1997 Fiscal Year Final Research Report Summary
Development of a novel method for genetic diagnosis of hereditary colon cancer syndromes using yeast.
| Project/Area Number |
08670549
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | Tohoku University |
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Principal Investigator |
ISHIOKA Chikashi Tohoku University, Institute of Development, Aging and Cancer, Associate Professor, 加齢医学研究所, 助教授 (60241577)
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| Co-Investigator(Kenkyū-buntansha) |
KANAMARU Ryunosuke Tohoku University, Institute of Development, Aging and Cancer, Professor, 加齢医学研究所, 教授 (70152783)
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| Project Period (FY) |
1996 – 1997
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| Keywords | hereditary colon cancer / genetic diagnosis / hMLH1 / hMSH2 / APC |
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| Research Abstract |
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
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[Publications] Ishioka, C., Suzuki, T., FitzGerald, M., Krainer, M., Shimodaira, H., Shimada, A., Nomizu, T., Isselbacher, K.J., Haber, D., and Kanamaru, R.: "Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast." Proc.Natl.Acad.Sci., USA.94. 2449-53 (1997)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Ishioka, C., Shimodaira, H., Englert, C., Shimada, A., Osaka, M., Jia, L.Q., Suzuki, T., Gamo, M., and Kanamaru, R.: "Oligomerization is not essential for growth suppression by p53 in p53-deficient osteosarcoma Saos-2 cells." Biochem.Res.Commun.232. 54-60 (1997)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Delia, D., Goi, K., Mizutani, S., Yamada, T., Aiello, A., Fontanella, E., Lamorte, G., Iwata, S., Ishioka, C., Krajewski, S., Reed, J.C., and Pierotti, M.A.: "Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations." Oncogene. 14. 2137-2147 (1997)
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「研究成果報告書概要(欧文)」より
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[Publications] Goi, K., Takagi, M., Iwata, S., Delia, D., Asada, M., Donghi, R., Tsunematsu, Y., Nakazawa, S., Yamamoto, T., Yokota, J., Tamura, K., Saeki, Y., Utsunomiya, J., Takahashi, T., Ishioka, C., Eguchi, M., and Kamata, N., Mizutani, S.: "DNA damage-associated dysreguration of the cell-cycle and apoptosis control in cells with germ-line p53 mutation" Cancer Res.57. 1895-1902 (1997)
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「研究成果報告書概要(欧文)」より
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[Publications] Krainer, M., Silva-Arrieta, S., FitzGerald, M.G., Shimada, A., Ishioka, C., Kanamaru, R., MacDonald, D.J., Unsal, H., Finkelstein, D.M., Bowcock, A., Isselbacher, K.J., and Haber, D.A.: "Differential contributions of BRCA1 and BRCA2 to early-onset breat cancer" N.Eng1.J.Med.336. 1416-21 (1997)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Suzuki, T., Ishioka, C., Kato, S., Mitachi, Y., Asamura, M., and Kanamaru, R.: "Detection of APC mutations by a yeast-based protein truncation test (YPTT)." Genes Chromosomes & Cancer. (in press). (1998)
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「研究成果報告書概要(欧文)」より
