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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10216205/
• 关键词: Medaka fish; Mobile genetioc element; Transposable element; Tol2 element; Transposition mechanisms; Gene transfer; Transposase; 転移因子; 挿入; Tol2; 変異; 自然集団

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.

五年研究任期的前三年致力于有关TOL2元素的迁移式麦克纳主义的基础研究。获得了以下结果：（1）检测到转座反应的4.7-Kb元素本质的一部分。 （2）鉴定出具有转座酶基因的自主TOL2副本。 （3）从Medaka鱼细胞中分离出转座酶的mRNA。 （4）检测到新设计的实验系统（包括几种细菌抗药性基因），检测到了Medeka鱼细胞中的从头插入。 （5）事实证明，目标位点的重复正好为8 bp，不偏爱特定序列或核苷酸。在第四年，该研究被扩展到包括生物技术应用。结果如下：（1）在转座酶中添加核定位信号，导致换位频率高三倍。 （2）将带有GFP基因作为标记基因的TOL2副本成功地整合到Medaka鱼类染色体中。 （3）积分发生在种系细胞中，插入的副本继承为随后的世代。 （4）基因转移在斑马鱼中也成功。 （5）证明TOL2以与鱼类相同的方式在人和小鼠细胞中转置。 （6）建议在转座酶蛋白中存在核外定位信号。 （7）建议一个主要信号存在于转座酶蛋白的主要结构的中心。 （8）发现X射线照射可在高频下诱导换位。

项目成果

Iida A, Inagaki H, Suzuki M, Hori H, Koga A: "The tyrosinase gene of the i^b albino mutant of the medaka fish carries a transposable element insertion in the promoter region."Pigment Cell Research. 17(2). (2004)

Iida A、Inagaki H、Suzuki M、Hori H、Koga A：“青鳉鱼 i^b 白化突变体的酪氨酸酶基因在启动子区域携带转座元件插入。”色素细胞研究。

KOGA,Akihiko: "Detection of de novo insertion of the medaka fish transposable element Tol2."Genetics. 156(3). 1243-1247 (2000)

KOGA，Akihiko：“检测青鳉鱼转座因子 Tol2 的从头插入。”遗传学。

KOGA, Akihiko: "Amino acid sequence of a putative transposase protein of the medaka fish transposable element Tol2"FEBS Letters. 461(3). 295-298 (1999)

KOGA，Akihiko：“青鳉鱼转座元件 Tol2 的推定转座酶蛋白的氨基酸序列”FEBS Letters。

KOGA, Akihiko: "Gamera, a family of LINE-like repetitive sequences widely distributed in medaka and related fishes"Heredity. 89(6). 446-452 (2002)

KOGA，Akihiko：“Gamera，一个广泛分布在青鳉和相关鱼类中的类 LINE 重复序列家族”遗传。

KOGA,Akihiko: "Homogeneity in the structure of the medaka fish transposable element Tol2." Genetical Research. 73(1). 7-14 (1999)

KOGA,Akihiko：“青鳉鱼转座因子 Tol2 结构的同质性。”