Formation of Extrachromosomal Circular Amplicons with Direct or Inverted Duplications in Drug-Resistant Leishmania tarentolae

Abstract

Selection for methotrexate resistance in Leishmania spp. is often associated with amplification of the H locus short-chain dehydrogenase-reductase gene ptr1 as part of extrachromosomal elements. Extensive sequences are always coamplified and often contain inverted duplications, most likely formed by the annealing of inverted repeats present at the H locus. By gene targeting mediated by homologous recombination, several repeated sequences were introduced in the vicinity of ptr1. Selection for methotrexate resistance in these transfectants led to ptr1 amplification as part of small circles with direct or inverted duplications whether the integrated sequences consisted of direct or inverted repeats. Hence, for a region to be amplified in L. tarentolae during drug selection, a drug resistance gene is required and must be flanked by (any) homologous repeated sequences. The distance between these repeats and their orientation will determine the length of the amplicon and whether it contains direct or inverted duplications.

Intrastrand Annealing Leads to the Formation of a Large DNA Palindrome and Determines the Boundaries of Genomic Amplification in Human Cancer

“…Second, a DNA IR also serves either as an "at-risk-motif" or a "fragile site" to regulate large palindrome formation in S. cerevisiae when DNA replication, repair, and checkpoint function are compromised (14,18). Furthermore, IR sequences preexisting in the genome are found at the centers of palindromes in Leishmania and Schizosaccharomyces pombe (2,11). This DNA IR-facilitated model of gene amplification predicts that palindrome formation is in part regulated by the presence of DNA IRs and occurs nonrandomly in the genome, which may provide a mechanistic basis for the nonrandom distribution of DNA palindromes in human cancer cells (36).…”

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…The copy number of its chromosome may vary either in wild-type (WT) cells or in drug-resistant mutants (31)(32)(33)(34), and the ploidy of specific chromosomes of individual cells may differ within a population, a concept known as mosaic aneuploidy (35). Leishmania also amplifies specific portions of its genome by gene rearrangements at the level of direct or inverted repeated sequences, leading to small extrachromosomal circular or linear amplicons (32,34,36,37). Recently, we found that repeated sequences are widespread in the Leishmania genome and that there is constitutive amplification at the level of these repeated sequences (J. M. Ubeda et al, submitted for publication).…”

Linear amplicons as precursors of amplified circles in methotrexate- resistant Leishmania tarentolae

“…The circular and linear amplicons in MTX-resistant Leishmania tropica and Leishmania tarentolae were derived from the H locus. Furthermore, analysis of the MTX-resistant mutants at different steps of drug selection showed that the linear amplicons were formed during the first steps of selection, while the circles appeared later, thus suggesting a linear precursor-circular product relationship (11,12). A similar precursor-product relationship has also been proposed for formation of the CD1 circles derived from the linear amplicon LD1 (13).…”

“…Amplification of the H locus upon MTX selection has been described in several Leishmania species (21)(22)(23). The extrachromosomal circular amplicons derived from the L.tarentolae H locus have been characterized and were shown to be created at the level of direct or inverted repeated sequences flanking ptr1 (9,12,24). In this study, by tagging the individual alleles of the H locus with different dominant markers, we were able to follow the formation of amplicons at different steps of MTX selection.…”

“…Extrachromosomal linear amplicons generated while selecting for drug resistance were described more recently (8,9). Co-existence of circular and linear amplicons were described in α-difluoromethylornithine-resistant Leishmania donovani (8), in arsenite-resistant Leishmania species (10) and in methotrexate (MTX)-resistant Leishmania (11,12). The circular and linear amplicons in MTX-resistant Leishmania tropica and Leishmania tarentolae were derived from the H locus.…”