Kinetoplastidae display naturally occurring ancillary DNA-containing structures

Abstract

Kinetoplast-derived, DNA-containing structures were found in several members of the order Kinetoplastida. The structures, for which we propose the name ancillary DNA-containing structures (aDNA), were discovered during the course of low-light-level video fluorescence microscopy studies using several nucleic acid-specific fluorescent reagents. DNase treatment and supravital stain with Höechst 33342 confirmed that aDNA is not an artifact of specimen preparation. Fluorescent in situ hybridization using either a 122-bp kinetoplast DNA-specific probe derived from a conserved region of minicircle DNA or a 188-bp nuclear DNA-specific probe derived from highly repetitive nuclear DNA demonstrated that aDNA is derived from the kinetoplast and not the nucleus. However, the structures do not contain minicircle DNA replication intermediates. Immunofluorescence assays using an anti-mitochondrial protein antibody, anti-mtp70, demonstrated that the structures contain mitochondrial protein and confirmed their kinetoplast origin. The frequency of occurrence of aDNA varies markedly between members of the Kinetoplastida. In the case of Trypanosoma cruzi stocks, the percentage of cells with aDNA was positively correlated to the population doubling time of the stock. However, there is no statistically significant relationship between the developmental or replicative stage of the parasite and the frequency of aDNA. An inhibitor of DNA topoisomerase I had no effect upon the frequency of aDNA. An inhibitor of DNA topoisomerase II gave equivocal results depending upon the parasite stock used. We speculate that aDNA may be the morphological consequence of a yet-to-be-determined biological process intrinsic to but variable within the Kinetoplastida.