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Research

One- plus Two-hybrid System, a Novel Yeast Genetic Selection for Specific Missense Mutations Disrupting Protein/Protein Interactions^*

Ji Young Kim^,,¶, Ok Gu Park^,, Jae Woon Lee^|| and Young Chul Lee^,**

From the Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, South Korea and ^|| Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030

To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.

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