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目的 利用同源性分析 ,克隆定位新的人类间隙连接蛋白基因 ,并探讨该基因和遗传性耳聋的关系。方法 将小鼠 Cx5 7基因的编码区与美国国家生物信息中心 (National Center for BiotechnologyInformation,NCBI)的表达序列标签数据库 (database of expressed sequence tags,db EST)进行 BL AST(basic local alignment search tool)分析 ,在得到的代表人类新基因的 EST序列构建重叠群上设计引物CX5 8F1/CX5 8F2和 CX5 8R1/CX5 8R2 ,分别与 c DNA文库及 Ready c DNA载体臂上引物行巢式聚合酶链反应 (polymerase chain reaction,PCR)、c DNA末端快速扩增 (rapid amplification of c DNA ends,RACE) ,获得 c DNA后与大规模测序结果比较 ,定位该基因 ,并在常染色体显性遗传性耳聋家系中进行突变分析。结果 将利用同源性分析得到的 9个人类新的 EST,构建重叠群设计引物行巢式 PCR、RACE,在肝、肾的Ready c DNA和胎盘 c DNA文库中得到 1个全长 c DNA并命名为 CX5 8。通过与大规模测序结果比较 ,将该基因定位于 1p32 .3- p34 .1。12个常染色体显性遗传性耳聋家系中未检测到突变。结论 通过同源性分析 ,我们克隆定位了 1个新的人类间隙连接蛋白基因 CX5 8。该基因突变可能不是引起常染色体显性遗传性耳聋的常见原
OBJECTIVE: To clone and locate a new human gap junction protein gene by homology analysis and to explore the relationship between this gene and hereditary deafness. Methods The coding region of mouse Cx5 7 gene was analyzed by BLAST (basic local alignment search tool) with the database of expressed sequence tags (db EST) of the National Center for Biotechnology Information (NCBI) The primers CX5 8F1 / CX5 8F2 and CX5 8R1 / CX5 8R2 were designed on the contigs of the constructed EST sequences representing human new genes. The primers were nested with the c DNA library and the primers of Ready c DNA vector respectively. (PCR), rapid amplification of c DNA ends (RACE), c DNA was obtained and compared with large-scale sequencing results, locate the gene, and autosomal dominant inherited deafness in the family Perform mutation analysis. Results Nine new human ESTs were obtained using homology analysis. The contigs were designed to be nested PCR and RACE. One full-length cDNA was obtained from the liver and kidney Ready c DNA and placental c DNA libraries Named CX5 8. Compared with the results of large-scale sequencing, the gene was located in 1p32 .3-p34. 1.12 autosomal dominant inherited deafness family did not detect mutations. Conclusion By homology analysis, we cloned a new human gap junction protein gene CX5 8. This gene mutation may not be a common cause of autosomal dominant deafness