Differential detection of KRAS mutations in codons 12 and 13 with a modified loop-hybrid (LH) mobility shift assay using an insert-type LH-generator

2011 ◽  
Vol 412 (19-20) ◽  
pp. 1874-1878 ◽  
Author(s):  
Shoichi Matsukuma ◽  
Mitsuyo Yoshihara ◽  
Tetsuji Suda ◽  
Manabu Shiozawa ◽  
Makoto Akaike ◽  
...  
Keyword(s):  
Differential Detection ◽  
Mobility Shift ◽  
Kras Mutations ◽  
Mobility Shift Assay

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

MicroRNA319a regulates plant resistance to Sclerotinia stem rot

2021 ◽  
Author(s):  
Weiguo Dong ◽  
Wenqing Ren ◽  
Xuan Wang ◽  
He Yuke
Keyword(s):  
Plant Resistance ◽  
Host Susceptibility ◽  
Stem Rot ◽  
Sclerotinia Stem Rot ◽  
Sequencing Data ◽  
Mobility Shift ◽  
Expression Levels ◽  
Affected Plant ◽  
Mobility Shift Assay ◽  
Rot Disease

Abstract MicroRNA319a (miR319a) controls cell division arrest in plant leaves by inhibiting the expression of TCP (TEOSINTE BRANCHED 1/CYCLOIDEA/PCF) family genes. However, it is unclear whether miR319a influences infections by necrotrophic pathogens and host susceptibility. In this study, we revealed that miR319a affected plant resistance to stem rot disease of Sclerotinia sclerotiorum. In the plants of Brassica rapa infected with S. sclerotiorum, miR319a levels increased while expression levels of several BraTCP genes significantly decreased compared with those of the uninfected plants. The overexpression of BraMIR319a in B. rapa increased the susceptibility of the plants to S. sclerotiorum and aggravated stem rot disease, whereas the overexpression of BraTCP4-1 promoted the plant resistance. Our RNA-sequencing data revealed a potential relationship between miR319a and pathogen-related WRKY genes. Chromatin immunoprecipitation (ChIP) assay, electrophoretic mobility shift assay (EMSA) and reporter transaction assay showed that BraTCP4-1 was bound to the promoters of WRKY75, WRKY70, and WRKY33 genes and directly activated these pathogen-related genes. Moreover, the expression levels of WRKY75, WRKY70, and WRKY33 in the plants overexpressing BraMIR319a declined significantly whereas those of the plants overexpressing BraTCP4-1 increased significantly. These results suggest that miR319a and its targeted gene BraTCP4 regulate stem rot resistance through pathways of WRKY genes.

Amifostine Inhibits Hematopoietic Progenitor Cell Apoptosis by Activating NF-κB/Rel Transcription Factors

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4060-4066 ◽  
Author(s):  
Maria Fiammetta Romano ◽  
Annalisa Lamberti ◽  
Rita Bisogni ◽  
Corrado Garbi ◽  
Antonio M. Pagnano ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Apoptosis ◽  
Progenitor Cell ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Mobility Shift ◽  
Human Cord Blood ◽  
Mobility Shift Assay ◽  
Induced Apoptosis

Abstract We investigated the involvement of NF-κB/Rel transcription factors that reportedly can inhibit apoptosis in various cell types in the antiapoptotic mechanism of the cytoprotectant amifostine. In the nontumorigenic murine myeloid progenitor 32D cells incubated with amifostine, we detected a reduction of the IκB cytoplasmic levels by Western blotting and a raising of nuclear NF-κB/Rel complexes by electrophoretic mobility shift assay. Amifostine inhibited by more than 30% the growth factor deprivation-induced apoptosis, whereas its effect failed when we blocked the NF-κB/Rel activity with an NF-κB/Rel-binding phosphorothioate decoy oligodeoxynucleotide. In human cord blood CD34+ cells, the NF-κB/Rel p65 subunit was detectable (using immunofluorescence analysis) mainly in the cytoplasm in the absence of amifostine, whereas its presence was appreciable in the nuclei of cells incubated with the cytoprotectant. In 4 CD34+ samples incubated for 3 days in cytokine-deficient conditions, cell apoptosis was reduced by more than 30% in the presence of amifostine (or amifostine plus a control oligo); the effect of amifostine was abolished in cultures with the decoy oligo. These findings indicate that the inhibition of hematopoietic progenitor cell apoptosis by amifostine requires the induction of NF-κB/Rel factors and that the latter can therefore exert an antiapoptotic activity in the hematopoietic progenitor cell compartment. Furthermore, the identification of this specific mechanism underlying the survival-promoting activity of amifostine lends support to the possible use of this agent in apoptosis-related pathologies, such as myelodysplasias.

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

A fluorescence based non-radioactive electrophoretic mobility shift assay

2000 ◽  
Vol 78 (2) ◽  
pp. 163-170 ◽  
Author(s):  
K Ruscher ◽  
M Reuter ◽  
D Kupper ◽  
G Trendelenburg ◽  
U Dirnagl ◽  
...  
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Mobility Shift Assay

scholarly journals Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes

2004 ◽  
Vol 384 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Heiner KOESSLER ◽  
Joerg KAHLE ◽  
Christa BODE ◽  
Detlef DOENECKE ◽  
Werner ALBIG
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Histone H3 ◽  
Maximal Activity ◽  
Significant Loss ◽  
Tata Box ◽  
Mobility Shift ◽  
Nuclear Factor Y ◽  
Binding Factor ◽  
Mobility Shift Assay

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133–227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

scholarly journals RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains

2014 ◽  
Vol 42 (15) ◽  
pp. 10185-10195 ◽  
Author(s):  
Constanze Schelhorn ◽  
James M.B. Gordon ◽  
Lidia Ruiz ◽  
Javier Alguacil ◽  
Enrique Pedroso ◽  
...  
Keyword(s):  
Rna Binding ◽  
Titration Calorimetry ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Mobility Shift ◽  
Cytoplasmic Polyadenylation ◽  
C Terminus ◽  
Self Association ◽  
Mobility Shift Assay ◽  
A Minor

Abstract Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

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