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

人APC基因在结直肠肿瘤的发生中起着重要作用，因此我们利用芽殖酵母来研究其失活机制。我们推测了两种失活机制：一种是DNA复制错误导致APC的突变，另一种是化学致癌物引起的突变。我们有一种很好的方法来检测长DNA片段的突变，我们称之为酵母终止密码子试验。酵母终止密码子分析是检测移码突变和无义突变等蛋白质截短突变的重要手段。结果表明，MSH2缺陷细胞系是检测酵母细胞中人APC基因突变的最好模型。其次，我们对人APC基因突变簇区进行了分析，发现APC基因突变频率高于野生型酵母MSH2细胞系。大多数…缺失突变在MSH2缺陷酵母细胞中的插入较多，在APC简单重复序列中突变最多。另一方面，到目前为止，我们检测到的野生型酵母细胞中的大多数突变都是无义突变。因此，APC基因的失活机制具有APC基因突变谱的特征，因此我们可以在大肠肿瘤的临床资料中推测其失活机制。大肠息肉超过6个的患者总体上有较大的结直肠癌发生的风险。为此，我们在相关医院收集了大量有关大肠息肉的临床资料。从所有肿瘤中提取核甘酸，分析APC基因的MCR区。用终止密码子试验或改良终止密码子试验检测APC基因突变。同一个体的每个息肉的APC突变都不是APC基因的相同部分，因此我们不能检测到会导致DNA复制错误的罕见的多态性。目前尚不能在酵母模型中完成APC突变数据库的建立，因此需要尝试对更多的酵母样本和人类临床资料进行APC基因的分析。较少

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