Sequence characterization and comparative expression profile of buffalo WNT10B gene in adult and fetal tissues.

In this study, Wingless-type MMTV (mouse mammary tumor virus) integration site family member (WNT10B) gene was sequence characterized in the Indian water buffalo. Sequence analysis revealed an open reading frame of 1176 nucleotides in buffalo, encoding 391 amino acids long protein. Nineteen nucleotide variations were observed between cattle and buffalo resulting in six amino acid changes. Phylogenetic analysis showed the clustering of ruminant species together. Real-time expression analysis of WNT10B in tissues collected from different organs of fetal and adult buffalo, revealed, the gene being abundantly expressed in the rumen and liver of the fetus. The fetal ovary, heart, kidney, lung, testis and mammary gland showed moderate expression, while in adult tissues, expression was high in the ovary, testis, brain, kidney, small intestine and liver, whereas lower expression was observed in the adult rumen. Significant differences in WNT10B expression levels were found for the brain, small intestine, testes, kidney, heart, rumen, and ovary when adult and fetal tissues were compared. A moderate level of genetic variation was found between cattle and buffalo WNT10B and expression patterns in a variety of tissues in adult buffalo implies that in addition to possible roles in adipogenesis and hematopoiesis, the WNT10B gene might be playing a significant role in other regulatory pathways as well.

Sequence-based structural analysis and evaluation of polymorphism in buffalo Nod-like receptor-1 gene.

In this study, we have sequence characterized and analyzed the polymorphism in buffalo NOD1 (nucleotide-binding oligomerization domain 1) gene as well as its expression analysis. Full-length sequence analysis of NOD1 revealed this gene in buffalo being conserved with respect to the domain structures, similar to other species. Alternate splice variants having exon3 skipping also identified for the first time in the gene expressed in buffalo-purified peripheral blood mononuclear cells (PBMCs). Phylogenetically ruminant species were found to be clustering together and buffalo displaying maximum similarity with cattle. Sequencing of NOD1 across 12 Indian buffalo breeds identified 23 polymorphic sites within coding region, among which 16 were synonymous and 7 changes found to be non-synonymous. Four SNPs (single nucleotide polymorphisms) of them were genotyped in 393 animals belonging to 12 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, showing variable allelic frequencies. Principal component analysis revealed, riverine and swamp buffaloes being distinctly placed with the distribution of breeds within the group based on the geographical isolation. Further, quantitative real-time PCR detected NOD1 expression in multiple tissues with PBMCs and lungs showing highest expression among the tissues examined. Structural analysis based on the translated amino acid sequence of buffalo NOD1 identified four protein interaction motifs LxxLL important for ligand binding. Molecular interaction analysis of iE-DAP and NOD1-LRR and their complex stability and binding-free energy studies indicated variable binding energies in buffalo and cattle NOD1. Overall, the study reveals unique structural features in buffalo NOD1, important for species-specific ligand interaction.