Figure 2. Overexpression of transcription activator Mit in transcription repressors-deficiency strains highly derepressed AOX and GFP expression by P_AOX1.

(A) Studies on the mRNA expression levels of MIT1, PRM1 and MXR1 in WT, Δmig1, Δmig2, Δnrg1, Δmig1Δmig2 and Δmig1Δmig2Δnrg1 strains when grown in 1% glycerol. Quantitative RT-PCR (qRT-PCR) was performed to determine the levels of mRNA. The mRNA levels were normalized to the levels of ACT1 mRNA in each sample, and relative to the levels of each gene mRNA in WT. The error bars represent the mean of three biological replicates assayed in duplicate. (B) Colorimetric reaction of AOX enzyme assay of WT, WT-Mit1 and WT-Prm1 strains on different carbon sources. Cells were grown for 2 days on YND agar medium and then replica plated onto medium containing different carbon sources. Twelve hours later, AOX activity was visualized by overlaying the AOX activity reaction mixture along with a permeabilizing agent. Western blot detection of AOX protein in WT, WT-Mit1 and WT-Prm1 strains induced on different carbon sources. Original gels/blots are provided in Supplementary Fig. S2. (C) Relative AOX activity levels of WT, WT-Mit1, Δmig1Δmig2, Δmig1Δmig2-Mit1, Δmig1Δmig2Δnrg1 and Δmig1Δmig2Δnrg1-Mit1 strains cultured in different carbon sources. Cells of each strain were grown for 24 hours in YND medium and transferred into fresh YNB medium supplemented with different carbon sources (0.5% methanol, 1% glucose and 1% glycerol) at an initial OD₆₀₀ of 1.0. Cells were collected every 3 hours and AOX specific activity was measured in cell extracts prepared as described in materials and methods. The highest AOX specific activity of each strain in different carbon sources was presented. (D) Relative GFP expression levels of WT-GFP, Δmig1Δmig2-Mit1-GFP, Δmig1Δmig2Δnrg1-Mit1-GFP strains during growth on different carbon sources. Cells of each strain were grown for 24 hours in YND medium and transferred into fresh YNB medium supplemented with different carbon sources (0.5% methanol, 1% glucose and 1% glycerol) at an initial OD₆₀₀ of 1.0. Cells were collected every 3 hours and GFP expression was monitored. The highest GFP expression of each strain in different carbon sources was presented.