A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae
A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae
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DOI：
10.1038/35001009
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发表时间：
2000-02-10
期刊：
影响因子：
64.8
通讯作者：
Rothberg, JM
中科院分区：
文献类型：
--
作者：
Uetz, P;Giot, L;Rothberg, JM
进行了两个大规模的酵母双杂交筛选，以鉴定从酿酒酵母基因组序列预测的全长开放阅读框之间的蛋白质 - 蛋白质相互作用。在一种方法中，我们构建了一个约6,000个酵母转化体的蛋白质阵列，每个变换剂表达一个开放式阅读帧之一，作为与激活结构域的融合。该阵列是通过192个酵母蛋白的简单且自动化的过程筛选的，并通过其在数组中的位置确定了正面响应，在第二种方法中，我们汇总了表达大约6,000个激活域融合中的一个单元格，以生成库。我们使用高通量筛选程序来筛选几乎所有预测的酵母蛋白中的所有，以GAL4 DNA结合结构域融合蛋白表示，并针对文库进行筛选，并通过序列分析表征了阳性。这些方法导致检测到涉及1,004 s的957个推定的相互作用，这些数据揭示了在生物学环境中将功能上未分类的蛋白质的相互作用，在同一生物学功能中涉及的蛋白质之间的相互作用，以及将生物学功能连接到较大生物学功能中的相互作用之间的相互作用细胞过程。这些屏幕的结果在此处显示。
Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array, In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins, These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.