Characterization of the Ets-domain of Human SPI-C: Gene Construction, Cloning e Expression.
Scholles, A.B.; Renard, G.; Werlang, I.C.R.; Batista, E.; Basso, L.A.; Santos, D.S.
Centro de Pesquisas em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontificia Universidade Católica do Rio Grande do Sul / TECNOPUC. Avenida Ipiranga, 6681, Prédio 92A – Porto Alegre-RS, CEP 90619-900 – Brazil.
Cancer can be defined as a genetic disease, resulting from multiple events associated with initiation, promotion and metastatic growth. Cancer results from the loss of control of cellular homeostasis. Cell homeostasis is the result of the balance between proliferation and cell death, while cellular transformation can be viewed as a loss of relationship between these events. ETS proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis (Carlsson R. et al., 2005, European Journal of Cancer, 41, 2462-2478). SPI-C protein belongs to SPI subfamily of the ETS family. Members of SPI subfamily seem to play an important role in lymphocyte development (Shintani S. et al., 2000, PNAS, 97, 7417-7422). Human SPI-C mRNA is preferentially detected in foetal and adult spleen, lymph nodes and lower levels in bone marrow and foetal liver (Carlsson R. et al., Gene, 299, 271-278). This work reports the construction of the DNA sequence that codes for 105 amino-acids of human SPI-C. The work have presented is a step towards the understanding of molecular mechanisms of DNA recognition by members of the Ets-domain family of eukaryotic transcription factors. The region of the gene that codes for Ets-domain of SPI-C was synthetically constructed by the PCR extension of overlapping oligonucleotides by a DNA polymerase (patent deposited on INPI – 016050005891). This fragment was cloned into pCR-Blunt vector and subcloned into pET- 23a(+) expression vector using NdeI e HindIII restriction enzymes. The latter construction was sequenced and are currently underway expression assays with different strains of Escherichia coli. The biochemical and structural data will be used to classify the DNA binding activities aiming at building a DNA binding domain knowledge database to develop new analysis software and techniques for efficient and effective interpretation of experimental protein-DNA interaction data.
Supported by CNPq.
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