title: Isolation, characterization and functional analysis of full length p53 cDNA from Bubalus bubalis.
abstract: p53 plays a pivotal role in maintaining the genomic integrity of the cell and has an important role in cellular transformation. We isolated and cloned a full length p53 cDNA (Bp53) from water buffalo in expression vectors designed to generate tagged proteins with FLAG or GFP. Bp53 was found to be 1161 nucleotide long and codes for 386 amino acid residues with 79% homology with human p53 containing 393 amino acids. Although Bp53 has some inherent differences in amino acid composition in different functional domains as compared to human p53 but the total electrostatic charge of amino acids has been maintained. Bp53 cDNA was transiently transfected in a p53 null human NSCLC cell line and as expected, it was predominantly localized in the nucleus. Besides, Bp53 effectively transactivates a number of target genes similar to human p53 and exerts most of its anti-tumorigenic potential in culture as observed in clonogenic and cell viability assays. Like human p53 mutants, core domain mutant version of Bp53 was found to be mis-localized to cytoplasm with diminished tumor suppressor activity. However, Bp53 appeared to be more sensitive to mdm2 mediated degradation and as a result, this protein was less stable as compared to human p53. For the first time we have characterized a functionally efficient wild-type p53 from buffalo having lower stability than human p53 and thus, buffalo p53 could be used as a model system for further insight to the molecular basis of wild-type p53 instability.
authors.affiliation: National Centre for Human Genome Studies and Research, Panjab University, Chandigarh 160014, India.
authors.forename: Minu
authors.initials: M
authors.lastname: Singh
authors.affiliation: National Centre for Human Genome Studies and Research, Panjab University, Chandigarh 160014, India.
authors.forename: Suruchi
authors.initials: S
authors.lastname: Aggarwal
authors.affiliation: National Dairy Research Institute (NDRI), Karnal, Haryana 132001, India.
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Mohanty
authors.affiliation: National Centre for Human Genome Studies and Research, Panjab University, Chandigarh 160014, India. Electronic address: tmukhopa@hotmail.com.
authors.forename: Tapas
authors.initials: T
authors.lastname: Mukhopadhyay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002020:Buffaloes; D002460:Cell Line; D003001:Cloning, Molecular; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D055550:Protein Stability; D021381:Protein Transport; D059748:Proteolysis; D051736:Proto-Oncogene Proteins c-mdm2 / Q000502:physiology; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D018076:DNA, Complementary; D016159:Tumor Suppressor Protein p53; D051736:Proto-Oncogene Proteins c-mdm2
keywords: Cloning; Tumor suppressor; Water buffalo; mdm2; p21