Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Dec 1988; 8(12): 5188–5199.
PMCID: PMC365621

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.

Abstract

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major (S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431-439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.

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 (2.9M), 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

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Beverley SM. Characterization of the 'unusual' mobility of large circular DNAs in pulsed field-gradient electrophoresis. Nucleic Acids Res. 1988 Feb 11;16(3):925–939. [PMC free article] [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]
  • Beverley SM, Ellenberger TE, Cordingley JS. 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. [PMC free article] [PubMed]
  • Beverley SM, Ismach RB, Pratt DM. Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns. Proc Natl Acad Sci U S A. 1987 Jan;84(2):484–488. [PMC free article] [PubMed]
  • Carroll SM, Gaudray P, De Rose ML, Emery JF, Meinkoth JL, Nakkim E, Subler M, Von Hoff DD, Wahl GM. Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin. Mol Cell Biol. 1987 May;7(5):1740–1750. [PMC free article] [PubMed]
  • Carroll SM, DeRose ML, Gaudray P, Moore CM, Needham-Vandevanter DR, Von Hoff DD, Wahl GM. Double minute chromosomes can be produced from precursors derived from a chromosomal deletion. Mol Cell Biol. 1988 Apr;8(4):1525–1533. [PMC free article] [PubMed]
  • Coderre JA, Beverley SM, Schimke RT, Santi DV. Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2132–2136. [PMC free article] [PubMed]
  • Detke S, Chaudhuri G, Kink JA, Chang KP. DNA amplification in tunicamycin-resistant Leishmania mexicana. Multicopies of a single 63-kilobase supercoiled molecule and their expression. J Biol Chem. 1988 Mar 5;263(7):3418–3424. [PubMed]
  • Dewes H, Ostergaard HL, Simpson L. Impaired drug uptake in methotrexate resistant Crithidia fasciculata without changes in dihydrofolate reductase activity or gene amplification. Mol Biochem Parasitol. 1986 May;19(2):149–161. [PubMed]
  • Ellenberger TE, Beverley SM. Biochemistry and regulation of folate and methotrexate transport in Leishmania major. J Biol Chem. 1987 Jul 25;262(21):10053–10058. [PubMed]
  • Ellenberger TE, Beverley SM. Reductions in methotrexate and folate influx in methotrexate-resistant lines of Leishmania major are independent of R or H region amplification. J Biol Chem. 1987 Oct 5;262(28):13501–13506. [PubMed]
  • Federspiel NA, Beverley SM, Schilling JW, Schimke RT. Novel DNA rearrangements are associated with dihydrofolate reductase gene amplification. J Biol Chem. 1984 Jul 25;259(14):9127–9140. [PubMed]
  • Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. [PubMed]
  • Fojo A, Akiyama S, Gottesman MM, Pastan I. Reduced drug accumulation in multiply drug-resistant human KB carcinoma cell lines. Cancer Res. 1985 Jul;45(7):3002–3007. [PubMed]
  • Ford M, Fried M. Large inverted duplications are associated with gene amplification. Cell. 1986 May 9;45(3):425–430. [PubMed]
  • Gadler H. Nucleic acid hybridization for measurement of effects of antiviral compounds on human cytomegalovirus DNA replication. Antimicrob Agents Chemother. 1983 Sep;24(3):370–374. [PMC free article] [PubMed]
  • Garvey EP, Coderre JA, Santi DV. Selection and properties of Leishmania tropica resistant to 10-propargyl-5,8-dideazafolate, an inhibitor of thymidylate synthetase. Mol Biochem Parasitol. 1985 Oct;17(1):79–91. [PubMed]
  • Garvey EP, Santi DV. Stable amplified DNA in drug-resistant Leishmania exists as extrachromosomal circles. Science. 1986 Aug 1;233(4763):535–540. [PubMed]
  • Gomez-Eichelmann MC, Holz G, Jr, Beach D, Simpson AM, Simpson L. Comparison of several lizard Leishmania species and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids. Mol Biochem Parasitol. 1988 Jan 15;27(2-3):143–158. [PubMed]
  • Grumont R, Washtien WL, Caput D, Santi DV. 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. [PMC free article] [PubMed]
  • Hamlin JL, Milbrandt JD, Heintz NH, Azizkhan JC. DNA sequence amplification in mammalian cells. Int Rev Cytol. 1984;90:31–82. [PubMed]
  • Hightower RC, Metge DW, Santi DV. Plasmid migration using orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1987 Oct 26;15(20):8387–8398. [PMC free article] [PubMed]
  • Hughes DE, Simpson L. Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6058–6062. [PMC free article] [PubMed]
  • Hutchinson F. Chemical changes induced in DNA by ionizing radiation. Prog Nucleic Acid Res Mol Biol. 1985;32:115–154. [PubMed]
  • Iovannisci DM, Goebel D, Allen K, Kaur K, Ullman B. Genetic analysis of adenine metabolism in Leishmania donovani promastigotes. Evidence for diploidy at the adenine phosphoribosyltransferase locus. J Biol Chem. 1984 Dec 10;259(23):14617–14623. [PubMed]
  • Kink JA, Chang KP. Tunicamycin-resistant Leishmania mexicana amazonensis: expression of virulence associated with an increased activity of N-acetylglucosaminyltransferase and amplification of its presumptive gene. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1253–1257. [PMC free article] [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]
  • Looney JE, Hamlin JL. Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells. Mol Cell Biol. 1987 Feb;7(2):569–577. [PMC free article] [PubMed]
  • Maurer BJ, Lai E, Hamkalo BA, Hood L, Attardi G. Novel submicroscopic extrachromosomal elements containing amplified genes in human cells. Nature. 1987 Jun 4;327(6121):434–437. [PubMed]
  • Riordan JR, Ling V. Genetic and biochemical characterization of multidrug resistance. Pharmacol Ther. 1985;28(1):51–75. [PubMed]
  • Schimke RT. Gene amplification in cultured animal cells. Cell. 1984 Jul;37(3):705–713. [PubMed]
  • Schimke RT. Gene amplification in cultured cells. J Biol Chem. 1988 May 5;263(13):5989–5992. [PubMed]
  • Schimke RT, Sherwood SW, Hill AB, Johnston RN. Overreplication and recombination of DNA in higher eukaryotes: potential consequences and biological implications. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2157–2161. [PMC free article] [PubMed]
  • Schwartz DC, Cantor CR. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. [PubMed]
  • Shaw JM, Feagin JE, Stuart K, Simpson L. Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons. Cell. 1988 May 6;53(3):401–411. [PubMed]
  • Simpson L. The mitochondrial genome of kinetoplastid protozoa: genomic organization, transcription, replication, and evolution. Annu Rev Microbiol. 1987;41:363–382. [PubMed]
  • Stark GR. DNA amplification in drug resistant cells and in tumours. Cancer Surv. 1986;5(1):1–23. [PubMed]
  • Stark GR, Wahl GM. Gene amplification. Annu Rev Biochem. 1984;53:447–491. [PubMed]
  • Tait A. Sexual processes in the kinetoplastida. Parasitology. 1983 Apr;86(Pt 4):29–57. [PubMed]
  • Van der Ploeg LH, Cornelissen AW, Barry JD, Borst P. Chromosomes of kinetoplastida. EMBO J. 1984 Dec 20;3(13):3109–3115. [PMC free article] [PubMed]
  • Van der Ploeg LH, Schwartz DC, Cantor CR, Borst P. Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules. Cell. 1984 May;37(1):77–84. [PubMed]
  • Varshavsky A. On the possibility of metabolic control of replicon "misfiring": relationship to emergence of malignant phenotypes in mammalian cell lineages. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3673–3677. [PMC free article] [PubMed]
  • Wallbanks KR, Maazoun R, Canning EU, Rioux JA. The identity of Leishmania tarentolae Wenyon 1921. Parasitology. 1985 Feb;90(Pt 1):67–78. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...