INDUCTION OF GENE AMPLIFICATION IN PLASMODIUM FALCIPARUM (METHOTREXATE, DIHYDROFOLATE REDUCTASE, MALARIA, THYMIDYLATE SYNTHETASE, RESISTANCE)

Human erythrocytic in vitro cultures of the Honduras I strain of the malaria parasite Plasmodium falciparum have been stressed stepwise with increasing concentrations of methotrexate (MTX), a folate antagonist. This selection has produced a strain that is 450 times more resistant to the drug than the original culture. Uptake of sublethal doses of radiolabeled MTX by infected red blood cells was 6-36 times greater in the resistant cultures than in the nonresistant controls. DNA isolated from all of the parasites was probed by hybridization with ('35)S-labeled DNA derived from a clone of the yeast thymidylate synthetase (TS) gene. This showed 50 to 100 times more copies of the gene in the resistant parasites than in the control. The increased hybridization of the TS probe to the DNA from the resistant parasites is direct evidence of gene amplification because DHFR and TS are actually one and the same bifunctional enzyme in P. falciparum. Hence, the evidence presented indicates that induced resistance of the malaria parasite to MTX in this case is due to overproduction of DHFR resulting from amplification of the DHFR-TS gene.