Specific A + T-Rich Repetitive DNA-Sequences in Maxicircles from Wildtype Leishmania mexicana Amazonensis and Variants with DNA Amplification

Abstract

To explain the low cross-hybridization between kinetoplast DNA maxicircles of Leishmania parasites that show DNA amplification and those of parasites without DNA amplification, we isolated and cloned two maxicircle fragments, one specific to each group of parasites. The cloned fragment from wildtype L. m. amazonensis (MbpW94) and that from an arsenite-resistant variant with DNA amplification (MpbA29) hybridized only to maxicircles from parasites of the group from which the fragment was originally derived. Both fragments were A+T-rich, tandemly repeated, and lacked long conserved open reading frames and transcriptional products. MpbW94 (685 bp) was harbored in a segment of roughly 12 kb in maxicircles of wildtype parasites and of an arsenite-resistant variant without DNA amplification, while MbpA29 (1121 bp) occupied a 6- to 7-kb segment of maxicircle DNA in arsenite- and tunicamycin-resistant variants with DNA amplification. These maxicircle DNA segments appear to resemble previously described maxicircle divergent regions of other kinetoplastids. The presence of these specific sequences allows differentiation between maxicircles of drug-resistant L. m. amazonensis with DNA amplification and those of parasites without DNA amplification and helps explain the low cross-hybridization between maxicircles of the two parasite groups. Furthermore, these sequences allow the study of the kinetics of the changeover of A+T-rich regions of maxicircles during the transition period from one maxicircle type to the other.