TAILIEUCHUNG - Báo cáo khoa học: Molecular cloning of the Matrix Gla Protein gene from Xenopus laevis Functional analysis of the promoter identifies a calcium sensitive region required for basal activity

To analyze the regulation of Matrix Gla Protein (MGP) gene expression inXenopus laevis, we cloned the xMGPgene and its 5¢ region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. TheXenopusMGP (xMGP) gene is organized into ®ve exons, one more as its mammalian counterparts. | Eur. J. Biochem. 269 1947-1956 2002 FEBS 2002 doi Molecular cloning of the Matrix Gla Protein gene from Xenopus laevis Functional analysis of the promoter identifies a calcium sensitive region required for basal activity Natercia Conceicao1 Nuno M. Henriques1 Marc C. P. Ohresser1 I Philip Hublitz2 Roland Schiile2 and M. Leonor Cancela1 1University of Algarve-CCMAR Campus de Gambelas Faro Portugal 2Universitats-Frauenklinik Abteilungfur Geburtshilfe und Gyndkologie Zentrum fur Klinische Forschung Albert Ludwigs-Universitdt Freiburg Germany To analyze the regulation of Matrix Gla Protein MGP gene expression in Xenopus laevis we cloned the xMGP gene and its 5 region determined their molecular organization and characterized the transcriptional properties of the core promoter. The Xenopus MGP xMGP gene is organized into five exons one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5 end of the xMGP promoter revealed a minimal activating element in the sequence from -70 to -36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from -180 to -36 the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach .

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