| Research Abstract |
1. Conservation of DnaA protein and DnaA-box in bacteria and their evolution (Yoshikawa): (1) Conservation of the combination of dnaA gene and DnaA-boxes was confirmed in Pseudomonas and Micrococcus in addition to E. coli and B. subtilis, suggesting its central role in initiation of chromosomal replication in bacteria. (2) We succeeded in cloning a dnaA homologue from Mycoplasma, cells containing the smallest genome, suggesting ubiquitous presence of DnaA protein in enbacteria. (3) A clone containing a possible dnaA homologue was obtained from yeast, a lower eukaryote.
2. Function of DnaA protein and regulation of its expression (Moriya): (1) Involvement of DnaA protein and DnaA-box in initiation of replication and its regulation was demonstrated using a dnaA-ts mutant cell of B. subthis. (2) DnaA protein was purified, and the mode of its specific binding to DnaA-box was analyzed in vitro. (3) An anti-DnaA antibody was prepared to determine cellular content of DnaA which determines the initiation frequency under certain physiological conditions. (4) In steady state growth, other factors seem to be limiting in the initiation in addition to DnaA.
3. Structure and function of replication machinery in B. subtilis (Ogasawara): (1) Structure of B. subtilis dnaG and dnaH genes were determined to identify them as homologous wtih E. coli dnaN and dnaZX, respectively. They code for two subunits of polymerase III. (2) Structure of a second gene for initiation of replication, dnaB, was determined. Its product protein was purified, and an antibody was prepared. So far, DNA binding activity of the protein was not detected in vitro.
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