Mapping sequences that are required for PHF1b DNA recognition. Depicted is the yeast strain with chromosomally integrated reporters carrying three tandem β1 initiator sites that was used for one-hybrid assays. Candidates identified by one-hybrid assays were used to construct a number of 5′ and 3′ sequential deletions of the PHF1b gene to define the minimal β1-INR binding domain. The GAL4 AD was fused to all of the PHF1b fragments and tested for its ability to activate three tandem β1-INR sites linked to either His3 or LacZ. Full length PHF1b is shown for comparison to relate relative sizes of AD fused PHF1b fragments (#2-14 with the exception of #9, which is a derivative of PHF1a) and one-hybrid candidates B37 and A4. The DNA binding activities both positive (+) and negative (−) are as indicated. The criterion for DNA binding is measured by the ability of the constructs to activate both reporter genes (His3 and LacZ ) at levels comparable to B37 and A4 isolates. His3 expression was measured by the survival of the yeast colonies that could grow on plates that contained 10 mM 3-aminotriazole and β-galactosidase activity from the second reporter, measured by β-galactosidase assays. The results from both assays were the same and are indicated by one column to the right.
Saha et al. BMC Pharmacology and Toxicology 2013 14:37 doi:10.1186/2050-6511-14-37