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)

Pregnancy-specific glycoproteins (PSGs) are the major placental glycoproteins, that together with the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. In order to develop an animal model for understanding the molecular mechanisms underlying the control of PSG expression, we isolated and characterized cDNA and genomic clones encoding a rodent PSG, rnCGM3. The rnCGM3 cDNA is 2761 bp in length and contains an open reading frame that encodes a 475 amino acid polypeptide with a domain arrangement of L1N1-L2N2-L3N3-A. The sequence in 5'-untranslated and L1 regions of rnCGM3 is identical to a previously identified cDNA, rnCGM6. The transcription initiation sites of both genes are located at nucleotide -197 upstream of the translation start site. In transient transfection assays using a chloramphenicol acetyltransferase (CAT) reporter gene, we demonstrated that DNA elements at nucleotides -326 to -185 (PI) and -147 and -86 (PII) relative to the translation start site of rnCGM3 could both function as promoters. The downstream promoter, PII, which is located within the first exon, shares high sequence identity with the minimal promoters of human PSG genes. Electrophoretic mobility shift assays (EMSAs) showed that protein factors in placental cell extracts formed three complexes (PIICI, PIICII, and PIICIII) with the PII promoter element. The PIICIII complex was also observed by DNase I footprinting analysis. Unlike PII, the upstream promoter, PI, contains a TATA box. DNase I footprinting analysis revealed two nuclear protein binding sites at nucleotides -311 to -290 (PISI) and -257 to -239 (PISII) in PI. EMSAs showed that protein factors in placental cell extracts bound to both sites and deletion of either site markedly reduced CAT expression. PISII contains a palindromic motif, TGTTGCTCAACA, and protein cross-linking and Southwestern hybridization analyses demonstrated that the protein factor binding to PISII had an apparent molecular mass of 40 kDa.
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PMID:Characterization of two promoters of a rat pregnancy-specific glycoprotein gene. 806 38

Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes. Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein. To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays. These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells. Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein. The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters. Such conservation suggests that this sequence may have an important role in mdr gene expression. The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity. DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein. These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.
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PMID:Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene. 809 13

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

Nuclear protein binding sites in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV) were identified by the method of DNase I footprinting. Using nuclear protein extracts from a feline T lymphoma cell line, several discrete footprints were generated upstream of the transcriptional initiation site (-50 to -150). The specificity of protein binding was examined by competition with oligonucleotides representing consensus DNA binding sites for known transcription factors. Binding to AP-1 (-124) and ATF (-58) motifs was observed, with cross-competition between these sites. A strong footprint signal was also detected over a tandemly repeated C/EBP motif (-94, -86) and an adjacent weaker footprint was found to be specific for an NF1 motif (-72/-63). The effect on FIV LTR promoter activity of progressively deleting these nuclear factor binding sites was examined by linking LTR deletion mutants to the chloramphenicol acetyltransferase (CAT) gene. Deletion of the AP-1 site caused a 10- to 25-fold loss of CAT activity whereas deletion past the ATF site reduced activity virtually to background levels. The effects of deleting the C/EBP and NF1 sites were less marked and varied according to cell type. Transactivation of the LTR was assayed using constructs linked to a CAT reporter gene. The full-length FIV LTR was not significantly trans-activated. However, the expression of a deleted LTR construct lacking the AP-4/AP-1 site but retaining C/EBP and ATF sites was partially restored by co-infection with FIV or by co-transfection with an infectious molecular clone of FIV (FIV-PPR). These results show that host transcription factors responsive to cellular activation have a major role in regulating FIV expression, and suggest that virus-coded trans-activators acting through U3 may play a role in some cellular environments.
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PMID:Cis- and trans-regulation of feline immunodeficiency virus: identification of functional binding sites in the long terminal repeat. 812 51

The mouse epidermal cell line 308 contains an activated Ha-ras gene and forms benign papillomas when transplanted to the skin of athymic nude mice. A radiation-associated malignant variant of this cell line, 308-10Gy5, has been isolated and shown to form squamous cell carcinomas in nude mice. To further examine the molecular events involved in malignant conversion of 308-10Gy5, we assessed the activator protein-1 (AP-1) binding and transactivating ability of 308 and 308-10Gy5. In nuclear protein extracts of 308, AP-1 sequence-specific binding to an oligonucleotide containing a single high-affinity AP-1 binding site was induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, as determined by gel shift analysis. Nuclear extracts of 308-10Gy5 bound to the AP-1 oligonucleotide without treatment with tumor promoters. Not only was sequence-specific AP-1 DNA binding constitutively active in malignant versus benign tumor cells, but so was transactivation of a unique AP-1-responsive chloramphenicol acetyltransferase reporter construct, pTiCTaK. Constitutive transactivation of this AP-1-responsive reporter construct was observed in the malignant but not the benign tumor cells. Furthermore, steady-state transcript levels of the tumor-associated AP-1-responsive genes stromelysin, urokinase-type plasminogen activator, c-jun, and c-fos were higher in malignant 308-10Gy5 cells than in benign 308 cells. These results suggest that acquisition of constitutive AP-1 DNA binding and transactivation can result in sustained deregulation of gene expression. While malignant progression in keratinocytes is probably not due solely to the acquisition of constitutive cellular AP-1 activity, the effect of deregulated expression of AP-1-regulated genes, especially basement membrane-degrading enzymes, may be functionally related to malignant conversion.
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PMID:Constitutive AP-1 DNA binding and transactivating ability of malignant but not benign mouse epidermal cells. 814 9

Hemopexin (Hx) is an abundant acute-phase protein (APP) that binds heme with high affinity. In rat hepatic cells, the transcription rate of the Hx gene is increased by interleukin (IL)-1 and IL-6. To investigate the cis-acting regulatory elements (REs) responsive to these hormones, chloramphenicol acetyltransferase constructs of rat and human Hx gene sequences were tested in transiently transfected hepatoma cells. An IL-6-RE was identified in the promoter of both rat and human Hx genes, the function of which was dependent on the core sequence (CCGGGAA) common in other APP genes. The previously characterized Hx A element mediated a relatively minor cytokine response as compared with the Hx IL-6-RE. The human Hx A element, in contrast to the rat and human Hx IL-6-REs, was strongly trans-activated by cotransfected CAAT enhancer-binding proteins (C/EBP)-beta and -delta. The rat gene homolog of the human Hx A element was inactive as a cytokine RE and was minimally trans-activated by C/EBP isoforms. Results of electrophoretic mobility shift assays indicated that the Hx IL-6-RE is a binding site for the IL-6-inducible nuclear protein IL-6 RE-BP, which also binds to the conserved IL-6-REs of other APP genes and is distinct from C/EBP beta.
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PMID:The rat and human hemopexin genes contain an identical interleukin-6 response element that is not a target of CAAT enhancer-binding protein isoforms. 817 75

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to < 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites. 822 15

In an effort to identify regulatory elements of the serum amyloid A (SAA) gene that play a major role in its expression under acute-phase conditions, we studied the expression of a set of chimeric SAA-chloramphenicol acetyltransferase (CAT) plasmids containing a progressively deleted upstream 5' sequence of the SAA gene. Two regulatory regions (-314 to -135 and -135 to -31) capable of driving cytokine-induced transcription have been identified. Gel retardation assays revealed that the regulatory region located between positions -314 and -135 is a major site of interaction for highly inducible and constitutive nuclear proteins in acute-phase rabbit liver. DNase I footprint and competition analyses showed that this region contains two adjacent nuclear protein binding sites (between -191 and -140) with varying affinity for protein binding. Both of these binding sites are capable of driving cytokine-induced transcription of a reporter gene containing a minimal promoter. Detailed analyses of the inducible nuclear proteins that bind to this promoter element showed that they are homologues of the CCAAT/enhancer binding protein (C/EBP) family. Accumulation of the inducible nuclear factors under acute conditions, when maximal transcription activity has been reported, suggests a critical role for these proteins in the expression of the SAA gene.
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PMID:Analysis of the promoter element of the serum amyloid A gene and its interaction with constitutive and inducible nuclear factors from rabbit liver. 826 19

Transcription of the J6 gene (a member of the serpin family) is induced in murine F9 teratocarcinoma cells 24-48 h after retinoic acid (RA) treatment. Previously, we have identified a region of the J6 5'-flanking region (-1050 to -738) that is involved in regulating transcription of this gene. In this report, we show that a RA-induced nuclear protein in the F9 cell extract recognizes the GAGATAG sequence which is repeated four times in this regulatory region of the J6 gene. The kinetics of RA induction of the GAGATAG-binding protein correlates with that of J6 mRNA, suggesting that the GAGATAG-binding protein may be involved in the transactivation of the J6 gene in RA-treated F9 cells. Competition experiments demonstrate further that the RA- induced GAGATAG-binding protein is related to the transcription factors GATA-1 and GATA-2. Furthermore, insertion of the RA regulatory region of the J6 gene into a thymidine kinase promoter/chloramphenicol acetyltransferase expression vector causes an increase in chloramphenicol acetyltransferase expression by 5-8-fold in human HeLa cells when co-transfected with human GATA-2 or GATA-3 expression vectors. This suggests that the J6 gene is likely to be transactivated by the GATA-2, GATA-3, or related transcription factor, which is activated by retinoic acid during F9 cell differentiation.
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PMID:A retinoic acid-inducible GATA-binding protein binds to the regulatory region of J6 serpin gene. 827 58

rSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt -102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5' upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additional cis element (-645/-506). The positive and negative cis-elements together drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (-44/-29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (-44/-29) are competed by unlabeled -44/-29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides -40 to -38 and -34 to -32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-specific core promoter elements.
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PMID:Molecular cloning and functional analysis of the promoter of rat skeletal muscle voltage-sensitive sodium channel subtype 2 (rSkM2): evidence for muscle-specific nuclear protein binding to the core promoter. 828 44


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