Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transcriptional and translational signals required for efficient expression of the chloramphenicol acetyltransferase, beta-galactosidase, and tissue plasminogen activator genes, under the control of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus, were investigated by SDS-PAGE and RNA dot blot analysis. The recombinant baculoviruses all contained alterations in the leader sequence or 5' proximal coding region of the polyhedrin gene. Highest levels of foreign proteins and polyhedrin-linked mRNAs were observed when portions of the coding sequence of the polyhedrin gene were fused in phase with the foreign gene. Recombinant viruses in which the foreign gene was inserted upstream from the polyhedrin ATG start codon expressed nonfused products but at lower levels than contructs which produced fusion proteins. A corresponding decrease in the levels of mRNAs produced by such constructs was also observed. Some constructs in which the foreign gene was inserted out of phase downstream from the polyhedrin start codon expressed nonfused protein products at low levels but produced polyhedrin-linked mRNA at levels comparable to vectors which produced protein fusions. These data suggest that reinitiation of translation can take place at AUG start codons a short distance downstream from the primary polyhedrin start codon. These results indicate that sequences immediately upstream from the polyhedrin start codon are important for regulation of transcription and that additional sequences near the AUG start codon can have a dramatic influence on the levels of translation observed.
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PMID:Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. 314 47

In order to identify a possible enhancer-like regulatory sequence for the human tissue plasminogen activator (tPA) gene, various DNA segments in the 5'-flanking region were ligated to the chloramphenicol acetyltransferase (CAT) reporter gene driven by the tPA gene promoter, and their CAT enhancing activities were measured following transfection to human melanoma-derived Bowes cells that highly express the gene. Major and minor activities were detected in two adjacent upstream sequences, 160 base pairs (bp) (-2288 to -2129) and 102 bp (-2390 to -2289), respectively, and the former was subjected to further analysis. The CAT enhancing activity of the 160-bp sequence was greatly affected by its position and orientation in the constructs and the sequence also functioned weakly with the SV40 promoter. Deletion of any small portion from the sequence abolished the CAT enhancing activity, suggesting that the entire sequence is required for the activity. This sequence did not show a further CAT enhancing activity in Bowes cells treated with the inducers phorbol 12 myristate 13-acetate and dexamethasone and did not function in HeLa or HT1080 cells under any conditions. Taken together, the 160-bp sequence is likely to be responsible for the constitutive and/or cell type-specific expression of the tPA gene in human cells.
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PMID:A novel regulatory sequence affecting the constitutive expression of tissue plasminogen activator (tPA) gene in human melanoma (Bowes) cells. 803 4

F9 embryonal carcinoma cells differentiate in response to retinoic acid (RA). To investigate the regulation of RA receptors (RARs) expression during this process, cDNA probes specific for the major RAR isoforms were used. In contrast to the level of RAR beta 2 mRNA which was high in cells treated 5 days with RA and below detection in untreated cells, as previously described, the steady state levels of RAR alpha 1, alpha 2, gamma 1, and gamma 2 mRNAs were markedly decreased in the RA-differentiated cells as compared to untreated cells. The down-regulation of the RA-responsive system in differentiated cells was also evident in gel shift assays as a marked decrease in binding capacity to a retinoid acid response element (beta 2RARE), as well as in chloramphenicol acetyltransferase (CAT) assays as a sixfold decrease in RA-mediated transacting activity via this element. The down-regulation of RAR DNA-binding and transacting activity coincided with the burst in tissue plasminogen activator secretion and thus, occurred at the hinge between early and late differentiation. The down-regulation of RA responsiveness may constitute an important event in the transition between early and late differentiation stage in F9 cells.
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PMID:Down-regulation of retinoic acid receptor activity associated with decreased alpha and gamma isoforms expression in F9 embryonal carcinoma cells differentiated by retinoic acid. 840 46

The endothelial lining of blood vessels is constantly exposed to fluid mechanical forces generated by flowing blood. In vitro application of fluid shear stresses to cultured endothelial cells influences the expression of multiple genes, as reflected by changes in their steady-state mRNA levels. We have utilized the B chain of platelet-derived growth factor (PDGF-B) as a model to investigate the mechanisms of shear-stress-induced gene regulation in cultured bovine aortic endothelial cells (BAECs). Northern blot analysis revealed elevated endogenous PDGF-B transcript levels in BAECs, after exposure to a physiological level of laminar shear stress (10 dynes/cm2; 1 dyne = 100 mN) for 4 h. A transfected reporter gene, consisting of a 1.3-kb fragment of the human PDGF-B promoter coupled to chloramphenicol acetyltransferase (CAT), indicated a direct effect on transcriptional activity. Transfection of a series of PDGF-B-CAT deletion mutants led to the characterization of a cis-acting component within the PDGF-B promoter that was necessary for shear-stress responsiveness. In gel-shift assays, overlapping oligonucleotide probes of this region formed several protein-DNA complexes with nuclear extracts prepared from both static and shear-stressed BAECs. A 12-bp component (CTCTCAGAGACC) was identified that formed a distinct pattern of complexes with nuclear proteins extracted from shear-stressed BAECs. This shear-stress-responsive element does not encode binding sites for any known transcription factor but does contain a core binding sequence (GAGACC), as defined by deletion mutation in gel-shift assays. Interestingly, this putative transcription factor binding site is also present in the promoters of certain other endothelial genes, including tissue plasminogen activator, intercellular adhesion molecule 1, and transforming growth factor beta 1, that also are induced by shear stress. Thus, the expression of PDGF-B and other pathophysiologically relevant genes in vascular endothelium appears to be regulated, in part, by shear-stress-induced transcription factors interacting with a common promoter element.
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PMID:Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element. 835

We have previously reported that exposure of cultured bovine aortic endothelial cells (EC) to 10% average strain resulted in an increase in tissue plasminogen activator (tPA) mRNA, immunoreactive tPA protein, and tPA activity in the medium. The present study was designed to examine the regulation of tPA gene expression in EC by cyclic strain. We performed a functional analysis of the tPA promoter by transfecting bovine aortic EC with a 1.4-kilobase (kb) construct of the human tPA promoter coupled to chloramphenicol acetyltransferase. We found that subjecting the EC to 10% average strain (and not 6% average strain) resulted in a 2.6-fold increase in activity of the 1.4-kb tPA promoter by 4 h. Analysis of deletion mutants of the promoter transfected into EC demonstrated a 60% drop-off in activity between position -145 and -105. Deoxyribonuclease I protection analysis of the segment downstream of position -196 suggested involvement of activator protein-2 (AP-2) and adenosine 3',5'-cyclic monophosphate-responsive element (CRE)-like binding sites, which was confirmed by electrophoretic mobility shift assays. Site-directed mutants of either the AP-2 or CRE-like regions resulted in a 65% decrease in activity compared with the wild type. Double mutations abolished basal transcription and any strain-induced activity. A shear stress responsive element (SSRE) binding site is present at -945, but site-directed mutants did not show any drop in activity compared with wild type by cyclic strain. These studies demonstrate that cyclic strain regulates tPA gene transcription in bovine aortic EC and that this transcriptional activation is dependent on factors that are similar to those activated with phorbol ester.
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PMID:Regulation of tPA in endothelial cells exposed to cyclic strain: role of CRE, AP-2, and SSRE binding sites. 937 27