KINETOPLASTIDAE DISPLAY NATURALLY-OCCURRING ANCILLARY DNA-CONTAINING STRUCTURES

Kinetoplast-derived, DNA-containing structures were found in several m embers of the order Kinetoplastida. The structures, for which we propo se the name ancillary DNA-containing structures (aDNA), were discovere d during the course of low-light-level video fluorescence microscopy s tudies using several nucleic acid-specific fluorescent reagents. DNase treatment and supravital stain with Hoechst 33342 confirmed that aDNA is not an artifact of specimen preparation. Fluorescent in situ hybri dization using either a 122-bp kinetoplast DNA-specific probe derived from a conserved region of minicircle DNA or a 188-bp nuclear DNA-spec ific probe derived from highly repetitive nuclear DNA demonstrated tha t aDNA is derived from the kinetoplast and not the nucleus. However, t he structures do not contain minicircle DNA replication intermediates. Immunofluorescence assays using an anti-mitochondrial protein antibod y, anti-mtp70, demonstrated that the structures contain mitochondrial protein and confirmed their kinetoplast origin. The frequency of occur rence of aDNA varies markedly between members of the Kinetoplastida. I n the case of Trypanosoma cruzi stocks, the percentage of cells with a DNA was positively correlated to the population doubling time of the s tock. However, there is no statistically significant relationship betw een the developmental or replicative stage of the parasite and the fre quency of aDNA. An inhibitor of DNA topoisomerase I had no effect upon the frequency of aDNA. An inhibitor of DNA topoisomerase II gave equi vocal results depending upon the parasite stock used. We speculate tha t aDNA may be the morphological consequence of a yet-to-be-determined biological process intrinsic to but variable within the Kinetoplastida .