MINT蛋白(1-365氨基酸)相互作用分子的筛选

Screening and detecting proteins interacting with the Nterminal of MINT

目的 :用酵母双杂交技术筛选与MINT(Msx2 in teractingnucleartargetprotein) F1片段相互作用的分子 ,对MINT转录抑制的机制进行探讨 .方法 :以Mint F1片段 (1～ 36 5氨基酸残基 )连入载体pGBKT7构建的质粒为诱饵 ,利用酵母双杂交技术对人淋巴结cDNA文库进行筛选 ,并分析所获得的阳性克隆 .结果 :从 6× 1 0 7个克隆中共获得 1 2个不重复的阳性克隆 .经序列分析 ,得到 3个有意义的基因 ,分别为人小核RNA 蛋白复合体多肽G(SNRPG)、癌基因Vav和人微球蛋白 1 (MCRS1 ) .结论 :从筛选到的这 3个分子的定位与功能来看 ,MINT分子的N端片段可以与小核RNA 蛋白复合体 (snRNPs) ,Vav,MCRS1和RBP Jκ等相互作用 ,参与细胞内的信号传递 ,调控细胞周期 。

AIM: To study the mechanism of the repression of MINT, a 3576aa protein in nuclear matrix, and to further clarify the regulation of the pathway of Notch, yeast two hybrid assay was used to screen molecules which can interact with MINT F1(amino acids 1 365). METHODS: The bait vector was constructed by inserting Mint F1 into pGBKT7 to screen the cDNA library of human lymph node. The positive clones were restrictively digested and sequenced for further analysis. RESULTS: Seventy three clones were obtained after 6×10 7 clones were yielded by the four kinds of nutrition limitation and β galactosidase assays. Twelve positive clones were obtained after restriction of positive clones. Sequencing of the clones showed that there were 3 significative molecules: small nuclear ribonucleoprotein polypeptide G (SNRPG), Vav oncogene and microspherule protein 1 (MCRS1). CONCLUSION: Analysing with the 3 molecules obtained from the human cDNA library, we can see that the function of MINT may be complex, it may participate in the splicing of RNA by affecting the spliceosomal small nuclear ribonucleoproteins (snRNPs), or regulate the cellular signal transduction pathway by interacting with some signal transduction factors, such as Vav1 and RBP Jκ. It may also affect the size and shape of the nucleolus through MCRS1.