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.

*  Site-directed mutagenesis: maximum substitution - PCR, RT-PCR and Real-Time PCR

Site-directed mutagenesis: maximum substitution - (Mar/31/2014 ). Hi, I would like to change a 5 amino acid (max 15nt) sequence ... I was thinking of using the NEB Q5 site-directed mutagenesis kit. ... instance if you wanted to change all the bases in a 15 nt site ...
protocol-online.org/biology-forums-2/posts/32039.html

*  Richard Lab:Site Directed Mutagenesis - OpenWetWare

An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004; 32(14): e115. ... Richard Lab:Site Directed Mutagenesis. From OpenWetWare. Revision as of 13:31, 26 June 2011 by Michael A. Speer. (talk , ... Retrieved from "https://openwetware.org/mediawiki/index.php?title=Richard_Lab:Site_Directed_Mutagenesis&oldid=519140" ... 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. ...
https://openwetware.org/wiki/?title=Richard_Lab:Site_Directed_Mutagenesis&oldid=519140

*  Site-directed mutagenesis for the introduction of mutations

Site-directed mutagenesis is a PCR-based technique ... Site-directed mutagenesis is a molecular biology tool to ... Site-Directed Mutagenesis System. However, you need to optimize it as per your conditions. GENEART® Site-Directed Mutagenesis ... Site-directed mutagenesis is a PCR-based mutagenesis that combines some extra steps such as methylation and recombination. It ... Site-directed mutagenesis for the introduction of mutations. Biochempages May 1, 2017 No Comments ...
biochempages.com/2017/05/site-directed-mutagenesis-for-the-introduction-of-mutations.html

*  Phosphorylation site-directed mutagenesis of NPSR1-A an | Open-i

Phosphorylation site-directed mutagenesis of NPSR1-A and NPSR1-B C-termini. Relative CGA mRNA expression after 1, 6 and 24 h ... Figure 7: Phosphorylation site-directed mutagenesis of NPSR1-A and NPSR1-B C-termini. Relative CGA mRNA expression after 1, 6 ... Figure 7: Phosphorylation site-directed mutagenesis of NPSR1-A and NPSR1-B C-termini. Relative CGA mRNA expression after 1, 6 ... we performed phosphorylation site-directed mutagenesis. The NPSR1-A and -B isoforms possess distinct C-termini which, together ...
https://openi.nlm.nih.gov/detailedresult.php?img=PMC3142248_1471-2466-11-39-7&req=4

*  Are Purified Primers Really Necessary For Site-Directed Mutagenesis? - Bitesize Bio

Most site-directed mutagenesis protocols strongly recommend that you use only PAGE- or HPLC-purified primers to mutate plasmid ... Are Purified Primers Really Necessary For Site-Directed Mutagenesis? By Emily Crow ... However, for mutagenesis primers, the mismatched bases will be at the center of the oligo; thus, shorter versions of the oligo ... I have found that if you take the time to carefully design your primers, you can achieve SDM at most sites with near 90% ...
https://bitesizebio.com/8077/are-purified-primers-really-necessary-for-site-directed-mutagenesis/

*  site-directed-mutagenesis | NEB

Site-directed mutagenesis (SDM) is a method to create specific, targeted changes in double stranded plasmid DNA. ... site-directed-mutagenesis site-directed-mutagenesis. Product Listing Application Overview Site-directed mutagenesis (SDM) is a ... the mutagenesis reaction is complete in less than two hours.. Figure 2: Q5 Site-Directed Mutagenesis Kit Overview.. This kit is ... Unlike kits that rely on linear amplification, primers designed for the Q5 Site-Directed Mutagenesis Kit should not overlap to ...
https://neb.com/applications/dna-amplification-and-pcr/site-directed-mutagenesis

*  Site-directed mutagenesis definition | Drugs.com

Definition of site-directed mutagenesis. Provided by Stedman's medical dictionary and Drugs.com. Includes medical terms and ... Disclaimer: This site is designed to offer information for general educational purposes only. The health information furnished ... on this site and the interactive responses are not intended to be professional advice and are not intended to replace personal ...
https://drugs.com/dict/site-directed-mutagenesis.html

*  'Round-the-horn site-directed mutagenesis -...

Retrieved from "http://www.openwetware.org/wiki/%27Round-the-horn_site-directed_mutagenesis" ... Round-the-horn is a PCR-based mutagenesis. The muations are contained in one or both of the primers. The primers are ... You can amplify a low-copy plasmid with primers that introduce cloning sites for receiving an insert. The PCR product becomes ... The reverse primer in theis example sits on the complementary strand and abuts the site of mutation. ...
openwetware.org/index.php?title=

*  Changes related to 'Richard Lab:Site Directed Mutagenesis' - OpenWetWare

Richard Lab:Site Directed Mutagenesis. No changes on linked pages during the given period. ...
openwetware.org/index.php?title=Special:RecentChangesLinked&from=20130316041958&hideminor=0&target=Richard_Lab:Site_Directed_Mutagenesis

*  Sauer:Site-directed mutagenesis - OpenWetWare

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Sauer:Site-directed_mutagenesis&oldid=17848" ...
https://openwetware.org/wiki/?title=Sauer:Site-directed_mutagenesis&oldid=17848

*  How do I design primers to use with the Q5® Site-Directed Mutagenesis Kit? | NEB

FAQ: How do I design primers to use with the Q5® Site-Directed Mutagenesis Kit? For best results, back-to-back primers should ...
https://neb.com/faqs/2014/03/21/how-do-i-design-primers-to-use-with-the-q5-site-directed-mutagenesis-kit1

*  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 ...
mdpi.com/1422-0067/17/5/764

*  Catalysts | Free Full-Text | Improving the Indigo Carmine Decolorization Ability of a Bacillus amyloliquefaciens Laccase by...

Here we describe the improvement of the decolorization ability of a bacterial laccase through site-directed mutagenesis. A ... site-directed mutagenesis; extracellular expression; indigo carmine; decolorization laccase; site-directed mutagenesis; ... Improving the Indigo Carmine Decolorization Ability of a Bacillus amyloliquefaciens Laccase by Site-Directed Mutagenesis. Jiayi ... Here we describe the improvement of the decolorization ability of a bacterial laccase through site-directed mutagenesis. A ...
mdpi.com/2073-4344/7/9/275

*  RCSB PDB - 1MAC: CRYSTAL STRUCTURE AND SITE-DIRECTED MUTAGENESIS OF BACILLUS MACERANS ENDO-1,3-1,4-BETA-GLUCANASE...

Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase. ... CRYSTAL STRUCTURE AND SITE-DIRECTED MUTAGENESIS OF BACILLUS MACERANS ENDO-1,3-1,4-BETA-GLUCANASE. ...
rcsb.org/pdb/explore/materialsAndMethods.do?structureId=1MAC

*  Sequence Similarity - 1MAC: CRYSTAL STRUCTURE AND SITE-DIRECTED MUTAGENESIS OF BACILLUS MACERANS ENDO-1,3-1,4-BETA...

Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase. ... CRYSTAL STRUCTURE AND SITE-DIRECTED MUTAGENESIS OF BACILLUS MACERANS ENDO-1,3-1,4-BETA-GLUCANASE. ...
rcsb.org/pdb/explore/sequenceCluster.do?structureId=1MAC

*  Metabolites | Free Full-Text | Changes in Primary and Secondary Metabolite Levels in Response to Gene Targeting-Mediated Site...

Gene targeting (GT) via homologous recombination allows precise modification of a target gene of interest. In a previous study, we successfully used GT to produce rice plants accumulating high levels of free tryptophan (Trp) in mature seeds and young leaves via targeted modification of a gene encoding anthranilate synthase-a key enzyme of Trp biosynthesis. Here, we performed metabolome analysis in the leaves and mature seeds of GT plants. Of 72 metabolites detected in both organs, a total of 13, including Trp, involved in amino acid metabolism, accumulated to levels |1.5-fold higher than controls in both leaves and mature seeds of GT plants. Surprisingly, the contents of certain metabolites valuable for both humans and livestock, such as γ-aminobutyric acid and vitamin B, were significantly increased in mature seeds of GT plants. Moreover, untargeted analysis using LC-MS revealed that secondary metabolites, including an indole alkaloid, 2-[2-hydroxy-3-β-d-glucopyranosyloxy-1-(1H-indol-3-yl)propyl]
mdpi.com/2218-1989/2/4/1123/notes

*  Targeted site-directed mutagenesis of a heme oxygenase locus by gene replacement in the mossCeratodon purpureus | SpringerLink

Targeted site-directed mutagenesis of a heme oxygenase locus by gene replacement in the moss Ceratodon purpureus. ... and thus provides the first demonstration of efficient site-directed mutagenesis in a plant nuclear genome. Our studies also ... Schaefer DG (2002) A new moss genetics: targeted mutagenesis in Physcomitrella patens. Annu Rev Plant Biol 53:477-501CrossRef ... We use cookies to improve your experience with our site. More information Accept. ...
https://link.springer.com/article/10.1007/s00425-004-1411-6

*  Gene Expression and Molecular Cloning > Mutagenesis > Site-Directed Mutagenesis Kits Laboratory Product Directory and...

Mutagenesis > Site-Directed Mutagenesis Kits products in the SelectScience products and suppliers directory ... Site-Directed Mutagenesis (3). Agilent Technologies. QuikChange I The original version or our QuikChange technique that ... QuikChange II XL Site-Directed Mutagenesis Kit, 10 rxn. Agilent Technologies. Our second generation QuikChange kit has improved ... QuikChange II Site-Directed Mutagenesis Kit, 10 rxn. Agilent Technologies. Our second generation QuikChange kit has improved ...
selectscience.net/gene-expression-and-molecular-cloning/product-directory/site-directed-mutagenesis-kits/?catID=4876&u=E359D75A-8F61-4E1F-8B14-CB13D0F4F2E8

*  Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype...

... and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. ... indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand ... indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand ... carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site ...
https://garvan.org.au/research/publications/1540

*  Lirias: Protein engineering of xylose (glucose) isomerase from actinoplanes-missouriensis .2. site-directed mutagenesis of the...

Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate ... site-directed mutagenesis of the xylose binding-site. Authors: Lambeir, Am ×. Lauwereys, M. Stanssens, P. Mrabet, Nt. Snauwaert ... Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of ... With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is ...
https://lirias.kuleuven.be/handle/123456789/32458

*  P Blume-Jensen

site directed mutagenesis*protein serine threonine kinases*kidney*carrier proteins*cell survival*neoplasms*phosphorylation*nude ... Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact ... Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact ...
https://labome.org/expert/usa/the/blume-jensen/p-blume-jensen-307087.html

*  DNA Clean-Up Buffers

Mutagenesis * Random Mutagenesis Kit * Site-Directed Mutagenesis Kit * Mutan-Super Express Km ...
clontech.com/US/Products/Nucleic_Acid_Purification/Accessories/Buffers_and_Enzymes/DNA_Clean-Up?sitex=10020:22372:US&PROD=sqFQ121JCqKKoivj5WBi_nix:S&PROD_pses=ZGC487EBFB2F906FB7E118CE951380B235928F9D77FFAEFABAD84A1AEF8951C90B3A96A406F1954B59496D028746E62EB1DFC11F2ECC9EA160

*  Protein Regulation with Rapid Kinetics - ProteoTuner Systems

Mutagenesis * Random Mutagenesis Kit * Site-Directed Mutagenesis Kit * Mutan-Super Express Km ...
clontech.com/US/Products/Inducible_Systems/Inducible_Protein_Stabilization/Plasmid?sitex=10020:22372:US&PROD=DqpwMTqeJQ5r5xVAuIiNXaJH:S&PROD_pses=ZG486D26EEC0ADA36092B4B856F56E9BA53C981297B3729D9A0197CD6499B255130CDC6A4FBF50C70EF836362FF08CE3780DA7E5E44F3461ED

*  RACE-Ready Rat cDNA

Mutagenesis * Random Mutagenesis Kit * Site-Directed Mutagenesis Kit * Mutan-Super Express Km ...
clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_and_Genomic_DNA/RACE-Ready_cDNA/Rat?sitex=10020:22372:US&PROD=XKF-IH73XrncSN9wSWJRY7qq:S&PROD_pses=ZG5E71C7493DB592C4EF866A2EB462B5A5B3073F18A361198CAC1A7C8585E1310C51E7F1CD019EF03335683564535FB4B2F47078FD5A99AEDE

*  Ofd1, a human disease gene, regulates the length and distal structure of centrioles

Missense mutations were created using Quik Change II XL site directed mutagenesis kit (Stratagene). Final products were ... Expression of the disease alleles from the endogenous Ofd1 locus allows for direct comparison of the effects of the mutation on ... The proximal end of the mother and daughter centrioles is the site at which a single new centriole, termed a procentriole, ... Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits. J Biol ...
pubmedcentralcanada.ca/pmcc/articles/PMC2841064/?lang=en-ca

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)



primers


  • For these methods, primers can be designed in either an overlapping (QuikChange®, Agilent) or a back-to-back orientation ( Q5® Site-Directed Mutagenesis Kit ) (Figure 1). (neb.com)
  • Before primers are designed, it is important to determine which mutagenesis workflow is to be used. (neb.com)
  • 1. Design mutagenesis primers. (openwetware.org)
  • How do I design primers to use with the Q5® Site-Directed Mutagenesis Kit? (neb.com)
  • Are Purified Primers Really Necessary For Site-Directed Mutagenesis? (bitesizebio.com)
  • Most site-directed mutagenesis protocols strongly recommend that you use only PAGE- or HPLC-purified primers to mutate plasmid templates. (bitesizebio.com)
  • What do you think - would you try mutagenesis with un-purified primers? (bitesizebio.com)

mutation


  • 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. (openwetware.org)
  • I don't have experience with large ones, but I thought the limitation was related to the size of the mutation - for instance if you wanted to change all the bases in a 15 nt site, that would probably be fine, but much more and it would start to get a bit "floppy" and you could get some re-annealing in the mismatched regions. (protocol-online.org)
  • The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. (garvan.org.au)
  • It means that we can direct the mutation according to our interest. (biochempages.com)
  • But we can also commercially available site-directed mutagenesis kits to introduce a mutation. (biochempages.com)

GeneArt


  • Currently, there are a number of commercially available kits that also require specific modification and/or unique E. coli strains (for example, the Phusion Site-Directed Mutagenesis® from Thermo and the GeneArt® system from Life). (neb.com)
  • Here, I am going to explain a simplified protocol for the site-directed mutagenesis that uses GENEART ® Site-Directed Mutagenesis System. (biochempages.com)
  • GENEART ® Site-Directed Mutagenesis System comes with components necessary for mutagenesis reaction, recombination as well as transformation. (biochempages.com)

Mutations


  • Site-directed mutagenesis is a procedure for the introduction of mutations in a target DNA sequence. (biochempages.com)
  • Using the site-directed mutagenesis, it is possible to introduce three different kinds of mutations into the target DNA. (biochempages.com)
  • Relative CGA mRNA expression after 1, 6 and 24 h NPS stimulation of HEK-293 cells overexpressing NPSR1-A or -B containing phosphorylation site mutations in the C-termini. (nih.gov)

oligo


  • thus, shorter versions of the oligo will not necessarily contain the appropriate site, and an unmutated copy of the plasmid will be amplified instead. (bitesizebio.com)

amino acid


  • 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)
  • GeneMorph II Random Mutagenesis Kits Error-prone PCR is a random mutagenesis technique for introducing amino acid changes into proteins. (selectscience.net)

polymerase


  • The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most plasmids, the mutagenesis reaction is complete in less than two hours. (neb.com)
  • Basically, PCR-based mutagenesis involves the use of normal PCR using Taq-Polymerase and other necessary components. (biochempages.com)

residues


  • We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. (garvan.org.au)
  • Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate the structural and functional role of specific residues. (kuleuven.be)
  • Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of the substrate binding site while the role of Phe 26 seems to be purely structural. (kuleuven.be)
  • Phosphorylation occurs predominantly on serine (S) and threonine (T) residues and, as illustrated in Figure 1b, NPSR1-A carries five unique C-terminal phosphorylation sites, whereas NPSR1-B only two. (nih.gov)

bases


  • This gene repair led to the exchange of single bases, and thus provides the first demonstration of efficient site-directed mutagenesis in a plant nuclear genome. (springer.com)

QuikChange


  • QuikChange I The original version or our QuikChange technique that introduced a fast, easy, and efficient non-PCR alternative to other site-directed mutagenesis techniques. (selectscience.net)
  • Our second generation QuikChange kit has improved fidelity through the addition of a high-fidelity PCR enzyme (Pfu Ultra) to eliminate unwanted second-site errors. (selectscience.net)

Gene


  • Kochevenko A, Willmitzer L (2003) Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate synthase gene. (springer.com)
  • The results showed that the removal of phosphorylation sites did not have any effect on downstream gene expression (representative gene shown in Figure 7), suggesting that the significant differential effect between NPSR1-A and -B involves mechanism(s) distinct from simple phosphorylation differences. (nih.gov)

primer


method


  • Site-directed mutagenesis (SDM) is a method to create specific, targeted changes in double stranded plasmid DNA. (neb.com)
  • Emanuelsson O, Nielsen H, von Heije G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. (springer.com)

work


  • The mutagenesis work together with the crystallographic studies presented in detail in two accompanying papers adds significantly to the understanding of the catalytic mechanism of this enzyme. (kuleuven.be)

efficient


  • 2004. An efficient one-step site-directed and site-saturation mutagenesis protocol. (openwetware.org)

important


  • The methylation is an important step in the mutagenesis. (biochempages.com)
  • The NPSR1-A and -B isoforms possess distinct C-termini which, together with the third intracellular loop, contain phosphorylation sites potentially important for arrestin docking . (nih.gov)

specific


  • Figure 1: Site-specific mutagenesis proceeds in less than 2 hours. (neb.com)

comes


  • When it comes to mutagenesis, it can be a tricky choice: unpurified oligos will not eliminate the possibility of getting a correctly mutated product, they will only decrease your chances. (bitesizebio.com)

replacement


  • 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)

create


base


  • With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is confirmed. (kuleuven.be)

unique


  • We generated NPSR1-A constructs with all five unique phosphorylation sites in the C-terminal mutated to alanine (AΔ5p), three sites mutated (AΔ3p) and two sites mutated (AΔ2p) and NPSR1-B constructs with the only two unique sites mutated (BΔ2p) (Additional file 2, Figure S1). (nih.gov)

general


  • Disclaimer: This site is designed to offer information for general educational purposes only. (drugs.com)