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Binding Sequence (binding + sequence)
Selected AbstractsAn enhancer sequence directs LacZ expression to developing pharyngeal endoderm in transgenic miceGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2001Hema Parmar Abstract Summary: The murine Hoxc-6 homeobox gene comprises three exons with two distinct promoters (PRI and PRII) located 9 kb apart. To characterise the PRII promoter, a region 3 kb upstream of the transcription start site was sequenced, and an Antananapedia-like consensus binding sequence was found (Coletta et al., 1991). A LacZ reporter gene construct, containing three copies of this sequence, directs highly specific expression in cells forming pharyngeal endoderm in transgenic mice. Expression was first detected in a few individual anterior endoderm cells at E7.5, which increase in number up to E9.5, where expression was clearly visible in the pharyngeal endoderm. Expression of the endodermal genes HNF3,, Pax-9, Shh, and Nkx2.5 showed colocalization with the LacZ -positive cells in the foregut and pharyngeal endoderm. This novel enhancer provides a means of tracking the morphogenetic movement of endodermal cells fated to form the foregut. genesis 31:57,63, 2001. © 2001 Wiley-Liss, Inc. [source] IFN-,-induced BACE1 expression is mediated by activation of JAK2 and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytesGLIA, Issue 3 2007Hyun Jin Cho Abstract ,-Site APP cleaving enzyme 1 (BACE1) is an essential enzyme for the production of , amyloid. Since we found that injection of interferon-, (IFN-,) into young mouse brains increased BACE1 expression in astrocytes, we investigated molecular mechanisms underlying this process by cloning a putative BACE1 promoter. BACE1 promoter activity was differentially regulated by IFN-, in a region specific manner and down-regulated by an inhibitor of Janus kinase 2 (JAK2). A dominant negative mutant of signal transducer and activator of transcription 1 (STAT1) expression suppressed BACE1 promoter activity, and this was rescued by transfecting wild type STAT1. Electrophoretic mobility shift assay and promoter activity assays indicated that STAT1 binds directly to the putative STAT1 binding sequence of BACE1 promoter. Because IFN-, treatment induced STAT1 phosphorylation, we examined whether the expression of a suppressor of cytokine signaling (SOCS), negative regulator of JAK2, suppresses BACE1 promoter activity. The results show that SOCS1 or SOCS3 expression suppressed BACE1 promoter by blocking phosphorylation of Tyr701 residue in STAT1. Also, because IFN-, treatment specifically potentiated extracellular signal regulated MAP kinase (ERK) 1/2 activation, pretreatment of mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitor, PD98059, significantly attenuated IFN-,-induced BACE1 promoter activity and protein expression through blocking phosphorylation of Ser727 residue in STAT1, suggesting that ERK1/2 is associated with IFN-,-induced STAT1 signaling cascade. Taken together, our results suggest that IFN-, activates JAK2 and ERK1/2 and then phosphorylated STAT1 binds to the putative STAT1 binding sequences in BACE1 promoter region to modulate BACE1 protein expression in astrocytes. © 2006 Wiley-Liss, Inc. [source] Constitutive androstane receptor (CAR) ligand, TCPOBOP, attenuates Fas-induced murine liver injury by altering Bcl-2 proteins,HEPATOLOGY, Issue 1 2006Edwina S. Baskin-Bey The constitutive androstane receptor (CAR) modulates xeno- and endobiotic hepatotoxicity by regulating detoxification pathways. Whether activation of CAR may also protect against liver injury by directly blocking apoptosis is unknown. To address this question, CAR wild-type (CAR+/+) and CAR knockout (CAR,/,) mice were treated with the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and then with the Fas agonist Jo2 or with concanavalin A (ConA). Following the administration of Jo2, hepatocyte apoptosis, liver injury, and animal fatalities were abated in TCPOBOP-treated CAR+/+ but not in CAR,/, mice. Likewise, acute and chronic ConA-mediated liver injury and fibrosis were also reduced in wild-type versus CAR,/, TCPOBOP-treated mice. The proapoptotic proteins Bak (Bcl-2 antagonistic killer) and Bax (Bcl-2-associated X protein) were depleted in livers from TCPOBOP-treated CAR+/+ mice. In contrast, mRNA expression of the antiapoptotic effector myeloid cell leukemia factor-1 (Mcl-1) was increased fourfold. Mcl-1 promoter activity was increased by transfection with CAR and administration of TCPOBOP in hepatoma cells, consistent with a direct CAR effect on Mcl-1 transcription. Indeed, site-directed mutagenesis of a putative CAR consensus binding sequence on the Mcl-1 promoter decreased Mcl-1 promoter activity. Mcl-1 transgenic animals demonstrated little to no acute liver injury after administration of Jo2, signifying Mcl-1 cytoprotection. In conclusion, these observations support a prominent role for CAR cytoprotection against Fas-mediated hepatocyte injury via a mechanism involving upregulation of Mcl-1 and, likely, downregulation of Bax and Bak. (HEPATOLOGY 2006;44:252,262.) [source] A novel repressor of nif and glnA expression in the methanogenic archaeon Methanococcus maripaludisMOLECULAR MICROBIOLOGY, Issue 1 2003Thomas J. Lie Summary Nitrogen assimilation in the methanogenic archaeon Methanococcus maripaludis is regulated by transcriptional repression involving a palindromic ,nitrogen operator' repressor binding sequence. Here we report the isolation of the nitrogen repressor, NrpR, from M. maripaludis using DNA affinity purification. Deletion of the nrpR gene resulted in loss of nitrogen operator binding activity in cell extracts and loss of repression of nif (nitrogen- fixation) and glnA (glutamine synthetase) gene expression in vivo. Genetic complementation of the nrpR mutation restored all functions. NrpR contained a putative N-terminal winged helix,turn,helix motif followed by two mutually homologous domains of unknown function. Comparison of the migration of NrpR in gel-filtration chromatography with its subunit molecular weight (60 kDa) suggested that NrpR was a tetramer. Several lines of evidence suggested that the level of NrpR itself is not regulated, and the binding affinity of NrpR to the nitrogen operator is controlled by an unknown mechanism. Homologues of NrpR were found only in certain species in the kingdom Euryarchaeota. Full length homologues were found in Methanocaldococcus jannaschii and Methanothermobacter thermoautotrophicus, and homologues lacking one or more of the three polypeptide domains were found in Archaeoglobus fulgidus, Methanopyrus kandleri, Methanosarcina acetivorans, and Methanosarcina mazei. NrpR represents a new family of regulators unique to the Euryarchaeota. [source] Identification of a novel cis -acting element conferring sulfur deficiency response in Arabidopsis rootsTHE PLANT JOURNAL, Issue 3 2005Akiko Maruyama-Nakashita Summary SULTR1;1 high-affinity sulfate transporter is highly regulated in the epidermis and cortex of Arabidopsis roots responding to sulfur deficiency (,S). We identified a novel cis -acting element involved in the ,S-inducible expression of sulfur-responsive genes in Arabidopsis. The promoter region of SULTR1;1 was dissected for deletion and gain-of-function analysis using luciferase (LUC) reporter gene in transgenic Arabidopsis. The 16-bp sulfur-responsive element (SURE) from ,2777 to ,2762 of SULTR1;1 promoter was sufficient and necessary for the ,S-responsive expression, which was reversed when supplied with cysteine and glutathione (GSH). The SURE sequence contained an auxin response factor (ARF) binding sequence (GAGACA). However, SURE was not responsive to naphthalene acetic acid, indicating its specific function in the sulfur response. The base substitution analysis indicated the significance of a 5-bp sequence (GAGAC) within the conserved ARF binding site as a core element for the ,S response. Microarray analysis of early ,S response in Arabidopsis roots indicated the presence of SURE core sequences in the promoter regions of ,S-inducible genes on a full genome GeneChip array. It is suggested that SURE core sequences may commonly regulate the expression of a gene set required for adaptation to the ,S environment. [source] Glutamate receptors on myelinated spinal cord axons: I. GluR6 kainate receptors,ANNALS OF NEUROLOGY, Issue 2 2009Mohamed Ouardouz PhD Objective The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Although overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptor,dependent axonal Ca2+ deregulation. Methods Dorsal column axons were loaded with a Ca2+ indicator and imaged in vitro using confocal laser-scanning microscopy. Results Activation of glutamate receptor 6 (GluR6) kainate receptors promoted a substantial increase in axonal [Ca2+]. This Ca2+ accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine-dependent intracellular stores, which, in turn, depended on activation of L-type Ca2+ channels: ryanodine, nimodipine, or nifedipine blocked the agonist-induced Ca2+ increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca2+ response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal N,-nitro-L-arginine methyl ester, blocked the Ca2+ increase. Loading axons with a peptide that mimics the C-terminal PDZ binding sequence of GluR6, thus interfering with the coupling of GluR6 to downstream effectors, greatly reduced the agonist-induced axonal Ca2+ increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Cav1.2. Interpretation Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal "nanocomplexes" to release toxic amounts of Ca2+ may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur. Ann Neurol 2009 [source] A Targeted Releasable Affinity Probe (TRAP) for In Vivo PhotocrosslinkingCHEMBIOCHEM, Issue 9 2009Ping Yan Dr. Abstract A protein TRAP: The in vivo photocrosslinking of TRAP after its intracellular targeting to a binding sequence on the bait protein stabilizes protein interactions. Because the crosslinker is releasable, simple mass spectrometry can be used to identify the protein binding sites after purification. Protein crosslinking, especially coupled to mass-spectrometric identification, is increasingly used to determine protein binding partners and protein,protein interfaces for isolated protein complexes. The modification of crosslinkers to permit their targeted use in living cells is of considerable importance for studying protein-interaction networks, which are commonly modulated through weak interactions that are formed transiently to permit rapid cellular response to environmental changes. We have therefore synthesized a targeted and releasable affinity probe (TRAP) consisting of a biarsenical fluorescein linked to benzophenone that binds to a tetracysteine sequence in a protein engineered for specific labeling. Here, the utility of TRAP for capturing protein binding partners upon photoactivation of the benzophenone moiety has been demonstrated in living bacteria and mammalian cells. In addition, ligand exchange of the arsenic,sulfur bonds between TRAP and the tetracysteine sequence to added dithiols results in fluorophore transfer to the crosslinked binding partner. In isolated protein complexes, this release from the original binding site permits the identification of the proximal binding interface through mass spectrometric fragmentation and computational sequence identification. [source] IFN-,-induced BACE1 expression is mediated by activation of JAK2 and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytesGLIA, Issue 3 2007Hyun Jin Cho Abstract ,-Site APP cleaving enzyme 1 (BACE1) is an essential enzyme for the production of , amyloid. Since we found that injection of interferon-, (IFN-,) into young mouse brains increased BACE1 expression in astrocytes, we investigated molecular mechanisms underlying this process by cloning a putative BACE1 promoter. BACE1 promoter activity was differentially regulated by IFN-, in a region specific manner and down-regulated by an inhibitor of Janus kinase 2 (JAK2). A dominant negative mutant of signal transducer and activator of transcription 1 (STAT1) expression suppressed BACE1 promoter activity, and this was rescued by transfecting wild type STAT1. Electrophoretic mobility shift assay and promoter activity assays indicated that STAT1 binds directly to the putative STAT1 binding sequence of BACE1 promoter. Because IFN-, treatment induced STAT1 phosphorylation, we examined whether the expression of a suppressor of cytokine signaling (SOCS), negative regulator of JAK2, suppresses BACE1 promoter activity. The results show that SOCS1 or SOCS3 expression suppressed BACE1 promoter by blocking phosphorylation of Tyr701 residue in STAT1. Also, because IFN-, treatment specifically potentiated extracellular signal regulated MAP kinase (ERK) 1/2 activation, pretreatment of mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitor, PD98059, significantly attenuated IFN-,-induced BACE1 promoter activity and protein expression through blocking phosphorylation of Ser727 residue in STAT1, suggesting that ERK1/2 is associated with IFN-,-induced STAT1 signaling cascade. Taken together, our results suggest that IFN-, activates JAK2 and ERK1/2 and then phosphorylated STAT1 binds to the putative STAT1 binding sequences in BACE1 promoter region to modulate BACE1 protein expression in astrocytes. © 2006 Wiley-Liss, Inc. [source] Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouseJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2001Edward D. Karoly Abstract The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5, flanking region, 1607 bases of coding region, and 309 bases of 3, flanking region. This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191. Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively. The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli. FMO activity in particulate fractions isolated from isopropyl-,-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45°C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E. coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:300,308, 2001 [source] The basis of asymmetry in IS2 transpositionMOLECULAR MICROBIOLOGY, Issue 4 2001Leslie A. Lewis In the first step of IS2 transposition, the formation of an IS2 minicircle, the roles of the two IS ends differ. Terminal cleavage initiates exclusively at the right inverted repeat (IRR) , the donor end , whereas IRL is always the target. At the resulting minicircle junction, the two abutted ends are separated by a spacer of 1 or 2 basepairs. In this study, we have identified the determinants of donor and target function. The inability of IRL to act as a donor results largely from two sequence differences between IRL and IRR , an extra basepair between the conserved transposase binding sequences and the end of the element, and a change of the terminal dinucleotide from CA-3, to TA-3,. These two changes also impose a characteristic size on the minicircle junction spacer. The only sequences required for the efficient target function of IRL appear to be contained within the segment from position 11,42. Although IRR can function as a target, its shorter length and additional contacts with transposase (positions 1,7) result in minicircles with longer, and inappropriate, spacers. We propose a model for the synaptic complex in which the terminus of IRL makes different contacts with the transposase for the initial and final strand transfer steps. The sequence differences between IRR and IRL, and the behavioural characteristics of IRL that result from them, have probably been selected because they optimize expression of transposase from the minicircle junction promoter, Pjunc. [source] Trex-1 deficiency in rheumatoid arthritis synovial fibroblastsARTHRITIS & RHEUMATISM, Issue 9 2010Michel Neidhart Objective To explore whether the increased expression of long interspersed nuclear element 1 (LINE-1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex-1, an exonuclease involved in the metabolization of L1 DNA:RNA hybrids. Methods Chromatin immunoprecipitation was used to detect L1-related p40 protein (L1-ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1-ORF1p and pGL3-TS3 carrying the target sequence for L1-ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex-1 measured by flow cytometry. The expression of Trex-1 mRNA and protein was also compared using real-time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex-1 in the L1-ORF1p,mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex-1 expression vector. Results Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3-TS3. L1-ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex-1. Levels of Trex-1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex-1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex-1, however, resulted in an enhancement of L1-ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex-1 inhibited 5-azacytidine,induced expression of p38, MAPK, a gene carrying the TS3 sequence. Conclusion The deficiency of Trex-1 in RASFs allows a longer half-life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a "spontaneous" aggressive behavior. [source] | ||||||||||||||||