FIG. 3.
Genomic analysis of the Δgra2 knockout and complemented clones. (A) Southern blot analysis showing that the knockout clones lack the GRA2 open reading frame, in contrast to the wild type (WT). The knockout clones were complemented by nonhomologous integration of plasmid pG43 into the T. gondii genome. Genomic DNA was digested with NcoI and hybridized with the GRA2 genomic fragment (SalI-BstEII) of 544 bp corresponding to most of the GRA2 open reading frame. Two fragments (a 4.5-kb fragment and a faintly hybridizing 5.4-kb fragment indicated by *) are detected in the wild type; in the complemented clones these fragments are of different sizes, reflecting their integration into different genomic locations. (B) Knockout clones B2 and C12 were derived from a single integration of the ble gene at the GRA2 locus, whereas knockout clone C1 integrated three additional copies of the selectable marker. Genomic DNA was digested with NsiI and hybridized with a 480-bp fragment (NsiI-PacI) corresponding to the ble open reading frame. Digestion of plasmid pGRA2/Ble/GRA2(8.9) (P) liberates a hybridizing fragment of 1 kb, due to the presence of an NsiI site in the 3′ flanking region of GRA2.