Mutagenesis, Site-Directed: Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.Mutagenesis: Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS.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.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.Binding Sites: The parts of a macromolecule that directly participate in its specific combination with another molecule.Mutagenesis, Insertional: Mutagenesis where the mutation is caused by the introduction of foreign DNA sequences into a gene or extragenic sequence. This may occur spontaneously in vivo or be experimentally induced in vivo or in vitro. Proviral DNA insertions into or adjacent to a cellular proto-oncogene can interrupt GENETIC TRANSLATION of the coding sequences or interfere with recognition of regulatory elements and cause unregulated expression of the proto-oncogene resulting in tumor formation.Base Sequence: The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.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.Models, Molecular: Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.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.Recombinant Proteins: Proteins prepared by recombinant DNA technology.Amino Acid Substitution: The naturally occurring or experimentally induced replacement of one or more AMINO ACIDS in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish, enhance, or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties.Structure-Activity Relationship: The relationship between the chemical structure of a compound and its biological or pharmacological activity. Compounds are often classed together because they have structural characteristics in common including shape, size, stereochemical arrangement, and distribution of functional groups.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.Kinetics: The rate dynamics in chemical or physical systems.Protein Conformation: The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).Cysteine: A thiol-containing non-essential amino acid that is oxidized to form CYSTINE.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.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.Catalysis: The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.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.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.Substrate Specificity: A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.Protein Structure, Secondary: The level of protein structure in which regular hydrogen-bond interactions within contiguous stretches of polypeptide chain give rise to alpha helices, beta strands (which align to form beta sheets) or other types of coils. This is the first folding level of protein conformation.Bacterial Proteins: Proteins found in any species of bacterium.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.Histidine: An essential amino acid that is required for the production of HISTAMINE.Point Mutation: A mutation caused by the substitution of one nucleotide for another. This results in the DNA molecule having a change in a single base pair.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.Catalytic Domain: The region of an enzyme that interacts with its substrate to cause the enzymatic reaction.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.Alanine: A non-essential amino acid that occurs in high levels in its free state in plasma. It is produced from pyruvate by transamination. It is involved in sugar and acid metabolism, increases IMMUNITY, and provides energy for muscle tissue, BRAIN, and the CENTRAL NERVOUS SYSTEM.Cell Line: Established cell cultures that have the potential to propagate indefinitely.Aspartic Acid: One of the non-essential amino acids commonly occurring in the L-form. It is found in animals and plants, especially in sugar cane and sugar beets. It may be a neurotransmitter.Crystallography, X-Ray: The study of crystal structure using X-RAY DIFFRACTION techniques. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)Lysine: An essential amino acid. It is often added to animal feed.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.Enzyme Stability: The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.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.Asparagine: A non-essential amino acid that is involved in the metabolic control of cell functions in nerve and brain tissue. It is biosynthesized from ASPARTIC ACID and AMMONIA by asparagine synthetase. (From Concise Encyclopedia Biochemistry and Molecular Biology, 3rd ed)Genes, Bacterial: The functional hereditary units of BACTERIA.DNA Mutational Analysis: Biochemical identification of mutational changes in a nucleotide sequence.DNA Transposable Elements: Discrete segments of DNA which can excise and reintegrate to another site in the genome. Most are inactive, i.e., have not been found to exist outside the integrated state. DNA transposable elements include bacterial IS (insertion sequence) elements, Tn elements, the maize controlling elements Ac and Ds, Drosophila P, gypsy, and pogo elements, the human Tigger elements and the Tc and mariner elements which are found throughout the animal kingdom.Escherichia coli Proteins: Proteins obtained from ESCHERICHIA COLI.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.Serine: A non-essential amino acid occurring in natural form as the L-isomer. It is synthesized from GLYCINE or THREONINE. It is involved in the biosynthesis of PURINES; PYRIMIDINES; and other amino acids.Amino Acid Motifs: Commonly observed structural components of proteins formed by simple combinations of adjacent secondary structures. A commonly observed structure may be composed of a CONSERVED SEQUENCE which can be represented by a CONSENSUS SEQUENCE.Mutant Proteins: Proteins produced from GENES that have acquired MUTATIONS.Restriction Mapping: Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.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.Sequence Deletion: Deletion of sequences of nucleic acids from the genetic material of an individual.Circular Dichroism: A change from planar to elliptic polarization when an initially plane-polarized light wave traverses an optically active medium. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)Protein Engineering: Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes.Arginine: An essential amino acid that is physiologically active in the L-form.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).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).)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.Cricetinae: A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS.Genetic Complementation Test: A test used to determine whether or not complementation (compensation in the form of dominance) will occur in a cell with a given mutant phenotype when another mutant genome, encoding the same mutant phenotype, is introduced into that cell.Saccharomyces cerevisiae: A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement.Tryptophan: An essential amino acid that is necessary for normal growth in infants and for NITROGEN balance in adults. It is a precursor of INDOLE ALKALOIDS in plants. It is a precursor of SEROTONIN (hence its use as an antidepressant and sleep aid). It can be a precursor to NIACIN, albeit inefficiently, in mammals.Hydrogen-Ion Concentration: The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH = log 1/2[1/(H+)], where (H+) is the hydrogen ion concentration in gram equivalents per liter of solution. (McGraw-Hill Dictionary of Scientific and Technical Terms, 6th ed)Electrophoresis, Polyacrylamide Gel: Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.Ligands: A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed)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.Glycosylation: The chemical or biochemical addition of carbohydrate or glycosyl groups to other chemicals, especially peptides or proteins. Glycosyl transferases are used in this biochemical reaction.DNA, Bacterial: Deoxyribonucleic acid that makes up the genetic material of bacteria.Hydrolysis: The process of cleaving a chemical compound by the addition of a molecule of water.Dimerization: The process by which two molecules of the same chemical composition form a condensation product or polymer.Mutagens: Chemical agents that increase the rate of genetic mutation by interfering with the function of nucleic acids. A clastogen is a specific mutagen that causes breaks in chromosomes.Electron Spin Resonance Spectroscopy: A technique applicable to the wide variety of substances which exhibit paramagnetism because of the magnetic moments of unpaired electrons. The spectra are useful for detection and identification, for determination of electron structure, for study of interactions between molecules, and for measurement of nuclear spins and moments. (From McGraw-Hill Encyclopedia of Science and Technology, 7th edition) Electron nuclear double resonance (ENDOR) spectroscopy is a variant of the technique which can give enhanced resolution. Electron spin resonance analysis can now be used in vivo, including imaging applications such as MAGNETIC RESONANCE IMAGING.Polymerase 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.Gene Expression Regulation, Bacterial: Any of the processes by which cytoplasmic or intercellular factors influence the differential control of gene action in bacteria.Ethylnitrosourea: A nitrosourea compound with alkylating, carcinogenic, and mutagenic properties.Glutamic Acid: A non-essential amino acid naturally occurring in the L-form. Glutamic acid is the most common excitatory neurotransmitter in the CENTRAL NERVOUS SYSTEM.CHO Cells: CELL LINE derived from the ovary of the Chinese hamster, Cricetulus griseus (CRICETULUS). The species is a favorite for cytogenetic studies because of its small chromosome number. The cell line has provided model systems for the study of genetic alterations in cultured mammalian cells.Amino Acids: Organic compounds that generally contain an amino (-NH2) and a carboxyl (-COOH) group. Twenty alpha-amino acids are the subunits which are polymerized to form proteins.Directed Molecular Evolution: The techniques used to produce molecules exhibiting properties that conform to the demands of the experimenter. These techniques combine methods of generating structural changes with methods of selection. They are also used to examine proposed mechanisms of evolution under in vitro selection conditions.Tyrosine: A non-essential amino acid. In animals it is synthesized from PHENYLALANINE. It is also the precursor of EPINEPHRINE; THYROID HORMONES; and melanin.Glycine: A non-essential amino acid. It is found primarily in gelatin and silk fibroin and used therapeutically as a nutrient. It is also a fast inhibitory neurotransmitter.Transcription Factors: Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process.Spin Labels: Molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin that can be detected by electron spin resonance spectroscopy and can be bonded to another molecule. (McGraw-Hill Dictionary of Chemical and Technical Terms, 4th ed)Phenotype: The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment.Genetic Vectors: DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.Carrier Proteins: Transport proteins that carry specific substances in the blood or across cell membranes.Phenylalanine: An essential aromatic amino acid that is a precursor of MELANIN; DOPAMINE; noradrenalin (NOREPINEPHRINE), and THYROXINE.Cercopithecus aethiops: A species of CERCOPITHECUS containing three subspecies: C. tantalus, C. pygerythrus, and C. sabeus. They are found in the forests and savannah of Africa. The African green monkey (C. pygerythrus) is the natural host of SIMIAN IMMUNODEFICIENCY VIRUS and is used in AIDS research.Disulfides: Chemical groups containing the covalent disulfide bonds -S-S-. The sulfur atoms can be bound to inorganic or organic moieties.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.Protein Processing, Post-Translational: Any of various enzymatically catalyzed post-translational modifications of PEPTIDES or PROTEINS in the cell of origin. These modifications include carboxylation; HYDROXYLATION; ACETYLATION; PHOSPHORYLATION; METHYLATION; GLYCOSYLATION; ubiquitination; oxidation; proteolysis; and crosslinking and result in changes in molecular weight and electrophoretic motility.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.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.Biocatalysis: The facilitation of biochemical reactions with the aid of naturally occurring catalysts such as ENZYMES.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.Phosphorylation: The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.Threonine: An essential amino acid occurring naturally in the L-form, which is the active form. It is found in eggs, milk, gelatin, and other proteins.Cell Membrane: The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.Hydrogen Bonding: A low-energy attractive force between hydrogen and another element. It plays a major role in determining the properties of water, proteins, and other compounds.Protein Folding: Processes involved in the formation of TERTIARY PROTEIN STRUCTURE.Protein Structure, Quaternary: The characteristic 3-dimensional shape and arrangement of multimeric proteins (aggregates of more than one polypeptide chain).Molecular Structure: The location of the atoms, groups or ions relative to one another in a molecule, as well as the number, type and location of covalent bonds.Codon: A set of three nucleotides in a protein coding sequence that specifies individual amino acids or a termination signal (CODON, TERMINATOR). Most codons are universal, but some organisms do not produce the transfer RNAs (RNA, TRANSFER) complementary to all codons. These codons are referred to as unassigned codons (CODONS, NONSENSE).SOS Response (Genetics): An error-prone mechanism or set of functions for repairing damaged microbial DNA. SOS functions (a concept reputedly derived from the SOS of the international distress signal) are involved in DNA repair and mutagenesis, in cell division inhibition, in recovery of normal physiological conditions after DNA repair, and possibly in cell death when DNA damage is extensive.Glutamine: A non-essential amino acid present abundantly throughout the body and is involved in many metabolic processes. It is synthesized from GLUTAMIC ACID and AMMONIA. It is the principal carrier of NITROGEN in the body and is an important energy source for many cells.Oxidation-Reduction: A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471).Mutation, Missense: A mutation in which a codon is mutated to one directing the incorporation of a different amino acid. This substitution may result in an inactive or unstable product. (From A Dictionary of Genetics, King & Stansfield, 5th ed)Models, Chemical: Theoretical representations that simulate the behavior or activity of chemical processes or phenomena; includes the use of mathematical equations, computers, and other electronic equipment.Ethyl Methanesulfonate: An antineoplastic agent with alkylating properties. It also acts as a mutagen by damaging DNA and is used experimentally for that effect.Temperature: The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.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.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.Ultraviolet Rays: That portion of the electromagnetic spectrum immediately below the visible range and extending into the x-ray frequencies. The longer wavelengths (near-UV or biotic or vital rays) are necessary for the endogenous synthesis of vitamin D and are also called antirachitic rays; the shorter, ionizing wavelengths (far-UV or abiotic or extravital rays) are viricidal, bactericidal, mutagenic, and carcinogenic and are used as disinfectants.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)Sequence Analysis, DNA: A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis.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.Nucleic Acid Conformation: The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.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.Models, Structural: A representation, generally small in scale, to show the structure, construction, or appearance of something. (From Random House Unabridged Dictionary, 2d ed)Proline: A non-essential amino acid that is synthesized from GLUTAMIC ACID. It is an essential component of COLLAGEN and is important for proper functioning of joints and tendons.Thermodynamics: A rigorously mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). It describes systems whose states are determined by thermal parameters, such as temperature, in addition to mechanical and electromagnetic parameters. (From Hawley's Condensed Chemical Dictionary, 12th ed)Peptide Mapping: Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases.Oxidoreductases: The class of all enzymes catalyzing oxidoreduction reactions. The substrate that is oxidized is regarded as a hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The recommended name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor. (Enzyme Nomenclature, 1992, p9)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.Leucine: An essential branched-chain amino acid important for hemoglobin formation.Peptide Fragments: Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.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.Isoleucine: An essential branched-chain aliphatic amino acid found in many proteins. It is an isomer of LEUCINE. It is important in hemoglobin synthesis and regulation of blood sugar and energy levels.Gene Expression: The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION.Spectrometry, Fluorescence: Measurement of the intensity and quality of fluorescence.Bacillus: A genus of BACILLACEAE that are spore-forming, rod-shaped cells. Most species are saprophytic soil forms with only a few species being pathogenic.Magnetic Resonance Spectroscopy: Spectroscopic method of measuring the magnetic moment of elementary particles such as atomic nuclei, protons or electrons. It is employed in clinical applications such as NMR Tomography (MAGNETIC RESONANCE IMAGING).Structural Homology, Protein: The degree of 3-dimensional shape similarity between proteins. It can be an indication of distant AMINO ACID SEQUENCE HOMOLOGY and used for rational DRUG DESIGN.Membrane Transport Proteins: Membrane proteins whose primary function is to facilitate the transport of molecules across a biological membrane. Included in this broad category are proteins involved in active transport (BIOLOGICAL TRANSPORT, ACTIVE), facilitated transport and ION CHANNELS.Peptides: Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are linear polypeptides that are normally synthesized on RIBOSOMES.Macromolecular Substances: Compounds and molecular complexes that consist of very large numbers of atoms and are generally over 500 kDa in size. In biological systems macromolecular substances usually can be visualized using ELECTRON MICROSCOPY and are distinguished from ORGANELLES by the lack of a membrane structure.Gene Library: A large collection of DNA fragments cloned (CLONING, MOLECULAR) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences.Serine Endopeptidases: Any member of the group of ENDOPEPTIDASES containing at the active site a serine residue involved in catalysis.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.DNA-Directed DNA Polymerase: DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair.Genetic Engineering: Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc.Diethyl Pyrocarbonate: Preservative for wines, soft drinks, and fruit juices and a gentle esterifying agent.DNA Repair: The reconstruction of a continuous two-stranded DNA molecule without mismatch from a molecule which contained damaged regions. The major repair mechanisms are excision repair, in which defective regions in one strand are excised and resynthesized using the complementary base pairing information in the intact strand; photoreactivation repair, in which the lethal and mutagenic effects of ultraviolet light are eliminated; and post-replication repair, in which the primary lesions are not repaired, but the gaps in one daughter duplex are filled in by incorporation of portions of the other (undamaged) daughter duplex. Excision repair and post-replication repair are sometimes referred to as "dark repair" because they do not require light.Viral Proteins: Proteins found in any species of virus.Transformation, Genetic: Change brought about to an organisms genetic composition by unidirectional transfer (TRANSFECTION; TRANSDUCTION, GENETIC; CONJUGATION, GENETIC, etc.) and incorporation of foreign DNA into prokaryotic or eukaryotic cells by recombination of part or all of that DNA into the cell's genome.Protein Subunits: Single chains of amino acids that are the units of multimeric PROTEINS. Multimeric proteins can be composed of identical or non-identical subunits. One or more monomeric subunits may compose a protomer which itself is a subunit structure of a larger assembly.Valine: A branched-chain essential amino acid that has stimulant activity. It promotes muscle growth and tissue repair. It is a precursor in the penicillin biosynthetic pathway.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.Genes, Reporter: Genes whose expression is easily detectable and therefore used to study promoter activity at many positions in a target genome. In recombinant DNA technology, these genes may be attached to a promoter region of interest.Fungal Proteins: Proteins found in any species of fungus.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.Saccharomyces cerevisiae Proteins: Proteins obtained from the species SACCHAROMYCES CEREVISIAE. The function of specific proteins from this organism are the subject of intense scientific interest and have been used to derive basic understanding of the functioning similar proteins in higher eukaryotes.Recombination, Genetic: Production of new arrangements of DNA by various mechanisms such as assortment and segregation, CROSSING OVER; GENE CONVERSION; GENETIC TRANSFORMATION; GENETIC CONJUGATION; GENETIC TRANSDUCTION; or mixed infection of viruses.Xenopus laevis: The commonest and widest ranging species of the clawed "frog" (Xenopus) in Africa. This species is used extensively in research. There is now a significant population in California derived from escaped laboratory animals.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.Cross-Linking Reagents: Reagents with two reactive groups, usually at opposite ends of the molecule, that are capable of reacting with and thereby forming bridges between side chains of amino acids in proteins; the locations of naturally reactive areas within proteins can thereby be identified; may also be used for other macromolecules, like glycoproteins, nucleic acids, or other.Alleles: Variant forms of the same gene, occupying the same locus on homologous CHROMOSOMES, and governing the variants in production of the same gene product.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.Geobacillus stearothermophilus: A species of GRAM-POSITIVE ENDOSPORE-FORMING BACTERIA in the family BACILLACEAE, found in soil, hot springs, Arctic waters, ocean sediments, and spoiled food products.Glycoside HydrolasesZinc: A metallic element of atomic number 30 and atomic weight 65.38. It is a necessary trace element in the diet, forming an essential part of many enzymes, and playing an important role in protein synthesis and in cell division. Zinc deficiency is associated with ANEMIA, short stature, HYPOGONADISM, impaired WOUND HEALING, and geophagia. It is known by the symbol Zn.Endopeptidases: A subclass of PEPTIDE HYDROLASES that catalyze the internal cleavage of PEPTIDES or PROTEINS.Glutathione Transferase: A transferase that catalyzes the addition of aliphatic, aromatic, or heterocyclic FREE RADICALS as well as EPOXIDES and arene oxides to GLUTATHIONE. Addition takes place at the SULFUR. It also catalyzes the reduction of polyol nitrate by glutathione to polyol and nitrite.Plant Proteins: Proteins found in plants (flowers, herbs, shrubs, trees, etc.). The concept does not include proteins found in vegetables for which VEGETABLE PROTEINS is available.Biological Transport: The movement of materials (including biochemical substances and drugs) through a biological system at the cellular level. The transport can be across cell membranes and epithelial layers. It also can occur within intracellular compartments and extracellular compartments.Protein Multimerization: The assembly of the QUATERNARY PROTEIN STRUCTURE of multimeric proteins (MULTIPROTEIN COMPLEXES) from their composite PROTEIN SUBUNITS.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.Streptomyces: A genus of bacteria that form a nonfragmented aerial mycelium. Many species have been identified with some being pathogenic. This genus is responsible for producing a majority of the ANTI-BACTERIAL AGENTS of practical value.Bacillus subtilis: A species of gram-positive bacteria that is a common soil and water saprophyte.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.Adenosine Triphosphate: An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.Transformation, Bacterial: The heritable modification of the properties of a competent bacterium by naked DNA from another source. The uptake of naked DNA is a naturally occuring phenomenon in some bacteria. It is often used as a GENE TRANSFER TECHNIQUE.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.Virulence: The degree of pathogenicity within a group or species of microorganisms or viruses as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenic capacity of an organism is determined by its VIRULENCE FACTORS.Hot Temperature: Presence of warmth or heat or a temperature notably higher than an accustomed norm.Molecular Conformation: The characteristic three-dimensional shape of a molecule.Models, Biological: Theoretical representations that simulate the behavior or activity of biological processes or diseases. For disease models in living animals, DISEASE MODELS, ANIMAL is available. Biological models include the use of mathematical equations, computers, and other electronic equipment.Cricetulus: A genus of the family Muridae consisting of eleven species. C. migratorius, the grey or Armenian hamster, and C. griseus, the Chinese hamster, are the two species used in biomedical research.Transcriptional Activation: Processes that stimulate the GENETIC TRANSCRIPTION of a gene or set of genes.Spectrophotometry: The art or process of comparing photometrically the relative intensities of the light in different parts of the spectrum.Open Reading Frames: A sequence of successive nucleotide triplets that are read as CODONS specifying AMINO ACIDS and begin with an INITIATOR CODON and end with a stop codon (CODON, TERMINATOR).Static Electricity: The accumulation of an electric charge on a objectOocytes: Female germ cells derived from OOGONIA and termed OOCYTES when they enter MEIOSIS. The primary oocytes begin meiosis but are arrested at the diplotene state until OVULATION at PUBERTY to give rise to haploid secondary oocytes or ova (OVUM).Regulatory Sequences, Nucleic Acid: Nucleic acid sequences involved in regulating the expression of genes.Surface Plasmon Resonance: A biosensing technique in which biomolecules capable of binding to specific analytes or ligands are first immobilized on one side of a metallic film. Light is then focused on the opposite side of the film to excite the surface plasmons, that is, the oscillations of free electrons propagating along the film's surface. The refractive index of light reflecting off this surface is measured. When the immobilized biomolecules are bound by their ligands, an alteration in surface plasmons on the opposite side of the film is created which is directly proportional to the change in bound, or adsorbed, mass. Binding is measured by changes in the refractive index. The technique is used to study biomolecular interactions, such as antigen-antibody binding.Isoenzymes: Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics.Hydrolases: Any member of the class of enzymes that catalyze the cleavage of the substrate and the addition of water to the resulting molecules, e.g., ESTERASES, glycosidases (GLYCOSIDE HYDROLASES), lipases, NUCLEOTIDASES, peptidases (PEPTIDE HYDROLASES), and phosphatases (PHOSPHORIC MONOESTER HYDROLASES). EC 3.Subtilisins: A family of SERINE ENDOPEPTIDASES isolated from Bacillus subtilis. EC 3.4.21.-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.DNA Replication: The process by which a DNA molecule is duplicated.Glutamates: Derivatives of GLUTAMIC ACID. Included under this heading are a broad variety of acid forms, salts, esters, and amides that contain the 2-aminopentanedioic acid structure.Adenosine Triphosphatases: A group of enzymes which catalyze the hydrolysis of ATP. The hydrolysis reaction is usually coupled with another function such as transporting Ca(2+) across a membrane. These enzymes may be dependent on Ca(2+), Mg(2+), anions, H+, or DNA.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.Spodoptera: A genus of owlet moths of the family Noctuidae. These insects are used in molecular biology studies during all stages of their life cycle.Phenylglyoxal: A reagent that is highly selective for the modification of arginyl residues. It is used to selectively inhibit various enzymes and acts as an energy transfer inhibitor in photophosphorylation.Hydrophobic and Hydrophilic Interactions: The thermodynamic interaction between a substance and WATER.Virus Replication: The process of intracellular viral multiplication, consisting of the synthesis of PROTEINS; NUCLEIC ACIDS; and sometimes LIPIDS, and their assembly into a new infectious particle.HEK293 Cells: A cell line generated from human embryonic kidney cells that were transformed with human adenovirus type 5.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.Pichia: Yeast-like ascomycetous fungi of the family Saccharomycetaceae, order SACCHAROMYCETALES isolated from exuded tree sap.Heme: The color-furnishing portion of hemoglobin. It is found free in tissues and as the prosthetic group in many hemeproteins.Protein Interaction Domains and Motifs: Protein modules with conserved ligand-binding surfaces which mediate specific interaction functions in SIGNAL TRANSDUCTION PATHWAYS and the specific BINDING SITES of their cognate protein LIGANDS.

*  Mutation of the ATP-Binding Pocket of SSA1 Indicates That a Functional Interaction Between Ssa1p and Ydj1p Is Required for Post...
Site-directed mutagenesis, cloning, strains, and media: Four mutant alleles of SSA1, ssa1-101 (K69Q), ssa1-102 (G198D), ssa1- ... 1991 Functional analysis of a conserved amino-terminal region of hsp70 by site-directed mutagenesis. Yeast 7: 699-716. ... were created using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). SSA1 inserted between the HindIII ... maintaining a unique NcoI site at the 3′ end and introducing a HindIII site, a new start codon, and six consecutive histidine ...
  http://www.genetics.org/content/156/2/501
*  Difference between revisions of "Richard Lab:Site Directed Mutagenesis" - OpenWetWare
An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004; 32(14): e115. ... This protocol only uses one oligo per mutation site (cuts the oligo cost in half) but can mutate more than one site at a time. ... This protocol only uses one oligo per mutation site (cuts the oligo cost in half) and can mutate more than one site at a time. ... Retrieved from "https://openwetware.org/mediawiki/index.php?title=Richard_Lab:Site_Directed_Mutagenesis&oldid=518978" ...
  https://openwetware.org/wiki/?title=Richard_Lab:Site_Directed_Mutagenesis&diff=518978&oldid=518976
*  Lidstrom:Site-Directed Mutagenesis - OpenWetWare
Lidstrom:Site-Directed Mutagenesis. From OpenWetWare. Revision as of 06:44, 29 February 2012 by Janet B. Matsen. (talk , ... Retrieved from "https://openwetware.org/mediawiki/index.php?title=Lidstrom:Site-Directed_Mutagenesis&oldid=588129" ... Kunkel Mutagenesis. *Justin Siegel at the Baker lab is our local expert. The UW iGem team uses this method. ...
  https://openwetware.org/wiki/?title=Lidstrom:Site-Directed_Mutagenesis&oldid=588129
*  Site-directed mutagenesis - Wikipedia
Nobel Lecture on Invention of Site-Directed Mutagenesis OpenWetWare Diagram summarizing site-directed mutagenesis. ... Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker Direct gene deletion and site-specific ... Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and ... "SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites". BMC Bioinformatics. 14 ...
  https://en.wikipedia.org/wiki/Site-directed_mutagenesis
*  Site directed mutagenesis
... WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de). Mon Aug 15 03:50:43 EST 2011 * ...
  http://www.bio.net/bionet/mm/methods/2011-August/105220.html
*  help for site directed mutagenesis
... philip hardwidge hardp3 at mayo.edu Fri Feb 23 09:16:01 EST 2001 *Previous message: help ... I would like undertaken site directed mutagenesis, but never did. : , : , Can somebody tell me if it is very difficult to do, ... Several papers from about 10 years ago offer facile PCR-based methods for making site-directed mutants. -Phil Hardwidge Mayo ... Previous message: help for site directed mutagenesis *Next message: help for site directed mutagenesis ...
  http://www.bio.net/bionet/mm/methods/2001-February/087575.html
*  Site Directed Mutagenesis Of Dienelactone Hydrolase - Digital Library
Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ... Chen, Wei, 1965-. Site Directed Mutagenesis Of Dienelactone Hydrolase, thesis, December 1992; Denton, Texas. (digital.library. ... Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ... Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ...
  https://digital.library.unt.edu/ark:/67531/metadc500900/
*  Efficient multi-site-directed mutagenesis directly from genomic template.
... the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated ... This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for ... In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved ...
  http://www.biomedsearch.com/nih/Efficient-multi-site-directed-mutagenesis/23151786.html
*  Knight:Site-directed mutagenesis/Single site - OpenWetWare
Knight:Site-directed mutagenesis/Single site. From OpenWetWare. Revision as of 10:59, 24 November 2008 by Tk. (talk , contribs) ... Back to Site-directed mutagenesis Note that this is a work in progress and needs to be verified against my lab notebooks. -- ... This procedure is primarily derived from the Stratagene QuikChange Site-Directed Mutagenesis manual with some modifications ... Retrieved from "https://openwetware.org/mediawiki/index.php?title=Knight:Site-directed_mutagenesis/Single_site&oldid=264831" ...
  https://openwetware.org/wiki/?title=Knight:Site-directed_mutagenesis/Single_site&oldid=264831
*  Knight:Site-directed mutagenesis/Multi site - OpenWetWare
Knight:Site-directed mutagenesis/Multi site. From OpenWetWare. Revision as of 12:14, 23 August 2007 by Reshma P. Shetty. (talk ... This procedure is primarily derived from the Stratagene QuikChange Multi Site-Directed Mutagenesis manual with some ... Retrieved from "https://openwetware.org/mediawiki/index.php?title=Knight:Site-directed_mutagenesis/Multi_site&oldid=144493" ... This protocol permits mutation at multiple sites simultaneously with only a single oligo per site. ...
  https://openwetware.org/wiki/?title=Knight:Site-directed_mutagenesis/Multi_site&oldid=144493
*  Site-Directed Mutagenesis - Cloning and Mutagenesis - GENEWIZ
SITE-DIRECTED MUTAGENESIS. GENEWIZ can increase your research productivity by performing your time-consuming site-directed ... Our customized mutagenesis services provide a fail-safe approach to obtain mutant constructs quickly, with 100% accuracy, thus ...
  https://www.genewiz.com/en/Public/Services/Cloning-Mutagenesis/Site-Directed-Mutagenesis
*  Expression and site-directed mutagenesis of hepatic glucokinase | Biochemical Journal
Expression and site-directed mutagenesis of hepatic glucokinase. A J Lange, L Z Xu, F Van Poelwijk, K Lin, D K Granner, S J ... Expression and site-directed mutagenesis of hepatic glucokinase. A J Lange, L Z Xu, F Van Poelwijk, K Lin, D K Granner, S J ... Expression and site-directed mutagenesis of hepatic glucokinase Message Subject (Your Name) has forwarded a page to you from ... Your Name) thought you would like to see this page from the Biochemical Journal web site. ...
  http://www.biochemj.org/content/277/1/159
*  Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ | SpringerLink
Puett D., Huang J., Xia H. (1994) Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ. In: ... Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ. ... Site-directed mutagensis of virtually any plasmid by eliminating a unique site. Anal Biochem 1992; 200: 81-8.PubMedCrossRef ... El-Deiry S, Kaetzel D, Kennedy G, Nilson J, Puett D. Site-directed muta-genesis of the human chorionic gonadotropin β-subunit: ...
  https://link.springer.com/chapter/10.1007/978-1-4613-8386-4_12
*  Difference between revisions of "Lidstrom:Site-Directed Mutagenesis" - OpenWetWare
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Lidstrom:Site-Directed_Mutagenesis&oldid=588128" ... Kunkel Mutagenesis. *Justin Siegel at the Baker lab is our local expert. The UW iGem team uses this method. ...
  https://openwetware.org/wiki/?title=Lidstrom:Site-Directed_Mutagenesis&diff=588128&oldid=588127
*  Problems with Quick change lightning multi site directed mutagenesis kit - Molecular Cloning - BioForum
I am using quick change lightning multi site directed mutagenesis kit from stratagene, i want to mutate 5nt , my plasmid+insert ... Problems with Quick change lightning multi site directed mutagenesis kit - posted in Molecular Cloning: ... Problems with Quick change lightning multi site directed mutagenesis kit. Started by Adnan, Sep 18 2009 07:13 AM ... I am using quick change multi site directed mutagenesis kit.........and am following its protocol only, nt a mixed protocol.. I ...
  http://www.protocol-online.org/forums/topic/10371-problems-with-quick-change-lightning-multi-site-directed-mutagenesis-kit/
*  IJMS | Free Full-Text | Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed...
... site-directed mutagenesis uricase; exon replacement/restoration; site-directed mutagenesis ... After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity ... Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis. Guangrong Xie †. ... In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis ...
  http://www.mdpi.com/1422-0067/17/5/764
*  Difference between revisions of "'Round-the-horn site-directed mutagenesis" - OpenWetWare
... this is the case for any site-directed protocol). Finally, as opposed to standard site-directed mutagenesis, this procedure ... this is the case for any site-directed protocol). Finally, as opposed to standard site-directed mutagenesis, this procedure ... this is the case for any site-directed protocol). Finally, as opposed to standard site-directed mutagenesis, this procedure ... Difference between revisions of "'Round-the-horn site-directed mutagenesis". From OpenWetWare ...
  https://openwetware.org/wiki/?title=
*  Site Directed Mutagenesis of the Murine Coronavirus Spike Protein | Springer for Research & Development
Bos E.C.W., Heijnen L., Spaan W.J.M. (1995) Site Directed Mutagenesis of the Murine Coronavirus Spike Protein. In: Talbot P.J ... Site Directed Mutagenesis of the Murine Coronavirus Spike Protein. Effects on Fusion ... Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus ... We use cookies to improve your experience with our site. More information Accept. ...
  https://rd.springer.com/chapter/10.1007/978-1-4615-1899-0_45
*  Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I | Biochemical Journal
Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I. S J Thornewell, S G Waley ... Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I Message Subject (Your Name) has forwarded a page ... A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala ... Your Name) thought you would like to see this page from the Biochemical Journal web site. ...
  http://www.biochemj.org/content/288/3/1045
*  Changing the Regioselectivity of the Tryptophan 7-Halogenase PrnA by Site-Directed Mutagenesis.
Changing the Regioselectivity of the Tryptophan 7-Halogenase PrnA by Site-Directed Mutagenesis.. код для вставки. код для ... 201007896 Enzymatic Halogenation Changing the Regioselectivity of the Tryptophan 7-Halogenase PrnA by Site-Directed Mutagenesis ... Figure 1. Active site of PrnA showing the binding of the substrate tryptophan. The isoalloxazine ring of FAD is to the left and ... KGaA, Weinheim 2951 Communications Figure 2. Model of the active site of PrnA (gray C, blue N, red O). The suggested change in ...
  https://www.docme.ru/doc/1914763/changing-the-regioselectivity-of-the-tryptophan-7-halogen.
*  Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C |...
Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C. Radha ... Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C ... Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C ... Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C ...
  http://www.biochemj.org/content/367/1/255
*  The paclitaxel site in tubulin probed by site-directed mutagenesis of Saccharomyces cerevisiae β-tubulin
... Ruth A. Entwistle,a ... We have used site-directed mutagenesis to examine the relative importance of the five residues in determining sensitivity of ... Home , About , Journals , Submit , Contact Us , Français. Enter Your Search:. Search tips. Search criteria. Articles. Journal ... Modeling of the paclitaxel site (Fig. 2) places the side chain of this residue far from paclitaxel (the closest K19 side chain ...
  http://pubmedcentralcanada.ca/pmcc/articles/PMC2577837/?lang=en-ca
*  Structure Cluster - 1CGU: CATALYTIC CENTER OF CYCLODEXTRIN GLYCOSYLTRANSFERASE DERIVED FROM X-RAY STRUCTURE ANALYSIS...
Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed ... CENTER OF CYCLODEXTRIN GLYCOSYLTRANSFERASE DERIVED FROM X-RAY STRUCTURE ANALYSIS COMBINED WITH SITE-DIRECTED MUTAGENESIS. ... Our website will not work properly. Please update to a newer version or download a new web browser, such as Chrome or Firefox. ... Pre-calculated protein structure alignments at the RCSB PDB website. Bioinformatics 26: 2983-2985 ...
  http://www.rcsb.org/pdb/explore/structureCluster.do?structureId=1CGU
*  Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes |...
The plasmids were subsequently subjected to sitedirected mutagenesis using the Quick‐Change mutagenesis kit (Stratagene). The ... This website is best viewed using the latest versions of all modern web browsers. Older browsers may not display correctly. ... we symbolized this direct interaction in the model by a direct contact between Ada2 and these two proteins. Furthermore, we ... furthermore identified direct interactions between the pairs Taf5-Taf6 and Taf6-Taf9; therefore, we permitted direct contact ...
  http://msb.embopress.org/content/7/1/503
*  Advanced Enzymes || About Us
The lab employs classical mutagenesis as well as site-directed mutagenesis methods to achieve its goals. ... Learn More About Enzymes , Site Map , Videos. Copyright © Advanced Enzymes. All Rights Reserved. Site Designed By Dotcom ... Advanced Enzymes establishes direct exports in over 59 countries globally. *Dr. Anil Gupta, General Manager - R&D, receives the ... Website: http://www.enzytech.in. Advanced Bio-Agro Tech Ltd., India (BioAgro). BioAgro, a subsidiary of Advanced Enzymes, ...
  http://advancedenzymes.com/abt_cert.aspx

Coles PhillipsProtein 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.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.Signature-tagged mutagenesis: Signature-tagged mutagenesis (STM) is a genetic technique used to study gene function. Recent advances in genome sequencing have allowed us to catalogue a large variety of organisms' genomes, but the function of the genes they contain is still largely unknown.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.Silent 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.Reaction coordinateList of strains of Escherichia coli: Escherichia coli is a well studied bacterium that was first identified by Theodor Escherich, after whom it was later named.Ethyl groupBurst kinetics: Burst kinetics is a form of enzyme kinetics that refers to an initial high velocity of enzymatic turnover when adding enzyme to substrate. This initial period of high velocity product formation is referred to as the "Burst Phase".Database of protein conformational diversity: The Database of protein conformational diversity (PCDB) is a database of diversity of protein tertiary structures within protein domains as determined by X-ray crystallography. Proteins are inherently flexible and this database collects information on this subject for use in molecular research.FERM domain: In molecular biology, the FERM domain (F for 4.1 protein, E for ezrin, R for radixin and M for moesin) is a widespread protein module involved in localising proteins to the plasma membrane.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.Acid catalysis: In acid catalysis and base catalysis a chemical reaction is catalyzed by an acid or a base. The acid is the proton donor and the base is the proton acceptor.Ligation-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.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.Specificity constant: In the field of biochemistry, the specificity constant (also called kinetic efficiency or k_{cat}/K_{M}), is a measure of how efficiently an enzyme converts substrates into products. A comparison of specificity constants can also be used as a measure of the preference of an enzyme for different substrates (i.Transmembrane domain: Transmembrane segment usually denotes a single transmembrane alpha helix of a transmembrane protein, also known as an integral protein.http://www.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.CS-BLASTHistidinePoint mutationPhase problem: In physics the phase problem is the name given to the problem of loss of information concerning the phase that can occur when making a physical measurement. The name itself comes from the field of x-ray crystallography, where the phase problem has to be solved for the determination of a structure from diffraction data.AcetyllysineRNA 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.CpG OligodeoxynucleotideAsparagineComposite transposon: A composite transposon is similar in function to simple transposons and Insertion Sequence (IS) elements in that it has protein coding DNA segments flanked by inverted, repeated sequences that can be recognized by transposase enzymes. A composite transposon, however, is flanked by two separate IS elements which may or may not be exact replicas.Escherichia coli (molecular biology): Escherichia coli (; commonly abbreviated E. coli) is a gammaproteobacterium commonly found in the lower intestine of warm-blooded organisms (endotherms).Rigerimod: Rigerimod (IPP-201101, Lupuzor) is a polypeptide corresponding to the sequence 131-151 of the 70k snRNP protein with a serine phosphorylated in position 140.http://www.Short linear motifGC 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.X-ray magnetic circular dichroismProtein engineering: Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles.Protein detoxification: Protein detoxification is the process by which proteins containing methylated arginine are broken down and removed from the body.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.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.Baby hamster kidney cell: Baby Hamster Kidney fibroblasts (aka BHK cells) are an adherent cell line used in molecular biology.Zuotin: Z-DNA binding protein 1, also known as Zuotin, is a Saccharomyces cerevisiae yeast gene.Tryptophan operon leaderAlkaliphile: Alkaliphiles are a class of extremophilic microbes capable of survival in alkaline (pH roughly 8.5-11) environments, growing optimally around a pH of 10.Margaret Jope: Margaret Jope (1913–2004) was a Scottish biochemist, born as Henrietta Margaret Halliday in Peterhead, Scotland.Ligand (biochemistry): In biochemistry and pharmacology, a ligand is a substance that forms a complex with a biomolecule to serve a biological purpose. In protein-ligand binding, the ligand is usually a signal-triggering molecule binding to a site on a target protein.DNA-binding proteinGlycosylation: Glycosylation (see also chemical glycosylation) is the reaction in which a carbohydrate, i.e.Chemically 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.Mutagen: In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations can cause cancer, mutagens are therefore also likely to be carcinogens.Zero field splitting: Zero field splitting describes various interactions of the energy levels of an electron spin (S>1/2) even in the absence of an applied magnetic field. It is important in the electron spin resonance of biological molecules.Thermal cyclerForward genetics: Forward genetics is the approach of determining the genetic basis responsible for a phenotype. This was initially done by generating mutants by using radiation, chemicals, or insertional mutagenesis (e.Excitotoxicity: Excitotoxicity is the pathological process by which nerve cells are damaged or killed by excessive stimulation by neurotransmitters such as glutamate and similar substances. This occurs when receptors for the excitatory neurotransmitter glutamate (glutamate receptors) such as the NMDA receptor and AMPA receptor are overactivated by glutamatergic storm.Proteinogenic amino acid: Proteinogenic amino acids are amino acids that are precursors to proteins, and are incorporated into proteins cotranslationally — that is, during translation. There are 23 proteinogenic amino acids in prokaryotes (including N-Formylmethionine, mainly used to initiate protein synthesis and often removed afterward), but only 21 are encoded by the nuclear genes of eukaryotes.Non-receptor tyrosine kinase: Non-receptor tyrosine kinases (nRTKs) are cytoplasmic enzymes that are responsible for catalysing the transfer of a phosphate group from a nucleoside triphosphate donor, such as ATP, to tyrosine residues in proteins. Non-receptor tyrosine kinases are a subgroup of protein family tyrosine kinases, enzymes that can transfer the phosphate group from ATP to a tyrosine residue of a protein (phosphorylation).Glycine (plant): Glycine is a genus in the bean family Fabaceae. The best known species is the soybean (Glycine max).Pituitary-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.Site-directed spin labeling: Site-directed spin labeling (SDSL) is a technique for investigating the structure and local dynamics of proteins using electron spin resonance. The theory of SDSL is based on the specific reaction of spin labels with amino acids.Phenotype microarray: The phenotype microarray approach is a technology for high-throughput phenotyping of cells.Multiple cloning site: A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid.Phenylalanine N-monooxygenase: Phenylalanine N-monooxygenase (, phenylalanine N-hydroxylase, CYP79A2) is an enzyme with system name L-phenylalanine,NADPH:oxygen oxidoreductase (N-hydroxylating). This enzyme catalyses the following chemical reactionDsbC protein family: DsbC (Disulfide bond C) is a prokaryotic disulfide bond isomerase. The formation of native disulfide bonds play an important role in the proper folding of proteins and stabilize tertiary structures of the protein.KIAA0895L: Uncharacterized protein KIAA0895-like also known as LOC653319, is a protein that in humans is encoded by the KIAA0895L gene.Membrane protein: Membrane proteins are proteins that interact with biological membranes. They are one of the common types of protein along with soluble globular proteins, fibrous proteins, and disordered proteins.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.Hyperphosphorylation: 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.Threonine proteaseCell membraneIce Ih: [showing details of an ice cube under magnification. Ice Ih is the form of ice commonly seen on Earth.Knotted protein: Knotted proteins are proteins whose backbones entangle themselves in a knot. One can imagine pulling a protein chain from both termini, as though pulling a string from both ends.

(1/31010) Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.

The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly.  (+info)

(2/31010) Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.

Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane.  (+info)

(3/31010) Regulation of p53 function and stability by phosphorylation.

The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21(Waf1/Cip1)-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.  (+info)

(4/31010) Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo.

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.  (+info)

(5/31010) Functional studies by site-directed mutagenesis on the role of Sp1 in the expression of the pyruvate kinase M and aldolase A genes.

During the cell cycle of mitogen stimulated rat thymocytes, an 8-10-fold induction of glycolytic enzymes and a corresponding increase in the mRNA levels has been observed. This prompted us to study the transcriptional regulation of the rat aldolase A and pyruvate kinase M genes. cis-Regulatory elements of both promoters were evaluated by site-directed mutagenesis of promoter/luciferase constructs and transient transfections of rat hepatoma FTO2B cells. Furthermore, the binding proteins were identified by mobility shift assays in the presence of specific antibodies. In the aldolase AH1 promoter, five binding sites for Sp1 and Sp3 and a TPA responsive element were identified as essential for transcriptional regulation. Most of the promoter activity can be attributed to these regulatory elements. In the pyruvate kinase M promoter three out of five binding sites of Sp1 and Sp3 (B box and GC boxes 1 and 3) turned out to be functional in the transfection assays whereas the disruption of GC box 2 had no effect, and the disruption of the GC box 4 had only a minor effect on the promoter activity. Both promoters are stimulated by Sp1 as well as Sp3, as judged by cotransfection experiments of Drosophila SL2 cells. Therefore, the Sp1- and Sp3-directed transcription provides a means for common regulatory mechanism of the aldolase A and the pyruvate kinase M genes.  (+info)

(6/31010) An antiviral mechanism of nitric oxide: inhibition of a viral protease.

Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses.  (+info)

(7/31010) Molecular dynamics of the sodium channel pore vary with gating: interactions between P-segment motions and inactivation.

Disulfide trapping studies have revealed that the pore-lining (P) segments of voltage-dependent sodium channels undergo sizable motions on a subsecond time scale. Such motions of the pore may be necessary for selective ion translocation. Although traditionally viewed as separable properties, gating and permeation are now known to interact extensively in various classes of channels. We have investigated the interaction of pore motions and voltage-dependent gating in micro1 sodium channels engineered to contain two cysteines within the P segments. Rates of catalyzed internal disulfide formation (kSS) were measured in K1237C+W1531C mutant channels expressed in oocytes. During repetitive voltage-clamp depolarizations, increasing the pulse duration had biphasic effects on the kSS, which first increased to a maximum at 200 msec and then decreased with longer depolarizations. This result suggested that occupancy of an intermediate inactivation state (IM) facilitates pore motions. Consistent with the known antagonism between alkali metals and a component of slow inactivation, kSS varied inversely with external [Na+]o. We examined the converse relationship, namely the effect of pore flexibility on gating, by measuring recovery from inactivation in Y401C+E758C (YC/EC) channels. Under oxidative conditions, recovery from inactivation was slower than in a reduced environment in which the spontaneous YC/EC cross-link is disrupted. The most prominent effects were slowing of a component with intermediate recovery kinetics, with diminution of its relative amplitude. We conclude that occupancy of an intermediate inactivation state facilitates motions of the P segments; conversely, flexibility of the P segments alters an intermediate component of inactivation.  (+info)

(8/31010) Functional consequences of mutations in the human alpha1A calcium channel subunit linked to familial hemiplegic migraine.

Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.  (+info)



  • genome
  • Since 2013, the development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing of the genome, and mutagenesis may be performed in vivo with relative ease. (wikipedia.org)
  • These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. (wikipedia.org)
  • The primary advantage of this technique is its ability to eliminate any foreign DNA from the genome after the mutagenesis process. (wikipedia.org)
  • Since 2013, development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing or mutagenesis of the genome in vivo. (wikipedia.org)
  • enzyme
  • The coenzymes bind to the active site of an enzyme to promote catalysis. (wikipedia.org)
  • HindIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis. (wikipedia.org)
  • The segment may be at an enzyme active site, or sequences that have structural significance or immunogenic property. (wikipedia.org)
  • specificity
  • Since the glycoprotein hormones contain a common α-subunit, and since both subunits are believed to participate in receptor binding (6, 7), interesting questions and models can be developed regarding subunit contact sites and hormone receptor contact sites, particularly those responsible for conferring specificity. (springer.com)
  • gene
  • This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. (wikipedia.org)
  • The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. (wikipedia.org)
  • restriction site
  • These protocols are used if you want to change one to three consecutive bases in a sequence (like removing a restriction site or changing an amino acid). (openwetware.org)
  • While restriction enzymes cleave at specific DNA sequences, they are first required to bind non-specifically with the DNA backbone before localizing to the restriction site. (wikipedia.org)
  • homology
  • In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. (mdpi.com)
  • One of the known DSB repair pathways that are essentially functional in all organisms[citation needed] are the non-homologous end joining (NHEJ) and homology directed repair (HDR). (wikipedia.org)
  • mouse
  • Insertional mutagenesis using transposons, retrovirus such as mouse mammary tumor virus and murine leukemia virus may be used to identify genes involved in carcinogenesis and to understand the biological pathways of specific cancer. (wikipedia.org)
  • active
  • 1, Haloperoxidases (hemeand vanadium-containing) catalyze the formation of hypohalous acids,[3, which diffuse out of the active site and then react with substrate. (docme.ru)
  • Perhydrolases catalyze the formation of peracids, which react outside of the active site with halide ions to form hypohalous acids. (docme.ru)
  • Active site of PrnA showing the binding of the substrate tryptophan. (docme.ru)
  • Defining the active site structure. (wikipedia.org)
  • A number of important active sites are still poorly defined. (wikipedia.org)
  • Understanding the active site function. (wikipedia.org)
  • The structure of some enzymes are very well characterized, however, the function of some component of the active site is poorly understood. (wikipedia.org)
  • enzymes
  • DOI: 10.1002/anie.201007896 Enzymatic Halogenation Changing the Regioselectivity of the Tryptophan 7-Halogenase PrnA by Site-Directed Mutagenesis** Alexander Lang, Stefan Polnick, Tristan Nicke, Peter William, Eugenio P. Patallo, James H. Naismith, and Karl-Heinz van Pe* Dedicated to Professor Franz Lingens on the occasion of his 85th birthday For many years, haloperoxidases were the only type of halogenating enzymes known. (docme.ru)
  • Type I restriction enzymes recognize specific sequences, but cleave DNA randomly at sites other than their recognition site whereas type II restriction enzymes cleave only at their specific recognition site. (wikipedia.org)
  • approach
  • One approach of this technique is to excise a portion of DNA and replaced with a library of sequences containing all possible combinations at the desired mutation sites. (wikipedia.org)
  • specific
  • Despite the fact that this residue is most likely responsible for the unwinding of DNA and coordination to water rather than direct interaction with the attacking nucleophile, its specific function is unknown. (wikipedia.org)
  • Many researchers seek to introduce selected changes to DNA in a site-specific manner. (wikipedia.org)
  • important
  • Upon comparing the amino acid sequences of mammalian brain and S. cerevisiae β-tubulin we observed five differences at sites in β-tubulin purported to be important in paclitaxel binding. (pubmedcentralcanada.ca)
  • function
  • As a result of the site-mutagenesis experiments previously outlined, it is thus proposed that Lys-125, Asp-123, and Asp-108 of HindIII function similarly to Lys-92, Asp-90, and Asp-74 in EcoRV, respectively. (wikipedia.org)