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

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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