Construction of C. albicans strains for Dox-inducing the expression of assorted CaCDC4 domains. (A) Schematic representation of Ca Cdc4 domains expressed from the Tet-on system. The strains with which they are derived and predicted Tet-on cassette size are shown (B) Generation of Tet-on cassettes for expressing assorted CaCdc4 domains. Different portions of CaCDC4 were PCR-generated with primer sets (Table 2) containing common Aat II and Xho I sites for replacing full-length CaCDC4 on pTET25M-CaCDC4-6HF as described in the Methods. By digestion with Sac II and Kpn I, each cassette was used to transform C. albicans strain JSCA0022 for integration into the CaADH1 locus. (C) Verification of Tet-on cassettes being integrated into CaADH1 locus by Southern blotting analysis. Organization of the CaADH1 locus with respect to Spe I sites is shown in Figure 3B. The relative positions of the probe used and the predicted Spe I-digested pattern of the CaADHI locus are indicated in Figure 3B. One Spe I-fragment of 3.3 kb specific to CaADH1 locus could be detected in genomic DNA from strain JSCA0022 and its derivatives digested with Spe I. The correctness of integration of the cassette into the CaADH1 locus of various strains was confirmed by alteration of the Spe I-fragment from size of 3.3 kb to 9.4 kb (Figure 3B) or various sizes as indicated in Figure 3A.