| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
The H1 histone kinase was purified from mouse mammary carcinoma cell line FM3A cells by use of several steps including ammonium sulfate precipitation, mono Q, hydroxylapatite, superose 12 and mono S coulumn chromatography. The-S1 peptide which contained the sequence of H1 histone phosphorylated at mitosis, was used as substrate. The purified enzyme was the same one as murine homolog to yeast CDC2^+ kinase. This was proven by immunological methods. This H1 histone/cdc2 kinase could phosphorylate specifically H1 histone and recognized Thr(Ser)-Pro-X-Lys sequence, that was proven by use of several synthetic peptides. The activity of H1 histone/cdc2 kinase was increased at G2/M phase and the activation was seemed to be regulated by the dephophorylation of the phosphorylated tyrosine residue of it. Furthermore, Okadaic acid, which was potent inhibitor of phophatase 1 and 2A, could activated the cdc2 kinase in vivo at G1/S and S phase, and it could inactivate same kinase in vivo at mitosis. This suggest that both activation and inactivation of this enzyme are requlated by the protein phosphorylation cascade. Next, it was proven that cdc2 kinase was necessary enzyme for the cells t
o enter mitosis by use of temperature sensitive mutant tsFT210 cells. The tsFT210 cells could grow at 33^ﾟC but could not grow at 390C. The cells were arrested at G2/M phase at 39^ﾟC.The activity of cdc2 kinase in vitro was temperature sensitive, compared to wild type enzyme. The the cDNA of cdc2 kinase was amplified by PCR method, and sequenced. One point mutation, C to T Change, was found in this mutant at the 905th base from the initial A in ATG codon. This mutation caused a change of proline to serine in the carboxyl teminal region of this enzyme. The wild type cDNA could com
pensate the temperature sensitivity of this mutant by the transfection into the mutant, it was clear that the mutant cells could not enter nitosis because of the defect of cdc2 kinase.
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