title: Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania.
abstract: Circular and linear amplicons were analyzed in detail in Leishmania tropica cells resistant to methotrexate (MTX). Both types of elements presented sequences related to the H locus and coexisted in resistant cells. The linear amplicons appeared first during the selection process (at 10 microM MTX) and varied with regard to size and structure in cells exposed to increasing concentrations of drug. The circular element was evident at higher concentrations (50 microMs) but was the major amplified DNA in cells resistant to 1000 microM MTX while the level of amplification of the linear elements remained low. The extrachromosomal DNAs were unstable in the absence of drug and their disappearance coincided with an increase in sensitivity to MTX. Mapping of the minichromosomes and the circular element showed that they were all constituted by inverted duplications. The circular amplicon contained an inverted repeat derived from the H locus that encompassed the pteridine reductase gene (PTR1) responsible for MTX resistance. The amplified segment in the linear amplicons was longer and included the pgpB and pgpC genes that encode P-glycoproteins of unknown function previously characterized in different Leishmania species.
authors.affiliation: Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, Granada, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Olmo
authors.forename: R
authors.initials: R
authors.lastname: Arrebola
authors.forename: V
authors.initials: V
authors.lastname: Bernier
authors.forename: D
authors.initials: D
authors.lastname: González-Pacanowska
authors.forename: L M
authors.initials: LM
authors.lastname: Ruiz-Pérez
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D004270:DNA, Circular / Q000235:genetics* / Q000378:metabolism; D016054:DNA, Protozoan / Q000235:genetics* / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D017125:Genes, Protozoan / Q000235:genetics; D007895:Leishmania tropica / Q000187:drug effects* / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics; D015183:Restriction Mapping
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015342:DNA Probes; D004270:DNA, Circular; D016054:DNA, Protozoan; D010088:Oxidoreductases; C085592:pteridine reductase; D008727:Methotrexate