This project is concerned with analyses of the genetic diversity of medically important parasitic protozoa and its implications to the epidemiology, course, and diagnosis of disease. The project has become increasingly involved in isolation of diversity at the DNA level. We have developed a method to synchronize the DNA synthetic cycle of trypanosomatids using very high levels of hydroxyurea. We are utilizing the technique for the production of large quantities of synchronized cells for elucidation and analyses of cell cycle-specific and developmental stage-specific substances. We have recently identified the presence in kinetoplastids of cytoplasmic DNA elements which contain kDNA minicircles and mitochondrial proteins. We are utilizing topoisomerase inhibitors to further analyze these DNA particles. It is possible that these particles represent the sites of guide RNA synthesis in the kinetoplastids. We have developed the technology to analyze the DNA synthetic cycle of amoeba by flow cytometry. The technique overcomes the problem of nonspecific background fluorescence which exists when amoeba are prepared for flow cytometry by conventional methods. The low-light- level video microscopy techniques developed for the study of the interaction of malaria merozoites with erythrocytes has been expanded to include the interaction of Trypanosoma cruzi and Toxoplasma gondii with vertebrate cells.
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