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At least 937 records · Page 52Linked to original sources

PubMed2004

Elicitor-induced activation of transcription via W box-related cis-acting elements from a basic chitinase gene by WRKY transcription factors in tobacco.

A putative elicitor responsive element with two W boxes (CTGACC/T) has been identified in the region between -125 and -69 of a tobacco class I basic chitinase gene CHN48. We generated transgenic tobacco calli that contained the -125/-69 region fused to a luciferase reporter gene. The expression of the reporter gene was induced upon treatment with an elicitor, xylanase from Trichoderma viride (TvX). This induction required protein kinase activity. We isolated three cDNA clones encoding DNA-binding proteins, designated as NtWRKY1, NtWRKY2, and NtWRKY4, from tobacco cultured cells. Gel mobility shift assays showed that in vitro translation products of NtWRKY1, NtWRKY2 and NtWRKY4 bound to W box of CHN48 gene. These NtWRKY proteins stimulated W box-mediated transcription of a luciferase reporter gene in the transient assay. In addition, the transactivation of W box-mediated transcription by NtWRKY1 and NtWRKY4 was enhanced in response to elicitor treatment, suggesting elicitor-induced posttranscriptional activation of these NtWRKYs. Northern blot analyses showed that mRNAs for NtWRKY1 and NtWRKY2 increased after treatment with the elicitor, whereas mRNAs for NtWRKY4 were expressed constitutively at a low level. These results suggested possible involvement of NtWRKYs in elicitor-responsive transcription of defense genes in tobacco.

Amino Acid Sequence↗

PubMed2004

Peroxisome proliferator-activated receptor alpha ligands activate transcription of lectin-like oxidized low density lipoprotein receptor-1 gene through GC box motif.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a receptor for oxidized LDL. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors regulating transcription of various genes. We examined effects of PPAR ligands on LOX-1 expression and their transcriptional regulation in vascular endothelial cells. PPARalpha-specific ligands, such as fenofibrate and WY-14643, but not PPARgamma-specific ligands induced LOX-1 expression. Reduced expression of PPARalpha by antisense oligonucleotides directed to PPARalpha blocked fenofibrate-induced LOX-1 expression. Luciferase reporter gene assays with deletion and point mutations in the LOX-1 promoter revealed that transcriptional activity of LOX-1 gene by fenofibrate was localized in the -114/-106 GC box. Electrophoretic mobility shift assays with the radiolabeled GC box sequence showed inducible bands by PPARalpha ligands, which is competitively suppressed by unlabeled GC box motif and by an antibody to PPARalpha. In conclusion, PPARalpha appears to be one of the key regulators that induce LOX-1 expression, utilizing the GC box as a promoter.

Animals↗

PubMed2004

Polo-box motif targets a centrosome regulator, RanGTPase.

Mammalian polo-like kinase (Plk) acts at various stages in early and late mitosis. Plk1 localizes in the centrosome, the central spindle, the midbody as well as the kinetochore. The non-catalytic region in the C-terminus of Plk1 has conserved sequence motifs, named polo-boxes. These motifs are important for Plk localization. GFP protein fused with the core sequences of polo-box (50 amino acids) localized Plk to target organelles. We screened for Plk interacting proteins by constructing a tandem repeat of the polo-box motif, and used it as bait in the two-hybrid system with HeLa cell cDNA library. RanGTPase was detected as a positive clone. Through in vitro and in vivo protein binding analysis in synchronized cells by thymidine block and by nocodazole treatment, we confirmed the interaction between endogenous Ran and Plk1. We showed that endogenous Ran and Plk1 proteins were co-localized to centrosomes, which is a major target organelle of endogenous Plk1, in early mitotic cells by immunofluorescence. Finally, we demonstrated that Plk1 phosphorylated RanBPM, a Ran-binding protein in microtubule organizing center, through the interaction with Ran. These data suggested that the core motif of polo-box is sufficient for Plk1-targeting, and that Plk1 may play roles in centrosome through recruitment and/or activation of Ran/RanBPM proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2005

SMRT has tissue-specific isoform profiles that include a form containing one CoRNR box.

SMRT acts as a corepressor for a range of transcription factors. The amino-terminal part of the protein includes domains that mainly mediate transcriptional repression whilst the carboxy-terminal part includes domains that interact with nuclear receptors using up to three motifs called CoRNR boxes. The region of the SMRT primary transcript encoding the interaction domains is subject to alternative splicing that varies the inclusion of the third CoRNR box. The profile in mice includes an abundant, novel SMRT isoform that possesses just one CoRNR box. Mouse tissues therefore express SMRT isoforms containing one, two or three CoRNR boxes. In frogs, the SMRT isoform profile is tissue-specific. The mouse also shows distinct profiles generated by differential expression levels of the SMRT transcript isoforms. The formation of multiple SMRT isoforms and their tissue-specific regulation indicates a mechanism, whereby cells can define the repertoire of transcription factors regulated by SMRT.

Alternative Splicing↗

PubMed2005

Cloning and characterization of a high mobility group box 1 (HMGB1) homologue protein from Schistosoma mansoni.

Mammalian homologue of high mobility group box chromatin protein (HMGB) 1 was identified and cloned from human parasites, Schistosoma mansoni and S. haematobium. Sequence analyses showed that the parasite HMGB1s has 35-40% identity to human and rodent HMGB1s, and 33% identity to Caenorhabditis elegans HMGB1. Parasite HMGB1s also contains an A box and B box domain similar to mammalian HMGB1, however, it lacks the C-terminal tail that is present in mammalian HMGB1s. Analysis of the expression of HMGB1 in various life cycle stages of S. mansoni reveal S. mansoni HMGB1 (SmHMGB1) as a stage-specific protein, expressed abundantly in egg and adult female stages and at moderate levels in skin-stage schistosomula. Significant levels of SmHMGB1 were also present in excretory secretions of egg stages. Subsequent characterization studies showed that SmHMGB1 is a potent inducer of pro-inflammatory cytokines such as TNFalpha, IL-1Ralpha, IL-2Ralpha, IL-6, IL-13, IL-13Ralpha1, IL-15 and MIP-1alpha from mouse peritoneal macrophages. Pro-inflammatory activity, especially production of TNFalpha-inducing activity, appears to be a function of the B box domain protein. This was confirmed by both real-time reverse transcription PCR and by cytokine ELISA. Thus, results presented in this study suggest that SmHMGB1 may be a key molecule in the development of host inflammatory immune responses associated with schistosomiasis.

Amino Acid Sequence↗

PubMed2005

Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box.

The anaphase-promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators. Cdh1p is an APC coactivator that directly binds APC substrates. A genetic screen in budding yeast identified residues within Cdh1p critical for its function. Cdh1p proteins containing mutations within the "C box" or the "IR" motif could bind substrate, but not the APC, whereas mutants that only bound the APC were not identified, suggesting an ordered assembly of the ternary APC-Cdh1p-substrate complex. Supporting this hypothesis, we found that substrate binding to wild-type Cdh1p enhanced its association with the APC in yeast cells. We used peptide competition assays to demonstrate that Cdh1p interacts directly with the D box and the KEN box, two motifs within APC substrates known to be required for APC-mediated degradation. Moreover, an intact D box domain within a substrate was required to stimulate the association between the Cdh1p-substrate complex and the APC.

Amino Acid Motifs↗

PubMed2004

Learning a new behavioral strategy in the shuttle-box increases prefrontal dopamine.

Using microdialysis from medial prefrontal cortex of gerbils during aversive auditory conditioning in the shuttle-box we have previously shown a transient increase of dopamine efflux correlated with the establishment of avoidance behavior. We hypothesized that the acquisition of a new behavioral strategy is generally accompanied by this extra prefrontal dopamine release. The present experiment aimed at further testing this hypothesis. In a pre-training period in the shuttle-box the gerbils acquired an active avoidance response by generalizing two different tone signals to a GO-meaning (change of shuttle-box compartment). Thereafter, they were subjected in relearning sessions to differentially associate the known tone stimuli with GO- and NOGO- (no change of shuttle-box compartment) conditions, respectively. The following formation of discrimination behavior led to a similar extra dopamine increase as found during establishment of the avoidance strategy. This significant enhancement was limited to rapidly relearning individuals. Furthermore, the dopamine increase attenuated in these animals with increasing performance during the course of the discrimination training, similar to the retrieval stage of the avoidance strategy. Therefore, the dopamine system seems to be critically involved in the initial formation of associations for new behavioral strategies, i.e. learning. We assume that the prefrontal dopamine increase during initial learning of the complex discrimination behavior indicates an involvement of working memory principles and a goal-directed formation of a behavioral strategy.

Animals↗

PubMed2005

A proximal E-box modulates NGF effects on rat PPT-A promoter activity in cultured dorsal root ganglia neurones.

The rat preprotachykinin A (rtPPTA) promoter fragment spanning -865+92, relative to the major transcriptional start, has previously been demonstrated to be nerve growth factor (NGF) responsive in primary cultures of rat dorsal root ganglion (DRG) neurones [Harrison, P.T., Dalziel, R.G., Ditchfield, N.A., Quinn, J.P., 1999. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector. Neuroscience 94, 997-1003]. In this communication, we demonstrate that an E box element at -60, in part, regulates the activity of this rtPPT-A promoter fragment in DRG neurones in response to NGF. Differential regulation of the promoter is observed in the presence or absence of NGF when the E Box site is present. Under basal conditions binding of proteins to this -60 element may antagonise promoter activity. Hence, in the absence of NGF, mutation of the -60 E box increased reporter gene expression. Further, comparison of levels of reporter gene expression supported by both WT and mutated promoter indicate that in the presence of NGF the -60 E box element also plays a role as an activator domain. This represents a novel mechanism for NGF regulation of rtPPT-A. Similarly, an important role for this signalling pathway was observed in neonate rat DRG neuronal cultures, which require NGF for their survival, namely mutation of the -60 element resulted in higher levels of reporter gene expression.

Animals↗

PubMed2006

Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition.

Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000-fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the beta-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate.

Crystallography, X-Ray↗

PubMed2002

The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23.

Properly regulated cyclin proteolysis is critical for normal cell cycle progression. A nine-amino acid peptide motif called the destruction box (D box) is present at the N terminus of the yeast mitotic cyclins. This short sequence is required for cyclin ubiquitination and subsequent proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 required for cyclin ubiquitination. We have tested the D box of five mitotic cyclins for interaction with six APC/C subunits. The APC/C subunit Cdc23, but not five other subunits tested, interacted by two-hybrid analysis with the N terminus of wild-type Clb2. None of these subunits interacted with the N termini of the cyclins Clb1, Clb3, or Clb5. Mutations in the D box sequences of Clb2 inhibited interaction with Cdc23 both in vivo and in vitro. Our results provide the first evidence for a direct interaction between an APC/C substrate (Clb2) and an APC/C subunit (Cdc23).

Amino Acid Motifs↗

PubMed2000

CDC6 interacts with c-Myc to inhibit E-box-dependent transcription by abrogating c-Myc/Max complex.

The c-myc oncogene product (c-Myc) is a transcription factor that dimerizes with Max and recognizes the E-box sequence. It plays key functions in cell proliferation, differentiation and apoptosis. It is generally thought that c-Myc transactivates genes encoding proteins essential to cell-cycle progression by binding to the E-boxes that control them. The functions of c-Myc are also thought to be modulated by its associated proteins, several of which have recently been identified. In this study, we found that c-Myc directly bound in vivo and in vitro to the N-terminal region of human CDC6, a component of the pre-replication complex, and that both co-localized in cell nuclei. CDC6 bound to the C-proximal region of c-Myc, thereby competing with Max on the E-box sequence and changing c-Myc/Max heterodimer to a Max/Max homodimer. In consequence, the E-box-dependent transcription activity of c-Myc was abrogated. These results suggest that, in addition to its DNA replication activity, CDC6 also has a role as a transcriptional suppressor of c-Myc.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2001

Overlapping CRE and E-box promoter elements can independently regulate COX-2 gene transcription in macrophages.

Macrophage cyclooxygenase-2 (COX-2) transcription is mediated through the collaboration of different promoter elements. Here, the role of an overlapping cyclic AMP responsive element (CRE)/E-box was investigated. Nuclear proteins bound both the CRE and E-box, which synergized with other promoter elements to induce COX-2 transcription. Endotoxin induced binding of nuclear proteins to the CRE and E-box and each element independently induced higher COX-2 transcription levels than the overlapping CRE/E-box. Transcription factors associated with the CRE binding complex included c-Jun and CRE binding protein and with the E-box binding complex USF-1; their overexpression significantly induced COX-2 transcription. Therefore, both CRE and E-box promoter elements regulate COX-2 transcription in macrophages.

Animals↗

PubMed2003

The variant 'his-box' of the C18-Delta9-PUFA-specific elongase IgASE1 from Isochrysis galbana is essential for optimum enzyme activity.

IgASE1, a C18-Delta9-polyunsaturated fatty acid-specific fatty acid elongase component from Isochrysis galbana, contains a variant histidine box (his-box) with glutamine replacing the first histidine of the conserved histidine-rich motif present in all other known equivalent proteins. The importance of glutamine and other variant amino acid residues in the his-box of IgASE1 was determined by site-directed mutagenesis. Results showed that all the variation in amino acid sequence between this motif in IgASE1 and the consensus sequences of other elongase components was required for optimum enzyme activity. The substrate specificity was shown to be unaffected by these changes suggesting that components of the his-box are not directly responsible for substrate specificity.

Acetyltransferases↗

PubMed1998

DNA binding of NF-Y: the effect of HMGI proteins depends upon the CCAAT box.

NF-Y is a conserved sequence-specific transcription factor binding to CCAAT boxes. The chromatin-associated HMGI proteins influence promoter activities through positive and negative effects on binding of transcription factors. It was previously shown that HMGI(Y) synergizes the binding of NF-Y to the alpha2-collagen CCAAT box [Currie, R.A. (1997) J. Biol Chem. 272, 30880-30888]. Using recombinant proteins, we confirm that at low concentrations of NF-Y, HMGI(Y) acts synergistically on the alpha2-collagen CCAAT and we extend this observation to HMGI and HMGI-C. However, enhancement of DNA binding to gamma-globin, alpha-globin and MHC class II Ea CCAAT boxes was not observed. At high concentrations, HMGI proteins inhibit binding to alpha2-collagen and to gamma-globin, but not to high affinity Ea or a-globin CCAAT. In none of our experiments did we see a ternary complex between NF-Y, HMGI(Y) and DNA. In protein competition experiments, NF-Y affinity was at least two orders of magnitude higher, even in the context of the suboptimal gamma-globin CCAAT. Our data prove that HMGI proteins have complex positive and negative effects on NF binding to some, but not to all CCAAT boxes, suggesting that this phenomenon is dictated by the sequences flanking the pentanucleotide rather than direct protein-protein interactions.

Animals↗

PubMed1999

Negative cooperativity between juxtaposed E-box and cAMP/TPA responsive elements in the cholecystokinin gene promoter.

The promoter of the cholecystokinin (CCK) gene possesses evolutionary conserved juxtaposed E-box and cAMP/TPA responsive elements (CRE/TRE). We have examined the functional interaction of these two sites. As previously noted, c-Jun/c-Fos heterodimers greatly increase promoter activity through association with the CRE/TRE. Mutation of the E-box enhanced the activation by c-Jun/c-Fos, as well as stimulation by forskolin and bFGF, that acts through the CRE/TRE site. Moreover, c-Jun/c-Fos stimulation was inhibited by co-expression of c-Myc and Max. The results indicate that factors associating with the E-box exhibit a negative cooperative effect on the activation via the CRE/TRE element. We propose that this mechanism plays a significant role in CCK gene transcription and other genes with juxtaposed E-box and CRE/TRE.

Animals↗

PubMed1999

The CCAAT-box binding factor NF-Y is required for the expression of phospholipid hydroperoxide glutathione peroxidase in human epidermoid carcinoma A431 cells.

Promoter activation in the expression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene in human epidermoid carcinoma A431 cells was studied in the present investigation. Luciferase reporter assays with plasmids carrying a 400 bp of the promoter DNA were performed to analyze the regulatory element in the proximal promoter of human PHGPx gene. Transient transfection with a series of 5'-deletion and internal truncation mutants showed that the 5'-flanking region spanning from -212 to -121 bp was important for the basal expression of PHGPx gene in A431 cells. A region from -170 to -140 bp was protected in DNase I footprinting assays and bound the nuclear proteins in electrophoretic mobility shift assays. This region, denoted FP3, contains the consensus recognition sites for AP-2, CCAAT-box and CRE. The oligonucleotide competitor with the mutation at CCAAT-box could not eliminate the nuclear protein binding in gel-shift assay and the site-directed mutagenesis at the CCAAT-box decreased the luciferase activity of PHGPx promoter for approximate 50% in reporter gene assays. Competition experiments indicate that the binding of nuclear factor to the FP3 region was abolished by oligodeoxyribonucleotide corresponding to NF-Y/CP1 binding site to a greater extent than by those corresponding to sites for CTF/NFI and C/EBP. Taken together, the CCAAT-box in the promoter ranging from -156 to -151 bp, bound to NF-Y/CP1, was essential for the basal expression of human PHGPx gene in A431 cells.

CCAAT-Enhancer-Binding Proteins↗

PubMed1999

The E-box DNA binding protein Sgc1p suppresses the gcr2 mutation, which is involved in transcriptional activation of glycolytic genes in Saccharomyces cerevisiae.

Glycolytic gene expression is mediated by the Gcr1p-Gcr2p transcriptional activation complex. A screen for multicopy suppressors of gcr2 yielded SGC1. SGC1's suppression activity was specific to gcr2, it did not extend to gcr1. Disruption of SGC1 moderately affected glycolytic enzyme activities, although no growth defect was evident. Sgc1p exhibits a bHLH motif which is characteristic of E-box DNA-binding proteins. DNA footprinting experiments demonstrated Sgc1p's ability to bind at an E-box. However, its binding specificity was less than 10-fold, which is also characteristic of E-box binding proteins. LexA fusion experiments demonstrated that Sgc1p has weak intrinsic activating activity independent of GCR1 and GCR2. We propose that Sgc1p binds at E-boxes of glycolytic genes and contributes to their activation.

DNA-Binding Proteins↗

PubMed1996

A simplified procedure for boxing elastomeric impressions.

Although it is taught in most dental schools, the boxing of impressions is often avoided by dentists after their graduation. This article presents a simple and efficient procedure that makes boxing impressions more acceptable. Irreversible hydrocolloid and the subsequent stone mix are confined with a magnetic boxing strip that can be easily removed and replaced. The procedure is described for boxing edentulous and partially edentulous altered cast impressions made with elastomeric impression materials.

Alginates↗