title: Mutant dihydrofolate reductase-thymidylate synthase genes in pyrimethamine-resistant Plasmodium falciparum with polymorphic chromosome duplications.
abstract: We have identified dihydrofolate reductase (DHFR) gene point mutations and chromosomal changes in pyrimethamine-resistant mutants selected in vitro of Plasmodium falciparum strain FCR3. A pyrimethamine-resistant derivative of the pyrimethamine-sensitive strain FCR3, FCR3-D8, that had been grown in the absence of pyrimethamine for an extended time, was grown in two concentrations of pyrimethamine, and surviving drug-resistant parasites were subcloned. One selected mutant, FCR3-D81, that grew at 1 X 10(-6) M pyrimethamine, contained a single point mutation in the DHFR domain which caused an amino acid change (Phe to Ser) at amino acid 223, whereas another mutant, FCR3-D85, that grew at 5 X 10(-6) M pyrimethamine had that same mutation and an additional point mutation that changed amino acid 54 (Asp to Asn). The selection of FCR3-D85, whose nucleotide sequence was identical to that previously reported for FCR3-D8, confirmed that the original FCR3-D8 parasite population had changed during extended growth in vitro in the absence of drug pressure. FCR3-D81 and FCR3-D85 cells contained different pairs of polymorphic chromosomes that hybridized to a DHFR-TS probe as well as to three other chromosome 4 specific DNAs, indicating that at least part of chromosome 4 had been duplicated and that these parasites were aneuploid with 15 rather than 14 chromosomes. The mutant DHFR-TS genes were diploid. We consider the roles of the polymorphic chromosome duplications and DHFR point mutation(s) as causes of pyrimethamine resistance.
authors.affiliation: Department of Microbiology, Dartmouth Medical School, Hanover, NH 03756.
authors.forename: M
authors.initials: M
authors.lastname: Tanaka
authors.forename: H M
authors.initials: HM
authors.lastname: Gu
authors.forename: D J
authors.initials: DJ
authors.lastname: Bzik
authors.forename: W B
authors.initials: WB
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Inselburg
mesh_terms: D000818:Animals; D001483:Base Sequence; D002875:Chromosomes / Q000648:ultrastructure; D016054:DNA, Protozoan / Q000737:chemistry; D004351:Drug Resistance; D007621:Karyotyping; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D011110:Polymorphism, Genetic; D011739:Pyrimethamine / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*
chemical_list: D016054:DNA, Protozoan; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D011739:Pyrimethamine