核基质MINTC端片段(2226-2959)相互作用分子的筛选与鉴定  

Screening and identification of molecules interacting with nuclear matrix protein MINT C-terminal (2226-2959)

作　　者：李军锋[1] 周鹏[1] 秦红[1] 韩骅[1] 

机构地区：[1]第四军医大学基础部医学遗传学与发育生物学教研室

出　　处：《第四军医大学学报》2003年第24期2220-2223,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金项目 (30 2 0 0 1 4 8) ;国家高技术发展规划项目 (0 0 1CB50 990 6)

摘　　要：目的 :为了研究MINT的功能及其转录抑制的机制 ,筛选与MINT相互作用的蛋白 .方法 :以MINT第 2 2 2 6 2 95 9氨基酸 (F5 )为诱饵 ,用酵母双杂交法筛选小鼠胚胎 9d的cDNA文库 .用诱饵质粒和文库先后转化酵母宿主后 ,对 4 .5× 1 0 6个酵母克隆进行了营养筛选和α 半乳糖苷酶筛选 ,获得 5 1个阳性克隆 .从中提取质粒进行酶切鉴定 ,获得 1 0个非重复克隆 ,对此 1 0个克隆进行序列分析 .结果 :筛选到的 3个基因分别为MINT ,α 晶状体蛋白结合蛋白 1 (AlphaA CRYBP1 )和核受体辅抑制因子 1 (N CoR1 ) .在酵母中分析了N CoR1与MINT的作用区段 ,N CoR1与MINT的 2 2 2 6 2 95 9和 2 96 0 35 76两个片段均有相互作用 .结论 :①MINT在细胞内可能形成二聚或多聚体 ;②除了RBP J和MSX2外 ,MINT还可能调节AlphaA CRYBP1的活性 ;③MINT可能通过N CoR1募集组蛋白去乙酰化酶 (HDAC)AIM: To screen proteins that interact with a fragment of MINT (F5, amino acids 2225 2955) in order to study the function and mechanism of MINT mediated transcription repression. METHODS: Yeast two hybrid assay was used to screen a cDNA library of 9 d mouse embryo. Among 4.5×10 6 yeast clones transformed with the bait plasmid and the cDNA library of 9 d mouse embryo, 51 were positive for nutritional screening and β galactosidase assay. Restriction digestion identified 10 independent positive clones and they were analyzed by DNA sequencing. RESULTS: These cDNA represented 3 correctly fused cDNA fragments, which were MINT, alpha A crystallin binding protein I (AlphaA CRYBP1), and nuclear receptor co repressor 1 (N CoR1), respectively. Furthermor, we analyzed the sites by which MINT interacts with N CoR1. We found that N CoR1 interacted with MINTF6 besides MINTF5. CONCLUSION: ① MINT may dimerize or multimerize in cells; ② MINT may regulate AlphaA CRYBP1 in addition to MSX2 and RBP J; ③ MINT may repress transcription by recruiting histone deacetylase (HDAC) through N CoR1.

关 键 词：MINT MSX2 N-CoR1 酵母菌杂交 

分 类 号：Q785[生物学—分子生物学] Q789