Development of yeast color assay for screening of genetic mutation

• Project/Area Number: 11557018
• Research Category: Grant-in-Aid for Scientific Research (B).
• Allocation Type: Single-year Grants
• Section: 展開研究
• Research Field: Experimental pathology
• Research Institution: HOKKAIDO UNIVERSITY
• Principal Investigator: MORIUCHI Tetsuya Hokkaido University Institute for Genetic Medicine, Div.Cancer-Related Genes, Professor, 遺伝子病制御研究所, 教授 (20174394)
• Co-Investigator(Kenkyū-buntansha): HAMADA Junichi Hokkaido University Institute for Genetic Medicine, Div.Cancer-Related Genes, Asso.Professor, 遺伝子病制御研究所, 助教授 (50192703) ; TADA Mitsuhiro Hokkaido University Institute for Genetic Medicine, Div.Cancer-Related Genes, Asso.Professor, 遺伝子病制御研究所, 助教授 (10241316)
• Project Period (FY): 1999 – 2000
• Project Status: Completed (Fiscal Year 2000)
• Budget Amount: ¥6,900,000 (Direct Cost: ¥6,900,000); Fiscal Year 2000: ¥2,000,000 (Direct Cost: ¥2,000,000); Fiscal Year 1999: ¥4,900,000 (Direct Cost: ¥4,900,000)
• Keywords: yeast / genetic diagnosis / p53 / APC / PTEN / AIE-75 / ADE2

Research Abstract

1. APC yeast color assay : We have developed a sensitive yeast-based stop codon assay for screening almost the entire coding region of the APC gene. By this method we have been able to detect somatic mutations of the APC gene in 57% of colorectal cancers, 18% of breast cancers, and none in nonsmall cell lung cancers. Mutant APC generated red yeast colonies.
2. p53 dominant-negative assay : We examined the dominant-negative potential of various p53 mutants using a yeast-based assay in which both wild-type and mutant p53 were efficiently expressed. We have tested a total of 106 p53 mutants. Dominant-negative p53 generated pink yeast colonies.
3. PTEN stop codon assay : We have developed a yeast-based assay for the detection of PTEN nonsense mutation (shown as red yeast colonies), and applied it to a total of 42 astrocytic tumors. Interestingly pink colonies were found in 10 of 18 glioblastomas. Sequence analysis of the pink colonies revealed a sequence of psi PTEN cDNA.

Research Products

• [Publications] Marutani,M: "Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme"Cancer Res.. 59. 4765-4769 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kobayashi,I.: "Identification of an autoimmune enteropathy-related 75-kilodalton antigen"Gastroenterology. 117. 823-830 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Zhang,CL.: "Detection of PTEN nonsense mutation and psiPTEN expression in high-grade astrocytic tumors by a yeast-based stop codon assay"Oncogene. 19. 4346-4353 (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Furuuchi,K.: "Somatic mutations of the APC gene in primary breast cancers"Am.J.Pathol.. 156. 1997-2005 (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hamada,J.: "Increased Oxidative DNA Damage in Mammary Tumor Cells by Continuous Epidermal Growth Factor Stimulation"J.Natl.Cancer Inst.. 93. 214-219 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Ozaki,T.: "Deletion of the C-terminal region of p73alpha enhances both its transactivation function and DNA-binding activity but inhibits induction of apoptosis"Cancer Res.. 59. 5902-5907 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Marutani M, Tonoki H, Tada M, Takahashi M, Kashiwazaki H, Hida Y, Hamada J, Asaka M and Moriuchi T: "Dominant negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme."Cancer Res.. 59. 4765-4769 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kobayashi I, Imamura K, Kubota M, Ishikawa S, Yamada M, Tonoki H, Okano M, Storch WB, Moriuchi T, Sakiyama Y, Kobayashi K: "Identification of an autoimmune enteropathyrelated 75-kilodalton antigen."Gastroenterology. 117. 823-830 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Zhang CL, Tada M, Kobayashi H, Nozaki M, Moriuchi T, Abe H.: "Detection of PTEN nonsense mutation and psiPTEN expression in central nervous system high-grade astrocytic tumors by a yeast-based stop codon assay."Oncogene.. 19. 4346-53 (2000)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Furuuchi K, Tada M, Yamada H, Kataoka A, Furuuchi N, Hamada J, Takahashi M, Todo S, Moriuchi T.: "Somatic mutations of the APC gene in primary breast cancers."Am J Pathol.. 56. 1997-2005 (2000)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hamada J, Nakata D, Nakae D, Kobayashi Y, Akai H, Konishi Y, Okada F, Shibata T, Hosokawa M, Moriuchi T.: "Increased Oxidative DNA Damage in Mammary Tumor Cells by Continuous Epidermal Growth Factor Stimulation."J Natl Cancer Inst.. 93. 214-219 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ozaki T, Naka M, Takada N, Tada M, Sakiyama S, Nakagawara A.: "Deletion of the COOH-terminal region of p73alpha enhances both its transactivation function and DNA-binding activity but inhibits induction of apoptosis in mammalian cells."Cancer Res.. 59. 5902-5907 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Zhang, CL.: "Detection of PTEN nonsense mutation and psiPTEN expression in high-grade astrocytic tumors by a yeast-based stop codon assay"Oncogene. 19. 4346-4353 (2000)
• [Publications] Furuuchi, K.: "Somatic mutations of the APC gene in primary breast cancers"Am.J.Pathol.. 156. 1997-2005 (2000)
• [Publications] Hamada, J.: "Increased Oxidative DNA Damage in Mammary Tumor Cells by Continuous Epidermal Growth Factor Stimulation"J.Natl.Cancer Inst.. 93. 214-219 (2001)
• [Publications] 守内哲也: "Identification of an autoimmune enteropathy-related 75-kilodalton antigen"Gastroenterology. 117. 823-830 (1999)
• [Publications] 守内哲也: "Somatic mutations of the APC gene in primary breast cancers"Am. J. Pathol.. (印刷中).
• [Publications] 守内哲也: "Dominant negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme"Cancer Res.. 59. 4765-4769 (1999)
• [Publications] 守内哲也: "A functional and quantitative mutational analysis of p53 mutations in yeast indicates strand biases and different roles of mutations in DMBA- and BBN-induced tumors"Int. J. Cancer. 83. 700-705 (1999)
• [Publications] 守内哲也: "Distinct prognostic values of p53 mutation and loss of estrogen receptor and their cumulative effect in primary breast cancers"Int.J.Cancer. 89. 92-99 (2000)
• [Publications] 浜田淳一: "Increased E1AF expression in mouse fibrosarcoma promotes metastasis through induction of MT1-MMP expression"Oncogene. 18. 1771-1776 (1999)