Expression of frameshifted PGK1 in tra1-L3733A and tra1-F3744A strains. A. PGK1fs-lacZ. The promoter and coding reading of PGK1 from-544 to 891 was synthesized by PCR, and the reading frame changed after codon 166 by endfilling of the Asp718 site (493 bp, solid line) and religation. This results in a stop 26 codons 3' to the frameshift (asterick). PGK1 is fused to residues 305-403 of HIS3 (white box) followed by the coding sequence of lacZ. B. Yeast strains CY4353 (TRA1 UPF1), CY5932 (TRA1 upf1Δ), CY4103 (tra1-L3733A UPF1), and CY5972 (tra1-L3733A upf1Δ) were transformed with a LEU2 centromeric plasmid containing PGK1fs-lacZ. Cultures were grown to stationary phase in media depleted of leucine, inoculated at a 100-fold dilution in YPD media, grown for 16 hr at 30°C and β-galactosidase activity determined, normalizing to cell density.
Kvas et al. BMC Genetics 2012 13:19 doi:10.1186/1471-2156-13-19