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)

In order to identify the mechanism by which cyclic AMP stimulates expression of the human renin gene (REN), the effect of forskolin was tested in transient expression analyses of REN 5'-flanking DNA-chloramphenicol acetyltransferase (CAT) reporter gene constructs in secondary cultures of human chorio-decidual cells, a major site of renin synthesis. Forskolin induced a mean 5-fold stimulation which was localized to DNA in the region -249 to -162 with respect to the transcription start site (+1). Such DNA also mediated a response to forskolin in heterologous (HSV thymidine kinase) promoter constructs. Strong cAMP-response element (CRE) homology at -222 to -218 resembled the target for members of the CRE binding protein (CREB) family. Gel shift assays demonstrated similarly migrating nucleoprotein complexes for oligonucleotides containing the putative REN CRE as for a canonical CRE, in chorio-decidual, JEG-3 and HeLa nuclear extracts. Mutation of residues critical for CREB attachment reduced binding. In conclusion, a CRE was identified at -222 to -218 that appears critical for cAMP-induced human renin gene transcription.
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PMID:Identification of cyclic AMP response element in the human renin gene. 816

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

The -1389 to +588 region of the human genomic glutathione peroxidase gene (hgpx1) was amplified using the polymerase chain reaction. This DNA fragment was cloned and sequenced, and various deletion constructs derived from the hgpx1 5'-flanking region were fused to the chloramphenicol acetyltransferase gene. These reporter genes were analyzed in transient transfection assays using primary cultured human ventricular cardiomyocytes obtained from patients with tetralogy of Fallot. Two distinct regions upstream from the transcription start site, which was determined using S1 nuclease analysis, were identified to be responsive to the oxygen tension in culture (pO2 values of 150 or 40 mm Hg). Methylation interference footprinting assays revealed proteins closely apposed to two sequences located at -1232 to -1213 and -282 to -275. We have designated these oxygen responsive elements ORE1 and ORE2, respectively. Gel mobility shift assays using double-stranded oligonucleotides corresponding to each site have demonstrated the formation of specific complexes using both cultured human cardiomyocyte and HeLa nuclear extracts. ORE1 and ORE2 bind disparate proteins with equal precision as bound complexes could be competed away with identical sequences but not with either the other ORE or an unrelated sequence. Insertion of these oxygen responsive elements into a reporter gene governed by a SV40 promoter similarly regulated chloramphenicol acetyltransferase activity according to the oxygen tension in culture.
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PMID:Identification of oxygen responsive elements in the 5'-flanking region of the human glutathione peroxidase gene. 826 24

In an effort to identify regulatory elements of the serum amyloid A (SAA) gene that play a major role in its expression under acute-phase conditions, we studied the expression of a set of chimeric SAA-chloramphenicol acetyltransferase (CAT) plasmids containing a progressively deleted upstream 5' sequence of the SAA gene. Two regulatory regions (-314 to -135 and -135 to -31) capable of driving cytokine-induced transcription have been identified. Gel retardation assays revealed that the regulatory region located between positions -314 and -135 is a major site of interaction for highly inducible and constitutive nuclear proteins in acute-phase rabbit liver. DNase I footprint and competition analyses showed that this region contains two adjacent nuclear protein binding sites (between -191 and -140) with varying affinity for protein binding. Both of these binding sites are capable of driving cytokine-induced transcription of a reporter gene containing a minimal promoter. Detailed analyses of the inducible nuclear proteins that bind to this promoter element showed that they are homologues of the CCAAT/enhancer binding protein (C/EBP) family. Accumulation of the inducible nuclear factors under acute conditions, when maximal transcription activity has been reported, suggests a critical role for these proteins in the expression of the SAA gene.
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PMID:Analysis of the promoter element of the serum amyloid A gene and its interaction with constitutive and inducible nuclear factors from rabbit liver. 826 19

Recently, we have shown that the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) gene is efficiently expressed in human hepatoblastoma HepG2 cells and these cells can support productive HIV-1 replication. In this study we show that HepG2 cells contain a nuclear factor that binds to the HIV-1 trans-activating region (TAR), which we named HepG2-derived TAR binding protein (HTBP). Gel retardation assays using synthetic oligonucleotide probes carrying different mutations in the TAR region and competition DNA mobility-shift experiments using these oligonucleotides revealed the binding site encompassing between +7 and +13 nucleotides (5'-TCTGGTT-3') in the HIV-1 LTR. An in vivo CAT competition assay using -65HIV-1 LTR CAT as a reporter plasmid and various competitor plasmids containing these mutated oligonucleotides also demonstrated that HTBP can influence the HIV-1 LTR-directed CAT gene expression in HepG2 cells by interaction with a specific sequence in the TAR region.
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PMID:Identification of a human immunodeficiency virus type 1 TAR binding protein in human hepatoblastoma HepG2 cells that trans-activates HIV-1 LTR-directed gene expression. 828 41

rSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt -102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5' upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additional cis element (-645/-506). The positive and negative cis-elements together drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (-44/-29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (-44/-29) are competed by unlabeled -44/-29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides -40 to -38 and -34 to -32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-specific core promoter elements.
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PMID:Molecular cloning and functional analysis of the promoter of rat skeletal muscle voltage-sensitive sodium channel subtype 2 (rSkM2): evidence for muscle-specific nuclear protein binding to the core promoter. 828 44

We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.
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PMID:Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells. 829 29

Identification of the factors controlling transcription of the epidermal growth factor (EGF) receptor gene is essential for understanding regulation of the EGF receptor and its overexpression in human carcinomas. In this study, we have identified a 60-base pair (bp) region (-919 to -860) relative to the AUG translation initiation codon in the EGF receptor 5' promoter that functions as a cis-acting EGF receptor transcriptional repressor (ETR). This fragment also acted as a repressor when linked to the thymidine kinase promoter. Gel mobility shift assays demonstrated that trans-acting factors bind to 60- and 19-bp fragments. Competition and chloramphenicol acetyltransferase assays with oligonucleotides containing mutations and deletions in this region indicate that the TTCGAGGG sequence (-877 to -870) is required for binding as well as repressor activity. While the ETR-protected region contains consensus sequences for the E2F binding site, no competition was observed with an E2F binding fragment. However, DNA-protein blot analysis indicates that both the 60- and 19-bp fragments specifically bind a 128-kDa polypeptide in extracts from HeLa or A431 human epidermoid carcinoma cells. These results suggest that a novel transcription factor(s) negatively regulates EGF receptor gene expression through binding to the ETR element.
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PMID:Identification of an epidermal growth factor receptor transcriptional repressor. 830 97

Angiotensinogen is abundantly expressed in adipose tissue as well as in liver where it is mainly produced. To address the mechanism of this adipogenic expression, promoter regions of the mouse angiotensinogen gene are fused to the chloramphenicol acetyltransferase reporter gene and stably transfected into 3T3-L1 preadipocytes. Promoter activity correlates well with an increase of mRNA levels during adipogenic differentiation, thereby demonstrating that the induction is primarily due to transcriptional activation. Deletion analysis indicates that the proximal promoter region from -96 to +22 is able to mediate the chloramphenicol acetyltransferase induction and identifies two transcriptionally active regions: AGE1 (position -399 to -139) and AGE2 (position -96 to -52). Heterologous promoter assay reveals that AGE1 behaves with a constitutive enhancer-like property and that AGE2 functions as a differentiation-inducible activator. Gel shift experiments show that AGE2 specifically binds a novel factor (AGF2), which is induced upon differentiation. Furthermore, a constitutive factor (AGF3) binds to the core promoter region including the exon 1 (from -6 to +22, AGE3). Mutations within either AGE2 or AGE3 that disrupt nuclear factors binding in vitro dramatically reduced the chloramphenicol acetyltransferase activation in the native promoter context. These results suggest that both AGE2 and AGE3 are necessary for mediating efficient activation of the mouse angiotensinogen promoter during adipogenic differentiation.
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PMID:Proximal and core DNA elements are required for efficient angiotensinogen promoter activation during adipogenic differentiation. 832 78

The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2) gene encodes a Ca2+ transport pump whose expression is regulated during skeletal/cardiac muscle development and by different pathophysiological states of the heart. This study was designed to delineate cis-acting regulatory elements involved in SERCA2 gene expression. A series of unidirectionally deleted fragments of the upstream 1,460 bp SERCA2 promoter were linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transient DNA transfection experiments performed with these constructs in C2C12 muscle cells and NIH3T3 fibroblasts revealed a 17 bp upstream promoter element (UPE) important for transcription of the SERCA2 gene in skeletal muscle cells. These studies have also identified a strong (muscle specific) negative regulatory region located upstream of nucleotide -658. Gel mobility shift and southwestern analyses using the 17 bp UPE have revealed a specific DNA binding complex referred to as Ca2+ ATPase promoter factor -1 (CaPF1). The binding factor has an approximate M(r) of 43 kDa. Comparison of CaPF1 with known transcription factors suggests that the CaPF1 complex may be a novel DNA-binding transcription factor which plays a role in SERCA2 gene regulation in vivo.
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PMID:Analysis of the rabbit cardiac/slow twitch muscle sarcoplasmic reticulum calcium ATPase (SERCA2) gene promoter. 833 69


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