Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii

Larry M C Chow, Alexander K C Wong, Buddy Ullman, Dyann F. Wirth

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 μg mg ml-1) of parental cells. The vinblastine-resistant parasites were also resistant to promycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug effiux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdrl, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr 1 and 83% identity with the L. donovani 1mdr 1 gene. The lemdr 1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.

Original languageEnglish (US)
Pages (from-to)195-208
Number of pages14
JournalMolecular and Biochemical Parasitology
Volume60
Issue number2
DOIs
StatePublished - 1993

Fingerprint

Leishmania enriettii
MDR Genes
Vinblastine
Organism Cloning
Drug Resistance
Genes
Parasites
P-Glycoproteins
Amino Acids
Parasitic Diseases
Proteins
Leishmaniasis
Multiple Drug Resistance
P-Glycoprotein
Microtubules
Pharmaceutical Preparations
Open Reading Frames
Inhibitory Concentration 50
Amino Acid Sequence
Clone Cells

Keywords

  • Amplification
  • Drug resistance
  • Leishmania
  • Transfection

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii. / Chow, Larry M C; Wong, Alexander K C; Ullman, Buddy; Wirth, Dyann F.

In: Molecular and Biochemical Parasitology, Vol. 60, No. 2, 1993, p. 195-208.

Research output: Contribution to journalArticle

Chow, Larry M C ; Wong, Alexander K C ; Ullman, Buddy ; Wirth, Dyann F. / Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii. In: Molecular and Biochemical Parasitology. 1993 ; Vol. 60, No. 2. pp. 195-208.
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