Meiosis in Saccharomyces cereuisiae Mutants Lacking the Centromere - Binding Protein CP 1 Daniel

نویسنده

  • Richard E. Baker
چکیده

CPl (encoded by the CEPl gene) is a centromere binding protein of Saccharomyces cerevisiae that binds to the conserved DNA element I (CDEI) of yeast centromeres. To investigate the function of CP1 in yeast meiosis, we analyzed the meiotic segregation of CEN plasmids, nonessential chromosome fragments (CFs) and chromosomes in cepl null mutants. Plasmids and CFs missegregated in 10-2076 of meioses with the most frequent type of aberrant event being precocious sister segregation at the first meiotic division; paired and unpaired CFs behaved similarly. An unpaired chromosome Z ho olog (2N + 1) also missegregated at high frequency in the cepl mutant (7.6%); however, missegregation of other chromosomes was not detected by tetrad analysis. Spore viability of cepl tetrads was significantly reduced, and the pattern of spore death was nonrandom. The inviability could not be explained solely by chromosome missegregation and is probably a pleiotropic effect of cepl. Mitotic chromosome loss in cepl strains was also analyzed. Both simple loss (1:0 segregation) and nondisjunction (2:O segregation) were increased, but the majority of loss events resulted from nondisjunction. We interpret the results to suggest that CP1 generally promotes chromatid-kinetochore adhesion. Y EAST (Saccharomyces cerevisiae) centromeres contain three highly conserved DNA sequence elements, named CDEI, CDEII and CDEIII (centromere CARBON 1982; HIETER et al. 1985). Together, these elements (about 120 bp) comprise the functional centromere and when used to replace the resident centromere of a yeast chromosome are sufficient to provide full meiotic and mitotic centromere function (COTTAREL et al. 1989; MURPHY and FITZGERALDHAYES 199 1). CDEI is the degenerate octanucleotide sequence RTCACRTG (R = purine), CDEIII is a partially palindromic sequence 25 bp in length, and CDEII is a 78-86-bp region of highly A + T-rich (>go%) DNA which seems to provide a spacer function (FITZGERALD-HAYES 1987). Mutational analyses have revealed that CDEIII is absolutely essential for mitotic centromere function, while mutations of CDEI and CDEII impair but do not abolish function (CUMBERLEDGE and CARBON 1987; GAUDET and FITZGERALD-HAYES 1987; HEGEMANN et al. 1988; MCGREW, DIEHL and FITZGERALD-HAYES 1986; PANZERI et al. 1985). Wild-type yeast chromosomes typically are lost about once in 100,000 mitoses (HARTWELLet al. 1982). Deletion of CDEI results in a 10-60-fold increase in the rate of mitotic loss (CUMBERLEDGE and CARBON 1987; GAUDET and FITZGERALD-HAYES 1989; PANZERI et al. 1985). Electron micrographs of mitotic yeast nuclei fail to reveal a differentiated kinetochore structure; spindle microtubules seem to attach directly to the chromatin Genetics 131: 43-53 (May, 1992) DNA element) (FITZGERALD-HAYES, CLARKE and fibers (PETERSON and RIS 1976). But nuclease sensitivity studies of native yeast chromatin show that the centromeric DNA is highly resistant to digestion and flanked on both sides by nuclease hypersensitive sites associated with highly phased nucleosome arrays, leading BLOOM and CARBON (1 982) to speculate that the nuclease-resistant core represents a structurally primitive kinetochore. Two centromere-specific DNA binding factors have been identified, one which binds to CDEI and one which binds to CDEIII. The CDEIIIbinding factor, CBF3, is actually a complex of three different proteins, at least one of which is phosphorylated (LECHNER and CARBON 1991). CP1 (also known as CBFl) is a relatively abundant protein which binds to CDEI (BAKER, FITZGERALD-HAYES and O’BRIEN 1989; BRAM and KORNBERG 1987; CAI and DAVIS 1989; JIANC and PHILIPPSEN 1989). Isolation of the gene encoding CPl has revealed that the protein has a molecular weight of 39,000 and is a member of the helix-loop-helix family of DNA-binding proteins (BAKER and MASISON 1990; CAI and DAVIS 1990; MELLOR et al. 1990). The CP 1 gene has been named CEPl (BAKER and MASISON 1990), CBFl (CAI and DAVIS 1990) and CPFl (MELLOR et al. 1990). Disrupting CEPl in haploid strains of yeast has pleiotropic effects (BAKER and MASISON 1990; CAI and DAVIS 1990; MELLOR et al. 1990). Mitotic chromosome loss rate is increased and growth rate is decreased. The magnitude of the chromosome loss rate increase is about 10-fold, consistent with the mitotic effect of CDEI deletion but insufficient to 44 D. C. Masison and R. E. Baker explain the 35% increase observed for cell doubling time. An unexpected phenotype of cep l null strains is that they require exogenously added methionine for growth. The basis of the methionine auxotrophy is not known, but it is likely that CP1, in addition to its function as a kinetochore protein, is also a transcription factor like other members of the helix-loop-helix family (MURRE, MCCAW and BALTIMORE 1989). Several genes encoding enzymes of the methionine biosynthetic pathway contain CDEI sites in their promoter regions (THOMAS, CHEREST and SURDIN-KERJAN 1989). The segregational defects in cep l strains appear to be caused directly by the lack of CP1 at the centromere. The effects of CDEI mutations on centromere function in vivo are quantitatively correlated with the affinity of CP1 binding in vitro (BAKER, FITZGERALD-HAYES and O'BRIEN 1989; CAI and DAVIS 1989), and the increases in chromosome loss rate attributable to cisand trans-acting mutations (CDEI deletion and cepl gene disruption, respectively) are equivalent and nonadditive (BAKER and MASISON CUMBERLEDGE and CARBON (1987) observed that plasmids containing CDEI-deleted centromeres displayed significantly increased rates of meiotic missegregation, more than would have been expected given the mitotic effects of the same mutations. The segregational defect observed was precocious sister segregation at the first meiotic division, suggesting that CDEI was somehow involved in maintaining sister chromatid cohesion during meiosis I (CUMBERLEDGE and CARBON 1987). [Similar findings had been obtained previously by PANZERI et al. (1985), but interpretation of the results was clouded by the fact that two very similar mutations gave very different meiotic segregation patterns.] The effect of deleting CDEl from the centromere of an endogenous chromosome (chromosome ZZZ) was also tested. When the mutation was heterozygous, segregation appeared normal, but when both homologs carried the CDEI-deleted centromere, sporulation and spore viability were poor, presumably due to missegregation of chromosome ZZZ (CUMBERLEDGE and CARBON 1987). A subsequent study by GAUDET and FITZGERALD-HAYES (1 989) confirmed the results of CUMBERLEDGE and CARBON with respect to plasmid missegregation, but in the later study no missegregation was observed when one or both chromosomes ZZZ carried the CDEI-deleted centromere. To study the meiotic role of CPl directly, we have analyzed meiosis in cep l null mutants. The results are quite consistent with the previous findings and indicate that CP1 is important for proper kinetochore function during meiosis. 1990). MATERIALS AND METHODS Plasmids: Plasmid pDK243 (KOSHLAND, KENT and HARTWELL 1985) was obtained from D. KOSHLAND, plasmid pJS2 (SHERO et al. 1991) from P. HIETER, plasmid PEL1 1 (LOUIS and HABER 1989) from E. LOUIS and plasmid pRIPl (PARKER and JACBOSON 1990) from R. PARKER, Plasmid pDM8 was constructed by inserting (after Klenow fill-in) the 5.4-kbp SalI-Snal ade3-2p fragment from pDK243 into the SmaI site of pJS2, inactivating the SUP11 gene. Plasmid pDM2 was derived from pRIPl by inserting a Klenowblunted 2.2-kbp LEU2 fragment into the EcoRV site in uRA3. Yeast strains and media: The diploid yeast strains used in this study are listed in Table 1 along with the haploid parents from which they were all derived. All except the chromosome I-marked strains (D77-R1, D92 and D93) are congenic to strain 38 1 G (HARTWELL 1980). Strain construction was carried out using standard methods (SHERMAN, FINK and HICKS 1986) and in all cases involved multiple backcrosses. The cepl::URA? alleles have been described (BAKER and MASISON 1990). Both are disruption alleles where CEPl sequences have been deleted and replaced with URA3; their cepl phenotypes are indistinguishable. A Uraderivative of cePl::URA3-10, designated cepl::ura3, was obtained by selecting for 5-fluoro-orotic acid resistance. The trpl::LEU2 allele was obtained by gene transplacement using plasmid pELl1 as described (LOUIS and HABER 1989). Chromosome Z trisomy was introduced via strain VG3 1-1 1C of GUACCI and KABACK (1 99 1). First, CEPl was disrupted to obtain strain D77-Rl (Table l), then D77-R1 was mated with two different 38 1 G-derived parents to obtain trisomic strains D92 and D93. D92 and D93 are thus congenic with each other, but not with the other strains used in the study. All media were as described (BAKER and MASISON 1990) except for color indicator plates which were synthetic complete medium containing only 6 rg/ml adenine (% normal concentration). Selection for ura3 mutants was carried out with uracil dropout plates supplemented with 50 Pg/ml of uracil and 1.0 mg/ml of 5-fluoro-orotic acid (PCR, Inc., Gainesville, Florida) (BOEKE, LACROUTE and FINK 1984). Sporulation medium was 1% potassium acetate supplemented with adenine, histidine, lysine, tryptophan, tyrosine, leucine, uracil and methionine at one half their normal concentrations. All strains were grown at 30" except for sporulation which was carried out at 22". Yeast transformations were performed by the lithium acetate procedure (IT0 et al. 1983). Generation of chromosome fragments: Chromosome fragment CFIII(D8B.d.D30-18B) was generated by transforming strain D30-18B with NotI-cut pDM8 and selecting for uracil prototrophy (GERRING, CONNELLY and HIETER 199 1). Several of the transformants had the expected phenotype. They formed pink colonies with rare white sectors, the result of mitotic loss of the chromosome fragment. The Ura and color phenotypes cosegregated. The expected structure of CFIII(D8B.d.D30-18B) is a long arm consisting of the left arm of chromosome ZZZ distal to the D8B sequence, the centromere region from chromosome VI, and a short arm consisting of vector sequences, URA3 and add2p (SHERO et al. 1991). Its size is about 150 kbp and for convenience we have designated it CF(URA3). A LEU2 derivative of CF(URA3) was obtained by marker change generating CFIII(D8B.d.D30-18B.LEUZ). The marker change was accomplished by one-step gene disruption using the uraj::LEU2 disruption allele of plasmid pDM2. Strain D64-61A was transformed with NsiI-NdeI-cut pDM2 DNA selecting for leucine prototrophy. Among the transformants were several that were phenotypically Leu+ and Uraand formed pink colonies with rare white sectors; the Leu and color phenotypes cosegregated. For convenience, CFIII(D8B.d.D30-18B.LEU2) is referred to as CF(LEU2). Meiosis in Strains Lacking CP1 45

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تاریخ انتشار 2002