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)

c-sis/platelet-derived growth factor 2 (PDGF-2) is a prototype growth factor with transforming potential. The c-sis/PDGF-2 transcript contains a long 5' untranslated sequence (UTS) that is highly G.C rich. To examine the influence of this sequence on sis/PDGF-2 expression, we localized the c-sis/PDGF-2 promoter and used this promoter or the simian virus 40 early promoter to drive expression of the bacterial chloramphenicol acetyltransferase or sis/PDGF-2 gene. The 5' UTS of c-sis/PDGF-2 mRNA had no effect on RNA expression but was shown to exert a potent inhibitory effect on translation. By deletion analysis, we demonstrated that the 5' UTS inhibited protein expression by as much as 40-fold. The inhibitory effect was independent of reporter gene, cell type, or promoter used. A highly G.C-rich 140-base-pair sequence immediately preceding the c-sis/PDGF-2 initiation codon was shown to be nearly as effective as the entire 5' UTS in translational inhibition. Transfection analysis demonstrated that the 5' UTS significantly reduced the transforming efficiency of the sis/PDGF-2 gene as well. Thus, our findings raise the possibility that changes in regulation at the level of sis/PDGF-2 translation may play a role in development of the neoplastic phenotype.
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PMID:The 5' untranslated sequence of the c-sis/platelet-derived growth factor 2 transcript is a potent translational inhibitor. 327 70

Vimentin is a growth-regulated gene whose mRNA levels increase severalfold after stimulation of quiescent cells. We have isolated and sequenced a genomic fragment of human DNA containing the vimentin 5'-flanking sequence and untranslated region. S1 nuclease analysis was used to determine the transcription initiation site. Deletion mutants of the promoter region were constructed, linked to a chloramphenicol acetyltransferase gene, and analyzed for transient expression by transfection into BALB/c 3T3 cells. These experiments revealed the presence in the human vimentin promoter region of a negative-regulatory element, flanked by positive elements. The most 5' of the positive elements is able to overcome the effects of the negative element. Analysis of these deletion constructs in stable cell lines confirmed the results of the transient assays. Using these stable cell lines, we can also demonstrate that the vimentin promoter region can confer platelet-derived growth factor inducibility to a linked chloramphenicol acetyltransferase gene and that the sequences required for this inducibility reside between positions -241 and +73.
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PMID:Functional analysis and growth factor regulation of the human vimentin promoter. 343 46

The state of induction of the platelet-derived growth factor (PDGF) A chain markedly differs among drugs and cells. The increase in A chain mRNA by serum was due to activation of transcription. Transcription was also activated by cycloheximide (CHX) even during serum starvation, indicating that the expression of the PDGF-A chain is inhibited by transcription suppressor factor with a short life during serum starvation. On the other hand, post-transcriptional regulation played a very important role in the increase in A chain mRNA by 12-O-tetradecanoylphorbol-13-acetate (TPA) and superinduction by TPA and CHX. We also analyzed the regions of PDGF-A chain gene that respond to serum and TPA by the chloramphenicol acetyltransferase (CAT) assay and the gel retardation assay. The region from TATA to -135 bp has the activity of the basal expression of PDGF-A chain gene and is considered to be involved in down regulation after the treatment with serum and TPA. Elements that respond to serum and increase the expression of PDGF-A chain gene are present in the region from -135 bp to -223 bp. Elements that inhibit the expression of PDGF-A chain gene during serum starvation are present in the region from -223 bp to -416 bp.
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PMID:Mechanism of regulation of PDGF-A chain gene expression by serum and TPA. 784 Nov 94

Vasoconstrictors such as arginine vasopressin (AVP) and angiotensin II (Ang II) have been shown to increase protein and mRNA levels of smooth muscle alpha-actin (SM-alpha-actin) in vascular smooth muscle cells. In the same cells, platelet-derived growth factor (PDGF) decreased SM-alpha-actin protein and mRNA. The rat SM-alpha-actin promoter that has recently been isolated contains two E-boxes and three CC(A/T)6GG (CArG) elements. To examine regulation of the SM-alpha-actin promoter, a 765-bp region of the rat SM-alpha-actin gene was ligated into chloramphenicol acetyltransferase (CAT)-containing vectors and transfected into rat aortic vascular smooth muscle cells. Stimulation of cells with either AVP or Ang II increased CAT activity 5- to 10-fold. PDGF was able to completely block the AVP-induced increase in CAT activity. To identify regions of the promoter responsible for both the AVP stimulation and PDGF inhibition of promoter activity, a series of truncation mutants were prepared and transfected into vascular smooth muscle cells. Truncation of both E-boxes and the most distal CArG element did not qualitatively alter either AVP-induced stimulation of CAT activity or PDGF inhibition. However, removal of the middle CArG element resulted in a loss of AVP stimulation. These studies indicate that the AVP-induced elevation and PDGF-induced inhibition of SM-alpha-actin levels in vascular smooth muscle cells are mediated at least in part through regulation of the SM-alpha-actin promoter. The critical region of the promoter mediating this effect involves at a minimum one of the CArG elements.
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PMID:Regulation of smooth muscle alpha-actin promoter by vasopressin and platelet-derived growth factor in rat aortic vascular smooth muscle cells. 795 49

We previously reported that in transformed mouse sarcoma cells of spontaneous origin and in revertants transfected with a fos-cat fusion, the 600-bp c-fos promoter region provides chloramphenicol acetyltransferase activity. In the present study, we investigated the binding of transcriptional factor protein(s) to a region (-503 to -361) upstream of the sis (platelet-derived growth factor)-inducible factor (SIF)-binding element. Gel electrophoresis retardation (GER) assay clearly demonstrated the appearance of strong binding activity to a newly described fragment in the 142-bp region studied. Further analysis using synthetic oligodeoxyribonucleotides and GER defined a binding region of 30 bp (AvaI-AvaII) from -503 to -472 that partially overlaps with a region known to bind fos promoter binding site 2 (FBS2). DNase I footprint analysis discovered a novel sequence in the upstream region of the c-fos promoter to which protein(s) in nuclear extracts from various mouse and human cells bind. This factor(s) is not identical to most known transcriptional factors present in the promoter region of nuclear oncogenes. A proximal part of this fragment is very conservative and contains several AP-2-like-binding sites.
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PMID:Characterization of a 142-bp fragment of the murine c-fos oncogene promoter upstream of the SIF-binding element. 819 66

Transcription of the junB gene is rapidly and transiently induced by a variety of extracellular signals. We report here that expression directed by a junB promoter/chloramphenicol acetyltransferase reporter construct (junB/CAT) is induced by fetal bovine serum, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), platelet-derived growth factor, and fibroblast growth factor in mouse fibroblast 3T6 cells. Deletion analysis of the promoter region of the junB gene indicates that there are at least two cis-regulatory elements that confer the capacity for serum-dependent induction. These two serum response elements (SRE1 and SRE2) are mapped between nucleotides -1451 and -1425 and between nucleotides -3100 and -2500, respectively, relative to the site of initiation of transcription. SRE1, the nucleotide sequence of which resembles that of the serum response element of the c-fos gene, is activated by TPA, platelet-derived growth factor, and fibroblast growth factor, but these growth-stimulating factors do not induce SRE2-mediated transcription. Pretreatment of the cells with phorbol dibutyrate, which reduces the level of protein kinase C activity in cells, almost completely abolishes the activation of SRE1 by TPA. Pretreatment with phorbol dibutyrate also reduces (but does not eliminate) the serum-dependent activation of SRE1. By contrast, the induction of SRE2 by serum is not affected by this pretreatment. Herbimycin A, an inhibitor of protein kinases, inhibits the activity of SRE2, but not that of SRE1. These results suggest that transcription of the junB gene can be induced by at least two distinct signaling pathways, which are mediated by SRE1 and SRE2, respectively. In addition, EGF induces expression of junB/CAT as strongly as does serum, but neither SRE1 nor SRE2 is sufficient for responsiveness to EGF.
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PMID:Two cis-regulatory elements that mediate different signaling pathways for serum-dependent activation of the junB gene. 831 5

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

Thrombin is a coagulation system protease that also serves as a potent stimulator of gene expression in several cell types, including endothelial cells (EC). We and others have previously demonstrated that the transcription of platelet-derived growth factor (PDGF) B-chain (c-sis) by EC is stimulated severalfold by thrombin. Here we examine the molecular mechanism of this regulatory process using bovine aortic EC transiently transfected with a vector containing the chloramphenicol acetyltransferase (CAT) gene under the control of a 400-base pair fragment of the human PDGF B-chain promoter. Thrombin treatment of these cells caused a severalfold increase in CAT expression. Deletion analysis and site-directed mutagenesis revealed that the region spanning nucleotides -61 to -53 from the transcription initiation site (referred to as the thrombin response, or ThR, region) was critical for the transcriptional response to thrombin. Electrophoretic mobility shift assays with an oligonucleotide corresponding to the region -64 to -44, which contained the ThR region, led to the identification of a thrombin-inducible nuclear factor (TINF) in extracts from thrombin-treated, but not control, EC. TINF was formed as early as 40 min post-thrombin treatment, persisted for at least 7 h, but was no longer present after 24 h. TINF appeared in the absence of de novo protein synthesis. The ThR region consists of a repeat of a CCACCC element in an ABBA configuration, which, based on mutation analysis and transfection assays, appears to be critical in mediating thrombin stimulation of the PDGF B-chain gene. The conservation of the ThR region in the promoter of the PDGF B-chain among three species (human, feline, and murine) further supports the importance of this region as a cis-acting regulatory element.
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PMID:Identification of a thrombin response element in the human platelet-derived growth factor B-chain (c-sis) promoter. 862 96

The PDGFbeta r gene has been implicated in many physiological processes including development and wound healing. Aberrant expression of the receptor is seen in many pathological conditions such as atherosclerosis and inflammatory diseases. To study the mechanisms of PDGFbeta r regulation, we identified the regulatory regions of the gene. We have cloned and characterized the promoter region of the platelet-derived growth factor beta receptor (PDGFbeta r). We isolated a 4.5 Kb genomic fragment which confers PDGFbeta r tissue-specific promoter activity. This fragment can direct transcription of a luciferase reporter gene in a cell-specific manner which correlates well with the known pattern of expression of the PDGFbeta r. The specificity of this clone was demonstrated by its high activity in NIH 3T3 fibroblasts and lack of activity in N-MUNG epithelial cells, a pattern that parallels the expression of the endogenous PDGFbeta r. We have defined a 614 bp region encompassing the 5' untranslated region of the gene which includes the basal promoter region. We generated transgenic mice that carry the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the 4.5 Kb promoter. The expression pattern of the reporter gene was compared to that of the endogenous PDGFbeta r gene. The promoter was able to direct reporter gene expression with the same temporal and spatial pattern as the endogenous PDGFbeta r. The most prominent expression was in condensing mesenchyme of developing blood vessels, bone and tissues adjacent to epithelium. We conclude that this clone contains the regulatory regions sufficient to direct expression of the PDGFbeta r. The further analysis of this promoter will help elucidate the transcriptional regulation of expression of the PDGFbeta r, and provide a useful tool for directing expression of heterologous genes.
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PMID:Isolation and characterization of the platelet-derived growth factor beta receptor promoter. 902 58

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun promoter. In this study, we asked whether signals activating JNK regulate the c-jun promoter. Using NIH 3T3 cells expressing G protein-coupled m1 acetylcholine receptors as an experimental model, we have recently shown that the cholinergic agonist carbachol, but not platelet-derived growth factor, potently elevates JNK activity. Consistent with these findings, carbachol, but not platelet-derived growth factor, increased the activity of a c-jun promoter-driven reporter gene (for chloramphenicol acetyltransferase). However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but resulted in surprisingly limited induction of the c-jun promoter. This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore which of its regulatory elements participate in transcriptional control. We observed that deletion of the 3' AP-1 site diminished chloramphenicol acetyltransferase activity in response to carbachol, but only to a limited extent. In contrast, deletion of a MEF2 site dramatically reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the c-jun promoter in response to carbachol, and stimulation of m1 receptors, but not direct JNK activation, induced expression of a MEF2-responsive plasmid. Taken together, these data strongly suggest that MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a yet to be identified pathway, distinct from that of JNK.
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PMID:Signaling from G protein-coupled receptors to the c-jun promoter involves the MEF2 transcription factor. Evidence for a novel c-jun amino-terminal kinase-independent pathway. 925 89


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