Identification of a growth-suppressing gene in normal human fibroblasts and mouse brain

正常人成纤维细胞和小鼠大脑中生长抑制基因的鉴定

• 批准号: 03454556
• 负责人: AYUSAWA Dai
• 金额: $ 3.26万
• 依托单位: Institute of Molecular and Cellular Biosciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454556/
• 关键词: Senescence; Immortalization; Gene; Chromosome 7; Signal transduction; Gene mapping; L1反復配列; 相補性群; 不死化細胞株; 微小核細胞融合法; 放射線雑種細胞; 分裂停止遺伝子; 老化遺伝子; 正常線維芽細胞; 不死化; RFLP解析; 微少核細胞融合法; 不死化遺伝子; 染色体導入; 染色体マッピング; 不死化細胞; アンチセンスcDNAライブラリ-

Normal somatic cells in mammals stop dividing in culture after a limited number of cell division. This phenomenon is called cellular senescence and governed by several genes. When these genes are inactivated in a cell, it acquires potential to divide indefinitely and immortalize. Genetic analysis revealed that there exist at least four genetic complementation groups (A-D) in immortal human cell lines. We have found that introduction of human chromosme 7 by microcell-mediated chromosome transfer specifically induces senescence in cell lines belonging to group D.Then we made a panel of radiation hybrids under the backgroud of mouse A9 cells. The hybrids were characterized by FISH analysis and PCR with STS markers to determine how fragments of human chromosme 7 are integrated on mouse A9 genome. Of these hybrids, we selected a clone that contains only 2-3 Mb human DNA and can induce senescence in group D cell lines. We rescued human DNA from this clone and used it as a probe to map the putative senescence gene on human chromosme 7. The mapping data are useful for us to identify the gene.

哺乳动物的正常体细胞在有限数量的细胞分裂后停止分裂。这种现象被称为细胞衰老，由几个基因控制。当这些基因在细胞中失活时，它就获得了无限分裂和永生的潜力。遗传分析表明，在不朽的人类细胞系中至少存在4个遗传互补群（A-D）。我们发现通过微细胞介导的染色体转移引入人7号染色体可特异性诱导d组细胞系的衰老。然后我们在小鼠A9细胞的背景下制作了一组辐射杂交种。利用FISH分析和带STS标记的PCR对杂种进行鉴定，以确定人类7号染色体片段如何整合到小鼠A9基因组上。在这些杂交中，我们选择了一个只含有2-3 Mb人类DNA的克隆，可以诱导D组细胞系衰老。我们从该克隆中提取了人类DNA，并将其作为探针来定位人类7号染色体上的假定衰老基因。图谱数据对我们鉴定基因很有用。

项目成果

Takayanagi,A.: "Intron 1 an the 5'-flanking region of the human thymidylate synthase gene as a regulatory determinant of growth-dependent expression." Nucleic Acid Res.20. 4021-4025 (1992)

Takayanagi,A.：“内含子 1 是人胸苷酸合成酶基因的 5 侧翼区域，是生长依赖性表达的调节决定因素。”

T.Hara et al.: "The UDP-galactose translocator gene is mapped to the Wiskott-Aldrich syndrome locuc of the X chromosome." Semat.Cell Mol.Genet.19. 571-575 (1993)

T.Hara 等人：“UDP-半乳糖易位基因被定位到 X 染色体的 Wiskott-Aldrich 综合征基因座。”

Imai,N.: "Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme El" Gene. 18. 279-282 (1992)

Imai,N.：“编码小鼠泛素激活酶 E1 的功能活性 cDNA 的克隆和序列”基因。

Kaneda,S.,Horie,N.,Takahashi,K.,Takayanagi,A.,Seno,T.& Ayusawa,D.: "Promoterーspecific regulatory sequence in the human thymidylate synthase gene clustered at the 5'ーend of the first intron." J.Biol.Chem.(1992)

Kaneda, S.、Horie, N.、Takahashi, K.、Takayanagi, A.、Seno, T. 和 Ayusawa, D.：“人类胸苷酸合成酶基因中的启动子特异性调控序列聚集在第一个内含子。”J.Biol.Chem.(1992)

N.Imai et al.: "Cloning and sequence of a functionally active cDNA for mouse ubiquitin activating enzyme E1." Gene. 118. 282-297 (1992)

N.Imai 等人：“小鼠泛素​​激活酶 E1 的功能活性 cDNA 的克隆和序列。”