Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum.
The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.
Zolg
J W
JW
Department of Molecular Biology, Biomedical Research Institute, Rockville, MD.
Plitt
J R
JR
Chen
G X
GX
Palmer
S
S
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004351:Drug Resistance / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011739:Pyrimethamine / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D011739:Pyrimethamine