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

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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 mu- tants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy de- fect. For all four mutants, the karyogamy defect and the cell cycle defect co- segregated, 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 de- scribed (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 prod- ucts 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 dur- ing 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 di- vision 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 oc- curs 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 de- fect 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 dis- tinguished 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.
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