Stable amplification of a linear extrachromosomal DNA in mycophenolic acid-resistant Leishmania donovani

Keith Wilson, Stephen M. Beverley, Buddy Ullman

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

Pulsed field gel electrophoretic analysis of chromosomes of MPA 100 cells, a strain of Leishmania donovani that possesses an approx. 15-fold amplified IMP dehydrogenase (IMPDH) gene copy number, revealed a new 280-kb extrachromosomal DNA, IMPDH-280, that was not present in wild type parental cells. Southern blots of these pulsed field gels revealed that the vast majority of the amplified impdh genes were localized on IMPDH-280. In addition to the 700-kb wild type chromosome, the impdh probe also recognized a 740-kb chromosome in the MPA100 genome. The pulse time-dependent relative mobility of IMPDH-280 in pulsed field gels, the failure of limited γ-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to λ-exonuclease digestion, demonstrated that IMPDH-280 was a linear molecule. IMPDH-280 was also recognized by a telomere probe but not by fragments derived from amplified DNAs found in other drug-resistant Leishmania. IMPDH-280 and the drug resistance phenotype remained stable when MPA100 cells were propagated in the absence of drug for 2 years. The appearance of IMPDH-280 in MPA 100 cells represents one of the first examples of an amplification of a linear extrachromosomal DNA element mediating drug resistance in Leishmania and the first instance of a linear DNA amplification that is stable in the absence of selective pressure.

Original languageEnglish (US)
Pages (from-to)197-206
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume55
Issue number1-2
DOIs
StatePublished - Oct 1992

Keywords

  • Drug resistance
  • Extrachromosomal element
  • Gene amplification
  • IMP dehydrogenase
  • Leishmania donovani
  • Pulsed field gel electrophoresis

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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