FIG. 2.
Disruption of PMR1. (A) Restriction map of pPD602 containing PMR1 and schematic diagram of disruption of PMR1. Black arrow, ORF of PMR1; open boxes, flanking regions of PMR1; bold striped boxes, Walker motifs; black lines, probes for Southern and Northern blot analysis; filled boxes, ORF. Disruption vector pUNE10 contains the hygromycin B resistance gene cassette (Hygr) (thin striped box) flanked by the 5′ upstream region and the 3′ end of the PMR1 gene. Double homologous recombination between the PMR1 locus and the disruption vector pUNE10 results in gene replacement. Restriction enzymes are as follows: At, ArtI; RI, EcoRI; RV, EcoRV; and Sm, SmaI. (B) Southern blot analysis of Δpmr1 mutants. The genomic DNAs extracted from ectopic transformant TF01 (lane 1); Δpmr1 mutants DIS07, DIS33, and DIS96 (lanes 2 to 4); parental strain LC2 (lane 5); and DMI-sensitive strain PD5 were digested with EcoRV and separated in a 0.8% agarose gel. The blot was probed with probe 1 (2.5-kb SmaI-ArtI fragment) and was reprobed with probe 2 (2.3-kb EcoRI fragment).