Figure 3.

Gap1p and KAR2, PDI1 and EUG1 transcript levels in strains grown on alternative nitrogen sources. Overnight cultures of FGY58 (SHR3) and FGY60 (shr3Δ6) transformed with pPL257 (GAP1-FLU1), pregrown at 30°C under GAP1-repressing conditions in SC (minus uracil), were harvested, washed once with H₂O, and used to inoculate SC (minus uracil), SD (plus adenine and lysine), SUD (plus adenine and lysine), and SPD (plus adenine and lysine) media at a starting OD₆₀₀ of 0.5. The cultures were incubated for 4 h at 30°C, and extracts of total cell protein and RNA were prepared. (A) Proteins from an equivalent of 0.2 OD₆₀₀ units were analyzed by immunoblotting as described in MATERIALS AND METHODS. Gap1p expression levels were quantitated by phosphorimaging. Values from two independent experiments have been corrected for background and normalized to the Gap1p expression level in the FGY58 strain grown in SC (minus uracil); error bars indicate 1 standard deviation. (B) Five-microgram RNA aliquots were separated by gel electrophoresis and analyzed by Northern analysis as described in Figure 2. Phosphorimager quantitations from two independent experiments are presented; transcript levels are normalized to the expression found in the SHR3 strain (FGY58) grown in SC (minus uracil), the medium with the highest basal transcription levels; error bars indicate 1 standard deviation.