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)

Tissue-type plasminogen activator (t-PA) gene expression is regulated by the tumor-promoting phorbol ester, phorbol-12-myristate 13-acetate (PMA), by cyclic AMP analogues, and the cAMP agonist, forskolin. Based on nuclear "run-on" transcription assays, t-PA expression is modulated by PMA on the level of transcription. 8-Bromo-cyclic AMP and forskolin do not induce t-PA gene transcription alone but act synergistically with PMA. These effects are confirmed by transient expression assays in HeLa cells employing deletion mutants of the t-PA gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Constitutive expression and most of the PMA-mediated induction requires sequences downstream of position -145. DNase I protection ("footprint") analysis of this region reveals two protein-binding sites: one between position -102 and -115, differing from the consensus sequence of the cAMP-responsive element (CRE) by the substitution of an adenine for a guanine in the middle of the core motif (TGACATCA), and another, located in the first exon (between position +60 and +74), displaying homology to the consensus sequence of the activator protein 2- (AP-2) binding site (CCCCACCCCC). Base substitutions in the core of either the CRE-like element or the AP-2 site suppress constitutive CAT expression by over 80%, whereas the relative PMA- and PMA plus cAMP-mediated responses are retained. CAT expression is below the detection limit when both elements are mutagenized together. Hence, the CRE-like element and the exon-located AP-2-binding site have a cooperative impact on basal transcription, but each element can independently convey the effect of activators of the protein kinase C- and A-dependent pathways of signal transduction. The results of band-shift analysis and competition titration experiments demonstrate that the CRE-like element acts as a low affinity binding site for the same proteins which recognize the authentic CRE.
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PMID:A DNA motif related to the cAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and cAMP. 216 21

Regulation of GH gene expression by GRF involves cAMP as a second messenger. We have demonstrated that a 500-basepair fragment of the human GH (hGH) gene 5' flanking region can confer cAMP inducibility upon the chloramphenicol acetyltransferase transcription unit in transient transfections of rat pituitary tumor cells treated with forskolin, an activator of adenyl cyclase. The same hGH construct is not induced by forskolin in nonpituitary-derived cells. Experiments with hGH deletion constructs reveal that binding sites for transcription factor AP-2 and the pituitary-specific factor GHF-1 are not required for forskolin stimulation, but that GHF-1 may potentiate the effect. RNA analyses reveal that forskolin also stimulates accumulation of transcripts initiated at the hGH promoter. Other agents that elevate cAMP levels also stimulate hGH expression. Since the hGH 5' flanking region contains no sequences homologous to the cAMP-responsive element of the somatostatin gene, and the AP-2 sites do not appear to be required for the forskolin response, these results suggest that a novel cAMP-responsive element exists within 82 basepairs upstream from the transcriptional start of the hGH gene and that hGH regulation by GRF may involve interaction between a tissue-specific element and a cAMP-inducible element.
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PMID:Induction of human growth hormone promoter activity by the adenosine 3',5'-monophosphate pathway involves a novel responsive element. 254 55

The role of the hepatitis B virus (HBV) X gene during virus infection has not been defined. We previously showed that expression of the HBV X gene in the human hepatocellular carcinoma cell line HepG2 trans-activates chloramphenicol acetyltransferase gene expression under control of the human immunodeficiency virus 1 (HIV-1) long terminal repeat and we have now identified a specific sequence in the HIV-1 long terminal repeat that is responsive to the HBV X gene. Plasmid constructs with the chloramphenicol acetyltransferase gene regulated by an isolated and twice-repeated 12-base-pair HIV-1 enhancer sequence homologous to the nucleotide sequence that binds the nuclear transcription factor NF-kappa B (the HIV-1 kappa B-like sequence) were trans-activated by the HBV X gene in HepG2 cells, indicating that the kappa B-like enhancer sequence in the HIV-1 long terminal repeat is responsive to the X gene. When eight copies of the HIV-1 kappa B-like sequence were used to regulate beta-globin gene expression, transcription of this gene was activated by the HBV X gene in HepG2 cells and no beta-globin gene transcription was detected in the absence of the HBV X gene. beta-globin gene expression regulated by the activator protein 2 (AP-2) binding sequence was not activated by the HBV X gene. Treatment of HepG2 cells with phorbol ester resulted in modest activation of the HIV-1 kappa B-like enhancer sequence suggesting that an NF-kappa B-like factor was induced in these cells as it is in T lymphocytes by phorbol ester; however, phorbol ester did not demonstrably enhance the activation of the HIV-1 enhancer observed with the HBV X gene. These experiments indicate that the HIV-1 kappa B-like transcriptional enhancer sequence is activated by the HBV X gene and suggest that the HBV X gene might play a role in regulating transcription of a gene under control of a kappa B-like enhancer during HBV infection. Since such a sequence has not been found in the HBV genome and HBV gene expression appears not to be regulated by the HBV X gene, a cellular gene that plays a role in HBV replication could be the target of the X gene during HBV infection.
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PMID:Hepatitis B virus X gene activates kappa B-like enhancer sequences in the long terminal repeat of human immunodeficiency virus 1. 274 Mar 49

Two overlapping cosmids have been isolated containing the entire murine gene for SPARC (osteonectin), a Ca2+-binding, phosphorylated glycoprotein associated with extracellular matrix synthesis and remodeling. The gene contains 10 exons and covers 26.5 kilobase pairs of DNA. Exon analysis shows that the two N-terminal glutamic acid-rich sequences which are predicted to undergo conformational change upon binding of calcium, as well as the C-terminal EF-hand Ca2+-binding domain are each encoded by a single exon. Comparative analysis of the exon sequence does not support the idea that the SPARC gene has evolved by shuffling of exons from other Ca2+-binding proteins. The 5' flanking region of the SPARC gene, which promotes transcription when placed in front of the bacterial chloramphenicol acetyltransferase gene, contains neither "TATA" nor "CAAT" box sequences. However, unlike most other genes lacking these motifs, mapping of the 5' end of the SPARC gene by RNase protection and primer extension analysis reveals only a single major and one minor transcription start site. The upstream region to -120 includes six repeats of the sequence GGAGG, two repeats of the sequence 5' GGAGG A/C GGAGGG 3', and a potential transcription factor AP-2 binding site.
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PMID:Characterization of the mouse SPARC/osteonectin gene. Intron/exon organization and an unusual promoter region. 316 75

A transforming growth factor-beta (TGF-beta) activating element (TAE), with a nuclear factor-1 (NF-1)-like sequence, was previously located 1.6 kilobases upstream from the transcription start site in the alpha 1(I) collagen promoter (Ritzenthaler, J. D., Goldstein, R. H., Fine, A., Lichtler, A., Rowe, D. W., and Smith, B. D. (1991) Biochem. J. 280, 157-162). Double-stranded TAE, but not NF-1 consensus sequences, abrogated TGF-beta stimulation of co-transfected collagen promoter-chloramphenicol acetyltransferase constructs. Mutations in non-NF-1 binding sites, located by methylation interference, eliminated activity of the TAE oligonucleotide. However, TAE sequences failed to bind in vitro expressed NF-1 protein, to compete for NF-1-binding proteins, and to bind with protein which reacts with antibodies to NF-1 family of proteins. Within the TAE there was an activator protein 2 (AP-2) binding site. Although AP-2 protein bound to TAE, antibodies to AP-2 did not react with nuclear protein-TAE complexes. TAE bound to a 34,000-Da protein on Southwestern analysis. However, the UV-cross-linked TAE-nuclear protein complex was 82,000 Da. Finally, a dose-response study demonstrated that TGF-beta increased TAE nuclear binding proteins at lower doses with a different response curve than NF-1 nuclear binding proteins. Taken together these data demonstrated that TGF-beta functions in human lung fibroblasts to activate collagen transcription through TAE sites by protein complexes independent of NF-1 or AP-2 protein.
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PMID:Regulation of the alpha 1(I) collagen promoter via a transforming growth factor-beta activation element. 851 94

DR-nm23 cDNA was cloned recently by differential screening of a cDNA library derived from chronic myelogenous leukemia-blast crisis primary cells. It is highly homologous to the putative metastasis suppressor nm23-H1 gene and the closely related nm23-H2 gene. When overexpressed in the myeloid precursor 32Dcl3 cell line, it inhibited granulocyte colony-stimulating factor-stimulated granulocytic differentiation and induced apoptosis. We have now found that the expression of DR-nm23 is not restricted to hematopoietic cells but is also detected in an array of solid tumor cell lines, including carcinoma of the breast, colon, and prostate, as well as the glioblastoma cell line T98G. We have also isolated both the gene and its 5'-flanking region and found that DR-nm23 localizes on chromosome 16q13. The gene consists of six exons and five introns. When fused in-frame to the nucleotide sequence for the green fluorescent protein and transfected in SAOS-2 cells, it generates a protein of the predicted size that localizes to the cytoplasm. The 5'-flanking region of DR-nm23 does not contain a canonical TATA box or a CAAT box, but it is G+C rich and contains two binding sites for the developmentally regulated transcription factor activator protein 2 (AP-2). Transient expression assays of DR-nm23 promoter-chloramphenicol acetyltransferase constructs demonstrated that the segment from nucleotides -1028 to +123 has the highest activity in hematopoietic K562 cells and in TK-ts13 hamster fibroblasts. Moreover, AP-2 induced a 3-fold transactivation of the DR-nm23 5'-flanking segment from nucleotides -1676 to +123 and interacted specifically with oligomers containing putative AP-2 binding sites (-936 to -909, and -548 to -519) as indicated by electrophoretic mobility shift assay. Furthermore, nuclear run-on assays from high and low DR-nm23-expressing cells (K562 and CCRF-CEM, respectively) revealed similar transcription rates. Therefore, the regulation of the DR-nm23 gene expression might involve other mechanisms occurring at posttranscriptional and/or translational levels.
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PMID:Gene structure, promoter activity, and chromosomal location of the DR-nm23 gene, a related member of the nm23 gene family. 906 90

MCAM/MUC18 is a cell-surface glycoprotein of 113 kDa, originally identified as a melanoma antigen, whose expression is associated with tumor progression and the development of metastatic potential. We have previously shown that enforced expression of MCAM/MUC18 in primary cutaneous melanoma led to increased tumor growth and metastatic potential in nude mice. The mechanism for up-regulation of MCAM/MUC18 during melanoma progression is unknown. Here we show that up-regulation of MCAM/MUC18 expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The MCAM/MUC18 promoter contains four binding sites for AP-2, and electrophoretic mobility shift assay gels demonstrated that the AP-2 protein bound directly to the MCAM/MUC18 promoter. Transfection of AP-2 into highly metastatic A375SM melanoma cells (AP-2-negative and MCAM/MUC18-positive) inhibited MCAM/MUC18 promoter-driven chloramphenicol acetyltransferase reporter gene in a dose-dependent manner. MCAM/MUC18 mRNA and protein expression were down-regulated in AP-2-transfected but not in control cells. In addition, re-expression of AP-2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of MCAM/MUC18 is regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly by down-regulating MCAM/MUC18 gene expression. Since AP-2 also regulates other genes that are involved in the progression of human melanoma such as c-KIT, E-cadherin, MMP-2, and p21(WAF-1), we propose that loss of AP-2 is a crucial event in the development of malignant melanoma.
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PMID:Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells. 963 18

Trophoblast cells are specialized extra-embryonic cells present only in eutherian mammals. They play a major role in the implantation and placentation processes. To understand better the molecular mechanisms that control the development and function of trophoblast cells, we sought to identify the transcription factors that regulate murine adenosine deaminase (ADA) gene expression in the placenta. Here we report a detailed characterization of a placenta-specific footprinting region (FP1) in the Ada placental regulatory element. The sequence of FP1 was mapped by DNase I footprinting and was found to match a consensus AP-2 transcription factor-binding site. Electrophoretic mobility shift assays demonstrated that FP1 interacted with AP-2-like proteins. Further analysis using AP-2 antibody confirmed that AP-2 protein was indeed present in the placenta and bound to FP1. Mutation at the AP-2 site in FP1 abolished the ability of the Ada placental regulatory element to bind AP-2 proteins and failed to target chloramphenicol acetyltransferase reporter gene expression to placentas in transgenic mice, indicating that AP-2 is required for Ada expression in the placenta. In addition, RNase protection assays demonstrated that AP-2gamma was the predominant AP-2 family member expressed in the placenta. In situ hybridization analysis revealed that AP-2gamma expression was enriched in the trophoblast lineage throughout development, suggesting that AP-2gamma may be critical for trophoblast development and differentiation.
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PMID:Transcription factor AP-2gamma regulates murine adenosine deaminase gene expression during placental development. 976 60

Pyrrolidine dithiocarbamate (PDTC) has been widely used as an inhibitor of the nuclear factor-kappa B, (NF-kappa B) signalling pathway. Here, we show that kappa B-dependent reporter gene expression induced by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) is potentiated by PDTC in the human pro-monocytic U937 cell line. The stimulatory effect of PDTC on kappa B-dependent gene expression was shown with a 4 x kappa B chloramphenicol acetyltransferase construct and required an intact kappa B element in the human immunodeficiency virus long terminal repeat (HIV-1 LTR). Unexpectedly, an HIV-1 LTR construct with a mutation of the activator protein 2 (AP-2) binding site located between the two kappa B elements was unresponsive to the stimulatory effect of PDTC with TPA. The stimulation or inhibition of kappa B-dependent gene expression was dependent on PDTC pre-treatment and the concentration of TPA. No stimulatory effect on HIV-1 LTR activity was observed with the metal chelator dipyridyl or the anti-oxidant N-acetyl-L-cysteine. These results are consistent with the hypothesis that PDTC treatment potentiated kappa B-dependent gene expression in a manner dependent on the concentration of TPA.
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PMID:Dual activity of pyrrolidine dithiocarbamate on kappa B-dependent gene expression in U937 cells: I. Regulation by the phorbol ester TPA. 1040 58

The transcriptional activator protein-2 (AP-2) has been suggested to participate in keratinocyte gene regulation. Cystatin A, a cysteine proteinase inhibitor, is one of the cornified cell envelope constituents and is expressed in the upper epidermis. We report AP-2-dependent transcriptional regulation of cystatin A gene expression of keratinocytes. At least three isoforms of AP-2 (AP-2 alpha, beta, gamma) have been described. Transfection of AP-2alpha, beta and gamma expression vectors into cultured normal human keratinocytes (NHK) resulted in increased cystatin A expression in both mRNA and protein levels. Among the three isoforms AP-2gamma was most potent in inducing cystatin A expression. In contrast, transfection of antisense oriented AP-2gamma expression vector decreased basal AP-2 expression, accompanied by decreased cystatin A mRNA. The fragment, +77 to -478 of 5'-flanking region of human cystatin A gene, was subcloned into chloramphenicol acetyltransferase (CAT) reporter vector (p478CAT). Cotransfection of p478CAT vector with AP-2alpha, beta, and gamma expression vectors resulted in three-, three-, and sixfold increase in the CAT activity, respectively. Transfection of the deleted construct (p478DeltaAP-2CAT, devoid of AP-2-like binding site (-75 to -84)) decreased CAT activity by one-third compared to p478CAT promoter activity. Cotransfection of p478DeltaAP-2CAT with AP-2alpha, beta, and gamma expression vectors had no effect on the decreased promoter activity. Immunohistochemical analysis of human skin showed that AP-2alpha is exclusively expressed in the nuclei of basal cell layer. AP-2gamma is expressed in the nuclei of basal, spinous, and granular cell layers. AP-2beta expression was not observed in the epidermis. Gel mobility shift assay revealed that the AP-2gamma protein specifically binds to oligonucleotides containing AP-2-like binding site of cystatin A gene. These results indicate that AP-2gamma regulates the cystatin A gene expression of epidermal keratinocytes at the transcriptional level.
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PMID:Transcriptional factor AP-2gamma increases human cystatin A gene transcription of keratinocytes. 1109 74


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