Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii
Identifieur interne : 002D54 ( Main/Exploration ); précédent : 002D53; suivant : 002D55Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii
Auteurs : Larry M. C. Chow [États-Unis] ; Alexander K. C. Wong [États-Unis] ; Buddy Ullman [États-Unis] ; Dyann F. Wirth [États-Unis]Source :
- Molecular & Biochemical Parasitology [ 0166-6851 ] ; 1993.
English descriptors
- Teeft :
- Acid sequence, Agarose, Agarose blocks, Amino, Amino acid identity, Amino acid level, Amino acid sequence, Amino acids, Amplification, Bacterial transport proteins, Bamhi, Bamhi fragment, Beverley, Binding sites, Biol, Bottom panel, Cell biol, Cell line, Cell lines, Channel activity, Chloroquine resistance, Chromosomal, Circular element, Donovani, Donovani ldmdrl gene, Double minutes, Drug concentration, Drug effiux, Drug efflux, Drug resistance, Drug resistance mechanisms, England biolabs, Enriettii, Enriettii cells, Ethidium bromide staining, Expression vector paltneo, Extra band, Extrachromosomal, Extrachromosomal circle, Extrachromosomal element, Extrachromosomal elements, Fastlane agarose, Functional analysis, Gene, Gene amplification, Gene family, Gene products, Genomic, Harvard school, Hincli fragment, Human mdrl, Hybridized, Hydropathy plot, Internal duplication, Ldmdrl, Ldmdrl gene, Leishmania, Leishmania cells, Leishmania donovani, Leishmania enriettii, Leishmania tarentolae, Lemdrl, Lemdrl gene, Lemdrl sequence, Loading control, Ltpgpa, Mammalian cells, Mammalian neoplastic cells, Model system, Multidrug, Multidrug resistance, Mutant cells, Noti fragment, Nucleic acids, Nucleotide, Nucleotide binding motif, Nucleotide binding sites, Nylon paper, Parasite, Parental cells, Plasmodium falciparum, Previous work, Public health, Pulse time, Puromycin, Putative, Reading frame, Resistant, Resistant cells, Restriction pattern, Same filter, Sequence analysis, Sequence identity, Signal intensity, Significant homology, Southern analysis, Southern blot, Tafe, Transfected, Transfection, Transfection analysis, Transfection experiments, Transmembrane domains, Unrelated drugs, Vinblastine, Vinblastine resistance, Wild type, Xhoi fragment.
Abstract
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.
Url:
DOI: 10.1016/0166-6851(93)90131-G
Affiliations:
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<profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acid sequence</term>
<term>Agarose</term>
<term>Agarose blocks</term>
<term>Amino</term>
<term>Amino acid identity</term>
<term>Amino acid level</term>
<term>Amino acid sequence</term>
<term>Amino acids</term>
<term>Amplification</term>
<term>Bacterial transport proteins</term>
<term>Bamhi</term>
<term>Bamhi fragment</term>
<term>Beverley</term>
<term>Binding sites</term>
<term>Biol</term>
<term>Bottom panel</term>
<term>Cell biol</term>
<term>Cell line</term>
<term>Cell lines</term>
<term>Channel activity</term>
<term>Chloroquine resistance</term>
<term>Chromosomal</term>
<term>Circular element</term>
<term>Donovani</term>
<term>Donovani ldmdrl gene</term>
<term>Double minutes</term>
<term>Drug concentration</term>
<term>Drug effiux</term>
<term>Drug efflux</term>
<term>Drug resistance</term>
<term>Drug resistance mechanisms</term>
<term>England biolabs</term>
<term>Enriettii</term>
<term>Enriettii cells</term>
<term>Ethidium bromide staining</term>
<term>Expression vector paltneo</term>
<term>Extra band</term>
<term>Extrachromosomal</term>
<term>Extrachromosomal circle</term>
<term>Extrachromosomal element</term>
<term>Extrachromosomal elements</term>
<term>Fastlane agarose</term>
<term>Functional analysis</term>
<term>Gene</term>
<term>Gene amplification</term>
<term>Gene family</term>
<term>Gene products</term>
<term>Genomic</term>
<term>Harvard school</term>
<term>Hincli fragment</term>
<term>Human mdrl</term>
<term>Hybridized</term>
<term>Hydropathy plot</term>
<term>Internal duplication</term>
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<term>Ldmdrl gene</term>
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<term>Leishmania cells</term>
<term>Leishmania donovani</term>
<term>Leishmania enriettii</term>
<term>Leishmania tarentolae</term>
<term>Lemdrl</term>
<term>Lemdrl gene</term>
<term>Lemdrl sequence</term>
<term>Loading control</term>
<term>Ltpgpa</term>
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<term>Multidrug resistance</term>
<term>Mutant cells</term>
<term>Noti fragment</term>
<term>Nucleic acids</term>
<term>Nucleotide</term>
<term>Nucleotide binding motif</term>
<term>Nucleotide binding sites</term>
<term>Nylon paper</term>
<term>Parasite</term>
<term>Parental cells</term>
<term>Plasmodium falciparum</term>
<term>Previous work</term>
<term>Public health</term>
<term>Pulse time</term>
<term>Puromycin</term>
<term>Putative</term>
<term>Reading frame</term>
<term>Resistant</term>
<term>Resistant cells</term>
<term>Restriction pattern</term>
<term>Same filter</term>
<term>Sequence analysis</term>
<term>Sequence identity</term>
<term>Signal intensity</term>
<term>Significant homology</term>
<term>Southern analysis</term>
<term>Southern blot</term>
<term>Tafe</term>
<term>Transfected</term>
<term>Transfection</term>
<term>Transfection analysis</term>
<term>Transfection experiments</term>
<term>Transmembrane domains</term>
<term>Unrelated drugs</term>
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<term>Vinblastine resistance</term>
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<front><div type="abstract" xml:lang="en">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.</div>
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