Intracellular localization of a Trypanosoma cruzi kDNA minicircle transcript using RNA: RNA in situ hybridization

Using RNA: RNA in situ hybridization, the intracellular location of a transcript encoded by and spanning the entire length of a Trypanosoma cruzi kinetoplast DNA minicircle was determined. In axenically cultured T. cruzi epimastigotes, the hybridization signal was restricted to the kinetoplast, which was situated in the perinuclear region of the cell. Following conversion of epimastigotes to culture-derived metacyclic trypomastigotes, the kinetoplast moved to an acentric position in the metacyclic trypomastigote. Again, the hybridization signal co-localized with the position of the kinetoplast. These results suggested that the transcript remained closely associated with the T. cruzi kinetoplast within the mitochondrion in each of the morphological forms. Using specific oligonucleotide probes derived from a cDNA encoding the transcript, the entire native kDNA minicircle encoding the transcript was cloned and its nucleotide sequence was determined. The nucleotide sequence of the intact native minicircle was identical to that of the full-length cDNA corresponding to the minicircle transcript, indicating that the transcript was not modified prior to the time of cDNA synthesis and cloning.