The identification of a drug-resistance gene from the PACv2 library made from pyrimethamine-resistant P. chabaudi. (A) Wild-type P. berghei was transfected with the PACv2 library made from pyrimethamine-resistant P. chabaudi. The parasitemia was monitored every 24 h after transfection. The arrows indicate the initiation and the termination of the drug treatment. (B) CHEF electrophoresis of the selected parasites by screening with pyrimethamine was performed, followed by Southern blot analysis using the gfp gene as the probe. (Lane L) PACv2 library in the total population of drug-resistant parasites. (Lanes 1–4) Clonal drug-resistant parasites with the PACv2 construct containing the resistance gene. (C) The inserted DNA fragment in the PACv2 from the drug-resistant parasites obtained from the PACv2 library is schematically shown. (Top) Genomic sequence surrounding PCHAS_072830, the dhfr-ts gene, on chromosome 7 of P. chabaudi, and the Sau3AI recognition sites are also shown as S. (Blue arrow) Genomic locus of the end of the inserted DNA fragment, which was identified by genome walking analysis.
