Joint study on the DNA mismatch repair system : Functional analysis of the DNA mismatch repair genes using Saccharomyces cerevisiae.

DNA错配修复系统的联合研究：利用酿酒酵母对DNA错配修复基因进行功能分析。

基本信息

批准号：
08044234
负责人：
ISHIOKA Chikashi
金额：
$ 3.84万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

1. We have developed the expression system of human mismatch repair genes, hMLH1 and hMSH2 in Saccharomyces cerevisiae. We have also constructed a series of yeast strains with mismatch repair defect. When hMLH1 cDNA was expressed in mismatch repair-deficient mlhl strain, the transformants demonstrated to suppress partially the mutator phenotype. When hMLH1 cDNA was also expressed in mismatch repair-proficient strain, the transformants demonstrated to be mutator phenotype, due to dominat mutator effect of the hMLH1. Using the later phenotype, we have analyzed 27 hMLH1 sequenceari ants including 21 amno acid substitutions, 2 nonsense mutations, 2 in-frame deletions and 2 carboxy-terminal frameshift mutations. Among these, 25 variants showed no effect on the dominant mutator effect, indicating that these hMLH1 are pathogenic mutations. Two variants reported as missense mutations retained the ability to indicate dominant mutator effect, suggesting that these variants may be polymorphisms. Remaining two variants reported as polymorphisms retained the dominant mutator effect, suggesting that this type of assay has potentially detect unknown loss-of-function mutations. The assay developed in this study could be helpful for better understanding of genetics of HNPCC.2. According to the muation data base of the International Collaborating Group of HNPCC (ICG-HNPCC) and our international survey of unpublished muatations through this joint study, nearly 200 different hMSH2 and hMLH1 mutations have been documented. Among these, approxymately 10% of hMSH2 mutations and 30% of hMLH1 mutations were missense mutations, indicating that there is a significant fraction of HNPCC mutations which need to clarify their functional significance on pathogenicity in order to discriminate from non-pathogenic polymorphisms.

1。我们已经开发了酿酒酵母中人类不匹配修复基因，HMLH1和HMSH2的表达系统。我们还构建了一系列具有不匹配修复缺陷的酵母菌菌株。当HMLH1 cDNA在不匹配修复缺陷的MLHL菌株中表达时，转化子被证明可以部分抑制突变器表型。当HMLH1 cDNA在不匹配修复菌株中也表达时，由于HMLH1的主导突变效应，该转化子被证明是突变器表型。使用后来的表型，我们分析了27个HMLH1 Sequenceari蚂蚁，其中包括21种氨基酸取代，2个废话突变，2个框内缺失和2个羧基末端移码突变。其中25种变体对显性突变效应没有影响，表明这些HMLH1是致病性突变。据报道，两种变体保留了表明显性突变效应的能力，表明这些变体可能是多态性。剩下的两个变体报道为多态性，保留了显性突变效应，这表明这种类型的测定可能检测到未知的功能丧失突变。这项研究中开发的测定可能有助于更好地理解HNPCC的遗传学2。根据HNPCC国际合作小组（ICG-HNPCC）的研究数据库，以及我们通过这项联合研究的未发表熔化的国际调查，已经记录了近200种不同的HMSH2和HMLH1突变。其中，大约10％的HMSH2突变和30％的HMLH1突变是错义突变，这表明HNPCC突变的一部分很大一部分需要阐明其对致病性的功能意义，以便歧视非肺病多态性。