Disruption of the pfeik1 gene. A. Strategy for gene disruption. The transfection plasmid contains a PCR fragment spanning positions 1467–2255 of the entire 4.8 kb pfeik1 coding sequence (as predicted on PlasmoDB). The fragment excludes two regions essential for catalytic activity, labelled 'ATP' (a glycine-rich region required for orientation of ATP) and 'E' (a glutamate residue required for structural stability of the enzyme). The positions of primers used for genotyping clones, and for nested PCR to genotype oocsyts are indicated by numbered arrows. B: PCR analysis. Genomic DNA isolated from pfeik1- clones C1 and C8, and from 3D7 wild-type parasites, was subjected to PCR using the indicated primers (see Figure 4A for primer locations). Lanes 1, 5, 9: primers 1 + 2 (diagnostic for the wild-type locus); lanes 2, 6, 10: primers 3 + 4 (diagnostic for the pCAM-BSD-PfeIK1 plasmid); lanes 3, 7, 11: primers 1 + 4 (diagnostic for 5' integration boundary); lanes 4,8,12: primers 3 + 2 (diagnostic for 3' integration boundary). M = co-migrating markers. C: Schematic of expected sizes on Southern blot analysis of wild-type 3D7 parasites and pfeik1- parasites. D: Southern blot analysis of the pfeik1 locus in wild-type 3D7 and pfeik1- clones C1 and C8. Genomic DNA was digested with Hind III, transferred to a Hybond membrane and probed with the pfeik1 fragment that was used as the insert in the pCAM-BSD-PfeIK1 plasmid. Positions of the bands corresponding to the wild-type locus (WT), 5' integration (5' int.), 3' integration (3' int.) and linearized plasmid (plasmid) are shown on the right. Sizes of co-migrating markers are indicated on the left.