Journal: bioRxiv

Article Title: Azole resistance is mediated by integration of sterol gene regulation and membrane transporter production by the zinc cluster-containing transcription factor Upc2A in Candida glabrata

doi: 10.1101/2021.05.07.443069

Figure Lengend Snippet: A. Normal expression of lacZ gene fusions requires the presence of intact SREs in Upc2A target promoters. Low-copy-number plasmids containing translational fusions between ERG1, CDR1 and PDR1 5’ regulatory region and E. coli lacZ were generated containing either the wild-type version of each promoter or the same fragment with the SRE mutant (mSRE) shown to reduce in vitro binding in . These plasmids were introduced into wild-type cells, grown in the absence or presence of fluconazole and then β-galactosidase activity determined. B. Western blot analysis of CDR1 and PDR1 expression upon loss of the wild-type SRE. All alleles of PDR1 were integrated into the chromosome while all alleles of CDR1 were carried on a low-copy-number plasmid. The presence of either the wild-type (wt) or mutant (m) SRE at each gene is indicated at the bottom of the panel. Each strain was grown in the presence or absence of fluconazole and levels of proteins of interest determined using western blotting with appropriate antibodies as described above. Erg11 was detected using an anti-peptide antiserum. The right hand panel shows the quantitation as described in . C. The strains described above were tested by serial dilution for their growth on rich medium (YPD) or the same medium containing fluconazole (FLC).

Article Snippet: Cultures were then 10-fold serially diluted and spotted onto YPD agar plates containing different concentrations [10 or 20 μg/ml] of fluconazole (LKT laboratories, St Paul, MN), caspofungin 100 ng/ml (Apexbio, Houston, TX), congo red 100 μg/ml (Sigma-Aldrich, St. Louis, MO), caffeine (Sigma-Aldrich).

Techniques: Expressing, Low Copy Number, Generated, Mutagenesis, In Vitro, Binding Assay, Activity Assay, Western Blot, Plasmid Preparation, Quantitation Assay, Serial Dilution