Expression and localization of the gene products necessary for cell proliferation

细胞增殖所需基因产物的表达和定位

基本信息

批准号：
04680254
负责人：
KIKUCHI Yoshiko
金额：
$ 1.28万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04680254/
关键词：
yeast ts mutants mouse TopoII complementation mutant isolation rice cdc2 マウスTOPII マウス遺伝子変異体 cdc2キナーゼ DNAトポイソメラーゼII 発現制御細胞内局在化

项目摘要

Fundamental processes of the regulation in cell proliferation are believed to be common in every eukaryote from yeast to humans. In this project, we isolate higher eukaryotic genes which can complement yeast mutations, mutate those genes and characterize the behavier of the gene products in yeast. The purpose of this project is to clarify their roles in cell proliferation by examining the expression of the gene in the cell cycle or localization of the gene products by using the specific antibodies.Mouse cDNA encoding topoisomeraseII was isolated. The gene was fused to the yeast GAL1 promoter and was introduced into yeast top2 deletion mutant. The transformant was able to grow in the medium containing galactose but not in glucose, indicating that the mouse TOPII complemented the yeast top2 mutation. We constructed various C-terminal deletion mutants and examined those enzymatic activities in vitro, localization in yeast cells by staining with specific antibodies and the complementation activities. We found that the 322 amino acid deletion mutant possessed the enzymatic activity but did not complement, because they were located in the cytoplasm. Furthermore, we isolated a temperature sensitive mutant of mouse TOPII gene by selecting in yeast. We are planning an introduction of the mutant gene into mouse cell lines or bodies.We isolated rice cdc2 and cognate genes by using PCR techniques. Those genes were fused to yeast GAP promoter and introduced to temperature sensitive cdc28 mutant. They complemented weakly.

人们认为，从酵母到人类的每个真核生物中，细胞增殖调节的基本过程都是常见的。在这个项目中，我们分离了较高的真核基因，这些真核基因可以补充酵母突变，突变这些基因并表征酵母中基因产物的行为。该项目的目的是通过使用特定的抗体在细胞周期或基因产物的定位中检查基因的表达来阐明其在细胞增殖中的作用。分离了编码拓扑异构体的小鼠cDNA。该基因被融合到酵母Gal1启动子上，并被引入酵母Top2缺失突变体中。转化剂能够在含半乳糖的培养基中生长，但不能在葡萄糖中生长，表明小鼠topii补充了酵母Top2突变。我们构建了各种C末端缺失突变体，并在体外检查了这些酶活性，通过用特定抗体染色和互补活性在酵母细胞中定位。我们发现322个氨基酸缺失突变体具有酶活性，但没有补体，因为它们位于细胞质中。此外，我们通过在酵母中选择了小鼠topii基因的温度敏感突变体。我们计划将突变基因引入小鼠细胞系或身体。我们使用PCR技术分离了水稻CDC2和同源基因。这些基因被融合到酵母间隙启动子上，并引入温度敏感的CDC28突变体。他们补充弱。