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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to the muation data base of the International Collaborating Group of HNPCC (ICG-HNPCC) and our international survey of unpublished muatations through this joint study, nearly 200 different hMSH2 and hMLH1 mutations have been documented.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. 我们开发了人类错配修复基因hMLH1和hMSH2在酿酒酵母中的表达系统。我们还构建了一系列具有错配修复缺陷的酵母菌株。当 hMLH1 cDNA 在错配修复缺陷的 mlhl 菌株中表达时，转化体表现出部分抑制突变表型。当 hMLH1 cDNA 也在错配修复熟练菌株中表达时，由于 hMLH1 的显性突变效应，转化体被证明具有突变表型。使用后来的表型，我们分析了 27 个 hMLH1 序列蚂蚁，包括 21 个氨基酸取代、2 个无义突变、2 个框内删除和 2 个羧基末端移码突变。其中，25个变异对显性突变效应没有影响，表明这些hMLH1是致病性突变。报告为错义突变的两个变体保留了指示显性突变效应的能力，表明这些变体可能是多态性。报告为多态性的其余两个变体保留了显性突变效应，表明这种类型的测定有可能检测未知的功能丧失突变。本研究开发的检测方法可能有助于更好地了解 HNPCC.2 的遗传学。根据 HNPCC 国际合作组 (ICG-HNPCC) 的突变数据库以及我们通过这项联合研究对未发表的突变进行的国际调查，已记录了近 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，这表明有很大一部分HNPCC突变需要阐明其致病性的功能意义，以便与非致病性多态性区别开来。