Beginning to characterize the L. amazonensis telomerase Giardini, M.A.1,3; Siqueira Neto, J.L.1,3; Lira, C.B.B.1,2; Cano, M.I.N.1,3
1Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP); 2Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron (LNLS); 3Departamento de Genética, Instituto de Biociências de Botucatu, Universidade Estadual Paulista "Júlio de Mesquita Filho".
Telomeres are the physical ends of eukaryotic chromosomes. They are specialized nucleoprotein structures responsible for maintaining the genome integrity and stability. In Leishmania spp., telomeres consist of conserved TTAGGG repeats, which are maintained by the action of telomerase. Telomerase is a ribonucleoprotein composed of a reverse transcriptase component (telomerase reverse transcriptase - TERT), an intrinsic RNA component (telomerase RNA - TER) and several other associated proteins. It ensures the complete DNA replication by adding new telomeric sequences to the G-rich strand and it also works as part of the higher order complex that regulates the capped/uncapped telomere states. The Leishmania amazonensis telomerase gene has recently been cloned and characterized in our lab (Giardini et al., 2006). This study aims the characterization of the biological features of L. amazonensis telomerase (LaTERT), including its subcellular localization and the role it plays in parasite telomeres. Recombinant LaTERT, as well as truncated versions of the protein, were obtained using the pET28(a)+ bacterial expression system. Western blotting experiments showed that a rabbit polyclonal antibody produced from the C-terminal half of the recombinant protein has recognized a native polypeptide of approximately 150 kDa in a total S100 L. amazonensis extract. A chromatin immunoprecipitation (ChIP) assay made possible to demonstrate that LaTERT immunoprecipitates with the telomeric DNA in vivo. An indirect immunofluorescence assay is underway and may help us to undercover the subcellular localization of L. amazonensis telomerase during parasite life span. Financial Support: FAPESP, UNICEF/ UNDP/World Bank/WHO (TDR).
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