Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

Abstract

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. cx-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.

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

“…Amplicons in Leishmania spp. can be found either as extrachromosomal circles (reviewed in references 2, 27, and 41) or as linear molecules with telomeric sequences (14,15,25,34,47). Circular and linear amplicons contain either direct (head-to-tail) or inverted (head-to-head) repeats (3,16,19,23,29,34,43), two types of structures also found in extrachromosomal circular amplicons of tumor cells (reviewed in references 39 and 40).…”

Drug Target Identification Using a Trypanosome Overexpression Library

“…It targets ornithine decarboxylase (ODC) (17), an enzyme involved in the production of trypanothione, which is essential for redox homeostasis in trypanosomes (18). It is known that overexpression of ODC can yield resistance in Leishmania donovani (19), and we showed that, in trypanosomes, it clearly confers a selective advantage to cells growing under DFMO pressure (Fig. 2).…”

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

“…These amplicons appear to be linear in nature by their pulse time-dependent migration on contourclamped homogeneous electric field gels (46). Leishmania species exhibit considerable chromosomal plasticity in response to stress and are known to amplify both circular and linear chromosomal elements in response to selective pressure (32,33,(47)(48)(49)(50). Although the L. donovani genome has not been reported, the genome of L. infantum, a species phylogenetically akin to L. donovani, reveals that APRT is localized ϳ29 kb from the end of chromosome 26 (36).…”

“…Pulse Field Gel Electrophoresis (PFGE)-InCert-agarose (Cambrex, Rockland, ME) plugs (5%) containing 2 ϫ 10 7 L. donovani promastigotes were prepared as described (32,33). Chromosomes of wild type, ⌬aprt, ⌬hgprt/⌬xprt, and two independent clones of ⌬hgprt/⌬xprt[Ino/Hyp] parasites were fractionated by PFGE using a contour-clamped homogeneous electric field gel apparatus (Bio-Rad) on a 1% agarose gel at 14°C for 24 h with a 60-s pulse time in 0.5ϫ Tris-borate-EDTA buffer as described (33).…”

“…Chromosomes of wild type, ⌬aprt, ⌬hgprt/⌬xprt, and two independent clones of ⌬hgprt/⌬xprt[Ino/Hyp] parasites were fractionated by PFGE using a contour-clamped homogeneous electric field gel apparatus (Bio-Rad) on a 1% agarose gel at 14°C for 24 h with a 60-s pulse time in 0.5ϫ Tris-borate-EDTA buffer as described (33). The gel was stained with ethidium bromide, and a conventional Southern blot was performed (26) using 32 Plabeled full-length APRT coding sequence as the probe (7).…”