Toward understanding of regulation of mRNA capping and functions of the cap structure.

旨在了解 mRNA 加帽的调节和帽结构的功能。

• 批准号: 06680673
• 负责人: YAMAGISHI Masahiro
• 金额: $ 1.41万
• 依托单位: Aichi Cancer Center (1995)National Institute of Genetics (1994)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680673/
• 关键词: Capping; Yeast; Guanylyltransferase; Temperature-sensitive mutants; キャッピング; 温度感受性変異株

The yeast mRAN capping enzyme is composed of alpha and beta subunits possessing guanylyltransferase and triphoshatase activities, respectively. In this study, we have isolated 10 recessive temperature-sensitive mutations of the CEG1 gene encoding the alpha subunit and determined the mutation sites. Nine of the mutations(ceg1-1 to ceg1-9)were isolated on a single-copy plasmid-and the remaining one(ceg1-10)on a multi-copy plasmid. The presence of ceg1-10 in multiple copies is essential for the viability of cells carrying the mutation, and a shift to the restrictive temperature resulted in quick growth arrest of ceg1-10 cells while other mutants decreased growth rates gradually upon the temperature up-shift. Intragenic complementation was not observed for pairwise combinations of the mutations.Guanylyltransferase activity was examined for each mutant protein by assaying of a covalent Ceglp-GMP complex formation. While most of the mutant protein showed clear heat-lability, Ceg1-8p and Ceg1-9p maintained substantial activities during incubation at a high temperature. Further characterization was carried out for isolated mutant proteins expressed in E.coli cells.Isolation of extragenic suppressors as well as detailed analyzes of the ceg1 mutant cells are in progress currently.

酵母mRAN加帽酶由分别具有鸟苷酰转移酶和三磷酸酶活性的α和β亚基组成。在这项研究中，我们已经分离出10个隐性温度敏感突变的CEG 1基因编码的α亚基，并确定突变位点。9个突变（ceg 1 -1到ceg 1 -9）是在单拷贝质粒上分离的，剩下的一个（ceg 1 -10）是在多拷贝质粒上分离的。ceg 1 -10以多个拷贝的形式存在对于携带突变的细胞的生存力是必不可少的，并且向限制性温度的转变导致ceg 1 -10细胞的快速生长停滞，而其他突变体在温度升高时逐渐降低生长速率。通过测定共价Ceglp-GMP复合物的形成，检测了每个突变蛋白的鸟苷酰转移酶活性。虽然大多数突变蛋白显示出明显的热不稳定性，Ceg 1 - 8 p和Ceg 1 - 9 p在高温下孵育期间保持了大量的活性。对ceg 1突变体蛋白在大肠杆菌细胞中的表达进行了进一步的鉴定，目前正在进行基因外抑制子的分离以及ceg 1突变体细胞的详细分析。

项目成果

M.Yamagishi: "Isolation and characterization of mutants for mRNA capping enzyme in yeast.(Japaniese)" Natl.Inst.Genet.Ann.Report. 45. 15-16 (1995)

M.Yamagishi：“酵母中 mRNA 加帽酶突变体的分离和表征。（日语）”Natl.Inst.Genet.Ann.Report。

山岸正裕: "酵母mRNAキャッピング酵素変異体の単離と解析" 遺伝学研究所年報. 45. 15-16 (1995)

Masahiro Yamagishi：“酵母mRNA加帽酶突变体的分离和分析”遗传学研究所年度报告45. 15-16 (1995)。

M.Yamagishi: "Yeast mRNA capping enzyme temperature-sensitive mutants : Isolation of the mutations and characterization of the altered enzymes." Aichi Cancer Center Res.Inst.Sci.Report. 14(in ress). (1996)

M.Yamagishi：“酵母 mRNA 加帽酶温度敏感突变体：突变的分离和改变的酶的表征。”

Masahiro Yamagishi: "Yeast mRNA capping enzyme temperature-sensitive mutants: Isolation of the mutations and characterization of the altered enzymes." Aichi Cancer Center Research Institute Scientific Report. 14(印刷中). (1996)

Masahiro Yamagishi 酶：“酵母 mRNA 加帽温度敏感突变体：突变酶的分离和特征分析。”爱知癌症中心研究所科学报告 14（出版中）。

Masahiro Yamagishi: "Isolation of temperature Sensitive mutants for mRNA capping enzyme in Saccharomyces cerevisiae." Mol. Gen. Genet.249. 147-154 (1995)

Masahiro Yamagishi：“酿酒酵母 mRNA 加帽酶温度敏感突变体的分离。”