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)

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.
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PMID:Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. 132 38

The gene for ornithine transcarbamylase (OTC; EC 2.1.3.3), a urea cycle enzyme, is expressed almost exclusively in the liver and small intestine. To identify DNA elements regulating transcription of the OTC gene in the liver, transient expression analysis was carried out by using hepatoma (HepG2) and nonhepatic (CHO) cell lines. The 1.3-kilobase 5'-flanking region of the rat OTC gene directed expression of the fused chloramphenicol acetyltransferase gene in HepG2 cells much more efficiently than in CHO cells. Analysis of deletion mutants of the 5'-flanking region in HepG2 cells revealed that there are at least one negative and two positive regulatory elements within the about 220-base-pair immediate 5'-flanking region. DNase I footprint analysis showed the presence of factors binding to these regulatory elements in nuclear extracts of rat liver and brain, and footprint profiles at the two positive elements exhibited liver-specific features. Transient expression analysis also revealed the existence of an enhancer region located 11 kilobases upstream of the transcription start site. The OTC enhancer was able to activate both its own and heterologous promoters in HepG2 but not in CHO cells. The enhancer was delimited to an about 230-base-pair region, and footprint analysis of this region revealed four protected areas. Footprint profiles at two of the four areas exhibited liver-specific features, and gel shift competition analysis showed that a factor(s) binding to the two liver-specific sites is related to C/EBP. These results suggest that both liver-specific promoter and enhancer elements regulate expression of the OTC gene through interaction with liver-specific factors binding to these elements.
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PMID:Promoter and 11-kilobase upstream enhancer elements responsible for hepatoma cell-specific expression of the rat ornithine transcarbamylase gene. 230 62

We describe a simple, rapid yet extremely sensitive assay for chloramphenicol acetyltransferase (CAT) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of CAT activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (CoA) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used CAT assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product.
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PMID:A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells. 275 94

Ornithine transcarbamylase (OTCase) is a mitochondrial matrix enzyme that catalyzes the 2nd step in the mammalian urea cycle. The gene encoding OTCase is located on the X chromosome and expression of OTCase is limited almost exclusively to hepatocytes. We have characterized a lambda phage recombinant, isolated from a mouse genomic library, that spans the first two exons of the mouse OTCase gene. Nuclease S1 mapping and primer extension analysis of this clone allowed us to determine that the transcription start site is 136 base pairs (bp) upstream from the translation initiation codon. Two TATA-like sequences were found 25 and 153 bp from the transcription initiation point. An 800-bp fragment containing the 5' flanking region of the OTCase gene was fused upstream to the coding sequence of the chloramphenicol acetyltransferase gene to assay promoter activity. This plasmid was introduced into mouse fibroblast NIH 3T3 cells and human hepatoma Hep G2 cells by the calcium phosphate co-precipitation method. After DNA transfection chloramphenicol acetyltransferase activity was observed only in Hep G2 cells. We conclude that this 800-bp fragment contains sufficient information to control OTCase gene expression in a tissue-specific manner, probably by interacting with trans-acting factor(s) which are not present in the other cell line.
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PMID:The 5' flanking region of the ornithine transcarbamylase gene contains DNA sequences regulating tissue-specific expression. 301 88

The control of the expression of the pilin gene (pilE) in Neisseria gonorrhoeae under a wide variety of growth conditions has been studied. The expression of pilE was measured using transcriptional fusions between pilE and the gene encoding chloramphenicol acetyltransferase (CAT), and the level of pilin production was measured by Western blot analysis. Many of the conditions tested affected both growth rate and pilin gene expression (e.g. isoleucine, high osmolarity, high temperature, anaerobic growth, pH 6, urea and iron depletion). Changes in the level of many other proteins were also observed, depending on the conditions, indicating that gonococci undergo an adaptive response to environmental variations. Moreover, environment-induced changes in the level of many proteins, including pilin, seem to involve the pilA/pilB regulatory system, which has been previously proposed to modulate the expression of the gonococcal pilin gene.
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PMID:Control of Neisseria gonorrhoeae pilin gene expression by environmental factors: involvement of the pilA/pilB regulatory genes. 916 25

The Drosophila proliferating cell nuclear antigen (PCNA) gene promoter contains at least three transcriptional regulatory elements, the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. In nuclear extracts of Drosophila Kc cells, we detected a novel protein factor(s), CFDD (common regulatory factor for DNA replication and DREF genes) that appeared to recognize two unique nucleotide sequences (5'-CGATA and 5'-CAATCA) and bind to three sites in the PCNA gene promoter. These sites were located at positions -84 to -77 (site 1), -100 to -93 (site 2) and -134 to -127 (site 3) with respect to the transcription initiation sites. Sites 2 and 3 overlapped with DRE and URE, respectively, and the 5'-CGATA matched with the reported recognition sequence of BEAF-32 (boundary element-associated factor of 32 kDa). Detailed analyses of CFDD recognition sequences and experiments with specific antibodies to DREF (DRE-binding factor) and BEAF-32 suggest that CFDD is different from DREF, UREF (URE-binding factor) and BEAF-32. A UV cross-linking experiment revealed that polypeptides of approximately 76 kDa in the nuclear extract interact directly with the CFDD site 1 sequence. Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying mutations in CFDD site 1 and examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying mutations in site 1, introduced into flies by germ line transformation, revealed that CFDD site 1 plays an important role for the promoter activity both in cultured cells and in living flies. In addition to the PCNA gene, multiple CFDD sites were found in promoters of the DNA polymerase alpha and DREF genes, and CFDD binding to the DREF promoter was confirmed. Therefore, CFDD may play important roles in regulation of Drosophila DNA replication-related genes.
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PMID:Identification of CFDD (common regulatory factor for DNA replication and DREF genes) and role of its binding site in regulation of the proliferating cell nuclear antigen gene promoter. 927 47

Production of the glycoprotein hormone alpha-subunit (GPH alpha) was enhanced by sodium butyrate (Btr) in HeLa cells. Induction of the HeLa alpha-subunit gene by Btr was inhibited by the simultaneous addition of cycloheximide (CHX), indicating a requirement for continued protein synthesis. Transient expression assays using plasmids containing the GPH alpha gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that the GPH alpha promoter is inducible by Btr in HeLa cells, and this induction could be prevented by 2-deoxyglucose (dGlc). CAT production driven by the SV-40 early promoter, the cytochrome P-450-IA1 promoter, and the Rous sarcoma virus long terminal repeat was also enhanced by Btr, but the augmented synthesis was not inhibited by the addition of dGlc, demonstrating that the effect is restricted only to some promoters. CAT synthesis could be induced by Btr when the GPH alpha promoter extended upstream to position -169 (relative to the transcription start site at +1) but not when the promoter terminated at -150, classifying the DNA between these termini as a Btr-responsive element (BRE). This region overlaps the composite trophoblast-specific enhancer. Inactivation of enhancer subdomains by site-directed mutagenesis confirmed the deletion analysis and ranked their response to Btr as CRE < TSE < URE < alpha ACT. Electrophoretic mobility shift analysis failed to detect any significant difference among several enhancer binding proteins in nuclear extracts from untreated and Btr-treated cells. Together, these results suggest that Btr-mediated induction of the alpha-subunit gene in HeLa cells is manifest either through the synthesis of a new transcription factor(s), which is inhibited by CHX but required for increased transcription from the GPH alpha gene promoter, or through the activity of existing factors that may require glycosylation or phosphorylation by a modification system that is inducible by Btr and inhibited by dGlc and CHX. These results further suggest that the factor is not an enhancer-binding protein or that Btr increases its transactivation potential without altering its DNA-binding activity.
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PMID:Sodium butyrate-mediated induction of the glycoprotein hormone alpha-subunit gene: requirement for continued protein synthesis, identification of a butyrate-responsive element, and inhibition of promoter activation by 2-deoxyglucose. 1040 94

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.
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PMID:Regulation of urease gene expression by Streptococcus salivarius growing in biofilms. 1122 Mar 3

MBD2 is the only member of a family of methyl-CpG-binding proteins that has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). To understand the apparently contradictory function of MBD2/dMTase, we studied the effects of dMTase overexpression on the activity of various in vitro methylated promoters transiently transfected into HEK293 cells. We found that forced expression of a MBD2/dMTase expression vector (His-dMTase) differentially activated two methylated reporters, pSV40-CAT (the SV40 enhancerless promoter adjacent to the chloramphenicol acetyltransferase (CAT) reporter gene) and pGL2T+I4xTBRE (a region of the p21 promoter next to the luciferase reporter gene), in a time- and dose-dependent manner. His-dMTase increased pSV40-CAT expression by 3-10-fold after 96 h, while pGL2T+I4xTBRE expression was increased by 2-3-fold after only 48 h and did not further increase at 96 h. Gene activation was not universal because no effect was seen with the p19-ARF promoter. We then assessed whether activation might be due to demethylation within the promoter region. Using bisulfite mapping, we found that exogenous expression of His-dMTase induced demethylation at 8 of the 10 CpG sites within the SV40 promoter. The observation that His-dMTase increases the demethylase activity in the cells was also confirmed using an in vitro CpG demethylase assay with a mC32pG oligonucleotide substrate and purified Q-Sepharose fractions from HEK293 cells transfected with His-dMTase or empty pcDNA3.1His vector. We propose that a single protein possessing both repressor and demethylase functions has evolved to coordinate a program that requires suppression of some methylated genes and activation of others.
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PMID:Promoter-specific activation and demethylation by MBD2/demethylase. 1217 48

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.
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PMID:Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution. 1606 32


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