Figure 1

Construction of a C. albicans strain for repressibly expressing CaCDC4 . (A) Strain construction (detailed in the Methods). The first CaCDC4 allele on BWP17 was deleted by mini-Ura-blaster to obtain JSCA0018. Plasmid pFA-HIS1-MET3p-CaCDC4 containing partial CaCDC4 coding sequence was linearized at a unique site for introducing into strain JSCA0018 to generate JSCA0021. 5-FOA was used to counter-select CaURA3 removal to obtain JSCA0022 for re-introducing the Tet-on plasmid with a CaURA3 marker. (B) Verification of constructed strains by Southern blotting analysis. Organization of the CaCDC4 locus with respect to Nde I sites is shown. The relative positions of the probe used and the predicted Nde I-digested pattern of the CaCDC4 locus are indicated. Two Nde I-fragments of 14 kb and 8.5 kb, specific to CaCDC4, could be detected in genomic DNA from BWP17 digested with Nde I; two Nde I-fragments of 8.5 kb and 4.5 kb, specific to CaCDC4 and Cacdc4::URA3-dpl200, respectively, could be detected in JCSA0018; two Nde I-fragments of 4.5 kb and 7.4 kb specific to Cacdc4::URA3-dpl200 and Cacdc4::P _MET3 -CDC4:HIS1, respectively, could be detected in JSCA0021; and two Nde I-fragments of 13.5 kb and 7.4 kb specific to Cacdc4::dpl200 and Cacdc4::P _MET3 -CDC4:HIS1, respectively, could be detected in JSCA0022. A non-specific Nde I-fragment is indicated as “*” can be detected in all strains tested.