Drug resistance and P-glycoprotein gene amplification in the protozoan parasite Leishmania.
Amplification of the H circle is often associated with methotrexate (MTX) selection in Leishmania species. We have shown that the H circle of Leishmania tarentolae contains an open reading frame, ItpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). H region amplification was also noted in some mutants selected for resistance to arsenite and vinblastine. Mutants having the complete 68-kb circles were cross-resistant to MTX, but two arsenite mutants having only part of the H region amplified, but including ItpgpA, were not cross-resistant to MTX. These results suggest that the putative determinant for MTX resistance present on the H circle is not ItpgpA. We have also determined how ItpgpA-containing plasmids were generated from the chromosomal copy. The H circle contains a 30-kb inverted duplication separated by two unique DNA segments. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We have shown that the two unique segments in chromosomal DNA are flanked by inverted repeats suggesting that H circles could be formed by a foldback mechanism (see fig. 2). Unexpectedly, a plasmid present in cells selected for arsenite resistance lacked part of the H region and the long inverted repeats. It appears to have been formed by intrachromosomal recombination between two P-glycoprotein genes, ItpgpA and ItpgpB, located adjacent to the H region. Our results show that under drug pressure, the same P-glycoprotein-encoding region in Leishmania may be amplified by very different mechanisms and yield different amplicons.
Ouellette
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M
Service d'Infectiologie, CHUL, Québec, Canada.
Borst
P
P
D000818:Animals; D001151:Arsenic / Q000494:pharmacology; D018053:Arsenites; D004351:Drug Resistance / Q000235:genetics*; D005784:Gene Amplification / Q000187:drug effects / Q000235:genetics; D066298:In Vitro Techniques; D007891:Leishmania / Q000187:drug effects* / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D008727:Methotrexate / Q000494:pharmacology*; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D011995:Recombination, Genetic
D018053:Arsenites; D008562:Membrane Glycoproteins; C015001:arsenite; D001151:Arsenic; D008727:Methotrexate