Cowman A F, Morry M J, Biggs B A, Cross G A, Foote S J
Walter and Eliza Hall Institute of Medical Research, Victoria, Australia.
Proc Natl Acad Sci U S A. 1988 Dec;85(23):9109-13. doi: 10.1073/pnas.85.23.9109.
We describe the isolation and the sequence of the gene for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS; EC 1.5.1.3 and EC 2.1.1.45, respectively) from two pyrimethamine-resistant clones of Plasmodium falciparum, HB3 and 7G8. We have also derived the sequence of the DHFR portion of the gene, by amplification using polymerase chain reaction, for the pyrimethamine-sensitive clone 3D7 and the pyrimethamine-resistant strains V-1, K-1, Csl-2, and Palo-alto. The deduced protein sequence of the resistant DHFR portion of the enzyme from HB3 contained a single amino acid difference from the pyrimethamine-sensitive clone 3D7. It is highly likely that this difference is involved in the mechanism of drug resistance in HB3. The sequence of the DHFR gene from other pyrimethamine-resistant strains contains the same amino acid difference from the sensitive clone 3D7. However, they all differ at one other site that may influence pyrimethamine resistance. The DHFR-TS gene is present as a single copy on chromosome 4 in all pyrimethamine-sensitive and pyrimethamine-resistant isolates tested. Therefore, the molecular basis of pyrimethamine resistance in the parasites tested is not amplification of the DHFR-TS gene.
我们描述了从恶性疟原虫的两个耐乙胺嘧啶克隆HB3和7G8中分离出双功能酶二氢叶酸还原酶-胸苷酸合成酶（DHFR-TS；分别为EC 1.5.1.3和EC 2.1.1.45）的基因并测定其序列。我们还通过聚合酶链反应扩增，得到了乙胺嘧啶敏感克隆3D7以及耐乙胺嘧啶菌株V-1、K-1、Csl-2和帕洛阿尔托的该基因DHFR部分的序列。HB3中该酶抗性DHFR部分的推导蛋白质序列与乙胺嘧啶敏感克隆3D7相比，有一个氨基酸差异。极有可能这个差异与HB3的耐药机制有关。其他耐乙胺嘧啶菌株的DHFR基因序列与敏感克隆3D7相比，都含有相同的氨基酸差异。然而，它们在另一个可能影响乙胺嘧啶抗性的位点上都有所不同。在所测试的所有乙胺嘧啶敏感和耐乙胺嘧啶分离株中，DHFR-TS基因在4号染色体上均以单拷贝形式存在。因此，所测试寄生虫中乙胺嘧啶抗性的分子基础不是DHFR-TS基因的扩增。
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