Transposition mechanisms and biotechnology application of the medaka fish To12 transposable element

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The first three years of the five-year research term were devoted to basic research on the transposition mecchanisms of the Tol2 element. The following results were obtained : (1)Portions of the 4.7-kb element esstential for the transposition reaction were detected. (2)An autonomous Tol2 copy, carrying a gene for the transposase, was identified. (3)An mRNA for the transposase was isolated from medaka fish cells. (4)De novo insertion in medeka fish cells was detected, with a newly devised experimental system including several bacterial drug-resistance genes. (5)Target site duplications were proved to be exactly 8 bp with no preference for specific sequences or nucleotides.In the fourth year, the research was expanded to include biotechnology applications. The results were as follows : (1)Addition of a nuclear localization signal to the transposase led to a three times higher transposition frequency. (2)A Tol2 copy carrying the GFP gene as a marker gene was successfully integrated into the medaka fish chromosomes. (3)The integration occurred in the germline cells, and the inserted copy was inherited to subsequent generations. (4)Gene transfer was also successful in zebrafish. (5)Tol2 was demonstrated to transpose in human and mouse cells in the same manner as that in fish. (6)An extranuclear localization signal was suggested to be present in the transposase protein. (7)One main signal was suggested to be present at about the center of the primary structure of the transposase protein. (8)X-ray irradiation was found to induce transposition at a high frequency.