Analysis of the functions of RCC1 gene involved in chromosomal condensation

染色体浓缩相关RCC1基因的功能分析

• 批准号: 05680615
• 负责人: HAYASHI Naoyuki
• 金额: $ 1.22万
• 依托单位: KYUSHU UINVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680615/
• 关键词: RCC1; G protein; protein-protein interaction; 2-hybrid method; nucleopore; guanine nucleotide exchange; chromosomal condensation; cell cycle; 2ハイブリッド法; phosphorylation; cdc2 Kinase; alternative splicing

The tsBN2 cell, which has a mutation in the RCC1 gene, causes condensation of prematurely replicated chromosomes by the temperature-shift to 39.5ﾟC after synchronization at S phase. RCC1 encodes a guanine nucleotide exchanging factor for the nuclear small G protein, Ran. We tried to isolate the factors interacting with RCC1 to investigate the mechanisms of RCC1 function to control the chromosomal condensation. By 2-hybrid screening, 5 cDNAs from RanBP1 and RanBP2 genes were obtained. RanBP1 formed complex with RCC1 depending upon presence of Ran and inhibited release of the guanine nucleotide from Ran by RCC1 in vitro. On the other hand, we found that RanBP1 localized cytosole in vivo by indirect immunofluorescence observation. RanBP2 encoded the 358 kDa nucleopore-protein, and it had the leucine-rich region in N-terminus, the region homologous to cyclophilin in C-terminus, and 8 times repeated Zn-finger in the middle region. We found that this protein localized at cytosolic filaments of the nucleopores by immunofluorescence and immuno-electron microscope observations. The polyclonal antibody against RanBP2 inhibited protein import of the nuclear protein, indicating that this was essential for the nuclear transport system.

tsBN2 细胞的 RCC1 基因发生突变，在 S 期同步后温度升高至 39.5°C，导致过早复制的染色体凝结，RCC1 编码核小 G 蛋白 Ran 的鸟嘌呤核苷酸交换因子。我们试图分离与 RCC1 相互作用的因素，以研究 RCC1 控制染色体浓缩的功能机制。 2-杂交筛选，获得了来自RanBP1和RanBP2基因的5个cDNA，其依赖于Ran的存在而与RCC1形成复合物，并在体外抑制RCC1从Ran释放鸟嘌呤核苷酸。通过间接免疫荧光观察体内RanBP2编码358kDa的核孔蛋白，并且其具有富含亮氨酸的区域。 N端、C端与亲环蛋白同源的区域以及中间区域的8次重复的Zn指通过免疫荧光和免疫电镜观察发现该蛋白定位于核孔的胞浆丝。 RanBP2 抑制了核蛋白的蛋白输入，表明这对于核运输系统至关重要。

项目成果

Nakashima,T.Sekiguchi,T.Sunamoto,H.Yura,K.Tomoda,S.GO.M.kere.J.Schlessnid.D Nishimoto,T.: "Stracture of the CCG1 genc,arelationship between the exon/intron and the foctieral domain / module of CCG1 protein" Gene. (発表予定). (1994)

Nakashima, T. Sekiguchi, T. Sunamoto, H. Yura, K. Tomoda, S. GO. J. Schlessnid, T.：“CCG1 基因的结构，外显子/内含子与内含子之间的关系。 CCG1 蛋白的功能域/模块”基因。（待出版）。（1994）

E. Noguchi, N. Hayashi, T. Nishimoto et al.: "Menimum essential of CCG1/TAF_<II> 250 required for Complementing the Tenperature-Sensitive Cell Cycle Mutants, tsBN462 and ts 13 Cells, of Hamster BHK21 Cell" Somatic Cell Molecular Genetics. 20. 505-513 (199

E. Noguchi、N. Hayashi、T. Nishimoto 等人：“补充仓鼠 BHK21 细胞的温度敏感细胞周期突变体、tsBN462 和 ts 13 细胞所需的 CCG1/TAF_<II> 250 的最低必需值”体细胞

E.Noguchi,N.Hayashi,T.Nishimoto et al.: "Minimum essential region of CCG1/TAFII250 reqiured for cell cycle specific promoter-selective transcription defective in tsBN462 and ts13 cells of hamster BHK21 cells." Somatic Cell Molecular Genetics. (印刷中).

E.Noguchi、N.Hayashi、T.Nishimoto 等人：“仓鼠 BHK21 细胞的 tsBN462 和 ts13 细胞中细胞周期特异性启动子选择性转录缺陷所需的 CCG1/TAFII250 最小必需区域。” （目前正在印刷中）。

N.Yokoyama,N.Hayashi,T.Nishimoto et al.: "A giant nucleopore protein that binds Ran/TC4" Nature. 376. 184-188 (1995)

N.Yokoyama、N.Hayashi、T.Nishimoto 等人：“结合 Ran/TC4 的巨型核孔蛋白” Nature。

N.Hayashi,T.Nishimoto et al.: "RanBP1,which is a protein binding to Ran/TC4,a Ras-like nuclear G protein, inhibits RCC1 through Ran/TC4." Molecular General Genetics. (印刷中).

N. Hayashi、T. Nishimoto 等人：“RanBP1 是一种与 Ran/TC4（一种 Ras 样核 G 蛋白）结合的蛋白质，通过分子通用遗传学抑制 RCC1。”