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

在为期五年的研究期限的前三年，致力于Tol2元素转位机制的基础研究。获得了以下结果：(1)检测到了转座反应所需的4.7kb片段。(2)鉴定出携带转座酶基因的自主型Tol2拷贝。(3)从青冈鱼细胞中分离到转座酶的mRNA。(4)利用一种新设计的包括几种细菌耐药基因的实验系统，在美第卡鱼细胞中检测到从头插入。(5)靶点复制被证明是准确的8个碱基，不偏爱特定的序列或核苷酸。在第四年，研究扩大到包括生物技术的应用。结果如下：(1)在转座酶中加入核定位信号后，转座酶的转座频率增加3倍。(2)以GFP基因为标记基因的Tol2拷贝已成功整合到青冈鱼的染色体中。(3)生殖系细胞发生整合，插入的拷贝可遗传给后代。(4)斑马鱼的基因转移也是成功的。(5)Tol2在人和小鼠细胞中的转座方式与在鱼类中相同。(6)转座酶蛋白中存在核外定位信号。(7)转座酶蛋白一级结构中心附近存在一个主要信号。(8)X射线照射可引起较高频率的移位。

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.