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258 Tikhmyanova et al.functionally characterized “interactors” for that protein. Numerous technicalextensions and derivatives of the two-hybrid system paradigm have been developed,and two-hybrid interaction screening can now be performed not only inyeast, but also in bacteria or other organisms (e.g., ref. 12).Despite these many accomplishments, there remain a number of technicallimitations that restrict the utility of two-hybrid-based protein interaction data.One issue is that of false-positives. Estimates of the frequency of nonspecific“positives” obtained for a protein used in a two-hybrid library screen vary, butmay be as high as 50% or more, in some particularly bad cases. A second issueis that of false negatives. Meta-analyses comparing the results of large- andsmall-scale two-hybrid screens, and studies comparing the results of two-hybridand other protein-interaction techniques, have led to the clear realization thatthe two-hybrid system probably substantially undersamples the interactor poolfor any given sample (11,13). There are various approaches to addressing theseproblems. In this and a companion chapter (16), one systematic approach,which is the development of a two-hybrid system variant with extendedscreening capacity and greater internal controls will be discussed.The basic yeast two-hybrid system paradigm is shown in Fig. 1A. In libraryscreening with this system, a “bait” protein is made, in which a protein of interestis expressed as a chimera with a DNA-binding domain (DBD) of knownsequence-binding specificity. For the “classic” two-hybrid system, this bait proteinmust be confirmed to lack transcriptional activating sequences; as suchautoactivation will make it unusable in a library screen. To measure baitdependenttranscription, the bait is expressed in strains of yeast with reportergenes, wherein a binding site for the bait DBD is located within the promoterregion of two reporter genes. These are typically a colorimetric reporter (LacZand GusA) and an auxotrophic selection gene (HIS3, LEU2, and LYS2). Inlibrary screening, a library of “preys,” representing a cDNA library expressedfrom a vector that fuses them to a transcriptional activation domain (AD), isintroduced into yeast containing transcriptionally inactive baits. Interaction ofan AD-fused library constituent with the bait turns on the reporter genes, allowingselection of positive clones.In 1999, the “dual bait” two-hybrid system, which can be used to simultaneouslyanalyze the interaction of two distinct baits with the same interactiveFig. 1. (Opposite page) Two-hybrid system and dual bait system. An AD-fusedprotein (prey) interacts with a LexA-fused protein (bait1) to drive transcription of LexAop-responsive LEU2 and LacZ reporters but does not interact with a cI-fused bait, andthus, does not turn on transcription of cI op-responsive LYS2 and GusA reporters.Note: as drawn herein, cI-fused bait is representing a nonspecific partner; the systemcan also be configured for the prey to interact with both baits. AD, activation domain.