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本文通过同源重组方法,构建了一系列单倍体缺失株:pho85△单缺失株YPH600、pho85△cln1△双缺失株YPH610、cln1△cln2△双缺失株YPH640和半乳糖诱导存活的pho85△chn1△cln2△(GAL1-10PHO85)三缺失株YPH630。比较不同缺失株的生长速度可以看出,PHO85单基因缺失比CLN1、CLN2双基因缺失对细胞生长速度的影响大。在去诱导的不同时间,取细胞进行光学显微镜形态学分析及DNA含量的流式细胞计量分析(FACS)。结果表明YPH630三缺失株的单倍体酵母细胞休止在G1期。 相似文献
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芽残酵母PHO85基因对细胞周期调控的影响 总被引:1,自引:0,他引:1
By homo-recombination with yeast intergrating plasmids, a serial of haploid mutants of budding yeast YPH499 had been constructed, which included pho85 delta strain YPH600, pho85 delta cln1 delta strain YPH610, cln1 delta cln2 delta strain YPH640 and galactose inducible strain YPH630 (pho85 delta cln1 delta cln2 delta (GAL1-10PHO85)). By analyzing the growth rate of different strains, we concluded that PHO85 gene have greater influence than CLN1 and CLN2 genes on cell growth control. After transferred from galactose media to glucose media, the tri-mutant cells collected at intervals were observed with microscope and analyzed by FACS. The results showed that the tri-mutant cells arrest in G1 phase when they were transferred to glucose media. 相似文献
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酵母PHO85结合蛋白PAP1基因的克隆和表达 总被引:3,自引:3,他引:0
酵母调探因了PHO85是一个依赖于细胞周期蛋白(cyclin)的蛋白酶(CDK),参与对细胞周期和酸性磷酸酯酶基因表达的调控。冯PHO85为靶分子,利用酵母的染色体基因文库中克隆到了一个新的与PHO85相结合的蛋白因子的基因,利用酵母双杂交(two-hybrid)系统从酵母的染色体基因文加中克隆到了一个新的与PHO85相结合的蛋白因子的基因,将此蛋白质命名为PAP1(PHO85associated 相似文献
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酵母PHO80基因的克隆,表达及功能分析 总被引:1,自引:0,他引:1
利用原位杂交方法从野生型酵母菌染色体DNA中克隆了PHO80基因,全长约4.2kb,包括1.1kb的上游序列和879bp的编码序列。以URA3基因为筛选标记,通过体内同源重组和营养互补筛选获得了PHO80基因缺失突变株。进一步研究了PHO80在细胞内的功能,它是酵母阻遏型酸性磷酸酯酶结构基因以及调控基因PHO81表达的负调控因子,但不影响PHO4和PHO85的表达。还构建了PHO80-LacZ融合基因,探索它在细胞内的表达规律。β-半乳糖苷酶活力测定结果表明,PHO80基因在细胞内呈低水平表达,并受自身产物和PHO85的抑制,推测它与PHO5和PHO81基因的调控模式可能相似。 相似文献
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酵母基因Pho85编码一个依赖于细胞周期蛋白 (cyclin)的蛋白激酶 (CDK) ,参与多种调控途径。PHO85功能的多效性归于其相关的细胞周期因子 ,现已经鉴定了 10个与PHO85相关的细胞周期因子 (PCL)。为了筛选PAP1 PHO85激酶复合物的特异底物 ,以PAP1为靶分子 ,利用酵母双杂交 (two hybrid)系统从酵母cDNA文库中克隆到一个与PAP1相互作用的蛋白质因子的基因 ,Ylr190w。Ylr190w编码 491个氨基酸的多肽链。体外翻译的YLR190w与纯化的融合蛋白GST PAP1可以被谷胱甘肽亲和柱共同吸附 ,这表明PAP1与YLR190w在体外也可以结合。用免疫沉淀获得的PAP1 PHO85复合物可以磷酸化在大肠杆菌中表达GST YLR190w ;并受到无机磷浓度影响 :高磷条件时磷酸化程度高 ,低磷条件时磷酸化程度低。它能与酵母细胞内YAF9结合 ,YAF9是人具有转录调控活性蛋白质因子AF9的酵母同源物。YLR190w与YAF9的相互作用受到磷条件影响 ,突变YLR190w蛋白S/TP位点的S和T后 ,它们的相互作用明显减弱 ,且不再受到磷条件影响 相似文献
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酵母PHO85基因的定点诱变和功能分析 总被引:2,自引:1,他引:1
克隆酵母PHO85基因,用URA3基因取代其部分编码序列,通过染色体同源重组的途径,构建PHO85缺失突变株。以细胞内酸性磷酸酯酶的活力水平显示PHO85的功能作用。完整PHO85基因转入缺陷型酵母菌,能使表达回复,位点专一诱变的PHO85基因(Gly13→ASP或LYS33→Glu)丧失此种功能。PHO85与CDC28编码的蛋白激酶有较高的同源性。PHO85的Gly13和Lys33相当于蛋白激酶 相似文献
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从酵母染色体DNA中获得1个约3.0kb的BamHI的克隆片段和1个约5.0kb的PstI片段,前者包含1745bp的PHO81基因编码序列及1244bp的上游序列,后者包含2236bp的编码序列及约2.8kb的下游序列,经拼接得到完整的PHO81基因。以URA3基因取代部分PHO81的编码序列,通过体内同源重组,获得PHO81基因缺陷的酵母细胞株。构建PHO81-LacZ融合基因,以β-半乳糖苷酶的活力表示PHO81基因的表达水平,研究了它的表达作用。PHO81基因为阻遏型表达,受无机磷浓度的控制,高磷使基因表达阻遏,低磷去阻遏。PHO81对其自身的表达有正调控作用,它与PHO5和PHO11基因的调控模式相似,但PHO81上游调控序列和PHO5及PHO11的同源性很低。 相似文献
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酵母pho80和pho85基因编码的蛋白质是阻遏型酸性磷酸酯酶基因表达调控系统中的2种负调控因子.其中pho85基因编码产物己被证明是1种类似于p34~(cdc2/CDC28)的蛋白激酶,磷酸化该调控系统中的正调控因子Pho4,并使之失活,用抗pho85抗体从啤酒酵母YPH499及其衍生菌株的细胞抽提液中得到Pho85免疫复合物,对大肠杆菌表达的Pho4蛋白进行了体外磷酸化分析,证实酵母Pho80是Pho85蛋白激酶活力所必需,pho80基因的表达水平直接影响Pho85免疫复合物对Pho4蛋白的磷酸化程度.根据基因序列推导的Pho80蛋白氨基酸序列中,含有一段与几种cyclin同源的区域,通过寡核苷酸的插入或小片段缺失而对该区域及邻近部位在基因水平进行的微小改变均可导致Pho80丧失阻遏酸性磷酸酯酶基因表达的能力. 相似文献
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探测了17种金属离子对嗜热菌BF80菌生长和降解苯酚的影响。结果表明:与对照相比, 0.01%的Cu2+、Zn2+、Co2+、Ba2+、Hg2+、Ni2+、Ag+ 和Al3+对嗜热菌BF80有强抑制作用; Cr2+对嗜热菌BF80的苯酚降解特性有强抑制作用, 而其生长量只受到一定的抑制作用; Sn2+、Fe2+、Fe3+和Pn2+ 对嗜热菌BF80的生长和苯酚降解有一定抑制作用, 该作用随金属粒子浓度的增加而增大; 低浓度Mn2+ 和Mo2+可以使其生长量增大且促进苯酚降解, 但超过0.1%的浓度则抑制其生长; Ca2+ 和Mg2+可以加速嗜热菌BF80的生长和降解苯酚的速率, 但对苯酚的最大降解率却几乎没有影响; Mo2+ 和Mn2+的复合作用使嗜热菌BF80的生长量更大, 但是苯酚降解率却比分别单独添加Mo2+ 和Mn2+时低。 相似文献
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芽殖酵母(Saccharomyces cerevisiae)和裂殖酵母(Schizosaccharomyces pombe)是用来研究异染色质形成、细胞周期、DNA复制等重要细胞功能的理想单细胞真核生物.本文主要介绍这2种酵母中异染色质形成的机制.异染色质是一种抑制基因转录和DNA重组的特殊染色质结构.尽管在芽殖酵母和裂殖酵母中异染色质形成都需要组蛋白修饰,但异染色质建立的机制不同.在芽殖酵母中参与异染色质形成的主要蛋白是Sir1-4蛋白(其中Sir2为组蛋白H3去乙酰化酶),而组蛋白H3赖氨酸9甲基化酶Clr4和异染色质蛋白Swi6在裂殖酵母异染色质形成中起关键的作用.在这两个酵母中,参与异染色质形成的组蛋白修饰蛋白由DNA结合蛋白招募到异染色质.此外,裂殖酵母也利用RNA干扰系统招募组蛋白修饰蛋白. 相似文献
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利用PHO81lacZ融合基因,对它的上游进行缺失分析,发现有两个区域对PHO81基因的表达是必需的:-401~-289bp和-1012~-801bp。比较PHO81和PHO5,PHO84基因上游,未发现有较高同源性的序列存在,但在-401~-289bp区域有PHO4蛋白结合位点的核心序列5′CACGTG/T3′,以及CACGTG/T两侧富含A/T的序列(可能是PHO2结合位点)。推测-401~-289bp包含了PHO81的上游激活序列(UAS),-1012~-801bp可能起增强的作用。用酵母总蛋白质对-1012~-801bp进行凝胶阻抑电泳分析,证明有未知的蛋白因子结合在这个区域。 相似文献
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Negative regulators of the PHO system in Saccharomyces cerevisiae: isolation and structural characterization of PHO85. 总被引:10,自引:1,他引:9
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One of the negative regulators of the PHO system of Saccharomyces cerevisiae, PHO85, has been isolated by transformation and complementation of a pho85 strain. The complementing activity was delimited within a 1258 bp DNA segment and this region has been sequenced. The largest open reading frame found in this region can encode a protein of 302 amino acid residues. A pho85 mutant resulted from disruption of the chromosomal counterpart of the open reading frame described above. Therefore, we concluded that the gene we have cloned is PHO85. This result also indicates that PHO85 is nonessential. Northern analysis revealed that the size of the PHO85 message is 1.1 kb. No similarity was found between the putative amino acid sequences of two negative regulators, the PHO80 and PHO85 proteins. 相似文献
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In yeast, the repression of acid phosphatase under high phosphate growth conditions requires the trans-acting factor PHO80. We have determined the DNA sequence of the PHO80 gene and found that it encodes a protein of 293 amino acids. The expression of the PHO80 gene, as measured by Northern analysis and level of a PHO80-LacZ fusion protein is independent of the level of phosphate in the growth medium. Disruption of the PHO80 gene is a non-lethal event and causes a derepressed phenotype, with acid phosphatase levels which are 3-4 fold higher than the level found in derepressed wild type cells. Furthermore, over-expression of the PHO80 gene causes a reduction in the level of acid phosphatase produced under derepressed growth conditions. Finally, we have cloned, localized and sequenced a temperature-sensitive allele of PHO80 and found the phenotype to be due to T to C transition causing a substitution of a Ser for a Leu at amino acid 163 in the protein product. 相似文献
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The Kluyveromyces lactis zymocin complex kills Saccharomyces cerevisiae cells in a process that involves tRNA cleavage by its tRNAse gamma-toxin subunit. In contrast to the gamma-toxin mode of action, the early steps of the zymocin response are less well characterized. Here, we present high-dosage suppressors of zymocin that encode a putative Pkc1-related kinase (ISR1) and UDP-glucose pyrophosphorylase (UGPase) (UGP1). Anti-UGPase Western blots and GAL10 - ISR1 overexpression suggest that zymocin suppression correlates with overproduction of UGPase or Isr1. As judged from protection against exo-zymocin and unaltered sensitivity to endogenous gamma-toxin, high-copy ISR1 and UGP1 operate in early, nontarget steps of the zymocin pathway. Consistent with a recent report on in vitro phosphorylation of Isr1 and UGPase by the CDK Pho85, high-copy ISR1 and UGP1 suppression of zymocin is abolished in a pho85 null mutant lacking CDK activity of Pho85. Moreover, suppression requires UGPase enzyme activity, and ISR1 overexpression also protects against CFW, a chitin-interfering poison. Our data agree with roles for UGPase in cell wall biosynthetic processes and for Isr1 in Pkc1-related cell wall integrity. In sum, high-copy ISR1 and UGP1 cells affect early steps of the zymocin response and potentially prevent the lethal K. lactis killer complex from establishing cell surface recognition and/or contact. 相似文献