Home About us Contact
|
Home About us Contact | ||
|
|
||
Gel Mobility Shift (gel + mobility_shift)
Selected AbstractsAlu-DNA repeat-binding protein p68 is a part of Alu-RNA containing ,-RNPFEBS JOURNAL, Issue 8 2000Dmitry V. Lukyanov An Alu-DNA repeat-binding protein with a molecular mass of 68 kDa (p68) is identified in the somatic human cell nucleoplasm. Gel mobility shift assay (GMSA), South-western blotting and affinity purification on DNA attached to the carrier were used in the identification. GMSA revealed multiple complexes with the exponential dependence of their relative mobility. A narrow binding site of the p68 was revealed using synthetic oligonucleotides. It is located between the A-box and B-box of the RNA polymerase III promoter and is identical to that reported for the Alu-binding protein from human spermatozoids. The same narrow binding site, the similarity of the isolation procedure from germ and somatic cells, and similar binding properties and molecular masses suggest homology of the two proteins. Antibodies raised against Alu-protein complexes led to hypershift of the complexes in GMSA and stained p68 in active fractions in human spermatozoids and in Alu-RNA-containing ,-RNP particles. Immunofluorescence of a HeLa cell monolayer revealed an intranuclear dot pattern with the dots corresponding to euchromatin areas and some dots located at the cell periphery in the cytoplasm. ,-RNP particles bound Alu-DNA in vitro and contained p68 as shown using the immunogold procedure. Alu-DNA binding activity was revealed in cytoplasm as well as in nucleoplasm. The possible nature of the main Alu-DNA binding protein and its involvement in the particle structure are discussed. [source] 2-oxoglutarate downregulates expression of vascular endothelial growth factor and erythropoietin through decreasing hypoxia-inducible factor-1, and inhibits angiogenesisJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Ken Matsumoto In oxygenated cells, hypoxia-inducible factor-1 (HIF-1) , subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel,Lindau tumor suppressor E3 ligase complex using 2-oxoglutarate as a substrate. We examined the effect of 2-oxoglutarate on the production of erythropoietin and vascular endothelial growth factor (VEGF). The expression of erythropoietin and VEGF protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The promoter activity of VEGF-luciferase was dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited the HIF-1, protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1, protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of angiogenesis. J. Cell. Physiol. 209: 333,340, 2006. © 2006 Wiley-Liss, Inc. [source] An ABA-responsive bZIP protein, OsBZ8, mediates sugar repression of , -amylase gene expressionPHYSIOLOGIA PLANTARUM, Issue 1 2003Yi-Ching Lee Expression of some , -amylase genes in cereals is suppressed by sugars and activated by sugar starvation. A 100-bp sugar response sequence (SRS) identified in the promoter of a rice , -amylase gene, ,Amy3, contains three essential motifs: the GC box, the G box, and the TATCCA element. To study the mechanism of sugar regulation of ,Amy3 transcription, an ABA-responsive bZIP protein, OsBZ8, which binds specifically to the G box in ,Amy3 SRS was characterized and function analysed. In sucrose-starved rice suspension cells and embryos, decline in OsBZ8 mRNA levels coincided with the induction of ,Amy3 mRNA accumulation. In vivo gain- and loss-of-function studies by transient expression assays in rice embryos revealed that OsBZ8 suppresses SRS activity through the G box and overrides the activity of an activator, OsMYBS1, which binds to the TATCCA element. Gel mobility shift assays revealed that OsBZ8 binds specifically to the G box in vitro. These studies suggest that OsBZ8 is a suppressor responsible for sugar repression of ,Amy3 expression, and OsMYBS1 is responsible for sugar starvation induced expression of ,Amy3. [source] Interaction between two cis -acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stressesTHE PLANT JOURNAL, Issue 2 2003Yoshihiro Narusaka Summary Many abiotic stress-inducible genes contain two cis -acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (,174 to ,55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis. [source] Isolation and characterization of the Xenopus HIVEP gene familyFEBS JOURNAL, Issue 6 2004Ulrike Dürr The HIVEP gene family encodes for very large sequence-specific DNA binding proteins containing multiple zinc fingers. Three mammalian paralogous genes have been identified, HIVEP1, - 2 and - 3, as well as the closely related Drosophila gene, Schnurri. These genes have been found to directly participate in the transcriptional regulation of a variety of genes. Mammalian HIVEP members have been implicated in signaling by TNF-, and in the positive selection of thymocytes, while Schnurri has been shown to be an essential component of the TGF-, signaling pathway. In this study, we describe the isolation of Xenopus HIVEP1, as well as partial cDNAs of HIVEP2 and - 3. Analysis of the temporal and spatial expression of the XHIVEP transcripts during early embryogenesis revealed ubiquitous expression of the transcripts. Assays using Xenopus oocytes mapped XHIVEP1 domains that are responsible for nuclear export and import activity. The DNA binding specificity of XHIVEP was characterized using a PCR-mediated selection and gel mobility shift assays. [source] Molecular cloning of the Matrix Gla Protein gene from Xenopus laevisFEBS JOURNAL, Issue 7 2002Functional analysis of the promoter identifies a calcium sensitive region required for basal activity To analyze the regulation of Matrix Gla Protein (MGP) gene expression in Xenopus laevis, we cloned the xMGP gene and its 5, region, determined their molecular organization, and characterized the transcriptional properties of the core promoter. The Xenopus MGP (xMGP) gene is organized into five exons, one more as its mammalian counterparts. The first two exons in the Xenopus gene encode the DNA sequence that corresponds to the first exon in mammals whereas the last three exons show homologous organization in the Xenopus MGP gene and in the mammalian orthologs. We characterized the transcriptional regulation of the xMGP gene in transient transfections using Xenopus A6 cells. In our assay system the identified promoter was shown to be transcriptionally active, resulting in a 12-fold induction of reporter gene expression. Deletional analysis of the 5, end of the xMGP promoter revealed a minimal activating element in the sequence from ,70 to ,36 bp. Synthetic reporter constructs containing three copies of the defined regulatory element delivered 400-fold superactivation, demonstrating its potential for the recruitment of transcriptional activators. In gel mobility shift assays we demonstrate binding of X. laevis nuclear factors to an extended regulatory element from ,180 to ,36, the specificity of the interaction was proven in competition experiments using different fragments of the xMGP promoter. By this approach the major site of factor binding was demonstrated to be included in the minimal activating promoter fragment from ,70 to ,36 bp. In addition, in transient transfection experiments we could show that this element mediates calcium dependent transcription and increasing concentrations of extracellular calcium lead to a significant dose dependent activation of reporter gene expression. [source] Visualization of the interaction between archaeal DNA polymerase and uracil-containing DNA by atomic force microscopyGENES TO CELLS, Issue 1 2006Yasuo Asami Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B-family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double-stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil-containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis. [source] Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expressionJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2010Y. Nakayama Nakayama Y, Yang L, Mezawa M, Araki S, Li Z, Wang Z, Sasaki Y, Takai H, Nakao S, Fukae M, Ogata Y. Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression. J Periodont Res 2010; 45: 602,611. © 2010 John Wiley & Sons A/S Background and Objective:, Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription. Material and Methods:, To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells. Results:, Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides ,116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides ,425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa). Conclusion:, These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways. [source] Common and distinct mechanisms of different redox-active carcinogens involved in the transformation of mouse JB6P+ cellsMOLECULAR CARCINOGENESIS, Issue 7 2008Sun Yang Abstract We transformed JB6P+ cells with prolonged intermittent low-dose UVB radiation or prolonged exposure to low-dose H2O2 or CdCl2. Stable transformation was confirmed by an anchorage-independence assay. The JB6P+ transformants formed more colonies (,six folds) in soft agar as compared to their JB6P+ parent cells and were associated with increased intracellular reactive oxygen species (ROS) levels. Activating protein-1 (AP-1) is a family of transcription factors that are rapidly activated by elevated intracellular ROS levels, and their composition is important in the process of cellular transformation and/or tumor progression. To investigate if carcinogenesis induced by distinct carcinogens was via similar molecular mechanisms in these transformants, gel mobility shift and immunoblot analyses were utilized to determine the distinct AP-1 compositions. Compared to parent JB6P+ cells, the gain of JunB and Fra-1 in AP-1 DNA binding complexes was markedly increased in all transformed cells, which might contribute to a more proliferative phenotype, while loss of Fra-2 occurred in JB6P+/H2O2 and JB6P+/Cd cells. Differential AP-1 components in the transformants suggested that their transformations might be mediated by distinct transcription signalings with distinct AP-1 dimer compositions. However, all three transformants exhibited increased activation of pathways involved in cell proliferation (ERK/Fra-1/AP-1 and JNK/c- jun/AP-1) and anti-apoptosis (Bcl-xl). The development of the JB6P+ transformants (JB6P+/UVB; JB6P+/H2O2; JB6P+/Cd) provides a unique tool to study the mechanisms that contribute to different redox-active carcinogens in a single model. © 2007 Wiley-Liss, Inc. [source] Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excisionMOLECULAR MICROBIOLOGY, Issue 6 2001Douglas Hinerfeld The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process. [source] Genomic organisation and regulation of murine alpha haemoglobin stabilising protein by erythroid Kruppel-like factorBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2007Janelle R. Keys Summary Alpha haemoglobin stabilising protein (AHSP) binds free , -globin chains and plays an important role in the protection of red cells, particularly during , -thalassaemia. Murine ASHP was discovered as a GATA-1 target gene and human AHSP is directly regulated by GATA-1. More recently, AHSP was rediscovered as a highly erythroid Kruppel-like factor (EKLF) -dependent transcript. We have determined the organisation of the murine AHSP gene and compared it to orthologs. There are two CACC box elements in the proximal promoter. The proximal element is absolutely conserved, but does not bind EKLF as it is not a canonical binding site. In rodents, the distal element contains a 3 bp insertion that disrupts the typical EKLF binding consensus region. Nevertheless, EKLF binds this atypical site by gel mobility shift assay, specifically occupies the AHSP promoter in vivo in a chromatin immunoprecipitation assay, and transactivates AHSP through this CACC site in promoter,reporter assays. Our results suggest EKLF can occupy CACC elements in vivo that are not predictable from the consensus binding site inferred from structural studies. We also propose that absence of AHSP in EKLF-null red cells exacerbates the toxicity of free , -globin chains, which exist because of the defect in , -globin gene activation. [source] | |||||||||||||