Inactivation model of human Adenomatous Polyposis Coli gene by using budding yeast in vivo.

人APC基因在大肠癌的发生发展中起着重要作用，因此本研究利用酵母菌对APC基因进行失活，以揭示其失活机制。我们推测APC失活机制有两种：一种是DNA复制错误导致APC突变，另一种是化学致癌物导致APC突变。我们有一种很好的方法来检测长片段DNA的突变，我们称之为酵母终止密码子分析。酵母终止密码子分析法是检测蛋白质截短突变如移码突变和无义突变的有效方法。因此，酵母突变试验最好的模型是msh2缺陷细胞系，用于检测酵母细胞中人APC基因的突变。其次，我们分析了人APC基因的突变簇区域，我们检测到APC突变的频率比野生型酵母msh2细胞系更高。最 关于我们 msh2缺陷酵母细胞中的突变是插入或缺失突变，最常见于APC简单重复序列中。另一方面，到目前为止，我们在野生型酵母细胞中检测到的大多数突变都是无义突变。因此，APC基因的失活机制具有突变谱的特点，我们可以推测大肠肿瘤的临床材料中存在APC基因的失活机制。为此，我们收集了相关医院大肠息肉的临床资料。提取肿瘤组织核酸，分析APC基因的MCR区。我们用终止密码子法或改良终止密码子法检测APC突变。同一个体的每个息肉的APC突变不是APC基因的同一部分，因此我们不能检测到罕见的多态性发生，会使DNA复制错误。目前我们还不能在酵母模型中完成APC基因突变数据库的建立，因此我们需要尝试对更多的酵母样本和人类临床资料中的APC基因进行分析。少

Human APC gene plays the most important role of colorectal tumorigenesis, so we investigate to reveal the inactivation mechanism by using budding yeast. We presumed 2 type of inactivation mechanism : one is DNA replication error will cause the mutation of APC and the next reason is chemical carcinogen will cause the mutation. We has a good method to detect the mutations of long size DNA fragment, what we call, yeast stop codon assay. Yeast stop codon assay is very useful to detect the protein truncating mutation such as frameshift mutation and nonsense mutation.On the first step, we performed some yeast cell lines those defects yeast mlh1, msh2, pms1, pms2 genes. At the result, the most good model of yeast mutation assay is msh2 defect cell lines to detect the mutation of human APC gene in yeast cells. The next, we analyzed the mutation cluster region of human APC gene and we detected more highly frequent mutation rate of APC mutation than that of wild type yeast msh2 cell lines. Most … More mutations in msh2 defect yeast cells were insertion of deletion mutations are most frequent in APC simple reapeat sequence. On the other hand, most mutations in wild type yeast cells were nonsense mutation we detected until now. So the inactivation mechanism has the feature of mutation spectrum of APC gene so that we might presume the inactivation mechanism in the clinical materials of colorectal tumors.The patients who has more than 6 polyps of colon has much risk of colorectal carcinogenesis in general. So we collected lots of chinical materials of colorectal polyps in our related hospitals. we extracted the nuculeic acid from all each tumors and analyzed the MCR region of APC gene. We easily detected APC mutation by using stop codon assay or modified stop codon assay. APC mutations of each polyps of one individual were not the same part of APC gene, so we cannot detect rare polymorphism to occur that would make the error of DNA replication. Now we cannot finish the APC mutation data base in the yeast models, so we need to attempt to analyze human APC gene of more number of yeast samples and human clinical materials. Less