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种真核生物中都是共同的。在这个项目中，我们分离了能够补充酵母突变的高等真核基因，对这些基因进行突变，并对基因产物在酵母中的行为进行表征。本项目的目的是通过检测拓扑异构酶II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GAL1启动子融合，并导入酵母top2缺失突变体中。转化子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。我们构建了不同的C末端缺失突变体，并检测了它们的体外酶活性、特异性抗体染色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，但不具有补体活性，因为它们位于细胞质中。此外，我们还通过酵母筛选获得了一个小鼠TOPII基因的温度敏感型突变体。我们正计划将突变基因导入小鼠细胞系或体内。我们利用聚合酶链式反应技术分离了水稻cdc2和同源基因。将这些基因与酵母GAP启动子融合，导入温度敏感型cdc28突变体。它们的互补性很弱。

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.