Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcriptional regulation of the glycoprotein hormone alpha-subunit gene has been studied extensively in placental cells, but much less is known about its regulation in the pituitary gland. In this study, transcriptional control of the human alpha-subunit gene by GnRH was analyzed using transient expression assays in primary cultures of rat pituitary cells using alpha promoter constructs linked to a luciferase reporter gene. Deletion mutants between -846 and -156 basepairs (bp) had little effect on basal expression, but further deletion to -132 bp reduced basal activity by approximately 50%. Deletion of a cAMP response element between -132 and -99 bp caused a marked loss of basal activity, reducing expression to that of background luciferase activity. The same constructs were analyzed for cAMP responsiveness in primary pituitary cells. The degree of stimulation with 1 mM 8-bromo-cAMP (3.6- to 6.0-fold) was relatively unaffected by deletions from -846 to -132 bp, whereas cAMP stimulation was decreased by further deletion to -99 bp, consistent with the presence of previously defined cAMP response elements in this region of the alpha promoter. GnRH stimulation of alpha promoter activity was highly dependent upon the time of hormone addition after transfection, being most effective when added soon after transfection. Under optimal conditions, GnRH stimulated -846 alpha LUC expression by 20-fold. GnRH responsiveness was retained with deletion to -346 bp, but it was decreased by 55% after deletion to -280 bp and by 79% with deletion to -244 bp.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter. 128 68

The LH/CG receptor is a G protein-coupled receptor present on gonadal cells whose levels are modulated by a number of hormones, growth factors, and second messenger analogs. With the recently cloned cDNA for the LH/CG receptor, it has been shown that changes in the levels of the cognate mRNA are involved, at least in part, in the observed changes in receptor density. In order to study the transcriptional regulation of the LH/CG receptor we have isolated a 2-kilobase region of the 5'-flanking region of the rat LH/CG receptor gene and subcloned nucleotide -1 (relative to the translational initiation codon) to -1370 into a luciferase reporter plasmid. We show here that this region of the LH/CG receptor gene is able to enhance luciferase activity in MA-10 cells, a line of Leydig tumor cells that normally express LH/CG receptors, as opposed to human kidney 293 cells, which do not. Furthermore, the addition of 8-bromo-cAMP to MA-10 cells, under conditions known to decrease LH/CG receptor numbers and receptor mRNA levels, decreases the relative luciferase activity to about 26% of control. This decrease in reporter gene activity is severely blunted in a subclone of MA-10 cells with a cAMP-resistant phenotype. Our studies show, for the first time, that sequence(s) present with 1370 base pairs of the translational start site of the rat LH/CG receptor gene are sufficient for conferring expression of this gene in Leydig cells and for the negative modulation of LH/CG receptor gene transcription by high concentrations of cAMP.
 ...
PMID:The 5'-flanking region of the rat luteinizing hormone/chorionic gonadotropin receptor gene confers Leydig cell expression and negative regulation of gene transcription by 3',5'-cyclic adenosine monophosphate. 131 38

The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.
 ...
PMID:Expression of recombinant human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts. 132 83

Cartilage breakdown, as seen in inflammatory and degenerative joint diseases, can be mediated by proteolytic enzymes, such as the metalloproteinase collagenase, the only enzyme able to digest collagen at neutral pH. In vitro collagenase gene expression can be stimulated by the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. We have investigated the effect of prostaglandin E1 (PGE1) on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated collagenase mRNA levels in the rabbit synoviocyte cell line HIG-82. PGE1, but not PGE2 or PGF2 alpha, was able to selectively reduce collagenase mRNA levels in a dose-dependent fashion. PGE1 markedly increased intracellular levels of cAMP, while PGE2 and PGF2 alpha had little or no effect on cAMP production in the HIG-82 synoviocytes. Agents known to increase intracellular cAMP levels, such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), mimicked the effect of PGE1, on collagenase mRNA levels. PGE1, forskolin, and IBMX also decreased collagenase mRNA levels in human skin fibroblasts, demonstrating that this observation was not unique to the HIG-82 cell line. Transient transfection experiments carried out in HIG-82 cells using a 1.2-kilobase portion of the 5'-flanking region of the human collagenase gene linked to the reporter gene luciferase demonstrated that PGE1, forskolin, and IBMX exert their inhibitory effect on the promoter region of the collagenase gene.
 ...
PMID:Prostaglandin E1 inhibits collagenase gene expression in rabbit synoviocytes and human fibroblasts. 137 21

The nature of a DNA element located in the -100 to -85 region of the rat PRL gene has been characterized. Previous studies demonstrated that this region may contribute to basal and hormonally regulated expression of the PRL gene. As this region contains a sequence with similarity to a consensus cAMP-responsive element (CRE), a possible role for the cAMP response element binding protein (CREB) has been explored. A point mutation which made the PRL CRE-like sequence less like a consensus CRE had little effect on basal or cAMP-stimulated expression of a PRL-luciferase reporter gene. DNase footprint studies demonstrated that the proximal region of the PRL gene does not contain a high affinity CREB binding site. Mobility shift experiments demonstrated that the major GH3 nuclear protein which interacts with the -100 to -85 region of the PRL gene in vitro is not CREB. Transfection of a dominant inhibitor of CREB action had little or no effect on expression of an indicator gene containing the PRL proximal region. Thus, the PRL proximal region does not contain a high affinity CREB binding site, and it is unlikely that CREB plays a major role in expression of the PRL gene. The functional capabilities of the -100 to -85 region of the PRL gene were then tested in a transfection assay. Synthetic multimers of this region were found to be sufficient to permit a transcriptional response to cAMP or TRH in GH3 cells and cAMP in Rat-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Characterization of a non-tissue-specific, 3',5'-cyclic adenosine monophosphate-responsive element in the proximal region of the rat prolactin gene. 138 49

We have determined the structural organization of the dihydrolipoyl transacylase (E2) gene of the human branched chain alpha-keto acid dehydrogenase complex. The single copy E2 gene spans approximately 68 kilobases of genomic DNA. The complete coding region consisting of the 5'- and 3'-untranslated regions, the mitochondrial targeting sequence (61 amino acids), and the mature E2 sequence (421 amino acids) are encoded by 11 exons ranging from 62 to 2239 base pairs. All the donor and acceptor splice sites conform to the gt-ag rule. Sequence analysis of the promoter-regulatory region showed the presence of a "CAAT box"-like sequence 537 bases upstream of the transcription initiation site. The "TATA box"-like sequence is absent. Also located in this region are sequences resembling glucocorticoid-responsive and cAMP-responsive elements, fat-specific elements, and Sp1- and AP-2-binding sites. Several sets of direct and inverted repeats are also present. Promoter assays using human hepatoma cells (Hep-G2) and Swiss mouse preadipocytes (3T3-L1) showed that a 4.1-kilobase PstI fragment upstream of the transcription start site confers high expression of the luciferase reporter gene. Moreover, an intronless E2 pseudogene was isolated. It corresponds to the complete mitochondrial presequence and the lipoyl-bearing domain that are encoded by exons I through IV of the functional E2 gene. However, the E2 pseudogene contains multiple base changes, deletions, and insertions, and is flanked by short direct repeats. The data indicate that the E2 pseudogene is a retroposon.
 ...
PMID:Structure of the gene encoding dihydrolipoyl transacylase (E2) component of human branched chain alpha-keto acid dehydrogenase complex and characterization of an E2 pseudogene. 142 40

Iron influences luminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB+ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICDABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di(o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB+ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8-9 fold each for tonB, 2-3 fold each for tonB+) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.
 ...
PMID:Iron control of the Vibrio fischeri luminescence system in Escherichia coli. 151 May 56

CG is encoded by separate alpha- and beta-subunit genes. Expression of both genes is stimulated by cAMP, but the kinetics of activation are different, with cAMP stimulation of the alpha gene preceding that of the beta gene. The cAMP response element (CRE) in the alpha gene contains a palindromic DNA sequence, TGACGTCA, that binds the transcription factor CREB, a nuclear phosphoprotein that is activated by protein kinase-A. Previously, detailed characterization of a CRE in the CG beta gene had been difficult due to low levels of expression in transfected cells. In this study the 5'-flanking sequence of the CG beta gene was fused to a sensitive luciferase (LUC) reporter gene, allowing delineation of a CG beta CRE in transient expression assays performed in JEG-3 choriocarcinoma cells. The full-length CG beta promoter, -3700 to 362 basepairs (bp), was stimulated 8- to 14-fold by treatment with 1 mM 8-bromo-cAMP. Analyses of a series of deletion mutants in the CG beta promoter demonstrated that -311 CG beta LUC retained nearly complete cAMP stimulation, but deletion to -187 bp eliminated cAMP responsiveness. Overlapping DNA fragments between -311 and -30 bp were fused to a heterologous promoter (-99 alpha LUC) to further define the locations of basal elements and CREs. Basal expression required a combination of at least two distinct elements between -311 and -30 bp, whereas cAMP responsiveness was conferred by sequences between -311 and -202 bp. Shorter DNA sequences within this region were insufficient for cAMP stimulation, suggesting that more than one element may be required. DNase-I footprinting and gel mobility shift studies demonstrated at least three distinct protein-binding sites within the CG beta CRE sequence. Recombinant CREB (expressed in E. coli) did not bind to these sites, and they share no sequence homology with the alpha gene CRE, indicating that a cAMP-responsive transcription factor other than CREB interacts with the CG beta promoter.
 ...
PMID:Novel cyclic adenosine 3',5'-monophosphate response element in the human chorionic gonadotropin beta-subunit gene. 164 92

Previously, 5' deletion analysis revealed three important upstream regions within the regulatory region of the cAMP-induced, prespore gene SP60 of D. discoidium, each of which contains a CA-rich sequence element (CAE: consensus CACACAYYYCACACAAA/T). In this study, we have made site-directed mutations within these CAEs and examined their effect on reporter gene activity (luciferase or lacZ). Point mutations within or deletion of the distal CAE (CAE-1), middle CAE (CAE-2) or proximal CAE (CAE-3) result in substantial decreases in promoter activity at 18 h of development or in response to cAMP. lacZ fusions made with the CAE mutant promoters produced novel beta-gal staining patterns that suggest the presence of one or more morphogen gradients within the prespore zone of the slug and indicate that the CAEs are also important in regulating the spatial patterning of SP60 expression in the multicellular aggregate. Gel mobility shift assays were used to identify activities from crude nuclear extracts that bind oligonucleotides containing the CAEs. One of the binding activities is not observed in extracts from vegetative cells or cells in early development and is induced during multicellular development with kinetics similar to those of SP60 gene expression. This activity is also induced in response to cAMP and specifically binds the wild-type CAE-1- and CAE-2-containing oligonucleotides. CAE-1 and CAE-2 oligonucleotides containing point mutations within the CAE core sequence neither bind to nor compete for the cAMP-induced, developmentally regulated factor(s) and result in substantial reductions in expression levels when substituted for the wild-type CAEs in vivo. The correlation between in vitro binding and in vivo function suggests that the CAE-1/CAE-2 binding activity may be involved in regulating cAMP and developmentally induced expression of SP60. A second, constitutive in vitro binding activity with high affinity to CAE-3 is also described. Models are proposed to relate the binding activities with the effects of the mutations on the spatial patterning of SP60-lacZ expression.
 ...
PMID:Developmental and spatial regulation of a Dictyostelium prespore gene: cis-acting elements and a cAMP-induced, developmentally regulated DNA binding activity. 166 78

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte function-associated Ag-1 (LFA-1), plays an important role in leukocyte-endothelial cell interactions. It is induced by proinflammatory cytokines such as IL-1, TNF-alpha, or IFN-gamma. However, little is known concerning the intracellular regulatory mechanisms which trigger ICAM-1 up-regulation. In order to study potential regulatory elements involved in ICAM-1 induction we have cloned the human ICAM-1 gene and 5 kb of its 5'-regulatory region. The sequence of the cDNA was found to be distributed over seven exons separated by six introns, whereby each of the five extracellular Ig-like domains of ICAM-1 is encoded by its own exon. The upstream sequence harbors a number of sequence motifs implicated in the regulation and expression of eukaryotic genes, including binding sites for the transcription factors SP-1, AP-1, and NF-kB. Primer extension and S1 nuclease analysis revealed two transcription initiation sites 319 bp and 41 bp upstream of the translation start site. Consensus TATA boxes were found at the expected positions about 25 bp upstream of both start sites. Reverse transcriptase polymerase chain reaction showed differential use of the two TATA boxes in A549 and HS913T cells. Both RNA seem to code for the same for of ICAM-1 protein. For regulation studies a 1.3-kb EcoRI/SalI fragment of the 5'-flanking region was used to promote transcription of a linked luciferase reporter gene in transient-transfection assays in A549 and HS913T cells. Treatment of A549 cells with IL-1 or TNF-alpha resulted in a two- or fourfold increase in luciferase activity. Furthermore, a sixfold induction could be achieved after treatment with the phorbol ester PMA. In contrast, agents that increase intracellular cAMP levels did not induce luciferase activity. Northern blot analysis was used to investigate the kinetics of ICAM-1 mRNA synthesis upon induction with TNF-alpha and PMA. These data suggest that the up-regulation of ICAM-1 by cytokines occurs at least partly at the transcriptional level. Deletion analysis of the 1.3-kb fragment of the 5'-flanking region revealed sequences responsible for promotion and inhibition of transcription. In particular, two functionally distinct regions have been characterized: a short fragment containing an NF-kB binding site has been shown to function as an activator, followed immediately downstream by a sequence acting as a silencer element. Therefore, ICAM-1 gene expression seems to be modulated by multiple cis-acting elements.
 ...
PMID:Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5'-regulatory region. Induction by cytokines and phorbol ester. 168 Sep 19


1 2 3 4 5 6 7 8 9 10 Next >>