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

五年研究期的前三年致力于Tol2元素转位机制的基础研究。得到以下结果：(1)检测到转位反应必需的4.7 kb元素的部分。(2)发现了一个携带转座酶基因的自主Tol2拷贝。(3)从medaka鱼细胞中分离到转座酶mRNA。(4)采用新设计的包含多种细菌耐药基因的实验系统，检测了medeka鱼细胞的De novo插入。(5)目标位点重复被证明是精确的8bp，没有特定的序列或核苷酸偏好。在第四年，研究扩展到包括生物技术应用。结果表明：(1)在转座酶中加入核定位信号可使转座频率提高3倍。(2)将携带GFP基因作为标记基因的Tol2拷贝成功整合到medaka鱼染色体中。(3)整合发生在生殖系细胞中，插入的拷贝遗传给后代。(4)斑马鱼的基因转移也成功。(5)结果表明，在人和小鼠细胞中，Tol2的转座方式与在鱼细胞中的转座方式相同。(6)转座酶蛋白中存在核外定位信号。(7)一个主要信号存在于转座酶蛋白初级结构的中心。(8) x射线照射可引起高频率的转位。

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.