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 Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-kappaB, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.
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PMID:Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA. 875 22

Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time-dependently, without a change in beta-actin mRNA level, in NG108-15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage-dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin-dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization-induced transactivation was also blocked by CaM kinase inhibitors. The 200-bp 5'-upstream region (-344/-145) was localized as a Ca2+/ calmodulin-responsive element (CaMRE), which confers depolarization-induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca-responsive elements such as CRE, SRE, NF-AT, or the C/EBP beta-binding site and was separated from a 64-bp cyclic AMP/ phorbol 12-myristate 13-acetate-responsive element (-144/-81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L-type Ca channels.
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PMID:Ca2+/calmodulin-dependent transcriptional activation of neuropeptide Y gene induced by membrane depolarization: determination of Ca(2+)- and cyclic AMP/phorbol 12-myristate 13-acetate-responsive elements. 878 4

Previously, we showed that surface expression of intercellular adhesion molecule 1 (ICAM-1) was strongly upregulated in T cells carrying proviral human T-cell leukemia virus type 1 (HTLV-1) and that the viral transactivator protein Tax1 was capable of inducing the ICAM-1 gene. To determine the responsive elements in the human ICAM-1 gene promoter, a reporter construct in which the 5'-flanking 4.4-kb region of the ICAM-1 gene was linked to the promoterless chloramphenicol acetyltransferase (CAT) gene was cotransfected with expression vectors for Tax1 and Tax2, both of which were separately confirmed to be potent transactivators of the HTLV-1 long terminal repeat (LTR). Tax1 strongly activated the ICAM-1 promoter in all the cell lines tested: three T-cell lines (Jurkat, MOLT-4, and CEM), one monocytoid cell line (U937), and HeLa. Unexpectedly, Tax2 activated the ICAM-1 promoter only in HeLa. By deletion and mutation analyses of the 1.3-kb 5'-flanking region, we found that Tax1 transactivated the ICAM-1 promoter mainly via a cyclic AMP-responsive element (CRE)-like site at -630 to -624 in the Jurkat T-cell line and via an NF-kappaB site at -185 to -177 and an SP-1 site at -59 to -54 in HeLa. On the other hand, Tax2 was totally inactive on the ICAM-1 promoter in Jurkat but transactivated the promoter via the NF-kappaB site at -185 to -177 in HeLa. Gel mobility shift assays demonstrated proteins specifically binding to the CRE-like site at -630 to -624 in Tax1-expressing T-cell lines. Stable expression of Tax1 but not Tax2 in Jurkat subclones enhanced the surface expression of ICAM-1. The differential ability of Tax1 and Tax2 in transactivation of the ICAM-1 gene may be related to the differential pathogenicity of HTLV-1 and HTLV-2.
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PMID:Differential transactivation of the intercellular adhesion molecule 1 gene promoter by Tax1 and Tax2 of human T-cell leukemia viruses. 897 Sep 74

Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages. We have shown elsewhere that both the major immediate-early gene (MIE) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV (HCMV) or simian CMV (SCMV) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding sites referred to as the SNE (SRF/NFkappaB-like element), as well as four classical NFkappaB sites within the HCMV version, contribute to TPA responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF, Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha. Therefore, to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE (SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid. However, the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS binding motifs (TTCC), which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins. This protein complex was more abundant in U-937, K-562, and HeLa cell extracts than in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered only twofold after TPA treatment. A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in DNA-transfected U-937 cells, indicating that the stimulation appeared likely to be a true protein kinase C-mediated signal transduction event rather than a differentiation response. Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element, together with differences in the spacing between the SRF and ETS motifs, appear to account for the inability of the SCMV SNEs to respond to serum induction.
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PMID:Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types. 897 Sep 84

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.
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PMID:Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation. 900 Dec 14

Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca2+. In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5'-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca2+ is primarily dependent on both the TH cAMP-responsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response.
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PMID:Tyrosine hydroxylase gene promoter activity is regulated by both cyclic AMP-responsive element and AP1 sites following calcium influx. Evidence for cyclic amp-responsive element binding protein-independent regulation. 903 29

We used a catecholaminergic neuron-like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase-activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase-PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so-called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose-dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 x 10(-10) and 2 x 10(-9) M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was approximately 1,000-fold less potent in stimulating cyclic AMP (with EC50 = 2 x 10(-7) M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c-fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p < 0.01 vs. controls). Induction of CAT activity linked to both TH and c-fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt-RAB) of cyclic AMP-dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c-fos transcription according to a process that is primarily dependent on the cyclic AMP-PKA pathway.
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PMID:Pituitary adenylate cyclase-activating polypeptide triggers dual transduction signaling in CATH.a cells and transcriptionally activates tyrosine hydroxylase and c-fos expression. 908 43

The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation.
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PMID:Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression. 919 27

Paclitaxel (Taxol) is a novel chemotherapeutic drug that is effective against breast and ovarian cancers. Although the primary target of paclitaxel is microtubules, its efficacy exceeds that of conventional microtubule-disrupting agents, suggesting that it may have additional cellular effects. Previously, we demonstrated that paclitaxel can induce interleukin-8 (IL-8) gene expression at the transcriptional level in subsets of human ovarian cancer lines. In this as well as the previous report, we present evidence that this ability is not linked to the lipopolysaccharide pathway of IL-8 gene induction. The present study identifies the cis-acting elements and trans-acting factors involved in this induction by transfecting DNA constructs containing the 5'-flanking region of the IL-8 gene linked to the chloramphenicol acetyltransferase reporter gene into paclitaxel-responsive and nonresponsive ovarian cancer cells (responsiveness refers to the IL-8 response). Paclitaxel only activated the IL-8 promoter in responsive cells. The AP-1 and NF-kappaB binding sites in the IL-8 promoter are required for activation by paclitaxel; in contrast, a C/EBP site required for IL-8 promoter activation in other cell types is not involved. Gel shift assays demonstrate that paclitaxel causes a marked increase in protein binding to the NF-kappaB and AP-1 consensus binding sequences in the paclitaxel-responsive ovarian cells, but not the nonresponsive cells. The induction of NF-kappaB and AP-1 binding is reduced by the addition of protein kinase C inhibitors and cyclic AMP effector, respectively. These results demonstrate a molecular mechanism for cell-specific paclitaxel-induced IL-8 gene expression which may have clinical relevance.
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PMID:Identification of tumor-specific paclitaxel (Taxol)-responsive regulatory elements in the interleukin-8 promoter. 927 87

Protein kinase C (PKC) activation after treatment of human neuroblastoma SK-N-BE(2)C cells with phorbol 12-myristate 13-acetate (PMA) was found to enhance transcription of the human dopamine beta-hydroxylase (DBH) in those cells. To identify which cis-acting element is responsive to the PMA treatment during DBH gene expression, we employed transient transfection assays with serially deleted constructs of the human DBH gene's 5' upstream region fused to the chloramphenicol acetyltransferase (CAT) gene. Treatment of transfected cells with PMA resulted in an approximate threefold increase in CAT expression for all deletion constructs ranging from -978 bp to -262 bp, while the enhancement did not occur with a construct shortened to -114 bp. The region between -262 and -114 bp from the initiation site of transcription contains several cis-regulatory elements including a cyclic AMP response element (CRE) and putative AP1 and YY1 sequences. Site-directed mutagenesis of those cis-acting elements were performed to identify which of the elements mediated the PMA-induced transcriptional enhancement. Substitution of bases in the putative AP1 site containing in part a putative YY1 sequence did not effect the PMA inducibility. However, specific mutations in the CRE sequence abolished the PMA-inducible effect. Changing the CRE sequence into an authentic AP1 sequence (TGACGTCC --> TGACTCA) did not affect the PMA inducibility, suggesting that AP1 factors might interact with the new AP1 site upon PKC activation. A specific PKC inhibitor, GF109203X, completely inhibited the stimulatory effect of PMA on the expression of the human DBH gene. PMA induced an increase in the DBH mRNA level as detected by Northern blot analysis. Gel retardation showed that the binding of nuclear factors to CRE, putative YY1, and AP1 was sequence specific. Our data suggest that the enhancement of the human DBH gene expression by PMA treatment is mediated by the CRE motif in the 5' upstream region of the gene, and occurs via a PKC-dependent pathway.
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PMID:A protein kinase C-activating phorbol ester enhances transcription of the human DBH gene through a cyclic AMP response element in SK-N-BE(2)C cells. 942 17


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