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)

Expression of the human chorionic gonadotropin (hCG)-alpha gene in placental trophoblasts is markedly stimulated by cAMP, a property preserved in a reporter plasmid containing its cAMP response elements (CREs) linked to the chloramphenicol acetyltransferase coding sequence (CRE alpha CAT). In search of a potential physiologic regulator of hCG gene expression via cAMP, we found that JEG-3 syncytial trophoblast cells have specific binding sites for vasoactive intestinal peptide (VIP) with dissociation constant of 1 nM. VIP maximally increased the transient expression of CRE alpha CAT and the expression of endogenous hCG-alpha mRNA in JEG-3 cells by 4- and 9-fold, respectively. Exposure of JEG-3 cells to 30 nM VIP increased cAMP levels 60-fold after 10-30 min, but cAMP rapidly declined thereafter. As a consequence of this desensitization, the effect of VIP on stimulation of both CRE alpha CAT and endogenous hCG-alpha and hCG-beta mRNA levels more closely resembled that of forskolin or 8-br-cAMP at time points much less than 24 h. Moreover, transient exposure to 8-br-cAMP was much less effective than 24 h of continuous incubation on CRE alpha CAT activity. We conclude that VIP rapidly increases cAMP content and activates hCG-alpha gene expression in JEG-3 cells, but sustained elevations in cAMP are necessary for maximal accumulation of this CRE-regulated gene product.
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PMID:Vasoactive intestinal peptide increases intracellular cAMP and gonadotropin-alpha gene activity in JEG-3 syncytial trophoblasts. Constraints posed by desensitization. 169 18

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.
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PMID:Expression of a chimeric VIP gene is targeted to the intestine in transgenic mice. 207 11

The regulation of gastrin gene transcription was studied in GH4 pituitary cells transfected with constructs comprised of the first exon of the human gastrin gene and various lengths of 5' regulatory sequences ligated upstream of the reporter gene chloramphenicol acetyltransferase. Gastrin reporter gene activity in GH4 cells was equal to the activity of a reporter gene transcribed from the endogenously expressed growth hormone promoter. The effect of a variety of peptides on gastrin gene transcription including epidermal growth factor (normally present in the gastric lumen), gastrin-releasing peptide, vasoactive intestinal peptide, and somatostatin (present in gastric nerves) was assessed. Epidermal growth factor increased the rate of gastrin transcription almost 3-fold, whereas thyrotropin-releasing hormone and vasoactive intestinal peptide increased gastrin transcription 2- and 1.5-fold, respectively. Gastrin-releasing peptide, a peptide that strongly stimulates gastrin release, weakly increased gastrin transcription (1.3-fold). Somatostatin inhibited the increase in gastrin transcription induced by epidermal growth factor, thyrotropin-releasing hormone, and vasoactive intestinal peptide. Constructs containing various lengths of 5' regulatory sequences defined a response element -40 to -82 base pairs (bp) 5' to the transcription initiation site. This 40-bp sequence contains Sp1 and AP2 binding sites, which suggests that epidermal growth factor and thyrotropin-releasing hormone stimulate gastrin gene transcription through transcription factors that bind to Sp1 and/or AP2 motifs.
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PMID:Regulation of the gastrin promoter by epidermal growth factor and neuropeptides. 256 64

A transcriptional cAMP-responsive enhancer element (CRE) consisting of the 8-base pair (bp) palindrome, 5' TGACGTCA 3', is found in several eukaryotic genes. We analyzed the effects on gene transcription of point mutations within the CRE, the influence of the bases surrounding the CRE, and the requirements for transcriptional synergism of tandemly repeated CREs. When inserted as an oligonucleotide with restriction enzyme linker sites, the 8-bp CRE itself is as active in conferring cAMP responsivity on an enhancerless chloramphenicol acetyltransferase reporter plasmid as is a single copy of the choriogonadotropin alpha (CG alpha), twice repeated 18-bp sequence containing the CRE. Point mutations in the first (T to A), fourth (C to G), or eighth (A to T) positions of the CRE, when contained within the CG alpha 18-bp sequence, each inhibited transcriptional activity greater than 90%. However, the identical eighth position A to T mutation occurs in the cAMP-responsive sequence of the vasoactive intestinal peptide (VIP) gene, and that mutant sequence in the context of the adjacent bases of the native VIP sequence is maximally cAMP responsive when inserted in the reporter plasmid. The substantially reduced activity of the core 8-bp CRE when synthesized as a cassette including the adjacent bases of the rat glucagon or bovine parathyroid hormone gene further emphasizes the restrictive influence of particular surrounding sequences. Active oligonucleotides containing the 8-bp palindrome and different but equally permissive contexts have comparable properties in transfected reporter genes and gel mobility-shift assays. The pair of tandemly repeated 18-bp elements containing the CRE in the CG alpha gene synergistically stimulate transcription either with paired native CREs or when one native CRE is paired with one mutant CRE, suggesting the presence of cooperative interactions. Tandem insertion of more than two 18-bp sequences, or insertion of additional sequences between the two CREs, inhibits transcription. These observations indicate that the contexts of the bases adjacent to CREs exert profound influences on the transcriptional activities mediated by the cAMP-responsive elements.
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PMID:Structural determinants for transcriptional activation by cAMP-responsive DNA elements. 284 37

The expression of a transfected plasmid containing 5.2 kilobases (kb) of 5' regulatory DNA sequence of the human vasoactive intestinal peptide (VIP) gene attached to coding sequences of the reporter gene chloramphenicol acetyltransferase (CAT) was compared with endogenous VIP expression in subclones of the human neuroblastoma cell line SK-N-SH. These subclones vary widely in basal and inducible quantities of VIP and its precursor mRNA and can be interconverted under specified culture conditions. Endogenous VIP immunoreactivity, detectable in all subclones, was lowest in the neuronal subclone SH-SY-5Y, whereas 15- to 25-fold higher levels were observed in the epithelial-appearing SH-EP and intermediate SH-IN subclones. Treatment with 10 nM phorbol 12-myristate 13-acetate (PMA) stimulated VIP peptide levels approximately 5-fold in SH-SY-5Y cells but did not increase appreciably VIP levels in the other subclones. Treatment with 2.5 microM forskolin resulted in less than 50% stimulation of VIP expression in all subclones. Levels of mRNA encoding the VIP precursor generally paralleled these differences in VIP immunoreactivity. In cells transfected with the VIP/CAT fusion gene, CAT activity reflected closely these differences in basal VIP expression and the changes in response to PMA and forskolin. Deletion of 2.7 kb of the most upstream sequences resulted in an 80-90% reduction in basal CAT activity in SH-IN, but not SH-SY-5Y cells, and resulted in an 80% reduction in PMA stimulation in SH-SY-5Y cells. Deletion to within 74 nucleotides of the transcription start site resulted in CAT expression in SH-IN cells that was only 3% of that seen with the full 5.2-kb flanking sequences and further diminished the remaining PMA responsiveness in SH-SY-5Y cells. The data indicate that important cell-type-specific transcription regulatory sequences reside greater than 2.5 kb upstream from the VIP transcription start site.
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PMID:Lineage-specific regulation of the vasoactive intestinal peptide gene in neuroblastoma cells is conferred by 5.2 kilobases of 5'-flanking sequence. 320 Aug 39

Signals responsible for expression of the vasoactive intestinal peptide (VIP)-stimulated prolactin gene in GH3 pituitary tumor cells were examined. Transfection with a deoxyribonucleic acid (DNA) construct containing the chloramphenicol acetyltransferase (CAT) gene fused to the 2.5-kb prolactin 5'-upstream regulatory sequence indicated that VIP stimulated CAT expression. However, this effect could not be mimicked by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and was inhibited by the L-type Ca(2+)-channel blocker verapamil. While KCl had little effect on CAT activity, combined treatment with KCl and 8-Br-cAMP synergistically activated CAT expression. Potentiation between KCl and 8-Br-c-AMP was also seen with c-fos messenger ribonucleic acid (mRNA) expression. In addition, KCl and 8-Br-cAMP synergistically activated cAMP response element (CRE)-mediated CAT expression, and the synergism was abolished by verapamil. In the presence of okadaic acid, cAMP had no significant activation on CRE-driven CAT expression, whereas KCl-stimulated CAT expression was greatly potentiated. These results indicate that cAMP and Ca2+ synergistically activated CRE-driven gene expression through non-overlapping phosphorylation events in GH3 cells.
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PMID:Synergistic activation of cAMP and calcium on cAMP-response-element-mediated gene expression in GH3 pituitary tumor cells. 873 May 12