The Transcription Factor Mrr1p Controls Expression of the MDR1 Efflux Pump and Mediates Multidrug Resistance in Candida albicans
Figure 2
Construction of mrr1Δ Mutants and Complemented Strains
(A) Structure of the deletion cassette from plasmid pZCF36M2 (top), which was used to delete the MRR1 ORF in strains SC5314, F5, G5, and CAG48B, and genomic structure of the MRR1 locus in the parental strains (bottom). The MRR1 coding region is represented by the white arrow and the upstream and downstream regions (5′MRR1 and 3′MRR1) by the solid lines. The SAT1 flipper cassette (SAT1-FLIP), in which the caFLP gene is expressed from the inducible SAP2 promoter [30], is represented by the grey rectangle bordered by FRT sites (black arrows). The 34 bp FRT sites are not drawn to scale. The probes used for Southern hybridization analysis of the mutants are indicated by the black bars.
(B) Structure of the DNA fragments from plasmids pZCF36K2, pZCF36K3, pZCF36K4, and pZCF36K5 (top), which were used for integration the MRR1F2–1, MRR1F5, MRR1G2–2, and MRR1G5 alleles, respectively, into the disrupted mrr1 locus of homozygous and heterozygous mrr1Δ mutants (bottom) using the caSAT1 selection marker (grey arrow). TACT1, transcription termination sequence of the ACT1 gene.
Only relevant restriction sites are given in (A) and (B): A, ApaI; B, BglII; E, EcoRI; N, NsiI; Nd, NdeI; P, PstI; S, SalI; ScI, SacI; ScII, SacII; X, XhoI. The PstI and NsiI sites shown in parenthesis were destroyed by the cloning procedure.
(C) Southern hybridization of NsiI-digested genomic DNA of mrr1Δ mutants derived from strain SC5314 and of strains with reinserted MRR1 alleles with the MRR1-specific probe 1. The sizes of the hybridizing fragments (in kb) are given on the left side of the blot and their identities are indicated on the right. The genotype of the strains is given above the respective lanes. Only one of the two independently constructed series of strains is shown. Inactivation of MRR1 in the clinical isolates F5 and G5 and the reporter strain CAG48B and reinsertion of different MRR1 alleles in mrr1 mutants of the reporter strain occurred in an analogous fashion.