Abstract
BackgroundThe Homeobox (Hox) family complex contains 39 genes, clustered into four groups (A-D) all expressing in sequential manner. The HOX proteins are transcriptional factors involved in regulation of pattern formation of the anterio-posterior body axis across the species. Most of the Hox family genes have been studied with respect to their organization and expression during the embryonic stages. However, expression pattern of Homeobox C11 (Hoxc11) gene in the 5′ region, particularly in higher mammals remains largely unexplored.ResultsWe cloned and expressed Homeobox C11 (Hoxc11) gene from water buffalo Bubalus bubalis. The recombinant HOXC11 protein expressed as inclusion bodies was solubilized in Tris buffer (10 mM, pH-6.5) and purified using Ni-NTA affinity column. The purity and molecular weight of HOXC11 protein (~33 kDa) were confirmed by SDS-PAGE and western blot analysis. Employing immunohistochemistry approach, we localized HOXC11 protein in the nuclei across the tissues of buffalo. Western blot analysis showed highest expression of HOXC11 protein in kidney and lung although its possible renal and respiratory roles are not yet established. Electrophoretic mobility shift assay (EMSA) demonstrated the specific binding of HOXC11 protein with the promoter element, CE-LPH1 of lactase-phlorizin hydrolase (LPH) gene showing reduced mobility of the protein-DNA complex, corroborating with earlier report on the possible role of this protein in intestinal functions. In silico analysis of HOXC11 showed predominance of α helices and presence of six conserved domains. We deduced the putative 3D structure of HOXC11 protein and fifteen possible DNA interacting residues within the homeodomain.ConclusionsPresent study augments our understanding on the specific expression of HOXC11 protein in kidney and lung in water buffalo. The fifteen DNA interacting residues reported herein provide an opportunity to establish much broader structural and functional perspectives of HOXC11 protein in the context of genome analysis in general and animal biotechnology in particular.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-638) contains supplementary material, which is available to authorized users.
Highlights
The Homeobox (Hox) family complex contains 39 genes, clustered into four groups (A-D) all expressing in sequential manner
Expressional validation of HOXC11 protein Recombinant HOXC11 protein expression was induced with 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) at 37°C for 4 hours
UV fluorescence spectra showed a maxima at 330 nm evidencing HOXC11 protein has buried tryptophan residues in the non-polar environment (Figure 1D)
Summary
The Homeobox (Hox) family complex contains 39 genes, clustered into four groups (A-D) all expressing in sequential manner. Most of the Hox family genes have been studied with respect to their organization and expression during the embryonic stages. 39 Hox genes organized into four distinct clusters (A, B, C and D) are located on chromosomes 7, 17, 12 and 2, respectively [2]. Each cluster in turn is composed of 9–13 closely related genes giving rise to distinct paralogs and often, not always, have overlapping functions. These paralogs arranged in a collinear manner are successively activated, from ‘head’ to ‘tail’ orchestrating sequential expression regulating development [3,4]
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