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Development of a novel method for genetic diagnosis of hereditary colon cancer syndromes using yeast.

开发一种使用酵母对遗传性结肠癌综合征进行基因诊断的新方法。

基本信息

批准号：
08670549
负责人：
ISHIOKA Chikashi
金额：
$ 1.47万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670549/
关键词：
hereditary colon cancer genetic diagnosis hMLH1 hMSH2 APC HNPCC

项目摘要

1.Development of methods for detection of heterozygous mutations in the mismatch repair genes and the APC gene.(1) Development of methods for detection of heterozygous mutations in the mismatch repair genes. We developed a novel method which has the ability to detect heterozygous loss-of-function mutations. Using this method, RNA was extracted from patient lymphocytes and the hMLH1 cDNA was amplified from the RNA by RT-PCR technique. The amplified hMLH1 cDNA was introduced, homologously combined into a gap vector in vivo and was expressed in yeast Saccharomyces cerevisiae. When the wild-type hMLH1 was expressed, yeast transformants showed mutator phenotype, possibly, due to the iterferance of host mismatch repair system (dominant mutator effect). We have shown that most of the hMLH1 germline mutations detected in HNPCC families lose this function, suggesting that this system has potentially detect unknown heterozygous hMLH1 loss-of-function mutations.(2) Development of methods for dete … More ction of heterozygous APC mutations.We also developed a novel method, called stop codon (SC) assay which has the ability to detect ptotein truncating mutations in APC gene, a gene mutated in familial adenomatous polyposis (FAP), from patient lymphocytes. Using the SC assay, an APC fragment was amplified by PCR and the fragment was introduced into a URA3 gene-tagged gap vector in vivo. The plasmid expressed the fusion protein consisted of the APC-derived polypeptide at the amino terminus and the URA3-derived polypeptide at the carboxy terminus. The fosion protein was able to compliment yeast ura3-phenotype therefore the transformant was able to grow on the plate lacking Uracil. Any mutations that disrupt the reading frame of the APC sequence, such as nonsense and frameshift mutations, could detect as inability to compliment yeast ura3-phenotype and the yeast transformants cannot grow on the same plates.2.Using the new methods described in (1) and (2), we have detected heterozygous loss-of-function hMLH1 mutation and heterozygous protein truncating mutations from FAP as HNPCC families. Less

1.错配修复基因和APC基因杂合突变检测方法的开发。(1)错配修复基因杂合突变检测方法的开发。我们开发了一种能够检测杂合功能丧失突变的新方法。使用这种方法，从患者淋巴细胞中提取RNA，并通过RT-PCR技术从RNA中扩增hMLH1 cDNA。将扩增的 hMLH1 cDNA 引入体内，同源组合到缺口载体中，并在酿酒酵母中表达。当野生型hMLH1表达时，酵母转化体表现出突变表型，这可能是由于宿主错配修复系统的干扰（显性突变效应）所致。我们已经证明，在 HNPCC 家族中检测到的大多数 hMLH1 种系突变都失去了此功能，这表明该系统有可能检测未知的杂合 hMLH1 功能丧失突变。(2) 开发检测杂合 APC 突变的方法。我们还开发了一种称为终止密码子 (SC) 测定的新方法，该方法能够检测蛋白截断突变 APC 基因是一种在家族性腺瘤性息肉病 (FAP) 中突变的基因，来自患者淋巴细胞。使用 SC 测定，通过 PCR 扩增 APC 片段，并将该片段引入体内 URA3 基因标记的间隙载体中。该质粒表达的融合蛋白由氨基末端的APC衍生的多肽和羧基末端的URA3衍生的多肽组成。 fosion蛋白能够补充酵母ura3表型，因此转化体能够在缺乏尿嘧啶的平板上生长。任何破坏 APC 序列阅读框的突变，例如无义突变和移码突变，都可能被检测为无法补充酵母 ura3 表型，并且酵母转化体不能在同一平板上生长。 2.使用 (1) 和 (2) 中描述的新方法，我们检测到来自 FAP 的杂合功能丧失 hMLH1 突变和杂合蛋白截短突变：全国人大家庭。较少的