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)

We have studied the effect of several environmental chemicals on the transient expression of a chloramphenicol acetyltransferase (cat) reporter gene linked to the promoter sequences in the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1). Aflatoxin B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and benzo[a]pyrene cause a significant increases in CAT expression in mouse hepatoma Hepa-1 cells. The induction of CAT after TCDD treatment is abolished by administration of N-acetyl-L-cysteine or 2-mercaptoethanol and does not take place in a mutant cell line that lacks CYP1A1 enzymatic activity. Linker-scanning mutational analysis of transcription factor binding sites in the promoter revealed that both the NF kappa B and an adjacent aromatic hydrocarbon response element (AhRE) are required for TCDD-dependent CAT expression. In addition, mutation of the NFAT/AP-1 binding sites in the negative regulatory region of the promoter increases the magnitude of the TCDD effect. We conclude that induction of a functional CYP1A1 monooxygenase by TCDD stimulates a pathway that generates thiol-sensitive reactive oxygen intermediates which, in turn, are responsible for the TCDD-dependent activation of genes linked to the LTR. These data might provide an explanation for findings that TCDD increases infectious HIV-1 titers in experimental systems and for epidemiologic reports suggesting that exposure to aromatic hydrocarbons, such as found in cigarette smoke, is associated with an acceleration in AIDS progression.
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PMID:Dioxin activates HIV-1 gene expression by an oxidative stress pathway requiring a functional cytochrome P450 CYP1A1 enzyme. 760 37

The AP-1 consensus sequences (TGAGTCA) are the major 12-O-tetradecanoylphorbol113-acetate (TPA) responsive elements shared by several TPA inducible genes, such as c-sis, c-fos, c-myc, collagenase, stromelysin, hMTIIA and SV40. However, the role of AP-1 binding sites, which are present in the introns 3, 5, and 11 of ODC gene, in the regulation of TPA-induced ornithine decarboxylase (ODC) gene transcription are unknown. We determined the TPA responsiveness of the AP-1 sequences in the introns of ODC gene in CV-1 cells which induce ODC activity and mRNA in response to TPA treatment. ODC introns containing AP-1 sequences were inserted into the chloramphenicol acetyltransferase (CAT) reporter gene. Transient transfection of CV-1 cells with the intron-CAT constructs followed by TPA treatment did not induce CAT activity. However, when flanking regions of the AP-1 site in intron 3 were narrowed down to 74 bp, TPA induced CAT activity by 5- to 7-fold. The TPA-inducibility could be eliminated by mutation of the AP-1 site (TGAGTCA-->TGATGCCA or TGATGA) in 74 bp of intron 3. These results indicate that the AP-1 sequences in the intact ODC introns may not be responsive to TPA. The flanking sequences of the AP-1 site may be crucial to determine whether the AP-1 site is accessible to the TPA-induced transcriptional factor(s).
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PMID:Lack of 12-O-tetradecanoylphorbol-13-acetate responsiveness of ornithine decarboxylase introns which have AP-1 consensus sequences. 765 80

To define DNA regulatory elements that mediate the response of the keratin 1 (K1) gene to Ca(2+)-induced differentiation, regions spanning the 5'- and 3'-flanking sequences, coding regions, and introns from the human K1 gene were cloned into vectors containing the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into cultured mouse keratinocytes. A 4.3-kilobase (kb) region located 3' to the K1 gene stimulated CAT activity in response to increasing Ca2+ concentrations from 0.05 mM (basal cells) to 1.2 mM (differentiated cells). The 4.3-kb fragment was also active in human epidermal cells but inactive in NIH 3T3 cells and primary mouse fibroblasts. Deletion analysis localized the activity to the terminal 1682 base pairs (bp) of the flanking sequence which retained Ca2+ sensitivity in epidermal cells but was not active in mesenchymal cells. Removal of a 207-base pair element created an enhancer which was active in both epidermal and mesenchymal cells but was still Ca(2+)-inducible. Further deletions identified two elements which functioned synergistically to give maximal Ca(2+)-sensitive activity. Stably transfected epidermal cell lines expressed CAT under the direction of these elements when grafted onto nude mice to reconstitute an intact epidermis. Previously reported keratin regulatory motifs were not contained in the 1682-bp fragment, but an AP-1 site was identified in one of the synergistic subunits.
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PMID:Identification of control elements 3' to the human keratin 1 gene that regulate cell type and differentiation-specific expression. 767 99

Decay accelerating factor (DAF) is a complement regulatory protein that protects host tissue from complement-mediated damage by preventing the assembly and/or promoting the dissociation of C3 and C5 convertases. To identify and analyze the DNA sequence elements responsible for controlling DAF expression, the 5'-flanking region of the human DAF gene was cloned. Sequencing of 880 nucleotides upstream from the ATG codon revealed the absence of classic TATA or CAAT boxes. RNase protection and primer extension assays revealed a series of transcription start sites in a 10 nucleotide region located 86 nucleotides upstream from the ATG (the first of these start sites is numbered +1). Using HeLa, K562, EBV, and Molt 4 cells, DAF mRNA and protein were analyzed by Northern and Western blot. The DAF mRNA and protein levels roughly correlated, suggesting transcriptional control of gene expression. K562 and HeLa expressed high levels, EBV expressed intermediate levels, and Molt 4 expressed essentially no detectible DAF mRNA or protein. Regions of DAF 5'-flanking DNA were subcloned into plasmids containing the chloramphenicol acetyltransferase reporter gene and tested in transient transfection assays. The construct extending from -206 to +84 (-206/+84) had transcriptional activity in the DAF-positive HeLa, K562, and EBV lines, but no activity in the DAF-negative Molt 4 line. In the three DAF-positive lines, major enhancer activity was demonstrated between -206 and -77, and between -77 and -54 (containing cAMP responsive element and AP-1 binding site). Additional deletion of the region between -54 and -34 (containing an Sp1 binding site) reduces chloramphenicol acetyltransferase activity further but the low numerical values preclude statistical significance. The identification of transcription start sites and enhancer regions in the DAF gene will be important for studies of the mechanisms whereby cytokines and other factors may modulate DAF expression.
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PMID:Identification of 5'-flanking regions affecting the expression of the human decay accelerating factor gene and their role in tissue-specific expression. 767 27

We have analyzed the transcriptional regulation of the fra-2 gene in chicken embryo fibroblasts. Like c-fos, fra-2 was inducible by phorbol ester, cAMP and calcium ionophore, as well as serum. In all three cases, the induction of two species of fra-2 transcript (5.7 kb and 6.8 kb) was delayed and prolonged compared with that of c-fos mRNA. The size difference between the two transcripts was attributable to the heterogeneity of the 3'-end, probably reflecting utilization of different polyadenylation sites. The major transcriptional start point is located at 30 bp downstream of a TATA-like sequence. In the fra-2 promoter region, which is located in a typical CpG island, enhancer consensus sequences such as SCM, SRE, GC boxes and CRE-like sequences were detected upstream of the TATA-like sequence in the same order as that in the 5'-upstream region of the chicken c-fos gene. Fibroblast transfection studies with a series of promoter deletion constructs positioned upstream of bacterial chloramphenicol acetyltransferase indicated, however, that SRE-like sequence is not the sole responsible element for the serum induction, and that a minimal fragment containing no SRE-like sequence is sufficient for this induction. Two typical AP-1 sequences are located between the major transcriptional initiation site and the coding sequence, and the binding activity of protein complexes to these sequences was induced by serum.
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PMID:Analysis of fra-2 gene expression. 768 44

Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.
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PMID:Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene. 770 74

Previous studies suggested that individual components of the activator protein 1 (AP-1) complex behave in a highly idiosyncratic fashion at the level of the human atrial natriuretic peptide (ANP) gene promoter. ANP gene transcription is activated by c-jun and is generally suppressed by c-fos. In the present study, fra-1, a close relative of the c-fos gene product in terms of its structure and functional activity, behaved like fos in cardiac atriocytes, effecting an approximately 50% reduction in c-jun-activatable expression of a human ANP chloramphenicol acetyltransferase (CAT) reporter. In cardiac ventriculocytes, however, fra-1 effected a synergistic amplification of the c-jun response (a 2.5-fold increase over c-jun alone). In atrial cells, fos-like proteins were not uniformly inhibitory in that a carboxy terminal deletion mutant of c-fos activated a human ANP-CAT reporter in the atriocyte cultures. Finally, using a series of domain-swap mutations in the fos/fra structural sequences, we showed that sequences at both the amino and the carboxy termini are required to realize the full fra-1-dependent stimulatory effect as well as the c-fos-dependent inhibition of ANP gene transcription. These findings suggest considerable heterogeneity in the response of the ANP promoter to different components of the AP-1 complex. Such heterogeneity may serve to broaden the range of biological responses available to this promoter as the cardiac cell attempts to adapt to perturbations in the extracellular environment.
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PMID:Fra-1, a Fos gene family member that activates atrial natriuretic peptide gene transcription. 772 15

An Ets-related E1A-F has been characterized as an enhancer-binding protein for the adenovirus E1A gene. Here we show, in transient expression assays, that E1A-F can activate three different subclasses of the matrix metalloproteinase gene promoters. Expressions of the chloramphenicol acetyltransferase (CAT) reporter gene under the control of stromelysin, type I collagenase and 92 kD type IV collagenase promoters were increased approximately 10- to 20-fold by co-transfection with the E1A-F expression vector. Activation levels were as much high as those obtained by exogenous expression of AP-1 transcription factor. These results suggest that E1A-F positively regulates transcriptions from matrix metalloproteinase genes that are associated with invasion and metastasis of tumor cells.
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PMID:Ets-related protein E1A-F can activate three different matrix metalloproteinase gene promoters. 773

Loricrin gene expression is limited to terminally differentiating keratinocytes of stratified squamous epithelia. To define the regulatory elements that mediate the expression of the loricrin gene, we replaced the loricrin coding sequences from a 6.5-kilobase genomic fragment with the chloramphenicol acetyltransferase gene and transfected this construct into cultured mouse keratinocytes. High expression levels were observed in both undifferentiated as well as differentiating cells. Transgenic mice bearing a similar construct, but with beta-galactosidase as the reporter gene, corroborated these in vitro findings and showed tissue- and cell type-specific, but not differentiation-specific expression. Deletion analysis of the promoter region determined that sequences up to -60 base pairs from the start of transcription could be removed without significant loss of promoter activity. Within these proximal 60 base pairs is an evolutionarily conserved AP-1 element that is recognized by both purified c-Jun and AP-1 factors from keratinocytes in vitro. Mutation of this AP-1 site abolished the activity of the loricrin promoter. These studies show that elements directing expression of the loricrin gene to the stratified squamous epithelia are contained within a 6.5-kilobase genomic fragment, and those elements required to restrict expression to differentiated keratinocytes lie outside this region.
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PMID:The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiation-specific expression. 773 16

Regulation of lactate dehydrogenase (LDH) (EC 1.1.1.27) isozymes occurs through a multitude of physiological signals. Here, we show that modulation of LDH A subunit occurs via the protein kinase C pathway. Activators of protein kinase C, such as tetradecanoylphorbol acetate (TPA) and dioctanoylglycerol (DG), caused a 3-4-fold accumulation of LDH A subunit mRNA in rat C6 glioma cells. The specific protein kinase C inhibitor bisindolylmaleimide GF 109203X prevented the TPA-induced increase of LDH A subunit mRNA. To analyze the molecular basis of these effects in more detail, the transcription-modulatory effects of TPA and DG were evaluated in transient transfection assays using plasmids which contain LDH A subunit promoter fragments fused to a chloramphenicol acetyltransferase reporter gene. Both effector agents caused a marked increase of the transcriptional activity of an LDH -830/+25 bp promoter/CAT construct. In contrast, a phorbol ester which fails to activate protein kinase C, phorbol 12 beta,13 alpha-didecanoate, had no effect on the LDH promoter activity. Transient transfection analysis of LDH promoter deletion/CAT constructs, DNA/protein binding assays, including footprint and gel shift analyses, identified a TRE/AP-1 enhancer module at position -294 bp which was the target for the protein kinase C-mediated signal transduction pathway. Thus, our data demonstrate an active role of the protein kinase C signal pathway in regulating LDH A subunit gene expression which may be significant in regulating LDH isozyme patterns under various physiologic conditions.
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PMID:Transcriptional regulation of the lactate dehydrogenase A subunit gene by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. 775 43


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