Two Hybrid Systems

Two-Hybrid Systems

Paul N. MacDonald has assembled a collection of powerful molecular tools for examining and characterizing protein-protein, protein-DNA, and protein-RNA interactions. The techniques range from the most basic (introducing plasmids into yeasts, interaction assays, and recovering the plasmids from yeast), to the most advanced alternative strategies (involving one-hybrid, split two-hybrid, three-hybrid, membrane recruitment systems, and mammalian systems). Methods are also provided for dealing with the well-known problem of artifacts and false positives and for identifying the interacting partners in important biological systems, including the SMAD and nuclear receptor pathways. To ensure ready reproducibility and robust results, each technique is described in step-by-step detail by researchers who employ it regularly.

The Yeast Two-hybrid System

Biologists have already identified thousands of new gene sequences, and genome sequencing efforts are speeding up the discovery process even further. With this explosion of sequence information comes the need to understand how genes work in concert in order to fulfill the cells functions. The yeast two-hybrid system--used to identify protein-protein interactions--is one of the most powerful and versatile methods for characterizing a protein's function. It has become an essential tool for both academic researchers and those in biotechnology and pharmaceutical companies. This volume presents work by pioneers in the field and is the first publication devoted solely to the yeast two-hybrid system. It includes detailed protocols, practical advice on troubleshooting, and suggestions for future development. In addition, it explains how to construct an activation domain hybrid library, how to identify mutations that disrupt an interaction, and how to use the system in mammalian cells. Many of the contributors have developed new applications and variations of the technique. Chapter topics include characterizing hormone/receptor complexes, identifying peptide ligands, analyzing interactions mediated by protein modifications, and dissecting the cell cycle and other complex genetic networks. The Yeast Two-Hybrid System is the single complete resource for scientists interested in this powerful research method.

Methodologies for Intelligent Systems, ...