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

青鳉鱼To12转座因子的转座机制及生物技术应用

基本信息

批准号：
10216205
负责人：
KOGA Akihiko
金额：
$ 17.92万
依托单位：
Grant-in-aid for Scientific Research on Priority Areas (2)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2002
项目状态：
已结题

项目摘要

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.

五年研究期的前三年致力于Tol2元素转位机制的基础研究。获得以下结果： (1)检测到转座反应必需的4.7-kb元件的部分。 (2)鉴定出携带转座酶基因的自主Tol2拷贝。 (3)从青鳉鱼细胞中分离出转座酶的mRNA。 (4)利用新设计的包含多个细菌耐药基因的实验系统，检测到了青鳉鱼细胞中的从头插入。 (5)靶位点重复被证明正好是8 bp，对特定序列或核苷酸没有偏好。第四年，研究范围扩大到包括生物技术应用。结果如下： (1)向转座酶添加核定位信号导致转座频率提高了三倍。 (2)以GFP基因为标记基因的Tol2拷贝成功整合到青鳉鱼染色体中。 (3)整合发生在生殖系细胞中，插入的拷贝遗传给后代。 (4)基因转移在斑马鱼中也取得了成功。 (5)Tol2被证明以与鱼类相同的方式在人和小鼠细胞中转座。 (6)转座酶蛋白中存在核外定位信号。 (7)一个主要信号被认为存在于转座酶蛋白一级结构的中心附近。 (8)X射线照射被发现可诱导高频转位。