Expression and localization of the gene products necessary for cell proliferation

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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.