Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.
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PMID:Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells. 216 40

Tissue-type plasminogen activator (t-PA) gene expression is regulated by the tumor-promoting phorbol ester, phorbol-12-myristate 13-acetate (PMA), by cyclic AMP analogues, and the cAMP agonist, forskolin. Based on nuclear "run-on" transcription assays, t-PA expression is modulated by PMA on the level of transcription. 8-Bromo-cyclic AMP and forskolin do not induce t-PA gene transcription alone but act synergistically with PMA. These effects are confirmed by transient expression assays in HeLa cells employing deletion mutants of the t-PA gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Constitutive expression and most of the PMA-mediated induction requires sequences downstream of position -145. DNase I protection ("footprint") analysis of this region reveals two protein-binding sites: one between position -102 and -115, differing from the consensus sequence of the cAMP-responsive element (CRE) by the substitution of an adenine for a guanine in the middle of the core motif (TGACATCA), and another, located in the first exon (between position +60 and +74), displaying homology to the consensus sequence of the activator protein 2- (AP-2) binding site (CCCCACCCCC). Base substitutions in the core of either the CRE-like element or the AP-2 site suppress constitutive CAT expression by over 80%, whereas the relative PMA- and PMA plus cAMP-mediated responses are retained. CAT expression is below the detection limit when both elements are mutagenized together. Hence, the CRE-like element and the exon-located AP-2-binding site have a cooperative impact on basal transcription, but each element can independently convey the effect of activators of the protein kinase C- and A-dependent pathways of signal transduction. The results of band-shift analysis and competition titration experiments demonstrate that the CRE-like element acts as a low affinity binding site for the same proteins which recognize the authentic CRE.
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PMID:A DNA motif related to the cAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and cAMP. 216 21

By using gel mobility shift assay, it was shown that the nuclear extract from F9 embryonal carcinoma (EC) cells contains a novel transcriptional regulatory factor, BF-H, that binds to the 5' upstream region of the early gene of polyoma virus. Two binding sites were located in the transcriptional enhancer domain "A" (nucleotide 5034-5041) and in the 5' upstream of the domain "A" (4998-5005), having a consensus motif (AAPuATGG) between them. Combination of in vitro mutagenesis with chloramphenicol acetyltransferase (CAT) assay revealed that BF-H is a positive transcriptional factor. Interestingly, the binding of BF-H disappeared after differentiation of F9 cells by treatment with retinoic acid, whereas BF-H was present in the F9 cells differentiated with both retinoic acid and dibutyryl cyclic AMP (dbcAMP). These observations suggest that BF-H regulates the expression of genes in a developmental stage-specific manner in early embryos of the mouse.
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PMID:A novel transcriptional regulatory factor that binds to the polyoma virus enhancer in a developmental stage-specific manner. 216 3

The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.
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PMID:On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion. 223 15

The alpha and beta subunit genes encoding chorionic gonadotropin (CG) are regulated transcriptionally in placental cells by cyclic AMP (cAMP). The regulatory response sequences of the alpha gene have been studied extensively. Similar studies of the CG beta subunit (CG beta) gene have not been possible because transcriptionally active sequences have not been identified in the clones isolated to date. The CG beta subunit genes form a complex cluster of seven structurally similar genes that include six CG beta-like genes and a single luteinizing hormone beta subunit (LH beta) gene. We isolated overlapping clones containing the entire CG beta/LH beta gene cluster (68 kilobases) from a human genomic cosmid library. The organization of the gene cluster was similar to that found in previous analyses, as determined by Southern blots of genomic DNA, but differed from some of the gene assignments, as determined by fragments cloned in lambda phage. The 5'-flanking sequence of the most active CG beta gene (CG beta 5) was linked to the chloramphenicol acetyltransferase (CAT) coding sequence for analyses of transient expression in different cell types. CG beta CAT was expressed preferentially in JEG-3 choriocarcinoma cells, and expression was markedly stimulated by treatment with 8-bromo-cAMP. Deletion mutagenesis of the CG beta 5'-flanking sequence revealed that multiple regions were required for maximal expression. The kinetics for cAMP stimulation of alpha CAT and CG beta CAT expression were different, suggesting that different pathways may be involved in cAMP-stimulated expression of the alpha and CG beta genes.
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PMID:Isolation and characterization of the human chorionic gonadotropin beta subunit (CG beta) gene cluster: regulation of transcriptionally active CG beta gene by cyclic AMP. 246 94

The role of the cyclic-AMP (cAMP) responsive element (CRE) in eukaryotic gene transcription was investigated in several cell lines transfected by constructs containing the chloramphenicol acetyltransferase (CAT) gene linked to the three different promoters, simian virus (SV) 40, human c-Ha-ras-1, or chicken beta-actin promoter, with or without CRE. CRE had inducible enhancer activity only when it was linked to the SV40 promoter and in a few cell lines such as PC12. CRE functioned as a constitutive enhancer with the human c-Ha-ras-1 promoter in all cell lines examined. CRE also had constitutive enhancer activity when it was linked to the chicken beta-actin promoter, but this activity was observed only in KB, HeLa, and A431 cells. The different types of enhancer activities of CRE depending on the cell and promoter may be caused by interaction with different trans-acting factors that were demonstrated by gel retardation analyses.
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PMID:Dual enhancer activities of the cyclic-AMP responsive element with cell type and promoter specificity. 253 60

Prior studies have demonstrated that a small proportion of blood lymphocytes from patients with human cytomegalovirus (HCMV) infection express only the viral immediate-early (IE) genes (L. Einhorn and A. Ost, J. Infect. Dis. 149:207-214, 1984; G. P. A. Rice, R. D. Schrier, and M. B. A. Oldstone, Proc. Natl. Acad. Sci. USA 81:6134-6138, 1984). The present studies demonstrate that the IE genes of HCMV are transcribed in Jurkat cells (T lymphocytes) only after activation of the cells with mitogens. Transcription of the IE genes is from an upstream enhancer promoter-regulatory region containing several different repeated sequence motifs. Chimeric plasmids were constructed with just a single copy or three copies of a synthetic oligonucleotide sequence of either the 16-, 18-, 19-, or 21-base-pair (bp) repeat elements upstream of the minimal wild-type promoter sequence to drive expression of the indicator gene, chloramphenicol acetyltransferase (CAT). The 18- or 19-bp motifs in the enhancer region were found to be important in mediating the effect of the mitogens. However, the CAT activity detected with the 19-bp repeat was always significantly higher than that found with the 18-bp repeat. There was an additive effect by multiple copies of the 18- or 19-bp repeat sequences on gene expression. The 19-bp repeat contains a sequence identical to that described for a cyclic AMP (cAMP) response element, and plasmids containing only this sequence and the minimal promoter sequences upstream of the CAT gene respond to agents which increase intracellular cAMP. Functional cAMP response elements are present in the wild-type promoter-regulatory region and are associated with the 19-bp repeat sequences. It is proposed that activation of lymphocytes results in expression of the IE genes of HCMV, in part via the activation of cellular trans-acting factors which interact with the 18- and 19-bp motifs in the HCMV IE promoter-regulatory region. The 19-bp repeat is the major contributor to the strength of this enhancer-containing promoter-regulatory region.
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PMID:The promoter-regulatory region of the major immediate-early gene of human cytomegalovirus responds to T-lymphocyte stimulation and contains functional cyclic AMP-response elements. 254 10

Interleukin 1 (IL-1) induces the synthesis of kappa immunoglobulin light chains and the expression of surface immunoglobulin in the murine pre-B-cell line 70Z/3 (J. G. Giri, P. W. Kincade, and S. B. Mizel, J. Immunol. 132:223-228, 1984). In the present study, we found that these effects of IL-1 are mimicked by cyclic AMP (cAMP) analogs and cAMP-elevating drugs. The induction of kappa immunoglobulin light-chain gene expression by IL-1 was associated with an increase in intracellular cAMP levels. Incubation of 70Z/3 cells with IL-1 or cAMP resulted in the activation of the kappa immunoglobulin enhancer, as detected by the induction of chloramphenicol acetyltransferase (CAT) in cells transfected with a kappa enhancer-CAT expression plasmid. In contrast, CAT plasmids lacking a kappa immunoglobulin enhancer were inactive in the presence of IL-1 or cAMP. Furthermore, IL-1 and cAMP analogs and inducers were found to induce the activation of a NF-kappa B-like DNA-binding protein that exhibited specificity for the kappa immunoglobulin enhancer. These results suggest that cAMP may play an important role as a second messenger for IL-1 in the induction of kappa immunoglobulin light-chain synthesis in pre-B cells via the activation of a DNA-binding protein that is similar or identical to NF-kappa B.
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PMID:Interleukin 1 and cyclic AMP induce kappa immunoglobulin light-chain expression via activation of an NF-kappa B-like DNA-binding protein. 254 70

The mechanism of cyclic AMP (cAMP) induction of fibronectin (FN) in HT-1080 and JEG-3 cells differs (D. C. Dean, R. F. Newby, and S. Bourgeois, J. Cell Biol. 106:2159-2170, 1988). In the fibrosarcoma cell line HT-1080, induction requires both protein synthesis and a lag period of 12 to 24 h. In the choriocarcinoma cell line JEG-3, protein synthesis is not required and induction peaks before 24 h, declining thereafter. We show that the FN promoter is transcribed in vitro and that the transcripts initiate at the proper site. Based on transfection experiments with these cells and FN promoter constructions, a cAMP-responsive element (CRE) was identified between -157 and -188 base pairs upstream of the human FN gene. This sequence also conferred cAMP inducibility in both cell lines on the herpesvirus thymidine kinase promoter when it was placed upstream of a thymidine kinase-chloramphenicol acetyltransferase fusion gene. DNase I protection analysis and gel retardation experiments revealed that the CRE was bound by a protein(s) that was present in both HT-1080 and JEG-3 cells as well as in NIH 3T3 cells. Multiple protein-CRE complexes were resolved by gel retardation with extracts of both cell lines. Forskolin treatment of these cells did not alter qualitatively or quantitatively the pattern of CRE-binding proteins that was observed. The FN promoter was at least 10 times more active in HT-1080 than in JEG-3 cells, even though in JEG-3 cells both the rate of FN biosynthesis and the level of accumulated FN mRNA were greater than those in HT-1080 cells. The difference in promoter activity in HT-1080 and JEG-3 cell was mediated by sequences that were located between positions -510 and -56. Deletion of the FN promoter from positions -510 to -56 resulted in an ~30-fold decrease in promoter activity when this construction was transfected into HT-1080 cells, and similar results were observed in NIH 3T3 cells; however, less than a 2-fold effect was observed in JEG-3 cells. Results of these studies suggest that there is some degree of tissue specificity of FN gene expression and reveal that cAMP induction is mediated, in part, by the same element (CRE) in both HT-1080 and JEG-3 cells.
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PMID:Forskolin inducibility and tissue-specific expression of the fibronectin promoter. 254 72

Transcription of the human proopiomelanocortin (POMC) gene is regulated by cAMP. To identify the region in the human POMC gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5'-flanking region of the human POMC gene fused to the structural sequence encoding the bacterial reporter enzyme chloramphenicol acetyltransferase (CAT). The transcriptional activity of the fusion genes introduced into the rat glial cell line C6 was assayed by measuring CAT activity in the cell lysate. Forskolin, an adenylate cyclase-activating agent, stimulated the expression of POMC-CAT fusion genes. Deletion analysis demonstrated that the region between -417 and -97 bp from the transcriptional origin of the human POMC gene was responsible for regulation by cyclic AMP.
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PMID:Cyclic AMP-responsive region of the human proopiomelanocortin (POMC) gene. 254 84


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