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)

It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cold-induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene. 136 May 41

The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA. This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion.
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PMID:Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS. 196 61

Human DNA-binding proteins, dbpA and dbpB (YB-1), are members of a protein family containing a cold-shock domain, and are regarded as transcriptional regulators. Here, we isolated genomic fragments of these genes and characterized their transcriptional regulation. Analysis of lambda phage genomic clones revealed that the dbpA gene consists of 10 exons spanning a 24-kb genomic region. The cold-shock domain, composed of about 70 amino acid residues, is encoded separately by exons 2-5. The exon 6, encoding 69 amino acid residues, was found to be an alternative exon. Northern-blot analysis showed that both genes were highly expressed in skeletal muscle and heart compared with in other tissues. The dbpA gene contains no typical TATA box or CAAT box at the immediate 5' region, but a sequence similar to an initiator consensus sequence was revealed at a major transcription-start site. A transient expression assay using the chloramphenicol acetyltransferase reporter gene revealed that the sequence located at positions -17 to +70 relative to the major transcription-start site was critical for promoter function. Within this region, the consensus sequence for serum-response element, CC(A/T)6GG, is present at positions -13 to -4 in addition to the initiator sequence. Immunofluorescence showed the cellular localization of dbpA to be both in the cytoplasm and nucleus, particularly at the perinuclear region. In situ hybridization demonstrated the localization of the dbpA gene on chromosome 12 band p13.1, whereas dbpB-(YB-1)-related genes were dispersed on many chromosomes with strongest hybridization signals on chromosome 1. All 16 dbpB (YB-1) clones, isolated from the same genomic library used for dbpA genomic cloning, were processed genes because of their intronless structures and multiple mutations. One of these processed genes possesses an open reading frame, which encodes most of the amino acid residues of dbpB (YB-1). These results indicate that dbpA and dbpB (YB-1) genes evolved in different fashions after deviation from a common ancestral gene.
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PMID:Characterization of the gene for dbpA, a family member of the nucleic-acid-binding proteins containing a cold-shock domain. 762 87

Mouse calbindin-D28k expression is regulated in vivo by estradiol in ovaries, uterus, and oviduct. To determine whether estrogen can have an effect on the transcription of the calbindin-D28k gene, the human breast cancer cells T47D were transiently transfected with a plasmid containing a 1.1 kilobase (kb) PstI/SacII fragment (-1075/+34) of the mouse calbindin gene ligated to the chloramphenicol acetyltransferase (CAT) gene and cotransfected with human estrogen receptor expression vector. T47D cells, transfected and treated with estradiol (10(-11) - 10(-7) M for 64-65 h), exhibited a dose-dependent increase in CAT activity (up to 6.2-fold). Transfection of MCF-7 breast cancer cells with the chimeric gene construct alone also resulted in an estradiol-dependent induction in CAT activity. Deletion mutant analysis demonstrated that there are two regions of the mouse calbindin-D28k promoter (between -1075/-702 and between -175/-78) that contribute to the induction by estradiol. These fragments, when linked to the thymidine kinase promoter to construct a heterologous promoter chimera, were able to convert the thymidine kinase promoter to estrogen responsiveness. In these regions there are multiple imperfect half-palindromic estrogen-responsive elements. Gel retardation assays demonstrated weak protein-DNA interactions that were competed with cold oligonucleotide containing the vitellogenin estrogen-response element. These findings indicate that the mouse calbindin-D28k promoter is capable of conferring estrogen responsiveness, which may be mediated by several imperfect half-palindromic estrogen-responsive elements, and suggest, in light of previous studies concerning 1,25-dihydroxyvitamin D regulation, multiple steroid regulation of the calbindin-D28k gene.
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PMID:Regulation by estrogen through the 5'-flanking region of the mouse calbindin-D28k gene. 777 78

Uncoupling protein (UCP) gene expression is tightly restricted to thermogenic brown adipocytes and is rapidly activated by norepinephrine released after cold exposure. To identify cis-acting regulatory elements controlling this gene, a region encompassing 4.5 kilobases of DNA upstream of the transcription start site was analyzed using hybrid UCP-chloramphenicol acetyltransferase reporter gene constructs. Evidence for the presence of both tissue-specific and beta-adrenergic response elements in this 4.5-kilobase region was obtained by comparing the expression of these reporter genes in transfected brown adipocytes (in vitro differentiated), brown preadipocytes, white adipocytes, and Chinese hamster ovary (CHO) cells and from experiments in transgenic animals. Deletion analyses in transfected cells indicated that the minimal region exhibiting promoter activity and tissue specificity is located between -157 and -57 base pairs (bp). A 211-bp activator element located between -2494 and -2283 bp was necessary for full expression in brown adipocytes. This element also activated expression of the homologous -157-bp promoter and expression of a heterologous promoter in both brown adipocytes and CHO cells. A second region, downstream of the activator and possibly located between positions -400 and -157 bp, inhibited the UCP promoter in CHO cells. In mice transgenic for a chloramphenicol acetyltransferase reporter gene containing these elements, expression was both tissue specific and regulatable by environmental temperature changes. These results indicate that both positive and negative cis-acting elements participate in the regulation of UCP gene expression.
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PMID:Tissue-specific and beta-adrenergic regulation of the mitochondrial uncoupling protein gene: control by cis-acting elements in the 5'-flanking region. 838 95

Transgenic mice were generated with a transgene containing the 211-base pair (bp) enhancer and 0.4 kilobase pairs of 5'-flanking DNA of the uncoupling protein (ucp) gene. Expression of this transgene was restricted to brown adipose tissue and was inducible by cold exposure or treatment of transgenic mice by norepinephrine, retinoic acid (RA), or CL-316,243 beta3-adrenoreceptor agonist. A search for retinoic acid response elements in the ucp gene enhancer was undertaken using mutagenesis and transfection of cultured cells with chloramphenicol acetyltransferase constructs. Deletion or mutations of several putative retinoic acid response elements were ineffective. Mutations of a TGAATCA region dramatically decreased the transcriptional activity in the presence of RA. In vitro this region was able to bind a complex containing proteins recognized by antibodies against Jun or Fos. Mutations of an adjacent region related to an inverted repeat of type 2 also markedly decreased RA effect. This region was able to bind in vitro retinoid X receptor alpha and retinoic acid receptor beta. The two regions form an activating region between bp -2421 and -2402 (referred to as the ucp gene-activating region), which has an enhancer activity but cannot confer RA response to a promoter. This response was obtained with a larger DNA fragment (bp -2489 to -2398) constituting a complex RA response domain.
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PMID:Essential cis-acting elements in rat uncoupling protein gene are in an enhancer containing a complex retinoic acid response domain. 894 Jan 69

Prolonged stress is associated with the induction of tyrosine hydroxylase (TH) gene expression in rat adrenal medulla. We have used transgenic mice expressing a transgene encoding 4.5 kb of rat TH gene 5' flanking region fused upstream of the structural gene encoding chloramphenicol acetyltransferase (CAT) to test whether cold exposure or immobilization stress regulates TH gene expression in mouse adrenal gland. Exposure of mice to cold for 3 days increases adrenal TH protein and enzymatic activity. Cold exposure also increases adrenal TH-CAT expression two- to threefold. Immobilization stress induces mouse adrenal TH-CAT expression after either one immobilization or multiple immobilizations. TH-CAT expression increases transiently after a single immobilization, but after multiple immobilizations the induction of TH-CAT is sustained for at least 24 h. TH protein and TH enzymatic activity in mouse adrenal gland are elevated 2.8-fold and 1.5-fold, respectively, after seven immobilizations, but are not increased after either one, two, or three immobilizations. These results indicate that cold exposure and immobilization stress induce adrenal TH gene expression at least partially by stimulating the transcription rate of the TH gene. Furthermore, as observed in the rat, multiple mechanisms apparently regulate adrenal TH gene transcription rate and TH enzyme induction depending on whether mice are subjected to a single immobilization or multiple immobilizations. Our results indicate that these transgenic mice are an excellent model system to study the molecular mechanisms regulating TH gene expression in adrenal medulla.
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PMID:Induction of tyrosine hydroxylase protein and a transgene containing tyrosine hydroxylase 5' flanking sequences by stress in mouse adrenal gland. 904 52

The uncoupling protein-1 gene is uniquely expressed in brown adipose tissue (BAT) and is positively regulated by cold exposure of animals and the sympathetic nervous system. To analyse the importance of a previously identified 211-bp enhancer [Cassard-Doulcier, Gelly, Fox, Schrementi, Raimbault, Klaus, Forest, Bouillaud and Ricquier (1993) Mol. Endocrinol. 7, 497-506] in the tissue-specific expression of this gene, transgenic mice were generated using the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. One out of fourteen lines of the control transgenic mice bearing the Herpes simplex thymidine kinase (TK) promoter expressed weakly the CAT reporter gene in several tissues, whereas the other lines did not express CAT. Eight founders bearing the 211-bp enhancer-TK transgene were obtained. In six lines, no expression of CAT was detected. In one line, the expression of CAT was restricted to BAT. In another line, the expression of CAT was found in BAT and, to a lesser extent, in testis. Moreover, in these lines a marked and specific increase in the expression of the reporter gene in BAT was observed either after exposure of mice to the cold or by treating them with a beta-adrenoceptor agonist drug. These results demonstrate that the 211-bp enhancer alone is sufficient to both direct and restrict expression to BAT. This enhancer also mediates the transcriptional response of the gene to beta-adrenergic stimulation, although it does not contain conserved cAMP response element.
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PMID:A 211-bp enhancer of the rat uncoupling protein-1 (UCP-1) gene controls specific and regulated expression in brown adipose tissue. 965 61

1 ,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] negatively regulates expression of the avian PTH (aPTH) gene transcript, and a vitamin D response element (VDRE) near the promoter of the aPTH gene had previously been identified. The present report assessed whether the negative activity imparted by the aPTH VDRE could be converted to a positive transcriptional response through selective mutations introduced into the element. The tested sequences were derived from individual and combined mutations to 2 bp in the 3'-half of the direct repeat element, GGGTCAggaGGGTGT. Cold competition experiments using mutant and wild-type oligonucleotides in the mobility shift assay revealed minor differences in the ability of any of these sequences to compete for binding to a heterodimer complex comprised of recombinant proteins. Ethylation interference footprint analysis for each of the mutants produced unique patterns over the 3'-half-sites that were distinct from the weak, wild-type footprint. Transcriptional outcomes evaluated from a chloramphenicol acetyltransferase reporter construct utilizing the aPTH promoter found that the individual T-->A mutant produced an attenuated negative transcriptional response while the G-->C mutant resulted in a reproducibly weak positive transcriptional outcome. The double mutant, however, yielded a 4-fold increase in transcription, similar to the 7-fold increase observed from an analogous construct using the human osteocalcin VDRE. UV light crosslinking to gapped oligonucleotides assessed the polarity of heterodimer binding to the wild-type and double mutant sequences and was consistent with the vitamin D receptor preferentially binding to the 5'-half of both elements. Finally, DNA affinity chromatography was used to immobilize heterodimer complexes bound to the wild-type and double mutant sequences as bait to identify proteins that may preferentially interact with these DNA-bound heterodimers. This analysis revealed the presence of a p160 protein that specifically interacted with the heterodimer bound to the wild-type VDRE, but was absent from complexes bound to response elements associated with positive transcriptional activity. Thus, the sequence of the individual VDRE appears to play an active role in dictating transcriptional responses that may be mediated by altering the ability of a vitamin D receptor heterodimer to interact with accessory factor proteins.
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PMID:Turning a negative into a positive: vitamin D receptor interactions with the avian parathyroid hormone response element. 1007 2

Cold shock proteins (CSPs) form a family of highly conserved bacterial proteins capable of single-stranded nucleic acid binding. They are suggested to act as RNA chaperones during cold shock inhibiting the formation of RNA secondary structures, which are unfavourable for transcription and translation. To test this commonly accepted theory, isolated CSPs from a mesophilic, thermophilic and a hyperthermophilic bacterium (Bacillus subtilis, Bacillus caldolyticus and Thermotoga maritima) were studied in an Escherichia coli based cell free expression system on their capability of enhancing protein expression by reduction of mRNA secondary structures. The E. coli based expression of chloramphenicol acetyltransferase and of H-Ras served as model systems. We observed a concentration-dependent suppression of transcription and translation by the different CSPs which makes the considered addition of CSPs for enhancing the protein expression in in vitro translation systems obsolete. Protein expression was completely inhibited at CSP concentrations present under cold shock conditions. The CSP concentrations necessary for 50% inhibition were lowest (140 microm) for the protein of the hyperthermophilic and increased when the thermophilic (215 microm) or even the mesophilic protein (451 microm) was used. Isolated in vitro transcription under the influence of CSPs showed that the transcriptory effect is independent from the rest of the cell. It could be shown in a control experiment that the inhibition of protein expression can be removed by addition of hepta-2'-desoxy-thymidylate (dT7); a heptanucleotide that competitively binds to CSP. The data are in line with a hypothesis that CSPs act on bulk protein expression not as RNA chaperones but inhibit their transcription and translation by rather unspecific nucleic acid binding.
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PMID:The influence of cold shock proteins on transcription and translation studied in cell-free model systems. 1615 90


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