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)

DNA sequences required for expression of the mouse cytochrome c oxidase subunit IV (COXIV) promoter were identified by transient expression of recombinant COXIV-chloramphenicol acetyltransferase constructs in COS and NIH-3T3 cells. Activity of the COXIV promoter is shown to depend upon upstream Sp1 binding sequences and two tandemly repeated 21-base pair sequence elements each mapping to sites of mRNA initiation. Each initiation region repeat contains a binding site for an ets-related transcription factor which demonstrates specificity for the characteristic GGAA ets sequence motif and reactivity with an ets domain-directed monoclonal pan ets antibody. The two 21-base pair repeats are sufficient for transcriptional activity suggesting that the ets-related factor may be involved in both transcriptional activation and start site positioning. The ets-related protein found in COS nuclear extracts is shown to be identical or closely related to the GA-binding protein (GABP) by comparison of electrophoretic mobilities and immunological reactivities of DNA-protein complexes formed with purified recombinant expressed GABP alpha and beta subunits. Sp1 and the GABP-related factors also bind to another mouse cytochrome oxidase subunit gene COXVb. The similar promoter features of these two genes suggests a possible means of coordinate transcriptional regulation among such respiratory proteins.
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PMID:The basal promoter elements of murine cytochrome c oxidase subunit IV gene consist of tandemly duplicated ets motifs that bind to GABP-related transcription factors. 133 Oct 86

We have investigated the functional elements involved in cAMP-stimulated transcription of the human ferredoxin gene. Unlike the bovine gene, the human gene lacked a second upstream RNA initiation site as demonstrated by sequence analysis of the exon boundary, lack of upstream RNA, and analysis of the promoter. The presence of a single promoter was determined by testing the ability of various gene segments to drive the expression of the chloramphenicol acetyltransferase gene after transfection into a mouse adrenal cell line Y1. Full promoter activity was conferred by a DNA fragment spanning -209 to +55, although the -94 to +55 fragment already provided some promoter activity. Transcription from the -94 to +55 segment was stimulated by 2-fold when 8-bromo-cAMP was added to the cell. Footprinting analyses showed two GC boxes at -50 to -70 and -87 to -108 were protected by proteins from both Y1 and HeLa cells. Competition experiments showed that a protein with a recognition sequence indistinguishable from Sp1 bound to these sites. When connected to a heterologous TATA box, the sequence at -76 to -42, which contained the proximal GC box, was able to confer a high level of basal transcription and cAMP stimulation. This sequence does not show sequence homology with the known cAMP-responsive element. Mutations or deletion of the Sp 1-binding site showed diminished basal transcription and defined the cAMP responsive sequence to be from -76 to -62. Therefore the cAMP-responsive sequence of the human ferredoxin gene was located at -76 to -62, which was adjacent to the Sp 1-binding site.
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PMID:Transcription of the human ferredoxin gene through a single promoter which contains the 3',5'-cyclic adenosine monophosphate-responsive sequence and Sp 1-binding site. 133 72

Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. DNA sequence analysis of the human aromatase gene has revealed that a putative promoter sequence exists immediately up-stream of the second exon. Chloramphenicol acetyltransferase functional analyses of cells transfected with chloramphenicol acetyltransferase expression plasmids containing various DNA fragments derived from the 3'-end of the first intron of the aromatase gene were performed to show that a promoter indeed exists in this region. However, in all of the cell lines used in this study, MCF-7, JAR, OVCAR-3, and skin fibroblast, the function of this promoter was inhibited by a negative regulatory element situated up-stream from the promoter. The results further suggest that this inhibitory element behaves as a silencer element, in that it could inhibit a simian virus-40 promoter from a distance of several kilobases. This negative element worked in both orientations and inhibited the functions of several promoters, including the newly identified promoter situated in the 3'-end of the first intron of the human aromatase gene. Primer extension analysis has been performed to determine the potential transcription start site. The mechanism of the regulation of aromatase expression is known to be very complex. The presence of a promoter and a silencer at the 3'-end of the first intron may represent one additional way that aromatase expression is controlled in estrogen-producing cells.
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PMID:Identification of a promoter and a silencer at the 3'-end of the first intron of the human aromatase gene. 133 79

Dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4) is the common component of the three mammalian alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. To study regulation of E3 gene expression, a 12-kilobase clone from a human leukocyte genomic library was isolated, and a 1.8-kilobase fragment containing part of the first intron, the first exon, and 1.5 kilobases of the 5' flanking region of the E3 gene was sequenced. The nucleotide sequence of the E3 promoter region revealed consensus sequences for several DNA binding proteins but no apparent TATA box or Sp1 sites. Although the 1.6-kilobase 5' flanking region has a low percentage of G+C (44%), the nucleotide sequence between +1 and -150 base pairs has a G+C content of 67%. Primer extension analysis showed a major transcriptional start site located 95 nucleotides upstream from the translation initiation codon. A series of 5' deletions from the E3 promoter-regulatory region were ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting constructs were transfected into HepG2 cells. The longest E3 promoter-CAT construct had a relatively high level of CAT enzyme activity, and deletion of a promoter element between -769 and -1223 base pairs resulted in a 3-fold increase in reporter gene expression. These results suggest that the human E3 promoter has characteristics of housekeeping and facultative promoters and that a negative regulatory element is present between 769 and 1223 base pairs upstream from the transcription start site.
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PMID:Characterization of the transcriptional regulatory region of the human dihydrolipoamide dehydrogenase gene. 133 63

Transcription of interleukin-6 (IL-6) gene in human HepG2 and HeLa cells was induced by treatment with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), phorbol 12-myristate 13-acetate, or dibutyryl cyclic AMP. These agents enhanced the expression of chloramphenicol acetyltransferase (CAT) activity in cells transfected with chimeric CAT genes driven by the transcriptional regulatory regions of human IL-6 gene. Both induced and basal levels of CAT expression were severely repressed upon co-transfection of expression vectors encoding the adenoviral E1A289R or E1A243R protein. The conserved region 1 of E1A proteins was required for this activity. IL-6-CAT expression could also be induced by co-transfecting expression vectors containing cDNAs of the catalytic subunit of protein kinase A or c-jun. E1A repressed transcriptional induction by these agents as well. Similar inhibition was observed when a CAT gene driven by the NF kappa B element of the IL-6 gene was used as a reporter plasmid. In a cell line stably transfected with the E1A gene, IL-1 or TNF-alpha failed to induce IL-6 mRNA. Electrophoretic mobility shift assays were carried out with nuclear extracts of these cells using, as probes, the NF kappa B element or the multiple regulatory element of the IL-6 gene. With either probe, additional faster migrating DNA-protein complexes were formed in the extracts of E1A-expressing cells as compared with the extracts of the corresponding control cells. Experiments with NF kappa B antibody revealed differences between the different DNA-protein complexes formed in the extract of E1A-expressing cells. These observations suggest that E1A represses IL-6 gene transcription by interfering with the formation of appropriate DNA-protein complexes.
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PMID:Transcriptional repression of interleukin-6 gene by adenoviral E1A proteins. 133 71

The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60% reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40% reduction in TSH-stimulated thymidine incorporation, a 31% increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells tranfected with wild type CREB.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:3',5'-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line. 133 55

The goal of this study was to determine whether alpha 2(1) procollagen gene expression is modulated by positive or negative trans-acting DNA-binding proteins. Previous studies have shown that a clone of SV40-transformed WI-38 fibroblasts (SVWI-38) does not produce any alpha 2(1) procollagen mRNA (Parker et al (1989), J. Biol Chem. 264, 7147-7152). In order to elucidate the mechanism(s) responsible for such inactivation, we have examined the activity of a transfected wild type COL1A2 promoter in SVWI-38 cells. A set of 5' promoter deletions was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into SVWI-38 and other cell lines expression type I collagen. The resulting CAT assays confirmed the importance of several upstream regions for promoter activity and documented the decreased transcriptional activity from an exogenous COL1A2 promoter in the SVWI-38 cell line. Competition experiments with an excess of COL1A2 promoter DNA fragment and a constant amount of COL1A2/CAT construct displayed a linear relationship between excess COL1A2 fragment and CAT activity in SVWI-38 cells, suggesting the involvement of a titratable negative effector. Electrophoretic mobility shift assays revealed the presence of a specific DNA-protein complex which was present in SVWI-38 cells and almost absent in control fibroblasts. Methylation interference analysis mapped the region of binding of this factor between nucleotides -80 and -72, relative to the transcription start site. Thus the data presented provide strong evidence for the existence of a negative trans-acting factor that may play a role in the repression of COL1A2 expression in SVWI-38 fibroblasts.
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PMID:The abolition of collagen gene expression in SV40-transformed fibroblasts is associated with trans-acting factor switching. 133 88

We report the fish use of an exponential decay electroporation system to introduce foreign DNA into fertilized zebrafish embryos. The plasmid RSVCAT (Rous sarcoma viral promoter (RSV) upstream from the chloramphenicol acetyltransferase gene (CAT)) was linearized and introduced into fertile zebrafish embryos by electroporation no later than the four-cell stage. Conditions for the procedure were empirically derived, and 68% of the treated animals survived through hatching to at least 6 days after fertilization and well beyond. Dot-blot analysis on DNA extracted from individual hatching fry demonstrated that 65% of the animals tested carried the foreign construct. Enzyme assays on the soluble proteins of treated animals were positive for chloramphenicol acetyltransferase activity. These data demonstrate that the foreign construct was being transiently expressed in the developing tissues of the embryo. The simplicity of this technique will greatly enhance the ability to analyze gene promoter regulation in vivo in transgenic zebrafish. The ability of the electroporated DNA to integrate into the host genome and to generate stable lines of transgenic fish is discussed.
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PMID:Transient expression of RSVCAT in transgenic zebrafish made by electroporation. 133 27

The type 1 glucose transporter (GLUT1) gene encodes an integral membrane glycoprotein responsible for facilitating transfer of glucose across plasma membrane and is rapidly activated by serum, growth factors, and by oncogenic transformation. To elucidate the molecular mechanisms of regulation of GLUT1 gene expression, we isolated and characterized the mouse GLUT1 gene. DNA elements regulating transcription of the gene were analyzed in transient expression assays after transfection of NIH/3T3 cells with a low background chloramphenicol acetyltransferase (CAT) vector system pSVOOCAT. We identified two enhancer elements; the first one is located 2.7 kilobases upstream of the cap site of the gene which contains the homologous sequences with two 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs), a serum response element, a cyclic AMP-responsive element (CRE) and three GC boxes, and the second one is located in the second intron of the gene which contains the homologous sequences with two TREs and one CRE. With the promoter alone the transcription of the gene is activated by src, only slightly activated by ras and is not activated by serum and platelet-derived growth factor. When the gene is accompanied by one of these enhancers, the transcription is activated by all these stimuli.
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PMID:Identification of two enhancer elements in the gene encoding the type 1 glucose transporter from the mouse which are responsive to serum, growth factor, and oncogenes. 133 57

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.
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PMID:Construction and expression of nonsense suppressor tRNAs which function in plant cells. 134 92


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