Yeast strains designed for screening of reversal agents and genetic suppressors of multidrug resistance

A yeast cell-based system for screening Candida glabrata multidrug resistance reversal agents and selection of loss-of-function pdr1 mutants

“…This allele was PCR amplified from a plasmid-borne CgPDR1 sequence of Cg4672 (Tsai et al, 2006), digested with BamHI plus EcoRI and ligated into a truncated pYES2-P GAL1 -PDR3 plasmid (Kozovska et al, 2004) from which the BamHI-EcoRI DNA fragment containing the entire PDR3 gene had been removed. After growth for 12 generations under nonselective conditions in a glucose-rich medium, 26% of the cells retained the plasmid-borne P GAL1 -CgPDR1 gene, indicating the mitotic stability of this newly constructed plasmid was comparable to that of P GAL1 -PDR3 (Kozovska et al, 2004). As shown in Table 1, the galactose-induced expression of CgPDR1 from the P GAL1 promoter rendered yeast cells resistant to cycloheximide [minimum inhibitory concentrations (MIC) = 1.5 mg mL À1 ] and fluconazole (MIC = 8 mg mL À1 ), thus indicating their functional expression in the heterologous genetic background.…”

“…Plasmid DNA from yeast cells was extracted according to Ward (1990). Plasmids pYES2-P GAL1 -PDR3 (2 mm URA3) and pYCp-P PDR5 -pma1(D378N) have been described previously (Kozovska et al, 2004). Plasmids pYES2-P GAL1 -PDR3 (2 mm URA3) and pYCp-P PDR5 -pma1(D378N) have been described previously (Kozovska et al, 2004).…”

“…Yeast transformation was carried out using the modified lithium acetate protocol (Nourani et al, 1997) or by electroporation (Thompson et al, 1997). Plasmids pYES2-P GAL1 -PDR3 (2 mm URA3) and pYCp-P PDR5 -pma1(D378N) have been described previously (Kozovska et al, 2004). Plasmid pYES2 (2 mm URA3) was used as an empty vector to introduce a plasmid-borne URA3 gene into host strains and to clone the CgPDR1 (Cg4672) gain-offunction allele expressed from the P GAL1 promoter.…”

“…Some of them are based on the inhibition of drug efflux transporter activities (Conseil et al, 2000;Monk et al, 2005) or suppression of gene expression preventing their biosynthesis (Gottesman et al, 2002;Sidorova et al, 2007;Stepanov et al, 2008). Recently, we have described a screening system for identification of compounds interfering with the function of the ScPdr3p multidrug resistance transcription factor in S. cerevisiae (Kozovska & Subik, 2003;Kozovska et al, 2004). In this study, we report the development of a heterologous screening system for multidrug resistance reversal agents specifically inhibiting the CgPdr1p function that is essential for the development of multidrug resistance in pathogenic C. glabrata.…”

Site‐directed mutagenesis of Asp853 in Pdr3p transcriptional activator from Saccharomyces cerevisiae

“…Plasmids pFL38-PDR3, pFL38-pdr3-7 (Nourani et al, 1997), pFL38-pdr3-18 (Simonics et al, 2000), pFL38-pdr3-Site-directed mutagenesis of Asp853 in Pdr3p 279 D853Y (Sidorova et al, 2007) and pYES2-PDR3 (Kozovska et al, 2004) served as sources of the PDR3 alleles. Mutations were introduced into pYES2-PDR3 from pFL38-borne pdr3 mutant alleles by exchanging the 2308 bp PacI-XhoI DNA fragment in the PDR3 gene.…”