Single stranded DNA binding proteins in trypanosoma cruzi

DESCRIPTION (provided by applicant): American trypanosomiasis, or Chagas' disease is a major public health problem among poor rural populations in Latin America, where 16-18 million persons suffer from this disease, and 90 million are at risk. Sharp differences in regulation of gene expression between trypanosomatids and their mammalian hosts have been described, suggesting this process as an interesting target for drug design. In these parasites, the presence of polycistronic transcription means that post-transcriptional regulation of gene expression is the main step in determining the abundance of mRNAs. Using nuclear protein fractions from Trypanosoma cruzi epimastigotes, the group in Uruguay demonstrated the formation of at least three complexes that specifically recognize two dinucleotide repeated sequences (poly[dT-dG] and poly[dC-dA]) which are abundant in the vicinity of coding regions of T. cruzi genes. The specificity and preliminary estimation of equilibrium constants of these complexes are in good agreement with reported data for gene regulatory proteins. The goal of this project is to determine whether these dinucleotide repeats and the proteins that bind to them have a role in regulation of gene expression, transcription or in selective transcript processing, degradation or stabilization or recombination. The specific aims of this FIRCA project are: 1. to characterize the proteins interacting with the cis target (purification and microsequencing), to determine the fine subcellular localization of these proteins by immunofluorescence microscopy and to determine if they show stage specific expression by western analysis. 2. to characterize the genes encoding these proteins (cloning, sequencing and analysis by southern blot and chromosomal location as well as northern blot analysis of their expression). 3. to examine the dinucleotide sequence for effect on expression of reporter constructs. The location and the minimum length of the repeat sequence and, if it corresponds, the stage at which expression is altered [posttranscriptional, translational, etc.] will be examined. These experimental approaches will lead to an understanding of the role of these dinucleotide repeated sequences and the function and significance of the proteins that specifically recognize them providing insights into the regulation of gene expression in T. cruzi.