The DHFR PCA. (A) DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate, an early step in the synthesis of purine. Methotrexate is a potent and highly specific competitive inhibitor of DHFR. (B) Bait and prey proteins are fused to complementary fragments of methotrexate-insensitive murine DHFR. No DHFR PCA selection is observed if there is no bait–prey interaction. If there is a bait–prey interaction, DHFR activity is reconstituted, allowing cells to proliferate in the presence of methotrexate. (C) Schematic representation of a DHFR PCA screen. The bait strain is incubated in liquid culture and the prey reporter strains are printed on solid medium. A mating plate is created through sequential printing of a bait to the prey strain array grown on agar in nonselection medium. Haploids and diploid mixture strains are transferred to agar plates containing a medium for the selection of diploids. The resulting diploid array is then transferred onto DHFR PCA selection plates containing methotrexate.