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.在不匹配修复基因和APC基因中检测杂合突变的方法的开发。（1）开发不匹配修复基因中杂合突变的方法的发展。我们开发了一种新型方法，该方法具有检测杂合功能丧失突变的能力。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扩增HMLH1 cDNA。引入了放大器HMLH1 cDNA，同源地合并为体内间隙载体，并在酿酒酵母中表达。当表达野生型HMLH1时，由于宿主不匹配修复系统的迭代（主要突变效应），酵母转化剂可能显示出突变体表型。 We have shown that most of the hMLH1 germline mutations detected in HNPCC families lose this function, suggesting that this system has potentially detected 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在APC基因中，患者淋巴细胞的腺瘤性息肉病（FAP）中突变的基因。使用SC分析，通过PCR扩增APC片段，并将片段引入体内URA3基因标记的GAP载体中。质粒表达的融合蛋白由氨基末端的APC衍生多肽和羧基末端的URA3衍生的多肽组成。 Fosion蛋白能够补充酵母URA3-表型，因此转化剂能够在缺乏尿嘧啶的板上生长。 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家庭。较少的