• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Development of a novel method for genetic diagnosis of hereditary colon cancer syndromes using yeast.

Research Project

Project/Area Number 08670549
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Gastroenterology
Research InstitutionTohoku University

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)

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)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordshereditary colon cancer / genetic diagnosis / hMLH1 / hMSH2 / APC / HNPCC
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

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] Chikashi, Ishioka: "Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast." Proc.Natl.Acad.,USA. 94. 2449-2453 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Chikashi, Ishioka: "Oligomerization is not essential for growth suppression by p53 in p53-dificient osteosarcoma Saos-2 cells." Biochem.Biophys.Res.Commun.232. 54-60 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Domenico, Delia: "Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations." Oncogene. 14. 2137-2147 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kumiko, Goi: "DNA damage-associated dysregulation of the cell cycle and apoptosis control in cells with germ-line p53 mutation." Cancer Res.57. 1895-1902 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Michael, Krainer: "Differential contributions of BRCA1 and BRCA2 to early-omset breast cancer." N.Engl.J.Med.336. 1416-1421 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takao, Suzuki: "Detection of APC mutations by a yeast-based protein truncation test(YPTT)." Genes Chromosomes & Cancer. (in press). (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [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
    • Related Report
      1997 Final Research Report Summary
  • [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
    • Related Report
      1997 Final Research Report Summary
  • [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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [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
    • Related Report
      1997 Final Research Report Summary
  • [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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Chikashi Ishioka: "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. 2449-2453 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Chikashi Ishioka: "Oligomerization is not essential for growth suppression by p53 in p53-deficient osteosarcoma Saos-2 cells." Biochem.Biophys.Res.Commun.232. 54-60 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Domenico Delia: "Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations." Oncogene. 14. 2137-2147 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kumiko Goi: "DNA damage-associated dysregulation of the cell cycle and apoptosis coontrol in cells with germ-line p53 mutation." Cancer Res.57. 1895-1902 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Michael Krainer: "Differential contributions of BRCA1 and BRCA2 to early-onset breast cancer." N.Engl.J.Med.336. 1416-1421 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Takao Suzuki: "Detection of APC mutations by a yeast-based protein truncation test (YPTT)." Genes Chromosomes & Cancer. (in press). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Chikashi Ishioka: "Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast." Proc. Natl. Acad. Sci., USA. (in press). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Li-Qun Jia: "Screening of the p53 status of human cell lines using a yeast functional assay." Molecular Carcinogenesis. (in press). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Chikashi Ishioka: "Oligom erization is not essential for growth sppression by p53 in p53-deficient osteosarcoma Saos-2 cells" Biochem. Biophys. Res. Commun.(in press). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Domenico Delia: "Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations." Oncogene. (in press). (1997)

    • Related Report
      1996 Annual Research Report

URL: 

Published: 1996-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi