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Proc Natl Acad Sci U S A. Feb 15, 1993; 90(4): 1599–1603.
PMCID: PMC45922

Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.

Abstract

We attempted to generate homozygous dhfr-ts (dihydrofolate reductase-thymidylate synthase) knockouts in virulent Leishmania major, an asexual diploid protozoan parasite. Transfection of a neo (neomycin phosphotransferase) targeting fragment yielded heterozygous replacement lines with high efficiency. However, second transfections with a hyg (hygromycin B phosphotransferase) targeting fragment in the presence of metabolites shown to rescue homozygous knockouts in attenuated Leishmania did not yield the expected dhfr-ts- thymidine auxotrophs obtained previously with attenuated lines. Molecular karyotype, Southern blot, and flow cytometric DNA content analysis of clonal transfectants revealed three classes: (i) genomic tetraploids, containing two wild-type dhfr-ts chromosomes and one neo and one hyg replacement chromosome; (ii) aneuploid trisomic lines with one wild-type dhfr-ts and one neo and one hyg replacement chromosome; (iii) diploids bearing homologous integration of the targeting fragment without replacement. Aneuploid and tetraploid lines predominated. This confirms the common impression that natural populations of Leishmania are often aneuploid. The remarkable ability of these parasites to undergo and tolerate changes in chromosome number suggests a general method for testing whether genes are essential for growth in vitro, as the ability of Leishmania to simultaneously undergo homologous gene replacement while retaining wild-type genes by increasing chromosome number provides a diagnostic and positive experimental result. Our results show that virulent Leishmania require at least one copy of dhfr-ts and argue that DHFR-TS plays an unanticipated role in addition to its role in the de novo synthesis of thymidine. These results also have implications for genetic tests of the organization of Leishmania populations.

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  • Cruz A, Coburn CM, Beverley SM. Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7170–7174. [PMC free article] [PubMed]
  • King DL, Turco SJ. A ricin agglutinin-resistant clone of Leishmania donovani deficient in lipophosphoglycan. Mol Biochem Parasitol. 1988 Apr;28(3):285–293. [PubMed]
  • Elhay M, Kelleher M, Bacic A, McConville MJ, Tolson DL, Pearson TW, Handman E. Lipophosphoglycan expression and virulence in ricin-resistant variants of Leishmania major. Mol Biochem Parasitol. 1990 May;40(2):255–267. [PubMed]
  • Cruz A, Beverley SM. Gene replacement in parasitic protozoa. Nature. 1990 Nov 8;348(6297):171–173. [PubMed]
  • Kapler GM, Coburn CM, Beverley SM. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol. 1990 Mar;10(3):1084–1094. [PMC free article] [PubMed]
  • Titus RG, Müller I, Kimsey P, Cerny A, Behin R, Zinkernagel RM, Louis JA. Exacerbation of experimental murine cutaneous leishmaniasis with CD4+ Leishmania major-specific T cell lines or clones which secrete interferon-gamma and mediate parasite-specific delayed-type hypersensitivity. Eur J Immunol. 1991 Mar;21(3):559–567. [PubMed]
  • Kapler GM, Beverley SM. Transcriptional mapping of the amplified region encoding the dihydrofolate reductase-thymidylate synthase of Leishmania major reveals a high density of transcripts, including overlapping and antisense RNAs. Mol Cell Biol. 1989 Sep;9(9):3959–3972. [PMC free article] [PubMed]
  • Shapiro SZ, Naessens J, Liesegang B, Moloo SK, Magondu J. Analysis by flow cytometry of DNA synthesis during the life cycle of African trypanosomes. Acta Trop. 1984 Dec;41(4):313–323. [PubMed]
  • Iovannisci DM, Beverley SM. Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array. Mol Biochem Parasitol. 1989 May 1;34(2):177–188. [PubMed]
  • Urlaub G, Käs E, Carothers AM, Chasin LA. Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell. 1983 Jun;33(2):405–412. [PubMed]
  • Howell EE, Foster PG, Foster LM. Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement. J Bacteriol. 1988 Jul;170(7):3040–3045. [PMC free article] [PubMed]
  • Bastien P, Blaineau C, Pages M. Leishmania: sex, lies and karyotype. Parasitol Today. 1992 May;8(5):174–177. [PubMed]
  • Sternberg J, Tait A. Genetic exchange in African trypanosomes. Trends Genet. 1990 Oct;6(10):317–322. [PubMed]
  • Gibson W, Garside L, Bailey M. Trisomy and chromosome size changes in hybrid trypanosomes from a genetic cross between Trypanosoma brucei rhodesiense and T. b. brucei. Mol Biochem Parasitol. 1992 Apr;51(2):189–199. [PubMed]
  • Lanotte G, Rioux JA. Fusion cellulaire chez les Leishmania (Kinetoplastida, Trypanosomatidae). C R Acad Sci III. 1990;310(7):285–288. [PubMed]
  • Panton LJ, Tesh RB, Nadeau KC, Beverley SM. A test for genetic exchange in mixed infections of Leishmania major in the sand fly Phlebotomus papatasi. J Protozool. 1991 May-Jun;38(3):224–228. [PubMed]
  • Kelly JM, Law JM, Chapman CJ, Van Eys GJ, Evans DA. Evidence of genetic recombination in Leishmania. Mol Biochem Parasitol. 1991 Jun;46(2):253–263. [PubMed]
  • Evans DA, Kennedy WP, Elbihari S, Chapman CJ, Smith V, Peters W. Hybrid formation within the genus Leishmania? Parassitologia. 1987 May-Dec;29(2-3):165–173. [PubMed]
  • Grögl M, Oduola AM, Cordero LD, Kyle DE. Leishmania spp.: development of pentostam-resistant clones in vitro by discontinuous drug exposure. Exp Parasitol. 1989 Jul;69(1):78–90. [PubMed]
  • Turco SJ, Descoteaux A. The lipophosphoglycan of Leishmania parasites. Annu Rev Microbiol. 1992;46:65–94. [PubMed]
  • McConville MJ, Bacic A. A family of glycoinositol phospholipids from Leishmania major. Isolation, characterization, and antigenicity. J Biol Chem. 1989 Jan 15;264(2):757–766. [PubMed]
  • Achterberg V, Gercken G. Cytotoxicity of ester and ether lysophospholipids on Leishmania donovani promastigotes. Mol Biochem Parasitol. 1987 Mar;23(2):117–122. [PubMed]
  • Herrmann HO, Gercken G. Metabolism of 1-0-[1'-14C]octadecyl-sn-glycerol in Leishmania donovani promastigotes. Ether lipid synthesis and degradation of the ether bond. Mol Biochem Parasitol. 1982 Feb;5(2):65–76. [PubMed]
  • Ishibashi T, Imai Y. Solubilization and partial characterization of alkylglycerol monooxygenase from rat liver microsomes. Eur J Biochem. 1983 Apr 15;132(1):23–27. [PubMed]
  • TIETZ A, LINDBERG M, KENNEDY EP. A NEW PTERIDINE-REQUIRING ENZYME SYSTEM FOR THE OXIDATION OF GLYCERYL ETHERS. J Biol Chem. 1964 Dec;239:4081–4090. [PubMed]
  • Beck JT, Ullman B. Nutritional requirements of wild-type and folate transport-deficient Leishmania donovani for pterins and folates. Mol Biochem Parasitol. 1990 Dec;43(2):221–230. [PubMed]
  • Beck JT, Ullman B. Biopterin conversion to reduced folates by Leishmania donovani promastigotes. Mol Biochem Parasitol. 1991 Nov;49(1):21–28. [PubMed]
  • Peixoto MP, Beverley SM. In vitro activity of sulfonamides and sulfones against Leishmania major promastigotes. Antimicrob Agents Chemother. 1987 Oct;31(10):1575–1578. [PMC free article] [PubMed]
  • Tait A. Sexual processes in the kinetoplastida. Parasitology. 1983 Apr;86(Pt 4):29–57. [PubMed]
  • Tibayrenc M, Kjellberg F, Ayala FJ. A clonal theory of parasitic protozoa: the population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas, and Trypanosoma and their medical and taxonomical consequences. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2414–2418. [PMC free article] [PubMed]

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