| Co-Investigator(Kenkyū-buntansha) |
MURAMATSU Masa-aki Institute of Medical Science, The University of Tokyo Assistant Professor, 医科学研究所, 教務職員 (50230008)
MASAI Hisao Institute of Medical Science, The University of Tokyo Assistant Professor, 医科学研究所, 助手 (40229349)
NAKAYAMA Naoki Institute of Medical Science, The University of Tokyo Assistant Professor, 医科学研究所, 助手 (80227967)
YOKOTA Takashi Institute of Medical Science, The University of Tokyo Associate Professor, 医科学研究所, 助教授 (50134622)
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| Research Abstract |
We intended to clarify the intracellular mechanisms of the cell cycle transition from G1 to S. 1) Control of replication in E. coli : G1 to S phase transition is represented by the initiation of chromosomal DNA replication. The new DNA strand synthesis in E. coli, the most thoroughly studied organism, is initiated by the action of primosome, a protein complex essential for priming DNA templates for DNA polymerase. We demonstrated that two types of primosomes, phiXI74 type (priA-dependent) and ABC type (dnaA-dependent), are functionally interchangeable in the ColEl replication. Furthermore, replication of F, R6K, and Rstl plasmids requires dnaA but not priA, whereas recA-dependent and dnaA-independent stable replicafion in E coli requires priA. We proposed that there are two types of replicons in E. coli, one dependrnt on the ABC primosome, and the other dependent on the phiXI74 type primosome. 2) GI to S transition in yeast : Mating pheromone is a yeast peptide that inhibits the initia
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tion of DNA replicafion, thereby arrests the cell cycle of target cells at Gl phase. We have previously demonstrated that its effect is exerted through a specific membrane receptor and a Gprotein. We further showed that a beta-gamma fusion polypeptide is as active as the natural beta/gamma subunit of the G-protein, so that, as a complex, both subunit are the key elements to introduce the mating pheromone signal. We have been looking for proteins that interact with the core sequence of putative chromosomal replication origins of yeast, which are likely to be the initiation factors for DNA replication. We have found several proteins and purified one of them, 100kD protein, to homogeneity. The CDC7 kinase is implicated to be involved in the initiation of DNA replication in yeast. We have constructed an overproducer of the CDC7 protein, and made antibodies against CDC7. Purification and characterization of these proteins are underway. 3) G1 to S transition in hematopoietic cells : Cytokines are a set of polypeptides that are mainly secreted from activated T cells, and act on various types of cells including hematopoietic cells. They not only support cell's viability and cell cycle progression from Gl to S, but also promote subsequent differentiation. Our research has been focusing on the role of IL-3 and GM-CSF on hematopoietic progenitor cells. Both act on very early progenitor cells to stimulate their proliferation and differentiation. Such similarities in the effects of these factors can be explained by our recent finding that receptors for both cytokines are composed of two polypeptides, alpha and beta, of which beta chain is shared. Analysis of the downstream events initiated upon binding of these cytokines to their receptors are underway. T cells are largely in GO/GI phase during the cell cycle. Upon the activation by antigen, they secrete a number of cytokines including IL-3 and GM-CSF, then they start to initiate DNA replication. We have been analyzing promoter Less
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