Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1984 Jun;4(6):1013–1019. doi: 10.1128/mcb.4.6.1013

Genetic analysis of nucleoside transport in Leishmania donovani.

D M Iovannisci, K Kaur, L Young, B Ullman
PMCID: PMC368868  PMID: 6234454

Abstract

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Full text

PDF
1013

Selected References

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

  1. Berens R. L., Marr J. J., Nelson D. J., LaFon S. W. Antileishmanial effect of allopurinol and allopurinol ribonucleoside on intracellular forms of Leishmania donovani. Biochem Pharmacol. 1980 Sep 1;29(17):2397–2398. doi: 10.1016/0006-2952(80)90275-0. [DOI] [PubMed] [Google Scholar]
  2. Carson D. A., Chang K. P. Phosphorylation and anti-leishmanial activity of formycin B. Biochem Biophys Res Commun. 1981 Jun 16;100(3):1377–1383. doi: 10.1016/0006-291x(81)91976-8. [DOI] [PubMed] [Google Scholar]
  3. Cohen A., Ullman B., Martin D. W., Jr Characterization of a mutant mouse lymphoma cell with deficient transport of purine and pyrimidine nucleosides. J Biol Chem. 1979 Jan 10;254(1):112–116. [PubMed] [Google Scholar]
  4. Cybulski R. L., Fry D. W., Goldman I. D. Adenosine stimulation of uphill adenine transport in L1210 leukemia cells. Evidence for a novel countertransport mechanism. J Biol Chem. 1981 May 10;256(9):4455–4459. [PubMed] [Google Scholar]
  5. Gottlieb M., Dwyer D. M. Identification and partial characterization of an extracellular acid phosphatase activity of Leishmania donovani promastigotes. Mol Cell Biol. 1982 Jan;2(1):76–81. doi: 10.1128/mcb.2.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gottlieb M., Dwyer D. M. Protozoan parasite of humans: surface membrane with externally disposed acid phosphatase. Science. 1981 May 22;212(4497):939–941. doi: 10.1126/science.7233189. [DOI] [PubMed] [Google Scholar]
  7. Green C. D., Martin D. W., Jr Characterization of a feedback-resistant phosphoribosylpyrophosphate synthetase from cultured, mutagenized hepatoma cells that overproduce purines. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3698–3702. doi: 10.1073/pnas.70.12.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gudas L. J., Cohen A., Ullman B., Martin D. W., Jr Analysis of adenosine-mediated pyrimidine starvation using cultured wild-type and mutant mouse T-lymphoma cells. Somatic Cell Genet. 1978 Mar;4(2):201–219. doi: 10.1007/BF01538985. [DOI] [PubMed] [Google Scholar]
  9. Heidelberger C. Fluorinated pyrimidines. Prog Nucleic Acid Res Mol Biol. 1965;4:1–50. doi: 10.1016/s0079-6603(08)60783-7. [DOI] [PubMed] [Google Scholar]
  10. Iovannisci D. M., Ullman B. High efficiency plating method for Leishmania promastigotes in semidefined or completely-defined medium. J Parasitol. 1983 Aug;69(4):633–636. [PubMed] [Google Scholar]
  11. Koszalka G. W., Krenitsky T. A. Nucleosidases from Leishmania donovani. Pyrimidine ribonucleosidase, purine ribonucleosidase, and a novel purine 2'-deoxyribonucleosidase. J Biol Chem. 1979 Sep 10;254(17):8185–8193. [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Marr J. J., Berens R. L. Antileishmanial effect of allopurinol. II. Relationship of adenine metabolism in Leishmania species to the action of allopurinol. J Infect Dis. 1977 Dec;136(6):724–732. doi: 10.1093/infdis/136.6.724. [DOI] [PubMed] [Google Scholar]
  14. Marr J. J., Berens R. L., Nelson D. J., Krenitsky T. A., Spector T., LaFon S. W., Elion G. B. Antileishmanial action of 4-thiopyrazolo (3.4-d) pyrimidine and its ribonucleoside. Biological effects and metabolism. Biochem Pharmacol. 1982 Jan 15;31(2):143–148. doi: 10.1016/0006-2952(82)90203-9. [DOI] [PubMed] [Google Scholar]
  15. Marr J. J., Berens R. L., Nelson D. J. Purine metabolism in Leishmania donovani and Leishmania braziliensis. Biochim Biophys Acta. 1978 Dec 1;544(2):360–371. doi: 10.1016/0304-4165(78)90104-6. [DOI] [PubMed] [Google Scholar]
  16. Munch-Petersen A., Mygind B., Nicolaisen A., Pihl N. J. Nucleoside transport in cells and membrane vesicles from Escherichia coli K12. J Biol Chem. 1979 May 25;254(10):3730–3737. [PubMed] [Google Scholar]
  17. Munch-Petersen A., Mygind B. Nucleoside transport systems in Escherichia coli K12: specificity and regulation. J Cell Physiol. 1976 Dec;89(4):551–559. doi: 10.1002/jcp.1040890410. [DOI] [PubMed] [Google Scholar]
  18. Nelson D. J., LaFon S. W., Tuttle J. V., Miller W. H., Miller R. L., Krenitsky T. A., Elion G. B., Berens R. L., Marr J. J. Allopurinol ribonucleoside as an antileishmanial agent. Biological effects, metabolism, and enzymatic phosphorylation. J Biol Chem. 1979 Nov 25;254(22):11544–11549. [PubMed] [Google Scholar]
  19. Nelson D. J., Lafon S. W., Jones T. E., Spector T., Berens R. L., Marr J. J. The metabolism of formycin B in Leishmania donovani. Biochem Biophys Res Commun. 1982 Sep 16;108(1):349–354. doi: 10.1016/0006-291x(82)91873-3. [DOI] [PubMed] [Google Scholar]
  20. Paterson A. R., Oliver J. M. Nucleoside transport. II. Inhibition by p-nitrobenzylthioguanosine and related compounds. Can J Biochem. 1971 Feb;49(2):271–274. doi: 10.1139/o71-039. [DOI] [PubMed] [Google Scholar]
  21. Paterson A. R., Simpson A. I. Inhibition of ribonucleoside metabolism in Ehrlich ascites tumor cells by purine analogue ribonucleosides. Can J Biochem. 1965 Oct;43(10):1701–1710. doi: 10.1139/o65-188. [DOI] [PubMed] [Google Scholar]
  22. Puziss M. B., Wohlhueter R. M., Plagemann P. G. Adenine transport and binding in cultured mammalian cells deficient in adenine phosphoribosyltransferase. Mol Cell Biol. 1983 Jan;3(1):82–90. doi: 10.1128/mcb.3.1.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rainey P., Santi D. V. Metabolism and mechanism of action of formycin B in Leishmania. Proc Natl Acad Sci U S A. 1983 Jan;80(1):288–292. doi: 10.1073/pnas.80.1.288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Scholtissek C. Studies on the uptake of nucleic acid precursors into cells in tissue culture. Biochim Biophys Acta. 1968 Jun 24;158(3):435–447. doi: 10.1016/0304-4165(68)90297-3. [DOI] [PubMed] [Google Scholar]
  25. Taube R. A., Berlin R. D. Membrane transport of nucleosides in rabbit polymorphonuclear leukocytes. Biochim Biophys Acta. 1972 Jan 17;255(1):6–18. doi: 10.1016/0005-2736(72)90003-x. [DOI] [PubMed] [Google Scholar]
  26. Tuttle J. V., Krenitsky T. A. Purine phosphoribosyltransferases from Leishmania donovani. J Biol Chem. 1980 Feb 10;255(3):909–916. [PubMed] [Google Scholar]
  27. Ullman B., Kaur K., Watts T. Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis. Mol Cell Biol. 1983 Jul;3(7):1187–1196. doi: 10.1128/mcb.3.7.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Voorheis H. P., Gale J. S., Owen M. J., Edwards W. The isolation and partial characterization of the plasma membrane from Trypanosoma brucei. Biochem J. 1979 Apr 15;180(1):11–24. doi: 10.1042/bj1800011. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES