Fig 4.

Construction of conditional mutants of endogenous ERG1 or TRP5. P_CTR4 was inserted directly upstream of target genes by ATMT and confirmed by Southern blotting. (A) Schematic representation of homologous recombination at the ERG1 locus. Restriction sites and sizes of expected fragments on Southern blotting are shown. Sp, SpeI; K, KpnI; Sa, SacI; H, HindIII; Hyg^r, hygromycin B resistance cassette; L arm, 5′ homologous sequence 2 kb upstream of the start codon of ERG1; R arm, 2-kb 3′ homologous sequence beginning at the start codon of ERG1; ORF, open reading frame. Parts of host DNA which are homologous to the L and R arms are shown as a thick line. (B) Total DNA from each strain was digested with HindIII and separated by gel electrophoresis. Southern blotting hybridization was performed as described in Materials and Methods. A partial fragment of ERG1 (length, 415 bp) or (C) TRP5 (length, 414 bp) was used as a hybridization probe.