FIG. 1.

Construction of upc2Δ mutants and strains expressing the UPC2^S1-1 and UPC2^S2-1 alleles. (A) Structure of the deletion cassette from plasmid pUPC2M2 (top), which was used to delete the UPC2 ORF in strain SC5314, and genomic structure of the UPC2 locus in the parental strain (bottom). The UPC2 coding region is represented by the white arrow and the upstream (UPC2_up) and downstream (UPC2_down) regions by the solid lines. The SAT1 flipper cassette (SAT1-FLIP) is represented by the gray rectangle bordered by FRT sites (black arrows). The 34-bp FRT sites are not drawn to scale. The probes used for Southern hybridization analysis of the mutants are indicated by the black bars. (B) Structure of the DNA fragments from plasmids pUPC2K2 and pUPC2K3 (top), which were used for integration of the UPC2^S1-1 and UPC2^S2-1 alleles, respectively, into the remaining wild-type UPC2 locus (not shown) or the disrupted upc2Δ locus of the heterozygous upc2 mutants (bottom) using the caSAT1 selection marker (gray arrow). T_ACT1, transcription termination sequence of the ACT1 gene. A, ApaI; Bg, BglII; N, NdeI; P, PstI; S, SpeI; ScI, SacI; ScII, SacII; X, XhoI. Only relevant restriction sites are given for panels A and B. The SpeI site shown in parentheses is present only on the chromosome containing the UPC2-1 allele in strain SC5314. (C) Southern hybridization of SpeI-digested genomic DNA of the wild-type strain SC5314 (lane 1), heterozygous (lanes 2 and 3) and homozygous (lanes 4 and 5) upc2Δ mutants, and strains with reinserted UPC2 alleles (lanes 6 to 13) with the UPC2-specific probe 1. The sizes (in kb) of the 1-kb ladder (lane M), which was also labeled, are shown on the left side of the blot. The positions of the original wild-type UPC2 alleles and the inactivated upc2Δ alleles are shown on the right side of the blot.