• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Apr 1983; 80(8): 2132–2136.
PMCID: PMC393771

Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica.


Leishmania tropica promastigotes that are highly resistant to methotrexate, a dihydrofolate reductase inhibitor, have been developed. Organisms resistant to 1 mM methotrexate have a 40-fold increase in dihydrofolate reductase which is associated with thymidylate synthetase, and they contain amplified regions of DNA that may be directly visualized on stained gels of restriction digests. The amplified DNA in these organisms is about 56 kilobases in length, has a copy number about 80-fold higher than that of wild-type organisms, and constitutes about 10% of the nuclear DNA. When the methotrexate-resistant L. tropica are propagated in drug-free medium, the dihydrofolate reductase-thymidylate synthetase protein and the amplified DNA decrease in a parallel fashion until they are indistinguishable from the levels in wild-type organisms. However, when these apparent revertants are again challenged with 1 mM methotrexate, enzyme overproduction and DNA amplification occur rapidly. As in mammalian cells, it appears that drug resistance in parasitic protozoa may be mediated by gene amplification.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • BROWNING CH. The chemotherapy of trypanosome infections. Ann N Y Acad Sci. 1954 Sep;59(2):198–213. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Santi DV, McHenry CS, Sommer H. Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate. Biochemistry. 1974 Jan 29;13(3):471–481. [PubMed]
  • Washtien WL, Santi DV. Assay of intracellular free and macromolecular-bound metabolites of 5-fluorodeoxyuridine and 5-fluorouracil. Cancer Res. 1979 Sep;39(9):3397–3404. [PubMed]
  • Santi DV, McHenry CS, Perriard ER. A filter assay for thymidylate synthetase using 5-fluoro-2'-deoxyuridylate as an active site titrant. Biochemistry. 1974 Jan 29;13(3):467–470. [PubMed]
  • Pogolotti AL, Jr, Weill C, Santi DV. Thymidylate synthetase catalyzed exchange of tritiumfrom [5-3H]-2'-deoxyuridylate for protons of water. Biochemistry. 1979 Jun 26;18(13):2794–2798. [PubMed]
  • Baccanari D, Phillips A, Smith S, Sinski D, Burchall J. Purification and properties of Escherichia coli dihydrofolate reductase. Biochemistry. 1975 Dec 2;14(24):5267–5273. [PubMed]
  • Brown PC, Beverley SM, Schimke RT. Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines. Mol Cell Biol. 1981 Dec;1(12):1077–1083. [PMC free article] [PubMed]
  • Simpson L, Da Silva A. Isolation and characterization of kinetoplast DNA from Leishmania tarentolae. J Mol Biol. 1971 Mar 28;56(3):443–473. [PubMed]
  • Crouse GF, Simonsen CC, McEwan RN, Schimke RT. Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. J Biol Chem. 1982 Jul 10;257(13):7887–7897. [PubMed]
  • Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. [PubMed]
  • Soberon X, Covarrubias L, Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • McCuen RW, Sirotnak FM. Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs. Biochim Biophys Acta. 1975 Apr 19;384(2):369–380. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Schimke RT, Kaufman RJ, Alt FW, Kellems RF. Gene amplification and drug resistance in cultured murine cells. Science. 1978 Dec 8;202(4372):1051–1055. [PubMed]
  • Chang AC, Nunberg JH, Kaufman RJ, Erlich HA, Schimke RT, Cohen SN. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. [PubMed]
  • Leon W, Fouts DL, Manning J. Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani. Nucleic Acids Res. 1978 Feb;5(2):491–504. [PMC free article] [PubMed]
  • Kaufman RJ, Brown PC, Schimke RT. Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5669–5673. [PMC free article] [PubMed]
  • Schimke RT, Brown PC, Kaufman RJ, McGrogan M, Slate DL. Chromosomal and extrachromosomal localization of amplified dihydrofolate reductase genes in cultured mammalian cells. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 2):785–797. [PubMed]
  • Bostock CJ, Tyler-Smith C. Gene amplification in methotrexate-resistant mouse cells. II. Rearrangement and amplification of non-dihydrofolate reductase gene sequences accompany chromosomal changes. J Mol Biol. 1981 Dec 5;153(2):219–236. [PubMed]
  • Milbrandt JD, Heintz NH, White WC, Rothman SM, Hamlin JL. Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6043–6047. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...