FIG 2.

Overexpression of TUP1 promotes ALS3 and ECE1 transcription. TET promoter-driven constructs of NRG1 and TUP1 were integrated into the ADH1 loci of wild-type strain SC5314 and the tup1Δ/nrg1Δ double mutant. (A) The morphology of the resulting strains was studied by microscopy after 6 h of incubation at 37°C in SDG or SDG with 10% human serum with or without the addition of 50 μg/ml doxycycline. Scale bar, 20 μm. (B) Total RNA of strains expressing either the pTET-TUP1 or the pTET-NRG1 construct was isolated after 6 h growth in SDG with 10% human serum. The pTET constructs were activated by the addition of 50 μg/ml doxycycline to the medium. The isolated RNA was used for RT-qPCR to determine the relative levels of gene expression of ALS3 and ECE1. A control RNA (wild type, 5 h YPD, 37°C) and the housekeeping gene ACT1 were used for normalization. Asterisks mark significant differences after overexpression of either NRG1 or TUP1 compared to the same strain without overexpression (P ≤ 0.05, two-tailed, unpaired Student's t test).