Two-Hybrid Systems: Methods and Protocols (Methods in Molecular Biology)

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9781617372100: Two-Hybrid Systems: Methods and Protocols (Methods in Molecular Biology)

Many, if not all, essential biological processes require selective interactions between proteins. Complex signaling systems require sequential, ordered protein–protein interactions at essentially all levels of the signaling cascade. For example, peptide hormones interact with selective membrane receptor proteins, and autophosphorylation of the receptor then recruits other key regulatory proteins that initiate kinase cascades in which each phosphorylation event requires selective recognition of the protein substrate. The ultimate signaling effect, in many cases, is the regulation of RNA polymerase II-directed transcr- tion in the nucleus, a process that involves numerous, multiprotein complexes important for transcription initiation, elongation, termination, and reinitiation. Defining, characterizing, and understanding the relevance of these protein– protein interactions is an arduous task, but substantial inroads have been made over the past 20 years. The development of more recent methodologies, such as mammalian expression systems, immunopurification schemes, expression cloning strategies, surface plasmon resonance (BiaCore), and nanosequencing technologies, has contributed a wealth of new insights into these complex multiprotein mechanisms and clearly accelerated the discovery process. Arguably, the yeast two-hybrid system has been one of the predominant and most powerful tools in this discovery process.

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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.

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The yeast two-hybrid system is one of the most widely used and productive techniques available for investigating the macromolecular interactions that affect virtually all biological processes. In Two-Hybrid Systems: Methods and Protocols, Paul N. MacDonald has assembled a collection of these 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 problems 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.
Comprehensive and highly practical, Two-Hybrid Systems: Methods and Protocols not only reveals how the great variety of plasmid vectors and approaches may be optimally deployed, but also quickly empowers novices to establish two-hybrid systems in their laboratories, and experienced researchers to expand their repertoire of techniques.

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