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Characterization of a TBP-like factor from Trypanosoma cruzi. Cribb, Pamela; Zelus, Dominique; Girardini, Javier; Zemzoumi, Khalid and Serra, Esteban. Instituto de Biología Molecular y Celular de Rosario. Suipacha 531. 2000. Rosario. <pcribb@fbioyf.unr.edu.ar> Gene expression of Trypanosoma cruzi and other kinetoplastid protozoa is believed to be regulated mostly at the post-transcriptional level since numerous post transcriptional regulation events but no RNApol II-dependent transcriptional regulation mechanism have been described. The TBPs are basal transcription factors that participate in the assemble of the initiation complex of the three RNA polymerases of eukariotes and in archeabacteria. Based on sequence alignment of TATA binding proteins (TBPs) from different organisms, we designed degenerate oligonucleotides and amplified a 300 bp fragment by PCR with DNA from T. cruzi . This fragment was later used as a probe for the screening of a genomic library, what allowed us to identify a clone containing an ORF of 789 bp that encodes a 28.7 kDa protein with an average identity of 45% with other TBPs. The analysis of the sequence obtained suggest that it codifies for a protein with intermediate characteristics between eukariotic TBPs and TBP-like factors (TLFs). We called this protein TcTLF-1. We cloned the coding region in pGEX and pQE vectors and were capable of express in E.coli and purify TcTLF-1 both as a fusion to GST and with a poli-His tail. Antibodies obtained against recombinant TcTLF-1 were used in western blot, indicating that the protein is present in both epimastigotes and trypomastigotes. In order to determine the DNA sequence to which TcTLF-1 binds we used a "Selex" strategy, and after five cycles we determined that our protein binds to sequences rich in G+C. Area temática: Parasitología Poster

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