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.

五年研究期的前三年致力于Tol2元件转座机制的基础研究。结果表明：（1）检测到了4.7kb转座反应必需元件的部分片段。(2)An鉴定了携带转座酶基因的自主Tol2拷贝。(3)An从青鳉鱼细胞中分离转座酶的mRNA。(4)De利用一种新设计的实验系统，包括几种细菌耐药基因，检测到medeka鱼细胞中的novo插入。（5）靶位点重复被证明是精确的8bp，没有对特定序列或核苷酸的偏好。在第四年，研究扩展到生物技术应用。结果表明：（1）在转座酶中加入核定位信号后，转座频率提高了3倍。(2)A将携带GFP基因的Tol2拷贝作为标记基因成功整合到青鳉染色体中。（3）整合发生在生殖细胞中，插入的拷贝遗传给后代。（4）在斑马鱼中也成功地进行了基因转移。（5）证明Tol2在人和小鼠细胞中以与在鱼中相同的方式转座。(6)An转座酶蛋白中可能存在核定位信号。（7）在转座酶蛋白一级结构的中心附近存在一个主要信号。（8）X射线照射可引起高频率的转座。

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。

INAGAKI,Hidehito: "The tyrosinase gene from medakafish:Transgenic expression rescues albino mutation." Pigment Cell Research. 11(5). 283-290 (1998)

稻垣秀人：“来自青鳉鱼的酪氨酸酶基因：转基因表达拯救了白化突变。”