Study on Nuclear Protein Involved in DNA Replication of Yeast

酵母DNA复制相关核蛋白的研究

• 批准号: 01560125
• 负责人: TSUCHIYA Eiko
• 金额: $ 1.09万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01560125/
• 关键词: S. cerevisiae; DNA replication; Nuclear protein; Cell cycle control; Sporulation; DNA-binding protein; Protein kinase; プロテインキナ-ゼ; Saccharomyces cerevisial

I have cloned the gene, NPS1, which encodes a novel nuclear protein involved in cell cycle regulation in yeast Saccharomyces cerevisiae. DNA sequence of 5,211 bp yeast genomic DNA fragment containing NPS1 was determined. In this fragment, one possible open deading frame composed of 4,077 bp was existed. The sequence was searched for similarity on DNA and protein databases, and NPS1 was found to be a novel gene.NPS1 transcript was found as 4-5 Kb mRNA in total RNA preparation from exponentially growing yeast cells. On the other hand, little transcript was detected in stationary-phase cells. One step gene disruption was carried out on NPS1, and the gene was found to be essential for yeast cell growth. A kimeric gene which have 0.8 Kb GAL1 promoter/enhancer sequence at 65 bp upstream of first ATG of NPS1 was constructed and transformed to DELTAnps1/NPS1 diploid. A haploid DELTAnps1 conditional lethal strain, YET101, was obtained from the meiotic segregants of the above diploid and phenotyopes of YET101 in the absence of NPS1 transcription was studied. YET101 stopped its growth in glucose medium and most of the cells in this medium was found to have a mononucleated large bud. The result suggested that NPS1 is involved in S to M phase control in cell division cycle. MATa/MATalpha, DELTAnps1/DELTAnpsl diploid carint pGAL1 : : NPS1 plasmid grew normally in galactose medium, but formed little spore in 1% K-asetate. NPS1 was found to play an important role in sporulating process.In order to analyze the function of NPS1 product, I prepared antibody against nps1 by immunizing mice with purified beta-galactosidase-nps1 fusion protein. Immunofluorecent staining of yeast cells with anti-nps1 antibody revealed the localization of nps1 in nucleus. Affinity purified nps1 possessed both DNA-binding- and protein kinase activities.

我克隆了 NPS1 基因，它编码一种参与酿酒酵母细胞周期调节的新型核蛋白。确定了含有 NPS1 的 5,211 bp 酵母基因组 DNA 片段的 DNA 序列。在该片段中，存在一个可能的由4,077 bp组成的开放死亡框架。在 DNA 和蛋白质数据库中搜索该序列的相似性，发现 NPS1 是一个新基因。在指数生长的酵母细胞制备的总 RNA 中，发现 NPS1 转录物为 4-5 Kb mRNA。另一方面，在稳定期细胞中检测到很少的转录物。对 NPS1 进行一步基因破坏，发现该基因对于酵母细胞生长至关重要。构建了NPS1第一个ATG上游65 bp处具有0.8 Kb GAL1启动子/增强子序列的kimeric基因，并将其转化为DELTAnps1/NPS1二倍体。从上述二倍体的减数分裂分离子中获得单倍体DELTAnps1条件致死菌株YET101，并研究了在NPS1转录缺失的情况下YET101的表型。 YET101在葡萄糖培养基中停止生长，并且发现该培养基中的大多数细胞具有单核大芽。结果表明NPS1参与细胞分裂周期S到M期的控制。 MATa/MATα、DELTAnps1/DELTAnpsl 二倍体 carint pGAL1 : : NPS1 质粒在半乳糖培养基中生长正常，但在 1% K-asetate 中形成小孢子。研究发现NPS1在孢子形成过程中发挥着重要作用。为了分析NPS1产物的功能，我用纯化的β-半乳糖苷酶-nps1融合蛋白免疫小鼠，制备了针对nps1的抗体。用抗 nps1 抗体对酵母细胞进行免疫荧光染色，揭示了 nps1 在细胞核中的定位。亲和纯化的 nps1 具有 DNA 结合活性和蛋白激酶活性。

项目成果

E.Tsuchiua: "Identification of a wheatーgerm agglutinin sensitive ATPase in yeast nuclei" FEBS Letters. 250. 285-288 (1989)

E.Tsuchiua：“酵母细胞核中小麦胚芽凝集素敏感 ATP 酶的鉴定”FEBS Letters 250. 285-288 (1989)。

E.Tsuchiya: "A 70-Kd nuclear protein involved in yeast DNA synthesis:Immunological identification and its analogy to hsp70" J.Bacteriol.

E.Tsuchiya：“参与酵母 DNA 合成的 70-Kd 核蛋白：免疫学鉴定及其与 hsp70 的类比”J.Bacteriol。

E.Tsuchiya: "Molecular cloning of NPS1,a novel CDC gene controlling S phase in Saccharomyces cerevisiae"

E.Tsuchiya：“NPS1 的分子克隆，一种控制酿酒酵母 S 期的新型 CDC 基因”

E.Tsuchiya: "A 70ーKd nuclear protein involved in yeast DNA synthesis Immunological identification and its analogy to hsp70" Biochem.Biophys.Res.Commn.

E.Tsuchiya：“参与酵母 DNA 合成的 70-Kd 核蛋白免疫学鉴定及其与 hsp70 的类比”Biochem.Biophys.Res.Commn。

E.Tsuchiya: "Identification of a wheat germ agglutinin-sensitive ATPase in yeast nuclei" FEBS Letters. 250. 285-288 (1989)

E.Tsuchiya：“酵母细胞核中小麦胚芽凝集素敏感 ATP 酶的鉴定”FEBS Letters。