Members of the order Kinetoplastida which include Trypanosoma and Leishmania are responsible for major tropical diseases. These flagellated protozoa share one of the most unusual mitochondrial DNA structures found in nature. The single mitochondria present in these organisms contains a relatively large proportion of the cellular DNA, kinetoplast DNA (kDNA), organized in a disk-shaped structure and made up of a population of two types of topologically linked circular molecules. In spite of recent advances made in our understanding of kDNA replication, outstanding questions remain about this complex DNA structure. The major goal of our research is to contribute to a better understanding about the structure and function of kDNA networks. We wish to argue that specific protein-kDNA interactions play a key role in the maintenance and segregation of these structures. Therefore, the identification and characterization of kDNA-associated proteins (KAPS) may provide some information about the function of these unusual DNA structures. We have focused our attention on a set of non-histone-like proteins that remain associated to Trypanosoma cruzi kDNA after a 2M NaCl extraction of kDNA in the presence of a mild detergent. Using immuno-electron microscopy and fluorescent microscopy we have observed that rabbit antisera raised again these proteins, recognize epitopes localized in the kinetoplast of T. cruzi cells. More interestingly, the same antibody preparation cross-reacts with similarly localized epitopes in Leishmania amazonensis and Trypanosoma brucei. Based on these observations, the present research proposal attempts to identify and characterize T. cruzi KAPS. We propose first, to screen a T. cruzi cDNA expression library with the antibody preparation described above and second, to select and characterize clones encoding putative T. cruzi KAPs. By understanding the structure and function of T. cruzi KAPs we may learn a great deal about how kDNA networks are maintained and propagated. This information may also provide us with a more rational approach toward the complete eradication of T. cruzi the causative agent of American trypanosomiasis.
