ANALYSIS OF THE PLASMODIUM-VIVAX DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE GENE SEQUENCE

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifuncti onal gene encoding dihydrofolate reductase-thymidylate synthase (DHFR- TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P . vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino aci ds (aa). Alignment with other malarial DHFR-TS genes showed that a 237 -residue DHFR domain and a 286-residue TS domain were separated by a 1 00-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respec tively, whereas an extensive isology was observed in the TS domain. Th e characteristic features of the P. vivax DHFR-TS gene sequence includ e an insertion of a short repetitive tandem array within the DHFR doma in that is absent in another human malaria parasite, P. falciparum, an d a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8 %), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5' noncoding region revealed the p resence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at -636. Comparison of the DH FR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser much less than Arg -58 and Ser much less than Asn-117. These aa residues correspond to co dons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys cr Arg-59 and Ser cr Asn-108) are associated wi th pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine. (C) 1998 Elsevi er Science B.V. All rights reserved.