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Author: Archana.S × End date: 2011 × Start date: 2010 ×
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    ThesisItemOpen Access
    Molecular characterisation of the growth hormone gene of Vechur cattle
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) Archana.S; T.V. Aravindakshan
    The study was undertaken with the objective of molecular characterization of the growth hormone (GH) gene of Vechur cattle. It is a 22,000 dalton single-chain polypeptide hormone that stimulates growth, cell reproduction and regeneration in humans and other animals. The production of growth hormone is tissue specific and occurs predominantly within a specialized subset of cells in the anterior pituitary gland. The genomic DNA was isolated from blood samples and a 1895 bp fragment of the entire transcriptional unit of the GH was amplified by PCR using synthetic oligonucleotide primer pairs designed based on the bovine sequence (Accession # M57764; Gordon et. al., 1983) downloaded from the National Centre for Biotechnology Information (NCBI) site . The gel purified PCR product was ligated into the pGEM®-T Easy cloning 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 ~1.9 kb GH gene insert in the vector was sequenced by the dideoxynucleotide sequencing method with primer walking using an automated DNA sequencer. The nucleotide sequence showed 99 per cent homology with Bos taurus Bos indicus ,Yak and Gayal The exon-intron boundaries in the Vechur occurred at the codons of the amino acid residues, Ala-4 (intron 1), Glu-57 (intron 2), Lys-96 (intron 3) and Met150 (intron 4). All these four residues were conserved in Bos taurus and Bos grunniens as well as in Giraffa camelopardalis. The positions of the exon-intron boundaries are also conserved in various species as evidenced from similar sizes of the exons. 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 Vechur cattle GH gene has an open reading frame (ORF) of 654 nucleotides encoding a signal peptide of 26 amino acid residues and a mature protein of 191 amino acid residues. The 191 amino acid residues of the mature polypeptide chain was found to be 100 per cent homologous to that of Bos taurus (Accession #M57764.1). The mature amino acid sequence of growth hormone of Vechur cattle Starts with Ala at NH2- terminal and Phe at COOH-terminal. The predicted secondary structure showed that the larger α-helical lobe was formed by four sections of the polypeptide chain (residues 1-35,66-86, 111-129 and 149-191) while the smaller lobe, which encompassed a small anti-parallel beta-sheet and a small irregular structure formed the remaining structure of the polypeptide chain. The predicted 3D protein structure obtained from the Swiss Model Server and EsyPred3D and the 3D structure of human GH protein revealed that the α-helices, β-sheets and random coils in the predicted protein are similar to that of the human GH structure. This work seems to be the first of its kind in establishing the DNA and protein sequence data and structural organization of growth hormone gene in Vechur cattle. The analysis of sequence data revealed that there was high similarity of the protein at the amino acid sequence level (100 per cent identity) with Bos taurus and Bos indicus. The signal peptide and intron sequences seem to be evolved more rapidly as there was less homology as compared to exonic sequences. Overall, the nucleotide sequence of Vechur GH gene was found to be similar (99 per cent identity) to that of Bos taurus and other Bos indicus. The regulatory sequences and the amino acid sequence shows 100 per cent identity with that of Bos taurus and other Bos indicus. The study reveals that the dwarf character of this breed is not due to any change in the nucleotide sequence of GH gene. The protein sequence analysis data denoted the slow basal rate of evolution shown by mammalian GHs and also showed that the GH gene is strongly conserved among species. Signal peptide sequences seems to be evolved more rapidly as there was less homology among them as compared to mature GH sequences and there was no correlation between the rates of evolution of these two sequences.