FIGURE 3.

Disruption and complementation of Tggnt1 and TgpgtA. A, strategy for deletion of Tggnt1 and its subsequent complementation. The plasmid-derived disruption of DNA with homologous targeting sequences was electroporated into parasites. Recovery of hxgprt-positive clones that were resistant to MPA and xanthine and were GFP-negative were candidates for double crossover gene replacement. B, gene replacement was confirmed by PCR-1, which demonstrated loss of gnt1 coding DNA, and PCR-2 and -3, which demonstrated that the inserted hxgprt DNA was flanked by neighboring gnt1 DNA. To complement Tggnt1 in the disruption strain, a plasmid containing an ∼7-kb genomic locus, including Tggnt1 coding region and 5′- and 3′-untranslated regions (A), was transfected. Complemented strains where the hxgprt is replaced by Tggnt1 locus were counter-selected under 6-thioxanthine. C, gnt1 replacement was confirmed by the positive PCR-1 and negative reactions for PCR-2 and PCR-3, which depended on the presence of hxgprt. D–F, TgpgtA was similarly targeted for disruption and complementation. Characteristics of the above strains are summarized in Table 1.