The Role of S. Cerevzszae Cell Division Cycle Genes in Nuclear Fusion

THE ROLE OF S. CEREVZSZAE CELL DIVISION CYCLE GENES IN NUCLEAR FUSION

SUSAN K. DUTCHER1 AND LELAND H. HARTWELL Department of Genetics, University of Washington, Seattle, Washington 98195 Manuscript received September 22, 1980 Revised copy received July 7, 1981

ABSTRACT Forty temperature-sensitive cell division cycle (cdc) mutants of Sac- charomyces cerevisiae were examined for their ability to complete nuclear fusion during conjugation in crosses to a CDC parent strain at the restrictive temperature. Most of the cdc mutant alleles behaved as the CDC parent strain from which they were derived, in that zygotes produced predominantly diploid progeny with only a small fraction of zygotes giving rise to haploid progeny (cytoductants) that signalled a failure in nuclear fusion. However, cdc4 mutants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy defect. For all four mutants, the karyogamy defect and the cell cycle defect cosegregated, suggesting that both defects resulted from a single lesion for each of these cdc mutants. Therefore, the cdc 4, 28, 34 and 37 gene products are required in both cell division and karyogamy.

ONJUGATION in the yeast Saccharomyces cerevisiue occurs between cells that differ from one another at the mating type locus on chromosome ZZZ. Cells of opposite mating type when mixed together respond to mating hormones by inducing agglutination (SHIMODAand YANAGISHIMA1975) and by arresting progress through the cell division cycle at a specific interval of the cycle (BUCK- ING-THROMet al. 1973; WILKERSONand PRINGLE1974). Cytoplasmic fusion occurs between unbudded, arrested cells to form a zygote (HARTWELL1973) fol- lowed by fusion of the nuclei in the isthmus of the new zygote. The cytological events of nuclear fusion during conjugation have been described (BYERSand GOETSCH1975). The initial contact between the two nuclei occurs at the organelle known as the spindle pole body. After the two haploid spindle pole bodies fuse, the single diploid spindle pole body duplicates and separates to form a mitotic spindle. The newly formed diploid cell then repro- duces by mitotic cell divisions. Only recently has a genetic analysis of the products that mediate the process of nuclear fusion been begun. A mutation in one gene, KARI, has been identified that prevents nuclear fusion ( CONDEand FINK 1976). We reasoned that some gene products that function in nuclear processes during cell division might also perform functions essential for the fusion of nuclei during conjugation. The cell division cycle has been delineated by the use of tem-

Piesent address: The Rockefeller University, New York, New York, lOMl

Genetics 100: 175-184 February, 1982 176 S. K. DUTCHER AND L. H. HARTWELL perature-sensitive mutants that block the progress of cells through the cell division cycle (cdc) at specific stages (HARTWELLet al. 1973; HEREFORDand HARTWELL1974; HARTWELL1976). Of particular interest in this study were cdc mutants that arrest the development of the spindle pole body, since this organelle appears centrally involved in both cell division and nuclear fusion. The spindle pole body serves as the microtubule organizing center for the mitotic and meiotic spindles and for extranuclear microtubules (ROBINOWand MARAK1966; BYEKS and GOETSCH1975). Its development in the cell division cycle has been cor- related with the arrest points of the cdc mutants. In cells arrested by the mating hormone, a-factor, or by temperature-sensitive mutations in eight genes, the spindle pole body remains as a single densely staining body with a satellite (BYERSand GOETSCH1974; B. BYERSand L. GOETSCH,personal communica- tion). In two mutants, cdc4 and cdc34, duplication of the spindle pole body occurs but the two duplicated halves fail to separate to form a complete spindle. Other cdc mutants arrest at points in the cell cycle at which nuclear migration or spindle elongation is blocked. To determine whether any CDC gene products functioned in nuclear fusion during conjugation, we tested the cdc mutants for defects in nuclear fusion. A defect in nuclear fusion would be expected to generate heterokaryotic zygotes rather than diploid zygotes. Heterokaryons have been identified and monitored by their production of haploid as well as heterokaryotic progeny (WRIGHTand LEDERBERG1957; ZAKHAROV,YURCHENKO and YAROVOY1969; CONDEand FINK 1976). Haploid progeny (cytoductants) produced by heterokaryons can be distinguished from the parental haploid cells because they may obtain cytoplasmic genes contributed by the second parent of the heterokaryon.

MATERIALS AND METHODS

Strains: The strains used in the the majority of these experiments were derived by muta- genesis from A364A and have been described (HARTWELLet al. 1973). Other strains used are listed in Table 1. Each cdc strain employed was used to construct a second strain, which was resistant to the drug cycloheximide and was a cytoplasmic petite. The cycloheximide resistance marker was selected as a spontaneously arising mutation on YEPD medium containing 10 pg/ml cycloheximide (Sigma Chemical Co., St. Louis, MO). Complementation tests were performed on all drug resistant mutants and only strains carrying cyhZ mutations were used. These strains were rcndered petite by overnight growth in YM-1 containing 10 pg/ml ethidium bromide (Sigma Chem:cal Co., St. Louis, MO). Colonies were subsequently tested and only nonsup- pressive petites (EPHRUSSIand GRANDCHAMP1965) were used. Coniugation: An aliquot of 3 x 106 cells of each parent was mixed and filtered onto 0.45 pm, 25 mm Millipore filters (Millipore Corp., New Bedford, MA). The filters were then placed onto YM-1 medium containing 20 g/liter Noble agar (Difco Laboratories, Detroit, MI) at 21" or at 34". The 34" plates were prewarmed and then maintained at 34" by immersing the plate in a 34 k 005O water bath. Aiter 5 hr of incubation (sufficient time for conjugation to occur and for the majority of the newly formed zygotes to complete one cell cycle), the cells on the filters were resuspended in 1 M sorbitol, subjected to sonic vibration to disperse clumps, and plated onto selective nutritional medium, YEPD and YEPG containing 3 pg/ml cycloheximide. DAPI staining: Approximately 1 x 106 cells were washed with 1% saline solution and centrifuged to a pellet. They were slowly resuspended in 4 ml of Carnoy's solution (3:l methanol . glacial acetic acid) and were left in the fixative for at least 1 hr. The cells were S. cereuisiae: NUCLEAR FUSION 177 TABLE 1 Strains used in experiments

Strains Source Genotype E420 J. CULOTTI' MATa adel ade2 ural lys2 tyrl his7 cdc2S-2 621.1 S. REED MATa tyrl cdc284 El 7 J. CULOTTI* MATa adel ade2 ural lys2 tyrl his7 cdc33-I E3-16 J. CULOTTI* MATa adel de2 ural lys2 tyrl his7 cdc34-1 BR214.4a B. REID* MATa adel ade2 ural lys2 tyrl his7 cdc35-1 531.11.2 This study MATa met2 tyrl cdc36-3 626.1 S. REED MATa adel ural cdc36-5 624.1 S. REED MATa met2 cdc37-l 532.1.4 This study MATa adel tyrl cdc38-I 533.4.2 This study MATa tyrl met2 cdc39-1 El 89 B. REID' MATa adel ade2 ural lys2 tyrl his7 cdcll-l 174.7.2 This laboratory MATa trpl urd 500.19.1 This study MATa leu1 thrl karl-1 2335.3.1 This study MATa lysl0 ade5,7 his5 pha2 met2 241 This laboratory MATa adel ade2 ural lys2 tyrl his7 prt2-1

* This laboratory. then centrifuged, washed twice with 1% saline, and resuspended in 1 ml of 1 yg/ml DAPI (4', 6'-diamidino-2-phenyl-indole2 HCI; Serva Fein, Heidelberg, Germany) for 30 min. The stained cells were washed once in 1% saline and examined with a Leitz Fluorescence Microscope at a magnification of 600~. Micromanipulation of newly formed zygotes: Two hours after mixing MATa and MATa cells, a sample of the conjugation mixture was spread onto dissection agar. Newly formed zygotes were separated from the bulk of the cells with a Cailloux Micromanipulator (C. H. Stoelting CO., Chicago, IL). The first bud was separated from the zygote following cytokinesis. Zygotes and first buds were allowed to grow into colonies at 21". Dissection performed at 34" was accomplished in a 34" hood. Dissection agar was made with YEPD or YEPG medium. TWOtypes of first buds from zygotes were distinguished microscopically: medial buds that arose at the isthmus of the zygote and end buds that arose at the poles of the zygotes. Of the first buds 58% were medial buds and 42% were end buds among 500 zygotes formed between MATa karl-2 (500.19.1) and MATa KARl (A364A) at 21.. Cytological examinations showed that medial buds are primarily binucleate (89% had two nuclei, n = 3001) and end buds are mainly mononucleate (93% had one nucleus, n = 300). Both showed considerable inviability at 21" and 34"; 33% and 31% of the medial buds failed to grow into colonies at 21" and 34" re- spectively, while 38% and 37% of the end buds failed to form colonies at 21" and 34". The lethality is not a result of micromanipulation of the cells because zygotes without a karl-l parent showed only two percent inviability following micromanipulation (DUTCHER1981 ) . Genetic techniques: Standard techniques were used and have been described elsewhere (MORTIMERand HAWTHORNE1969). The karyogamy phenotype was detected in tetrads as fol- lows: Each spore clone was tested with a MATa and a MAT& strain that carried three nuclear recessive drug resistance alleles, canl, tcm, cry1 and was a cytoplasmic petite (p-). After 12 hr, the mated cells were transferred by replica-plating to gylcerol medium with canavanine (GRENSONet al. 1966), trichodermin (SCHINDLER,GRANT and DAVIES1974) and cryptopleurim (SKOGERSON,MCLAUGHLIN and WAXATAMA1973). Cells with the karl-l mutation produced cytoductants and thus produced visible colonies, while KARl cells failed to generate colonies on this medium. Efficiency of test for cytoductant production: The test for cytoductant production was examined using the mutant karl-l to verify that only haploid progeny produced at the restrictive 178 S. K. DUTCHER AND L. H. HARTWELL temperature with the desired genotype will form colonies on the selective medium. The assay for cytoductants utilizes cycloheximide to select against CYH haploids and CYH/cyh2 diploids or heterolraryon? and glycerol to select for haploids that have received mitochondria from the heterokaryon. The following pedigree experiments demonstrate the efficacy of this assay. Buds were micromanipulated from zygotes of the cross .MATol karl-I CYH2 pf X MATa I'IARI cyh2 p-. Three types of colonies arose from buds-pure colonies with cells of only one parental genotype, mixed colonies with cells of both parental genotypes as well as some diploid cells and inviable microcolonios. End buds generally produced colonies with only one parental genotype (98% of viable colonies, n = 176) and the two parental types of colonies occurred with equal frequency. End buds are therefore nearly always haploid. Medial buds frequently produced mixed colonies (27% of viable colonies, n = 211) and are often heterokaryons or diploids. The formation of colonies by buds and zygotes on YEPG medium and YEPG medium with cycloheximide was compared to the YEPD medium control (Table 2). In general, zygotes and medial buds fail to divide on medium with cycloheximide but produce equal numbers of colonies on YEPD and YEPG media. Twenty-eight percent of end buds or one-half of the viable end buds on YEPD medium produced colonies on medium with cycloheximide, and the same pro- portion was seen on YEPD and YEPG medqa. These tests show that nearly all first buds receive and retain functional mitochondria and hence produce colonies on glycerol medium. Only end buds (mononucleate buds) containing the cyh2 allele and not heterokaryotic buds or zygotes will produce colonies on Cycloheximide medium. This selection scheme allows only buds that are mononucleate, have completed cytokinesis before imposition of selection, and are genotypically cycloheximide resistant to divide on glycerol medium containing cycloheximide. Frequencies of cytoductants and diploids observed by micromanipulation were comparable with the ratios of cytoductants to diploid cells of 5.4 k 0.45 obtained when mating mixtures were placed directly onto selective medium. Media: Rich media (YEPD, YEPG and YM-1) and minimal medium have been described previously (HARTWELL1967). Cycloheximide was used at a concentration of 3 pg/ml in YEPG plates and 10 pg/ml in YEPD plates. Canavanine sulfate was used at a concentration of 60 pg/ml, cryptopleurine was used at a concentration of 1 pg/ml, and trichodermin was used at a concentration of 20 pg/ml on medium with dextrose. Ten-fold lower concentrations of each of these three drugs was used on medium with glycerol as the carbon source.

RESULTS We tested cdc mutants for defects in nuclear fusion using logarithmically growing cultures of a MAT@CDC ,cyh2 p- tester strain and a MATa cdcx CYH

TABLE 2 Frequency of viable colonies produced by first buds and unbudded zygotes in crosses of MATol karl-I p+ (500.19.1) to MATa KAR cyh2 p- (A364A cyh2 p-) asdetermined by micromanipulation onto uarious media

Unbudded zygotes 0.71 (58/8&)a 0.69(29/42) 0.07(1/14) 0 (0/12) Medial first buds 0.67(28/42) 0.75(15/20) 0 (0/11) 0 (0/18) End first buds 0.64(21/33) 0.59 (13/22) 0.28 (7/25)b 0.28(11/39)

a Numbers in parentheses indicate number of viable cells divided by the number examined. Frequency Frequency b Expected frequency of colonies on Number of of viable KAR cyh2 cycloheximide containing = end buds x colonies x colonies medium examined on YEPD on YEPD - - 25 x 0.64 x 0.48 - 0.31 S. cerevisiae: NUCLEAR FUSION 179 p+ mutant strain. One half of the mating mixture was immediately placed at the restrictive temperature of 34"; the other half was left at 21 ". Five hours after mixing, the frequency of cytoductants and of diploids was determined by plating onto appropriate selective medium at 21". Cytoductants were detected on YEPG plates with cycloheximide. Diploids were detected on medium lacking appropriate nutrients. The configuration of markers in this experiment allows for the detection of cytoductants with the genotype of the CDC cyh2 parent. In crosses with the parent strain of the cdc mutants, A364A, cytoductants are produced at a low level at 21 O and at 34" (Table 3). The ratio of cytoductants to diploids ranged from 0.001 to 0.006 in 12 experiments at both tempera- tures. Cytological studies revealed less than one in 500 unbudded zygotes with more than one nucleus. Four CDC genes function in nuclear fusion: A thousand-fold increase in the number of cytoductants produced in 34" matings as compared to 21" matings was observed for three alleles of cdc4 (Table 3, Part A). Cytology of MATa cdc4 X MATa CDC4 zygotes formed at 34" showed that about 82% of the unbudded zygotes (n = 350) had two nuclei at three hours, while zygotes formed at 21 " showed less than one percent binucleate unbudded zygotes (n = 350). Since all three independently isolated alleles of cdc4 tested in these experiments displayed a temperature-sensitive karyogamy phenotype, it is almost certain that the temperature-sensitive mitotic defect and the temperature-sensitive karyogamy defect are pleiotropic manifestations of the same mutation. Nev- ertheless, linkage between the two phenotypes was investigated. In all 17 four- spored tetrads examined from a diploid heterozygous for the cdc4-I mutation, the two phenotypes cosegregated and each tetrad displayed two mutant and two wild-type progeny spores (data not shown). Thus, it is necessary for both par- ents to contribute functional CDC4 gene product for nuclear fusion to occur in the zygote at wild-type levels. Crosses involving mutations in the cdc28 gene showed an increase of 150-fold in the ratio of cytoductants to diploids produced in the 34" mating as compared to the 21" mating (Table 3, Part A). Cytology revealed that 8% of the MATa: CDC28 X MATa cdc28-4 zygotes (n = 300) formed at the restrictive temperature had two nuclei while less than 1% of the zygotes formed at 21 " had two nuclei (n = 300). Three alleles of cdc28 all display a temperature-sensitive karyogamy phenotype and the two phenotypes were linked. In l l of l l tetrads examined from a diploid heterozygous for the cdc28-4 mutation, the temperature-sensitive mitotic and karyogamy defects cosegregated and each tetrad displayed two mutant and two wild-type progeny spore clones. A 20-fold increase in the number of cytoductants produced at 34" compared to 21 O conjugation was observed for the only isolate of cdc34 (Table 3, Part A). This mutant arrests division after two cell cycles at the restrictive temperature. This mutant was preincubated at 34" for 3 hr and 6 hr prior to the standard conjugation procedure. After a 3 hr incubation, a 30-fold increase in the number of cytoductants per diploid was observed compared to that at the 21 " conjugation. 180 S. K. DUTCHER AND L. H. HARTWELL TABLE 3 Frequencies of cytoductants with the genotype of the CDC parent in cdc x CDC (174.7.2) crosses at 21" and 34"

Number of Ratio of CDC cgtod:ctants per lo6 cells cytwlucpts to diploid, produced Strain cdc mutant 21 340 ?.I 34" A364A - 42 55 0.001 0.005 A. cdc mutants with kar phenotype 314 cdc4-1 11 2.4 x 104 0.0007 22 458 cdc4-3 21 3 1.8 x 104 0.003 18 19036 cdc4-5 72 1.5x 104 0.003 12 185.3.4 odc28-1b 28 5.8 x 103 o.ooai 0.10 E420 ~dc28-2' 1 50 0.0002 0.01 621.4 cdc28-4b 177 1.3x 104 0.001 0.15 E3-16 cdc34-I b 11 5 0.001 0.05 624.1 cdc37-lb 111 387 0.001 0.09 B. cdc mutants without kar phenotype 369 cdci-1 28 15 0.002 0.002 370 cdc2-1a 37 28 0.002 0.005 104 cdc3-1 a 22 16 0.005 0.006 473 cdc5-1" 27 17 0.002 0.003 327 cdc6-1a 34 3 0.006 0.007 4008 cdc7-4" 368 378 0.002 0.001 13052 cdc8-3a 64 133 0.001 0.002 280 cdc9-2" 14-5 1.1 x 103 0.007 0.006 17012 cdc10-1" 440 82 0.004 0.002 332 cdcll-1" 81 26 0.003 0.002 471 cdc12-1" 7 2 0.0007 0.0002 428 cdc13-1a 123 128 0.003 0.004 7041 cdc14-1" 280 340 0.0004 0.002 17017 cdc15-1a 168 420 0.003 0.002 28 1 cd~26-1" 120 76 0.001 0.0009 4028 cdc17-1 105 62 0.005 0.006 14028 cdd8-1 67 30 0.007 0.004 3 95 cdcl9-1 150 31 0.002 0.001 127 cacao-1 71 74 0.008 0.001 146.2.3 cdc21-1" 479 180 0.005 0.003 248 cdc22-1" 19 32 0.0005 0.0007 9013 cdc23-1" 29 51 0.003 0.002 E185 cdc24-2 60 0.4 0.003 0.007 E187 cdc24-3 106 0.1 0.002 0.005 321 cdc25-1 27 271 0.002 0.004 7027 cdc26-1" 40 1.5 0.007 0.002 9002 cdc27-1" 301 81 7 0.002 0.003 17048 cdc29-1 36 21 0.001 0.0001 230 15 cdc30-1 122 25 7 0.004 0.006 12021 cdc31-1 28 24 0.0001 0.008 212 cdc32-1 6 2 0.002 0.006 El 7 cdc33-1" 11 0.5 0.001 0.005 S. cerevisiae: NUCLEAR FUSION 181

TABLE 3-Continued

Number of Ratio of CDC cytoductants per loo cells cytoductants to diploids produced Strain cdc mutant 210 34- 210 34" BR214.h cdc35-la 93 99 0.0009 0.001 531.11.2 cdc36-3a~b 831 2.1 x 103 0.003 0.005 626.1 cdc36-5a.b 465 1.1 x 103 0.002 0.002 532.1.4 cdc38-lasb 177 636 0.001 0.002 533.4.2 cdc39-la.b 237 1.2 x 103 0.002 0.003 E187 CdC41-I 61 1 1.4 X lo3 0.037 0.008

a No nuclear fusion defect in cdc x cdc crosses at 21" or 34". Other mutants produced in- sufficient frequencies of zygotes. Preincubation at 34" for 3 hr.

After a 6 hr incubation, few zygotes were produced and the ratio of cytoductants per diploid did not increase. Cytological examination of MATa cdc34-2 by MATa CDC34 zygotes revealed 4% of the unbudded zygotes were binucleate at 34" (n = 300) and less than one percent of the zygotes produced at 21 were binucleate (n = 300). In six tetrads from a diploid cdc34-ZJCDC34 heterozygote, the cell division defect cosegregated with the weak karyogamy defect and both showed 2+:2- segregation for the two defects. Crosses involving one parent with a mutation at the cdc37 locus showed an increase in the ratio of cytoductants to diploids of ninety-fold at 34" over the ratio observed in the 21" conjugation mixtures (Table 3, Part A). Cytology showed that 5% of the MATa CDC37 X MATa cdc37-1 unbudded zygotes (n = 350) formed at 34" were binucleate while only one of 350 (0.2%) unbudded zygotes formed at 21 o was binucleate. In 12 of 12 tetrads examined from a diploid heterozygous at the cdc37-1 locus, the two phenotypes cosegregated and both showed 2+:2- segregation for the two defects at 34". The four karyogamy defective loci are different: Linkage analyses of the cdc mutations that cause a karyogamy defect and karl-2 were performed and it was found that they are not linked to one another (Table 4). Recently, they have been mapped to different linkage groups (HARTWELLet at. 1973; REED1981; Table4). Most cdc mutants are not defective in nuclear fusion: The majority of the cdc mutants produce cytoductants at the low level characteristic of A364A. The ratio of cytoductants to diploids ranged from 0.0001 to 0.008 at both tempera- tures (Table 3, Part B) . Cytological examination of the zygotes confirmed these data. Because the tests reported here involve one cdc and one CDC nucleus, a failure of nuclear fusion would be detected only in those cases in which both nuclei must be wild type at the CDC locus for fusion to occur. For these 37 cdc genes, nuclear fusion does not require the contribution of CDC gene product from both parent cells. A cytological examination of zygotes from homozygous cdc x cdc crosses was made to determine if nuclear fusion was defective in cdc x cdc crosses. No nuclear fusion defect was observed for the 24 cdc genes tested (Table 3, Part B). 182 S. K. DUTCHER AND L. H. HARTWELL TABLE 4

Linkage unalysis of mutants with kar phenotype

Tetrad types Gene pair PD NPD T Map distance* cdc4-l- kurl-f 8 6 31 unlinked cdc28-4 - kzrl 9 6 23 unlinked cdc37-1- karl 11 12 42 unlinked kari-l - met2 21 0 31 30 centimorgans kari-1 - prt2 7 1 11 45 centimorgans prt2 - met2 10 1 7 36 centimorgans karl-l- pha2 16 1 34 39 centimorgans pha2 - met2 24 7 42 63 centimorgans

* Calculated from the formula of PERKINS(1949). -) Chromosome XVZI prt2 met2 knrl pha2 O---[-- 1__-1___- I

Seven of the 37 cdc mutant alleles (cdcl8, 19, 25, 27, 30, 31, 32) tested fail to arrest in the first cycle when shifted to the restrictive temperature (HARTWELL et al. 1973). To further test if these genes are required for nuclear fusion, each mutant was preincubated at 34" for 3 hr and 6 hr prior to the standard conjugation procedure. No increase in the ratio of cytoductants to diploids was observed for any of the multicycle mutants after this preincubation (data not shown).

DISCUSSION The protocol used to assess defects in karyogamy (nuclear fusion) imposed three requirements that limit the type of defects that could have been detected. First, if heterokaryotic zygotes are formed they must produce progeny that con- tained a single haploid nucleus rather than producing only heterokaryotic or diploid progeny. The requirement is probably not unduly restrictive since heterokaryons produced with the karl-1 mutation are unstable and produce many haploid progeny. Second, the mixing of cytoplasms must have been sufficient to allow transfer of mitochondria to most buds. That the transfer of mitochondria is efficient has been demonstrated in wild-type diploid zygotes (EPHRUSSI, MARGERIE-HOTTINGUERand ROMAN 1955) and in karl-1 generated zygotes (METHODS). Third, the karyogamy defect must have been expressed in the heterokaryon when only one nucleus was mutant. This requirement demands that the wild-type gene product contributed by the other parent be unable to rescue the mutant nucleus for its fusion defect. Although all of the cdc mutations tested here were known to be recessive for their cell cycle defect (HARTWELLet al. 1973), we were encouraged to proceed with the analysis because of the fact that the karl-l mutant exhibited just this type of behavior (FINKand CONDE1976). Most of the cdc mutants exhibit no defect in karyogamy when assayed by the methods used here. However a defect would have been missed if the cdc mutant produced only stable heterokaryons, inhibited mitochondrion transfer, or was S. cereuisiae: NUCLEAR FUSION 183 complemented by gene product contributed by the other parent. An attempt to detect defects of the latter type by cytological examination of cdc X cdc matings was unsuccessful. Of greater intercst is the fact that mutations in four cdc genes (4,28, 34,37) do produce defects in nuclear fusion in cdc by CDC zygotes. For all four of these cdc genes it was demonstrated that the lesion producing the defect in karyogamy is closely linked and therefore probably identical to the lesion producing the defect in cell division. It appears that these four gene products fumtion both in nuclear division and in nuclear fusion. It may be relevant that all four genes control early steps in the duplication (cdc28 and 37) or separation of the spindle pole bodies (cdc4 and 34) during the mitotic cycle (BYERSand GOETSCH1974, 1975) since nuclear fusion occurs at the site of the spindle pole body (BYERSand GOETSCH1975). An attractive hypothesis is that these gene products are com- ponents of or act upon the spindle pole body. REIDand HARTWELL(1978) have shown previously that the CDC4 gene was indispensable to the formation of diploid cells. That is, the number of diploids formed in an asynchronous cdc4-2 x CDC4 mating at 34O was depressed when compared to the number of diploids produced in the mating at 21 ". This depres- sion can now be accounted for by considering the number of cytoductants produced. The number of cytoductants plus the number of diploids formed at 34" is approximately equivalent to the number of diploids formed at 21". REID and HARTWELL(1978) also demonstrated that conjugation occurred only in the interval of the cell cycle defined by the cdc28 mutation. Synchronous cultures of cdc mutants were challenged to conjugate at their respective arrest points and most failed to form zygotes. Because the CDC4 gene product was required for diploid formation, the competence of cells arrested at the cdc4 arrest point to conjugate was not tested. By assaying both cytoductants and diploids, we have shown that conjugation does not occur at the position of the cell cycle defined by the cdc4 lesion. No zygotes were observed in the 5000 cells counted and few diploids or cytoductants (1 per 106 total cells) were produced as assayed on selective media.

We thank LARRYGOLDSTEIN for helpful discussions and for advice on this manuscript We are grateful to JAIMECONDE and GERRYFINK for supplying the karl-I mutant and to STEVEREED for supplying the cdc28-4, cdc36-I, cdc37-I, cdc38-I and cdc39-1 mutants. We thank NANCY GAMBLE,PATTI RIDGWAY and BARBARAHLAVIN for typing of this manuscript. This work was supported by National Institutes of Health Predoctoral Training Grants GM00182 and GM07735 and by a research grant GM17709 from the National Institutes of Health.

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