Tamanoi F, Walsh M, Kataoka T, Wigler M
Proc Natl Acad Sci U S A. 1984 Nov;81(22):6924-8. doi: 10.1073/pnas.81.22.6924.
Yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, which show remarkable homology to mammalian ras genes. To characterize these gene products, we have expressed the RAS2 gene in yeast using an inducible GAL10 promoter. After labeling with [35S]methionine and immunoprecipitating with a monoclonal antibody Y13-259, which reacts with p21 encoded by mammalian ras genes, a major band having an apparent molecular weight of 41,000 is detected. This band has also been identified in cell-free translation products of polyadenylated RNA extracted from yeast cells grown in the presence of galactose. Crude extracts of cells expressing the RAS2 gene exhibit guanine nucleotide binding activity. This is detected by incubation with [3H]GDP followed by immunoprecipitation with the antibody Y13-259. The binding of labeled GDP is inhibited by a 20-fold excess of GDP, GTP, and, to a lesser extent, by UTP, a characteristic similar to that possessed by the mammalian ras proteins. However, the activity of the yeast protein differs from that of the mammalian proteins in its strong dependence on temperature. The guanine nucleotide binding activity provides an assay to purify the yeast protein.
酿酒酵母含有两个基因,RAS1和RAS2,它们与哺乳动物的ras基因具有显著的同源性。为了表征这些基因产物,我们使用可诱导的GAL10启动子在酵母中表达了RAS2基因。用[35S]甲硫氨酸标记并使用与哺乳动物ras基因编码的p21反应的单克隆抗体Y13 - 259进行免疫沉淀后,检测到一条表观分子量为41,000的主要条带。在从在半乳糖存在下生长的酵母细胞中提取的聚腺苷酸化RNA的无细胞翻译产物中也鉴定出了这条带。表达RAS2基因的细胞粗提物表现出鸟嘌呤核苷酸结合活性。这通过与[3H]GDP孵育然后用抗体Y13 - 259进行免疫沉淀来检测。标记的GDP的结合受到20倍过量的GDP、GTP的抑制,并且在较小程度上受到UTP的抑制,这一特性与哺乳动物ras蛋白相似。然而,酵母蛋白的活性在对温度的强烈依赖性方面与哺乳动物蛋白不同。鸟嘌呤核苷酸结合活性为纯化酵母蛋白提供了一种检测方法。