Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.
Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.
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发表时间：
1996-12
期刊：
影响因子：
3.3
通讯作者：
P. James;J. Halladay;E. Craig
中科院分区：
文献类型：
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作者：
P. James;J. Halladay;E. Craig
双杂交系统是利用酿酒酵母成熟的分子遗传学技术检测蛋白质-蛋白质相互作用的有效技术。然而，由于系统中可用的组件所施加的限制，该技术的全部潜力尚未实现。这些限制包括笨重的质粒载体，不完整或设计不良的双杂交文库，以及导致选择大量假阳性的宿主菌株。我们使用了一种新的多酶方法来产生一组高度代表性的S.啤酒。此外，创建了一种独特的宿主菌株，其含有三种容易测定的报告基因，每种基因均在不同的诱导型启动子的控制下。该宿主菌株对弱相互作用极其敏感，并使用简单的平板测定法消除了几乎所有假阳性。还构建了改进的载体，其简化了双杂交系统所必需的基因融合体的构建。我们的分析表明，文库和宿主菌株提供了显着的改善，在确定的相互作用的克隆的数量和双杂交选择的效率。
The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.