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. 2013 Mar 6;8(3):e58956. doi: 10.1371/journal.pone.0058956

Figure 2. Kar2/BiP is essential for the viability of C. neoformans.

Figure 2

(A) The scheme for the construction of the PCTR4:KAR2 promoter insertion strain. A construct containing the CTR4 promoter and the NAT dominant selectable marker was inserted 8 bp upstream of the ATG start site of the KAR2 gene. Black boxes indicate exons of the KAR2 gene. (B) The correct genotypes of the PCTR4:KAR2 promoter strains [four independent strains (YSB1637, YSB1638, YSB1639, and YSB1640 as labeled 1 to 4, respectively)] compared to the WT H99 strain were confirmed by Southern blot analysis using genomic DNAs digested with the restriction enzyme EcoRΙ. The membrane was hybridized with a KAR2-specific probe, washed, and developed. (C) The WT H99, the PCTR4:KAR2 (YSB1637), and PCTR4:YPD1 (YSB859, a positive control) strains were cultured overnight at 30°C in a liquid YPD medium, 10-fold serially diluted, and spotted onto yeast nitrogen base agar (YNB) medium containing 12.5 µM CuSO4, 100 µM BCS, or 300 µM BCS. Cells were incubated at 30°C for 4 days and photographed.