Figure 1.—

Construction of the P_CTR4∷CAN2 promoter replacement strain. (A) The strategy for construction of the CAN2 promoter replacement allele with the CTR4 promoter. The first five exons of the CAN2 gene are illustrated as open boxes and an arrow for exon 6 depicts the direction of transcription. Primers for overlap PCR and diagnostic PCR are indicated as bent arrows. The shaded arrow box illustrates a NAT selectable marker, which consists of ACT1 promoter, NAT (nourseothricin acetyltransferase) gene, and TRP1 terminator, and the shaded, striped arrow box illustrates the CTR4 promoter as previously described (Ory et al. 2004). (B) Verification of the P_CTR4∷CAN2 strain by Southern hybridization. Each genomic DNA was digested with XbaI and XhoI and blotted membrane was probed with CAN2-specific probe that was PCR amplified with primers B359 and B93. WT indicates the H99 strain and lanes 1 to 4 indicate the independently isolated P_CTR4∷CAN2 strains (YSB733, YSB734, YSB735, and YSB736, respectively). (C) Northern hybridization of the controlled CAN2 expression by the CTR4 promoter. The WT H99 strain and the P_CTR4∷CAN2 strain (YSB734) were grown overnight at 30° in liquid YPD medium and subcultured into a fresh YNB liquid medium containing 200 μm BCS (+BCS) and 25 μm CuSO4 (+Cu). After a 12 hr-incubation, a portion of cultures was sampled and its total RNAs was isolated for Northern blot analysis as described in materials and methods.