Differential expression of Homeobox C11 protein in water buffalo Bubalus bubalis and its putative 3D structure

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.

Transcriptional Dynamics of Homeobox C11 Gene in Water Buffalo Bubalus bubalis

“…In a recent study, from our laboratory, we localized the HOXC11 protein in the nuclei of gonads and different somatic tissues of buffalo employing immunohistochemistry. Subsequently, immunoblotting using specific antibodies showed differential expression of buffalo HOXC11 protein reflecting its possible tissue-specific function (Sharma et al, 2014). In the present study, we have demonstrated varying levels of Hoxc11 gene expression in an adult buffalo.…”

Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis

“…Total RNA was isolated from the ovary following standard protocols and stored at—80°C, quality and integrity of RNA was tested on 1% formaldehyde agarose gel [ 20 ]. Further, RNA was quantified using a NanoDrop (ND-1000 Spectrophotometer, Thermo Fisher Scientific, USA) and tested for genomic DNA presence with β-actin primers ( ACTB ) (forward: and reverse: ).…”

“…The PCR reaction conditions involved initial denaturation at 95°C for 3 minutes, followed by 35 cycles each with a subsequent denaturation at 95°C for 1 minute, annealing at 52.0°C ( KGF ) and 54.0°C ( KITLG ) for 1.5 minutes and extension at 72°C for 2 minutes followed by a final extension at 72°C for 10 minutes. Each PCR product was electrophoresed on 1% agarose gel in 1x TAE buffer, sliced and DNA was eluted using gel extraction kit (Qiagen, Germany) [ 20 ]. Subsequently, eluted DNA fragments corresponding to KGF and KITLG were cloned between the respective restriction enzyme sites of His-tagged pET28a (Novagen, USA) and GST-tagged pGEX-4T1 (Amersham Bioscience) vectors, respectively.…”

Major Histocompatibility Complex Dmα/β Genes and Their Expressional Diversity in Healthy and Diseased Water Buffalo Bubalus bubalis

“…Screening for the positive clones were done by colony-PCR. The recombinant clones were then sequenced on 3130xl genetic analyzer using BigDye® Terminator v3.1 cycle sequencing kits (Applied Biosystems, USA) [33]. Then the deduced mRNA sequences of BuLA-DMα and DMβ genes were submitted to the NCBI database and their corresponding accession numbers were obtained.…”