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Development of Universal Yeast Assay to Identify Mutations of Tumor Suppressor Genes

开发通用酵母检测方法来鉴定肿瘤抑制基因的突变

基本信息

批准号：
13470229
负责人：
OKADA Futoshi
金额：
$ 7.49万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470229/
关键词：
universal stop codon assay yeast-based genetic diagnosis ADE2 truncating type mutation tumor suppressor gene yest-based genetic diagnosis 腫瘍抑制遺伝子汎用型酵母アッセイ法ストップコドンアッセイ

项目摘要

In the present study, we aimed to develop a yeast based stop codon assay which is readily applicable to any genes of interest. For this purpose, we studied the following issues : 1) We optimized the method to add sequences to both ends of the PCR-amplified product of tested genes for homologous recombination into the yeast expression vector, and verified efficiency and accuracy of recombination. 2) We found that protein product of some tested gene interfered ADE2 enzymatic activity by hiding the activity center. We added an appropriate spacer sequence to the vector. 3) We verified that the developed stop codon assay could detect truncating type mutations of various genes. Through these process we established and assay called 'universal stop codon assay' (Am J Pathol).We applied this assay to human cancer sampels, and detected mutation of E-cadherin in ovarian cancers. We studied potential candidate genes for testing by this assay, and identified HOXD3 in lung cancers (Int J Cancer), p73 in brain tumors (Brain Pathol), thymosine-beta4 in soft tissue sarcomas (Am J Pathol), VEGF in osteosarcomas (Br J Cancer), and TGF-beta receptor II in hepatic cancer (Cancer Immunol Immunother). We are now testing these genes with the universal stop codon assay.

在本研究中，我们的目的是开发一种基于酵母的终止密码子检测，这是很容易适用于任何感兴趣的基因。为此，我们研究了以下几个问题：1）优化了将待测基因PCR扩增产物两端加序列同源重组到酵母表达载体中的方法，并验证了重组的效率和准确性。2)我们发现，一些被测基因的蛋白产物通过隐藏活性中心来干扰ADE 2的酶活性。我们向载体中添加了适当的间隔区序列。3)我们验证了所开发的终止密码子检测可以检测各种基因的截短型突变。通过这些过程，我们建立了通用终止密码子分析法（AmJPathol），并将其应用于人类肿瘤标本，检测卵巢癌中E-cadherin的突变。我们研究了通过该测定进行测试的潜在候选基因，并鉴定了肺癌中的HOXD 3（Int J Cancer）、脑肿瘤中的p73（Brain Pathol）、软组织肉瘤中的胸腺素-β 4（Am J Pathol）、骨肉瘤中的VEGF（Br J Cancer）和肝癌中的TGF-β受体II（Cancer Immunol Immunother）。我们现在正在用通用终止密码子试验来检测这些基因。