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)

Carnitine palmitoyltransferase (CPT) regulates the flux of long-chain fatty acids into the mitochondria for subsequent beta-oxidation. A 485 bp segment of the promoter for the gene encoding the 68 kDa CPT was isolated from a rat lambda DASH genomic library using the polymerase chain reaction. The promoter contained a consensus binding sequence for CREB (cyclic AMP response element binding protein) at -153 to -166, and for C/EBP alpha (CCAAT/enhancer binding protein) at -115 to -128. DNAase I footprinting using proteins isolated from rat liver nuclei indicated the presence of several regions of nuclear protein binding, most notably at -95 to -130, at -273 to -295, and at a wide region encompassing -395 to -465. DNAase I footprinting studies with purified CREB and C/EBP alpha confirmed that protein binding to DNA occurred at the sites predicted by the consensus sequences. The segment containing 481 bp of 5' flanking sequence plus 181 bp of untranslated mRNA was ligated to the structural gene for chloramphenicol acetyltransferase (CAT). When this plasmid was transfected into Hep G2 cells, CAT activity was stimulated 7-fold by addition of 1 mM-8-bromo-cyclic AMP (8-Br-cAMP) or co-transfection of the expression vector coding for the catalytic subunit of protein kinase A (PKA). The ability of several known second messengers and transcription factors to stimulate transcription of 68 kDa CPT promoter-CAT reporter was tested in co-transfection experiments. 68 kDa CPT promoter-CAT reporter transcription activity was stimulated 7-fold by addition of 8-Br-cAMP, and this induction was depressed 50% by the addition of phorbol esters. When the 68 kDa CPT promoter-CAT reporter was co-transfected with an expression vector for CREB or C/EBP alpha, transcription was increased 3- and 10-fold respectively. 8-Br-cAMP caused an additional 8-fold induction in the presence of each factor to yield 25- and 80-fold induction respectively. Co-transfection of the expression vector for c-jun also increased the CAT activity driven by the 68 kDa CPT promoter, while co-transfection with the expression vector for c-fos had no effect. When expression vectors for both c-jun and c-fos were co-transfected with the 68 kDa CPT promoter, c-fos depressed the induction seen with c-jun alone.
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PMID:Isolation and characterization of the promoter for the gene coding for the 68 kDa carnitine palmitoyltransferase from the rat. 825 Aug 54

Lactogenic hormone-dependent expression of the rat beta-casein gene in mammary epithelial cells is controlled via a complex regulatory region in the promoter. The sequence between -176 and -82 is the minimal region to confer the response to glucocorticoid hormone and prolactin on a heterologous promoter. The response is further enhanced by the region between -282 and -176. DNase I footprinting experiments and electromobility shift assays revealed the presence of four binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms in the hormone response region between -220 and -132. In nuclear extracts from mammary epithelial cells, the prevalent C/EBP isoform binding to these sites is beta (C/EBP-beta). C/EBP-delta is also present in mammary epithelial cells, whereas C/EBP-alpha is not detectable. The C/EBP sites are located in close proximity to the previously characterized binding sites for the prolactin-inducible mammary gland factor/signal transducer and activator of transcription-5, the nuclear factor YY1, and the glucocorticoid receptor. The importance of the two proximal C/EBP binding sites at the 5' border of the minimal region was tested by mutational analysis. Mutations of each site were found to inhibit strongly both the basal and the lactogenic hormone-induced transcription of a beta-casein gene promoter chloramphenicol acetyltransferase construct. The results implicate C/EBPs as important regulators of beta-casein gene expression in the mammary epithelium.
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PMID:CCAAT/enhancer-binding protein isoforms beta and delta are expressed in mammary epithelial cells and bind to multiple sites in the beta-casein gene promoter. 762 3

The interleukin-6 (IL-6) gene expression in bovine monocytes is highly induced following bacterial lipopolysaccharide (LPS) stimulation. To identify the promoter element(s) involved in the inducible transcription of IL-6, a 5'-flanking region containing 230 bp of the bovine IL-6 gene was linked to a reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) and analyzed for its ability to confer LPS-responsiveness to the reporter CAT gene in monocytic cells. Using mutant reporter genes, we demonstrate that although mutation in the NF-kappa B element produces the major loss of induction, both NF-kappa B and C/EBP elements are necessary for maximal transcriptional activation of the bovine IL-6 gene. Gel electrophoretic mobility-shift assays have detected induced DNA-binding activities in the LPS-stimulated monocytes. Further characterization has revealed the activation and interaction of C/EBP-alpha, C/EBP-beta (NF-IL6), NFKB1 (p50), and RelA (p65) to their specific binding elements present in the bovine IL-6 gene. These results suggest a model in which induction of C/EBP-alpha in differentiating monocytes contributes and synergizes with induced C/EBP-beta and NF-kappa B, which are activated following LPS stimulation, to mediate a high rate of IL-6 transcription under inflammatory conditions.
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PMID:Lipopolysaccharide-mediated induction of the bovine interleukin-6 gene in monocytes requires both NF-kappa B and C/EBP binding sites. 766 56

A 268-base pair 5' distal fragment, SX2, which mediates basal level and inducer-dependent activation of the mouse heme oxygenase-1 gene, contains two activator protein-1 (AP-1) binding sites (Alam, J., and Zhining, D. (1992) J. Biol. Chem. 267, 21894-21900). Mutation of both AP-1 binding elements diminishes (by 50-70%), but does not abolish, the enhancer activity of SX2 in transient expression assays, suggesting that other sequences contribute to enhancer function. Directly upstream of the AP-1 binding sites are two copies of a sequence motif, TGAGGAAAT, which resemble elements found in cellular and viral genes that are known to interact with the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. These SX2 sequences bind specifically to liver-enriched, heat-stable nuclear proteins and confer C/EBP alpha-dependent transactivation of the heterologous chloramphenicol acetyltransferase (CAT) gene. Site-directed mutagenesis of these 9-base pair elements abolishes protein binding and transactivation, establishing these sequences as functional C/EBP binding sites. Stably transfected SX2/CAT fusion genes are induced between 37- and 44-fold in mouse hepatoma, Hepa, cells and between 52- and 111-fold in mouse fibroblast L929 cells in response to CdCl2 treatment. Subfragments of SX2 lacking the AP-1 binding elements do not mediate cadmium-dependent activation of the CAT gene, whereas subfragments containing the AP-1 binding elements, but lacking the C/EBP binding sites, exhibit only partial transcriptional activity. Site-directed mutagenesis of one or more of the C/EBP and AP-1 binding sites indicates that each of these elements is required for optimal activity of the SX2 enhancer fragment. The AP-1 binding elements, however, appear to be more important for induction as constructs containing multiple copies of either of the AP-1 binding elements, but not the C/EBP binding sequences, are readily activated by CdCl2. Treatment of Hepa cells with cadmium or heme does not alter the nuclear concentration of AP-1 or C/EBP binding activity.
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PMID:Multiple elements within the 5' distal enhancer of the mouse heme oxygenase-1 gene mediate induction by heavy metals. 792 91

We herein demonstrate competence of the 5' upstream region -1374 to +16 of the human growth factor-activatable Na+/H+ exchanger (NHE-1) gene to promote transcription of the chloramphenicol acetyltransferase gene in cells of hepatic origin (HepG2), vascular-smooth-muscle origin (VSM A7r5) and fibroblasts (3T3). We also describe the mapping of the regulatory elements required for such transcription. Sequential 5' end-deletions indicated that the 5' boundary of the positive regulatory elements of NHE-1 transcription is localized downstream of nucleotide -252 in both HepG2 and VSM A7r5 cells but downstream of nucleotide -654 in 3T3 cells. Footprinting analysis of the 0.25-kb promoter fragment using rat liver nuclear extracts identified 4 protected regions as follows: A, -31 to -9; B, -108 to -65; C, -124 to -111; and D, -239 to -215. Internal deletion and nucleotide substitutions within regulatory element D revealed its essential role for transcription of the human NHE-1 gene in HepG2 and VSM A7r5 cells. DNA binding and competition assays using rat liver nuclear extracts indicated that regulatory element D is recognized by 5 nuclear activities. Four of these activities (designated as NHE-1D1-4) are competed out completely by oligonucleotides containing the binding sites of transcription factors CREB, AP3, NFY, and other CCAAT box-binding proteins (C/EBP alpha or related proteins). This competition profile might be explained by the presence of homology between regulatory element D and the consensus sequence of C/EBP as well as the other competitor oligonucleotides. The actual relationship between these nuclear activities and the C/EBP family of proteins (or other transcription factors) remains to be determined.
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PMID:Proximal regulatory elements and nuclear activities required for transcription of the human Na+/H+ exchanger (NHE-1) gene. 828 17

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.
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PMID:A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor. 828 14

Primary brown adipocytes differentiated in culture were transiently transfected with plasmids containing different extensions of the 5'-flanking region of the rat uncoupling protein gene placed upstream of the bacterial chloramphenicol acetyltransferase reporter gene. Co-transfection of expression vectors for CCAAT/enhancer binding protein (C/EBP) alpha and C/EBP beta trans-activated the rat uncoupling protein gene promoter due to sequences in the 5' proximal region. DNAse I footprint analysis showed the presence of two C/EBP binding sites at positions -457/-440 and -335/-318, which interact with purified C/EBP beta as well as with C/EBP proteins present in brown fat or liver nuclear extracts. Two copies of each site placed upstream of the enhancerless SV40 promoter confer C/EBP alpha and C/EBP beta responsiveness to this heterologous promoter when co-transfected into HepG2 cells. It is concluded that the UCP gene is a target for C/EBP-dependent transcriptional regulation. This suggests that the C/EBP family of transcription factors is involved in the establishment of the characteristic phenotype of the brown adipocyte.
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PMID:CCAAT/enhancer binding proteins alpha and beta are transcriptional activators of the brown fat uncoupling protein gene promoter. 829 76

To determine the mechanisms of expression of the rat caeruloplasmin gene, the promoter region was analysed by DNAase I footprinting. Using nuclear extract from rat liver, a prominent site of protein-DNA interaction was detected from -93 to -48 upstream of the caeruloplasmin gene transcription start and sequence analysis of this region revealed three potential CCAAT/enhancer-binding protein (C/EBP) consensus elements. Mobility-shift analysis using an oligonucleotide encoding this region identified specific binding of proteins from rat liver nuclear extract, and some of these complexes were supershifted using antisera to the C/EBP alpha and beta family members. Mobility-shift studies using a polypeptide encoding the DNA-binding domain of C/EBP alpha also revealed a specific interaction with this region of the caeruloplasmin promoter, and DNAase I footprinting using this polypeptide protected the identical region from -93 to -48. Co-transfection of expression plasmids encoding C/EBP alpha or a related leucine-zipper factor D-binding protein (DBP) revealed a C/EBP-specific increase in reporter gene activity in HepG2 cells transfected with caeruloplasmin-chloramphenicol acetyltransferase containing the -93 to -48 region. A similar result was obtained when these constructs were co-transfected into mouse L cells which were shown not to express the endogenous caeruloplasmin gene. Taken together, these data indicate a role for C/EBP alpha and beta in mediating transcription from the caeruloplasmin gene promoter and suggest that this region of the promoter is not responsible for tissue-specific expression.
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PMID:Interaction of CCAAT/enhancer-binding protein alpha and beta with the rat caeruloplasmin gene promoter. 837 62

We have recently shown that regulatory element D (nucleotides -239 to -215) of the 0.25-kb promoter of the human growth factor-activatable Na+/H+ exchanger (NHE1) is important for gene transcription in cells of hepatic origin (Hep G2) and vascular smooth muscle origin (VSM A7r5). This element contains a sequence (nucleotides -230 to -222) with complete homology to the C/EBP binding site. We now demonstrate that nucleotide substitution mutations disrupting this C/EBP site suppressed transcription in Hep G2 cells, VSM A7r5 cells, and Sprague-Dawley VSM cells in primary culture. These mutations abolished the binding of rat liver nuclear activities as well as transcription factors C/EBP alpha, C/EBP beta, and C/EBP delta expressed in COS-1 cell lysates to element D. Anti-C/EBP antibodies supershifted DNA-protein complexes formed between hepatic nuclear activities or C/EBP proteins expressed in COS-1 cell lysates and regulatory element D. Finally, cotransfection experiments of NHE1 0.25-kb promoter-chloramphenicol acetyltransferase (CAT) construct and C/EBP expression vectors showed that C/EBP alpha and C/EBP delta are transactivators of the NHE1 proximal promoter in Hep G2 and VSM A7r5 cells. These results indicate that members of the C/EBP family of transcription factors are involved in the regulation of hepatic and vascular smooth muscle transcription of the human NHE1 gene.
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PMID:Role of C/EBP proteins in hepatic and vascular smooth muscle transcription of human NHE1 gene. 857 70

A construct comprising three tandemly repeated copies of the kappaB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-kappaBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-kappaB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-kappaBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein alpha (C/EBP-alpha) (but not C/EBP-beta or C/EBP-delta). Stimulation by C/EBP-alpha required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-kappaBCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-kappaB proteins, we co-transfected C/EBP-alpha with NF-kappaB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP- alpha reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a kappaB probe revealed that the addition of recombinant C/EBP-alpha protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappaB proteins had no effect. Our findings show that C/EBP-alpha may cause activation of some kappaB element-containing genes lacking C/EBP binding sites.
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PMID:CCAAT box enhancer binding protein alpha (C/EBP-alpha) stimulates kappaB element-mediated transcription in transfected cells. 862 20


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