TRYPANOSOMA-CRUZI - FLOW CYTOMETRIC ANALYSIS OF DEVELOPMENTAL STAGE DIFFERENCES IN DNA

Flow cytometry and DNA binding-specific fluorescent reagents were used to compare the total DNA, G-C, and A-T content of the epimastigote and trypomastigote stages of Trypanosoma cruzi stocks. Significant total DNA differences of 2-12% between epimastigotes and trypomastigotes were found in three of six stocks studied. The epimastigote G-C content of five of six stocks was 4-8% higher than trypomastigotes, whereas the trypomastigote A-T content was 2.5-13% higher than the epimastigote A-T content. Although no obvious developmental stage association between total DNA and base composition was found, intrastage associations do exist. These observations were unaffected by nucleoprotein extraction implying that the observed differences between trypomastigotes and epimastigotes are not a consequence of nucleoprotein interference with DNA-binding fluorochromes. The nuclei and kinetoplasts of four T. cruzi stocks were isolated and analyzed. Developmental stage differences in nuclear and kinetoplast DNA are stock-dependent and base composition-dependent; both organelles contribute to the observed differences in DNA of intact cells. We found a nearly linear association between the percentage of total kinetoplast DNA, G-C, and A-T content. During metacyclogenesis, the G-C content decreases by approximately 7% as epimastigotes transform into metacyclic trypomastigotes. The decrease in G-C content precedes changes in morphology or in complement resistance. If the DNA changes are causally connected to developmental stage transformations in T. cruzi remains to be determined. However, our results could facilitate studies of the molecular genetic processes the parasite uses to successfully complete various phases of its life cycle and, consequently, the disease process it evokes.