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. 1987 Dec;84(23):8360–8364. doi: 10.1073/pnas.84.23.8360

Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

D J Bzik 1, W B Li 1, T Horii 1, J Inselburg 1
PMCID: PMC299542  PMID: 2825189

Abstract

Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banyal H. S., Inselburg J. Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants. Exp Parasitol. 1986 Aug;62(1):61–70. doi: 10.1016/0014-4894(86)90008-1. [DOI] [PubMed] [Google Scholar]
  2. Belfort M., Maley G., Pedersen-Lane J., Maley F. Primary structure of the Escherichia coli thyA gene and its thymidylate synthase product. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4914–4918. doi: 10.1073/pnas.80.16.4914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bellisario R. L., Maley G. F., Galivan J. H., Maley F. Amino acid sequence at the FdUMP binding site of thymidylate synthetase. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1848–1852. doi: 10.1073/pnas.73.6.1848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beverley S. M., Ellenberger T. E., Cordingley J. S. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2584–2588. doi: 10.1073/pnas.83.8.2584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen M. J., Shimada T., Moulton A. D., Cline A., Humphries R. K., Maizel J., Nienhuis A. W. The functional human dihydrofolate reductase gene. J Biol Chem. 1984 Mar 25;259(6):3933–3943. [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  7. Chu F. K., Maley G. F., Maley F., Belfort M. Intervening sequence in the thymidylate synthase gene of bacteriophage T4. Proc Natl Acad Sci U S A. 1984 May;81(10):3049–3053. doi: 10.1073/pnas.81.10.3049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deininger P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem. 1983 Feb 15;129(1):216–223. doi: 10.1016/0003-2697(83)90072-6. [DOI] [PubMed] [Google Scholar]
  9. Diggens S. M., Gutteridge W. E., Trigg P. I. Altered dihydrofolate reductase associated with a pyrimethamine-resistant Plasmodium berghei berghei produced in a single step. Nature. 1970 Nov 7;228(5271):579–580. doi: 10.1038/228579a0. [DOI] [PubMed] [Google Scholar]
  10. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Ferone R., Burchall J. J., Hitchings G. H. Plasmodium berghei dihydrofolate reductase. Isolation, properties, and inhibition by antifolates. Mol Pharmacol. 1969 Jan;5(1):49–59. [PubMed] [Google Scholar]
  13. Ferone R., Roland S. Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5802–5806. doi: 10.1073/pnas.77.10.5802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freisheim J. H., Bitar K. G., Reddy A. V., Blankenship D. T. Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme. J Biol Chem. 1978 Sep 25;253(18):6437–6444. [PubMed] [Google Scholar]
  15. Garrett C. E., Coderre J. A., Meek T. D., Garvey E. P., Claman D. M., Beverley S. M., Santi D. V. A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. Mol Biochem Parasitol. 1984 Apr;11:257–265. doi: 10.1016/0166-6851(84)90070-7. [DOI] [PubMed] [Google Scholar]
  16. Gross R. H. A DNA sequence analysis program for the Apple Macintosh. Nucleic Acids Res. 1986 Jan 10;14(1):591–596. doi: 10.1093/nar/14.1.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grumont R., Washtien W. L., Caput D., Santi D. V. Bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica: sequence homology with the corresponding monofunctional proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5387–5391. doi: 10.1073/pnas.83.15.5387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hardy L. W., Finer-Moore J. S., Montfort W. R., Jones M. O., Santi D. V., Stroud R. M. Atomic structure of thymidylate synthase: target for rational drug design. Science. 1987 Jan 23;235(4787):448–455. doi: 10.1126/science.3099389. [DOI] [PubMed] [Google Scholar]
  19. Honess R. W., Bodemer W., Cameron K. R., Niller H. H., Fleckenstein B., Randall R. E. The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3604–3608. doi: 10.1073/pnas.83.11.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ikemura T. Codon usage and tRNA content in unicellular and multicellular organisms. Mol Biol Evol. 1985 Jan;2(1):13–34. doi: 10.1093/oxfordjournals.molbev.a040335. [DOI] [PubMed] [Google Scholar]
  21. Kenny E., Atkinson T., Hartley B. S. Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T. Gene. 1985;34(2-3):335–342. doi: 10.1016/0378-1119(85)90142-8. [DOI] [PubMed] [Google Scholar]
  22. Maley G. F., Bellisario R. L., Guarino D. U., Maley F. The primary structure of Lactobacillus casei thymidylate synthetase. III. The use of 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine and limited tryptic peptides to establish the complete amino acid sequence of the enzyme. J Biol Chem. 1979 Feb 25;254(4):1301–1304. [PubMed] [Google Scholar]
  23. Maley G. F., Maley F., Baugh C. M. Studies on identifying the folylpolyglutamate binding sites of Lactobacillus casei thymidylate synthetase. Arch Biochem Biophys. 1982 Jul;216(2):551–558. doi: 10.1016/0003-9861(82)90244-2. [DOI] [PubMed] [Google Scholar]
  24. Masters J. N., Attardi G. The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase. Gene. 1983 Jan-Feb;21(1-2):59–63. doi: 10.1016/0378-1119(83)90147-6. [DOI] [PubMed] [Google Scholar]
  25. McCutchan T. F., Welsh J. A., Dame J. B., Quakyi I. A., Graves P. M., Drake J. C., Allegra C. J. Mechanism of pyrimethamine resistance in recent isolates of Plasmodium falciparum. Antimicrob Agents Chemother. 1984 Nov;26(5):656–659. doi: 10.1128/aac.26.5.656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  27. Perryman S. M., Rossana C., Deng T. L., Vanin E. F., Johnson L. F. Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme. Mol Biol Evol. 1986 Jul;3(4):313–321. doi: 10.1093/oxfordjournals.molbev.a040400. [DOI] [PubMed] [Google Scholar]
  28. Purohit S., Mathews C. K. Nucleotide sequence reveals overlap between T4 phage genes encoding dihydrofolate reductase and thymidylate synthase. J Biol Chem. 1984 May 25;259(10):6261–6266. [PubMed] [Google Scholar]
  29. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sherman I. W. Biochemistry of Plasmodium (malarial parasites). Microbiol Rev. 1979 Dec;43(4):453–495. doi: 10.1128/mr.43.4.453-495.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sirawaraporn W., Yuthavong Y. Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi. Mol Biochem Parasitol. 1984 Mar;10(3):355–367. doi: 10.1016/0166-6851(84)90033-1. [DOI] [PubMed] [Google Scholar]
  33. Smith D. R., Calvo J. M. Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. Nucleic Acids Res. 1980 May 24;8(10):2255–2274. doi: 10.1093/nar/8.10.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  35. Takeishi K., Kaneda S., Ayusawa D., Shimizu K., Gotoh O., Seno T. Nucleotide sequence of a functional cDNA for human thymidylate synthase. Nucleic Acids Res. 1985 Mar 25;13(6):2035–2043. doi: 10.1093/nar/13.6.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thompson R., Honess R. W., Taylor L., Morran J., Davison A. J. Varicella-zoster virus specifies a thymidylate synthetase. J Gen Virol. 1987 May;68(Pt 5):1449–1455. doi: 10.1099/0022-1317-68-5-1449. [DOI] [PubMed] [Google Scholar]
  37. Walter R. D. Altered dihydrofolate reductase in pyrimethamine-resistant Plasmodium falciparum. Mol Biochem Parasitol. 1986 Apr;19(1):61–66. doi: 10.1016/0166-6851(86)90066-6. [DOI] [PubMed] [Google Scholar]
  38. Weber J. L. Analysis of sequences from the extremely A + T-rich genome of Plasmodium falciparum. Gene. 1987;52(1):103–109. doi: 10.1016/0378-1119(87)90399-4. [DOI] [PubMed] [Google Scholar]
  39. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  40. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

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