Figure 2.

Gene replacement construct and targeted disruption of the pakB gene. (A) Schematic diagram shows the pakB gene replacement construct, the wild-type pakB locus and the altered structure of the pakB gene expected if a double crossover, gene replacement event occurs. Black segments correspond to pakB sequences, the white segment to the thy gene, and wavy lines to extragenic sequences. The locations of probes A and B used in the genomic Southern analysis are indicated. (B) Southern blot analysis of JH10 cells and two clones (T1 and T2) that grew in the absence of thymidine. Genomic DNA was digested with EcoRV and hybridized with either probe A or probe B. The increased size of the EcoRV fragment detected with Probe A and the absence of bands hybridizing to Probe B are consistent with the scheme illustrated in A. Molecular size standards are indicated in kilobases. An equal amount of DNA was loaded on each lane of the gel. (C) Extracts from 1 × 10⁶ JH10, T1, or T2 cells were subjected to SDS-PAGE and stained with Coomassie Blue (CB) or transferred to Immobilon-P and probed with an anti-PakB antibody (PakB). Molecular size standards are indicated in kilodaltons.