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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.

细胞增殖调控的基本过程被认为在从酵母到人类的每一种真核生物中是共同的。在本项目中，我们分离了能补充酵母突变的高等真核基因，突变这些基因并表征基因产物在酵母中的行为。本研究的目的是通过检测该基因在细胞周期中的表达或利用特异性抗体定位基因产物来阐明它们在细胞增殖中的作用。将该基因与酵母GAL 1启动子融合，并导入酵母top2缺失突变体中。酵母菌能够在含有半乳糖的培养基中生长，但不能在葡萄糖中生长，这表明小鼠TOPII补充了酵母菌的top2突变。我们构建了各种C-末端缺失突变体，并在体外检测了这些酶的活性，通过特异性抗体染色和互补活性在酵母细胞中的定位。我们发现，322个氨基酸缺失突变体具有酶活性，但不互补，因为它们位于细胞质中。此外，我们还在酵母中筛选到了一个小鼠TOPII基因的温度敏感突变体。我们正计划将突变基因导入小鼠细胞系或小鼠体内，并利用PCR技术分离水稻cdc 2及其同源基因。将这些基因与酵母GAP启动子融合，并导入温度敏感突变体cdc 28中。他们的互补性很弱。