Amplification and molecular cloning of the ornithine decarboxylase gene of Leishmania donovani

Sheri Hanson, John Adelman, Buddy Ullman

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

A strain of Leishmania donovani has been described that is resistant to DL-α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (OD-Case) activity, and contains 15-fold greater amounts of ODCase activity and protein than the wild type strain from which it was derived (Coons, T., Hanson, S., Bitonti, A. J., McCann, P. P., and Ullman, B. (1990) Mol. Biochem. Pharmacol. 39, 77-90). From this mutant strain, another ODCase overproducing L. donovani strain, DFMO16, was generated by virtue of its ability to proliferate under even higher concentrations of DFMO. To investigate the mechanism by which DFMO-resistant cells overexpress ODCase, the leishnianial ODCase gene was isolated by hybridization to a fragment of the L. donovani ODCase gene that was generated by the polymerase chain reaction. The nucleotide sequence of a 4.5-kilobase DNA fragment encompassed an open reading frame encoding 707 amino acids (Mr = 77,350). The leishmanial protein contained an extra ∼200 amino acid NH2-terminal extension and lacked the COOH terminus of the mammalian ODCase. Northern blot analysis revealed two leishmanial ODCase transcripts of 4.8 and 6.5 kilobases, both of which were amplified 10-20-fold in the DFMO16 cells. Genomic Southern blot analysis established that the augmented amount of ODCase activity and ODCase mRNA in the DFMO16 strain could be attributed to a ∼10-20-fold amplification of the ODCase gene copy number. DFMO16 cells exhibited an unstable phenotype in that the amplification of the ODCase gene, the increased amount of ODCase transcript, the overproduction of ODCase activity, and the DFMO-resistance growth phenotype all reverted synchronously in the absence of selective pressure.

Original languageEnglish (US)
Pages (from-to)2350-2359
Number of pages10
JournalJournal of Biological Chemistry
Volume267
Issue number4
StatePublished - Feb 5 1992

Fingerprint

Eflornithine
Leishmania donovani
Ornithine Decarboxylase
Cloning
Molecular Cloning
Amplification
Genes
Phenotype
Amino Acids
Gene Dosage
Gene Amplification
Polymerase chain reaction
Southern Blotting
Northern Blotting
Open Reading Frames
Proteins
Nucleotides
Polymerase Chain Reaction
Messenger RNA
DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Amplification and molecular cloning of the ornithine decarboxylase gene of Leishmania donovani. / Hanson, Sheri; Adelman, John; Ullman, Buddy.

In: Journal of Biological Chemistry, Vol. 267, No. 4, 05.02.1992, p. 2350-2359.

Research output: Contribution to journalArticle

Hanson, Sheri ; Adelman, John ; Ullman, Buddy. / Amplification and molecular cloning of the ornithine decarboxylase gene of Leishmania donovani. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 4. pp. 2350-2359.
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abstract = "A strain of Leishmania donovani has been described that is resistant to DL-α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (OD-Case) activity, and contains 15-fold greater amounts of ODCase activity and protein than the wild type strain from which it was derived (Coons, T., Hanson, S., Bitonti, A. J., McCann, P. P., and Ullman, B. (1990) Mol. Biochem. Pharmacol. 39, 77-90). From this mutant strain, another ODCase overproducing L. donovani strain, DFMO16, was generated by virtue of its ability to proliferate under even higher concentrations of DFMO. To investigate the mechanism by which DFMO-resistant cells overexpress ODCase, the leishnianial ODCase gene was isolated by hybridization to a fragment of the L. donovani ODCase gene that was generated by the polymerase chain reaction. The nucleotide sequence of a 4.5-kilobase DNA fragment encompassed an open reading frame encoding 707 amino acids (Mr = 77,350). The leishmanial protein contained an extra ∼200 amino acid NH2-terminal extension and lacked the COOH terminus of the mammalian ODCase. Northern blot analysis revealed two leishmanial ODCase transcripts of 4.8 and 6.5 kilobases, both of which were amplified 10-20-fold in the DFMO16 cells. Genomic Southern blot analysis established that the augmented amount of ODCase activity and ODCase mRNA in the DFMO16 strain could be attributed to a ∼10-20-fold amplification of the ODCase gene copy number. DFMO16 cells exhibited an unstable phenotype in that the amplification of the ODCase gene, the increased amount of ODCase transcript, the overproduction of ODCase activity, and the DFMO-resistance growth phenotype all reverted synchronously in the absence of selective pressure.",
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