Figure 9.
2-hybrid assay to analyze subunit interaction. (A) Schematic illustration of the basic 2-hybrid constructs. The 8992 bp pGBD-MSH2 construct contains a modification of the alcohol dehydrogenase promoter (PADH) allowing for constitutive expression of an in-frame fusion between MSH2, including the hemagglutinin epitope and the GAL4 DNA Binding Domain (GBD). The vector is pGBD-C2 described previously (James et al., 1996). These plasmids are replicated in yeast via the high-copy 2 μ origin of replication and are selected for in trp1 defective yeast strains using medium lacking tryptophan (–TRP). The GAD constructs also contain the PADH promoter allowing for constitutive expression of an in-frame fusion between the open reading frames for the functional partners of Msh2p and the GAL4 Activating Domain (GAD). The functional partners include MSH6, MSH3, MLH1, PMS1, EXO1 and POL30. The vector is pGAD-C2 (James et al., 1996). The fusion constructs were made by PCR amplification of the partner open reading frames (partner) from yeast genomic DNA and the in frame fusion constructs were formed by in vivo recombination techniques (Oldenburg et al., 1997). The GAD plasmids are replicated in yeast via the high-copy 2 μ origin of replication and are selected for by growing leu2 defective yeast strains containing the constructs in medium lacking leucine (–LEU). Each haploid yeast strain contains a plasmid that will express a hybrid protein. After mating the haploid cells and the resulting diploids will carry both constructs necessary for assaying the 2-hybrid protein-protein interaction. (B) Student results from the 2-hybrid experiments to assay MMR subunit formation. MATa cells were transformed with pGBD-C2, pGBD-MSH2, and pGBD-msh2-M707I and mated on YEPD with individual MATÿstrains harboring pGAD-MSH6, pGAD-MSH3, pGAD-MLH1, pGAD-PMS1, pGAD-EXO1, and pGAD-POL30. Cells were sequentially replica plated onto –LEU –TRP –HIS, and –LEU –TRP and incubated at 30ÿC for 4 days. –LEU –TRP selects for diploids harboring both pGAD and pGBD constructs. Growth on –LEU –TRP –HIS indicates a 2-hybrid interaction. GBD fusions are listed to the left of each plate; GAD fusions are listed above each plate. GBD indicates cells transformed with the pGBD-C2 vector control. (Panel B figure and legend courtesy of Ruth Tennen, Princeton University. Reproduced with permission.)