Functional mapping of missens mutations in hMLH1 gene

hMLH1 基因错义突变的功能图谱

• 批准号: 11470503
• 负责人: ISHIOKA Chikashi
• 金额: $ 4.93万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470503/
• 关键词: 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

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.

为了开发检测和评估人类错配修复基因hMLH1错义突变的方法，我们进行了几项实验。(1)改进了以往报道的hMLH1基因的酵母功能检测方法，并分析了一些胚系来源的错义突变。我们还尝试利用hMLH1缺失的人成纤维细胞进行hMLH1功能检测，结果表明，无论有无错义突变，都可以在基于GFP的报告质粒的哺乳动物细胞中监测hMLH1表达的功能。(2)我们开发了一种在目的基因的开放阅读框架内产生全面错义突变的方法。该检测使用了一种基于酵母的P53功能分析方法，该方法使用了一种改进的巨型引物法，采用了96孔格式化的定点突变技术。该分析有效地产生了2314个错义突变，覆盖了95%以上已报道的错义突变以及以前未报道的突变。从表达的P53错义突变文库中，我们能够绘制出P53蛋白NH2和COO末端的高分辨率功能图谱。由于该方法对一些点突变是有效的，我们现在采用它来生成全面的hMLH1错义突变文库。

项目成果

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Bell D. 等人：“RAD52 中常见的无义突变”癌症研究。

Han, S.Y. et al.: "Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay"Cancer Research. 60. 3147-3151 (2000)

韩S.Y.

Kato, S., Shimada, A., Osada, M., Ikawa, S., Obinata, M., Nakagawara, A., Kanamaru, R., Ishioka, C.: "Effects of p51/p63 missense mutations on transcriptional activities of p53 downstream gene promoters"Cancer Res.. 59. 5908-5911 (1999)

Kato, S.、Shimada, A.、Osada, M.、Ikawa, S.、Obinata, M.、Nakakawara, A.、Kanamaru, R.、Ishioka, C.：“p51/p63 错义突变对转录的影响

Shuan-Yin Han et.al.: "Functional evaluation of PTEN missense mutations using in Vitro phosphoinositide phasphatase assay"Cancer Research. 60. 3147-3151 (2000)

Shuan-Yin Han 等人：“使用体外磷酸肌醇磷酸酶测定法对 PTEN 错义突变进行功能评估”癌症研究。

Kato, H. et al.: "Functional evaluation of p53 and PTEN gene mutations in gliomas"Clinical Cancer Research. 6. 3937-3943 (2000)

Kato, H. 等人：“神经胶质瘤中 p53 和 PTEN 基因突变的功能评估”临床癌症研究。