FIG. 2.

Construction of CAT1 null mutants and a revertant. The CAT1 genes of the wt and the tsa1Δ mutant (30, 31) were disrupted using URA3-dpl200 (32), yielding the cat1Δ and tsa1Δ cat1Δ mutants, respectively. The sense and antisense primers were nucleotide positions 754 to 823 and 2312 to 2381, respectively, of the CAT1 open reading frame (ORF). To construct a revertant, the DNA fragment containing its own promoter, ORF, and terminator was cloned into pLUX, linearized with NheI, and transformed into the cat1Δ mutant. Southern (A) and Northern (B) analyses were performed to confirm the authenticity of the constructed strains, using the ³²P-labeled probe prepared from the MfeI fragment of the CAT1 ORF. For the Southern analyses, genomic DNA was digested with NsiI and NcoI. Lanes 1, parental strains (CAI4 and the tsa1Δ mutant in panels A and B, respectively); lanes 2, strains with one allele disrupted; lanes 3, strains with URA3 popped out from the lane 2 strains; lanes 4, null mutants (the cat1Δ and tsa1Δ cat1Δ mutants in panels A and B, respectively); lanes 5, strains with URA3 popped out from the cat1Δ mutant; lanes 6, CAT1-reintroduced strains of the cat1Δ mutant.