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 IL-3 gene, located on chromosome 5, contains several cis-acting DNA sequences, i.e. CLE (conserved lymphokine element) and a GC-rich region, similar to the GM-CSF gene. To investigate the role of these elements, the 5' flanking region of the IL-3 gene was attached to a bacterial chloramphenicol acetyltransferase (CAT) gene. The fusion plasmids were analyzed by an in vitro transcription system using Jurkat cell nuclear extract prepared from cells stimulated with phorbol-12-myristate-13-acetate and calcium ionophore (PMA/A23187), introduced into Jurkat cells, expressed transiently, and stimulated by co-transfection of human T cell leukemia virus type I (HTLV-I) encoded transactivator, p40tax. The GC-rich region enhanced TATA-dependent transcription in the in vitro transcription system and also strongly responded to p40tax stimulation in the in vivo cotransfection assay. Using this GC-rich region as a probe, we identified a constitutive DNA-protein complex, alpha, whose binding specificity correlates with transcription activity. However, this element is not sufficient for the expression of the IL-3 gene in response to T cell activation signals (PMA/A23187) and no sequence was found within the IL-3 gene which mediates the response to PMA/A23187. The enhancer sequence which responds to T cell activation signals may be located outside the IL-3 gene and may be shared by other lymphokines, possibly by GM-CSF. We propose that the GM-CSF enhancer (CLE2/GC box) which mediates the response to T cell activation signals may stimulate the expression of the IL-3 gene.
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PMID:Definition of a GC-rich motif as regulatory sequence of the human IL-3 gene: coordinate regulation of the IL-3 gene by CLE2/GC box of the GM-CSF gene in T cell activation. 204 40

cAMP and phorbol esters mediate cellular metabolism by the activation of distinct signal transduction pathways consisting of a cascade of sequential protein phosphorylations. An important consequence of the activation of these pathways is the stimulation of gene transcription by way of interactions of specific proteins with DNA control elements. The 8-base-pair (bp) DNA consensus sequence TGACGTCA [cAMP response element (cAMP-RE)] has been shown to confer cAMP responsivity on transcription from various promoters, and the closely related 7-bp consensus sequence TGA-(C or G)TCA [phorbol 12-myristate 13-acetate response element (PMA-RE)] lends transcriptional responsiveness to phorbol esters. In the JEG-3 placental cell line we find that several variants of the cAMP-REs fused to a gonadotropin alpha promoter chloramphenicol acetyltransferase reporter gene mediate responsiveness to cAMP but not to phorbol esters. The PMA-RE is responsive to phorbol esters but also imparts submaximal sensitivity to cAMP in the JEG-3 cells and in the Hep G2 hepatoma cell line. The transcriptional activities of cAMP-RE and PMA-RE are markedly influenced by the composition of the neighboring bases, but different sequences are permissive for the activity of the cAMP-RE versus the PMA-RE. The two signaling agents together display a supraadditive effect on reporter genes containing active PMA-REs but not cAMP-REs. Gel-mobility-shift and UV cross-linking analyses show that distinct proteins bind to the two control elements. One protein of 38 kDa binds to the cAMP-RE and several proteins of 48-84 kDa bind to the PMA-RE.
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PMID:Cyclic AMP and phorbol ester-stimulated transcription mediated by similar DNA elements that bind distinct proteins. 284 47

Ultraviolet light (UV) exposure of cells infected with human immunodeficiency virus type I (HIV) or transfected with HIV reporter genes increases virus-directed gene expression. Here we report the mapping of the UV response on the long terminal repeat (LTR) by using human cells stably transfected with HIV promoter plasmids harboring different mutations and controlling the expression of the chloramphenicol acetyltransferase (cat) reporter gene. Promoter mutation analysis revealed that no specific upstream region of the LTR was associated with UV activation, although a significant decrease was observed with mutations in the basal promoter elements Spl and TATA. Most importantly, UV activation was not diminished by removal of the - 119 to -69 region encompassing the LTR enhancer region or, more specifically, by point mutations in the NF -kappa B binding elements. Consistent with this result, we found that the phorbol ester (PMA) response, which is known to act through the enhancer, occurred independently and was synergistic with the UV response. Removal of the -119 to -69 region did not affect UV activation; however, it resulted in total abrogation of the PMA response. These results suggest that UV activation is distinct from NF -kappa B activation and does not act through the enhancer in stably transfected cells. This is in dramatic contrast to what is found with transient expression analysis of these responses. Lastly, RNA protection experiments revealed that UV may act on preassembled basal transcription complexes by allowing elongation of nascent short mRNAs generated from the LTR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation of human immunodeficiency virus gene expression by ultraviolet light in stably transfected human cells does not require the enhancer element. 749 7

An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.
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PMID:Cyclic AMP signaling pathways are important in IL-1 beta transcriptional regulation. 759 50

The functional induction of c-fos in the sodium butyrate-induced differentiation of F-98 glioma cells was studied. Fos protein level was increased by butyrate. In contrast, c-Jun protein was constitutively expressed and was not affected by butyrate. Gel-retardation assay indicates Fos as a component of the complex formed between the consensus oligonucleotide of the TPA (PMA, phorbol 12-myristate 13-acetate) response element (TRE) and nuclear extract prepared from butyrate-treated cells. Transfection studies showed that butyrate increased transcription from a multimeric TRE-driven reporter construct, and the effect was mimicked by transfecting cells with fos-expression plasmid. Furthermore, under conditions of c-fos over-expression, transactivation by butyrate was essentially abolished. These data suggest that Fos induction had a functional role in gene activation. Characterization of stable c-fos transfectants demonstrated that these cells displayed alterations in morphology, showed serum-dependent growth, had slower growth rates and grew to lower saturation densities than did untransfected F-98 cells or transfected cells that did not express c-fos. Immunofluorescent staining indicated that fos transfectants also had elevated glial fibrillary acidic protein ('GFAP') expression. Transfection of the c-fos promoter-chloramphenicol acetyltransferase fusion gene into F-98 cells revealed that activation of c-fos by butyrate was exerted at the promoter level, and sequences located within nucleotides -757 to -402 of the c-fos promoter were responsible for butyrate induction. Our data indicate that transcriptional activation of c-fos through its promoter by butyrate resulted in increased Fos protein expression. Transfection studies show that both c-fos and butyrate activate TRE-containing genes, and fos may be a downstream mediator of butyrate. Furthermore, expression of c-fos plays a major role in modulating the growth properties of F-98 cells.
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PMID:Analysis of c-fos expression in the butyrate-induced F-98 glioma cell differentiation. 786 28

We have analyzed in various human leukemic cell lines a previously unrecognized region within the human TNF gene promoter that contains the sequence motif 5'-CCGCCCCCGCG-3'. This GC-rich sequence maps to bps -170 and -160 of the TNF gene. Electrophoretic mobility shift assays (EMSA) combined with methylation interference analysis revealed the binding of two distinct proteins with overlapping recognition sites. Supershift assays identified the constitutive transcription factor Sp1 and the immediate-early growth-response transcription factor Egr-1/Krox-24. Interestingly, this Egr-1-related factor was induced by PMA but not by TNF. The TNF gene GC-rich sequence conferred PMA responsiveness when linked to a heterologous minimal c-fos promoter. To examine the involvement of Egr-1/Krox-24 in TNF gene regulation, a Krox-24 expression vector was used, pSCTKr24. In Jurkat T cells pSCTKr24 stimulated pTNF-286CAT that contains sequences -286 to +34 of the human TNF gene fused to the chloramphenicol acetyltransferase (CAT) gene. Moreover, pSCTKr24 also stimulated the TNF gene GC-rich sequence linked to the minimal c-fos promoter. However, deletion of this site did not result in markedly reduced TNF promoter activity, suggesting that the Egr-1/Krox-24 response element may play an auxiliary role in TNF gene regulation.
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PMID:Characterization of an Krox-24/Egr-1-responsive element in the human tumor necrosis factor promoter. 791 37

Ribonucleotide reductase catalyses the reaction that eventually provides the four deoxyribonucleotides required for the synthesis and repair of DNA. U.v.-cross-linking and band-shift experiments have identified in COS 7 monkey cells an approx. 57 kDa ribonucleotide reductase R1 mRNA-binding protein called R1BP, which binds specifically to a 49-nt region of the R1 mRNA 3'-untranslated region (3'UTR). The R1BP-RNA binding activity was down-regulated by the tumour promoters phorbol 12-myristate 13-acetate (PMA; 'TPA') and okadaic acid, and up-regulated by the protein kinase C inhibitor staurosporine, in a dose-dependent fashion. Furthermore, staurosporine treatment decreased the stability of R1 and CAT (chloramphenicol acetyltransferase)/R1 hybrid mRNAs, whereas PMA and okadaic acid increased the stability of these messages, in a dose-dependent manner. In contrast, treatment of cells with forskolin, a protein kinase A inhibitor, did not alter either R1BP-RNA binding or R1 mRNA-stability characteristics. Transfectants containing R1 or CAT/R1 cDNA constructs with a deletion of the 49-nt 3'UTR sequence failed to respond in message-stability studies to the effects of PMA, staurosporine or okadaic acid. These observations indicate that a protein kinase C signal pathway regulates ribonucleotide reductase R1 gene expression post-transcriptionally, through a mechanism involving a specific cis-trans interaction at a 49-nt region within the R1 mRNA 3'UTR.
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PMID:Regulation of mammalian ribonucleotide reductase R1 mRNA stability is mediated by a ribonucleotide reductase R1 mRNA 3'-untranslated region cis-trans interaction through a protein kinase C-controlled pathway. 806 98

Protein tyrosine kinase p59fyn (Fyn) associates with the TCR-CD3 complex, which suggests that Fyn plays a significant role in the signal transduction involving TCR complex. In addition to cellular genes, viral promoters such as the HIV long terminal repeat (LTR) are also activated upon T cell activation. To elucidate the functional significance of Fyn in the expression of viral promoters, we transfected a Fyn-expression vector together with a reporter plasmid containing the chloramphenicol acetyltransferase gene driven by HIV LTR into a human T cell line, Jurkat. In this assay, Fyn stimulated the promoter in HIV LTR when the transfected cells were treated with both concanavalin A and PMA as an antigen-mimic stimulation. This activation required the intact SH2 domain of Fyn. Mutational analysis of HIV LTR showed that the NF kappa B binding sites were responsible for this effect. Electrophoretic mobility shift assays and UV cross-linking experiments showed that activation of T cells by anti-CD3 antibody induced four kappa B-binding proteins (50, 60, 65 and 100 kDa) in Fyn-overexpressing cells more efficiently than in the parental cells. Our results suggested that Fyn was able to regulate expression of a subset of genes via kappa B-binding proteins upon T cell activation.
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PMID:The protein tyrosine kinase Fyn activates transcription from the HIV promoter via activation of NF kappa B-like DNA-binding proteins. 858 83

Membrane lymphotoxin (LT) complex is a trimer composed of two subunits , LT-alpha and LT-beta of which the latter is a 33-kDa transmembrane protein. The LT-beta gene is expressed in lymphoid cells and organs, but little is known about its inducible regulation. Previously, the surface expression of LT-beta in Jurkat cells has been shown to increase in response to PMA. In this report, we used this model to study the transcriptional control of the human and murine LT-beta genes. PMA strongly induced the expression of LT-beta mRNA, and the level of induction was not changed markedly by cycloheximide (CHX) treatment. The LT-beta promoter region contains conserved Egr-1, nuclear factor (NF)-kappaB, and Ets binding sites, and PMA-inducible factors bound to these sites were detected by the gel-retardation technique (electrophoretic mobility shift assay (EMSA)). To identify sequences involved in transcriptional control, sets of human and mouse promoter-chloramphenicol acetyltransferase (CAT) constructs were generated and assayed by transient transfections. The PMA response was lost after deletion of the distal Ets binding site at -110. Mutations at either the Ets or NF-kappaB sites that prevented factor binding dramatically reduced PMA-inducible promoter activity, suggesting cooperative interaction between corresponding transcription factors in PMA activation. Mutation at the Egr-1 site also resulted in substantial loss of promoter activity, and the residual activity may be attributed to binding of constitutively expressed Sp-1 to the same site. We propose that the interaction between the members of NF-kappaB and Ets families of transcription factors and their cognate sites in the promoter is the major determinant of inducible expression of the LT-beta gene in Jurkat cells.
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PMID:Functional analysis of the lymphotoxin-beta promoter. Sequence requirements for PMA activation. 878 6

IL-4 and IL-10 inhibit the cytokine production and mRNA expression by monocytes/macrophages. To investigate the molecular mechanism of the inhibitory effect on transcriptional or post-transcriptional regulation of IL-6 gene expression by IL-4 and IL-10, we studied IL-6 production, expression level of IL-6 mRNA, IL-6 promoter activity, transcriptional activity of NF-kappaB and NF-IL-6, and IL-6 mRNA stability in human monocytic cell lines, THP-1 and U937, stimulated by PMA and LPS in the absence or the presence of IL-4 or IL-10. Both IL-4 and IL-10 were seen to inhibit IL-6 production and the expression of IL-6 mRNA in both monocytic cell lines studied. In chloramphenicol acetyltransferase assays, utilizing the transient transfection of a chloramphenicol acetyltransferase reporter plasmid containing the IL-6 gene promoter, IL-4, but not IL-10, suppressed the transcriptional activity of the IL-6 gene promoter stimulated by PMA and LPS. Electrophoretic mobility shift assays showed that IL-4, but not IL-10, inhibited nuclear NF-kappaB activity, and that IL-4 and IL-10 did not affect NF-IL-6 activity. On the other hand, IL-10 enhanced the degradation of IL-6 mRNA in a mRNA stability assay. These results suggest that IL-4 may inhibit the transcription of the IL-6 gene by affecting NF-kappaB binding activity, while IL-10 may inhibit the IL-6 mRNA levels post-transcriptionally, without suppressing promoter activity. Therefore, we conclude that IL-4 and IL-10 inhibit IL-6 production by different mechanisms in human monocytic cell lines.
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PMID:Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines. 878 24


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