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 mouse lactoferrin gene responded to forskolin, 12-O-tetradecanoyl phorbol-13-acetate, and epidermal growth factor (EGF) stimulation via two adjacent enhancer elements, the cAMP response element (CRE) and EGF response element (EGFRE), collectively referred to as the mitogen response unit. In this report, we examined the minimal promoter and enhancer elements of the mouse lactoferrin gene that are required for EGF-induced transcriptional activation. We found that the CRE and noncanonical TATA box (ATAAA) are the minimal promoter elements for basal activity of the chloramphenicol acetyltransferase (CAT) reporter construct whereas the EGFRE is needed for an additional activity induced by EGF in transiently transfected human endometrial carcinoma RL95-2 cells (RL95-2). The EGFRE, however, did not function in heterologous promoters [SV 40 and thymidine kinase (TK)]. Therefore, EGF-stimulated lactoferrin gene activity is promoter specific in RL95-2 cells. In transiently transfected cells, EGF and forskolin showed synergistic effects on the CAT reporter that contained both response elements. Mutation made at either element or insertion of extra nucleotides between the two elements severely affected EGF-stimulated activity. Nuclear protein prepared from RL95-2 cells formed three complexes (A, B, and C) with the oligonucleotides containing both EGFRE and CRE in electrophoretic mobility shift assay. A new complex (E) was detected with the nuclear protein of EGF-treated cells. By oligonucleotide competition experiments, we demonstrated that the complex E was generated by protein bound to CRE. EGF-induced binding activity could be abolished by calf intestinal alkaline phosphatase but not by the protein synthesis inhibitor, cycloheximide. Therefore, binding of a preexisting phosphoprotein to the CRE region could be one of the requirements for EGF-induced mouse lactoferrin gene promoter activity.
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PMID:Promoter-specific activation of mouse lactoferrin gene by epidermal growth factor involves two adjacent regulatory elements. 877 33

Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin-expressing cells, express high levels of renin mRNA from the endogenous Ren-1(c) gene. We have used these cells to characterize the role of the Ren-1(c) proximal promoter (+6 to -117) in the regulation of renin gene transcription. It was found that 4.1 kilobases (kb) of Ren-1(c) 5'-flanking sequence, in combination with the proximal promoter, are required for strong activation (approximately 2 orders of magnitude over the basal level of the promoter alone) of the chloramphenicol acetyltransferase reporter in transfection assays. Within the 4.1-kb fragment, a 241-base pair region was identified that retains full activity in an orientation-independent manner in combination with the promoter. The resulting transcripts initiate at the normal renin start site. Electrophoretic mobility shift assays identified a sequence at approximately position -60 in the promoter region that binds nuclear proteins specific for renin-expressing As4.1 cells. Mutations in this sequence, which disrupt binding of nuclear protein(s), completely abolish activation of transcription by the 4. 1-kb fragment. Activation of transcription by the 241-base pair enhancer was still observed, although it was diminished in magnitude (60-fold over the mutated promoter alone). We present a model derived from the current data that suggests that regulation of renin expression is achieved through cooperation of transcription factors binding at the proximal promoter element and a distal enhancer element to abrogate or override the effects of an intervening negative regulatory region.
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PMID:Role of proximal promoter elements in regulation of renin gene transcription. 879 16

Developmental studies of oncogene expression implicate the Fos and Jun family of transcription factors in the regulation of bone growth and differentiation. Promoters of many developmentally regulated genes, including osteocalcin, a marker of osteoblast differentiation, contain AP-1 sites that bind Fos/Jun dimers. Here, we demonstrate that the selective expression of fos- and jun-related genes is functionally related to the stage of osteoblast growth and differentiation in vitro. During osteoblast proliferation, nuclear protein levels of all seven activating protein-1 (AP-1) members are maximal. Subsequently, during the period of extracellular matrix maturation, levels decline. In fully differentiated osteoblasts, Fra-2 and (to a lesser extent) Jun D are the principal AP-1 members detectable by Western blot analysis. AP-1 complex composition and binding activity also exhibit developmental changes. All Fos and Jun family members are involved in AP-1 complex formation in proliferating cells, whereas Fra-2 and Jun D predominate in AP-1 complexes in differentiated osteoblasts. Overexpression of Fos and Jun family members in ROS 17/2.8 cells markedly affects the expression of an osteocalcin promoter-chloramphenicol acetyltransferase construct. Coexpression of only one AP-1 pair, Fra-2 and Jun D, stimulated reporter expression, whereas coexpression of other AP-1 pairs down-regulated expression (i.e. c-jun and any Fos family member) or had no effect (i.e. Fra-1 and Jun B). Promoter deletion analyses indicate that these effects are site specific. Consequential effects of Fra-2 on osteoblast differentiation are further demonstrated by antisense studies in which osteoblast differentiation and the development of a bone tissue-like organization were suppressed. Consistent with recent findings suggesting that AP-1 complex composition can selectively regulate gene transcription, our findings demonstrate that differential expression of Fos and Jun family members could play a role in the developmental regulation of bone-specific gene expression and, as a result, may be functionally significant for osteoblast differentiation.
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PMID:Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation. 882 1

Factor B (Bf) is a constituent of the alternative pathway of complement activation encoded within the major histocompatibility complex. Transcription of the murine gene from two initiation sites generates two Bf mRNA species differing in size and tissue distribution. Striking genetic, tissue-specific differences in Bf mRNA levels at extrahepatic sites (kidney and intestine) among mouse strains correlate with a DNA sequence polymorphism in the 5'-flanking region of the gene and differential nuclear protein binding at the Bf upstream transcriptional initiation site (UIS). To ascertain the functional consequences of this polymorphism in the Bf promoter, we analyzed the effects of strain-specific sequences in the Bf 5' region on the expression of a chloramphenicol acetyltransferase (CAT) reporter gene transfected in human and mouse hepatoma cells. The CAT activity and mRNA level produced when transcription was driven by the sequence of strains with high extrahepatic expression were reduced to background levels when the sequence specific to the low expressor strains was used. Eighty percent of this difference was accounted for by a point substitution that affects DNA-protein interaction at the UIS, the sequence of higher affinity conferring higher expression. Hepatocyte nuclear factor 4 (HNF-4), derived from HepG2, mouse liver and kidney or cell-free translation of HNF-4 RNA, is the nuclear protein that preferentially binds to the high expressor UIS. Bf-CAT is not expressed in cells that lack HNF-4 (CV-1). However, co-transfection of HNF-4 into CV-1 cells drives Bf-CAT expression and reproduces the differences derived from the substitution that affect HNF-4 binding in vitro. These data show that interaction of HNF-4 with polymorphic variants of the upstream Bf promoter is the major determinant of strain-specific extrahepatic factor B expression.
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PMID:Constitutive expression of murine complement factor B gene is regulated by the interaction of its upstream promoter with hepatocyte nuclear factor 4. 893 72

We previously identified a DNA binding element termed the upstream regulatory element (URE) that contains the consensus initiator sequence (Inr) in the upstream promoter of the rat prodynorphin gene. The URE displays specific binding to the upstream regulatory element binding protein (UREB1), a novel transcription regulator. Here, we report that the URE functions as a suppressor element. A series of chloramphenicol acetyltransferase reporters (pCAT) were constructed by subcloning either wild-type or mutated URE sequences into a pCAT reporter plasmid 5' of bases -135 to +58 of the prodynorphin gene. The basal -135 to +58 dynorphin promoter (pCAT 0.2) has robust transcriptional activity in Chinese hamster ovary (CHO) cells but not in rat pheochromocytoma PC12 cells. This robust transcriptional activity was completely inhibited in the presence of wildtype URE, whereas the mutations of the URE had no effect. Gel mobility shift assays showed that the complex formed by the URE and nuclear protein extracts can be competed by addition of wild-type URE oligonucleotide but not by specific mutations of the URE, defining particular bases required for protein interaction with the URE. The identical URE sequence is also found upstream in the promoter of human macrophage inflammatory protein 1 beta (hMIP 1 beta). The suppressive activity of the rat dynorphin URE can be replaced by the hMIP 1 beta URE. These data suggest that the URE may serve as a suppressor element in the regulation of dynorphin and hMIP 1 beta gene transcription.
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PMID:URE, an initiator (Inr)-like site, suppresses the promoter of the rat dynorphin gene. 907 Feb 43

The current study was undertaken to determine the mechanism by which the retinoid all-trans-retinoic acid regulates pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts. FRS fibroblasts were stably transfected with the ColCat3.6 plasmid, which contains a portion of the 5' flanking region of the rat pro alpha1(I) collagen gene linked to a reporter gene, chloramphenicol acetyltransferase. The effect of t-RA on CAT activity was determined as a function of concentration and incubation time. Maximal inhibition of CAT activity by t-RA occurred at 10(-8) M after 48 h of treatment. Transforming growth factor-beta1 did not block the inhibitory effect of t-RA on CAT activity. Computer sequence analysis of the 3.6-kb DNA fragment that contains the promoter for the rat pro alpha1(I) collagen gene identified a direct repeat RARE sequence composed of one diverse (5'-AGTAGA-3') and one idealized (5'-GGGTCA-3') half site located at positions -1345 and -1335, respectively. Two nuclear retinoid receptors that were expressed in bacteria, retinoic acid receptor-gamma and retinoid X receptor-alpha, were found to bind specifically to a double-stranded oligonucleotide containing the RARE in gel mobility shift assays. Mutation of the idealized half-site eliminated the binding of receptor proteins to the oligonucleotide. Gel mobility shift assays using nuclear protein extracts prepared from t-RA-treated FRS fibroblasts showed that binding to the oligonucleotide containing the RARE was decreased from control values. The same assays performed with the mutated oligonucleotide resulted in only slight binding. These studies indicate that t-RA downregulates the promoter activity of the rat pro alpha1(I) collagen gene by decreasing the binding of nuclear protein to the RARE sequence in the 5' flanking region of the gene.
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PMID:All-trans-retinoic acid inhibition of Pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts: identification of a retinoic acid response element in the Pro alpha1(I) collagen gene. 907 77

During cardiac hypertrophy, the chief myocardial energy source switches from fatty acid beta-oxidation (FAO) to glycolysis-a reversion to fetal metabolism. The expression of genes encoding myocardial FAO enzymes was delineated in a murine ventricular pressure overload preparation to characterize the molecular regulatory events involved in the alteration of energy substrate utilization during cardiac hypertrophy. Expression of genes involved in the thioesterification, mitochondrial import, and beta-oxidation of fatty acids was coordinately down-regulated after 7 days of right ventricular (RV) pressure overload. Results of RV pressure overload studies in mice transgenic for the promoter region of the gene encoding human medium-chain acyl-CoA dehydrogenase (MCAD, which catalyzes a rate-limiting step in the FAO cycle) fused to a chloramphenicol acetyltransferase reporter confirmed that repression of MCAD gene expression in the hypertrophied ventricle occurred at the transcriptional level. Electrophoretic mobility-shift assays performed with MCAD promoter fragments and nuclear protein extracts prepared from hypertrophied and control RV identified pressure overload-induced protein/DNA interactions at a regulatory unit shown previously to confer control of MCAD gene transcription during cardiac development. Antibody "supershift" studies demonstrated that members of the Sp (Sp1, Sp3) and nuclear hormone receptor [chicken ovalbumin upstream promoter transcription factor (COUP-TF)/erbA-related protein 3] families interact with the pressure overload-responsive unit. Cardiomyocyte transfection studies confirmed that COUP-TF repressed the transcriptional activity of the MCAD promoter. The DNA binding activities and nuclear expression of Sp1/3 and COUP-TF in normal fetal mouse heart were similar to those in the hypertrophied adult heart. These results identify a transcriptional regulatory mechanism involved in the reinduction of a fetal metabolic program during pressure overload-induced cardiac hypertrophy.
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PMID:A role for Sp and nuclear receptor transcription factors in a cardiac hypertrophic growth program. 917 36

A monomeric androgen responsive element (ARE) is not sufficient to mediate significant androgen induction of the prostate-specific antigen (PSA) gene. Co-transfection experiments using a series of 5'deletion fragments of the proximal promoter region of the PSA gene linked to bacterial chloramphenicol acetyltransferase (CAT) as a reporter have identified two motif sequences which are indispensable for androgen receptor (AR)-mediated transactivation of the PSA promoter and have been designated as motifs A and B respectively. Of note, motif B alone has very little independent enhancer activity regardless of the presence or absence of androgen, whereas multi-copies of motif A exert androgenic inducibility for a heterologous promoter independent of the presence of ARE. Nucleotide substitutions in either motif significantly decrease the androgen inducibility and the nuclear protein binding ability. Furthermore, gel band shift experiments consistently demonstrate that nuclear proteins can bind these motifs, and they are non-receptor factors. Our data indicate that these two DNA motifs are novel cis -regulatory elements and exhibit different mechanisms in cooperation with ARE for AR-mediated transactivation.
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PMID:Identification of two novel cis-elements in the promoter of the prostate-specific antigen gene that are required to enhance androgen receptor-mediated transactivation. 922 17

High levels of expression for the rat growth hormone (rGH) gene are restricted to the somatotroph cells of the anterior pituitary. Previously, we have shown that rGH cell-specific repression results in part from the recognition of negatively acting silencers by a number of nuclear proteins that repress basal promoter activity. Examination of these silencers revealed the presence of binding sites for proteins that belong to the NF1 family of transcription factors. Indeed, proteins from this family were shown to bind the rGH proximal silencer (designated silencer-1) in in vitro assays. Furthermore, this silencer site is capable of repressing chloramphenicol acetyltransferase (CAT) gene expression driven by an heterologous promoter (that of the mouse p12 gene), even in pituitary cells. Recently, we identified in the 5' untranslated region of the gene encoding human cellular retinol binding protein 1 (hCRBP1) a negative regulatory element (Fp1) that also bears an NF1 binding site very similar to that of rGH silencer-1. However, although deletion of Fp1 in the hCRBP1 gene yielded increased CAT activity, pointing toward a negative regulatory function exerted by this element, its insertion upstream of the p12 basal promoter results in an impressive positive stimulation of CAT gene expression. By exploiting NaDodSO4 gel protein fractionation and renaturation, we identified a 40-kD nuclear protein (designated Bp1) present in GH4C1 cells that binds very strongly to rGH silencer-1 but only weakly to hCRBP1 Fp1. Similarly, we also detected a 29-kD nuclear factor (designated Bp2) that recognizes exclusively the Fp1 element as its target site, therefore suggesting that different, but likely related, proteins bind these homologous elements to either activate or repress gene transcription. Although they bind DNA through the recognition of the NF1-like target sequence contained on these elements, competition and supershift experiments in electrophoretic mobility shift assays provided evidence that neither of these proteins belong to the NF1 family.
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PMID:The rat growth hormone and human cellular retinol binding protein 1 genes share homologous NF1-like binding sites that exert either positive or negative influences on gene expression in vitro. 930 37

The testis-specific histone gene H1t is expressed only in mammalian testis at the stage of pachytene spermatocytes. The tissue-specific regulation of the mouse H1t gene was examined in mouse testicular primary culture cells with gene constructs consisting of H1t promoter elements fused to the chloramphenicol acetyltransferase or the firefly luciferase reporter gene. Our experiments demonstrate that expression of the mouse H1t gene is enhanced by a conserved H1 histone gene-specific TG box 452 base pairs upstream of the transcription start site. The transcription of the H1t gene appears to be reduced by sequences between -1999 and -1506. No regulatory effect could be shown for the H1 box in the expression of the mouse H1t gene. Binding of nuclear protein extracted from mouse testis to these consensus elements was shown by electrophoretic mobility-shift assays with mouse testicular nuclear proteins and labeled oligonucleotides containing the upstream TG box sequences or the two testis-specific elements of the H1t gene.
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PMID:Testis-specific expression of the mouse histone gene H1t is regulated by several promoter elements. 940 43


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