| Project/Area Number |
11470503
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Human genetics
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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) |
SUZUKI Takao Tohoku University, School of Medicine and Hospital, Assistant Professor, 医学部・付属病院, 助手 (90292276)
SHIBATA Hiroyuki Tohoku University, Institute of Development Aging and Cancer, Assistant Professor, 加齢医学研究所, 助手 (50260071)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 2001: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥3,800,000 (Direct Cost: ¥3,800,000)
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| Keywords | hMLH1 / comprehensive mutation library / missense mutation / DNAミスマッチ修復 / MLH1 / 機能 / 診断 / ミスマッチ修復 / 大腸癌 |
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| Research Abstract |
To develop methods for detection and evaluation of missense mutations in human mismatch repair gene hMLH1, we performed several experiments. (1) We improved the previously reported yeast assay for functional evaluation of hMLH1 gene and analyzed a number of germ-line derived missense mutations. We also tried to develop hMLH1 functional assay using hMLH1 deficient human fibroblast cells, and the results suggested that function of cDNA-expressed hMLH1 with or without a missense mutation could be monitored in mammalian cells with a GFP-based reporter plasmid. (2) We have developed the method to generate comprehensive missense mutations within an open reading frame of a gene of interest. The assay used a yeast-based p53 functional assay with 96-well formatted site-directed mutagenesis technique using a modified mega-primer method. The assay is efficient for generating 2314 missense mutations that cover more than 95% of reported missense mutations as well as previously unreported mutations. From the expressed p53 missense mutation libraries, we were able to draw high-resolution map of p53 function throughout NH_2-and COO-terminus of the protein. Since the method was efficient to a number of point mutations, we are now adopted it to generate comprehensive hMLH1 missense mutation libraries.
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