Molecular mining of GGAA tagged transcripts and their expression in water buffalo Bubalus bubalis

Rawal, Leena; Ali, Safdar; Ali, Sher

doi:10.1016/j.gene.2011.10.004

Cited by 7 publications

(7 citation statements)

References 34 publications

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“…Buffalo is an economically important livestock species in the Indian sub-continent and South Asian countries [20]. We characterized HOXC11 protein from water buffalo Bubalus bubalis focusing particularly on its tissue specific expression, demonstrating its localization in the nuclei and finally deducing its putative 3D structure.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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“…Reaction specificity was confirmed with the analysis of melting curves. The copies of DMα and DMβ genes in buffalo genome were then extrapolated as per established protocols [35]. …”

Section: Methodsmentioning

confidence: 99%

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

Rawal

Panwar

Ali

2018

Cell Physiol Biochem

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Background/Aims: The major histocompatibility complex (MHC) categorized into three (I, II and III) classes elicits the immunogenic response by presenting exogenous peptides to T cells. The MHC-II DM is composed of DMα and DMβ, two polypeptide chains, both are encoded by separate MHC genes involved in antigen processing and presentation. Despite the acknowledged role of MHC complex in humans, the literature is silent on the organization and expression of these genes in water buffalo Bubalus bubalis, an agriculturally important animal species. Methods: We deduced the full-length mRNA sequences of DMα and DMβ genes, localized them onto the chromosome 2, assessed their copy number per haploid genome and studied tissue and disease specific expression. Results: The Real Time PCR showed higher expression of both the genes and their seven interacting partners in spleen, gonads and spermatozoa. Significantly, upregulation of DMα and DMβ genes and their interacting partners were detected in diseased group of buffaloes as compared to that in healthy ones. Conclusion: The upregulation of Bubalus bubalis (BuLA)-DMα and DMβ genes and their interacting partners reflect their role in regulating immune responses towards the amelioration of diseases. Work on this line would enhance our understanding on the overall roles of MHC locus, allowing development of possible therapeutic treatment strategies.

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“…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. The positive clones screened by colony-PCR and restriction digestion were then sequenced on Applied Biosystems 3130xl genetic analyzer on a 50 cm, 16 capillary array using BigDye Terminator v3.1 cycle sequencing kits (Applied Biosystems, Foster city, CA, USA) employing standard protocols [22]. …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

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Molecular mining of GGAA tagged transcripts and their expression in water buffalo Bubalus bubalis

Cited by 7 publications

References 34 publications

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

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

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

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

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