Pyrimethamine resistant Plasmodium falciparum: overproduction of dihydrofolate reductase by a gene duplication.
The accumulation of [3H]pyrimethamine by pyrimethamine-resistant (Pyrr) mutants of the Plasmodium falciparum strain FCR3 was examined by measuring the accumulation of drug in infected red blood cells. [3H]Pyrimethamine was stage specifically accumulated in trophozoites and schizont infected red blood cells. The mutant parasites accumulated drug as efficiently as FCR3. Pyrimethamine was associated with a high molecular weight protein that eluted from a Sephadex G200 column exactly as [3H]fluorodeoxyuridinemonophosphate (FdUMP) labeled parasite dihydrofolate reductase-thymidylate synthetase (DHFR-TS) enzyme. These results suggested that the pyrimethamine resistance was not associated with decreased drug permeability of the membrane. DHFR-TS-[3H]FdUMP enzyme complex of all the Pyrr mutants and FCR3 had a monomer of 70 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One highly resistant mutant, FCR3-D7, exhibited a 5-10 fold higher uptake of pyrimethamine and a proportionately higher amount of DHFR-TS protein than FCR3 but only a normal level of DHFR activity. The genomic DNA of FCR3-D7 was shown to contain at least twice as much DHFR-TS specific DNA than either FCR3-D8, another Pyrr mutant, or FCR3. Preliminary results suggested some of the DHFR-TS genetic material in FCR3-D7 is associated with a gene duplication.
Inselburg
J
J
Department of Microbiology, Dartmouth Medical School, Hanover, NH 03756.
Bzik
D J
DJ
Horii
T
T
D000818:Animals; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D005810:Multigene Family; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine