Fig. 1.
(A) Map of the insert from plasmid pZCF36DBH2, in which the 3× HA-tagged GAL4 activation domain (caGAL4AD-HA) is fused to the MRR1 DNA binding domain (MRR1DB, codons 1 to 128 of MRR1). Relevant restriction sites used for the construction of the plasmid and excision of the cassette (see Materials and Methods) are indicated. PADH1, ADH1 promoter; TACT1, transcription termination sequence of the ACT gene; caSAT1, Candida-adapted nourseothricin resistance marker; 3′ADH1, sequence from the C-terminal part of the ADH1 gene. The flanking ADH1 sequences (PADH1 and 3′ADH1) served for integration into the ADH1 locus by homologous recombination. (B) MDR1 promoter activity in an mrr1Δ mutant carrying a PMDR1-caGFP reporter fusion (strain CAG48MRR1M4B) and in transformants expressing wild-type Mrr1, an HA-tagged, hyperactive Mrr1, or the indicated Mrr1DB-Gal4AD fusion proteins. Cells were grown to log phase, and their mean fluorescence was determined by flow cytometry. The results obtained with two independent transformants are shown in each case (means and standard deviations from three experiments). (C) MICs of fluconazole for the mrr1Δ mutants SCMRR1M4A and SCMRR1M4B and transformants expressing wild-type Mrr1, HA-tagged Mrr1, or the indicated Mrr1DB-Gal4AD fusion proteins. Identical results were obtained with two independent series of strains. (D) Detection of the indicated HA-tagged Mrr1DB-Gal4AD fusion proteins in cell extracts of the corresponding strains by Western immunoblotting with an anti-HA antibody. Cell extracts of strains expressing untagged, wild-type Mrr1 were used as negative controls. A and B denote transformants of strains SCMRR1M4A and SCMRR1M4B, respectively. All samples were analyzed on the same gel, but some irrelevant lanes between lanes 2 and 3 have been removed from the figure.