S. M. Deb

Allelic diversity at MHC class II DQ loci in buffalo (Bubalus bubalis): evidence for duplication.

The genetic diversity of MHC class II DQ genes was investigated in riverine buffalo (Bubalus bubalis) by PCR-RFLP and sequencing. Highly variable regions (exons 2-3) of DQ genes were amplified from 152 buffaloes and genotyped by PCR-RFLP. Alleles identified by differential restriction patterns were sequenced for the characterization. PCR-RFLP was a rapid method to discriminate between DQA1 and duplicated DQA2 genes in buffalo, however, the method appeared to be inadequate for determining the more complicated DQB genotypes. A total of 7 and 10 alleles were identified for DQA and DQB loci, respectively. Nucleotide as well as amino acid variations among DQ alleles particularly at peptide binding regions were high. Such variations were as expected higher in DQB than DQA alleles. The phylogenetic analysis for both genes revealed the grouping of alleles into two major sub-groups with higher genetic divergence. High divergence among DQ allelic families and the isolation of two diverse DQA and DQB sequences from individual samples indicated duplication of DQ loci was similar in buffalo to other ruminants.

Isolation of two cDNAs encoding MHC-DQA1 and -DQA2 from the water buffalo, Bubalus bubalis.

In the present study, we explored structural and functional variations and possible duplication of the major histocompatibility complex (MHC)-DQA gene in water buffalo (Bubalus bubalis). Two cDNA sequences, amplified from one individual water buffalo, were designated as Bubu-DQA1 (DQA*0101) and -DQA2 (DQA*2001). The percentage of nucleotide and amino acid similarity between Bubu-DQA1 and -DQA2 revealed that these sequences display more similarity to alleles of respective DQA1 and DQA2 genes from other ruminant species than to each other. The phylogenetic analysis also revealed a considerably larger genetic distance between these two genes than between homologous genes from other species. The larger genetic distance between DQA*0101 and DQA*2001, and the presence of different bovine DQA putative locus specific amino acid motifs, suggests these sequences are non-allelic. This finding is consistent with DQA gene duplication in other ruminants.

cDNA characterization and molecular analysis of buffalo MHC class II gene, DRA (Bubu-DRA).

Abstract Sakaram, D., Niranjan, S.K., Kumar, S., Naskar, S., Deb, S.M., Mitra, A., Sharma, A. and Sharma, D. 2010. cDNA characterization and molecular analysis of buffalo MHC class II gene, DRA (Bubu-DRA). J. Appl. Anim. Res., 37: 73–76. Full cDNA of DRA gene was amplified in Murrah buffalo (Bubalus bubalis) and analysed to identify the genetic variability. Buffalo DRA (Bubu-DRA) sequence showed highest similarity with cattle (98%) followed by goat (96.5%) and sheep (96.1%). DRA gene was highly conserved among all the species especially ruminants. The residues involved in antigen binding at peptide binding site (PBS), N-linked glycosylation and heterodimer formation were highly conserved across the species. Unlike other MHC molecules, PBS sites of Bubu-DRA were highly conserved in al domain among all species. The absence of much variation in Bubu-DRA supports the notion that gene is mostly conserved among all mammalian species. However, highly conserved DRA gene among ruminants including buffalo may be attributed to its recent separation in evolutionary process.

Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis)

In the present study, water buffalo MHC (Bubu)-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

Genetic characterisation of buffalo MHC (Bubu)-DQB cDNA molecule

A 771 nucleotide long cDNA molecule corresponding to major histocompatibility complex (MHC)-DQB gene was amplified, cloned and sequenced from water buffalo (Bubalus bubalis). Open reading frame of the Bubu-DQB sequence was short by three nucleotides from the DQB genes of the other ruminants. Nucleotide similarity of Bubu-DQB was highest with cattle DQB alleles (84–97%), followed by goat (88.8%) and sheep (87.5%). The sequence comparison revealed that the DQB gene was highly variable in buffalo particularly, in exon 2 (β1 domain). A total of 45 amino acid substitutions were identified in Bubu-DQB compared to cattle DQB*0101 sequence, with a maximum (27) in β1 domain. The residues involved in antigen binding and heterodimer formation were found to be different in buffalo DQB sequence compared to other species. Phylogenetic study showed that the Bubu-DQB has evolved prior to the diversification of common DQB alleles in ruminants. Our study revealed the high genetic variation in the DQB gene in buffalo, which might have generated to recognise species specific antigens.

Genetic Differentiation Among Goats Using Randomly Amplified Polymorphic DNA (RAPD) Markers

Abstract Anbarasan, K., Sharma, A.K., Singh, R.K., Deb, S.M. and Sharma, D. 2001. Genetic differentiation among goats using randomly amplified polymorphic DNA (RAPD) markers. J. Appl. Anim. Res., 20: 83–88. To develop population specific markers using randomly amplified polymorphic DNA—ploymerase chain reaction (RAPD—PCR) in meat (Black Bengal and Non-descript Rohilkhand Local), milk (Barbari) and pashmina (Chegu) producing goats, a total of 35 random sequence decamer primers were tested. Nine primers generated polymorphism in one or more goat populations. Out of 117 bands scored, 46 (39%) were observed to be polymorphic. One of the primers (OPM-18) was found effective in differentiating the Pashmina (Chegu) from meat or milk type of goats. While the primer OPI-05 generated one band which might be specific for meat type goats i.e. Black Bengal and Non-descript (Local). However, population specificity and sequence homology for these bands need further investigation by hybridization, cloning and sequencing experiments.

Molecular Characterization of Bubaline Integrin β2 (ITGB2) cDNA

Abstract Sharma, D., Niranjan, S.K., Kumar, S., Deb, S.M., Naskar, S., Sharma, A. and Mitra, A. 2010. Molecular characterization of bubaline integrin β2 (ITGB2) cDNA. J. Appl. Anim. Res., 37: 217–220. A 2310-bp long buffalo (Bubalus bubalis) cDNA sequence of Integrin b2 (ITGB2) gene was characterized. At nucleotide sequence level, buffalo ITGB2 exhibited 96–98% homologies with other ruminants like cattle, bison, sheep, goat and deer. In coding region, total thirty two nucleotide differences have been observed between buffalo and cattle sequences including 15 non-homologous substitutions. Importantly, a conserved site (22) in PSI domain of the ruminants (Thr) was substituted in buffalo (Ala) indicating normal function of the integrin unless the second mutation in CD lie molecule coexists. Buffalo ITGB2 has seven differences in I-like domain region as compared to cattle. The transmembrane and cytoplasmic regions were highly conserved among buffalo and other ruminants. High homology and close phylogenetic association among ITGB2 sequences of the ruminants including buffalo indicates highly conserved nature of the gene.

Identification of Novel Allelic Variants of Integrin Beta 2 (ITGB2) Gene and Screening for Bubaline Leukocyte Adhesion Deficiency Syndrome in Indian Water Buffaloes (Bubalus bubalis)

A fragment of 570 bp corresponding to exon 5 and 6 of integrin beta 2 (ITGB2) gene was amplified for screening D128G mutation in one hundred and fifty two buffaloes (Bubalus bubalis) which causes bovine leukocyte adhesion deficiency syndrome (BLAD) in cattle, as well as to ascertain polymorphism. TaqI PCR-RFLP revealed no such mutation thus indicating the absence of bubaline leukocyte adhesion deficiency (BuLAD) allele in animals under study. However, the polymorphism studies using MspI restriction enzyme revealed two genotypic patterns viz. AA pattern (bands of 293, 141, 105, and 31 bp) and BB pattern (bands of 293, 105, 77, 64, and 31 bp). The sequences of A and B alleles were submitted to the GenBank (EU853307 and AY821799).

Molecular Variability of Somatotropin Releasing Hormone (SRH) Gene in Mithun (Bos frontalis)

Abstract Kumar, S., Manohar, R.P.V., Deb, S.M., Mitra, A., John, B., Sharma, A. and Bujarbaruah, K.M. 2008. Genetic variability of somatotropin releasing hormone (SRH) gene in Mithun (Bos frontalis). J. Appl. Anim. Res., 33: 205–207. A fragment of 452 bp corresponding to exon 2 and 3 of somatotropin releasing hormone (SRH) gene on amplification in 90 mithun (Bos frontalis) revealed two genotypie patterns viz. AA genotypie pattern (bands of 292 and 160 bp) and AB genotypie pattern (452, 292, and 160 bp) with respect to Saul restriction enzyme. The sequence of the A allele, which was the first report on mithun SRH gene, was submitted to GenBank (Accession No. EF014289).

Molecular characterisation of T cell receptor-zeta subunit (CD247) gene in buffalo (Bubalus bubalis)

The cDNA encoding T cell receptor-zeta (TCR-ζ; CD247) molecule of water buffalo (Bubalus bubalis) was isolated, cloned and sequenced in the present study. The CD247 cDNA comprised 1078 nucleotides including a 30 nucleotide 5′-untranslated region (UTR), 495 nucleotide single open reading frame (ORF) and 553 nucleotide 3′-UTR. Deduced amino acid of buffalo CD247 sequence was two residues shorter than the corresponding cattle and sheep sequences. However, ruminant-specific insertions and substitutions in intra-cytoplasmic (IC) domain were present in buffalo. Immunoreceptor tyrosine-based activation motifs (ITAMs), the important motifs for TCR signalling, were totally conserved among ruminants including buffalo. The 3′-UTR region of the buffalo CD247 was highly homologous to the corresponding region in the cattle sequence and showed lack of polymorphism after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using HaeIII and MseI restriction enzymes in buffalo population. Phylogenetically, buffalo sequence was closer to cattle sequence under the ruminants lineage. The conserved nature of this gene ensures TCR integrity which is vital for induction of optimal and efficient immune response.