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)

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the alpha-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of beta-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.
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PMID:Hepatocyte growth factor down-regulates the alpha-fetoprotein gene expression in PLC/PRF/5 human hepatoma cells. 128 Apr 22

A variety of gene constructs containing carp beta-actin regulatory sequences were tested for their ability to drive transient expression of the chloramphenicol acetyltransferase reporter gene in 3 fish cell lines: carp epithelial cells (EPC), rainbow trout hepatoma cells (RTH149), and rainbow trout fibroblasts (RTG2). The constructs showed a wide variation in their levels of expression, and there were significant differences in the effects of transcriptional elements in the 3 cell lines. Sequences that enhanced expression in EPC cells were inhibitory in RTH149 and RTG2 cells. All cell lines exhibited the presence of nuclear trans-acting factors that could bind to implicated transcriptional control elements. On the basis of the cell culture results, selected constructs were examined for activity in early carp development. Constructs active in embryos and fry were further tested and found to express transgenes in adult fish.
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PMID:Selection of promoters for gene transfer into fish. 130 23

The proportion of hexokinase (HK; EC 2.7.1.1) isozyme 1 (HK1) that is bound to the outer mitochondrial membrane is tissue specific and developmentally regulated. HK activity is known to be markedly elevated in many cancer cells and a significant fraction is mitochondrial bound. This study examined the role of the 15-amino acid N-terminal domain of HK1 in binding to liver and hepatoma mitochondria. A chimeric reporter construct, pCMVHKCAT, encoding this HK1 domain coupled to the chloramphenicol acetyltransferase (CAT) gene was electroporated into mouse Hepa 1-6 hepatoma cells. After digitonin treatment, cell fractions were assayed for HK, lactate dehydrogenase, and CAT activities. Digitonin (75 micrograms/mg of protein) caused cytosolic leak but 70% of HK remained with the pellet. HKCAT, like HK, remained predominantly with the pellet; CAT form the control, pCMVCAT, remained mostly unbound. Binding of membrane-free cell extracts to rat liver mitochondria in vitro showed 91% of the HKCAT bound, whereas only 12% of CAT bound. Specificity of HKCAT binding to mitochondria was demonstrated by competition of HK1 for HKCAT binding sites on rat liver mitochondria as well as by blockage of HKCAT binding by N,N'-dicyclohexylcarbodiimide, which covalently binds to porin and blocks HK1 binding. Deletional mutant constructs of HKCAT showed reduced binding with increasing deletion size. In summary, these studies demonstrate that the 15-amino acid N-terminal domain of HK1 is necessary and sufficient to confer mitochondrial binding properties to CAT and that there is specificity for this binding to the mitochondria.
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PMID:Targeting of hexokinase 1 to liver and hepatoma mitochondria. 130 5

Adenovirus infection of hepatoma cells inhibited transcription of the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) gene and virtually eliminated transcription of a chimeric gene which contained the PEPCK promoter linked to the structural gene for chloramphenicol acetyltransferase (CAT). This effect is due to the viral protein E1A, since adenovirus containing a deletion in the E1A gene did not repress transcription from the PEPCK promoter. Both the 243R and 283R products of the E1A gene were effective. The conserved region 1 (CR-1) domain of E1A was required for this effect. Treatment of hepatoma cells with 8-bromo-cAMP or transfection with plasmids coding for the catalytic subunit of protein kinase A, CAAT/enhancer binding protein alpha (C/EBP), or Jun, all potent inducers of PEPCK gene transcription, did not relieve the inhibition caused by E1A. This inhibition does not appear to be mediated by major enhancer elements and in the PEPCK gene since transcription from the PEPCK promoter containing block mutations in binding domains for C/EBP and cAMP regulatory element binding protein (CREB) was also inhibited by E1A. Transcription of chimeric genes containing two copies each of the major cAMP response domains (CRE-1 and P-3) linked to a neutral promoter and fused to the CAT structural gene was stimulated by the catalytic subunit of protein kinase A, but this effect was totally inhibited by E1A. The strong repressive effect of E1A on PEPCK gene transcription seems to involve an interruption of an obligatory interaction between factors which bind to the cAMP response element in the PEPCK promoter and the TATA box.
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PMID:Adenovirus E1A represses the cyclic AMP-induced transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) in hepatoma cells. 131 Mar 18

As an initial step toward understanding the transcriptional regulation of cholesterol 7 alpha-hydroxylase (CYP7) in man, we isolated and functionally characterized the 5'-flanking region of the human CYP7 gene. The nucleotide sequences of the first exon and 1.6 kb preceding the exon were determined and found to contain a TATA box at position -30, a modified CAAT box at position -92, three potential hepatocyte nuclear factor 3 (HNF-3) recognition sites at nucleotides -316, -288, and -255, respectively, and a modified sterol response element at position -271. DNA sequences containing 1.3 kb of the 5'-flanking region and 29 nucleotides of the first exon were linked to the chloramphenicol acetyltransferase gene and transiently transfected into several cell lines. Promoter activity was very strong in the human hepatoma cell line HepG2 but absent in cells of nonhepatic origin. Mutational analysis of the promoter identified several regions that function in the transcriptional regulation of CYP7. Introduction of a fragment containing the region from -432 to -220 upstream of a heterologous promoter, in either orientation, resulted in a tremendous stimulation of activity in HepG2 cells. DNase I footprint analysis identified three regions within this fragment which were protected from digestion. The overexpression of HNF-3 in HepG2 cells resulted in a 4-fold stimulation of CYP7 transcriptional activity. We suggest that the region between -432 and -220 functions as a cell-specific enhancer whose activity is controlled, in part, by HNF-3.
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PMID:Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene. 131 51

The interaction of IFN-alpha with IL-1 beta or TNF-alpha on hepatitis B surface antigen (HBsAg) expression was analysed in hepatitis B virus (HBV)-DNA integrated PLC/PRF/5 and non-integrated HuH-7 human hepatoma cells. Secretion of HBsAg in PLC/PRF/5 cells was reduced by IFN-alpha, IL-1 beta or TNF-alpha, and synergistically depressed when IFN-alpha was used in combination with IL-1 beta or TNF-alpha. By Northern blot analysis, the levels of HBsAg mRNA were suppressed by IFN-alpha in combination with IL-1 beta or TNF-alpha. In the chloramphenicol acetyltransferase plasmid transfection assay, IFN-alpha in combination with IL-1 beta or TNF-alpha caused a much greater suppression of HBV enhancer activity than IFN-alpha, IL-1 beta or TNF-alpha alone in both hepatoma cells. These findings suggest that the interaction of IFN-alpha with IL-1 beta or TNF-alpha synergistically represses HBV enhancer activity, resulting in depressed expression of HBsAg.
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PMID:Interaction of interferon-alpha with interleukin-1 beta or tumor necrosis factor-alpha on hepatitis B virus enhancer activity. 131 44

The isolation and characterization of the rat genomic clone encoding the cholesterogenic enzyme farnesyl diphosphate (FPP) synthase is reported. The gene is localized on a 15-kilobase (kb) genomic fragment, spans approximately 12 kb and contains eight exons. Sequences containing from 3.9 kb to 132 base pairs (bp) of the putative promoter were joined to the coding region of the bacterial reporter gene chloramphenicol acetyltransferase (CAT). The CAT activities or CAT mRNA levels of the hybrid genes were determined following either transient transfections into human hepatoma HepG2 cells or stable transfections into Chinese hamster ovary cells. The transient transfections identified a 319-bp fragment that was required for a 4-fold induction in the absence of sterols. Sequence analysis of this region showed it contained five potential copies of the sterol regulatory element (SRE-1) (Smith, J.R., Osborne, T.F., Brown, M.S., Goldstein, J.L., and Gil, G. (1988) J. Biol. Chem. 263, 18480-18487) previously identified in the promoters of the 3-hydroxy-3-methyl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein receptor genes. Further mutational and deletion analysis of the FPP synthase promoter-CAT constructs followed by stable transfection and primer extension of the CAT mRNA levels indicated that these potential SRE-1 regulatory elements were not involved in the sterol-mediated transcriptional regulation of the gene. Our analyses have identified a 115-bp region that is required for the transcriptional induction of FPP synthase in the absence of sterols. These results suggest that the FPP synthase gene may be regulated at the transcriptional level by a different mechanism than other sterol regulated genes.
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PMID:Molecular cloning and promoter analysis of the rat liver farnesyl diphosphate synthase gene. 132 Nov 49

Transcription of the rat serine dehydratase (SDH) gene is induced by glucagon, mediated by the action of cAMP. To identify the nucleotide sequences in the SDH gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5' flanking region of the rat SDH gene fused to the structural sequence encoding the bacterial reporter enzyme, chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat hepatoma cell line 7AD-7 were assayed by measuring CAT activity in the cell lysates. Chlorophenylthio-cyclic AMP (CPT-cAMP), a potent protein kinase A activating agent, stimulated the expression of SDH-CAT fusion genes, and these inductions could be enhanced further by the addition of dexamethasone, although the glucocorticoid alone had no effect on CAT activity. Deletion analysis demonstrated that an 80 bp region located approximately 3.5 kb upstream from the transcription initiation site of the rat SDH gene was responsible for stimulation of transcription by CPT-cAMP, whereas the 120 bp region immediately upstream of the cAMP responsive element (CRE)-containing sequences is essential for the enhancement of CPT-cAMP induction by the glucocorticoid.
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PMID:Identification of regions in the rat serine dehydratase gene responsible for regulation by cyclic AMP alone and in the presence of glucocorticoids. 133 28

Deletion mutagenesis and transfection studies into hepatic (mouse hepatoma (Hepa-1) and human hepatoblastoma (Hep-G2)) and nonhepatic (HeLa) cells indicated that high levels of expression of the human NAD(P)H:quinone oxidoreductase gene in tumor cells and its induction by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole are mediated by human antioxidant response element (hARE) located in the region between -470 and -445. The hARE, when attached to the thymidine kinase promoter and transfected into several mammalian cells, expressed high levels of the chloramphenicol acetyltransferase gene that was inducible by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole. Nucleotide sequence analysis of the hARE revealed the presence of a recognition site for binding to the AP1 protein. Mutation of the AP1 binding site located within the hARE resulted in the loss of expression and induction upon transfection into various cell types. Band shift and competition assays with hARE and nuclear extracts from control and beta-naphthoflavone-treated Hepa-1, Hep-G2 and HeLa cells indicated specific interaction of regulatory protein(s) to the hARE. The supershift assays using antibodies against specific proteins of the AP1 family identified Jun-D and c-Fos as two members in the hARE-protein complex observed in band shift assays.
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PMID:Regulation of human NAD(P)H:quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element. 840 91

Previous studies have documented that the amount of agonist activity expressed by the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) for tyrosine aminotransferase (TAT) induction in two rat hepatoma cell lines (Fu5-5 and HTC) is greater in Fu5-5 cells and could be varied in each cell line with changes in cell density. We have proposed that both phenomena are mediated by the binding of a trans-acting factor, the concentration or activity of which is lower in HTC cells. We have now used DNase-I hypersensitivity studies to identify a possible binding site for this factor at around -3.6 kilobases (kb) of the TAT gene. Fu5-5 and HTC cells were then stably transfected with hybrid constructs either with (3.9TATCAT) or without (2.9TATCAT) this region of the TAT gene fused up-stream of a chloramphenicol acetyltransferase (CAT) reporter gene. High levels of Dex-Mes agonist activity for the induction of CAT activity in Fu5-5 cells were seen only with the 3.9TATCAT construct, indicating that the 0.97-kb region unique to this construct controlled the high levels of Dex-Mes agonist activity. Furthermore, variations in Fu5-5 cell density caused major quantitative changes in the amount of Dex-Mes agonist activity only in cells containing the 3.9TATCAT construct, consistent with the same 0.97-kb sequences also controlling the variations in Dex-Mes agonist activity. Additional studies at high and low cell densities revealed that the modulation of Dex-Mes agonist activity for both the endogenous TAT gene and the transfected TAT/CAT gene was not due to changes in the start site of gene transcription. These studies both support our previous hypothesis that modulation of Dex-Mes agonist activity results from changes in a trans-acting factor and localize a necessary cis-acting element to sequences between -3.9 and -2.9 kb of the TAT gene. These studies, thus, define a potentially new element for glucocorticoid regulation of TAT gene transcription.
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PMID:Modulation of glucocorticoid induction of stably transfected tyrosine aminotransferase gene constructs involves elements up-stream of the glucocorticoid-responsive element. 135 Jul 62


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