Generation of P. falciparum D10Δgdha as confirmed by Southern blot, western blot and enzymatic activity measurement. (A) Schematic diagram of the endogenous gdha gene locus, the pHH1-Δgdha plasmid and the recombined gdha locus, following single cross-over recombination between the plasmid and an 841 bp region of gdha (D10-Δgdha). The plasmid contains a human dihydrofolate reductase (hDHFR) selectable marker under control of the P. falciparum calmodulin promoter (5') and flanked by the P. falciparum histidine rich protein II 3' UTR (3'), a region homologous to gdha (Δgdha) and an artificial 3' UTR (P. berghei dihydrofolate reductase/thymidylate synthase 3'UTR, PbDT-3'). NdeI restriction sites and sizes of the resulting diagnostic DNA fragments are indicated (in bold). (B) Southern blot of D10 wild type parasites (lane 1), D10Δgdha(lane 2) and two D10Δgdhaclones (lanes 3 and 4). The 5.3 kb endogenous gdha fragment is visible in the wild type and D10Δgdhamixed population of parasites before cloning (lanes 1 and 2). In the latter, plasmid (5.8 kb) and the two integration fragments (7.2 kb and 4.0 kb) are also detected. These fragments are still detected in the two clones, D10Δgdha-1 and D10Δgdha-2 (lanes 3 and 4). (C) Western blot of D10, D10Δgdha-1 and D10Δgdha-2 protein extracts verifying the absence of GDHa (49 kDa) in D10Δgdha-1 and D10Δgdha-2. The protein reacting non-specifically with the anti-GDHa antibody (~32 kDa) serves as a loading control. (D) Activity gel of D10, D10Δgdha-1 and D10Δgdha-2 protein extracts showing GDHa activity in D10 only. Purified recombinant GDHa was included as control.