We are interested in understanding the molecular mechanisms controlling gene expression in Trypanosoma cruzi. We have attempted to identify, in T. cruzi, genes which might share DNA homology with known transcriptional activators in yeast (e.g., GCN4) and mammalian cells (e.g., c-fos and c-myc). We found that T. cruzi DNA, in dot-blot hybridization experiments, hybridizes with the c-fos- and GCN4-specific probes, but not with a probe specific for c-myc. Further analysis by Southern hybridization revealed that the c-fos probe hybridizes to a single 8.1 kb band in EcoRl-digested T. cruzi DNA. The cloning and sequencing of this 8.1 kb DNA fragment should allow the identification of any c-fos-related genes contained within it. Moreover, we would like to study the regulation of this putative fos-related gene in vivo. Towards this end, we have initiated experiments with the ultimate goal of developing a plasmid-based transformation system in T. cruzi. We have been able to grow the parasite on a solid medium, which allows us to detect foreign DNA in the parasite by colony hybridization experiments. We have successfully transformed T. cruzi with plasmid DNA, and reisolated this DNA from parasite nuclei. Finally, we have determined that T. cruzi protein synthesis and parasite multiplication ceases in the presence of the antibiotic Geneticin. Parasites resistant to the drug have not been apparent. Thus, the gene (neo) conferring resistance to this drug may be useful as a resistance marker in a T. cruzi-based plasmid vector.
