Amplification of the inosinate dehydrogenase gene in Trypanosoma brucei gambiense due to an increase in chromosome copy number

Keith Wilson, Randolph L. Berens, C. David Sifri, Buddy Ullman

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

Drug resistance is a significant impediment to the therapy of African sleeping sickness in humans. To evaluate molecular mechanisms that contribute to drug resistance in African trypanosomes, a clonal strain of Trypanosoma brucei gambiense, MPA10, was selected in medium containing mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (IMPDH) activity. IMPDH activity in MPA10 cells was ~6-fold higher than that of wild type parental cells, although the enzymes in both strains were equally sensitive to inhibition by MPA. To evaluate the mechanism of IMPDH overexpression in MPA10 cells, the gene encoding IMPDH (impdh) was isolated from a T. b. brucei library by cross-hybridization to the Leishmania donovani impdh. Sequence analysis indicated that the T. brucei IMPDH was 76% identical with the L. donovani IMPDH. The T. brucei impdh hybridized to a 2.7-kb transcript that was expressed at ~10-fold greater levels in the MPA10 cells, and this impdh overexpression could be ascribed to an ~10-fold amplification of the impdh copy number. Pulsed field gel electrophoresis revealed that the extra impdh copies in MPA10 cells were localized to an ~6.0-Mb chromosome that comigrated with the wild type chromosome encompassing impdh. The amplification of impdh could be ascribed to an increased copy number of this 6.0-Mb chromosome, and a predicted augmented DNA content in MPA10 cells was confirmed by flow cytometry. This is the first demonstration that DNA amplification can serve as a molecular mechanism by which T. brucei become resistant to cytotoxic drugs, and the amplification of the 6.0-Mb chromosome represents a novel mechanism of drug resistance in parasitic protozoa.

Original languageEnglish (US)
Pages (from-to)28979-28987
Number of pages9
JournalJournal of Biological Chemistry
Volume269
Issue number46
StatePublished - Nov 18 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Amplification of the inosinate dehydrogenase gene in Trypanosoma brucei gambiense due to an increase in chromosome copy number'. Together they form a unique fingerprint.

  • Cite this