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 mammals, the apolipoprotein (apo) A-I gene is expressed predominantly in liver and intestine, while in avian species it is expressed in all tissues. Although liver and intestine are the major sites of chicken apoA-I mRNA synthesis, there are appreciable amounts of apoA-I mRNA in kidney, ovary/testes, brain, lung, skeletal, and heart muscle. In this study, the nucleotide sequences of the chicken apoA-I gene and its 5' flanking region, as well as the sequences involved in the expression of this gene, have been determined. The gene spans 1.5 kilobases and contains 4 exons and 3 introns, closely resembling the mammalian apoA-I gene. To determine the sequences involved in the expression of the chicken apoA-I gene, plasmid constructs containing serial deletions of the 5' flanking region of the chicken apoA-I gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected in human hepatoma (HepG2), colon carcinoma (Caco2), epithelial (Hela), mouse embryonal fibroblast (NIH3T3) cells, and quail myoblasts (QMLA29). The shortest deletion construct, containing 60 bp of the 5' upstream region, was sufficient for maximal transcriptional activity in all cell lines tested. This region contains a short sequence (nucleotides -60 to -54) that is highly conserved in birds and mammals, and an Sp1 binding site. Although the sequence between nucleotides -232 and -101 of the 5' region of the chicken apoA-I gene is partially homologous to the hepatic cell-specific enhancer of the mammalian apoA-I gene (located between nucleotides -222 and -110 upstream of the human apoA-I gene transcription start site), this chicken sequence is transcriptionally inactive in HepG2 cells. These results suggest that differences in the cis-acting regulatory elements of the apoA-I gene play a fundamental role in determining the differences in the tissue-specific expression of this gene in avian and mammalian species.
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PMID:Evolutionary distinct mechanisms regulate apolipoprotein A-I gene expression: differences between avian and mammalian apoA-I gene transcription control regions. 151 10

Several hormones, including insulin, glucagon, and glucocorticoids, regulate the expression of the rate-limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase [GTP: oxaloacetate carboxy-lyase (transphosphorylating); EC 4.1.1.32; PEPCK] in liver. In this report we demonstrate that retinoic acid (RA) also regulates PEPCK expression by inducing a 3-fold increase in the rate of transcription of the PEPCK gene. A RA response element located between -468 and -431 in the PEPCK promoter mediates a 7-fold increase in expression of a chimeric construct containing the basal PEPCK promoter ligated to the chloramphenicol acetyltransferase reporter gene. This element confers RA responsiveness through the heterologous thymidine kinase promoter and functions relatively independent of position and orientation. An 18-base-pair core sequence (-451 to -434) (i) mediates an effect of RA on PEPCK gene expression and contains motifs found in two other RA response elements; (ii) corresponds to AF1, an accessory factor element that is an integral component of the complex glucocorticoid response unit in the PEPCK gene promoter; (iii) is in a region involved in the developmental expression of the PEPCK gene; and (iv) shows homology to elements involved in the tissue-specific regulation of genes, including the hepatic apolipoprotein genes and the alpha 1-antitrypsin gene.
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PMID:A retinoic acid response element is part of a pleiotropic domain in the phosphoenolpyruvate carboxykinase gene. 184 96

Apolipoprotein (apo) B is the principal apolipoprotein of chylomicrons, very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Patients with homozygous hypobetalipoproteinemia (HBL), characterized by apoB deficiency, have markedly decreased levels of hepatocyte mRNA as well as intracellular B apolipoprotein, and a virtual absence of plasma apoB. We have cloned, sequenced and analyzed the 5' regulatory region of the human apoB gene from -899 to +121 bp in normal and hypobetalipoproteinemic subjects. TATA and CAAT boxes were located at -30 and -61, respectively, and two GC-like boxes were identified at positions +56 and +108. The analysis of the HBL sequence revealed two substitutions at positions -838 and -517, when compared to the normal sequence. These substitutions were not present in any known apoB regulatory elements. The transcriptional activities of the homozygous hypobetalipoproteinemic and normal regulatory regions were compared by chloramphenicol acetyltransferase (CAT) assays in Hep G2 cells, and were found to be the same. Therefore, we conclude that the 5' regulatory region of the HBL apoB gene in this kindred is normal, and the two base substitutions do not affect promoter activity of the apoB gene. These studies suggest that a coding region abnormality in the apoB gene may lead to HBL.
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PMID:Homozygous hypobetalipoproteinemia: transcriptional regulation and 5'-flanking sequence analysis in an apolipoprotein B deficiency state. 274 71

We have isolated the human apolipoprotein (apo) A-IV gene from a cosmid library and determined its complete nucleotide sequence. The gene contains three exons of 162, 127, and 1180 nucleotides separated by two introns of 357 and 777 nucleotides. A sequence polymorphism has been identified in the 3' noncoding portion of the third exon. The human apoA-IV gene lacks an intron in the area encoding the 5' nontranslated region of its mRNA, which distinguishes it from all the other human apolipoprotein genes whose sequences are known. Comparison matrix analysis of the human apoA-IV gene sequence revealed evidence for an ancestral 11-nucleotide repeat unit that spans the third exon. These repeated sequences are much more highly conserved than those present in either rat apoA-IV or in any other human apolipoprotein. Optimal alignments of the 5' flanking regions of the rat and human apoA-IV genes disclosed multiple deletions in the rat sequence as well as a highly conserved region of 90 nucleotides (90% sequence identity) located within 170 nucleotides of the start site of transcription. The 5' flanking regions of the human and rat apoA-IV genes were ligated to the bacterial chloramphenicol acetyltransferase gene, then transfected into different cultured cells. The apoA-IV gene sequences elicited preferential expression of chloramphenicol acetyltransferase activity when introduced into intestinally derived Caco-2 cells and liver-derived Hep-G2 cells, consistent with the tissue specificity of the native gene. Analysis of deletion mutants of the human apoA-IV 5' flanking region indicated that regions from -293 to -233 and from -127 to -60 upstream of the transcription start site contain sequences required for maximum gene expression. These findings on the structure and expression of rat and human apoA-IV should prove useful in studying the control of the apoA-IV gene.
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PMID:Structure and expression of the human apolipoprotein A-IV gene. 303 93

We have isolated and characterized a 2.5-kilobase pairs genomic DNA fragment which includes the 5'-flanking region and the first and second exons of the human apolipoprotein (apo) A-I gene. The major transcriptional start site was determined by primer extension analysis and is 235 base pairs (bp) upstream from the AUG translational start codon in liver and 234 bp upstream in the intestine. TATA box-like and CAT box-like sequences and two GC box sequences are present in the intestine 30, 108, 220, and 440 bp upstream, respectively, from the transcriptional start site. Fragments of 570 bp (-487 to +71) and 2.15 kilobase pairs (-2067 to +99) containing the 5'-flanking region of the apoA-I gene were fused upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene. These constructs, designated pA-I(0.6)CAT and pA-I(2.2)CAT, respectively, were introduced into human oral epithelial cells (KB), mouse NIH 3T3 cells, Chinese hamster ovary (CHO) cells, human hepatoma cells (Hep G2), human duodenal epithelial cells (Hutu 80), and human colonic epithelial cells (Caco-2) by calcium phosphate coprecipitation. When compared with control vectors, highly efficient CAT expression of both the pA-I(0.6)CAT and pA-I(2.2)CAT constructs were observed only in cells derived from the liver (Hep G2) and intestine (Caco-2), which is consistent with the tissue specificity of expression of the native gene. Analysis of deletion mutants of the human apoA-I 5'-flanking region revealed that: 1) the region from -250 to -199 bp, from -487 to -413 bp, and -1021 to -691 bp upstream from the transcriptional start site contain sequences required for maximum gene expression; and 2) the regions from -2067 to -1476 bp and -199 to -80 bp contain the sequences required for tissue-specific repression of apoA-I gene expression in non-apoA-I producing cells.
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PMID:Tissue-specific expression of apolipoprotein A-I (ApoA-I) is regulated by the 5'-flanking region of the human ApoA-I gene. 314 80

The human apolipoprotein B (apoB) gene codes for two related proteins, apoB-100 and apoB-48. ApoB-100 is synthesized in the liver, is the major protein constituent of low density lipoprotein, and serves as the ligand for the LDL receptor. cis-acting DNA sequence elements required for hepatic specific apoB transcription were identified in hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines transfected with apoB/CAT (chloramphenicol acetyltransferase) hybrid constructions. HepG2 cells express the transfected apoB constructions at high levels relative to expression in HeLa cells. Mutational analysis of the 5'-flanking region of the apoB gene revealed the presence of positive and negative regulatory regions. The most distal of these regions, located from -261 to -128 (with respect to the start site of transcription), was found to have a roughly equivalent negative activity in both cell types. However, sequences located from -128 to -86 showed a positive activity in HepG2 cells and a negative activity in HeLa cells. Finally, a sequence element located between positions -86 and -70 was found to have a very strong positive effect in HepG2 cells and only a mild positive effect in HeLa cells. These two proximal regions located between -128 and -70 appear to act together to determine the cell type-specific expression of the apoB gene in HepG2 and HeLa cells. Using the gel mobility shift assay and the DNase I footprinting technique, we demonstrated that DNA binding proteins from HepG2 and mouse liver nuclear extracts interact with the crucial positive region located between -86 and -70. This region was also found to contain sequence elements similar to sequences found in the promoters of other apolipoprotein genes, as well as other genes that are expressed in the liver, suggesting that these genes may share some transcriptional regulatory components.
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PMID:Cell type-specific expression of the human apoB gene is controlled by two cis-acting regulatory regions. 316 76

The mouse apolipoprotein (apo) E gene from strain C57BL/6 was isolated from a genomic DNA library and its complete nucleotide sequence, together with 1.3 kilobase of 5' flanking DNA and 300 base pairs of the 3' flanking DNA, was determined. Regulatory sequences in the proximal 5' flanking region of the gene were identified. Using a chloramphenicol acetyltransferase transient assay system, positive and negative cis-acting sequences were mapped within 380 base pairs of the 5' flanking region of the mouse apoE gene. Two nuclear protein binding sites were identified within this region by DNase I footprinting. We have characterized one of these regions, termed mouse apoE regulatory sequence (MARS-2), which spans nucleotides -151 to -133. Gel mobility shift assays using oligonucleotides of the MARS-2 sequence having specific deletions or substitutions as probes or competitors showed that the essential sequence of MARS-2 required for nuclear protein binding consists of 16 nucleotides encompassing -151 to -136. When nuclear extracts from different cells were examined, L cells and mouse liver nuclear protein contained the highest levels of binding protein for the MARS-2 probe. This protein, termed MARS-2 binding protein, was purified from mouse liver nuclear extracts to homogeneity using gel filtration and MARS-2 oligonucleotide-specific column chromatographic procedures. The Mr = 66,000 binding protein showed a gel mobility shift band that was identical to that of crude nuclear extracts.
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PMID:Characterization of an upstream regulatory sequence and its binding protein in the mouse apolipoprotein E gene. 759 86

Fibrates have been reported to modulate plasma high density lipoprotein cholesterol and apolipoprotein (apo) A-I concentrations. Therefore, the molecular mechanisms underlying the regulation of human apoA-I gene expression by fibrates was investigated. Fenofibrate reduced the expression of a reporter gene driven by the DNA sequences between -192 and +91 (BC-P-chloramphenicol acetyltransferase; CAT) relative to the apoA-I gene transcription start site approximately 3-fold. The sequences involved in the down-regulation of apoA-I gene transcription by fenofibrate were localized between -41 and +91 (P-CAT) relative to the transcription start site. The reduction of the expression of BC-P-CAT was dose-dependent and maximal at 500 microM (20 +/- 7%). Different peroxisome proliferators showed different levels of repression varying from 39 +/- 4% for fenofibrate, 43 +/- 5% for tetradecylthioacetic acid, 48 +/- 4% for bezafibrate, 54 +/- 2% for 5,8,11,14-eicotetraynoic acid, 76 +/- 2% for ciprofibrate, whereas Wy 14643 only marginally inhibited the expression of BC-P-CAT. By contrast, inclusion of sequences between -256 and -192 (ABC-P-CAT) attenuated the repression by fenofibrate. Furthermore, the apoA-IA site (-214 to -192; Awt-P-CAT) could counteract the repression of P-CAT by fenofibrate in the presence of cotransfected mPPAR alpha (peroxisome proliferator-activated receptor). In addition, the acyl-CoA oxidase-peroxisome proliferator response element (PPRE) could substitute the wild-type A-site in blocking the fenofibrate-induced reduction of the apoA-I promoter by mPPAR alpha. The protective effect of PPAR on fenofibrate induced inhibition of apoA-I expression was abolished after mutation of the direct repeat in the A site (Am-P-CAT). Consistent with these functional data only the wild-type, but not the mutated A site bound PPAR/retinoic X receptor heterodimers in gel shift assays. These data suggest that certain peroxisome proliferators can reduce the expression of the apoA-I promoter in a PPAR-independent fashion, through modulation of factors interacting with sequences localized between -41 and +91 of the apoA-I gene transcription initiation site. This inhibitory effect can be overcome when PPAR interacts with a functional PPRE, such as the apoA-I A site or the acyl-CoA oxidase-PPRE.
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PMID:Negative regulation of the human apolipoprotein A-I promoter by fibrates can be attenuated by the interaction of the peroxisome proliferator-activated receptor with its response element. 798 38

Expression of the very low density apolipoprotein II (apoVLDLII) gene in the chicken is absolutely dependent on estrogen. ApoVLDLII mRNA is expressed in the Leghorn male hepatoma (LMH) cell line in response to estrogen in completely defined medium. Addition of serum to these cultures results in a decrease in apoVLDLII mRNA. Data in this report demonstrate that 1 nM insulin has the same inhibitory effect as 10% serum. Insulin inhibits apoVLDLII mRNA in a dose-dependent manner; 100 fM insulin inhibits the estrogen-dependent response by 76%. After transfection of LMH cells with apoVLDLII sequences from an 8.9-kilobase (kb) genomic clone (pApo107) that contains the entire 2.9-kb coding sequence along with approximately 3 kb each of 5'- and 3'-flanking DNA, the estrogen-dependent expression of apoVLDLII mRNA from both the endogenous gene and transfected DNA is reduced by insulin. Furthermore, insulin reduces by more than 90% the estrogen-dependent expression from a chimeric construct, pApoCAT, which contains apoVLDLII sequences -900/+1455 cloned 5' of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. To determine the specificity of the response, expression of the pApoCAT construct was tested with insulin-like growth factor-I and insulin. Three hundred picomolar insulin inhibits the estrogen-mediated CAT activity by 50%. Insulin-like growth factor-I at this concentration has no effect or slightly increases the estrogen-dependent expression of pApoCAT, suggesting that the observed inhibitory action is mediated by the insulin receptor. Consequently, the LMH cells provide an excellent model system in which to study the molecular mechanism of insulin and estrogen interaction in the regulation of gene expression.
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PMID:Insulin inhibits the estrogen-dependent expression of the chicken very low density apolipoprotein II gene in Leghorn male hepatoma cells. 850 36

Although L-triiodothyronine (L-T3) lowers cholesterol, this hormone is not used to treat hypercholesterolemia because of its cardiotoxic effects. Thyromimetics, such as the novel compound CGS 23425, that mimic the beneficial but lack the detrimental effects of T3, may be useful in the treatment of hypercholesterolemia. To show that CGS 23425 has no cardiotoxicity, atrial contractility and force were both measured and found to be unchanged in rats treated with up to 10 mg/kg drug. The lipid lowering actions of this drug resulted in a 44% decrease in low-density lipoprotein (LDL) cholesterol in hypercholesterolemic rats treated with 10 microg/kg of the compound. Normal rats required a higher dose of 1000 microg/kg to elicit a similar 50% reduction in LDL cholesterol. Both CGS 23425 or T3 (10 nM) increased the specific binding of 125I-labeled LDL to Hep G2 cells and increased LDL receptor number by 44 and 49%, respectively. These data indicate that CGS 23425 enhances hepatic clearance of serum LDL cholesterol. Normal and fat-fed animals treated with the drug showed a dose-dependent increase in apolipoprotein AI, a protein that promotes the efflux of cholesterol from peripheral tissues. Transient transfection of a rat apolipoprotein AI promoter-chloramphenicol acetyltransferase construct, in human hepatoma cells, showed a dose-dependent increase in chloramphenicol acetyltransferase activity with EC50 values of 2 x 10(-12) M and 10(-10) M for thyroid hormone receptors beta1 and alpha1, respectively, with maximal responses at 10(-7) M. These data indicate that CGS 23425 is a thyromimetic that increases apolipoprotein AI expression via thyroid hormone receptor. In summary, CGS 23425 ameliorates hypercholesterolemia by increasing apolipoprotein A1 and the clearance of LDL cholesterol. Therefore, a compound like CGS 23425 may be useful for the prevention and reversal of atherosclerosis.
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PMID:Beneficial effects of a novel thyromimetic on lipoprotein metabolism. 928 17


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