FIG. 3.

One-step disruption of the DAS1 gene in C. boidinii. (A) Restriction map of the cloned fragment and disruption strategy. The lightly shaded boxes and arrows at both ends of URA3 show repeated sequences for homologous recombination to remove the URA3 gene after the gene disruption. (B) Genomic Southern analysis of EcoRI-digested total DNAs (3 μg of each) from the host strain TK62 (lane 1), the das1Δ strain (lane 2), and the das1Δ ura3 strain (lane 3) probed with the ³²P-labeled 1.8-kb EcoRI-EcoRV fragment, including the 5′-flanking region of DAS1. (C) Immunoblot analysis of strain TK62 (lane 1), the das1Δ strain (lane 2), and the das1Δ ura3 strain (lane 3) with extracts of methanol-induced cells and anti-DHAS polyclonal antibody.