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Nucleic Acids Res. Jul 15, 1998; 26(14): 3372–3378.
PMCID: PMC147699

Linear amplicons as precursors of amplified circles in methotrexate-resistant Leishmania tarentolae.

Abstract

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.

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Selected References

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  • Beverley SM. Gene amplification in Leishmania. Annu Rev Microbiol. 1991;45:417–444. [PubMed]
  • Ouellette M, Borst P. Drug resistance and P-glycoprotein gene amplification in the protozoan parasite Leishmania. Res Microbiol. 1991 Jul-Aug;142(6):737–746. [PubMed]
  • Stuart KD. Circular and linear multicopy DNAs in Leishmania. Parasitol Today. 1991 Jul;7(7):158–159. [PubMed]
  • Segovia M. Leishmania gene amplification: a mechanism of drug resistance. Ann Trop Med Parasitol. 1994 Apr;88(2):123–130. [PubMed]
  • Segovia M, Ortiz G. LD1 amplifications in Leishmania. Parasitol Today. 1997 Sep;13(9):342–348. [PubMed]
  • Rovai L, Tripp C, Stuart K, Simpson L. Recurrent polymorphisms in small chromosomes of Leishmania tarentolae after nutrient stress or subcloning. Mol Biochem Parasitol. 1992 Jan;50(1):115–125. [PubMed]
  • Beverley SM, Coburn CM. Recurrent de novo appearance of small linear DNAs in Leishmania major and relationship to extra-chromosomal DNAs in other species. Mol Biochem Parasitol. 1990 Aug;42(1):133–141. [PubMed]
  • Hanson S, Beverley SM, Wagner W, Ullman B. Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani. Mol Cell Biol. 1992 Dec;12(12):5499–5507. [PMC free article] [PubMed]
  • Papadopoulou B, Roy G, Ouellette M. Frequent amplification of a short chain dehydrogenase gene as part of circular and linear amplicons in methotrexate resistant Leishmania. Nucleic Acids Res. 1993 Sep 11;21(18):4305–4312. [PMC free article] [PubMed]
  • Grondin K, Papadopoulou B, Ouellette M. Homologous recombination between direct repeat sequences yields P-glycoprotein containing amplicons in arsenite resistant Leishmania. Nucleic Acids Res. 1993 Apr 25;21(8):1895–1901. [PMC free article] [PubMed]
  • Olmo A, Arrebola R, Bernier V, González-Pacanowska D, Ruiz-Pérez LM. Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania. Nucleic Acids Res. 1995 Aug 11;23(15):2856–2864. [PMC free article] [PubMed]
  • Grondin K, Roy G, Ouellette M. Formation of extrachromosomal circular amplicons with direct or inverted duplications in drug-resistant Leishmania tarentolae. Mol Cell Biol. 1996 Jul;16(7):3587–3595. [PMC free article] [PubMed]
  • Navarro M, Liu J, Muthui D, Ortiz G, Segovia M, Hamers R. Inverted repeat structure and homologous sequences in the LD1 amplicons of Leishmania spp. Mol Biochem Parasitol. 1994 Nov;68(1):69–80. [PubMed]
  • Papadopoulou B, Roy G, Ouellette M. A novel antifolate resistance gene on the amplified H circle of Leishmania. EMBO J. 1992 Oct;11(10):3601–3608. [PMC free article] [PubMed]
  • Callahan HL, Beverley SM. A member of the aldoketo reductase family confers methotrexate resistance in Leishmania. J Biol Chem. 1992 Dec 5;267(34):24165–24168. [PubMed]
  • Bello AR, Nare B, Freedman D, Hardy L, Beverley SM. PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11442–11446. [PMC free article] [PubMed]
  • Wang J, Leblanc E, Chang CF, Papadopoulou B, Bray T, Whiteley JM, Lin SX, Ouellette M. Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1. Arch Biochem Biophys. 1997 Jun 15;342(2):197–202. [PubMed]
  • Nare B, Hardy LW, Beverley SM. The roles of pteridine reductase 1 and dihydrofolate reductase-thymidylate synthase in pteridine metabolism in the protozoan parasite Leishmania major. J Biol Chem. 1997 May 23;272(21):13883–13891. [PubMed]
  • Nare B, Luba J, Hardy LW, Beverley S. New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity. Parasitology. 1997;114 (Suppl):S101–S110. [PubMed]
  • Borst P, Ouellette M. New mechanisms of drug resistance in parasitic protozoa. Annu Rev Microbiol. 1995;49:427–460. [PubMed]
  • Beverley SM, Coderre JA, Santi DV, Schimke RT. Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization. Cell. 1984 Sep;38(2):431–439. [PubMed]
  • White TC, Fase-Fowler F, van Luenen H, Calafat J, Borst P. The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance. J Biol Chem. 1988 Nov 15;263(32):16977–16983. [PubMed]
  • Chiquero MJ, Olmo A, Navarro P, Ruiz-Perez LM, Castanys S, Gonzalez-Pacanowska D, Gamarro F. Amplification of the H locus in Leishmania infantum. Biochim Biophys Acta. 1994 Nov 29;1227(3):188–194. [PubMed]
  • Ouellette M, Hettema E, Wüst D, Fase-Fowler F, Borst P. Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania. EMBO J. 1991 Apr;10(4):1009–1016. [PMC free article] [PubMed]
  • Brun R, Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed]
  • Papadopoulou B, Roy G, Ouellette M. Autonomous replication of bacterial DNA plasmid oligomers in Leishmania. Mol Biochem Parasitol. 1994 May;65(1):39–49. [PubMed]
  • Papadopoulou B, Roy G, Dey S, Rosen BP, Olivier M, Ouellette M. Gene disruption of the P-glycoprotein related gene pgpa of Leishmania tarentolae. Biochem Biophys Res Commun. 1996 Jul 25;224(3):772–778. [PubMed]
  • Papadopoulou B, Roy G, Mourad W, Leblanc E, Ouellette M. Changes in folate and pterin metabolism after disruption of the Leishmania H locus short chain dehydrogenase gene. J Biol Chem. 1994 Mar 11;269(10):7310–7315. [PubMed]
  • Tripp CA, Wisdom WA, Myler PJ, Stuart KD. A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts. Mol Biochem Parasitol. 1992 Oct;55(1-2):39–50. [PubMed]
  • Ortiz G, Navarro M, Segovia M. Location in the source chromosome of the 180-kb minichromosome of Leishmania major and characterization of the novel junction. Mol Biochem Parasitol. 1995 May;71(2):153–161. [PubMed]
  • Ortiz G, Segovia M. Characterisation of the novel junctions of two minichromosomes of Leishmania major. Mol Biochem Parasitol. 1996 Nov 25;82(2):137–144. [PubMed]
  • Petrillo-Peixoto ML, Beverley SM. Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major. Mol Cell Biol. 1988 Dec;8(12):5188–5199. [PMC free article] [PubMed]
  • Hightower RC, Ruiz-Perez LM, Wong ML, Santi DV. Extrachromosomal elements in the lower eukaryote Leishmania. J Biol Chem. 1988 Nov 15;263(32):16970–16976. [PubMed]
  • Papadopoulou B, Dumas C. Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania. Nucleic Acids Res. 1997 Nov 1;25(21):4278–4286. [PMC free article] [PubMed]

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