FIG. 2.

Construction of a tetracycline-sensitive mutant of CgKRE9 (Tet^s CgKRE9). (A) Scheme for replacement of the cognate CgKRE9 promoter region with the tetracycline-responsive promoter. A PCR-amplified fragment (double-headed arrow) from pCGK9tetAB (Materials and Methods) was used for the one-step gene replacement. The solid arrow indicates the ORF of CgKRE9. Open and shaded boxes indicate the S. cerevisiae URA3 gene and the tetracycline-responsive promoter, 97t, respectively. Homologous recombination between the two regions (hatched boxes) resulted in generation of the Tet^s CgKRE9 mutant. (B) Growth inhibition by tetracycline on the Tet^s CgKRE9 mutants. A total of 10⁴ cells were inoculated and were cultured on YPD (solid bars) or on YPGal (open bars) for 20 h at 30°C. Growth of cells with tetracycline (50 μg/ml) is expressed as percent of optical density at 600 nm of cells without tetracycline. As the wild type (WT), strain ACG22 (Table 1) was used. Error bars, standard deviations.