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)

Autoregulation of the human thyroid hormone receptor beta 1 (hTR beta 1) promoter was assessed by chloramphenicol acetyltransferase and luciferase reporter assays of transient transfections into COS1 and GH3 cells, DNase I footprinting, and gel shift assays. A 5'-deletional analysis of the promoter showed that the region between -906 and -839 and the sequence from -438 to -130 were positively regulated by T3 in COS1 cells cotransfected with an hTR beta 1 expression vector. We also transfected deletion constructs into GH3 cells and showed similar effects of T3 on the trans-activation of the reporters. DNase I footprinting showed a protected inverted palindromic thyroid response element (TRE) at position -890 to -866 in the distal fragment and a direct repeat at position -190 to -166 in the proximal fragment, which were protected by TRs. Mutation of each TRE significantly decreased the trans-activation of the promoter by T3. Gel mobility shift assays showed both proximal and distal TREs formed a retarded band with hTR alpha 1 or hTR beta 1 expressed in COS1 cells and reticulocyte lysates. The bands formed on the distal TRE and the proximal TRE appear to be preferentially formed by a TR homodimer and a heterodimer, respectively. Furthermore, the band formed on the distal TRE disappeared after adding T3 but that on the proximal TRE did not. These results indicate that hTR beta 1 expression is directly regulated by hTR alpha 1, beta 1, and their ligand through two TREs. The different structure of the TREs in this promoter suggests their physiological role in transcriptional regulation may be different.
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PMID:Two thyroid hormone response elements are present in the promoter of human thyroid hormone receptor beta 1. 801 48

Differentiation of THP1 monocytes to a macrophage phenotype is accompanied by increased apolipoprotein E gene transcription. Using transfection analysis with 5' deletion mutations of the 5' control region of the apo E gene in THP1 cells, we show that the -651 to +86 chloramphenicol acetyltransferase (CAT) construct is efficiently expressed in the monocyte; as has been reported for other cell types. Further, we found that an 176 bp region between -623 to -447 was required for the induction of apolipoprotein E gene transcription during 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of monocytes to macrophages. Gel-retardation patterns of the apolipoprotein E promoter region using nuclear extracts from differentiated or undifferentiated THP1 cells revealed altered binding of Ap1-like nuclear factor/s to the -620 to -583 bp region after macrophage differentiation. Mutation of an Ap1 element at position -602 abolished specific binding of Ap1-like proteins to the -620 to -583 bp fragment of the apo E gene and significantly reduced expression of a -623 to +86 apo E-CAT construct during differentiation. These data indicate that differentiation-related expression of the apolipoprotein E gene following phorbol ester stimulation is transduced by gene elements between -623 and -447. Furthermore, the data indicate that transcriptional activation of the apo E gene during macrophage differentiation is associated with induction of Ap1-like proteins which bind to the Ap1 response element present at -602 in the apolipoprotein E gene and importantly contribute to enhanced gene expression.
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PMID:Evaluation of the role of Ap1-like proteins in the enhanced apolipoprotein E gene transcription accompanying phorbol ester induced macrophage differentiation. 801 31

The cytokine interleukin-8 (IL-8) is an important mediator of neutrophil, lymphocyte, and basophil chemotaxis and activation. Earlier we demonstrated that beta interferon (IFN-beta) can inhibit tumor necrosis factor (TNF)-induced IL-8 gene expression at the transcriptional level, apparently by a novel mechanism. To define the cis-acting elements and trans-acting factors involved in this inhibition, DNA constructs containing portions of the 5'-flanking region of the IL-8 gene were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into human diploid FS-4 fibroblasts. The region spanning positions -98 to +44 was sufficient to confer both inducibility by TNF and inhibition by simultaneous treatment with IFN-beta. Inhibition of TNF- or IL-1-induced CAT activity by IFN-beta or IFN-alpha was also observed when a DNA fragment containing only the NF-IL-6 and NF-kappa B sites (positions -94 to -70) was placed upstream of the homologous or a heterologous minimal promoter. A construct containing three copies of the NF-kappa B element in front of the CAT gene also was inducible by TNF, and this stimulatory effect too was inhibited by IFN-beta, indicating that the NF-kappa B element is sufficient to confer inhibition by IFN-beta. This inhibitory effect was specific for the NF-kappa B site of the IL-8 gene since it was less marked with constructs containing three copies of the NF-kappa B site from the HLA-B7 gene. Gel shift assays with a probe containing the NF-kappa B and NF-IL-6 binding sites of the IL-8 gene (positions -101 to -63) showed that IFN-beta treatment did not block the activation of NF-kappa B proteins or their ability to bind to the NF-kappa B site. However, nuclear extracts from cells treated with TNF in the presence of IFN-beta gave rise to an additional band that appears to contain protein components from the NF-kappa B and NF-IL-6 families. NF-kappa B site-mediated suppression of IL-8 gene expression by IFN-beta represents a hitherto unknown mechanism and target of IFN action.
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PMID:Transcriptional inhibition of the interleukin-8 gene by interferon is mediated by the NF-kappa B site. 803 8

To define the mechanisms of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transcription of the ornithine decarboxylase (ODC) gene, we isolated a genomic clone (hODC41B) of ODC from a human leukocyte genomic DNA library. The restriction endonuclease map, in comparison with the previously published sequences of the human ODC gene, indicated that hODC41B contained a 15.7-kb sequence that extended from the sixth exon to about 10 kb upstream of the ODC gene. A 2.5-kb genomic fragment containing the 5' flanking region and the first exon was subcloned and sequenced. Sequence analysis revealed multiple putative promoter/enhancer elements (a TATA box, a CAAT box, 17 GC boxes, and a cAMP-responsive element) but no consensus AP-1 sequences (TGAGTCA) in the 2.5-kb 5' flanking region. However, three AP-1 sequences were located in introns 3, 5, and 11. We constructed a series of chimeric genes containing part of the first exon and increasingly longer 5' flanking sequences of the ODC gene fused to either bacterial chloramphenicol acetyltransferase (CAT) or luciferase reporter genes. TPA inducibility was determined by transient transfection and measurement of CAT or luciferase expression in HeLa cells. The induction of CAT activity by TPA decreased with decreasing lengths of the 5' flanking sequences up to nt -82. The TPA induction from the construct -72 ODC CAT was threefold to sevenfold, and the TPA inducibility of the same fragment was about ninefold to 30-fold with the luciferase reporter gene. Further deletion analysis revealed TPA-responsive sequences in ODC nt -42 to +54. Gel mobility shift assays using alpha-32P-end labeled ODC nt -42 to +60 revealed that nt -42 to +60 specifically bound HeLa cell nuclear proteins. HeLa cell nuclear protein binding to ODC nt -42 to +60 could not be completely competed by AP-1-, AP-2-, AP-3-, or SP1-responsive sequences.
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PMID:Non-AP-1 tumor promoter 12-O-tetradecanoylphorbol-13-acetate-responsive sequences in the human ornithine decarboxylase gene. 804 98

The androgen receptor (AR) mediates the biological functions of androgens and is essential for normal growth and differentiation of urogenital organs as well as initiation and maintenance of spermatogenesis. Withdrawal of androgens by castration or other methods has been shown to cause a marked, although often temporary, regression of many prostate cancers. In order to gain a better understanding of the transcriptional regulation of the AR, a series of truncation mutants derived from the 5'-region of the mouse AR (mAR) were inserted into the promoter-less plasmid pBLCAT3 and transiently expressed in the mouse alpha T3-1 and GT1-7 cell lines. The results of these experiments indicate the presence of a negative regulatory element in the 5'-untranslated region of the gene, which is able to reduce chloramphenicol acetyltransferase (CAT) activity by 77-89%. We have named this element the mAR suppressor (mARS). DNase-I protection assays of the 5'-untranslated region disclosed a protected domain. Gel mobility assays using the mARS revealed the presence of three protein-DNA complexes that could specifically bind to this protected domain. Insertion of the mARS into the thymidine kinase promoter containing pBLCAT2 vector resulted in a 2- to 10-fold decrease in CAT activity, but only if the insert was 3' to the start of transcription initiation. Finally, point mutations within the mARS were able to increase transcription of the AR promoter by 2.3-fold. The results of these experiments indicate that the mAR 5'-untranslated region contains a suppressor element.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The mouse androgen receptor is suppressed by the 5'-untranslated region of the gene. 805 66

Human leukosialin (CD43) is expressed on the surface of hematopoietic cells in cell-type specific and differentiation-stage-specific manners. Previously we found that the sequence from -53 to -40 was critically involved in the promoter function [Kudo, S. & Fukuda, M. (1991) J. Biol. Chem. 266, 8483-8489]. A transient-expression assay using a chloramphenicol acetyltransferase reporter gene revealed that the promoter could confer a high basal transcriptional activity in both leukosialin-producing and non-producing cells. The transcription factor interacting with the promoter sequence was determined by DNase I footprinting and gel-mobility-shift assays. The nuclear extracts from both leukosialin-producing Jurkat cells and non-producing Hela cells showed a footprint on the 5' flanking region from -58 to -34. Gel-mobility-shift assays revealed that DNA-protein complexes were formed with both nuclear extracts, and these complex formations were inhibited by an oligonucleotide containing the Sp1-binding consensus sequence. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in the supershift of the band for the DNA-protein complex. In addition, the footprint produced by the purified Sp1 transcription factor was identical to those produced by nuclear extracts of Jurkat and Hela cells. The mutational analyses revealed that the binding affinities of Sp1 to mutated promoter sequences were parallel to the transcriptional activity of these promoter sequences. Transient expression analyses in Drosophila Schneider cells demonstrated that cotransfection with Sp1 expression plasmid increased the transcriptional activity. These results establish that Sp1 can bind to the promoter and positively regulates the expression of the leukosialin gene. Even the stable expression of CAT constructs in non-producing Hela cells showed high transcriptional activity. The leukosialin expression thus appears to be regulated by the unique mechanism, that is the repression of high basal transcriptional activity rather than the activation of the basal transcriptional level. Tissue-specific expression is probably achieved by suppression of the basal transcriptional activity in non-producing cells.
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PMID:Transcriptional activation of human leukosialin (CD43) gene by Sp1 through binding to a GGGTGG motif. 805 99

The housekeeping enzyme 5-aminolevulinate synthase (ALAS) regulates the supply of heme for respiratory cytochromes. Here we report on the isolation of a genomic clone for the rat ALAS gene. The 5'-flanking region was fused to the chloramphenicol acetyltransferase gene and transient expression analysis revealed the presence of both positive and negative cis-acting sequences. Expression was substantially increased by the inclusion of the first intron located in the 5'-untranslated region. Sequence analysis of the promoter identified two elements at positions -59 and -88 bp with strong similarity to the binding site for nuclear respiratory factor 1 (NRF-1). Gel shift analysis revealed that both NRF-1 elements formed nucleoprotein complexes which could be abolished by an authentic NRF-1 oligomer. Mutagenesis of each NRF-1 motif in the ALAS promoter gave substantially lowered levels of chloramphenicol acetyltransferase expression, whereas mutagenesis of both NRF-1 motifs resulted in the almost complete loss of expression. These results establish that the NRF-1 motifs in the ALAS promoter are critical for promoter activity. NRF-1 binding sites have been identified in the promoters of several nuclear genes encoding mitochondrial proteins concerned with oxidative phosphorylation. The present studies suggest that NRF-1 may co-ordinate the supply of mitochondrial heme with the synthesis of respiratory cytochromes by regulating expression of ALAS. In erythroid cells, NRF-1 may be less important for controlling heme levels since an erythroid ALAS gene is strongly expressed and the promoter for this gene apparently lacks NRF-1 binding sites.
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PMID:Identification of regulatory sequences in the gene for 5-aminolevulinate synthase from rat. 809 50

The gene encoding a folate-binding protein (FBP) expressed in human placenta has been cloned by screening a genomic library with the KB cell FBP complementary DNA. This gene, contained in a 10-kilobase EcoRI fragment of this genomic clone, has 5 exons, 4 introns, the AATAA polyadenylation signal in the 3'-untranslated region, and a 5'-flanking sequence which contains the promoter elements, all of which span approximately 5 kilobases. Transcription initiation was mapped by RNase protection to a site 73 base pairs downstream from a G-rich sequence linked to a tandemly repeated GGAAG sequence which is a motif that the ets oncogene encoded GA-binding protein (GABP) transcription factor binds. Gel-shift and supershift mobility assays indicate that the G-rich sequence and the ets motif bind specifically to SP1 and GABP, respectively. These cis regulatory elements in tandem drive expression of the chloramphenicol acetyltransferase reporter gene in transiently transfected mouse 3T3 cells. The location of these elements upstream of transcription initiation in this gene, which lacks an appropriately located TATA box promoter, indicates that this SP1-GA binding region most probably regulates expression of this placental FBP. The gene encoding this placental FBP has been assigned the FBP/PL-1 gene because it is a member of a multigene family that includes a gene encoding a FBP expressed in both KB cells and placenta and its unprocessed pseudogene.
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PMID:Characterization of the gene encoding a folate-binding protein expressed in human placenta. Identification of promoter activity in a G-rich SP1 site linked with the tandemly repeated GGAAG motif for the ets encoded GA-binding protein. 810 41

Tumor necrosis factor-alpha (TNF alpha) is one of several autocrine/paracrine factors known to exert potent inhibitory effects on bone. We have shown that TNF alpha inhibition of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-stimulated synthesis of the bone-specific protein osteocalcin (OC) occurs by decreasing steady state levels of OC mRNA, suggesting a pretranslational mechanism. In many genes, TNF alpha action is mediated by the transcription factor NF kappa B. Analysis of OC 5'-flanking DNA revealed a sequence structurally homologous to the previously described NF kappa B-binding site and, thus, a potential TNF alpha response element. Deletion analysis was performed to identify the sequences mediating the response to TNF alpha in osteoblastic ROS 17/2.8 cells by transient transfection with reporter constructs containing rat OC 5'-flanking DNA [chloramphenicol acetyltransferase (CAT)] that retained or deleted homologous NF kappa B sites or a previously defined 1,25-(OH)2D3 response element (VDRE). Transfection with all reporter constructs resulted in low basal CAT activity, measured 72 h after transfection. 1,25-(OH)2D3 stimulated CAT activity 2.8- to 4.5-fold in cells transfected with constructs that included the VDRE. TNF alpha inhibited 1,25-(OH)2D3-stimulated, but not basal, CAT activity. Deletion analysis localized the effect of TNF alpha to a sequence between -522 and -306 relative to the OC transcription start site, an area that included the VDRE but deleted a homologous NF kappa B element. Transfection of cells with a heterologous reporter containing one copy of the OC VDRE inserted in correct orientation or two copies in inverse orientation was sufficient to confer a response to TNF alpha. Gel mobility shift analysis of DNA-nuclear protein interaction revealed that 1,25-(OH)2D3 stimulated an increase in binding of nuclear proteins to an OC 32P-VDRE probe. Preincubation of nuclear extract with specific monoclonal antibodies confirmed that the proteins binding the VDRE included the vitamin D receptor and retinoid-X receptor. TNF alpha treatment of cells inhibited the 1,25-(OH)2D3-stimulated increase in nuclear protein binding to the VDRE. These results suggest 1) the VDRE is sufficient to confer a response to the inhibitory effect of TNF alpha on 1,25-(OH)2D3-stimulated rat OC gene transcription; 2) the action of TNF alpha does not require homologous NF kappa B response elements; and 3) the mechanism of TNF alpha inhibition of 1,25-(OH)2D3-stimulated OC gene expression includes modulation of binding of the vitamin D receptor/retinoid-X receptor heterodimer to the VDRE.
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PMID:A single up-stream element confers responsiveness to 1,25-dihydroxyvitamin D3 and tumor necrosis factor-alpha in the rat osteocalcin gene. 811 49

The rat D2 dopamine receptor gene is transcribed from a TATA-less promoter that has an initiator-like sequence and several putative Sp1 binding sites. The main activator of this gene is between nucleotides -75 and -29, and a strong negative modulator is located between bases -217 and -76 (Minowa, T., Minowa, M. T., and Mouradian, M. M. (1992) Biochemistry 31, 8389-8396). In the present investigation, a small deletion series within this negative modulator fused with the reporter gene for chloramphenicol acetyltransferase was used to transfect the D2-expressing cells, NB41A3. Two cis-acting functional DNA sequences were identified: a 41-base pair segment between nucleotides -116 and -76 (D2Neg-B), which decreased transcription from the D2 promoter by about 45%, and a 26-base pair segment between nucleotides -160 and -135 (D2Neg-A), which, in the presence of the downstream negative modulator, reduced transcription down to the level of a promoterless vector. DNase I footprinting, gel mobility shift, and competitive cotransfection experiments suggested that D2Neg-A functions without trans-acting factors, whereas D2Neg-B interacts with nuclear factors at its Sp1 binding sequences. Gel supershift with anti-Sp1 antibody and UV cross-linking experiments revealed that a novel 130-kDa factor as well as Sp1 interact with D2Neg-B in NB41A3 cells. This novel protein recognizing Sp1 binding sequences in the D2 gene negative modulator is also found in nuclear extract from the rat striatum.
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PMID:Negative modulator of the rat D2 dopamine receptor gene. 815 98


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