Electrophoretic Mobility Shift Assay: An electrophoretic technique for assaying the binding of one compound to another. Typically one compound is labeled to follow its mobility during electrophoresis. If the labeled compound is bound by the other compound, then the mobility of the labeled compound through the electrophoretic medium will be retarded.Promoter Regions, Genetic: DNA sequences which are recognized (directly or indirectly) and bound by a DNA-dependent RNA polymerase during the initiation of transcription. Highly conserved sequences within the promoter include the Pribnow box in bacteria and the TATA BOX in eukaryotes.Base Sequence: The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.DNA-Binding Proteins: Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.Molecular Sequence Data: Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.Transcription Factors: Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process.Transcription, Genetic: The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.Binding Sites: The parts of a macromolecule that directly participate in its specific combination with another molecule.Sp1 Transcription Factor: Promoter-specific RNA polymerase II transcription factor that binds to the GC box, one of the upstream promoter elements, in mammalian cells. The binding of Sp1 is necessary for the initiation of transcription in the promoters of a variety of cellular and viral GENES.Protein Binding: The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.NF-kappa B: Ubiquitous, inducible, nuclear transcriptional activator that binds to enhancer elements in many different cell types and is activated by pathogenic stimuli. The NF-kappa B complex is a heterodimer composed of two DNA-binding subunits: NF-kappa B1 and relA.DNA Footprinting: A method for determining the sequence specificity of DNA-binding proteins. DNA footprinting utilizes a DNA damaging agent (either a chemical reagent or a nuclease) which cleaves DNA at every base pair. DNA cleavage is inhibited where the ligand binds to DNA. (from Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed)Gene Expression Regulation: Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control (induction or repression) of gene action at the level of transcription or translation.DNA: A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).Transcriptional Activation: Processes that stimulate the GENETIC TRANSCRIPTION of a gene or set of genes.Response Elements: Nucleotide sequences, usually upstream, which are recognized by specific regulatory transcription factors, thereby causing gene response to various regulatory agents. These elements may be found in both promoter and enhancer regions.Ophthalmologic Surgical Procedures: Surgery performed on the eye or any of its parts.Transfection: The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES.Nuclear Proteins: Proteins found in the nucleus of a cell. Do not confuse with NUCLEOPROTEINS which are proteins conjugated with nucleic acids, that are not necessarily present in the nucleus.Transcription Factor AP-1: A multiprotein complex composed of the products of c-jun and c-fos proto-oncogenes. These proteins must dimerize in order to bind to the AP-1 recognition site, also known as the TPA-responsive element (TRE). AP-1 controls both basal and inducible transcription of several genes.RNA, Messenger: RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.Luciferases: Enzymes that oxidize certain LUMINESCENT AGENTS to emit light (PHYSICAL LUMINESCENCE). The luciferases from different organisms have evolved differently so have different structures and substrates.Cell Line: Established cell cultures that have the potential to propagate indefinitely.Trans-Activators: Diffusible gene products that act on homologous or heterologous molecules of viral or cellular DNA to regulate the expression of proteins.Sp3 Transcription Factor: A specificity protein transcription factor that regulates expression of a variety of genes including VASCULAR ENDOTHELIAL GROWTH FACTOR and CYCLIN-DEPENDENT KINASE INHIBITOR P27.Cell Nucleus: Within a eukaryotic cell, a membrane-limited body which contains chromosomes and one or more nucleoli (CELL NUCLEOLUS). The nuclear membrane consists of a double unit-type membrane which is perforated by a number of pores; the outermost membrane is continuous with the ENDOPLASMIC RETICULUM. A cell may contain more than one nucleus. (From Singleton & Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d ed)Regulatory Sequences, Nucleic Acid: Nucleic acid sequences involved in regulating the expression of genes.Electrophoresis: An electrochemical process in which macromolecules or colloidal particles with a net electric charge migrate in a solution under the influence of an electric current.Cells, Cultured: Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.Enhancer Elements, Genetic: Cis-acting DNA sequences which can increase transcription of genes. Enhancers can usually function in either orientation and at various distances from a promoter.Repressor Proteins: Proteins which maintain the transcriptional quiescence of specific GENES or OPERONS. Classical repressor proteins are DNA-binding proteins that are normally bound to the OPERATOR REGION of an operon, or the ENHANCER SEQUENCES of a gene until a signal occurs that causes their release.Consensus Sequence: A theoretical representative nucleotide or amino acid sequence in which each nucleotide or amino acid is the one which occurs most frequently at that site in the different sequences which occur in nature. The phrase also refers to an actual sequence which approximates the theoretical consensus. A known CONSERVED SEQUENCE set is represented by a consensus sequence. Commonly observed supersecondary protein structures (AMINO ACID MOTIFS) are often formed by conserved sequences.Gene Expression Regulation, Enzymologic: Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control of gene action in enzyme synthesis.5' Flanking Region: The region of DNA which borders the 5' end of a transcription unit and where a variety of regulatory sequences are located.Tumor Cells, Cultured: Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely.DNA Primers: Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques.Gene Expression Regulation, Bacterial: Any of the processes by which cytoplasmic or intercellular factors influence the differential control of gene action in bacteria.Oligodeoxyribonucleotides: A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.Chloramphenicol O-Acetyltransferase: An enzyme that catalyzes the acetylation of chloramphenicol to yield chloramphenicol 3-acetate. Since chloramphenicol 3-acetate does not bind to bacterial ribosomes and is not an inhibitor of peptidyltransferase, the enzyme is responsible for the naturally occurring chloramphenicol resistance in bacteria. The enzyme, for which variants are known, is found in both gram-negative and gram-positive bacteria. EC 2.3.1.28.Upstream Stimulatory Factors: Ubiquitously expressed basic HELIX-LOOP-HELIX MOTIF transcription factors. They bind CANNTG sequences in the promoters of a variety of GENES involved in carbohydrate and lipid metabolism.Mutation: Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.Deoxyribonuclease I: An enzyme capable of hydrolyzing highly polymerized DNA by splitting phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide. This catalyzes endonucleolytic cleavage of DNA yielding 5'-phosphodi- and oligonucleotide end-products. The enzyme has a preference for double-stranded DNA.CCAAT-Enhancer-Binding Proteins: A class of proteins that were originally identified by their ability to bind the DNA sequence CCAAT. The typical CCAAT-enhancer binding protein forms dimers and consists of an activation domain, a DNA-binding basic region, and a leucine-rich dimerization domain (LEUCINE ZIPPERS). CCAAT-BINDING FACTOR is structurally distinct type of CCAAT-enhancer binding protein consisting of a trimer of three different subunits.Mutagenesis, Site-Directed: Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.HeLa Cells: The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for VIRUS CULTIVATION and antitumor drug screening assays.Plasmids: Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.Amino Acid Sequence: The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.Blotting, Western: Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.Bacterial Proteins: Proteins found in any species of bacterium.Chromatin Immunoprecipitation: A technique for identifying specific DNA sequences that are bound, in vivo, to proteins of interest. It involves formaldehyde fixation of CHROMATIN to crosslink the DNA-BINDING PROTEINS to the DNA. After shearing the DNA into small fragments, specific DNA-protein complexes are isolated by immunoprecipitation with protein-specific ANTIBODIES. Then, the DNA isolated from the complex can be identified by PCR amplification and sequencing.I-kappa B Proteins: A family of inhibitory proteins which bind to the REL PROTO-ONCOGENE PROTEINS and modulate their activity. In the CYTOPLASM, I-kappa B proteins bind to the transcription factor NF-KAPPA B. Cell stimulation causes its dissociation and translocation of active NF-kappa B to the nucleus.NF-kappa B p50 Subunit: A component of NF-kappa B transcription factor. It is proteolytically processed from NF-kappa B p105 precursor protein and is capable of forming dimeric complexes with itself or with TRANSCRIPTION FACTOR RELA. It regulates expression of GENES involved in immune and inflammatory responses.Oligonucleotides: Polymers made up of a few (2-20) nucleotides. In molecular genetics, they refer to a short sequence synthesized to match a region where a mutation is known to occur, and then used as a probe (OLIGONUCLEOTIDE PROBES). (Dorland, 28th ed)CCAAT-Binding Factor: A heterotrimeric DNA-binding protein that binds to CCAAT motifs in the promoters of eukaryotic genes. It is composed of three subunits: A, B and C.Electrophoresis, Polyacrylamide Gel: Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.Recombinant Fusion Proteins: Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.Signal Transduction: The intracellular transfer of information (biological activation/inhibition) through a signal pathway. In each signal transduction system, an activation/inhibition signal from a biologically active molecule (hormone, neurotransmitter) is mediated via the coupling of a receptor/enzyme to a second messenger system or to an ion channel. Signal transduction plays an important role in activating cellular functions, cell differentiation, and cell proliferation. Examples of signal transduction systems are the GAMMA-AMINOBUTYRIC ACID-postsynaptic receptor-calcium ion channel system, the receptor-mediated T-cell activation pathway, and the receptor-mediated activation of phospholipases. Those coupled to membrane depolarization or intracellular release of calcium include the receptor-mediated activation of cytotoxic functions in granulocytes and the synaptic potentiation of protein kinase activation. Some signal transduction pathways may be part of larger signal transduction pathways; for example, protein kinase activation is part of the platelet activation signal pathway.Transcription Factor RelA: A subunit of NF-kappa B that is primarily responsible for its transactivation function. It contains a C-terminal transactivation domain and an N-terminal domain with homology to PROTO-ONCOGENE PROTEINS C-REL.Reverse Transcriptase Polymerase Chain Reaction: A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols.Sequence Deletion: Deletion of sequences of nucleic acids from the genetic material of an individual.Proto-Oncogene Proteins c-jun: Cellular DNA-binding proteins encoded by the c-jun genes (GENES, JUN). They are involved in growth-related transcriptional control. There appear to be three distinct functions: dimerization (with c-fos), DNA-binding, and transcriptional activation. Oncogenic transformation can take place by constitutive expression of c-jun.Octamer Transcription Factor-1: A ubiquitously expressed octamer transcription factor that regulates GENETIC TRANSCRIPTION of SMALL NUCLEAR RNA; IMMUNOGLOBULIN GENES; and HISTONE H2B genes.Homeodomain Proteins: Proteins encoded by homeobox genes (GENES, HOMEOBOX) that exhibit structural similarity to certain prokaryotic and eukaryotic DNA-binding proteins. Homeodomain proteins are involved in the control of gene expression during morphogenesis and development (GENE EXPRESSION REGULATION, DEVELOPMENTAL).Cyclic AMP Response Element-Binding Protein: A protein that has been shown to function as a calcium-regulated transcription factor as well as a substrate for depolarization-activated CALCIUM-CALMODULIN-DEPENDENT PROTEIN KINASES. This protein functions to integrate both calcium and cAMP signals.Cloning, Molecular: The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.Cell Line, Tumor: A cell line derived from cultured tumor cells.Up-Regulation: A positive regulatory effect on physiological processes at the molecular, cellular, or systemic level. At the molecular level, the major regulatory sites include membrane receptors, genes (GENE EXPRESSION REGULATION), mRNAs (RNA, MESSENGER), and proteins.Transcription Factor AP-2: A family of DNA binding proteins that regulate expression of a variety of GENES during CELL DIFFERENTIATION and APOPTOSIS. Family members contain a highly conserved carboxy-terminal basic HELIX-TURN-HELIX MOTIF involved in dimerization and sequence-specific DNA binding.Recombinant Proteins: Proteins prepared by recombinant DNA technology.Proto-Oncogene Proteins c-ets: A family of transcription factors that share a unique DNA-binding domain. The name derives from viral oncogene-derived protein oncogene protein v-ets of the AVIAN ERYTHROBLASTOSIS VIRUS.Sequence Homology, Nucleic Acid: The sequential correspondence of nucleotides in one nucleic acid molecule with those of another nucleic acid molecule. Sequence homology is an indication of the genetic relatedness of different organisms and gene function.Binding, Competitive: The interaction of two or more substrates or ligands with the same binding site. The displacement of one by the other is used in quantitative and selective affinity measurements.Transcription Initiation Site: The first nucleotide of a transcribed DNA sequence where RNA polymerase (DNA-DIRECTED RNA POLYMERASE) begins synthesizing the RNA transcript.Early Growth Response Protein 1: An early growth response transcription factor that has been implicated in regulation of CELL PROLIFERATION and APOPTOSIS.Gene Expression: The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION.Thiocarbamates: Carbamates in which the -CO- group has been replaced by a -CS- group.Phosphorylation: The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.Oligonucleotide Probes: Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin.Tumor Necrosis Factor-alpha: Serum glycoprotein produced by activated MACROPHAGES and other mammalian MONONUCLEAR LEUKOCYTES. It has necrotizing activity against tumor cell lines and increases ability to reject tumor transplants. Also known as TNF-alpha, it is only 30% homologous to TNF-beta (LYMPHOTOXIN), but they share TNF RECEPTORS.Proto-Oncogene Protein c-ets-1: An ets proto-oncogene expressed primarily in adult LYMPHOID TISSUE; BRAIN; and VASCULAR ENDOTHELIAL CELLS.Down-Regulation: A negative regulatory effect on physiological processes at the molecular, cellular, or systemic level. At the molecular level, the major regulatory sites include membrane receptors, genes (GENE EXPRESSION REGULATION), mRNAs (RNA, MESSENGER), and proteins.Blotting, Northern: Detection of RNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES.Dimerization: The process by which two molecules of the same chemical composition form a condensation product or polymer.Zinc Fingers: Motifs in DNA- and RNA-binding proteins whose amino acids are folded into a single structural unit around a zinc atom. In the classic zinc finger, one zinc atom is bound to two cysteines and two histidines. In between the cysteines and histidines are 12 residues which form a DNA binding fingertip. By variations in the composition of the sequences in the fingertip and the number and spacing of tandem repeats of the motif, zinc fingers can form a large number of different sequence specific binding sites.Host Cell Factor C1: A cellular transcriptional coactivator that was originally identified by its requirement for the stable assembly IMMEDIATE-EARLY PROTEINS of the HERPES SIMPLEX VIRUS. It is a nuclear protein that is a transcriptional coactivator for a number of transcription factors including VP16 PROTEIN; GA-BINDING PROTEIN; EARLY GROWTH RESPONSE PROTEIN 2; and E2F4 TRANSCRIPTION FACTOR. It also interacts with and stabilizes HERPES SIMPLEX VIRUS PROTEIN VMW65 and helps regulate GENETIC TRANSCRIPTION of IMMEDIATE-EARLY GENES in HERPES SIMPLEX VIRUS.Blotting, Southwestern: A method that is used to detect DNA-protein interactions. Proteins are separated by electrophoresis and blotted onto a nitrocellulose membrane similar to Western blotting (BLOTTING, WESTERN) but the proteins are identified when they bind labeled DNA PROBES (as with Southern blotting (BLOTTING, SOUTHERN)) instead of antibodies.Artificial Gene Fusion: The in vitro fusion of GENES by RECOMBINANT DNA techniques to analyze protein behavior or GENE EXPRESSION REGULATION, or to merge protein functions for specific medical or industrial uses.NFI Transcription Factors: Transcription factors that were originally identified as site-specific DNA-binding proteins essential for DNA REPLICATION by ADENOVIRUSES. They play important roles in MAMMARY GLAND function and development.Gene Deletion: A genetic rearrangement through loss of segments of DNA or RNA, bringing sequences which are normally separated into close proximity. This deletion may be detected using cytogenetic techniques and can also be inferred from the phenotype, indicating a deletion at one specific locus.YY1 Transcription Factor: A ubiquitously expressed zinc finger-containing protein that acts both as a repressor and activator of transcription. It interacts with key regulatory proteins such as TATA-BINDING PROTEIN; TFIIB; and ADENOVIRUS E1A PROTEINS.Proto-Oncogene Proteins: Products of proto-oncogenes. Normally they do not have oncogenic or transforming properties, but are involved in the regulation or differentiation of cell growth. They often have protein kinase activity.E-Box Elements: DNA locations with the consensus sequence CANNTG. ENHANCER ELEMENTS may contain multiple copies of this element. E-boxes play a regulatory role in the control of transcription. They bind with basic helix-loop-helix (bHLH) type TRANSCRIPTION FACTORS. Binding specificity is determined by the specific bHLH heterodimer or homodimer combination and by the specific nucleotides at the 3rd and 4th position of the E-box sequence.3T3 Cells: Cell lines whose original growing procedure consisted being transferred (T) every 3 days and plated at 300,000 cells per plate (J Cell Biol 17:299-313, 1963). Lines have been developed using several different strains of mice. Tissues are usually fibroblasts derived from mouse embryos but other types and sources have been developed as well. The 3T3 lines are valuable in vitro host systems for oncogenic virus transformation studies, since 3T3 cells possess a high sensitivity to CONTACT INHIBITION.Receptors, Cytoplasmic and Nuclear: Intracellular receptors that can be found in the cytoplasm or in the nucleus. They bind to extracellular signaling molecules that migrate through or are transported across the CELL MEMBRANE. Many members of this class of receptors occur in the cytoplasm and are transported to the CELL NUCLEUS upon ligand-binding where they signal via DNA-binding and transcription regulation. Also included in this category are receptors found on INTRACELLULAR MEMBRANES that act via mechanisms similar to CELL SURFACE RECEPTORS.Erythroid-Specific DNA-Binding Factors: A group of transcription factors that were originally described as being specific to ERYTHROID CELLS.Gene Expression Regulation, Viral: Any of the processes by which cytoplasmic factors influence the differential control of gene action in viruses.Immediate-Early Proteins: Proteins that are coded by immediate-early genes, in the absence of de novo protein synthesis. The term was originally used exclusively for viral regulatory proteins that were synthesized just after viral integration into the host cell. It is also used to describe cellular proteins which are synthesized immediately after the resting cell is stimulated by extracellular signals.High Mobility Group Proteins: A family of low-molecular weight, non-histone proteins found in chromatin.TATA Box: A conserved A-T rich sequence which is contained in promoters for RNA polymerase II. The segment is seven base pairs long and the nucleotides most commonly found are TATAAAA.GA-Binding Protein Transcription Factor: A heterotetrameric transcription factor composed of two distinct proteins. Its name refers to the fact it binds to DNA sequences rich in GUANINE and ADENINE. GA-binding protein integrates a variety of SIGNAL TRANSDUCTION PATHWAYS and regulates expression of GENES involved in CELL CYCLE control, PROTEIN BIOSYNTHESIS, and cellular METABOLISM.Interleukin-1: A soluble factor produced by MONOCYTES; MACROPHAGES, and other cells which activates T-lymphocytes and potentiates their response to mitogens or antigens. Interleukin-1 is a general term refers to either of the two distinct proteins, INTERLEUKIN-1ALPHA and INTERLEUKIN-1BETA. The biological effects of IL-1 include the ability to replace macrophage requirements for T-cell activation.Proto-Oncogene Proteins c-fos: Cellular DNA-binding proteins encoded by the c-fos genes (GENES, FOS). They are involved in growth-related transcriptional control. c-fos combines with c-jun (PROTO-ONCOGENE PROTEINS C-JUN) to form a c-fos/c-jun heterodimer (TRANSCRIPTION FACTOR AP-1) that binds to the TRE (TPA-responsive element) in promoters of certain genes.Regulon: In eukaryotes, a genetic unit consisting of a noncontiguous group of genes under the control of a single regulator gene. In bacteria, regulons are global regulatory systems involved in the interplay of pleiotropic regulatory domains and consist of several OPERONS.Gene Expression Regulation, Neoplastic: Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control of gene action in neoplastic tissue.DNA Mutational Analysis: Biochemical identification of mutational changes in a nucleotide sequence.RNA-Binding Proteins: Proteins that bind to RNA molecules. Included here are RIBONUCLEOPROTEINS and other proteins whose function is to bind specifically to RNA.CCAAT-Enhancer-Binding Protein-beta: A CCAAT-enhancer-binding protein found in LIVER; INTESTINES; LUNG and ADIPOSE TISSUE. It is an important mediator of INTERLEUKIN-6 signaling.5' Untranslated Regions: The sequence at the 5' end of the messenger RNA that does not code for product. This sequence contains the ribosome binding site and other transcription and translation regulating sequences.Activating Transcription Factor 1: An activating transcription factor that regulates expression of a variety of genes including C-JUN GENES and TRANSFORMING GROWTH FACTOR BETA2.DNA, Complementary: Single-stranded complementary DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase. cDNA (i.e., complementary DNA, not circular DNA, not C-DNA) is used in a variety of molecular cloning experiments as well as serving as a specific hybridization probe.Receptors, Steroid: Proteins found usually in the cytoplasm or nucleus that specifically bind steroid hormones and trigger changes influencing the behavior of cells. The steroid receptor-steroid hormone complex regulates the transcription of specific genes.Sequence Alignment: The arrangement of two or more amino acid or base sequences from an organism or organisms in such a way as to align areas of the sequences sharing common properties. The degree of relatedness or homology between the sequences is predicted computationally or statistically based on weights assigned to the elements aligned between the sequences. This in turn can serve as a potential indicator of the genetic relatedness between the organisms.Protein Structure, Tertiary: The level of protein structure in which combinations of secondary protein structures (alpha helices, beta sheets, loop regions, and motifs) pack together to form folded shapes called domains. Disulfide bridges between cysteines in two different parts of the polypeptide chain along with other interactions between the chains play a role in the formation and stabilization of tertiary structure. Small proteins usually consist of only one domain but larger proteins may contain a number of domains connected by segments of polypeptide chain which lack regular secondary structure.Repetitive Sequences, Nucleic Acid: Sequences of DNA or RNA that occur in multiple copies. There are several types: INTERSPERSED REPETITIVE SEQUENCES are copies of transposable elements (DNA TRANSPOSABLE ELEMENTS or RETROELEMENTS) dispersed throughout the genome. TERMINAL REPEAT SEQUENCES flank both ends of another sequence, for example, the long terminal repeats (LTRs) on RETROVIRUSES. Variations may be direct repeats, those occurring in the same direction, or inverted repeats, those opposite to each other in direction. TANDEM REPEAT SEQUENCES are copies which lie adjacent to each other, direct or inverted (INVERTED REPEAT SEQUENCES).DNA, Bacterial: Deoxyribonucleic acid that makes up the genetic material of bacteria.Plicamycin: A tricyclic pentaglycosidic antibiotic from Streptomyces strains that inhibits RNA and protein synthesis by adhering to DNA. It is used as a fluorescent dye and as an antineoplastic agent, especially in bone and testicular tumors. Plicamycin is also used to reduce hypercalcemia, especially that due to malignancies.Dose-Response Relationship, Drug: The relationship between the dose of an administered drug and the response of the organism to the drug.DNA Probes: Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure DNA-DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections.Mutagenesis: Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS.Tetradecanoylphorbol Acetate: A phorbol ester found in CROTON OIL with very effective tumor promoting activity. It stimulates the synthesis of both DNA and RNA.Nucleic Acid Conformation: The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.Hepatocyte Nuclear Factor 4: A subfamily of nuclear receptors that regulate GENETIC TRANSCRIPTION of a diverse group of GENES involved in the synthesis of BLOOD COAGULATION FACTORS; and in GLUCOSE; CHOLESTEROL; and FATTY ACIDS metabolism.Escherichia coli: A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.Rats, Sprague-Dawley: A strain of albino rat used widely for experimental purposes because of its calmness and ease of handling. It was developed by the Sprague-Dawley Animal Company.Kinetics: The rate dynamics in chemical or physical systems.RNA: A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed)COUP Transcription Factors: A sub-family of steroid receptor-related orphan nuclear receptors that have specificity for a variety of DNA sequences related to AGGTCA. COUP transcription factors can heterodimerize with a variety of factors including RETINOIC ACID RECEPTORS; THYROID HORMONE RECEPTORS; and VITAMIN D RECEPTORS.Retinoid X Receptors: A subtype of RETINOIC ACID RECEPTORS that are specific for 9-cis-retinoic acid which function as nuclear TRANSCRIPTION FACTORS that regulate multiple signaling pathways.Basic Helix-Loop-Helix Leucine Zipper Transcription Factors: A family of transcription factors that contain regions rich in basic residues, LEUCINE ZIPPER domains, and HELIX-LOOP-HELIX MOTIFS.Operon: In bacteria, a group of metabolically related genes, with a common promoter, whose transcription into a single polycistronic MESSENGER RNA is under the control of an OPERATOR REGION.Genes, Regulator: Genes which regulate or circumscribe the activity of other genes; specifically, genes which code for PROTEINS or RNAs which have GENE EXPRESSION REGULATION functions.Receptors, Retinoic Acid: Proteins in the nucleus or cytoplasm that specifically bind RETINOIC ACID or RETINOL and trigger changes in the behavior of cells. Retinoic acid receptors, like steroid receptors, are ligand-activated transcription regulators. Several types have been recognized.Lipopolysaccharides: Lipid-containing polysaccharides which are endotoxins and important group-specific antigens. They are often derived from the cell wall of gram-negative bacteria and induce immunoglobulin secretion. The lipopolysaccharide molecule consists of three parts: LIPID A, core polysaccharide, and O-specific chains (O ANTIGENS). When derived from Escherichia coli, lipopolysaccharides serve as polyclonal B-cell mitogens commonly used in laboratory immunology. (From Dorland, 28th ed)Enzyme Inhibitors: Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.Time Factors: Elements of limited time intervals, contributing to particular results or situations.Viral Proteins: Proteins found in any species of virus.Liver: A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances.Receptors, Thyroid Hormone: Specific high affinity binding proteins for THYROID HORMONES in target cells. They are usually found in the nucleus and regulate DNA transcription. These receptors are activated by hormones that leads to transcription, cell differentiation, and growth suppression. Thyroid hormone receptors are encoded by two genes (GENES, ERBA): erbA-alpha and erbA-beta for alpha and beta thyroid hormone receptors, respectively.PyrrolidinesPolymerase Chain Reaction: In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.STAT1 Transcription Factor: A signal transducer and activator of transcription that mediates cellular responses to INTERFERONS. Stat1 interacts with P53 TUMOR SUPPRESSOR PROTEIN and regulates expression of GENES involved in growth control and APOPTOSIS.Podophyllin: Caustic extract from the roots of Podophyllum peltatum and P. emodi. It contains PODOPHYLLOTOXIN and its congeners and is very irritating to mucous membranes and skin. Podophyllin is a violent purgative that may cause CNS damage and teratogenesis. It is used as a paint for warts, skin neoplasms, and senile keratoses.PhosphoproteinsInterferon Regulatory Factor-1: An interferon regulatory factor that binds upstream TRANSCRIPTIONAL REGULATORY ELEMENTS in the GENES for INTERFERON-ALPHA and INTERFERON-BETA. It functions as a transcriptional activator for the INTERFERON TYPE I genes.Conserved Sequence: A sequence of amino acids in a polypeptide or of nucleotides in DNA or RNA that is similar across multiple species. A known set of conserved sequences is represented by a CONSENSUS SEQUENCE. AMINO ACID MOTIFS are often composed of conserved sequences.RNA, Small Interfering: Small double-stranded, non-protein coding RNAs (21-31 nucleotides) involved in GENE SILENCING functions, especially RNA INTERFERENCE (RNAi). Endogenously, siRNAs are generated from dsRNAs (RNA, DOUBLE-STRANDED) by the same ribonuclease, Dicer, that generates miRNAs (MICRORNAS). The perfect match of the siRNAs' antisense strand to their target RNAs mediates RNAi by siRNA-guided RNA cleavage. siRNAs fall into different classes including trans-acting siRNA (tasiRNA), repeat-associated RNA (rasiRNA), small-scan RNA (scnRNA), and Piwi protein-interacting RNA (piRNA) and have different specific gene silencing functions.Hepatocyte Nuclear Factor 3-beta: A forkhead transcription factor that regulates expression of metabolic GENES and is involved in EMBRYONIC DEVELOPMENT. Mutations in HNF-3beta have been associated with CONGENITAL HYPERINSULINISM.Fibroblasts: Connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules.Enzyme Induction: An increase in the rate of synthesis of an enzyme due to the presence of an inducer which acts to derepress the gene responsible for enzyme synthesis.STAT3 Transcription Factor: A signal transducer and activator of transcription that mediates cellular responses to INTERLEUKIN-6 family members. STAT3 is constitutively activated in a variety of TUMORS and is a major downstream transducer for the CYTOKINE RECEPTOR GP130.Interleukin-8: A member of the CXC chemokine family that plays a role in the regulation of the acute inflammatory response. It is secreted by variety of cell types and induces CHEMOTAXIS of NEUTROPHILS and other inflammatory cells.Cattle: Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor.Operator Regions, Genetic: The regulatory elements of an OPERON to which activators or repressors bind thereby effecting the transcription of GENES in the operon.Y-Box-Binding Protein 1: Y-box-binding protein 1 was originally identified as a DNA-binding protein that interacts with Y-box PROMOTER REGIONS of MHC CLASS II GENES. It is a highly conserved transcription factor that regulates expression of a wide variety of GENES.Mobility Limitation: Difficulty in walking from place to place.Basic-Leucine Zipper Transcription Factors: A large superfamily of transcription factors that contain a region rich in BASIC AMINO ACID residues followed by a LEUCINE ZIPPER domain.Sequence Homology, Amino Acid: The degree of similarity between sequences of amino acids. This information is useful for the analyzing genetic relatedness of proteins and species.Immunoblotting: Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies.Models, Genetic: Theoretical representations that simulate the behavior or activity of genetic processes or phenomena. They include the use of mathematical equations, computers, and other electronic equipment.NFATC Transcription Factors: A family of transcription factors characterized by the presence of highly conserved calcineurin- and DNA-binding domains. NFAT proteins are activated in the CYTOPLASM by the calcium-dependent phosphatase CALCINEURIN. They transduce calcium signals to the nucleus where they can interact with TRANSCRIPTION FACTOR AP-1 or NF-KAPPA B and initiate GENETIC TRANSCRIPTION of GENES involved in CELL DIFFERENTIATION and development. NFAT proteins stimulate T-CELL activation through the induction of IMMEDIATE-EARLY GENES such as INTERLEUKIN-2.Precipitin Tests: Serologic tests in which a positive reaction manifested by visible CHEMICAL PRECIPITATION occurs when a soluble ANTIGEN reacts with its precipitins, i.e., ANTIBODIES that can form a precipitate.Cell Differentiation: Progressive restriction of the developmental potential and increasing specialization of function that leads to the formation of specialized cells, tissues, and organs.Carrier Proteins: Transport proteins that carry specific substances in the blood or across cell membranes.Mitogen-Activated Protein Kinases: A superfamily of PROTEIN-SERINE-THREONINE KINASES that are activated by diverse stimuli via protein kinase cascades. They are the final components of the cascades, activated by phosphorylation by MITOGEN-ACTIVATED PROTEIN KINASE KINASES, which in turn are activated by mitogen-activated protein kinase kinase kinases (MAP KINASE KINASE KINASES).GATA2 Transcription Factor: An essential GATA transcription factor that is expressed primarily in HEMATOPOIETIC STEM CELLS.Isoenzymes: Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics.Apoptosis: One of the mechanisms by which CELL DEATH occurs (compare with NECROSIS and AUTOPHAGOCYTOSIS). Apoptosis is the mechanism responsible for the physiological deletion of cells and appears to be intrinsically programmed. It is characterized by distinctive morphologic changes in the nucleus and cytoplasm, chromatin cleavage at regularly spaced sites, and the endonucleolytic cleavage of genomic DNA; (DNA FRAGMENTATION); at internucleosomal sites. This mode of cell death serves as a balance to mitosis in regulating the size of animal tissues and in mediating pathologic processes associated with tumor growth.Introns: Sequences of DNA in the genes that are located between the EXONS. They are transcribed along with the exons but are removed from the primary gene transcript by RNA SPLICING to leave mature RNA. Some introns code for separate genes.Epithelial Cells: Cells that line the inner and outer surfaces of the body by forming cellular layers (EPITHELIUM) or masses. Epithelial cells lining the SKIN; the MOUTH; the NOSE; and the ANAL CANAL derive from ectoderm; those lining the RESPIRATORY SYSTEM and the DIGESTIVE SYSTEM derive from endoderm; others (CARDIOVASCULAR SYSTEM and LYMPHATIC SYSTEM) derive from mesoderm. Epithelial cells can be classified mainly by cell shape and function into squamous, glandular and transitional epithelial cells.Nitric Oxide Synthase Type II: A CALCIUM-independent subtype of nitric oxide synthase that may play a role in immune function. It is an inducible enzyme whose expression is transcriptionally regulated by a variety of CYTOKINES.DNA, Viral: Deoxyribonucleic acid that makes up the genetic material of viruses.Endothelium, Vascular: Single pavement layer of cells which line the luminal surface of the entire vascular system and regulate the transport of macromolecules and blood components.COS Cells: CELL LINES derived from the CV-1 cell line by transformation with a replication origin defective mutant of SV40 VIRUS, which codes for wild type large T antigen (ANTIGENS, POLYOMAVIRUS TRANSFORMING). They are used for transfection and cloning. (The CV-1 cell line was derived from the kidney of an adult male African green monkey (CERCOPITHECUS AETHIOPS).)Activating Transcription Factor 2: An activating transcription factor that regulates expression of a variety of GENES including C-JUN GENES; CYCLIN A; CYCLIN D1; and ACTIVATING TRANSCRIPTION FACTOR 3.Cyclooxygenase 2: An inducibly-expressed subtype of prostaglandin-endoperoxide synthase. It plays an important role in many cellular processes and INFLAMMATION. It is the target of COX2 INHIBITORS.Kruppel-Like Transcription Factors: A family of zinc finger transcription factors that share homology with Kruppel protein, Drosophila. They contain a highly conserved seven amino acid spacer sequence in between their ZINC FINGER MOTIFS.Nuclear Respiratory Factor 1: A transcription factor that controls the expression of variety of proteins including CYTOCHROME C and 5-AMINOLEVULINATE SYNTHETASE. It plays an important role in maintenance of the RESPIRATORY CHAIN of MITOCHONDRIA.Hepatocyte Nuclear Factor 1-alpha: Hepatocyte nuclear factor 1-alpha is a transcription factor found in the LIVER; PANCREAS; and KIDNEY that regulates HOMEOSTASIS of GLUCOSE.Interleukin-6: A cytokine that stimulates the growth and differentiation of B-LYMPHOCYTES and is also a growth factor for HYBRIDOMAS and plasmacytomas. It is produced by many different cells including T-LYMPHOCYTES; MONOCYTES; and FIBROBLASTS.Two-Hybrid System Techniques: Screening techniques first developed in yeast to identify genes encoding interacting proteins. Variations are used to evaluate interplay between proteins and other molecules. Two-hybrid techniques refer to analysis for protein-protein interactions, one-hybrid for DNA-protein interactions, three-hybrid interactions for RNA-protein interactions or ligand-based interactions. Reverse n-hybrid techniques refer to analysis for mutations or other small molecules that dissociate known interactions.Protein Biosynthesis: The biosynthesis of PEPTIDES and PROTEINS on RIBOSOMES, directed by MESSENGER RNA, via TRANSFER RNA that is charged with standard proteinogenic AMINO ACIDS.Nuclear Respiratory Factors: A family of transcription factors that control expression of a variety of nuclear GENES encoding proteins that function in the RESPIRATORY CHAIN of the MITOCHONDRIA.Genes, jun: Retrovirus-associated DNA sequences (jun) originally isolated from the avian sarcoma virus 17 (ASV 17). The proto-oncogene jun (c-jun) codes for a nuclear protein which is involved in growth-related transcriptional control. Insertion of c-jun into ASV-17 or the constitutive expression of the c-jun protein produces tumorgenicity. The human c-jun gene is located at 1p31-32 on the short arm of chromosome 1.beta-Galactosidase: A group of enzymes that catalyzes the hydrolysis of terminal, non-reducing beta-D-galactose residues in beta-galactosides. Deficiency of beta-Galactosidase A1 may cause GANGLIOSIDOSIS, GM1.Proto-Oncogene Proteins c-myb: Cellular DNA-binding proteins encoded by the myb gene (GENES, MYB). They are expressed in a wide variety of cells including thymocytes and lymphocytes, and regulate cell differentiation. Overexpression of myb is associated with autoimmune diseases and malignancies.Fushi Tarazu Transcription Factors: Fushi tarazu transcription factors were originally identified in DROSOPHILA. They are found throughout ARTHROPODS and play important roles in segmentation and CENTRAL NERVOUS SYSTEM development.Molecular Weight: The sum of the weight of all the atoms in a molecule.Enzyme Activation: Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.Proteins: Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein.Membrane Proteins: Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.Steroidogenic Factor 1: A transcription factor and member of the nuclear receptor family NR5 that is expressed throughout the adrenal and reproductive axes during development. It plays an important role in sexual differentiation, formation of primary steroidogenic tissues, and their functions in post-natal and adult life. It regulates the expression of key steroidogenic enzymes.Helix-Loop-Helix Motifs: Recurring supersecondary structures characterized by 20 amino acids folding into two alpha helices connected by a non-helical "loop" segment. They are found in many sequence-specific DNA-BINDING PROTEINS and in CALCIUM-BINDING PROTEINS.POU Domain Factors: A family of transcription factors characterized by the presence of a bipartite DNA-binding domain known as the POU domain. The POU domain contains two subdomains, a POU-specific domain and a POU-homeodomain. The POU domain was originally identified as a region of approximately 150 amino acids shared between the Pit-1, Oct-1, Oct-2, and Unc-86 transcription factors.Cell Line, Transformed: Eukaryotic cell line obtained in a quiescent or stationary phase which undergoes conversion to a state of unregulated growth in culture, resembling an in vitro tumor. It occurs spontaneously or through interaction with viruses, oncogenes, radiation, or drugs/chemicals.Sequence Analysis, DNA: A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis.Protein Isoforms: Different forms of a protein that may be produced from different GENES, or from the same gene by ALTERNATIVE SPLICING.Restriction Mapping: Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.Neoplasm Proteins: Proteins whose abnormal expression (gain or loss) are associated with the development, growth, or progression of NEOPLASMS. Some neoplasm proteins are tumor antigens (ANTIGENS, NEOPLASM), i.e. they induce an immune reaction to their tumor. Many neoplasm proteins have been characterized and are used as tumor markers (BIOMARKERS, TUMOR) when they are detectable in cells and body fluids as monitors for the presence or growth of tumors. Abnormal expression of ONCOGENE PROTEINS is involved in neoplastic transformation, whereas the loss of expression of TUMOR SUPPRESSOR PROTEINS is involved with the loss of growth control and progression of the neoplasm.Jurkat Cells: A CELL LINE derived from human T-CELL LEUKEMIA and used to determine the mechanism of differential susceptibility to anti-cancer drugs and radiation.3' Untranslated Regions: The sequence at the 3' end of messenger RNA that does not code for product. This region contains transcription and translation regulating sequences.GATA4 Transcription Factor: A GATA transcription factor that is expressed in the MYOCARDIUM of developing heart and has been implicated in the differentiation of CARDIAC MYOCYTES. GATA4 is activated by PHOSPHORYLATION and regulates transcription of cardiac-specific genes.

*  Knight:Electrophoretic mobility shift assay - OpenWetWare

Retrieved from "http://www.openwetware.org/wiki/Knight:Electrophoretic_mobility_shift_assay" ... An assay to check for protein-DNA binding. Materials. Protein-DNA binding. *Binding buffer *Need to determine composition. See ... Knight:Electrophoretic mobility shift assay. From OpenWetWare. (Difference between revisions). Jump to: navigation, search ...
openwetware.org/index.php?title=Knight:Electrophoretic_mobility_shift_assay&diff=65453&oldid=65452

*  The Pluripotency Regulator Zic3 Is a Direct Activator of the Nanog Promoter in ESCs - Lim - 2010 - STEM CELLS - Wiley Online...

Electrophoretic Mobility Shift Assay. Electrophoretic mobility shift assay (EMSA) was performed as previously described [18]. ... Abbreviations: EMSA, electrophoretic mobility shift assay; HisMBP, histidine and maltose binding protein; Kd, dissociation ... Unlabeled sequences of the WT and mut sequences were used in the competitive gel shift assay. (B): EMSA demonstrating the ... C): Competitive gel shift assay using WT competitors. Unlabeled WT competitor was allowed to compete for binding to Zic3-DBD ...
onlinelibrary.wiley.com/doi/10.1002/stem.527/full

*  Pharmaceuticals | Free Full-Text | The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain...

Electrophoretic Mobility Shift assay (EMSA). Whole-cell extracts of Prl stimulated B9-HeLa cells, treated with S5-DBD-PA, hTRX ... For this purpose electrophoretic mobility shift experiments and promoter-reporter assays were conducted. We observed that S5- ... b) Evaluation of Stat5 DNA binding activity by electrophoretic mobility shift assay (EMSA). For assessing the therapeutic ... b) Evaluation of Stat5 DNA binding activity by electrophoretic mobility shift assay (EMSA). For assessing the therapeutic ...
mdpi.com/1424-8247/6/8/960/htm

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Electrophoretic mobility shift assay.. Nuclear proteins were harvested as described previously (10). The sequence of NF-κB ... Electrophoretic mobility shift assay (EMSA) were performed at 4°C in binding buffer containing 5 μg liver nuclear extracts, 2 ... Nuclear factor (NF)-κB-specific electromobility shift assay of liver nuclear extracts performed in nontransgenic littermate and ... TUNEL assays on liver sections of nontransgenic,PK-BCL-2, and. transgenic lines 120 min after systemic injection of anti-Fas ...
ajpgi.physiology.org/content/277/3/G702

*  GATA-6 Is Involved in PPARγ-Mediated Activation of Differentiated Phenotype in Human Vascular Smooth Muscle Cells |...

Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared from VSMCs as described previously.16 Double-stranded wild ... Electrophoretic mobility shift assays (EMSAs) were carried out as described previously.18 ... A colorimetric assay based on the mitochondrial activity of living cells to cleave the tetrazolium ring and reduce the ... Subsequent BrdU incorporation assays were carried out according to the protocols supplied by the manufacturer (Biotrak, ...
atvb.ahajournals.org/content/23/3/404

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Electrophoretic Mobility Shift Assay.. A fragment spanning the region-525/−444 of the COL7A1 promoter, corresponding to the ... Gel mobility shift assays were performed with a labeled oligonucleotide spanning the region from −524 to −444 of the COL7A1 ... A, CAT assays using the (SBS)2-TK/CAT construct transfected into 1205-LU melanoma cells were carried out in the presence and ... Growth Assay.. Cells were seeded in their respective growth medium. In the case of melanoma cells, medium was changed after 12 ...
cancerres.aacrjournals.org/content/59/3/547

*  Islet-1 is Required for the Maturation, Proliferation, and Survival of the Endocrine Pancreas | Diabetes

Electrophoretic mobility shift assays.. pCS2 rat Isl-1-Myc plasmid (gift from Dr. Pfaff) was used as a template for in vitro ... Next, we performed electrophoretic mobility shift assays with either in vitro translated Myc-tagged Isl-1 or Ins-1 β-cell line ... Insulin staining was performed on the same section as TUNEL assay to locate islets. For bromodeoxyuridine (BrdU) assays, ... 7,793 was incubated in gel shift binding assays with in vitro translated Myc-tagged Isl-1 protein or Ins-1 β-cell extract. The ...
diabetes.diabetesjournals.org/content/58/9/2059.full.print

*  A new mutation in the hepcidin promoter impairs its BMP response and contributes to a severe phenotype in HFE related...

Electrophoretic mobility shift assay. HepG2 cells were incubated with either 10 ng/mL of human BMP9 or 20 ng/mL of human BMP4 ... Gel shift assay with purified polyclonal rabbit IgG anti-SMAD4 or anti-SMAD1/5/8 (Santa Cruz) or non-relevant antibody were ... Transfection, luciferase assay. Cells were transfected, using transfectine (Bio-Rad) with 100 ng of pGL4-hepcidin promoter ... Then crude nuclear extracts were prepared as described.20 For gel retardation assays, nuclear extracts (10 μg) were ...
haematologica.org/content/94/5/720.long

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Nuclear extracts and electrophoretic mobility shift assay.. INS-1 cells were serum deprived overnight before stimulation with ... Electrophoretic mobility shift assay with oligonucleotides comprising putative STAT-binding sites within the rSOCS3 promoter (A ... Ten micrograms nuclear extract or 5 μl TNT in vitro translation reaction were added to 5× electrophoretic mobility shift assay ... Luciferase reporter gene assays in INS-1 β-cells. A: Cotransfection of the empty vector or rSOCS3 expression vector with the ...
diabetes.diabetesjournals.org/content/54/12/3410

*  Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9 | Development

Electrophoretic mobility shift assay (EMSA). The following oligonucleotide probes (5′-3′) were used: site A wild-type probe, ... First, because lhx9 induces few ectopic Hcrt neurons in our assay and the number of endogenous Hcrt neurons is variable, it is ... However, overexpressing two or more candidate genes using our assay was not feasible due to DNA toxicity. Reducing the ... Using microarray gene expression analysis and high-throughput gene overexpression assays in zebrafish, we found that the LIM ...
dev.biologists.org/content/142/6/1113.long

*  bHLH-O proteins are crucial for Drosophila neuroblast self-renewal and mediate Notch-induced overproliferation | Development

Electrophoretic mobility shift assay (EMSA). Full-length CDSs of dpn, m8 and mγ were cloned in pRSET-A. Proteins were produced ... using the electrophoretic mobility shift assay (EMSA). We tested an EB-box oligo [a high-affinity binding site for bHLH-O ... Upon mixing two bHLH-O proteins, we also observed new shifted complexes that point to the existence of all possible ... We made mosaics for null alleles of various components of the pathway and assayed lacZ-reporters of E(spl)mγ and E(spl)m8, as ...
dev.biologists.org/content/139/7/1258

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Electrophoretic mobility shift assay (A) and representative band density (B) for nuclear factor (NF)-κB DNA binding in rats ... Electrophoretic mobility shift assay.. NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′, binding site underlined) consensus oligonucleotide ... Binding of the antibody to the DNA-protein complex was indicated by a supershift in the electrophoretic mobility shift assay ( ... Nitrite/nitrate assay.. Nitrite plus nitrate (NOx) production was measured in rat plasma as previously described (39) with a ...
ajplung.physiology.org/content/280/3/L400

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Electrophoretic Mobility Shift Assay. To produce the WOX11 protein, the full-length cDNA amplified with primers WOX1132a-F and ... A) Gel shift assays of WOX11 protein binding to a promoter sequence (S1) of RR2 containing the WOX binding site (underlined) or ... In Vivo Binding Assay of WOX11. For ChIP assays, wild-type, WOX11-GFP transgenic lines, and WOX11-GR trangenic plants treated ... The shifted band is indicated by an arrow.. (B) ChIP analysis of transgenic plants expressing a WOX11-GFP fusion protein. ...
plantcell.org/content/21/3/736

*  MafG Sumoylation Is Required for Active Transcriptional Repression

Electrophoretic mobility shift assay (EMSA). To produce sumoylated MafG in Escherichia coli, pET15b-MafG1-123 (16) was ... since the shifted band of sumoylated heterodimer displayed a mobility similar to that of the nonsumoylated heterodimer, which ... Fig.3B,3B, lanes 4 to 6), one additional band of slower mobility appeared (Fig. ​(Fig.3C,3C, lanes 4 to 6). When MafG and the ... Fig.3C,3C, lanes 1 to 3). The intensities of the shifted bands paralleled the incubated amount of protein (Fig. ​(Fig.3B,3B, ...
pubmedcentralcanada.ca/pmcc/articles/PMC1489127/?lang=en-ca

*  Distinct transcriptional responses of RNA polymerases I, II and III to aptamers that bind TBP

Electrophoretic mobility shift assay (EMSA). For TFIIIB•DNA complex, the TATA-DNA used in the binding assays was produced by ... The transcripts were assayed by either S1 protection or primer extension assays. ... S1 nuclease protection assays were performed essentially according to (26), with a 50 nt DNA oligonucleotide probe ... The thin arrows indicate when the different components of the assay were added, and the thick arrow indicates when aptamers ...
pubmedcentralcanada.ca/pmcc/articles/PMC549393/?lang=en-ca

*  Dynein light chain rp3 acts as a nuclear matrix-associated transcriptional modulator in a dynein-independent pathway | Journal...

Bcl2 MAR DNA complexes using electrophoretic mobility shift assays (EMSA). In these assays, the radiolabeled Bcl2 MAR probe was ... Electrophoretic mobility shift assay (EMSA). EMSAs were performed essentially as described (Ramakrishnan et al., 2000) using 10 ... We thank Xiaojing Ma for advice on EMSA and luciferase assays and Lynn Wang for advice on the real-time PCR assay. This work ... In the reporter assays involving Bcl2, Bcl2-MAR-luc (1 μg) was co-transfected with pTK-RL (0.05 μg) and a combination of ...
jcs.biologists.org/content/118/15/3431.long

*  A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality

Electrophoretic mobility shift assays. Sense and antisense oligonucleotides corresponding to the "S" allele (AGGGAATGGGGGCGGGAT ... Gel shift assays showed that the COL1A1 "s" allele had increased affinity for Sp1 protein binding in vitro when compared with ... Gel shift assays showed increased binding affinity of the "s" allele for Sp1 protein, and primary RNA transcripts derived from ... Binding affinity of the Sp1 protein for the polymorphic DNA recognition site in COL1A1 was analyzed using gel shift assays ( ...
pubmedcentralcanada.ca/pmcc/articles/PMC199568/

*  Angiotensin II Induces Myocyte Enhancer Factor 2- and Calcineurin/Nuclear Factor of Activated T Cell-Dependent Transcriptional...

Electrophoretic Mobility Shift Assays. VSMCs were serum-starved for 3 days and stimulated with 10−7 mol/L Ang II for 8 hours. ... Electrophoretic mobility shift assays (EMSAs) were performed as previously described.14 A highly specific anti-MEF2A antibody ... VSMCs were then stimulated with 10−7 mol/L Ang II for 48 hours, and cells were harvested for the luciferase assay. The total ... VSMCs were then stimulated with 10−7 mol/L Ang II for 48 hours, and cells were harvested for the luciferase assay. The total ...
circres.ahajournals.org/content/90/9/1004

*  Anti-inflammatory effects of triptolide in human bronchial epithelial cells | Lung Cellular and Molecular Physiology

Nuclear extract preparation and electrophoretic mobility shift assays.. 16HBE cells were stimulated for 3 h, pelleted, and ... in nuclear extracts and its modulation by triptolide were characterized with the use of electrophoretic mobility shift assays. ... then cytoplasmic and nuclear extracts were prepared and used for Western immunoblot and electrophoretic mobility shift assay ( ... C: IL-8 nuclear factor (NF)-κB luciferase reporter gene assay. The induction of IL-8-NF-κB luciferase with TNF-α stimulation ...
ajplung.physiology.org/content/279/5/L958.full

*  JCI - Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair

Electrophoretic mobility-shift assay. HaCaT keratinocytes (DMEM/10% FCS) or human primary keratinocytes (KBM-2) were grown to ... Using the electrophoretic mobility-shift assay (EMSA) technique, we observed a rapid and strong increase in activated STAT3 ... This assay determines the number of viable cells in proliferation assays, because the total amount of the formed formazan end ... Twenty micrograms of total RNA from wounded or nonwounded skin were used for RNase protection assays. RNase protection assays ...
https://jci.org/articles/view/9148

*  VLDL and apolipoprotein CIII induce ER stress and inflammation and attenuate insulin signalling via Toll-like receptor 2 in...

Electrophoretic mobility shift assay. ER. Endoplasmic reticulum. ERK. Extracellular signal-regulated kinase ...
https://link.springer.com/article/10.1007/s00125-017-4401-5

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Electrophoretic mobility shift assays.. 32P-labeled, double-stranded oligonucleotides EbS2 (5′-CAAGTCGGGGCAGATGTGGACTAGAACAAT ... we performed electrophoretic mobility shift assays (EMSAs). A 39 bp double-stranded oligonucleotide containing Eb and S2 sites ... Luciferase assay.. Luciferase activity was measured using the Dual Luciferase Assay System (Promega). The amount of transfected ... ChIP assays were performed with 24 hpf, mRNA-injected embryos, fixed with formaldehyde. A, B, Mouse IgG and mouse anti-V5 IgG ( ...
jneurosci.org/content/27/52/14375

*  The Transcriptional Activator LdtR from 'Candidatus Liberibacter asiaticus' Mediates Osmotic Stress Tolerance

Electrophoretic mobility shift assay Is the Subject Area "Electrophoretic mobility shift assay" applicable to this article? Yes ...
journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004101

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... in electrophoretic mobility shift assays using LNCaP cell nuclear extracts. These data demonstrate that cytosine methylation ... electrophoretic mobility shift assay; MS-PCR, methylation-sensitive PCR; HDAC, histone deacetylase complex; RT-PCR, reverse ... Mobility shift assay for binding of a MeCP1-like complex to the methylated GSTP1 promoter. End-labeled methylated (M-GSTP1) and ... Luciferase Assays.. Luciferase assays were performed using the Dual-Luciferase reporter assay system protocols manual as ...
cancerres.aacrjournals.org/content/61/12/4820

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Through the use of an electrophoretic mobility shift assay, we demonstrated that Tax induced NF-κB binding to both MCP-1 κB ... electrophoretic mobility shift assay; PBMC, peripheral blood mononuclear cell. ... When the A1 site was used as a probe, a clear shifted band was observed with nuclear extracts from HTLV-I-infected T-cell lines ... 8A ⇓ , Lanes 1-7). This shifted complex was specific to the A1 fragment because complex formation was competed away by addition ...
cancerres.aacrjournals.org/content/60/17/4939

GC box: In molecular biology, a GC box is a distinct pattern of nucleotides found in the promoter region of some eukaryotic genes upstream of the TATA box and approximately 110 bases upstream from the transcription initiation site. It has a consensus sequence GGGCGG which is position dependent and orientation independent.Symmetry element: A symmetry element is a point of reference about which symmetry operations can take place. In particular, symmetry elements can be centers of inversion, axes of rotation and mirror planes.DNA-binding proteinColes PhillipsPituitary-specific positive transcription factor 1: POU domain, class 1, transcription factor 1 (Pit1, growth hormone factor 1), also known as POU1F1, is a transcription factor for growth hormone.Eukaryotic transcription: Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells.DNA binding site: DNA binding sites are a type of binding site found in DNA where other molecules may bind. DNA binding sites are distinct from other binding sites in that (1) they are part of a DNA sequence (e.Proximity ligation assay: Proximity ligation assay (in situ PLA) is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions and modifications with high specificity and sensitivity. Protein targets can be readily detected and localized with single molecule resolution and objectively quantified in unmodified cells and tissues.DNA condensation: DNA condensation refers to the process of compacting DNA molecules in vitro or in vivo. Mechanistic details of DNA packing are essential for its functioning in the process of gene regulation in living systems.Optimax: Optimax is a UK-based laser eye treatment specialist which has 38 locations nationwide. Optimax was established as one of the first private clinics to offer laser eye surgery.RNA transfection: RNA transfection is the process of deliberately introducing RNA into a living cell. RNA can be purified from cells after lysis or synthesized from free nucleotides either chemically, or enzymatically using an RNA polymerase to transcribe a DNA template.Mature messenger RNA: Mature messenger RNA, often abbreviated as mature mRNA is a eukaryotic RNA transcript that has been spliced and processed and is ready for translation in the course of protein synthesis. Unlike the eukaryotic RNA immediately after transcription known as precursor messenger RNA, it consists exclusively of exons, with all introns removed.Electrophoresis (disambiguation): Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.Enhancer (genetics)Repressor: In molecular genetics, a repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA.CpG OligodeoxynucleotideSilent mutation: Silent mutations are mutations in DNA that do not significantly alter the phenotype of the organism in which they occur. Silent mutations can occur in non-coding regions (outside of genes or within introns), or they may occur within exons.Hypersensitive site: In genetics a hypersensitive site is a short region of chromatin and is detected by its super sensitivity to cleavage by DNase I and other various nucleases (DNase II and micrococcal nucleases). In a hypersensitive site, the nucleosomal structure is less compacted, increasing the availability of the DNA to binding by proteins, such as transcription factors and DNase I.Triparental mating: Triparental mating is a form of Bacterial conjugation where a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid present in a second bacterial strain into a third bacterial strain. Plasmids are introduced into bacteria for such purposes as transformation, cloning, or transposon mutagenesis.Protein primary structure: The primary structure of a peptide or protein is the linear sequence of its amino acid structural units, and partly comprises its overall biomolecular structure. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end.Ferric uptake regulator family: In molecular biology, the ferric uptake regulator (FUR) family of proteins includes metal ion uptake regulator proteins. These are responsible for controlling the intracellular concentration of iron in many bacteria.ChIP-exo: ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest (transcription factor) binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair.Abscription: During normal transcription, RNA polymerase transcribes a number of short nonproductive oligonucleotides, and this process is called abortive transcription. The trapped RNAPs have been named abscriptases and the synthesis of specific length oligonucleotides called abscription.CAAT box: In molecular biology, a CCAAT box (also sometimes abbreviated a CAAT box or CAT box) is a distinct pattern of nucleotides with GGCCAATCT consensus sequence that occur upstream by 60-100 bases to the initial transcription site. The CAAT box signals the binding site for the RNA transcription factor, and is typically accompanied by a conserved consensus sequence.Margaret Jope: Margaret Jope (1913–2004) was a Scottish biochemist, born as Henrietta Margaret Halliday in Peterhead, Scotland.Iroquois homeobox factor: Iroquois homeobox factors are a family of homeodomain transcription factors that play a role in many developmental processes.HT-0712Ligation-independent cloning: Ligation-independent cloning (LIC) is a form of molecular cloning that is able to be performed without the use of restriction endonucleases or DNA ligase. This allows genes that have restriction sites to be cloned without worry of chopping up the insert.TFAP2C: Transcription factor AP-2 gamma also known as AP2-gamma is a protein that in humans is encoded by the TFAP2C gene. AP2-gamma is a member of the activating protein 2 family of transcription factors.WIN 56,098: WIN 56,098 is a chemical that is considered to be an aminoalkylindole derivative. It is a tricyclic aryl derivative that acts as a competitive antagonist at the CB2 cannabinoid receptor.Early growth response proteins: Early growth response proteins are a family of zinc finger transcription factors.Pyrrolidine dithiocarbamateHyperphosphorylation: Hyperphosphorylation occurs when a biochemical with multiple phosphorylation sites is fully saturated. Hyperphosphorylation is one of the signalling mechanisms used by the cell to regulate mitosis.Allele-specific oligonucleotide: An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay.G-CSF factor stem-loop destabilising elementChemically induced dimerization: Chemically Induced Dimerization (CID) is a biological mechanism in which two proteins bind only in the presence of a certain small molecule, enzyme or other dimerizing agent. Genetically engineered CID systems are used in biological research to control protein localization, to manipulate signalling pathways and to induce protein activation.Zinc fingerDead Ronin: Dead Ronin is a compilation album by The Pack, released by Yeaah! Records in 2000.Deletion (genetics)Ying Yang Forever: Ying Yang Forever is the sixth studio album by Atlanta-based rap duo Ying Yang Twins. All of the tracks were produced by Joe Traxx.Orphan receptor: An orphan receptor is an apparent receptor that has a similar structure to other identified receptors but whose endogenous ligand has not yet been identified. If a ligand for an orphan receptor is later discovered, the receptor is referred to as an "adopted orphan".High mobility group protein HMG14 and HMG17: High mobility group protein HMG14 and HMG17 also known as nucleosomal binding domain is a family of evolutionarily related proteins.Tata Tapes controversy: The "Tata Tapes" controversy was a political scandal in India that was the culmination of a series of allegations of anti-national conduct levied by the then Chief Minister of Assam, Prafulla Kumar Mahanta, against the Tatas - their company Tata Tea in particular.Tata-AGP face-off: Target Tata The controversy erupted when journalist Ritu Sarin of the Indian Express broke a story that involved the illegal tapping of the telephone calls of business tycoon Nusli Wadia and published, on 4–5 October 1997, transcripts of the telephone conversations he had with Keshub Mahindra, Field Marshal Sam Maneckshaw, then Rajya Sabha Member of Parliament Jayant Malhoutra and Ratan Tata about Tata Tea's problems with the Assam Government.

(1/4336) Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA.

The ordered assembly of immunoglobulin and TCR genes by V(D)J recombination depends on the regulated accessibility of individual loci. We show here that the histone tails and intrinsic nucleosome structure pose significant impediments to V(D)J cleavage. However, alterations to nucleosome structure via histone acetylation or by stable hSWI/SNF-dependent remodeling greatly increase the accessibility of nucleosomal DNA to V(D)J cleavage. Moreover, acetylation and hSWI/SNF remodeling can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. These results are consistent with a model in which regulated recruitment of chromatin modifying activities is involved in mediating the lineage and stage-specific control of V(D)J recombination.  (+info)

(2/4336) An RNA-binding chameleon.

The arginine-rich RNA binding motif is found in a wide variety of proteins, including several viral regulatory proteins. Although related at the primary sequence level, arginine-rich domains from different proteins adopt different conformations depending on the RNA site recognized, and in some cases fold only in the context of RNA. Here we show that the RNA binding domain of the Jembrana disease virus (JDV) Tat protein is able to recognize two different TAR RNA sites, from human and bovine immunodeficiency viruses (HIV and BIV, respectively), adopting different conformations in the two RNA contexts and using different amino acids for recognition. In addition to the conformational differences, the JDV domain requires the cyclin T1 protein for high-affinity binding to HIV TAR, but not to BIV TAR. The "chameleon-like" behavior of the JDV Tat RNA binding domain reinforces the concept that RNA molecules can provide structural scaffolds for protein folding, and suggests mechanisms for evolving distinct RNA binding specificities from a single multifunctional domain.  (+info)

(3/4336) Activation of telomerase rna gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3.

Expression of the human telomerase RNA component gene, hTERC is essential for telomerase activity. The hTERC gene is expressed during embryogenesis and then downregulated during normal development, leaving most adult somatic cells devoid of hTERC expression. During oncogenesis, however, hTERC is re-expressed consequently contributing to the unrestricted proliferative capacity of many human cancers. Thus the identification of the molecular basis for the regulation of the telomerase RNA component gene in normal cells and its deregulation in cancer cells is of immediate interest. We have previously cloned the hTERC promoter and in this study have identified several transcription factors that modulate the expression of hTERC. We demonstrate that NF-Y binding to the CCAAT region of the hTERC promoter is essential for promoter activity. Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. These factors appear to act in a species-specific manner. Whereas Sp1 and Sp3 act on the human, bovine, and mouse TERC promoters, pRb activates only the human and bovine promoter, and NF-Y is only essential for the human TERC gene.  (+info)

(4/4336) Opposing regulation of choline deficiency-induced apoptosis by p53 and nuclear factor kappaB.

We have previously shown that fetal rat brain cells, preneuronal (PC12), and hepatocyte (CWSV-1) cells undergo apoptosis during choline deficiency (CD). The PC12 and epithelial cell culture models were used to determine the molecular mechanism by which CD induces apoptosis. Our data indicate that CD leads to both growth arrest and apoptosis in a subpopulation of cells, which correlate with the up-regulation of the tumor suppressor protein p53 and concurrent up-regulation of the cyclin-dependent kinase-inhibitor p21(WAF1/CIP1). Additionally, CD induced both a G1/S and a G2/M arrest. Transient transfection of a dominant negative p53 (p53DN) construct into PC12 cells, which inhibited endogenous p53 activation, significantly reduced the induction of apoptosis associated with CD. Interestingly, CD also induced the persistent activation of the transcription factor NF-kappaB. Activation of NF-kappaB has been shown to promote cell survival and proposed to antagonize p53. Consistent with this, expression of a super-repressor form of IkappaBalpha (SR-IkappaBalpha) that functions to strongly inhibit NF-kappaB activation, profoundly enhanced cell death during CD. In summary, these results suggest that the effects of CD on apoptosis and subsequent cell survival are mediated through two different signaling pathways, p53 and NF-kappaB, respectively. Taken together, our data demonstrates the induction of opposing mechanisms associated with nutrient deficiency that may provide a molecular mechanism by which CD promotes carcinogenesis.  (+info)

(5/4336) Induction of the transcriptional repressor Yin Yang-1 by vascular cell injury. Autocrine/paracrine role of endogenous fibroblast growth factor-2.

Yin Yang-1 (YY1) is a multifunctional transcription factor that can repress the expression of many growth factor, hormone, and cytokine genes implicated in atherogenesis. YY1 expression is activated in rat vascular smooth muscle cells shortly after injury. YY1 DNA binding activity paralleled elevated protein levels in the nucleus. Smooth muscle cell injury triggered the rapid extracellular release of immunoreactive fibroblast growth factor-2 (FGF-2). YY1 induction after injury was blocked by neutralizing antibodies directed against FGF-2. This growth factor increased YY1 mRNA and protein expression and stimulated YY1 binding and transcriptional activity. Overexpression of YY1 inhibited smooth muscle cell replication. Immunohistochemical analysis demonstrated YY1 staining in medial smooth muscle cells, coincident with FGF-2 expression. Proliferating cell nuclear antigen staining, in contrast, was confined mainly to the atherosclerotic intima. This is the first demonstration that YY1 is induced by either injury or FGF-2, is differentially expressed in normal and diseased human arteries, and that its overexpression inhibits vascular smooth muscle but not endothelial cell replication.  (+info)

(6/4336) Ingenol esters induce apoptosis in Jurkat cells through an AP-1 and NF-kappaB independent pathway.

BACKGROUND: Ingenol derivatives have received constant and multidisciplinary attention on account of their pleiotropic pattern of biological activity. This includes activation of protein kinase C (PKC), tumour-promotion, anticancer, and anti-HIV properties, and the possibility of dissecting co-cancerogenic and clinically useful activities has been demonstrated. Certain ingenol esters show powerful anticancer activity, and a structure-activity relationship model to discriminate between their apoptotic and non-apoptotic properties has been developed. RESULTS: The polyhydroxylated southern region of ingenol was selectively modified, using the anticancer and PKC activator ingenol 3,20-dibenzoate (IDB) as a lead compound. The evaluation of IDB analogues in apoptosis assays showed strict structure-activity relationships, benzoylation of the 20-hydroxyl being required to trigger apoptosis through a pathway involving caspase-3 and occurring at the specific cell cycle checkpoint that controls the S-M phase transition. Conversely, a study on the activation of the PKC-dependent transcription factors AP-1 and NF-kappaB by IDB analogues showed significant molecular flexibility, including tolerance to changes at the 3- and 20-hydroxyls. IDB-induced apoptosis was independent of activation of PKC, since it was not affected by treatment with the non-isoform-selective PKC inhibitor GF 109230X0. CONCLUSIONS: Remarkable deviations from the tumour-promotion pharmacophore were observed for both the apoptotic and the PKC-activating properties of IDB analogues, showing that ingenol is a viable template to selectively target crucial pathways involved in tumour promotion and development. Since the apoptotic and the PKC-activating properties of ingenoids are mediated by different pathways and governed by distinct structure-activity relationships, it is possible to dissect them by suitable chemical modification. In this context, the esterification pattern of the 5- and 20-hydroxyls is critical.  (+info)

(7/4336) CCAAT/enhancer-binding proteins alpha and beta negatively influence the capacity of tumor necrosis factor alpha to up-regulate the human cytomegalovirus IE1/2 enhancer/promoter by nuclear factor kappaB during monocyte differentiation.

Recently we demonstrated that the ability of tumor necrosis factor alpha (TNFalpha) to stimulate the human cytomegalovirus (HCMV) IE1/2 enhancer/promoter activity in myeloid progenitor-like cells decreases when these cells differentiate into promonocytic cells. In addition, TNFalpha stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor kappaB (NF-kappaB) activation and its binding to the 18-base pair sequence motifs of the IE1/2 enhancer. We demonstrate here that the cell differentiation-dependent reduction of TNFalpha stimulation is not due to insufficient NF-kappaB activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta. Overexpression of C/EBPalpha/beta in HL-60 cells, which normally produce only very small amounts of C/EBP, stimulated the basal activity of the promoter in the absence of NF-kappaB but suppressed the stimulatory effect of TNFalpha. A novel C/EBP-binding site was identified in the IE1/2 enhancer directly downstream of a NF-kappaB site. In order to understand the mechanisms of interaction, we used an IE1/2 promoter mutant that failed to bind C/EBP at this position and several constructs that contained exclusively NF-kappaB- and/or C/EBP-binding sites upstream of the minimal IE1/2 promoter. We could demonstrate that C/EBPalpha/beta interacts with NF-kappaB p65 and displays inhibitory activity even in the absence of direct DNA binding by forming p65-C/EBP-containing protein complexes bound to the NF-kappaB site. Moreover, C/EBP binding to the DNA adjacent to NF-kappaB supports the down-regulatory effect of C/EBPs possibly due to stabilization of a multimeric NF-kappaB-C/EBP complex. Our results show that cell differentiation factors may interfere with TNFalpha-induced human cytomegalovirus gene (re)activation.  (+info)

(8/4336) Cloning and characterization of a novel mouse AP-2 transcription factor, AP-2delta, with unique DNA binding and transactivation properties.

AP-2 transcription factors are sequence-specific DNA-binding proteins expressed in neural crest and other tissues during mammalian development. Three mammalian genes, AP-2alpha, AP-2beta, and AP-2gamma, have been reported previously. A partial predicted AP-2 gene was identified in tandem with AP-2beta on human chromosome 6p12-p21.1. The orthologous mouse gene, which we named Ap-2delta, was identified from a fetal mouse head cDNA library. Northern analysis revealed two transcripts in embryonic and newborn mouse brain, with markedly higher steady-state levels in the former. The predicted Ap-2delta protein comprised 452 amino acids and was highly similar to other AP-2 proteins across the DNA-binding and dimerization domains. Ap-2delta formed homodimers and heterodimers in vitro, bound an optimized AP-2 consensus DNA sequence, and transactivated gene expression in eukaryotic cells. Ap-2delta dimers bound poorly to an AP-2 binding sequence from the human metallothionein IIa promoter in vitro, revealing a sequence specificity not previously observed among other AP-2 proteins. The PY motif and critical residues in the transactivation domain, which are highly conserved in the AP-2 family and believed necessary for transactivation, were divergent in Ap-2delta. The unique protein sequence and functional features of Ap-2delta suggest mechanisms, besides tissue-specific AP-2 gene expression, for specific control of target gene activation.  (+info)



affinity


  • ChIP results and in vitro DNA binding assays revealed that Zic3 binds with high affinity and specificity on the Nanog promoter. (wiley.com)
  • Gel shift assays showed increased binding affinity of the "s" allele for Sp1 protein, and primary RNA transcripts derived from the "s" allele were approximately three times more abundant than "S" allele-derived transcripts in "Ss" heterozygotes. (pubmedcentralcanada.ca)