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1.
The CDC13 gene encodes a protein that binds to the G-rich single-strand at yeast telomeres, and serves as a regulator of telomere replication. Cdc13 interacts with Est1 and DNA polymerase alpha, and cells carrying the temperature-sensitive allele cdc13-1 cannot complete telomere replication at the restrictive temperature and possess long telomeres. We attempted to isolate and characterize genes that interact with CDC13, in order to clarify the molecular mechanisms of telomere replication. A STM1 cDNA was isolated in a two-hybrid screen using CDC13 as a bait. The temperature-sensitive growth phenotype and the alteration in telomere size in cdc13-1 cells were corrected by introduction of the STM1 gene on a multicopy vector, but the extended G-rich single-strand overhangs which are also characteristic in the cdc13-1 mutant were not affected. Furthermore, we found that multiple copies of SGS1, a gene encoding a helicase that can unwind guanine quadruplexes, inhibited suppression of the cdc13-1 phenotype by STM1. We also demonstrate that a fusion protein consisting of the N-terminal region of Cdc13 and the C-terminal region of Stm1 (which shows similarity to the beta-subunit of the telomere binding complex in Oxytricha) could complement a cdc13 disruptant. Although STM1 itself is not essential for telomere replication, our findings suggest that STM1 genetically interacts with CDC13 to maintain telomere structure. 相似文献
2.
《Cell cycle (Georgetown, Tex.)》2013,12(21):3428-3439
Distinguishing telomeres from DNA double strand breaks is critical for genome stability. In S. cerevisiae, the Cdc13 single-strand telomere binding protein is critical for protecting chromosome ends. The C-rich telomere strand is lost at high temperatures in cdc13-1 strains, leading to activation of the DNA damage checkpoint and cell inviability. Through a screen performed to identify activities involved in telomere C-strand loss, we identified two new rad24 alleles. Rad24 is an alternate Rfc1 subunit, functioning to load the 9-1-1 checkpoint clamp. In each rad24 allele, a transposon inserted within the RAD24 coding region leads to expression of different carboxyl-terminal portions of Rad24, deleting or truncating the amino-terminus. We show that an intact Rad24 amino-terminus is necessary for its checkpoint function. Interestingly, the initial cdc13-1 rad24-2 strains grew at 36Ã?Â?Å¡C, but the extent of suppression associated with rad24-2 weakened in serial backcrosses, and cdc13-1 segregants from these crosses showed a modest increase in temperature resistance. Moreover, while a RAD24 plasmid suppressed the checkpoint defect in the initial cdc13-1 rad24-2 strain, the temperature resistance was only partially suppressed. These data suggest that the TG1-3 amplification observed in this strain contributes to the suppression phenotype. By recreating the rad24-2 allele in a strain with normal telomeres, we find that, relative to the rad24-Ã?¢Ã?Â?†allele, rad24-2 increases the frequency of obtaining cdc13-1 cells capable of growth at high temperatures. Our hypothesis is that the Rad24-2 truncation protein affects telomere structure or recombination in a manner distinct from rad24-Ã?¢Ã?Â?†. 相似文献
3.
Addinall SG Holstein EM Lawless C Yu M Chapman K Banks AP Ngo HP Maringele L Taschuk M Young A Ciesiolka A Lister AL Wipat A Wilkinson DJ Lydall D 《PLoS genetics》2011,7(4):e1001362
To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology. 相似文献
4.
CDC17: an essential gene that prevents telomere elongation in yeast 总被引:26,自引:0,他引:26
The CDC17 gene product performs an essential stage-specific function during the Saccharomyces cerevisiae cell cycle. When cdc17-1 strains are grown at the maximum permissive temperature, recombination is induced preferentially in the genetic interval of the chromosome closest to the telomere. Telomeres are longer in cdc17 strains than in CDC17 strains at the permissive temperature because of addition of sequence near or in the poly (C1-3A) telomeric DNA and become even longer when cells are propagated at elevated temperatures. The mitotic recombination events require RAD52 function, but telomere growth does not. Long telomeres are maintained for many generations when crossed into a CDC17+ background, suggesting that telomere length is largely conserved during replication. The altered telomere length phenotype of cdc17 mutations is recessive and coreverts and cosegregates with the temperature-sensitive lethal phenotype. 相似文献
5.
Involvement of cdc13+ in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules. 总被引:45,自引:17,他引:28
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Previous genetic studies have shown that the fission yeast cdc13+ gene product interacts closely with the cdc2+ protein kinase during mitosis. Here, we have cloned the cdc13+ gene from a S. pombe gene bank by complementation of the temperature-sensitive defect of a cdc13-117 mutant strain. The complementing activity was localized to a 1.9-kb XbaI-NsiI DNA fragment, and nucleotide sequencing revealed a 1446-bp open reading frame. The predicted amino acid sequence contained 482 residues and was not homologous to any protein in a protein database. The cdc13+ gene function was confirmed to be essential for cell division since cells carrying a cdc13 null allele arrested with a cdc phenotype. However, unlike any existing temperature-sensitive cdc13 mutants, cdc13 null mutants arrested in G2 without septa or condensed chromosomes indicating that cdc13+ gene function is required at or prior to the initiation of mitotis. cdc13-117 mutant strains were found to be hypersensitive to the tubulin inhibitor thiabendazole. This observation suggests that the cdc13+ gene product, which is required for mitotic initiation, may interact with microtubules. 相似文献
6.
Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13 总被引:8,自引:0,他引:8
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In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends. Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance. Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13. A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells. Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed. Several ten1 mutations were found to confer telomerase-dependent telomere lengthening. Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint. These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes. We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA. 相似文献
7.
A Temperature-Sensitive Mutation of the Temperature-Regulated Serh3 I-Antigen Gene of Tetrahymena Thermophila: Implications for Regulation of Mutual Exclusion
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The Ser genes of Tetrahymena thermophila specify alternative forms of a major cell surface glycoprotein, the immobilization or i-antigen (i-ag). Regulation of i-ag expression assures that at least one i-ag gene is expressed at all times. To learn more about the regulatory system and the possible role of i-ag itself, we studied SerH3-ts1, a temperature-sensitive allele of the temperature-regulated SerH3 gene normally expressed from 20-36°. In homozygotes grown at the nonpermissive temperature (>32°), H3 is not present on the cell surface, but the gene continues to be transcribed until its 36° cutoff. H3 formed at the permissive temperature is stable at nonpermissive temperatures, indicating that SerH3-ts1 is temperature-sensitive for synthesis rather than function. At nonpermissive temperatures, the S i-ag is expressed in place of H3. This result suggests that normal H protein may play a role in regulating S expression. SerH3-ts1 was isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sequencing of SerH3-ts1 revealed a single A -> G transition at nucleotide 473, resulting in the substitution of glycine for aspartate. The affected residue is conserved in the internal repeats comprising the H protein, and the charge difference correlates with changes in electrophoretic mobility of the H3 protein. 相似文献
8.
tsAF8 and ts13 cells are temperature-sensitive (ts) mutants of BHK cells that specifically arrest, at nonpermissive temperature, in the G1 phase of the cell cycle. These two mutants can complement each other. Both cell lines can be made quiescent by serum deprivation (G0). When subsequently stimulated by serum, they can enter S phase at 34 degrees C but not at 39.5 degrees-40.6 degrees C. We have used these mutants to determine whether the nucleus is needed during the G0 leads to S transition for the expression of the G1 ts functions. For this purpose, we fused cytoplasts of G0-tsAF8 with whole ts13 cells in G0, and cytoplasts of G0-ts13 with whole tsAF8 cells in G0. Serum stimulation at the nonpermissive temperature induced DNA synthesis in both types of such fusion products. No DNA synthesis was induced by serum stimulation at the nonpermissive temperature in fusion products constructed between either G0-tsAF8 cytoplasts and whole G0-tsAF8 cells or G0-ts13 cytoplasts and whole G0-ts13 cells. These results demonstrate that the information for these two ts functions, which are required for entry of serum-stimulated cells into the S phase, are already present in the cytoplasm of G0 cells--that is, before serum stimulation commits them to the transition from the nonproliferating to the proliferating state. 相似文献
9.
A genomewide suppressor and enhancer analysis of cdc13-1 reveals varied cellular processes influencing telomere capping in Saccharomyces cerevisiae
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Addinall SG Downey M Yu M Zubko MK Dewar J Leake A Hallinan J Shaw O James K Wilkinson DJ Wipat A Durocher D Lydall D 《Genetics》2008,180(4):2251-2266
In Saccharomyces cerevisiae, Cdc13 binds telomeric DNA to recruit telomerase and to "cap" chromosome ends. In temperature-sensitive cdc13-1 mutants telomeric DNA is degraded and cell-cycle progression is inhibited. To identify novel proteins and pathways that cap telomeres, or that respond to uncapped telomeres, we combined cdc13-1 with the yeast gene deletion collection and used high-throughput spot-test assays to measure growth. We identified 369 gene deletions, in eight different phenotypic classes, that reproducibly demonstrated subtle genetic interactions with the cdc13-1 mutation. As expected, we identified DNA damage checkpoint, nonsense-mediated decay and telomerase components in our screen. However, we also identified genes affecting casein kinase II activity, cell polarity, mRNA degradation, mitochondrial function, phosphate transport, iron transport, protein degradation, and other functions. We also identified a number of genes of previously unknown function that we term RTC, for restriction of telomere capping, or MTC, for maintenance of telomere capping. It seems likely that many of the newly identified pathways/processes that affect growth of budding yeast cdc13-1 mutants will play evolutionarily conserved roles at telomeres. The high-throughput spot-testing approach that we describe is generally applicable and could aid in understanding other aspects of eukaryotic cell biology. 相似文献
10.
CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast. 总被引:9,自引:2,他引:7
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Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit. 相似文献
11.
Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210
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J R Hamaguchi R A Tobey J Pines H A Crissman T Hunter E M Bradbury 《The Journal of cell biology》1992,117(5):1041-1053
The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells. 相似文献
12.
In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components. 相似文献
13.
Summary Among temperature-sensitive mutants which were defective in septum formation and formed nonseptate filaments at nonpermissive temperatures three (ts31, ts341, ts526) were identified among 434 temperature-sensitive mutants isolated at random from a mutagenized population of Bacillus subtilis 168. The results of morphological observations and characterization of these mutants showed that ts31 and ts341 were septum-initiation mutants and that ts526 was a DNA elongation mutant. The above mutations, and other mutations affecting septum initiation (div355 and tms12) were mapped by PBS1-mediated transduction on the chromosome in three separate regions as follows: pur A16-ts526-div355-cysA14; metC3-(ts31, tms12)-pyrD1-recA1; ebr-2-ts341-uvrA1-hisA1-cysB3. Our results suggest that the initiation process of septum formation requires at least four kinds of gene product. In addition, the sesult obtained with ts526 suggests an intimate connection between septum initiation and DNA replication. 相似文献
14.
Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae. 总被引:2,自引:1,他引:1
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To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length. 相似文献
15.
Identification of Residues in Fission Yeast and Human P34(cdc2) Required for S-M Checkpoint Control
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In fission yeast, regulation of p34(cdc2) plays an important role in the checkpoint coupling mitosis to completion of DNA replication. The cdc2 mutations cdc2-3w (C67Y) and cdc2-4w (C67F) abolish checkpoint control without seriously affecting normal cell proliferation. However the molecular basis of this phenotype is not known. To better understand the role of p34(cdc2) in checkpoint control, we have screened for more mutations in Schizosaccharomyces pombe cdc2 with this phenotype. We have isolated cdc2-3w and cdc2-4w, as well as three new cdc2 alleles: cdc2-6w (N66I), cdc2-7w (E8V) and cdc2-8w (K9E). The altered residues map to two different regions on opposite faces of the protein, suggesting that the interaction between p34(cdc2) and components of the checkpoint pathway may be complex. In contrast to cdc2-3w and cdc2-4w, the new mutations alter residues that are conserved between the fission yeast cdc2(+) and other cdks, including the human CDC2 protein. Expression of the equivalent human CDC2 mutants in fission yeast abolishes checkpoint control, suggesting that these residues could be involved in checkpoint-dependent regulation of other eukaryotic cdks. 相似文献
16.
Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae. 总被引:49,自引:8,他引:41
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cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision. 相似文献
17.
Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae. 总被引:14,自引:8,他引:6
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M Wittekind J Dodd L Vu J M Kolb J M Buhler A Sentenac M Nomura 《Molecular and cellular biology》1988,8(10):3997-4008
18.
Stf1: Non-Wee Mutations Epistatic to Cdc25 in the Fission Yeast Schizosaccharomyces Pombe 总被引:1,自引:1,他引:0
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In Schizosaccharomyces pombe, cdc25 is a cell cycle regulated inducer of mitosis. wee1 and phenotypically wee alleles of cdc2 are epistatic to cdc25. Mutant alleles of a new locus, stf1 (suppressor of twenty-five), identified in a reversion analysis of conditionally lethal cdr1-76 cdc25-22 and cdr2-96 cdc25-22 double mutant strains, also suppress both temperature-sensitive and gene disruption alleles of cdc25. These mutants, by themselves, are phenotypically indistinguishable from wild type strains; hence they represent the first known mutations that are epistatic to cdc25 and do not display a wee phenotype. stf1 genetically interacts with other elements of mitotic control in S. pombe. stf1-1 is additive with wee1-50, cdc2-1w and cdc2-3w for suppression of cdc25-22. Also, like wee1- and cdc2-w, stf1- suppression of cdc25 is reversed by overexpression of the putative type 1 protein phosphatase bws1+/dis2+. Interaction with various mutants and plasmid overexpression experiments suggest that stf1 does not operate either upstream or downstream of wee1. Similarly, it does not operate through cdc25 since it rescues the disruption. stf1 appears to encode an important new element of mitotic control. 相似文献
19.
p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. 总被引:66,自引:27,他引:39
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cdc2+ encodes a protein kinase that is required during both G1 and G2 phases of the cell division cycle in fission yeast. suc1+ is an essential gene that was originally identified as a plasmid-borne sequence that could rescue certain temperature-sensitive cdc2 mutants. To investigate the role of the suc1+ gene product in the cell cycle p13suc1 has been expressed in Escherichia coli and purified. An immunoaffinity purified anti-p13suc1 polyclonal serum has been prepared and used to identify p13suc1 in fission yeast. The abundance of this protein did not alter either during the cell cycle or during entry into stationary phase. p13suc1 was found in yeast lysates in a complex with the cdc2+ gene product. Approximately 5% of cellular p34cdc2 was associated with p13suc1, and this fraction of p34cdc2 was active as a protein kinase. The stability of the complex was disrupted in yeast strains carrying temperature-sensitive alleles of cdc2 that are suppressible by overexpression of suc1+. The level of association between p13suc1 and p34cdc2 was not affected by cell cycle arrest in adverse nutritional conditions. p13suc1 is not a substrate of the p34cdc2 protein kinase. We propose instead that it acts as a regulatory component of p34cdc2 that facilitates interaction with other proteins. 相似文献
20.
Fission yeast with DNA polymerase delta temperature-sensitive alleles exhibits cell division cycle phenotype. 总被引:9,自引:0,他引:9
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DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences. 相似文献