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 transcriptionally active RVL3-VL30 element contains a triple repeat of TGACTCC, a sequence nearly identical to the AP-1 binding site. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation was unable to elicit chloramphenicol acetyltransferase (CAT) expression from a construct containing these AP-1-like sequences upstream of the thymidine kinase promoter present in pTES. Endothelin, which activates protein kinase C (pkC) and elevates intracellular Ca2+ in Rat-1 cells, was effective in stimulating CAT expression from the VL30-pTES construct. We attempted to assess the relative importance of these second messenger systems by stimulating each pathway separately with exogenous agonists. We determined that neither stimulation of pkC by the tumor promoter TPA nor elevation of intracellular Ca2+ by the tumor promoter thapsigargin was sufficient to stimulate CAT expression from the VL30-pTES vector. When combined, the two tumor promoters induced a synergistic increase in CAT expression. Our data indicate that elevation of intracellular Ca2+ by thapsigargin was not required for full activation of pkC by TPA. First, TPA was able to stimulate expression of other genes in Rat-1 cells, indicating full activation of pkC. Second, thapsigargin synergized effectively with epidermal growth factor to stimulate CAT activity from the VL30-pTES construct in cells depleted of pkC activity by chronic TPA treatment. The permissive effects of thapsigargin on gene expression were also observed for an endogenous gene, transin/stromelysin. The permissive effects of elevated intracellular Ca2+ levels may represent a general mechanism for the stimulation of some genes by pkC-mediated pathways.
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PMID:Two tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and thapsigargin, act synergistically via distinct signaling pathways to stimulate gene expression. 212 50

Fibronectin (FN) mRNA levels increased when quiescent cells (serum starved) were stimulated to undergo the G0/G1 transition by the addition of 20% given fetal calf serum to the media. The 5'-flanking region of the FN gene (position +69 to -510 base pairs (bp] was fused to the coding region of the chloramphenicol acetyltransferase (CAT), and the fusion gene was used in transfection assays. Expression of FNCAT increased on serum treatment indicating that the region of the FN gene between positions +69 and -510 bp mediated serum responsiveness. Deletion of FN gene 5'-flanking sequences from position -510 to -122 bp eliminated serum responsiveness suggesting that an element between these positions was mediating the effect. Sequences between positions -122 and -510 bp of the FN gene were able to confer serum responsiveness on a herpes virus thymidine kinase promoter-CAT fusion gene (TKCAT) when the FN gene sequences were cloned upstream of TKCAT. The ability to confer serum responsiveness on TKCAT was retained with a smaller 100-bp sequence (position -122 to -222 bp). Both a cAMP response element (position -170 bp) and a nuclear factor-1 binding site (position -155 bp) have been identified within this sequence (Dean, D. C., Blakeley, M. S., Newby, R. F., Ghazal, P., Hennighausen, L., and Bourgeois, S. (1989) Mol. Cell. Biol. 9, 1498-1506). The cAMP response element was serum-responsive when cloned upstream of TKCAT or a minimal FN promoter (deleted to position -56 bp) while the nuclear factor-1 binding site was unresponsive. Therefore, the cAMP regulatory element (CRE) is the serum-responsive element between position -122 and -222 bp. Serum-induced binding of proteins to the CRE was detected in gel retardation assays with extracts from cell lines where FN expression was serum-responsive. However, no serum-induced binding was detected with extracts from the JEG-3 cell line where FN expression was not serum-responsive. Serum-induced binding occurred rapidly, within 15 min, and did not require protein synthesis. The decay of serum-induced binding was relatively slow as increased binding was still detectable 24 h after removal of serum. The CRE also mediates transcriptional stimulation by cAMP, but unlike serum stimulation increased CRE binding activity was not detectable in extracts from cAMP-treated cells (Dean, D. C., Blakeley, M. S., Newby, R. F., Ghazal, P., Hennighausen, L., and Bourgeois, S. (1989) Mol. Cell. Biol. 9, 1498-1506).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serum stimulation of fibronectin gene expression appears to result from rapid serum-induced binding of nuclear proteins to a cAMP response element. 213 58

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.
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PMID:Hormonal induction of transfected genes depends on DNA topology. 215 20

Immortalization of B lymphocytes by Epstein-Barr virus (EBV) is complex and poorly understood. However, some evidence suggests that glucocorticoids influence this process. We identified a glucocorticoid-responsive element in the BamHI C fragment of EBV which we call ES-1. In glucocorticoid-treated cells, ES-1 enhanced chloramphenicol acetyltransferase gene expression from the herpes simplex virus thymidine kinase promoter, as well as the EBV Bam-C promoter, from which several latent viral gene products are transcribed. By Northern blot analysis, glucocorticoid treatment enhanced transcription from the Bam-C promoter in Jijoye cells, a Burkitt's lymphoma cell line. In addition, the DNA-binding domain of the glucocorticoid receptor bound specifically to the ES-1 region. These glucocorticoid effects on the Bam-C promoter region may provide some insight into the process of EBV immortalization.
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PMID:Identification of a glucocorticoid-responsive element in Epstein-Barr virus. 215 66

We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were cotransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (alpha 0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.
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PMID:Herpes simplex type 1 activation by Epstein-Barr virus nuclear antigen 1. 215 38

Interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes. Three transcriptional control elements within 300 base pairs of the 5'-flanking region of the rat glucagon gene interact with regulatory cellular proteins and direct transcription only in glucagon-producing islet cells. Two islet cell-specific enhancer-like elements (G2, G3) act together with the glucagon promoter (including the G1 element), which confers A cell specificity of glucagon gene expression. In the present study, the G3 element was analyzed in detail by protein binding and in vivo and in vitro transcription assays. Mutational analyses showed that the sequence of the G3 element comprises two distinct protein-binding domains: a more upstream domain A (5'-CGCCTGA-3'), and a more downstream domain B (5'GATTGAAGGGTGTA-3'). Binding of proteins to these two domains is mutually exclusive. Domain A, but not domain B, is responsible for both functional protein binding and the enhancement of transcription from the glucagon or thymidine kinase gene promoter chloramphenicol acetyltransferase reporter gene transfected in vivo into glucagon-producing islet cells (InR1-G9) and transcribed in vitro in a HeLa cell-free transcription system. In islet cell extracts, the Southwestern blot technique labeled a protein of 45 kDa binding to domain A within G3. We conclude that although the G3 sequence contains two protein-binding motifs, the organization of the G3 enhancer-like element is not bipartite. The islet cell specificity of the G3 element is conferred by a tissue-specific transcription factor or protein complex interacting with domain A of G3. This protein or protein complex recognizes different DNA sequences and provides promoter as well as enhancer activity because it binds also to the apparently unrelated sequence of the G1 promoter element.
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PMID:A pancreatic islet cell-specific enhancer-like element in the glucagon gene contains two domains binding distinct cellular proteins. 216 Apr 64

The BamHI J fragment of human cytomegalovirus (HCMV) AD169 located at 0.815 to 0.855 map units in the unique short component of the genome was demonstrated to be responsive to the HCMV IE proteins by using a transient chloramphenicol acetyltransferase (cat) gene expression system. The BamHI J fragment was cloned into a cat gene expression plasmid and then cotransfected with a plasmid that expresses the immediate early (IE) genes of HCMV AD169 into the HCMV permissive cell line MRC-5. The results indicated that the BamHI J fragment enhanced cat gene expression 10-fold when the HCMV IE proteins were present. The BamHI J fragment was demonstrated to have properties of an inducible enhancer. In the presence of the HCMV IE proteins, it enhances cat gene expression when positioned in either orientation both upstream and downstream from the cat gene; it enhances transcription from the herpes simplex virus type 1 (HSV-1) thymidine kinase gene and the simian virus 40 (SV40) early gene promoters; and it requires a cis-positioned promoter for enhancer activity.
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PMID:Detection of an IE responsive element(s) in the BamHI J fragment of human cytomegalovirus AD169. 216 22

The rate of transcription of the beta 2-adrenergic receptor gene is increased in response to beta-adrenergic agonist stimulation of the receptor at the cell surface. This effect is mediated by stimulation of adenylyl cyclase and elevation of intracellular cAMP levels. We have previously shown that this responsiveness to cAMP resides in the 5'-flanking region of the human beta 2-adrenergic receptor gene (Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4853-4857). A 34-base pair sequence derived from the beta 2-adrenergic receptor promoter region (-70 to -37 base pairs), containing the sequence GTACGTCA, confers responsiveness to cAMP when present in either orientation 5' to the thymidine kinase promoter on the chloramphenicol acetyltransferase reporter gene. Overexpression of the catalytic subunit of protein kinase A fully substituted for forskolin in inducing expression through this sequence, indicating that the cAMP induction is mediated through this kinase. Mutations within the GTACGTCA sequence completely abolished the stimulation. A 43-kDa transcription factor (cAMP response element-binding protein) confers cAMP responsiveness through binding to specific sequences. In gel mobility shift assays, purified cAMP response element-binding protein bound to the 34-base pair oligonucleotide from the beta 2-adrenergic receptor gene with an affinity similar to that for the well-characterized cAMP response element from the human glycoprotein hormone alpha-subunit gene, and failed to bind to mutated elements. Thus, positive autoregulation of the beta 2-adrenergic receptor gene appears to occur through receptor-mediated stimulation of adenylyl cyclase, with consequent activation of cAMP response element-binding protein and stimulation of beta 2-adrenergic receptor gene transcription. These results demonstrate a novel mechanism by which a receptor (beta 2-adrenergic receptor) stimulatory for adenylyl cyclase can exert positive feedback regulation on its own expression.
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PMID:A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP. 217 52

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interactions of thyrotropin-releasing hormone, phorbol ester, and forskolin-sensitive regions of the rat thyrotropin-beta gene. 217 92

The interferon (IFN)-gamma-mediated induction of indoleamine 2,3-dioxygenase (IDO) enzyme, which converts tryptophan into N-formylkynurenine, has been implicated in the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, and in the antiproliferative effect of IFN-gamma on tumor cells. The IDO activity is induced strongly in many cell types by IFN-gamma but rather poorly by IFN-alpha or -beta. A genomic DNA clone containing part of the transcribed region of the IDO gene and approximately 13 kilobases (kb) of the 5'-upstream DNA sequence was isolated and analyzed. An approximately 1.4-kb fragment of this clone, containing 329 nucleotides of the transcribed sequence and approximately 1.1 kb of the 5'-upstream sequence, when ligated to chloramphenicol acetyltransferase (CAT) structural gene made its expression inducible by IFN-gamma, but this construct responded poorly, if at all, to IFN-alpha 2. Deletion constructs derived from this plasmid narrowed down the IFN-gamma-responsive region to a 151-nucleotide segment (-495/-344) which also contained a 14-nucleotide sequence (GGTTTCAGTTTTCC) highly homologous to the IFN(alpha)-stimulated response element (ISRE) that has been found so far in all cellular genes inducible with IFN-alpha or -beta. Expression of CAT activity was stimulated by IFN-gamma more effectively than by IFN-alpha 2 when a 155-nucleotide fragment (-495/-340) containing the 151-nucleotide segment required for IFN-gamma response was inserted before herpes simplex virus thymidine kinase promoter linked to CAT structural gene. The results indicate that despite the presence of an ISRE, the control region of the IDO gene can distinguish between IFN-gamma and IFN-alpha. This may account for the differential activation of IDO gene expression by IFN-gamma as against IFN-alpha or -beta in intact cells, and suggests that the response of ISRE to IFN-alpha or -beta may be governed by other features in the upstream control region of this gene.
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PMID:Regulation of indoleamine 2,3-dioxygenase gene expression in human fibroblasts by interferon-gamma. Upstream control region discriminates between interferon-gamma and interferon-alpha. 217 56


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