Functional mapping of missens mutations in hMLH1 gene

为了开发检测和评估人类错配修复基因hMLH1错义突变的方法，我们进行了几个实验。(1)我们改进了先前报道的用于hMLH1基因功能评估的酵母实验，并分析了许多种系衍生的错义突变。我们还尝试使用hMLH1缺陷的人成纤维细胞开发hMLH1功能测定，结果表明，在哺乳动物细胞中，使用基于gfp的报告质粒可以监测携带或不携带错义突变的cdna表达的hMLH1的功能。(2)我们已经开发了一种方法，在感兴趣的基因的开放阅读框架内产生全面的错义突变。该试验使用基于酵母的p53功能试验，采用96孔格式的位点定向诱变技术，使用改进的大引物方法。该分析可有效产生2314个错义突变，覆盖95%以上的已报告的错义突变以及以前未报告的突变。从表达的p53错义突变文库中，我们能够绘制出p53在蛋白nh_2和coo末端的高分辨率功能图谱。由于该方法对许多点突变都是有效的，我们现在采用它来生成综合的hMLH1错义突变文库。

To develop methods for detection and evaluation of missense mutations in human mismatch repair gene hMLH1, we performed several experiments. (1) We improved the previously reported yeast assay for functional evaluation of hMLH1 gene and analyzed a number of germ-line derived missense mutations. We also tried to develop hMLH1 functional assay using hMLH1 deficient human fibroblast cells, and the results suggested that function of cDNA-expressed hMLH1 with or without a missense mutation could be monitored in mammalian cells with a GFP-based reporter plasmid. (2) We have developed the method to generate comprehensive missense mutations within an open reading frame of a gene of interest. The assay used a yeast-based p53 functional assay with 96-well formatted site-directed mutagenesis technique using a modified mega-primer method. The assay is efficient for generating 2314 missense mutations that cover more than 95% of reported missense mutations as well as previously unreported mutations. From the expressed p53 missense mutation libraries, we were able to draw high-resolution map of p53 function throughout NH_2-and COO-terminus of the protein. Since the method was efficient to a number of point mutations, we are now adopted it to generate comprehensive hMLH1 missense mutation libraries.