MVSc

The study was undertaken with the objectives of cloning and characterization of the gene encoding the milk protein alpha-lactalbumin (α-LA) of Vechur cow of Kerala. The α-LA is a mammary gland specific protein found in high concentrations in milk of many species and has a role in regulating lactose synthase. Alpha-lactalbumin is homologous with the c-type lysozymes and provides an example of extreme functional divergence in homologous proteins with closely similar structures.
The genomic DNA was isolated from blood samples and a 1756 bp fragment of the entire transcriptional unit of the α-LA was amplified by PCR using synthetic oligonucleotide primer pair designed based on the bovine α-LA gene sequence. The gel purified PCR product was ligated in to a T-vector and was transformed by giving heat shock to competent E. coli cells prepared by CaCl2 treatment. The recombinant clones among the transformed cells were identified by Blue–White Screening and the recombinant plasmid carrying the insert gene was isolated from the white clones by a modified SDS-alkaline lysis method. The 1.756 kb α-LA gene insert in the vector was sequenced by the dideoxynucleotide sequencing method with primer walking using an automated DNA sequencer.
The sequence was found to be having 99 per cent homology with that of Bos taurus, 98 per cent with that of Yak and 95 per cent with that of sheep α-LA gene. Comparison of the human and Vechur α-LA genes showed identical structural organization and identifies extensive homology within the transcription unit. The exon-intron boundaries in the human α-LA gene occur at the codons of the amino acid residues, Leu-26 (intron 1), Lys-79 (intron 2) and Trp-104 (intron 3). The latter two residues are conserved in both species and also in Bos taurus, whereas Leu-26 is replaced by Trp-26 in the Vechur and bovine proteins. This strict homology in the sites of insertion of introns suggests that the exon-intron organization of these genes was established before the divergence of these species. The positions of the exon-intron boundaries are also conserved as evidenced from similar sizes of the exons. The introns sizes are also comparable except in the case of intron 1, which is much larger in the human gene as a consequence of the insertion of a Alu family repeat sequence.
The Vechur α-LA gene has an open reading frame of 426 nucleotides encoding a signal peptide of 19 amino acid residues and a mature protein of 123 amino acid residues with NH2 terminal glutamic acid and COOH- terminal leucine. Alignment of this sequence with bovine counterpart showed that 122 amino acid residues are identical and with human α-LA sequence showed 73 per cent identity.
The predicted secondary structure of Vechur α-LA showed that the larger α- helical lobe is formed by the amino- and carboxyl-terminal sections of the polypeptide chain while the smaller lobe, which encompasses a small three stranded antiparallel beta-sheet, and a small irregular structure, is formed by the central section of the polypeptide chain. The predicted tertiary structure of Vechur α-LA also showed high homology with the bovine and human α-LA structures.
Overall, the structures of Vechur α-LA was found to be very similar to that of Bos taurus and human reflecting their high degree of amino acid sequence identity. The present study did not reveal any higher degree of structural or functional similarity between Vechur and human α-LA proteins as compared to that of Bos taurus. The superiority of human milk and its high suitability to infants could be due to the higher content of α-LA and might not be attributed to any structural variations of the protein. Since the higher content of α-LA in human milk could be due to the high expression of this gene, further studies may be carried out to find out sequence variations, if any, occur in the regulatory sequences upstream of the gene. Gene expression studies are suggestive as α-LA locus can also be used as a genetic marker to increase milk production in Vechur cattle, as this marker may be directly responsible for increased milk production.