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260 Tikhmyanova et al.partner was first described (14,15). In the dual bait system (schematicallyshown in Fig. 1B), one protein of interest is expressed as a fusion to a DBDprovided by λ bacteriophage cI (bait 1), whereas another is expressed as afusion to a DBD provided by the bacterial protein LexA (bait 2). Four separatereporter genes are used to analyze the interaction between the two baits andpreys. GusA and LYS2 are transcriptionally responsive to an operator for cI(cI op-GusA and cI op-LYS2), whereas LacZ and LEU2 are transcriptionallyresponsive to an operator for LexA (LexA op-LacZ and LexA op-LEU2). Thereare many advantages and potential uses for such a system, discussed at length inrefs. 14–17. For the specific purpose of library screening, a major benefit is thata library can be screened to identify proteins that interact with bait 1, then immediatelycounterscreened to eliminate “positives” that also interact with bait 2,reducing the false-positive rate. “True-positives” would have a transcriptionalactivation phenotype such that expression of LYS2 = GusA >> LacZ = LEU2.In addressing the second problem, that of false-negatives, the authors andcollaborators have exploited the fact that a given bait can identify nonequivalentsets of interactors when used for two-hybrid screening in bacteria vs inyeast (18). There a set of vectors (represented herein by the prototypic vectorpGLS23) suitable for expressing baits in either bacteria or yeast have beendescribed. It is proposed that screening baits constructed in these vectors inboth organisms, and/or counterscreening using dual bait capacities in yeast, cansignificantly improve the power of two-hybrid library interrogation. The companionchapter (Chapter 16) describes the use of the bacterial system in greaterdetail. This chapter focuses on the use of the yeast system, using a counterscreenapproach. It is noted, space limitations do not allow detailed presentationof basic auxiliary protocols related to execution of the technique (e.g.,preparation of yeast medium, Western blotting, and so on). These are found instandard reference manuals, including refs. 19 and 20.2. MaterialsA complete table of reagents compatible with the bacterial/yeast and dual baittwo-hybrid systems, together with acknowledgments to the numerous investigatorswhose work has contributed to the development of these tools, is available athttp://www.fccc.edu/research/labs/golemis/interactiontrapinwork.html. Many ofthese reagents are available commercially, and also can be acquired by requestfrom IG_Serebriiskii@fccc.edu, (215) 728-3885 phone, (215) 728-3616 fax, atFox Chase Cancer Center (Philadelphia, PA).2.1. Plasmids1. pGLS23—the plasmid for making cI fusion protein (bait 1), Fig. 2A. Bait expressionis from the constitutive alcohol dehydrogenase (ADH1) promoter. The yeastselection marker is HIS5, and the bacterial selective marker is Cm R (Note 1).