Expression and localization of the gene products necessary for cell proliferation

细胞增殖调控的基本过程被认为在从酵母到人类的每一个真核生物中都是共同的。在这个项目中，我们分离出可以补充酵母突变的高级真核基因，使这些基因突变，并表征基因产物在酵母中的行为。本项目的目的是通过检测基因在细胞周期中的表达或使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。分离了小鼠拓扑异构酶ⅱ的cDNA编码。将该基因与酵母GAL1启动子融合，导入酵母top2缺失突变体。该转化体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top2突变。我们构建了多种c末端缺失突变体，并在体外检测了这些突变体的酶活性，用特异性抗体染色检测了它们在酵母细胞中的定位和互补活性。我们发现322个氨基酸缺失突变体具有酶活性但不补体，因为它们位于细胞质中。此外，我们还通过酵母筛选分离到了小鼠TOPII基因的温度敏感突变体。我们正计划将突变基因引入小鼠细胞系或机体。利用PCR技术分离水稻cdc2及其同源基因。这些基因被融合到酵母GAP启动子中，并被引入到温度敏感的cdc28突变体中。他们无力地互相补充。

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.