Fig. 6. Disruption of the LPG3 gene and analysis of OB1 LPG3 mutations. (A) Targeting construct for the disruption of LPG3. The open box represents the NEO resistance cassette, dark shading indicates the LPG3 ORF, and the hatched region represents 5′ flanking sequence. (B) Southern blot analysis of genomic DNA digested with NotI + AscI. The probe consisted of the 0.85 kb EcoRV–BamHI fragment from the LPG3 ORF (see Figure 3D). Lane 1, WT; lane 2, OB1; lanes 3–5, LPG-negative lpg3^– null mutants. (C) Genomic DNA from WT (lanes 1 and 3) or OB1 (lanes 2 and 4) were digested with either NotI + XmaI (lanes 1 and 2) or NotI + XmaI + SalI (lanes 3 and 4). The restriction fragments were electrophoresed in a 0.85% agarose gel and transferred onto a Gene Screen Plus membrane. The membrane was probed with the ³²P-labeled 1.0 kb NotI–BamHI fragment from the LPG3 locus (see Figure 3) spanning the mutated site (nucleotide 476) of the LPG3^OB1 allele.