Molecular characterization of exon 2-3 of major histocompatibility complex (MHC) class I in buffalo (Bubalus bubalis)

Abstract

Present investigation was undertaken to characterize the exon 2 to 3 of major histocompatibility complex (MHC) class I genes in buffaloes (BuLA-A) of Murrah, Mehsana and Bhadawari breeds using Polymearse chain reaction-restriction length fragment polymorphism (PCR-RFLP). Two primers corresponding to the region of exon 2 intron 2 and exon 3 of BoLA-A were used to amplify the fragment of 714 bp. The PCR-RFLP patterns with respect to DdeI, TaqI, and HinfI restriction enzymes produced distinct polymorphic patterns in all the breeds. Two new patterns with the fragment sizes as 614, 60, 39 bp and 417, 187, 78, 21, 12 bp with DdeI digestion and 608, 97, 9 bp and 609, 106 bp with TaqI digestion were observed. The selected genotypes were cloned and sequenced to find out any change in nucleotides or amino acids with respect to the Holstein Friesian sequence available in the NCBI GenBank. The sequencing results showed that the exon 2- exon 3 products varied from 713 to 715 bp in buffaloes. Polymorphism in exon 2 occurred mostly due to changes of nucleotides at the positions from 62 to 72, 121 to170 and 184 to 237. The exon 3 initiated at 468 position of nucleotide after intron 2, which had purine rich region conserve sequence CGGGTCA. The phylogenetic tree predicted changes of higher degree in nucleotides as well as amino acids of exon 2- exon 3 and the nucleotide dissimilarity ranged from 3.0 to 14.7%. The results of the present study revealed that the exon 2 to 3 was found polymorphic in the buffaloes.