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

人类APC基因在结直肠肿瘤发生过程中起着重要的作用，因此我们通过芽孢酵母研究APC基因的失活机制。我们推测有两种失活机制：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长度DNA片段的突变，我们称之为酵母终止密码子试验。酵母终止密码子检测对于检测移码突变和无义突变等蛋白质截断突变非常有用。在第一步，我们对一些有缺陷的酵母细胞系进行了mlh1， msh2, pms1， pms2基因的检测。结果表明，在酵母细胞中检测人APC基因突变的最佳模型是msh2缺陷细胞系。接下来，我们分析了人类APC基因的突变簇区，发现APC突变的频率高于野生型酵母msh2细胞株。在msh2缺陷酵母细胞中，插入缺失突变在APC简单重复序列中最为常见。另一方面，迄今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。因此其失活机制具有APC基因突变谱的特征，可以推测其失活机制在结直肠肿瘤的临床资料中。一般而言，6个以上结肠息肉的患者发生结直肠癌的风险较大。因此我们在相关医院收集了大量结肠息肉的临床资料。我们从所有肿瘤中提取了APC基因的核酸，分析了APC基因的MCR区。我们使用终止密码子试验或改良终止密码子试验，很容易检测到APC突变。单个个体的每个息肉的APC突变不是APC基因的同一部分，因此我们无法检测到罕见的多态性发生，从而导致DNA复制错误。目前我们无法完成酵母模型中的APC突变数据库，因此我们需要尝试分析更多酵母样本和人类临床材料中的人类APC基因。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