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)

Phosphoenolpyruvate carboxykinase (PEPCK) governs the rate-limiting step in gluconeogenesis. Glucocorticoids and cAMP increase PEPCK gene transcription and gluconeogenesis, whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant, since they prevent cAMP and glucocorticoid-stimulated transcription. Basal promoter elements and hormone response elements for cAMP, glucocorticoids, and insulin have been defined in previous studies. By using stable transfectants containing a variety of different PEPCK-chloramphenicol acetyltransferase fusion gene constructs, a phorbol ester response sequence, located between positions -437 and -402 relative to the transcription start site, was identified. This region coincides with the insulin response sequence that has recently been defined in the PEPCK promoter. Using a vector containing various wild-type and mutated sequences of this region ligated to the heterologous thymidine kinase promoter, we delineated the boundaries of both elements to the 10 base pairs between positions -416 through -407. Thus, although it has been previously shown that insulin and phorbol esters repress PEPCK gene transcription through distinct pathways, the final target of insulin and phorbol ester action is the same DNA element.
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PMID:Signal transduction convergence: phorbol esters and insulin inhibit phosphoenolpyruvate carboxykinase gene transcription through the same 10-base-pair sequence. 165 Apr 76

Previous studies have shown that the gonadotropins follicle-stimulating hormone and luteinizing hormone stimulate proopiomelanocortin (POMC) promoter activity and mRNA levels in ovarian granulosa cells. The objective of these studies was to determine the role of cAMP-dependent protein kinases (pKA) in gonadotropin-stimulated gene expression. Primary cultures of rat granulosa cells were transfected with a gene construct consisting of the POMC promoter (-150 to +63; designated pOMC-CAT) fused to the chloramphenicol acetyltransferase (CAT) reporter gene either alone or cotransfected with an expression plasmid (designated mutant RI), which overexpresses a mutant form of the murine RI subunit incapable of binding cAMP and serving as an irreversible inhibitor of the catalytic subunit of pKA. Follicle-stimulating hormone or isoproterenol caused a significant stimulation of pOMC-CAT activity in transfected cells. Cotransfection of pOMC-CAT with mutant RI caused a significant inhibition of basal pOMC-CAT activity and abolished the gonadotropin stimulation. As a control, transfection of the SV-40 viral enhancer-promoter fused to CAT (pSV2-CAT) was unresponsive to follicle-stimulating hormone stimulation and cotransfection with mutant RI had no significant effect on pSV2-CAT activity. These studies suggest that gonadotropin regulation of the POMC promoter is mediated by pKA and that promoter activity is stringently controlled by pKA.
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PMID:Intracellular mechanisms of gonadotropin-stimulated gene expression in granulosa cells. 165 68

To identify the cis-acting elements responsible for cAMP stimulation of human prolactin (hPRL) promoter activity, pituitary GC cells were transfected with 5'-deleted hPRL promoters fused to the chloramphenicol acetyltransferase reporter gene. The proximal regulatory region (coordinates -250 to -42) was sufficient to confer strong cAMP stimulation (+/- 25 fold). Further 5' and 3' deletions performed within this proximal region demonstrated that two types of cis-acting elements are involved in the cAMP regulation: (i) the binding sites of the pituitary-specific factor Pit-1, and (ii) the sequence between coordinates -115 and -85 (named fragment A), which contains a TGACG motif. We show by gel-shift and Southwestern experiments that fragment A binds Pit-1 monomer and also a ubiquitous factor that is neither cAMP-responsive element-binding protein nor activator protein-1. Strong cAMP induction was observed when fragment A was juxtaposed to a Pit-1 binding site. That Pit-1 plays an important role was supported further by the finding that the hPRL proximal region conferred cAMP regulation when linked to the herpes simplex virus thymidine kinase promoter only in pituitary GC cells and not in other heterologous cells, which do not express Pit-1. Furthermore, we observed that concatenated Pit-1 binding sites were able to confer cAMP responsiveness to the thymidine kinase promoter in GC cells.
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PMID:Transcriptional induction of the human prolactin gene by cAMP requires two cis-acting elements and at least the pituitary-specific factor Pit-1. 165 38

The cis elements involved in the cAMP regulation of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) were studied by introducing a series of block mutations (10-15 base pairs of random sequence) into eight of the protein binding domains in a region of the promoter between -490 and +73. Each mutant promoter was ligated to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected into HepG2 cells. Transcription of PEPCK-CAT was stimulated 4-fold by the addition of 8-bromo-cAMP (8-Br-cAMP), whereas overexpression of the catalytic subunit of protein kinase A in these cells increased transcription from the PEPCK promoter 30-fold. Several elements within the PEPCK promoter acted synergistically to mediate this effect. These include CRE-1 (-92 to -82) and a complex unit from -220 to -280 composed of multiple binding sites termed P3(I) (-250 to -234), P3(II) (-260 to -250), and P4 (-286 to -270). Mutation of both CRE-1 and P3(I) resulted in the complete elimination of transcriptional induction by either 8-Br-cAMP or the catalytic subunit of protein kinase A. To examine the proteins involved in this response, we replaced CRE-1, which binds both C/EBP and cAMP-responsive element binding protein (CREB), with an optimal C/EBP binding sequence which significantly decreased the binding of CREB, but maintained the affinity for C/EBP. Transcription from this modified promoter was induced by 8-Br-cAMP and the catalytic subunit of protein kinase A (PKA) to a similar extent as noted with the native PEPCK promoter. However, the results of experiments involving cotransfection of PEPCK-CAT with expression vectors for PKA and either C/EBP or CREB suggest that CREB is capable of mediating a greater responsiveness to PKA than C/EBP. Our results indicate that multiple cis elements are involved in the cAMP induction of PEPCK gene transcription and that C/EBP and CREB are potentially involved in this response.
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PMID:Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements. 165 70

Cyclic AMP regulates a variety of cellular responses through activation of the catalytic subunit of cAMP-dependent protein kinase. The cDNAs for two protein isoforms of the catalytic subunit, C alpha and C beta, were placed into expression vectors, and their ability to stimulate cAMP-dependent transcription of the human enkephalin promoter was examined in transiently transfected CV-1 cells. Expression vectors for C alpha and C beta that were directed by the human cytomegalovirus promoter produced up to 350- and 200-fold increases in chloramphenicol acetyltransferase activity, respectively, when cotransfected with the ENKAT-12 reporter plasmid. Transcriptional activation was shown to be dependent upon functional kinase activity by point mutations in catalytic subunit vectors which eliminated activation. Transcriptional activation by C alpha and C beta was eliminated when the cAMP response elements (CREs) were deleted from the native enkephalin promoter, but activation was recovered when this region was replaced with an oligonucleotide containing two copies of the somatostatin CRE consensus TGACGTCA. C alpha expression vectors were found to produce 2-fold greater transcriptional activation than C beta expression vectors. These results were most likely due to the cellular kinase activity produced by the catalytic subunit expression vectors and did not appear to be dependent on CRE motif or substrate specificity. In vitro mutagenesis indicates that neither C alpha nor C beta requires N-terminal myristylation for transcriptional activation, but threonine-197 is critical to subunit function.
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PMID:Regulation of the human enkephalin promoter by two isoforms of the catalytic subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase. 165 33

Glucocorticoids enhance proenkephalin gene expression in several cell types. To elucidate the mechanism(s) involved, we analyzed the potentiation by dexamethasone of the cAMP-dependent increase in proenkephalin mRNA levels elicited by forskolin in C6 rat glioma cells. This potentiation did not require ongoing protein synthesis. In nuclear run-on transcription assays, dexamethasone alone did not alter proenkephalin transcription, but strongly increased the magnitude and duration of transcriptional elevation by forskolin through a direct action not requiring ongoing protein synthesis. Dexamethasone did not alter basal or stimulated cAMP levels. To search for functionally cooperative glucocorticoid and cAMP regulatory elements, we transfected C6 cells with plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of rat proenkephalin sequences from bases -5800 to +703. Maximum stimulation of transiently expressed CAT activity by forskolin required more than 145 and 190 or fewer base pairs of 5'-flanking sequence, implicating sequences up-stream from the previously described cAMP-inducible enhancer. Dexamethasone reduced forskolin-stimulated CAT expression from plasmids with 190 or more base-pairs of 5'-flanking sequence, an effect apparently involving multiple up-stream regions. Dexamethasone also reduced forskolin-stimulated CAT mRNA levels in C6 cells stably transfected with proenkephalin/CAT chimeric genes in the presence or absence of proteins synthesis. In summary, we demonstrate that glucocorticoids and cAMP synergize positively in regulating transcription of the endogenous gene, but interact negatively in regulating the chimeric constructs, which may lack the context or distal element(s) required for positive synergism.
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PMID:Proenkephalin gene expression in C6 rat glioma cells: potentiation of cyclic adenosine 3',5'-monophosphate-dependent transcription by glucocorticoids. 165 36

In this study we determined the activity of the rat luteinising hormone-beta gene promoter in a heterologous rat pituitary cell line (GH3 cells). 1.7 kb of LH-beta 5' flanking sequence and the first 5 bp of the 5' untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-beta-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH3 cells. Basal low-level CAT activity was only detected in GH3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-beta CAP site, confirming that promoter activity reflected correctly initiated LH-beta-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3-5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-beta promoter region. Transfection of deletion mutants delta-615-CAT, delta-385-CAT and delta-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between -1.7 and -0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-beta 5' flanking sequences to delta-85-CAT restored forskolin induction to wild-type levels (3-5-fold), suggesting the presence of a weak inhibitory element between -600 and -85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-beta promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-beta gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-beta promoter enhancer.
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PMID:Expression of luteinising hormone-beta subunit chloramphenicol acetyltransferase (LH-beta-CAT) fusion gene in rat pituitary cells: induction by cyclic 3'-adenosine monophosphate (cAMP). 165 45

30A5 preadipocytes, derived from 10T1/2 mouse fibroblasts, can be induced to differentiate into adipocytes by hormone treatment. In this paper, we introduce a modified procedure to induce differentiation of 30A5 cells by pretreatment with cAMP for a brief period or by a "nutrition deprivation" pretreatment, followed by incubation in medium containing insulin. These procedures accelerate the differentiation of the preadipocytes, so that the cells are fully differentiated within 4 days instead of the 7-8 days normally required. This differentiation is accompanied by the early induction of acetyl-CoA carboxylase (ACC). ACC catalyzes the rate-limiting step in the biogenesis of long chain fatty acids. To analyze the relationship between cAMP and insulin action in the induction of ACC and cell differentiation, we identified the DNA sequences in promoter II of the ACC gene necessary for the action of insulin and cAMP. Chimeric genes between different fragments of the ACC promoter and the promoterless chloramphenicol transacetylase (CAT) gene were constructed, and stable clones containing these chimeric genes were obtained. By analyzing the CAT activities in these stable clones, we established that insulin action in inducing ACC and cell differentiation requires prior treatment of cells with cAMP and the presence of specific DNA regions in the ACC promoter for cAMP action. Stable clones containing a chimeric gene which consists of DNA sequences in promoter II that are required for insulin action, thymidine kinase promoter, and the CAT gene did not respond to insulin. However, when the DNA sequences required for cAMP action were placed in this chimeric gene, it responded to insulin upon prior treatment of 30A5 cells with cAMP. Thus, cAMP and insulin, whose physiological actions generally appear to be antagonistic, are synergistically interacting in the induction of ACC and the differentiation of 30A5 cells.
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PMID:Regulation of acetyl-CoA carboxylase gene expression. Insulin induction of acetyl-CoA carboxylase and differentiation of 30A5 preadipocytes require prior cAMP action on the gene. 167 99

Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes--i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.
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PMID:Cell-free transcription directed by the 422 adipose P2 gene promoter: activation by the CCAAT/enhancer binding protein. 168 37

Catecholamines appear to be involved in behavioral responses to acute and chronic ethanol consumption. Since tyrosine hydroxylase (TH) is the rate-limiting enzyme for catecholamine biosynthesis and is regulated by second messenger systems known to be modulated by ethanol, we studied ethanol-induced changes in TH gene expression. In the N1E-115 neural cell line, Northern and Western blot analyses showed that treatment with 25-200 mM ethanol for 3 days caused a dose-dependent increase in TH mRNA and protein levels. N1E-115 cells were also stably transfected with pTH5'CAT, a plasmid containing 773 base pairs of the TH promoter fused to a chloramphenicol acetyltransferase (CAT) reporter gene. Subclones expressing pTH5'CAT showed ethanol-induced increases in CAT activity, suggesting that ethanol modulates TH gene transcription. Furthermore, simultaneous treatment of transfected cells with 100 mM ethanol and 1 nM to 1 microM prostaglandin E1 increased prostaglandin E1-mediated stimulation of TH-promoter activity. Similarly, simultaneous treatment of transfected cells with 100 mM ethanol and either 10 mM (-)-N6-(R-phenylisopropyl)adenosine or 0.5 mM 8-bromo-cAMP also resulted in increased TH-promoter activity compared to treatment with these agents without ethanol. These results suggest that ethanol treatment of N1E-115 cells has a prominent effect on both basal and cAMP-regulated TH expression. Ethanol-induced changes in TH expression may be a critical molecular event in adaptation of the central nervous system to ethanol.
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PMID:Ethanol increases tyrosine hydroxylase gene expression in N1E-115 neuroblastoma cells. 168 18


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