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)

The human gene for the recently identified 5-lipoxy-genase-activating protein (FLAP) has been cloned. The gene was isolated from two different genomic libraries and is contained within four overlapping bacteriophage clones. The gene spans greater than 31 kilobases and consists of five small exons and four large introns. Southern blot analysis of human genomic DNA suggests the presence of a single FLAP gene per haploid genome. A restriction site polymorphism was identified in intron II of the gene. This restriction fragment length polymorphism appears to be present in the normal population at a fairly high frequency. The transcription initiation site was located, at an adenine residue, 74 base pairs upstream of the ATG initiation codon. Examination of the sequence of the gene 5' to the mRNA start site revealed the presence of a possible TATA box (TGTAAT) 22 base pairs upstream and potential AP-2 and glucocorticoid receptor binding sites. Functional analysis of the FLAP gene promoter was assayed by transient transfection of mouse P388D1 cells (macrophage) and human HepG2 cells (hepatoma) with 5'-flanking sequences of the FLAP gene fused upstream of the chloramphenicol acetyltransferase reporter gene. Expression in the mouse macrophage cell line of the various FLAP gene promoter constructs revealed both tissue specificity and enhancer-like activities whereas in the hepatoma cell line only a minimum level of activity was obtained.
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PMID:Gene characterization and promoter analysis of the human 5-lipoxygenase-activating protein (FLAP). 167 82

We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.
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PMID:Structural and functional analyses of the promoter of the murine multidrug resistance gene mdr3/mdr1a reveal a negative element containing the AP-1 binding site. 168 3

Cis-acting elements involved in the control of rat alpha-fetoprotein gene expression in the liver and its modulation by glucocorticoid hormones were detected after transfection of chloramphenicol acetyltransferase constructs and their transient expression into two hepatoma cell lines. The proximal promoter region (-324 to -15) was found to contain all the information necessary for tissue-specific expression. It is also involved in the negative gene modulation by glucocorticoids and includes an activating regulatory domain allowing efficient expression in the HepG2 cells. Three regions within 7 kilobase pairs of the 5' extragenic sequences are capable of stimulating the chloramphenicol acetyltransferase activity driven by the alpha-fetoprotein promoter sequence. One of these regions, at about -2.5 kilobase pairs, contains a short indivisible 170-base pair DNA element that fulfills all the criteria of a tissue-specific enhancer, i.e. orientation and position independence, as well as cell-specific stimulation of gene expression driven by a homologous or heterologous promoter. The enhancing properties of this element are totally abolished by glucocorticoids. DNase I footprinting experiments indicate that several rat liver nuclear proteins interact with this enhancer element.
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PMID:Regulation of the rat alpha-fetoprotein gene expression in liver. Both the promoter region and an enhancer element are liver-specific and negatively modulated by dexamethasone. 168 47

We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli.
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PMID:c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells. 169 Aug 41

We have isolated the human angiotensinogen gene from a genomic library and determined the exon-intron junction sequences. The gene is 12 kilobases long and consists of five exons interrupted by four introns, as a single copy in the human genome. Of particular interest are the positions of the introns in the human angiotensinogen gene which are identical to those in the highly homologous human alpha 1-antitrypsin and alpha 1-antichymotrypsin genes, as well as rat and mouse angiotensinogen genes. Northern blot analysis showed that human hepatoma cells (HepG2) produce a large amount of angiotensinogen mRNA but not human glioma cells (T98G). To assay the promoter activity, the 1.3-kilobase genomic fragment containing the 5'-flanking region, first exon, and a part of first intron at positions -1222 to +44 was fused upstream to the chloramphenicol acetyltransferase gene, then transfected into HepG2 and T98G cells. The gene sequence was active only in HepG2 cells, suggesting the presence of a functional promoter. Analysis of deletion mutants demonstrated that the 76-base pairs region from -32 to +44 containing the TATA box and first exon is the minimal promoter, whose activity is as high as that of the SV40 enhancer-promoter. Since the basal expression of the human angiotensinogen gene is much higher in HepG2 than T98G cells, these results may reflect cell-specific differences in the gene transcription.
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PMID:Structure and expression of the human angiotensinogen gene. Identification of a unique and highly active promoter. 169 23

Rat T-kininogen (T-KG), a cysteine protease inhibitor, is an acute phase reactant which is induced to high levels in response to inflammation. Both hormones and cytokines participate in this regulation. To investigate the cis-acting elements responsible for the induction of gene expression, various 5'-fragments of the rat T-KG gene were fused to a chloramphenicol acetyltransferase marker gene. These constructs were transfected into a rat hepatoma cell line which was then treated with tumor necrosis factor or interleukin-6 or both cytokines. Expression of the chloramphenicol acetyltransferase gene was induced with interleukin-6 treatment, but suppressed by tumor necrosis factor. The 5'-region of the T-KG gene responsible for conferring both of these effects was localized between nucleotides -404 to -210 upstream of the transcription start site. Fragments containing this region were found to be effective in either orientation, and could also regulate a heterologous promoter.
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PMID:Differential regulation of rat T-kininogen by tumor necrosis factor and interleukin-6. 170

Serum amyloid A (SAA) is a major acute-phase protein whose chronic production by the liver can lead to the fatal disorder of secondary amyloidosis. Control of SAA is mediated by several inflammatory cytokines, including interleukin 1 (IL-1). To study the cis-acting regulatory elements responsible for constitutive and IL-1-induced expression, DNA constructs containing varying lengths of the promoter region from the human SAA2 beta gene 5' to the bacterial reporter gene, chloramphenicol acetyltransferase (CAT), were generated and transfected into human hepatoma cells, HepG2. Both positive and negative regulatory elements were found in the 5' flanking region of the human SAA2 beta gene. The more proximal region contains an IL-1 enhancer sequence GGGACTTTCC (SAA kappa B1; between -82 and -91), the binding site for the ubiquitous transcription factor NF-kappa B. IL-1 induction of the binding of nuclear factor to this sequence is maximal between 5 min and 30 min after incubation with IL-1 and negative in cells incubated for 60 min or longer. Mutation of the SAA kappa B1 sequence to a nonbinding form of NF-kappa B (CTCACTTTCC) abolishes the IL-1 effect. The SAA 5' region also contained an upstream repressor element, shown by transfection experiments. Within this element, a second NF-kappa B binding site (SAA kappa B2; -626 to -635) was found, and mutation of SAA kappa B2 to a non-NF-kappa B-binding form results in an increase in both constitutive + IL-1 stimulated SAA transcription.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Constitutive and NF-kappa B-like proteins in the regulation of the serum amyloid A gene by interleukin 1. 175 75

We have demonstrated that synthetic oligonucleotide representing glucocorticoid responsive element (GRE I) of MMTV inserted into the enhancerless early promoter of SV40 in p delta SVE-CAT expression vector, enhances transient expression of chloramphenicol acetyltransferase gene in HeLa and hepatoma cells cultivated in the presence of dexamethasone. The following changes in the structure of the core sequences (GTTACAAACTGTTCT) of the synthesized GRE eliminated its enhancing ability: i, changes in the left end of the core sequences from GTTACAAAATGTTCT to TCTTCAAACTGTTCT or to TACTCAAACTGTTCT; ii, the increase of gap between TGTTCT and the inverted repeat of this sequence. The above changes did not eliminate specific binding of glucocorticoid receptor to the synthetic oligonucleotides studied.
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PMID:Introduction of the glucocorticoid binding sequences into the expression vector p delta SVE-CAT and its effect on the CAT gene expression in mammalian cells. 179 99

Numerous physiological agents and conditions modulate cellular insulin sensitivity by downregulating or upregulating total cellular insulin receptors. In this study, we examined the effects of replacing complete medium in the absence or presence of insulin on the regulation of insulin-receptor gene expression in cultured human hepatoma cells (HepG2). Failure to replace complete medium resulted in growth arrest of HepG2 cells and a six- to sevenfold increase in insulin-receptor mRNA due to the prolongation of insulin-receptor mRNA half-life. Northern analysis revealed multiple insulin-receptor mRNA species; the largest species (11 kilobases) was disproportionately increased in growth-arrested cells. High concentrations of insulin (500 ng/ml) induced a 33.8% decrease in the abundance of insulin-receptor mRNA (n = 14). At lower concentrations, a trend of inhibition was observed but was not statistically significant. Insulin (500 ng/ml) did not affect insulin-receptor mRNA stability. The effect of conditioned media, insulin, and dexamethasone on insulin-receptor promoter activity was also examined. Various constructs of the 5'-flanking region of the insulin-receptor gene were attached immediately upstream to a chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into HepG2 cells via a pBR322-derived plasmid (pCAT). In cells replaced with complete medium, 12 and 118% of the promoter activity was contained within 578 and 877 base pairs, respectively, from the major translational initiation site. Conditioned media from growth-arrested cells in culture for 7 days increased promoter activity approximately twofold in 48 h. However, this increase failed to localize to any specific region on the insulin-receptor promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of media conditions, insulin, and dexamethasone on insulin-receptor mRNA and promoter activity in HepG2 cells. 184 49

The gene encoding rat kallikrein-binding protein (RKBP), a serine protease inhibitor, has been isolated and analyzed with the aid of the polymerase chain reaction. The gene is approximately 10 kilobases in length with four introns of approximately 2.2, 1.8, 0.9, and 0.84 kilobases. This gene is composed of five exons and encodes a polypeptide of 416 amino acid residues. The reactive center region of RKBP is encoded by the fifth exon with the putative P1-P1' residues being Lys-Ser. The organization of the RKBP gene is homologous to those of human alpha 1-antitrypsin and alpha 1-antichymotrypsin in size and arrangement of exons and introns, suggesting that they belong to the same subgroup of serpins. In the 5'-flanking region of the RKBP gene, a variant TATA box sequence, ATAAATA, is found 20 base pairs upstream from the transcription initiation site. The 5'-flanking region of the RKBP gene was able to direct transcription of the reporter gene chloramphenicol acetyltransferase when transfected into a rat hepatoma cell line. An internal promoter-like region was found in the first intron of the RKBP gene, downstream from the transcription initiation site and upstream from the translation initiation codon, however, it was unable to direct expression of the chloramphenicol acetyltransferase reporter gene in our experiments. The expression of RKBP in rat liver was induced by sex hormone treatment as indicated by dot-blot analysis. A genomic Southern blot using an RKBP cDNA probe revealed multiple bands suggesting that the RKBP gene belongs to a family of highly conserved genes.
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PMID:Molecular cloning and analysis of the rat kallikrein-binding protein gene. 187 45


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