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Results for “RNA, Competitive Endogenous”

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PubMed1995

Glucocorticoid receptor beta, a potential endogenous inhibitor of glucocorticoid action in humans.

Alternative splicing of the human glucocorticoid receptor (hGR) pre-mRNA generates two highly homologous isoforms, termed hGR alpha and hGR beta. hGR alpha is a ligand-activated transcription factor which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element (GRE) DNA sequences. In contrast, hGR beta does not bind glucocorticoids and is transcriptionally inactive. We demonstrate here that hGR beta is able to inhibit the effects of hormone-activated hGR alpha on a glucocorticoid-responsive reporter gene in a concentration-dependent manner. [3H]-Dexamethasone binding studies indicate that hGR beta does not alter the affinity of hGR alpha for its hormonal ligand. The presence of hGR beta in nuclear extracts and its ability to bind to a radiolabeled GRE oligonucleotide suggest that its inhibitory effect may be due to competition for GRE target sites. Reverse transcription-PCR analysis shows expression of hGR beta mRNA in multiple human tissues. These results indicate that hGR beta may be a physiologically and pathophysiologically relevant endogenous inhibitor of glucocorticoid action, which may participate in defining the sensitivity of target tissues to glucocorticoids. They also underline the importance of distinguishing between the two receptor isoforms in all future studies of hGR function and the need to revisit old data.

Alternative Splicing↗

PubMed2002

Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch-repair proteins.

The mechanism of expansion of the (CTG)n repeat in myotonic dystrophy (DM1) patients and the cause of its pathobiological effects are still largely unknown. Most likely, long repeats exert toxicity at the level of nuclear RNA transport or splicing. Here, we analyse cis- and trans-acting parameters that determine repeat behaviour in novel mouse models for DM1. Our mice carry 'humanized' myotonic dystrophy protein kinase (Dmpk) allele(s) with either a (CTG)84 or a (CTG)11 repeat, inserted at the correct position into the endogenous DM locus. Unlike in the human situation, the (CTG)84 repeat in the syntenic mouse environment was relatively stable during intergenerational segregation. However, somatic tissues showed substantial repeat expansions which were progressive upon aging and prominent in kidney, and in stomach and small intestine, where it was cell-type restricted. Other tissues examined showed only marginal size changes. The (CTG)11 allele was completely stable, as anticipated. Introducing the (CTG)84 allele into an Msh3-deficient background completely blocked the somatic repeat instability. In contrast, Msh6 deficiency resulted in a significant increase in the frequency of somatic expansions. Competition of Msh3 and Msh6 for binding to Msh2 in functional complexes with different DNA mismatch-recognition specificity may explain why the somatic (CTG)n expansion rate is differentially affected by ablation of Msh3 and Msh6.

Animals↗

PubMed2003

Promoters of the murine embryonic beta-like globin genes Ey and betah1 do not compete for interaction with the beta-globin locus control region.

Mammalian beta-globin loci contain multiple beta-like genes that are expressed at different times during development. The murine beta-globin locus contains two genes expressed during the embryo stage, Ey and betah1, and two genes expressed at both the fetal and postnatal stages, beta-major and beta-minor. Studies of transgenic human beta-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or betah1 promoter in the endogenous murine beta-globin locus. Promoter deletion eliminated expression of the mutant gene but did not affect expression of the remaining embryonic gene or the fetal-adult beta-globin genes on the mutant allele. These results demonstrate a lack of competitive effects between individual mouse embryonic beta-globin gene promoters and other genes in the locus. The implication of these findings for models of beta-globin gene expression are discussed.

Alleles↗

PubMed1999

alpha2D/A-adrenergic receptor gene induction in the retina by phorbol ester: involvement of an AP-2 element.

BACKGROUND: The alpha2-adrenergic receptor (alpha2-AR) expressed in the bovine retina has been demonstrated to be of the alpha2D subtype. The bovine alpha2D-adrenergic receptor (alpha2D/A-AR) gene has been cloned and characterized. This report describes the induction of this gene by phorbol- 12, 13-myristate acetate (PMA), an activator of protein kinase C (PKC). RESULTS: Treatment of the bovine retina for 60 min with PMA (1 micrometer) resulted in significant and similar increases in alpha2D/A-AR mRNA level and gene transcription. This indicates that PMA causes alpha2D/A-AR gene induction and that this induction takes place directly at the transcriptional level. In C6 cells, treatment with PMA at a concentration which was as low as 0.1 micrometer induced endogenous alpha2D/A-AR mRNA after 60 min. Luciferase reporter assays in C6 cells mapped the PMA-responsive element to a region between -247 bp and -163 bp on the alpha2D/A-AR promoter. Electrophoretic mobility shift assays showed an increased binding of nuclear factor(s) from PMA-treated bovine retina to this promoter region. Competition assays indicate that an AP-2 element may be involved in the PMA-dependent induction. CONCLUSION: These findings demonstrate for the first time, the direct induction of the alpha2D/A-AR gene by PMA and support a role for an AP-2 element in the induction mechanism.

Animals↗

PubMed1974

Reversal of pactamycin inhibition of methionyl-puromycin synthesis and 80S initiation complex formation by a ribosomal joining factor.

A crude mixture of polypeptide chain initiation factors (0.5 M KCl ribosomal wash) from reticulocyte ribosomes was fractionated by DEAE-cellulose column chromatography. Among several initiation factors obtained from the column, one factor eluting at 0.22-0.25 M KCl showed a remarkable ability to overcome the inhibition of Met-puromycin and 80S initiation complex formation caused by the antibiotic, pactamycin. Earlier experiments had shown that pactamycin does not prevent the binding of Met-tRNA(f) to the small ribosomal subunit but does interfere with the joining of the 60S ribosomal subunit to form the 80S initiation complex. A Lineweaver-Burk plot of initial rates of Met-puromycin formation showed that the interaction of the factor and pactamycin was of a competitive type. In the absence of the factor, [(35)S]Met-puromycin was not synthesized and [(35)S]Met-tRNA(f) bound only to the small ribosomal subunit. The amount of [(35)S]Met-tRNA(f) bound to 80S ribosomes bearing endogenous mRNA and the amount of [(35)S]Met-puromycin formed were directly related to the amount of factor added. Thus, this factor can be termed a "joining factor," and a simple assay of its activity can be devised based on its ability to overcome the pactamycin inhibition of the puromycin reaction.

Antibiotics, Antineoplastic↗

PubMed2004

Replication protein A (RPA) phosphorylation prevents RPA association with replication centers.

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2(D)) or alanine (RPA2(A)). Although RPA2(D) was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2(D) mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2(D) again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2(D). In contrast, under DNA damage or replication stress conditions, RPA2(D), like RPA2(A) and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with gamma-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

Animals↗

PubMed1992

Characterization of a bifunctional peptidylglycine alpha-amidating enzyme expressed in Chinese hamster ovary cells.

Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.

Amino Acid Sequence↗

PubMed2000

Influence of vegetables on the expression of GSTP1 in humans--a pilot intervention study (Sweden).

BACKGROUND: There are indications that a diet rich in vegetables and/or fruit has a protective effect against several types of diseases, including cancer. Data from experimental and epidemiological studies suggest that antioxidant constituents may provide protection against environmental carcinogens. AIMS: This study investigated the effect of additional vegetables in the diet on the expression of the endogenous antioxidant enzyme GSTP1 in human lymphocytes. METHODS: Six subjects were given an addition of mixed vegetables to their normal diet for a period of three weeks. The expression of GSTP1 protein and mRNA in lymphocytes was measured by Western blot and RT competitive PCR. RESULTS: After the intervention all six subjects had lower levels of GSTP1 mRNA, and five of the six subjects had lower GSTP1 protein levels. This suggests that increased vegetable intake decreases GSTP1 expression, possibly through the supply of additional antioxidants.

Adult↗

PubMed1991

Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1.

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.

Amino Acid Sequence↗

PubMed1992

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.

Animals↗

PubMed1999

Subplate neuron ablation alters neurotrophin expression and ocular dominance column formation.

Ocular dominance column formation in visual cortex depends on both the presence of subplate neurons and the endogenous expression of neurotrophins. Here we show that deletion of subplate neurons, which supply glutamatergic inputs to visual cortex, leads to a paradoxical increase in brain-derived neurotrophic factor mRNA in the same region of visual cortex in which ocular dominance columns are absent. Subplate neuron ablation also increases glutamic acid decarboxylase-67 levels, indicating an alteration in cortical inhibition. These observations imply a role for this special class of neurons in modulating activity-dependent competition by regulating levels of neurotrophins and excitability within a developing cortical circuit.

Base Sequence↗

PubMed2004

Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway.

The effect of a noncanonical Wnt, Wnt11, on canonical Wnt signaling stimulated by Wnt1 and activated forms of LRP5 (low density lipoprotein receptor-related protein-5), Dishevelled1 (Dvl1), and beta-catenin was examined in NIH3T3 cells and P19 embryonic carcinoma cells. Wnt11 repressed Wnt1-mediated activation of LEF-1 reporter activity in both cell lines. However, Wnt11 was unable to inhibit canonical signaling activated by LRP5, Dvl1, or beta-catenin in NIH3T3 cells, although it could in P19 cells. In addition, Wnt11-mediated inhibition of canonical signaling in NIH3T3 cells is ligand-specific; Wnt11 could effectively repress canonical signaling activated by Wnt1, Wnt3, or Wnt3a but not by Wnt7a or Wnt7b. Co-culture experiments with NIH3T3 cells showed that the co-expression of Wnt11 with Wnt1 was not an essential requirement for the inhibition, suggesting receptor competition as a possible mechanism. Moreover, in both cell types, elevation of intracellular Ca(2+) levels, which can result from Wnt11 treatment, led to the inhibition of canonical signaling. This result suggests that Wnt11 might not be able to signal in NIH3T3. Furthermore, P19 cells were found to express both endogenous canonical Wnts and Wnt11. Knockdown of Wnt11 expression using siRNA resulted in increased LEF-1 reporter activity, thus indicating that Wnt11-mediated suppression of canonical signaling exists in vivo.

Animals↗

PubMed1995

The human apolipoprotein E gene is negatively regulated in human liver HepG2 cells by the transcription factor BEF-1.

Apolipoprotein E (apoE) is a major constituent of plasma lipoprotein that functions in lipid transport and redistribution (reverse cholesterol transport) and probably plays an important role in inhibiting the development and/or progression of atherosclerosis. While cis-acting regions involved in basal and tissue-specific control of the apoE gene have been identified by promoter mapping studies, much less is known about factors that regulate the gene. In this study, we demonstrate that the region between -94 and -84 upstream of transcriptional start site of the human apoE gene contains a binding site for the transcriptional repressor factor BEF-1, a tyrosine-phosphorylated nuclear protein that was first identified in HeLa cells. Using gel retardation assays, we show that HeLa cell-derived BEF-1 binds the apoE BEF-1 homology, and this binding can be competed with the prototype BEF-1 sequence, but not by a mutated sequence. Furthermore, we demonstrate that the apoE- producing human liver HepG2 cell produces significant levels of BEF-1, which could bind to both the prototype BEF-1 sequence and the apoE homology, and be competed equivalently with cold BEF-1 or apoE homology. To determine if BEF-1 affected the expression of apoE, we performed competition experiments using plasmids containing the intact or mutated BEF-1 homology. The introduction of the intact BEF-1 site into HepG2 cells resulted in an induction of apoE mRNA, whereas control and mutated BEF-1-containing plasmids had no significant effect. We also found that increasing the level of nuclear BEF-1 by treatment of cells with orthovanadate resulted in a reduction in the level of apoE mRNA. Overall, our data suggest that the endogenous apoE gene in the human HepG2 cell line is repressed by the trans-acting influence of nuclear factor BEF-1.

Apolipoproteins E↗

PubMed2000

Characterization of the promoter of human leukocyte-specific transcript 1. A small gene with a complex pattern of alternative transcripts.

The gene for the human leukocyte-specific transcript 1 (LST1) encodes a small protein that modulates immune responses and cellular morphogenesis. The LST1 transcripts are expressed at high levels in dendritic cells. Because of the complex splicing pattern, use of alternative 5'-untranslated exons, and a biologically interesting pattern of expression of LST1 mRNA, we studied the human LST1 gene promoter and regulatory elements. We identified an additional upstream 5'-untranslated exon in U937 monocytic cells. Transient transfection studies demonstrated that the combination of regions from -1363 to -621 with -112 to -54, relative to the translation start codon, produced the highest level of transcripts from among the various constructs tested, but the pattern of transcripts produced was only a subset of those produced from the endogenous gene. DNase I footprinting analysis and electrophoretic mobility shift assays showed that oligonucleotide probes corresponding to three regions, -1171 to -1142 (BI), -1136 to -1111 (BII), and -783 to -751 (BIV), bound proteins in U937 nuclear extracts. Competition and supershift electrophoretic mobility shift assay did not identify any known transcription factors responsible for BII probe binding. These studies suggest that a novel DNA-binding site and interaction of multiple regulatory elements may be involved in mediating the expression of the various forms of LST1 mRNA.

Alternative Splicing↗

PubMed1987

Expression of mouse histone genes: transcription into 3' intergenic DNA and cryptic processing sites downstream from the 3' end of the H3 gene.

Introduction of the mouse histone H3.1 gene into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene resulted in the production of two mRNAs from the transfected gene, one with a normal 3' end and the other one with a longer 3'-untranslated region, ending at site X, which was poly(A)+. In contrast, the endogenous histone H3.1 gene only produced a single mRNA. The cryptic poly(A)+ site was only used when the histone H3.1 gene was transfected. To localize possible downstream cryptic processing sites, the hairpin loop at the end of the histone gene was deleted and the resulting deletions were introduced into L cells. Two major mRNAs were produced from this gene, one ending at site X and the major one ending at site Y, which was located 150 nucleotides before site X. Transcription extended downstream of site X efficiently in the endogenous gene, as judged by the extent of transcription of downstream sequences in isolated nuclei. Transcription extended downstream of site X in the transfected gene because the placement of a normal histone 3' end downstream of site X resulted in transcripts that ended at site X and longer transcripts that ended with the new histone 3' end. These results indicate that transcription may normally proceed a substantial distance past the hairpin loop (greater than 500 bases). The formation of the different 3' ends in these transfected genes was due to competition between different processing mechanisms.

Animals↗

PubMed1996

Platelet activating factor and endotoxin increase the enzyme activity and gene expression of type II phospholipase A2 in the rat intestine. Role of polymorphonuclear leukocytes.

Type II phospholipase A2 (PLA2-II) may regulate eicosanoid synthesis; is involved in various inflammatory processes, septic shock, and inflammatory bowel disease; and is up-regulated by LPS and inflammatory cytokines in vitro. We have previously shown that the platelet-activating factor (PAF)-induced intestinal injury is attenuated by peptido-leukotriene antagonists, suggesting a role of PLA2. The purpose of this study is to examine the regulation of intestinal PLA2-II by PAF and LPS. Using synthesized competitor RNA as competitor, we quantified PLA2-II transcripts by competitive reverse transcription-PCR. We found that PLA2-II gene is constitutively expressed in the rat ileum. PAF at a dose (1.5 micrograms/kg) below that causing intestinal injury rapidly up-regulated intestinal PLA2-II at both transcriptional and post-transcriptional levels, almost tripling its transcripts and significantly increasing enzyme activity in 30 min. LPS (5 mg/kg) also up-regulated PLA2-II. This effect could not be blocked by PAF antagonist. Depletion of circulating polymorphonuclear leukocytes (PMNs) abolished the effect of PAF on PLA2-II gene expression and enzyme activation. In contrast, PMN depletion prevented LPS-induced PLA2-II enzyme activity but enhanced the gene expression. We conclude that: 1) both PAF and LPS induce gene transcription and enzyme activation of PLA2-II in the small intestine; 2) PAF up-regulates PLA2-II via PMN activation; 3) LPS effect is independent of endogenous PAF formation; and 4) different pathways exist for PAF and LPS in the regulation of intestinal PLA2-II gene expression in vivo.

Animals↗

PubMed1997

Competitive PCR quantification of pro- and anti-inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced by immunization with myelin basic protein (MBP)/complete Freund's adjuvant and serves as a model for multiple sclerosis. Recent studies have suggested that cytokines play a crucial role in the clinical course of EAE. To clarify the roles of cytokines in EAE, we examined levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) mRNA in isolates from infiltrating inflammatory cells in EAE lesions induced in Lewis rats. The non-radioactive and sensitive competitive PCR method was employed to quantify the relative amounts of cytokine mRNA. Levels of both IFN-gamma and TNF-alpha mRNA were increased at the early stage of EAE and rapidly decreased at the peak stage. On the other hand, TGF-beta1 mRNA was demonstrated throughout the course of EAE as well as under normal conditions and its amount paralleled the severity of EAE. IL-10 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) under normal conditions, but was below the level of detection of competitive PCR. IL-10 mRNA expression peaked at the early stage of EAE and declined gradually thereafter. Taken together, these results suggest that IFN-gamma and TNF-alpha might play a crucial role in the development of EAE. Furthermore, it appears that the peak expression of IL-10 mRNA at the early stage and the following marked TGF-beta1 expression at the peak stage might represent an important endogenous mechanism to limit the extent of inflammation and to prevent relapse in the course of acute monophasic EAE.

Animals↗

PubMed1994

Serine protease inhibitors block N-terminal arginylation of proteins by inhibiting the arginylation of tRNA in rat brains.

The tRNA mediated, posttranslational, N-terminal arginylation of proteins occurs in all eukaryotic cells. In nervous tissue, these reactions can be inhibited by endogenous molecules with a molecular weight of between one thousand and five thousand. In the present experiments, exogenous serine protease inhibitors (10(-5) M or less) but not other types of protease inhibitors, were found to be able to block the arginylation of protein in extracts of rat brain homogenates. Inhibition was not by the usual mode of action of protease inhibitors, but by interfering (non-competitively) with the charging of tRNA. Since arginylated proteins are rapidly ubiquitinated and degraded by cytosolic proteases, serine protease inhibitors may act to stabilize proteins by a dual mechanism of inhibiting arginylation as well as inhibiting serine proteases.

Animals↗