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Nucleic Acids Research, 1995, Vol. 23, No. 15 2856-2864
© 1995

GENOME STRUCTURE AND MAPPING

Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania

Asuncioón Olmo, Roasalia Arrebola, Victor Bernie, Dolores González-Pacanowska and Luis M. Ruiz-Pérez*

Instituto de Parasitologia y Biomedicina, Consejo Superior de Investigaciones Cientificas C/ Ventanilla 11, Granda 18001, Spain

*To Whom correspondence should be addressed

Received May 9, 1995. Accepted June 22, 1995.

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 µM 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 µM) but was the major amplified DNA in cells resistant to 1000 µM 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 pterldine reductase gene (PTR1) responsible for MTX resistance. The amplified segment in the linear amplicons was longer and included the pgpBand pgpCgenes that encode P-glycoproteins of unknown function previously characterized In different Leishmania species.


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