Estudos sobre os mecanismos de recombinação em trypanosoma cruzi: obtenção e análise de células superexpressando o gene TcRaD51

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

10/08/2006

RESUMO

Trypanosoma cruzi is the parasite that causes Chagas disease. Only a few genes involved in DNA metabolism have been described in this species. Recently, our group characterized a gene encoding one of the key proteins involved in homologous recombination in T. cruzi, TcRad51. To better understand this process, we over-expressed TcRad51 in the CL Brener strain of T. cruzi. Northern blot assays showed an increase in mRNA levels of TcRad51 in the population of transfected cells as well as in cloned transfected cell lines. When we submitted these cells to agents that cause double strand DNA breaks, such as gamma radiation and Zeocin, we observed that cells over-expressing TcRad51 show an increased resistance to both agents. In addition, using pulse field gel electroforesis (PFGE), we observed a difference in the level of fragmentation of genomic DNA after exposition to gamma radiation, with the kinetics of chromosomal reconstitution being faster in transfected cells than in wild type cells. These results indicate TcRad51 has a role in the maintenance of genomic stability in this parasite, and participates in the process of double strand break repair after exposure to genotoxic agents. In addition, we also investigate the involvement of TcRad51 in gene targeting by homologous recombination. Wild type parasites and cells over-expressing TcRad51 were transfected with plasmids containing a drug resistance gene, the GFP gene and sequences homologous to tubulin. It was observed that cells over-expressing TcRad51 shows an increase in the number of clones resistant to G418 and GFP positive, which have probably the plasmid integrated into the tubulin locus. We also obtained clones derived from transfected cultures over-expressing TcRad51. The clones show similar growth when compared with the transfected population and wild type cells but show variable levels of TcRad51 mRNA expression. Further analysis using these clones will allow us to better understand the role of the TcRad51 gene in the T. cruzi DNA metabolism

ASSUNTO(S)

bioquímica teses.

ACESSO AO ARTIGO

Documentos Relacionados