Characterization of amplicons that suppress the conditional lethal growth phenotype of a Leishmania donovani mutant lacking normal purine salvage mechanisms

A conditionally lethal mutant of Leishmania donovani that lacks both hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase exhibits a strikingly restricted growth phenotype, can only survive as the promastigote under pharmacological constraints, and is profoundly compromised in its ability to infect macrophages and mice. Interestingly, the conditionally lethal growth phenotype displayed by these mutant parasites can be suppressed in vitro by selection of strains that have markedly amplified the adenine phosphoribosyltransferase gene on extrachromosomal elements that are unique to these suppressor strains. Employing pulsed field gel electrophoresis, we have now determined that the amplicons in two of these suppressor lines are linear molecules by: (1) their pulse time-dependent mobility; (2) the failure of γ-irradiation to generate new discrete bands; (3) their susceptibility to λ exonuclease digestion; and (4) the presence of telomeric sequences. Pulsed field gel electrophoresis also shows these amplicons to be approximately 200–275 kb in size. However, quantitative polymerase chain reaction and Southern blot analyses demonstrated that the amplification units are ∼40 kb in length, implying that the formation of these amplicons involved additional chromosomal rearrangements or oligomerization.

Extrachromosomal elements in suppressor strains of purine salvage-deficient Leishmania donovani are linear molecules of 200–275 kb with a basic amplification unit ∼40 kb in length.Figure optionsDownload high-quality image (90 K)Download as PowerPoint slideResearch highlights▶ Amplified APRT sequences in L. donovani localize to extrachromosomal elements. ▶ Extrachromosomal elements in suppressor strains exhibit a linear topology. ▶ Amplification units consist of 36–43 kb of L. donovani chromosome 26 DNA. ▶ Amplified regions are smaller than the extrachromosomal elements.