FIG. 1.

Disruption of PRA1 (orf19.3111). (A) PCR-based cassette method for disruption of PRA1 in two steps. The thick black bar represents genomic DNA at the PRA1 locus, and the white rectangles represent the PRA1 gene coding sequence. The PCR cassettes used for the disruption are composed of a selectable marker (gray ovoid rectangle) flanked by two 80-nucleotide segments from the PRA1 5′ UTR or 3′ UTR locus (small white rectangles) for the homologous recombination of the cassettes. The first allele of PRA1 was replaced by the HIS1 marker, and the second allele was replaced by the URA3 marker. Small arrows represent the orientations and approximate positions of oligonucleotides (Table 2) used for PCR analysis and confirmation of the disruption. (B) Confirmation of disruption by PCR. The parent strain RM1000, the first-allele-disrupted strain CAM33, and the second-allele-disrupted strain CAM35 were analyzed by PCR using the oligonucleotides described for panel A. A 1-kb DNA ladder (Invitrogen, Carlsbad, CA) was used for size reference. PCR with oligonucleotides oAM86 and oAM87 produced a 2.8-kb DNA fragment for the PRA1 wild-type allele, as seen for strains RM1000 and CAM33 (lanes 3 and 8), and a 3.2-kb fragment when PRA1 was replaced by the HIS1 marker, as seen for strains CAM33 and CAM35 (lanes 8 and 13), or the URA3 marker, as seen for strain CAM35 (lane 13). The proper integration of the markers was confirmed using external oligonucleotides of the PRA1 locus (oAM86 or oAM87) together with internal oligonucleotides for the markers HIS1 (H2 or H1, respectively) (lanes 1,2, 6, 7, 11, and 12) and URA3 (U2 or U1, respectively) (lanes 4, 5, 9, 10, 14, and 15).