COEXISTENCE OF CIRCULAR AND MULTIPLE LINEAR AMPLICONS IN METHOTREXATE-RESISTANT LEISHMANIA

Circular and linear amplicons were analyzed in detail in Leishmania tr opica cells resistant to methotrexate (MTX). Both types of elements pr esented sequences related to the H locus and coexisted in resistant ce lls. The linear amplicons appeared first during the selection process (at 10 mu M MTX) and varied with regard to size and structure in cells exposed to increasing concentrations of drug. The circular element wa s evident at higher concentrations (50 mu M) but was the major amplifi ed DNA in cells resistant to 1000 mu M MTX while the level of amplific ation of the linear elements remained low. The extrachromosomal DNAs w ere 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 inv erted duplications. The circular amplicon contained an inverted repeat derived from the H locus that encompassed the pteridine reductase gen e (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.