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CLONING, CHARACTERIZATION AND IDENTIFICATION OF POLYMORPHISM IN DRA AND TCR ZETA GENES IN DEONI CATTLE

KrishiKosh

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Title CLONING, CHARACTERIZATION AND IDENTIFICATION OF POLYMORPHISM IN DRA AND TCR ZETA GENES IN DEONI CATTLE
 
Creator KOPPULA, SWATHI
 
Contributor Dr. M. GNANA PRAKASH
 
Subject area, regeneration, planting, byproducts, forestry, nitrogen, carbon, sexual reproduction, pinus roxburghii, altitude
 
Description ABSTRACT
Major histocompatability complex is an extensive genetic region of clustered genes that control immune response. The class II region designated as BoLA consists of DQ, DR, DY genes. The present study was undertaken to clone and characterize DRA and TCR Zeta genes in Deoni cattle and to identify polymorphism in DRA, TCR Zeta and DYA genes by PCR-RFLP. The cDNA for the DRA and TCR Zeta genes were amplified by using specific primers designed based on available cattle sequences and purified products were cloned in competent E.coli (DH5α) strain.
The full length 1013bp product of cDNA of DRA gene contained a single ORF of 762 nucleotides that coded for 253 amino acids translated product. 24 amino acids formed signal peptide while 229 constituted mature peptide. The deduced amino acid sequences resembled those of class II molecules of other species for all the conserved residues having critical functional role. But a single N-linked glycosylation site in α1 was observed in cattle and buffalo when compared to human and swine which contain a second site in α2 domain. The signal peptide was found more variable among the species compared. Comparison of nucleotide and amino acid sequences among related species and dendrogram constructed revealed that the cattle sequences are more similar to buffalo sequences. To study polymorphism, a 847bp product corresponding to exon 2, intron 2 and exon 3 was amplified and digested with TaqI and Hae III. Sixty animals were screened for polymorphism. Polymorphism was observed in DRA gene of cattle for these enzymes.
The full length cDNA of cattle TCR Zeta (CD3Z) gene was cloned and characterised. The 1078 bp TCR Zeta (CD3Z) had a coding region (single ORF) of 495 nucleotides for a translated product of 164 amino acids. Initial 63 nucleotides encoded for signal peptide while the remainder 429 nucleotides encoded for three different domains, excluding stop codon. The mature peptide consisted of extracellular domain (9 amino acids), transmembrane domain (20 amino acids) and intra-cytoplasmic domain (114amino acids). The deduced amino acid sequences were compared among related ruminants. Most of the potential consensus sequences were conserved among the ruminant species. The critical residues for dimer formation, lipid raft association, putative nucleotide binding were also present in cattle. Comparison of sequences and dendrogram revealed strong association among ruminants, suggesting that this molecule is unique for ruminants with some specific function. To study polymorphism in genomic sequence of TCR Zeta gene, a 298 bp region corresponding to 256 bp of 3ʹ UTR and the rest from the coding region, was amplified and digested with Hae III and Mse I enzymes. Both the enzymes revealed monomorphic pattern.
A 253bp fragment corresponding to exon 2 and 296 bp fragment of exon 3 of the DYA gene were amplified from genomic DNA of 60 Deoni cattle samples and digested with AluI and HaeIII and Ava II and HinfI enzymes respectively. Similar pattern was observed suggesting the monomorphic nature of the gene.
 
Date 2016-12-27T11:55:40Z
2016-12-27T11:55:40Z
2016-10-21
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/93148
 
Relation D;496
 
Format application/pdf
 
Publisher PVNR TVU