and rnei-S33ZR). ularly true for mei-S3327, since a high number of Previous analysis of meiotic chromosome behavior in prophase I1 cells in this mutant contained clumped homozygous mei-S332' males revealed that sister-chro- chromatin that may have obscured any separated sis- matid separation frequently occurred in mid-late an- ter chromatids. Rare prophase cells with PSSC were aphase I, but was only rarely seen earlier (GOLDSTEIN also observed in mei-S3325 homozygotes (data not 834 A. W. Kerrebrock et al.

TABLE 3 Cytological analysis of males homozygousfor the indicated alleles

Prophase I to Early to mid Mid to late anaphase Prophase I1 and metaphase I anaphase I 1 prometaphase I1 Anaphase I1

Allele NOPSSC PSSC NOPSSC PSSC NoPSSC PSSC NoPSSC PSSC Normal Aberrant"

mei-S332' go6 0 17 0 17 6 127 11 72 12 (74%) (26%) (92%)(26%) (74%) (8%) (14%)(86%) mei-S33z3 45 0 2 0 31 6 45 24 28 44 (84%) (35%)(65%)(38%).(62%) (16%) mei-S332' 23 1 21 0 7 3 20 21 26 24 (30%) (49%) (51%) (52%) (48%)(70%)(52%) (51%) (49%) (30%) rnei-S33P 37 0 2 0 12 0 56 108 4 1 (96%) (4%) (98%) (2%)(98%) (4%) (96%) meiS332' 65 0 14 0 17 12 74 37 4 35 (59%) (41%) (67%) (33%) (10%) (90%)(10%) (33%) (67%) (41%) (59%) mei-S332' 39 0 24 0 1 15 3 21 97 36 (6%) (94%)(6%) (18%) (82%) (8%) (92%) Aberrant anaphases are those in which nondisjunction or lagging chromosomeswere observed. Numbers indicate the number of cells observed. Only unambiguous cases of cells with precocious sister-chromatid cohesion of at least one dyad were scored as such, thus the number of cells with PSSC is underestimated. shown). No metaphase I1 cells were observed in males wereindicated by an excess of nullo-X relative to homozygous for the strongest alleles (data not shown; diplo-X exceptions. GOLDSTEIN1980). However,some metaphase I1 plates As had been observed in males, the alleles which were observed in males homozygous for the weaker hadthe highest nondisjunctionfrequencies when alleles. homozygous in females (Table 4) resulted primarily For all of the alleles, we observed aberrant anaphase in meiosis I1 nondisjunction (Table 5). The total non- I1 figures in which sister chromatids had segregated disjunction frequency of 43.3% that we observed for to the same pole or which had lagging chromosomes the mei-S332' allele in females was very similar to the that had not segregated to either pole (Figure 3F, value of 44.6%reported by DAVIS(i97 l), except that Table 3, anddata not shown). The frequencies of we observed less chromosome loss. Three of the anaphase I1 cells showing missegregation of sister strong alleles in males (mei-S332', mei-S3324 and mei- chromatids of the major autosomes and sex chromo- S3327; Table 1) werestrong in females, with total somes paralleled the strengths of the alleles in males nondisjunctionfrequencies of 36-40%. The mei- based on the genetic tests. The frequency of normal S332' and mei-S3326 alleles were also strong in fe- anaphase I1 cells seen in homozygous mei-S3327 and males, with total nondisjunction frequencies of 33- mei-S332* mutants (%lo%) is consistent with the ex- 34%.These five alleles when homozygous in females, pected number of cells showing normal segregation and the three strongest alleles (mei-S332', mei-S?324 of the major chromosomes if sister chromatids are and m~i-S332~)over a deficiency, gave 10-20-fold segregating randomly (0.53 = 12%). greater equational exceptional ova relative to reduc- The cytological phenotypes of the new alleles, tional exceptional ova (Table 5). Similar patterns were namely separation of sister chromatids in late ana- observed for the weaker meiS332* allele in females phase I and nondisjunction or lagging chromosomes (data not shown). The frequency of reductional ex- in anaphase 11, appeared to be identical to thedefects ceptions in mei-S332 homozygotes were consistently observed forthe original mei-S332' allele (DAVIS197 1 ; higher in females (5-1 4%)than in males (51 %). How- GOLDSTEIN1980). Thus these alleles also result in ever, the high numbers of equational relative to re- defects in sister-chromatid cohesion. ductionalexceptions demonstrate that these alleles Sex chromosome nondisjunction in females: We result primarily in nondisjunction during the second also assayed nondisjunction of the sex chromosomes meiotic division in females. in females (Figure 2B) in order to determinewhether The frequencies of total sex chromosome nondis- the seven newmei-S332 alleles produced similar effects junctionobtained for the mei-S332' and mei-S3327 in both sexes, as had been found for the mei-S332' alleles over adeficiency in females (Table 6) were very allele (DAVIS197 1; GOLDSTEIN1980). Meiosis I and similar to the frequencies obtained for these alleles I I nondisjunction events were distinguished among when homozygous (Table 4). These results are con- the diplo-X exceptional females by determining their sistent with the hypothesis that these alleles, which are genotypes with respect to the centromere-linked mu- strong in both sexes and which show little change in tation carnation. Chromosome loss events in these tests strength over a deficiency, may be nulls of the mei- The Drosophila mei-S332 Gene 835

TABLE 4

Sex chromosome nondisjunction in females homozygous for the indicatedallele

Sample 4- mci-Sj'32' mei4332' m&S332' meiS332' mei-S332' mei-S33z7mei-S33z6 mei-s332' Regular ova X 3821 477 1749 2948 1280 3405 31882329 2272 Exceptional ova 0 2131 101 13 29 1 5 391 420 27 xx 0 81 236 12 175 2255 394 14 38 22 659 2198 2973 1746 3415 1746Total 2973 progeny 2198 659 3822 2370 2917 3973 Adjusted total" 84 3823 2647 1 2998 2212 47583425 3562 241 1 % nullo-X 0.05 24.0 16.126.3 0.4 16.4 0.3 2.2 23.6 % diplo-X 0.00 19.3 17.8 0.4 15.8 0.3 16.6 12.6 1.2 Total X nondisjunction 0.05 43.3 33.9 0.8 42.133.0 0.6 3.4 36.2

a The progeny total is adjusted to correct for recovery of only half of theexceptional progeny.

TABLE 5 Frequencies of reductional and equationalexceptions among the diplo-X progeny of females homozygous or hemizygous for the indicated allele

meiS332' mei-S332' rnei-S332' Samole mei-S332*mei-S332' mei-S332' Df12R)X58-6meiS332'DfZRjX58-6mti-S3326Dj(2R)X58-6 Reductional (car+/car) 8 11 8 32 12 29 12 16 Equational (car+/car+) 23 83 75 94 157 84 70 70 (carlcar) 44 164104 76 98 93 124 115 Total 198 75 353 159 21 1 230 197 179 % Reductional 10.7 5.6 5.0 9.1 13.7 5.2 6.1 8.9 % Equational 89.3 94.4 95.0 90.9 86.3 94.8 93.9 91.1

TABLE 6 Sex chromosome nondisjunctionin females with the indicatedallele over Df2R)X58-6

~~~ - mei-S332' mei-S332' mei-Sjr3f7 mei-S332' Sample mei-S3323mei-S332' Test 1" Test 2" Test 1 Test 2b meiS332'

~ ~ __ Regular ova X 1117 6772637 714 19561171 191 Exceptional ova 0 245 33 130 152 13021 1 49 xx 254 42 133 96 187 25 151 Total progeny 1616 2712 977 925 15692237 265 Adjusted total 2115 2787 1240 1173 1967 339 2518 % nullo-X 2.4 23.2 21.010.3 25.928.9 21.5 % diplo-X 24.0 3.0 21.5 16.4 19.0 14.7 12.0 Total X nondisjunction5.4 47.2 42.3 42.522.3 43.6 40.5 * Tests 1 and 2 were performed at different times using the same chromosomes. Tests 1 and 2 were performed at different times using different chromosomes. S332 locus. Although the levels of chromosome loss specific differences(compare Tables 1 and 4). The seen for the meiS332' (and mei-S3324) allele over a mei-S332* allele, which was comparable in strength to deficiency varied somewhat from test to test, the thethree other strong alleles in males (27% total frequencies of total nondisjunction did not change nondisjunction), was weak in females (3.4%total non- (Table 6). We believe that the differences in levels of disjunction). Similarly, the mei-S332j allele, which was chromosome loss in females are due to the presence moderate in males (15% totalnondisjunction) was of modifiers, which may also explain why we observed very weak in females (only 0.8% total nondisjunction). less chromosome lossin mei-S332' homozygotes Conversely, the mei-S332' and mei-S3326 alleles, which (Table 4) than DAVIS(1971). were moderate (9% total nondisjunction) and weak Surprisingly, results from the female tests revealed (2% total nondisjunction) in males, respectively, were that some of the new mei-S332 alleles exhibited sex strong when homozygous in females (both gave about 836 A. W. Kerrebrock et al.

TABLE 7 Recombination in females homozygousor hemizygous for the indicatedallele

mei3332’ rneiS332‘ mei-S3327 Sample + mei-S332’ meiS332’ mei-S332‘ mei-S3326 mei-S3327 Dx2R)X58-6 Dx2R)X58-6 Dx2R)X58-6 Num ber scoredNumber 1555 229542 105277 11425 565 569 673 Map distances (cM) 1 y-cv 12.0 11.4 26.8 14.0 9.9 10.8 15.1 15.6 10.8 2 cv-v 21.9 17.9 18.4 21.2 18.0 15.7 18.8 20.7 19.5 3 v-f 19.1 16.2 20.6 17.7 18.8 16.2 21.2 19.5 21.5 4 f-car 5.7 3.1 4.4 4.8 4.5 5.8 3.5 5.3 3.7 Total map distance map Total (cM) 58.7 70.2 48.6 57.7 51.2 48.5 58.6 61.1 55.5 (1.00) (1.20)(0.83) (0.98) (0.83)(0.87) (1.04)(1.00) (0.95) Numbers in parentheses indicate values for the map distances normalized to the control map distance. Total map distances in bold print indicate those that areidentical to the control map distanceby the binomial distribution test (LINDREN,MCELRATH and BERRY1978).

33% total nondisjunction). The reasons for the sex ciency, the X chromosome map distance was statisti- specificity exhibited by these alleles will be further cally identical to that of the wild type control (Table addressed in the DISCUSSION. We will refer to these 7). These results argue that the slight decreases in alleles in subsequent sections as “male-predominant” recombination seen for the homozygous alleles are (mei-S3323, mei-S3328)or “female-predominant” (mei- due to other mutations on the second chromosome. S332’, mei-S3326), since their effects are notrestricted We conclude that since mutations in mei-S332 have to one sex. virtually no effect on recombination, the product of Mutations in mei-S332 do not affect recombina- the mei-S332 locus is not required for homolog pair- tion: Since few reductional exceptionswere recovered ing. among the progeny of females homozygous or hemi- Nondisjunction of the autosomes: We also exam- zygous for any of the mei-S332 mutant alleles, it ined whether the seven new mei-S332 alleles affected seemed unlikely that themei-S332 gene productwould segregation of the autosomes, as did the mei-S332’ be required to maintain homolog pairing in females. allele (DAVIS197 1; GOLDSTEIN1980). For the two However, we looked for more subtle effects on hom- major autosomes 2 and 3, nondisjunction was assayed olog pairing in these mutants by measuring recombi- by mating males or females homozygous for the new nation levelsin females. Inthe nondisjunction test mei-S332 alleles to stocks carrying the compound au- crosses, the mei-S332- female parents were hetero- tosomes C(2)EN or C(3)EN. In these crosses, only zygous for five recessive X-linked mutations in order progeny arising from nullo or diplo exceptional ga- to measure recombination within four intervals span- metes can survive (Figure 2C). Since the regular ga- ningthe X chromosome (y-co; cv-v; v-f; fcar). We mete classes were not recovered, the nondisjunction anticipated that defects in homolog pairing in these frequencies foreither major autosomewere unknown. mutants would result in decreasedrecombination, Therefore, we scored these tests solely for the pres- which we could observe as adecrease in the map ence or absence of nondisjunction by comparing the length of the X chromosome. numbers of progeny in the mei-S332- test crosses to In general, the eight mei-S332 alleles had little or thenumbers of progeny in the mei-S332+ control no effect on recombination between X homologs cross. (Table 7). The mei-S332’ allele was originally reported All mei-S332 alleles, except the weak allele mei- to have no effect on recombination (DAVIS197 l), but S3325, resulted in nondisjunction of chromosomes 2 the stock that we used hada slight recombination and 3 when homozygous in males (Table 8). In fe- defect (80% of wild type). The other alleles showed males, the alleles that gave high frequencies of sex from80% (mei-S3327) to 100% (mei-S3324) of the chromosomenondisjunction in females (mei-S332’, control map distance when homozygous. Similar map mei-S332’,mei-S3324, mei-S3326, mei-S3327; Table 4) distances were found for the remaining mei-S332 al- also showed nondisjunction of chromosome 3 (Table leles as homozygotes (data not shown). The abnor- 8), whereas alleles that were weak in females in the mally high map distance seen in mei-S332’ homozy- sex chromosome nondisjunction tests (mei-S3323,mei- gotes (70.2 cM) was due to the inclusion of triploid S3325, mei-S3328)did not. All alleles, except the weak intersex progeny(2X3A), whichphenotypically resem- mei-S3325 allele,resulted in chromosome2 nondis- bled recombinants in the y-cu interval, but which were junction (Table 8), possibly because chromosome 2 is distinguishable with practice. For three of the strong more sensitive to alterations in the mei-S332 product alleles (mei-S332’,mei-S3324, mei-S3327) over a defi- in females than are theX or thirdchromosome. These “+” and y-’’ indicate the presence and absence of nondisjunction,respectively. Numbers in parentheses indicate the numberof progeny obtained in the test cross. Vials with less thanfive progeny were considered negative. results demonstrate that mutations in mei-S332 affect or no fourth chromosome nondisjunction (0-0.3%; segregation of the major autosomesin both males and data notshown). The numbers in parentheses in Table females. 9 are theexpected numbers for each ova class, assum- Nondisjunction of the fourth chromosomes was as- ing independent segregation of the X and 4 chromo- sayed in males and females homozygous for the eight somes (MATERIALS AND METHODS). For all five strong mei-S332 alleles in order to determine whether this alleles in females, the observed numbers in each ova chromosome was affected by mutations in mei-S332. class were not significantly differentfrom the ex- We also examined whether chromosomes were seg- pected numbers by the contingency x* test (mei- regating independently in mei-S332- mutant females S332‘.2,697:P > 0.95; mei-S3324: 0.5 > P > 0.3) (LIN- in a cross in which nondisjunction of both the X and DREN, MCELRATHand BERRY1978). Although small fourth chromosomes could be monitored simultane- numbers of progeny were scored in these tests, the ously (MATERIALS AND METHODS). Forboth sets of results we obtained strongly indicate that the X and 4 tests, homozygous mei-S332 mutants were mated to chromosomes were segregating independently in mei- flies with a compound fourth chromosome(C(4)RM ci S332 mutants. ef).Regular fourth chromosome gametes and excep- Absence of semidominance in rneiS332 mutants: tional nullo-4 and diplo-4 gametes were recoverable The original mei-S332’ mutation was reported toshow and distinguishable in this cross; however, we did not a slight amount of sex chromosome and fourth chro- assay thefrequency of equational andreductional mosome nondisjunction as a heterozygote relative to exceptions in the diplo-4 exceptional class. the wild type control in both sexes (DAVIS197 1). We As expected, thelevels of fourth chromosome non- measured nondisjunction in siblings heterozygous for disjunction observed in either sex roughly paralleled all eight mei-S332 alleles as well as the DfT2R)X58-6 results fromthe sex chromosome nondisjunctiontests. deficiency as additional controls for the sex chromo- In homozygous males (data notshown), the frequency some and autosome nondisjunction tests. In the male of total nondisjunction of chromosome 4 was higher tests, we found that thefrequencies of nondisjunction for the mei-S332’ allele (37.9%) than for the other measured in mei-S332 heterozygotes were essentially strong mei-S3324, mei-S332’ and mei-S332’ alleles (9- identical to the frequency of nondisjunction in the 17%). Although these strong alleles gave low levels of wild-type control, showing that all eight alleles and chromosome 4 nondisjunction relativeto mei-S332’ in the deficiency were fully recessive in males (data not this experiment, it should be noted that only a few shown). All eight alleles and the deficiency were also progeny were scored for each test (80-100 total), so found to be fully recessive in females, except as trans- that these values may be inaccurate. Otherwise, it is heterozygotes with the second chromosome balancer possible that some of the mei-S332 alleles show chro- SMl (Table 10 and data not shown); the semidomi- mosome specificity in males. nance that DAVISobserved in females heterozygous In the female tests, frequencies of total chromosome for mei-S332’ was also in the presence of the SMl 4 nondisjunction werestrikingly similar to frequencies balancer. We did not see this effect in the presence of of X chromosomenondisjunction (Table 9). The the FM6 balancer (data not shown). It is possible that strong alleles mei-S332’,mei-S332’, mei-s33z4, mei- the slight levels of nondisjunction observed for alleles S3326 and mei-S3327 gave fairly high levels of total of mei-S332 over SMl result from a second-site non- fourth chromosome nondisjunction (34-54%; Table complementer on the SMl chromosome. 9), whereas the male-predominant alleles rnei-S33P3 Viability of mei-S332 mutants: We reasoned that and mei-S3328 and theweak allele mei-S3325 gave little if the mei-S332 gene product was required for proper 838 A. W. Kerrebrock et al.

TABLE 9 Nondisjunction of the X and fourth chromosomesin females homozygous for the indicatedalleles

mei-S332+ mei-S332’ meiS332’ mei-S332‘ meiS332’Ova mei-S332’ mei-S332+ meiS33Z6 mei-Sj32’ 370 98 60 x; 4 98 370 34 166 68 (1 00.0) (57.1) (36.0) (165.1) (66.2) x; 0 2 57 21 24 52 23 (57.0) (22.4) (20.8) (51.2) (24.2) x; 44 0 61 11 18 34 18 (59.0) (12.5) (19.3) (35.8) (18.5) 0; 4 0 20 10 14 21 13 (23.1) (11.8) (9.9) (20.9) (12.2) 0;0 0 15 5 4 5 4 (13.2) (4.6) (5.7) (6.5) (4.5) 0; 44 0 15 4 3 6 3 (13.6) (2.6) (5.3) (4.5) (3.4) xx; 4 0 38 12 4 16 12 (32.9) (1 3.0) (6.1) (17.0) (14.6) xx; 0 0 17 6 2 6 7 (18.8) (5.1) (3.6) (5.3) (5.3) xx; 44 0 16 3 7 4 5 (19.4) (2.9) (3.3) (3.7) (4.1) Adjusted total 372 458 172 144 368 197 % X exceptions 0.0 52.8 46.5 47.2 31.5 44.7 % 4 exceptions 0.5 53.7 37.9 52.7 34.5 39.2 Numbers in parentheses indicate the number of progeny expected if the X and fourth chromosomes segregate independently.

TABLE 10 viable. These results indicate that the mei-S332 gene Sex chromosome nondisjunctionin heterozygous females product is not absolutely required during mitosis.

-k mei-S332’mei-S332’ - “ DISCUSSION 0Vd SM 1 + SMl Regular ova Time of mei-S332 function: The mei-S332 gene is X 4220 2948 2197 intriguing because it is required for sister-chromatid Exceptional ova cohesion in meiosis in both sexes. Our genetic analysis 0 2 1 105 of multiple alleles of mei-S332 revealed that the gene xx 2 1 77 product encoded by this locus is not needed through- Total progeny 2952 4222 2379 out meiosis. Instead, the earliest onset of the require- Adjusted total“ 2956 4224 2561 ment for mei-S332 function does not occur until late % nullo-X 0.148.2 0.05 anaphase I. Cytological analysis of homozygous mei- % diplo-X 0.14 0.05 6.0 Total X nondisjunction 0.28 0.10 14.2 S332 mutants shows that the primary defect in these mutants is a failure to maintain sister-chromatid cohe- a The progeny total is adjusted to correct for recovery of only half of the exceptional progeny. sion prior to anaphase of the second meiotic division. We have demonstrated also that mei-S332 is not required for viability and therefore appears dispensible chromosomesegregation during mitosis as well as for mitosis. meiosis, then strong alleles of this gene would result Loss of function of mei-S332 results in equational in adecrease inviability. We thereforeperformed nondisjunction, and thelate onsetof the action of mei- crosses to compare the number progeny that had of S332 we observed is unlikely to result from residual each of the mei-S332 alleles overthe Dfl2R)X58-6 gene function. We identified two alleles of mei-S332 deficiency to the number of their heterozygous sib- that are candidates for null alleles of this locus. Both lings that had either the deficiency or the mei-S332 of these alleles, whether homozygous or over a defi- mutant chromosome overthe mei-S332+ allele. For all ciency, give no evidence for an early requirement for eight alleles, including the potential null alleles mei- mei-S332 function, as they have little or no effect on S332’ and mei-S332’, the viability of flies that had the eitherchromosome segregation or recombination mutant allele over the deficiency was not different during the first meiotic division. This result distin- from thatof their siblings that had thewild-type allele guishes mei-S332 from the Drosophila meiotic muta- overthe deficiency (Table 11). We didnot select tion ord, which is also defective in sister-chromatid against lethal alleles of mei-S332 in our noncomple- cohesion during meiosis, but has pronounced defects mentation screen, since mei-S332’ over a deficiency is in early meiotic events (MASON 1976). The Drosophila mei-S332 Gene 839 TABLE 11

Numbers of progeny from the cross meiS332lSMI 6 X DjT2R)X58-6/SMI 0

Genotype md-S332’ mdS332‘ mei-S332’ mei-5‘332‘ meiS332’ nrei-S33Z6 meiS3327 mei-S332’ 233 317 325 178 352 178 325 mei-S332317 233 193178 215 + (39.1) (36.7) (37.8) (36.3) (35.3) (37.9) (38.1) (39.4) 182 296 265 DfT2R)X58-6296 182 317 157 135 161 138 + (30.5) (34.3) (30.8) (32.0) (3 1.8) (28.7) (28.5) (28.2) 250 269 155 329 Df(ZR)X58-6155 269181 250 159 189 157 (30.4) (29.0) (31.3) (3 1.6) (33.0) (33.4) (33.4) (32.4) (33.4) (33.4) (33.0) 1.6) (3 meiS332 (31.3) (29.0) (30.4) 596 863 859 490 998 470 565 490 565 470 998 490 859 Total863 596 Numbers in parentheses indicate the percentage of the total.

The quantitative level of nondisjunction observed Regulation of sister-chromatid cohesion in mito- in mei-S332 mutants can be influenced by variability sis: Sister-chromatid cohesion must also be maintained in the genetic background. While we attempted to during mitosis prior to separation at anaphase. How- remove modifiers by recombination, it is possible that ever, we did not detect a decreasein viability for any some variability remained. However, our major con- of the mei-S332 alleles over adeficiency, implying that clusions are not compromised by the possible exist- mei-S332 function is not critical for mitosis. Consis- ence of modifiers. In preliminary nondisjunction tests tent with this, we observedgynandromorphs only of the seven EMS-induced alleles, as well as in all later infrequently in the progeny of homozygous mei-S332 tests of all eight mei-S332 alleles either as homozygotes females (data not shown). Our results differ somewhat or over a deficiency, we consistently observed much from those of BAKER, CARPENTER and RIPOLL(1 978), higher levels of meiosis I1 relative to meiosis I nondis- who found a slight (approximately sixfold) increase in junction. Thus, we think that timing of the defects in somatic clones arising either from mitotic recombi- mei-,9332 mutants reflects the earliest requirement for nation or nondisjunction in homozygous mei4332’ mea4332 function. mutants. One explanation which would reconcile Model for meiS332 action: The onset of the re- these results is that meiS332 function, although not quirement for mei-S332 function coincides with the critical for mitosis, does contribute to proper mitotic time of differentiation of sister-chromatidkineto- segregation, and thatthe assay forproduction of chores during the first meiotic division. GOLDSTEIN somatic clones was more sensitive in detecting a slight (198 1) has shown by EM analysis that the single kine- mitotic effect. Alternatively, since the mitotic pheno- tochore jointly organizedby the two sister chromatids type observed in theearlier experiments was not in early prometaphase I undergoes a transformation mapped to the meiS332 locus, it could have resulted in structure between late prometaphase I and early from a second mutation on the mei-S332’ chromo- anaphase I, giving rise to a “double-disc” kinetochore some. (Figure This process may involve the reorganiza- 4). If themei-S332 product is indeed meiosis-specific, it tionof material comprising the single kinetochore is possible that different mechanisms operate tomain- into the double-disc structure (GOLDSTEIN198 1). We tain sister-chromatid cohesion during meiosis and mi- propose that the function of the meiS332 gene product is to maintain sister-chromatid cohesion after ki- tosis. Cytologically, pairing of sister chromatids ap- netochore differentiation and that this product is not pears quite different in the two divisions. In mitosis, essential for cohesion when the two sister chromatids sister chromatids remain closely pairedalong their share a single kinetochore in early meiosis I. Since whole length until anaphase. The kinetochore region double-disc kinetochores occasionally were observed does not appear to be essential for sister cohesion in to occur as early as prometaphase (GOLDSTEINI 198 I), mitosis because acentric fragments remain paireduntil our model accounts for the low levels of reductional anaphase in irradiatedgrasshopper neuroblasts exceptions recovered in the genetic tests. The higher (CARLSON1938). However, in mammalian cells it has levels of reductional nondisjunction observed in fe- been observed that the centric heterochromatindoes males may reflect an earlier time of kinetochore dif- not split in two until anaphase (SUMNER1991). Thus ferentiation in female meiosis. While we think it most mitoticsister-chromatid cohesion may be mediated probable that the mei-S332 gene product is localized both by the components of the mitotic chromosome to the kinetochore and structurally promotes cohe- scaffold found along the length of the entire chro- sion, it is possible the mei-S332 product acts in a mosome and by undivided centric heterochromatin. regulatory manner to prevent dissociation of the ki- In contrast, from anaphase I until metaphase I1of netochores. meiosis in Drosophila males, the sister chromatids are 840 A. W. Kerrebrock et al. A 1990; CUMBERLEDGEand CARBON 1987; GAUDETand FITZGERALD-HAYES1989; HAHNENBERGER,CARBON and CLARKE1991). This suggests that thereare meiosis-specific components required to maintain sister-chromatid cohesion. Further studies will reveal whether the product of the mei-S332 locus is such a Kinetochore meiosis-specific component. Differentiation Sex specificity of the mei-S332 alleles: An unex- 1 pected result from the analysis of additional mei-S332 B alleles was that four of the hypomorphic alleles had noticeably different strengths in males and females. The mei-S332* and mei-S3326 alleles whenhomozy- gous were stronger in females than in males, whereas the meiS332j and mei-S332' alleles when homozygous were stronger inmales than in females. There are three possible explanationsfor different effects of 1 these allelesin the two sexes: (1) different dosage C requirements for mei-S332 function in the two sexes; (2) the mei-S332 gene product interacts with sex-specific proteins or protein complexes, and these male- andfemale-predominant mutations disrupt such an interaction; or (3) the mutations differentially affect the synthesis or stability of the mei-S332 transcript or 1 protein product in males and females. Our data make D the first possibility very unlikelybecause it can account for only one of the sex-specific set of alleles. Further- more,the strengths of themale-predominant mei- S33.2' and female-predominant mei-S3326 alleles are fairly similar to the strengths of the three strongest alleles in males and females, respectively, but each of these alleles is veryweak in the opposite sex. At present, we are unable to distinguish between the latter two possibilities. Molecular analysis of the mei- FIGURE 4."Schematic diagram of meiotic chromosome cytology. S332 locus will help in elucidating the basis for the Two pairs of homologs are diagrammed for the late prometaphase I through late anaphase I stages of meiosis, with only the chromo- sex specificity exhibited by these alleles. somes but no microtubulesshown. It hasbeen demonstrated in A caveat to our observations on the apparent sex malemeiosis that in late prometaphase to earlyanaphase I the specificity of some of the meiS332 alleles is that these kinetochore, which is initially a singlehemispherical structure phenotypes may have been subject to modifiers. Var- shared by the sister chromatids (A), goes through a stage with an amorphous morphology (B), and differentiates into a doubledisc iability in results obtained with different chromosomes structure (C) (GOLDSTEIN1981). In late anaphase the sister chro- suggests that the genetic background may affect the matids are joined only at their kinetochores(D). Since the onset of degree of sex specificity observed,but it does not the requirement for mei-S332 function is late anaphase, following eliminate the female or male predominance. For ex- kinetochore differentiation, meiS332 may act to promote cohesion ample, a second test using a different combination of of the doubled kinetochore. recombinantchromosomes containing the homozy- paired only at or near the kinetochore regions, and gous male-predominant mei-S332' allele gave a three- the chromatid arms are completely free of one an- fold increase in nondisjunction in females relative to other (COOPER1950). the data presented here (data not shown). Since both More direct evidence to support theview that sister- of themale-predominant allelesgave consistently chromatid cohesion in meiosis and mitosis is promoted higher levels of nondisjunction in males than in fe- by different mechanisms comes from mutational anal- males from the time that they were first isolated, we yses of centromeres in the yeasts Saccharomyces cere-uis- think that the sex specificity is linked to these muta- iae and Schizosaccharomycespombe. In both yeasts, a tions. The two female-predominant alleles were influ- set of deletions of sequences within the centromere enced to a greater extent by genetic background, so which had little or no effect on mitotic centromere their classification as female-predominant alleles function were found to result in precocious sister- should be considered preliminary. chromatid separation in meiosis (CLARKEand BAUM Our genetic and cytologicalanalysis has demon- The Drosophila mei-S332 Gene 84 1 strated that mei-S332 controls sister-chromatid cohe- GOLDSTEIN, L.S. B., 1981 Kinetochore structure and its role in chromosome orientation during the first meiotic divisionin sion. The molecular cloning and identification of the male D. melanogaster. Cell 25: 591-602. mei-S332 gene product willallow us todetermine GRELL,R. G., 1976 Distributive pairing, pp. 436-486 in The whether its localization is consistent with a structural Genetics and Biology of Drosophila, Vol. 1A, edited by M. ASH- role at or near the kinetochore between anaphases I BURNER and E. NOVITSKI.Academic Press, New York. HAHNENBERGER,K., J. CARBON and L. CLARKE, and 11 of meiosis. The characterization of the mei- 199 1 Identification of DNA regions required for mitotic and S332 gene product will define further any possible meiotic functions within the centromere ofSchizosaccharomyces mitotic function of the gene and will clarify the sex pombe chromosome I. Mol. Cell. Biol. 11: 2206-2215. specificity observed withsome alleles. Importantly, HALL,J., 1972 Chromosome segregation influenced by two alleles mei-S332 can be used to identify other genes involved of the meiotic mutant c(3)G in Drosophila melanogaster.Genetics 71: 367-400. in chromosome segregation, either through genetic KOSHLAND,D., and L. HARTWELL,1987 The structure of sister screens for second-site noncomplementers, or in bio- minichromosome DNA before anaphase in Saccharomyces cere- chemical assays for proteins that interact with the mei- visiae. Science 238: 17 13-1 7 16. S332 gene product. LEVIS,R., T. HAZELRICCand G. RUBIN, 1985 Effects of genomic position on the expression of transduced copies of the white We thank SCOTT HAWLEY,BOB LEVIS,RUTH LEHMANN and gene of Drosophila. Science 229 558-561. KATHYMATTHEWS at the Bloomington Stock Center for providing LEWIS,E., and F. BACHER,1968 Method of feeding ethyl methane stocks. We are grateful to SCOTTHAWLEY, LARRYCOLDSTEIN and sulfonate (EMS) to Drosophila males. Drosophila Inform. Serv. RUTH LEHMANNfor helpful discussions. FRANKSOLOMON, CHRIS 43: 193. KAISER,LARRY GOLDSTEIN, SIMA MISRA, DAN CURTISand members LINDREN,B. W., G. W. MCELRATHand D. A. BERRY, of this laboratory provided many useful comments on the manu- 1978 Probability and Statistics. Macmillan, New York. script. We thankJANE BENSELfor preparing figures. This work was LINDSLEY,D., and E. GRELL, 1968 Genetic Variations of Drosophila supported by National Institutes of Health Postdoctoral Training melanogaster. Carnegie Institution of Washington, Washington, grant GM12678 to A.K., a National Science Foundation (NSF) DC. Predoctoral Fellowship to W.M., the Searle Scholars Program/The MAGUIRE,M., 1990 Sister chromatid cohesiveness: Vital function, Chicago Community Trust, NSF grant DMB-9020672, and by a obscure mechanism. Biochem. Cell Biol. 68: 1231-1242. grant from the Lucille P. Markey Charitable Trust. MAGUIRE,M. P., 1978a Evidence for separate genetic control of crossing over and chiasma maintenance in maize. Chromosoma LITERATURECITED 65 173-183. ASHBURNER,M., 1989 Drosophila. A LaboratoryManual. Cold MAGUIRE,M. P., 197813 A possible role forthe synaptonemal Spring Harbor Laboratory, Cold Spring Harbor, N.Y. complex in chiasma maintenance. Exp. 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