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294 Serebriiskii et al.(3,9), yeast (4,5), and mammals (5,8,10) have all been shown to activate transcriptionin this system. In addition, the affinity of the interacting partnersapproximately correlates with the magnitude of transcriptional activationobserved (3,7), although it is subject to some transcriptional activation thresholdconsiderations that compress dynamic range (8).1.2. Selectable Marker Genes Used With the BacterialTwo-Hybrid SystemIn a “classic” yeast two-hybrid system used for library screening, a yeaststrain carries at least one auxotrophic reporter (e.g., LEU2, HIS3,orLYS2) andone colorimetric reporter (e.g., LacZ or GusA) (2). In contrast, the bacterialsystem’s auxotrophic (HIS3) and colorimetric (LacZ) reporters are separated intwo different strains (Fig. 1B,C), whereas the HIS3 auxotrophic selection issupplemented by a cocistronic antibiotic-resistance selection cassette. Forlibrary screening purposes, the HIS3 selection strain is used as a primary assaysystem. Positive clones are subsequently assessed by streptomycin/spectinomycinantibiotic resistance using the initial bacteria strain, and by β-galactosidaseassay in a second LacZ reporter strain.1.2.1. The HIS3/aadA Selection CassetteE. coli cells bearing a hisB gene deletion do not grow on medium lackinghistidine (His-deficient medium). However, expression of the yeast HIS3 genein bacteria is sufficient to complement such a hisB defect, permitting growthon His-deficient medium (11,12). The stringency of this selection can beraised or lowered by altering the concentration of 3-aminotriazole (3-AT), acompetitive inhibitor of the HIS3p enzyme, in the medium (13). In bacteria,the HIS3 selection marker exhibits a low spontaneous background frequency(~3 × 10 –8 breakthrough colonies with 20 mM 3-AT [5], and up to ~10 6 candidatescan be plated on a single 100-mm agar plate). As shown in Fig. 1B,the selection cassette also harbors a secondary selection gene that is expressedcocistronically with the primary HIS3 selectable marker (5). This secondaryreporter, the bacterial aadA gene (conferring resistance to the antibioticsstreptomycin and spectinomycin [14]), can provide a rapid independentmeans to verify potential positives that come through the initial selection onHis-deficient medium (see Subheading 3.), as a mutation affecting HIS3activity will not influence aadA expression.Two E. coli strains are currently available for HIS3/aadA-based bacterialtwo-hybrid screening: KJ1567 (8,15) and Bacteriomatch II (Stratagene). Bothstrains grow on the same minimal medium. The most essential differencebetween them is that the Bacteriomatch II strain is tetracycline (Tc) sensitive,and thus accepts Stratagene libraries constructed in (Tc R ) plasmids such as the