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

在目前的研究中，我们的目标是建立一种基于酵母的终止密码子分析，它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)优化了将待测基因的PCR扩增产物两端加入酵母表达载体进行同源重组的方法，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而干扰了ADE2的活性。我们在载体上添加了适当的间隔区序列。3)验证了建立的终止密码子分析方法可以检测各种基因的截断型突变。通过这些过程，我们建立了称为通用终止密码子试验(Am J Pathol)的方法，并将该方法应用于人类癌症标本，并检测了卵巢癌中E-钙粘附素的突变。我们通过这个实验研究了潜在的候选基因，在肺癌中发现了Hoxd3，在脑肿瘤中发现了p73，在软组织肉瘤中发现了胸腺素-β4，在骨肉瘤中发现了血管内皮生长因子，在肝癌中发现了转化生长因子-β受体II。我们现在正在用通用终止密码子试验测试这些基因。

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.