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Cloning of an immortality-suppressor gene on human chromosome 7

人类 7 号染色体上的永生抑制基因的克隆

基本信息

批准号：
09044234
负责人：
AYUSAWA Dai
金额：
$ 2.88万
依托单位：
Kihara Institute for Biological Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044234/
关键词：
chromosome 7 microcell-mediated chromosome transfer YAC BAC radiation hybrid senescence telomerase telomere STS marker 細胞不死化遺伝子

项目摘要

Approximately 50% of immortal human cell lines so far tested seem to have a mutation on a gene on human chromosome 7 since introduction of this chromosome by microcell-mediated chromosome transfer specifically suppress their division potential. To identify it, we prepared a panel of radiation hybrids which contain a small segment of human chromosome 7 under mouse cell background. By chromosome transfer and PCR with STS markers, we narrowed down the gene within a 2-3 Mb region of the chromosome. We planned to rescue human DNA form the hybrids with a newly developed TAR (Transformation Associated Recombination) cloning system in yeast. In collaboration with NIH that invented the cloning system, we prepared a YAC/BAC library from one radiation hybrid. These BAC clones were introduced to human Met5A cell line to examine their anti-proliferative activity against this cell line.As by-products of this study, we have shown that the immortality suppresser gene negatively regulates the telomere maintenance mechanisms in particular human cell lines. Upon introduction of the gene into the telomerase-positive lines, the telomerase catalytic subunit gene was down-regulated before entering senescence and with shortening of telomeres. In the negative lines, their usually long telomeres were immediately shortened.

到目前为止，大约50%的不朽人类细胞系似乎在人类7号染色体上的一个基因上发生了突变，因为通过微细胞介导的染色体转移引入该染色体特别抑制了它们的分裂潜力。为了鉴定它，我们在小鼠细胞背景下制备了一组含有人类7号染色体小片段的辐射杂交体。通过染色体转移和STS标记的聚合酶链式反应，我们将基因的范围缩小到了染色体的2-3Mb区域。我们计划用一种新开发的酵母转化相关重组(TAR)克隆系统从杂交种中拯救人类DNA。与发明克隆系统的美国国立卫生研究院合作，我们从一个辐射杂交种中制备了YAC/BAC文库。这些BAC克隆被引入到人类Met5A细胞系中，以检测它们对该细胞系的抗增殖活性。作为这项研究的副产品，我们发现永生抑制基因对特定的人类细胞系的端粒维持机制具有负面调节作用。将该基因导入端粒酶阳性细胞系后，端粒酶催化亚单位基因在进入衰老前表达下调，端粒缩短。在负线中，它们通常较长的端粒立即缩短。