Mutagenesis: Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS.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, 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.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.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.Base Sequence: The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.Binding Sites: The parts of a macromolecule that directly participate in its specific combination with another molecule.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.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.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.Recombinant Proteins: Proteins prepared by recombinant DNA technology.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.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.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.Bacterial Proteins: Proteins found in any species of bacterium.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).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.Cysteine: A thiol-containing non-essential amino acid that is oxidized to form CYSTINE.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.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.Genes, Bacterial: The functional hereditary units of BACTERIA.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.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.Ethylnitrosourea: A nitrosourea compound with alkylating, carcinogenic, and mutagenic properties.Substrate Specificity: A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.Catalysis: The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.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.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.Cell Line: Established cell cultures that have the potential to propagate indefinitely.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.Histidine: An essential amino acid that is required for the production of HISTAMINE.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.DNA Mutational Analysis: Biochemical identification of mutational changes in a nucleotide sequence.Catalytic Domain: The region of an enzyme that interacts with its substrate to cause the enzymatic reaction.Escherichia coli Proteins: Proteins obtained from ESCHERICHIA COLI.Crystallography, X-Ray: The study of crystal structure using X-RAY DIFFRACTION techniques. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)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.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.Restriction Mapping: Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.Ethyl Methanesulfonate: An antineoplastic agent with alkylating properties. It also acts as a mutagen by damaging DNA and is used experimentally for that effect.DNA, Bacterial: Deoxyribonucleic acid that makes up the genetic material of bacteria.Sequence Deletion: Deletion of sequences of nucleic acids from the genetic material of an individual.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.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.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.Lysine: An essential amino acid. It is often added to animal feed.Phenotype: The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment.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.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.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).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.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.Mutant Proteins: Proteins produced from GENES that have acquired MUTATIONS.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.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.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.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.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)Cricetinae: A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS.Gene Expression Regulation, Bacterial: Any of the processes by which cytoplasmic or intercellular factors influence the differential control of gene action in bacteria.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.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.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.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).)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.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.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.Arginine: An essential amino acid that is physiologically active in the L-form.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)Transcription Factors: Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process.Sequence Analysis, DNA: A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis.Dimerization: The process by which two molecules of the same chemical composition form a condensation product or polymer.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)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.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.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.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.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).Electrophoresis, Polyacrylamide Gel: Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.Mutagenicity Tests: Tests of chemical substances and physical agents for mutagenic potential. They include microbial, insect, mammalian cell, and whole animal tests.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.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.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)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.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.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.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.Carrier Proteins: Transport proteins that carry specific substances in the blood or across cell membranes.DNA Damage: Injuries to DNA that introduce deviations from its normal, intact structure and which may, if left unrepaired, result in a MUTATION or a block of DNA REPLICATION. These deviations may be caused by physical or chemical agents and occur by natural or unnatural, introduced circumstances. They include the introduction of illegitimate bases during replication or by deamination or other modification of bases; the loss of a base from the DNA backbone leaving an abasic site; single-strand breaks; double strand breaks; and intrastrand (PYRIMIDINE DIMERS) or interstrand crosslinking. Damage can often be repaired (DNA REPAIR). If the damage is extensive, it can induce APOPTOSIS.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.Hydrolysis: The process of cleaving a chemical compound by the addition of a molecule of water.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.Methylnitronitrosoguanidine: A nitrosoguanidine derivative with potent mutagenic and carcinogenic properties.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.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.Biocatalysis: The facilitation of biochemical reactions with the aid of naturally occurring catalysts such as ENZYMES.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.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.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.Chromosome Mapping: Any method used for determining the location of and relative distances between genes on a chromosome.DNA Replication: The process by which a DNA molecule is duplicated.Rec A Recombinases: A family of recombinases initially identified in BACTERIA. They catalyze the ATP-driven exchange of DNA strands in GENETIC RECOMBINATION. The product of the reaction consists of a duplex and a displaced single-stranded loop, which has the shape of the letter D and is therefore called a D-loop structure.Tyrosine: A non-essential amino acid. In animals it is synthesized from PHENYLALANINE. It is also the precursor of EPINEPHRINE; THYROID HORMONES; and melanin.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)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.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.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.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 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.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.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.Cell Membrane: The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.Phenylalanine: An essential aromatic amino acid that is a precursor of MELANIN; DOPAMINE; noradrenalin (NOREPINEPHRINE), and THYROXINE.Disulfides: Chemical groups containing the covalent disulfide bonds -S-S-. The sulfur atoms can be bound to inorganic or organic moieties.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).Nucleic Acid Conformation: The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.Transposases: Enzymes that recombine DNA segments by a process which involves the formation of a synapse between two DNA helices, the cleavage of single strands from each DNA helix and the ligation of a DNA strand from one DNA helix to the other. The resulting DNA structure is called a Holliday junction which can be resolved by DNA REPLICATION or by HOLLIDAY JUNCTION RESOLVASES.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.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.Frameshift Mutation: A type of mutation in which a number of NUCLEOTIDES deleted from or inserted into a protein coding sequence is not divisible by three, thereby causing an alteration in the READING FRAMES of the entire coding sequence downstream of the mutation. These mutations may be induced by certain types of MUTAGENS or may occur spontaneously.Alkylating Agents: Highly reactive chemicals that introduce alkyl radicals into biologically active molecules and thereby prevent their proper functioning. Many are used as antineoplastic agents, but most are very toxic, with carcinogenic, mutagenic, teratogenic, and immunosuppressant actions. They have also been used as components in poison gases.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).Phosphorylation: The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.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.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.Viral Proteins: Proteins found in any species of virus.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.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.Genetic Techniques: Chromosomal, biochemical, intracellular, and other methods used in the study of genetics.Lac Operon: The genetic unit consisting of three structural genes, an operator and a regulatory gene. The regulatory gene controls the synthesis of the three structural genes: BETA-GALACTOSIDASE and beta-galactoside permease (involved with the metabolism of lactose), and beta-thiogalactoside acetyltransferase.Gene Expression: The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION.Fungal Proteins: Proteins found in any species of fungus.Serine Endopeptidases: Any member of the group of ENDOPEPTIDASES containing at the active site a serine residue involved in catalysis.Genes: A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms.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.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).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.Hypoxanthine Phosphoribosyltransferase: An enzyme that catalyzes the conversion of 5-phosphoribosyl-1-pyrophosphate and hypoxanthine, guanine, or 6-mercaptopurine to the corresponding 5'-mononucleotides and pyrophosphate. The enzyme is important in purine biosynthesis as well as central nervous system functions. Complete lack of enzyme activity is associated with the LESCH-NYHAN SYNDROME, while partial deficiency results in overproduction of uric acid. EC 2.4.2.8.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.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.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.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.Leucine: An essential branched-chain amino acid important for hemoglobin formation.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.Bacillus subtilis: A species of gram-positive bacteria that is a common soil and water saprophyte.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.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.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.Models, Genetic: Theoretical representations that simulate the behavior or activity of genetic processes or phenomena. They include the use of mathematical equations, computers, and other electronic equipment.Gene Targeting: The integration of exogenous DNA into the genome of an organism at sites where its expression can be suitably controlled. This integration occurs as a result of homologous recombination.Protein Multimerization: The assembly of the QUATERNARY PROTEIN STRUCTURE of multimeric proteins (MULTIPROTEIN COMPLEXES) from their composite PROTEIN SUBUNITS.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.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.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)Chromosomes, Bacterial: Structures within the nucleus of bacterial cells consisting of or containing DNA, which carry genetic information essential to the cell.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).HEK293 Cells: A cell line generated from human embryonic kidney cells that were transformed with human adenovirus type 5.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)Genes, Fungal: The functional hereditary units of FUNGI.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.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.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.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.Blotting, Southern: A method (first developed by E.M. Southern) for detection of DNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES.Genes, Suppressor: Genes that have a suppressor allele or suppressor mutation (SUPPRESSION, GENETIC) which cancels the effect of a previous mutation, enabling the wild-type phenotype to be maintained or partially restored. For example, amber suppressors cancel the effect of an AMBER NONSENSE MUTATION.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.Suppression, Genetic: Mutation process that restores the wild-type PHENOTYPE in an organism possessing a mutationally altered GENOTYPE. The second "suppressor" mutation may be on a different gene, on the same gene but located at a distance from the site of the primary mutation, or in extrachromosomal genes (EXTRACHROMOSOMAL INHERITANCE).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.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.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.Mesylates: Organic salts or esters of methanesulfonic acid.Genes, Lethal: Genes whose loss of function or gain of function MUTATION leads to the death of the carrier prior to maturity. They may be essential genes (GENES, ESSENTIAL) required for viability, or genes which cause a block of function of an essential gene at a time when the essential gene function is required for viability.GuanineModels, Structural: A representation, generally small in scale, to show the structure, construction, or appearance of something. (From Random House Unabridged Dictionary, 2d ed)DNA, Recombinant: Biologically active DNA which has been formed by the in vitro joining of segments of DNA from different sources. It includes the recombination joint or edge of a heteroduplex region where two recombining DNA molecules are connected.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.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 Biosynthesis: The biosynthesis of PEPTIDES and PROTEINS on RIBOSOMES, directed by MESSENGER RNA, via TRANSFER RNA that is charged with standard proteinogenic AMINO ACIDS.Multigene Family: A set of genes descended by duplication and variation from some ancestral gene. Such genes may be clustered together on the same chromosome or dispersed on different chromosomes. Examples of multigene families include those that encode the hemoglobins, immunoglobulins, histocompatibility antigens, actins, tubulins, keratins, collagens, heat shock proteins, salivary glue proteins, chorion proteins, cuticle proteins, yolk proteins, and phaseolins, as well as histones, ribosomal RNA, and transfer RNA genes. The latter three are examples of reiterated genes, where hundreds of identical genes are present in a tandem array. (King & Stanfield, A Dictionary of Genetics, 4th ed)DNA, Viral: Deoxyribonucleic acid that makes up the genetic material of viruses.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.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.Peptide Fragments: Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.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.Species Specificity: The restriction of a characteristic behavior, anatomical structure or physical system, such as immune response; metabolic response, or gene or gene variant to the members of one species. It refers to that property which differentiates one species from another but it is also used for phylogenetic levels higher or lower than the species.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.Static Electricity: The accumulation of an electric charge on a objectChromosome Deletion: Actual loss of portion of a chromosome.Hydrophobic and Hydrophilic Interactions: The thermodynamic interaction between a substance and WATER.Transcriptional Activation: Processes that stimulate the GENETIC TRANSCRIPTION of a gene or set of genes.Salmonella typhimurium: A serotype of Salmonella enterica that is a frequent agent of Salmonella gastroenteritis in humans. It also causes PARATYPHOID FEVER.Bacterial Outer Membrane Proteins: Proteins isolated from the outer membrane of Gram-negative bacteria.Oocytes: 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).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.

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

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)

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

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)

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

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)

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

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)

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

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)

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

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)

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

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)

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

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)

... - Site-specific mutagenesis techniques, also known as site-directed mutagenesis .. GeneTailor Site-Directed mutagenesis system (Invitrogen. I have designed
Webb, Helen M. and Sixma, T.K. and Hol, W.G.J. and Hirst, Timothy R. (1994) Analysis by Site-Directed Mutagenesis of Important Residues Invoved in the Assembly of Escherichia-Coli Heart-Labile NALYSIS BY SITE-DIRECTED MUTAGENESIS OF IMPORTANT RESIDUES Enterotoxin. In: 6th European Workshop on Bacterial Protein Toxins, Stirling, Scotland. (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) ...
There is a lack of information about structural requirements for a functional transmembrane domain in eukaryotic membrane proteins. This led me to design a eukaryotic system to bring more information about this structure and the role of the positively charged residues situated at the cytoplasmic side of a transmembrane region. The CD2 molecule was chosen as a model for an integral membrane protein. I deleted the transmembrane domain (26 amino acids) by oligonucleotide site-directed mutagenesis and overlap extension using PCR mutagenesis. Truncated forms with transmembrane regions 14, 12, 10 and 8 amino acids long were created. The common positive cluster (Lys-Arg-Lys-Lys) at the cytoplasmic domain was disrupted by substituting it for polar residues (Gln-Gln-Gln-Gln). The effects of such mutagenesis was examined after expression of the mutant proteins in eukaryotic cells (COS-7 and CHO) . Cellular localization was determined by panning and immunostaining experiments. The functional state of the ...
Creative Biostructure develops comprehensive protein evolution and engineering platform to generate gene diversification by site-directed mutagenesis libraries.
what are the criteria for good primers in a pcr reaction?describe how you would use site-directed mutagenesis to change, Hire Biology Expert, Ask Academics Expert, Assignment Help, Homework Help, Textbooks Solutions
Biomigas site-saturation mutagenesis technology systematically substitutes wildtype amino acids with partial or all 19 non-wildtype mutants.. Advantages. ...
The ion selectivity of the Na+ channel is determined by a ring of amino acids created by the P-loops of all four domains (D1-D4), and the major determinants are the residues referred to as `DEKA (Asp372, Glu898, Lys1419, and Ala1711; following residue numbers are the equivalents of human Nav1.5) (Heinemann et al., 1992; Sun et al., 1997) (Fig. 6A). However, the contribution of each P-loop to the ion selectivity is not equal (Schlief et al., 1996). Site-directed mutagenesis studies have shown that the positive Lys residue in D3 is the most critical residue for ion selectivity (Chiamvimonvat et al., 1996). In contrast, the physiological role of the D4 selectivity filter region is less clear. Cysteine mutagenesis experiments have shown that the five residues between Ser1710 and Asp1714 around the selectivity filter residue Ala1711 are all accessible from outside (Chiamvimonvat et al., 1996), suggesting that these five residues face the hydrophilic ion-conducting pore. In fact, mutations at the ...
Amino acids within TMs have been shown to play important roles in substrate binding, maintenance of protein stability, and correct folding of proteins (Hong et al., 2004, 2010; Gui and Hagenbuch, 2009; Miyagawa et al., 2009; Li et al., 2012). Studies of single nucleotide polymorphisms have also pointed out that mutants located within TMs often result in functional changes (Kalliokoski and Niemi, 2009). In the present study, we used site-directed mutagenesis to study the involvement of amino acid residues within the putative TM6 of OATP1B1 in substrate transport. Of the 24 amino acids analyzed, F262A, V264A, L267A, S269A, F276A, and F278A showed a more than 60% reduction of transport activity. Further studies revealed that these mutants had much reduced transporter protein expression compared with the wild-type. If protein expression is considered in the comparison of the transport activity, E-3-S uptake by these mutants was more than 60% of that by wild-type OATP1B1 (data not shown). Therefore, ...
Site-directed mutagenesis, cloning, strains, and media: Four mutant alleles of SSA1, ssa1-101 (K69Q), ssa1-102 (G198D), ssa1-103 (G199D), and ssa1-104 (G226D), were created using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). SSA1 inserted between the HindIII and BamHI sites of YEp351 (Hillet al. 1986; YEp351-SSA1 plasmid provided by E. Craig, University of Wisconsin, Madison) served as the template. The primer pairs used were as follows: for ssa1-101, 5′-CCGTTTTCGACGCTCAGCGTTTGATCGG-3′ and 5′-CCGATCAAACGCTGAGCGTCGAAAACGG-3′; for ssa1-102, 5′-GTCTTGATTTTCGACTTGGATGGTGGTACTTTCG-3′ and 5′-CGAAAGTACCACCATCCAAGTCGAAAATCAA GAC-3′; for ssa1-103,5′-GATTTTCGACTTGGGTGATGG TACTTTC GATGTC-3′ and 5′-GACATCGAAAGTACCATCACCCAAGTC GAAAATC-3′; and for ssa1-104, 5′-GGTGACACCCATTTGGAT GGTGAAGATTTTGAC-3′ and 5′-GTCAAAATCTTCACCATC CAAATGGGTGTCACC-3′. In each primer the altered nucleotide is boldfaced and underlined. Mutagenesis resulted in plasmids ...
Our lab (at least Amanda and Janet) have not tried it yet. Our understanding is that it is for more substantial changes than just single amino acid changes ...
This page is beta. Please feel free to update it. The idea here is to create a flow of work to simulate the point mutation of a single amino acid in a protein. ...
you will bite yourself in the a** when you later discover that there was a mutation somewhere else in your sequence. Considering the cost of commercial sequencing (the cheapest way are those packages where they just run the reaction and you get the raw data and do the evaluation by yourself, there is free software available like finch tv), sequencing your constructs is a must before you derive any data from them that you want to publish/patent or which are important in any other context. I had learned my lesson once by finding out almost too late that one of the primers I had used was missing a base (still in the time of running your own DNA synthesizer and sequencing with gels and 35S) my 2c Wo On 15 Aug., 04:05, mnr mnr ,mnr... from gmail.com, wrote: , If I were to go down the Phusion road, will I need to sequence the entire of , my gene (4.5 kbp) or just the mutated region to make sure the protein , finally has mutated in the right places? ...
Ortsgerichtete Mutagenese: Punktmutationen, Aminosäure-Austausche und Insertionen einführen. Bis zu 150 bp. Kurze Lieferzeit. Absolute Vertraulichkeit.
Site-specific mutagenesis techniques are aimed at the precise substitution, insertion or deletion of any coding sequence in vitro. More recently, however, such precise alterations are also being developed for in vivo gene/genome modifications. These techniques are revolutionizing our understanding of the genetic and molecular mechanisms in several biological systems, which could lead to the development of new enzymes, therapeutics as well as improved agricultural applications. ...
Older research outputs will score higher simply because theyve had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 271,050 tracked outputs that were published within six weeks on either side of this one in any source. This one has gotten more attention than average, scoring higher than 61% of its contemporaries ...
G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N-glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C-terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular ...
Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover. DAGKα is activated in vitro by Ca2+ and by acidic phospholipids. The regulatory region of DAGKα includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation. DAGKα also contains tandem C1 protein kinase C homology domains. We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKα and to determine the enzymic activities of different mutant forms of pig DAGKα in vitro. Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis. Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases. Mutation of D434 (Asp434) or D650 abolished all DAGKα activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for ...
GENEWIZ can increase your research productivity by performing your time-consuming site-directed mutagenesis projects efficiently and cost-effectively. Our customized mutagenesis services provide a fail-safe approach to obtain mutant constructs quickly, with 100% accuracy, thus eliminating the possibility of undesired mutations in your gene.. ...
TY - JOUR. T1 - Plasmid-based one-pot saturation mutagenesis. AU - Wrenbeck,Emily E.. AU - Klesmith,Justin R.. AU - Stapleton,James A.. AU - Adeniran,Adebola. AU - Tyo,Keith E.J.. AU - Whitehead,Timothy A.. PY - 2016/11/1. Y1 - 2016/11/1. N2 - Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.. AB - Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, ...
Defining fibronectins cell adhesion synergy site by site-directed mutagenesis.s profile, publications, research topics, and co-authors
1RQL: X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis
Directed evolution has so far been used almost exclusively as a tool for engineering proteins. By mutation techniques such as site-directed mutagenesis, cassette-mutagenesis, random mutagenesis, and error prone PCR variants of protein functions have been generated and the libraries thus produced have been screened for their ability to perform a specific function. Recursive application of this procedure has been used successfully for the modification of physical and catalytic properties of enzymes such as pH optima, thermo-tolerance, solvent stability, stereoselectivity, catalytic activity and substrate specificity as well as toxicity resistance mechanisms in bacteria and the host range and stability of viruses.Traditional mutagenesis approaches for evolving new properties in enzymes have a number of limitations. These approaches are only applicable to genes or sequences that have been cloned and functionally characterized and that have a discrete function. Also, the traditional mutagenesis ...
mutagenesis expert come in~ - posted in Molecular Cloning: I want to do a site directed mutagenesis in a 6kb plasmid, the primer set is primerF: GCCTCTGCTTAGTGGaaTTcCCACCCCCACAACCCGCAGG primerR: CCTGCGGGTTGTGGGGGTGGgAAttCCACTAAGCAGAGGC, The reaction system was designed as following: template 1ul (100ng) primer 1+1 ul (10mM each) dNTPs 1 ul (25mM each) 10Xpfu buffer 5 ul pfu 1 ul H2O...
G. DeSantis and J.B. Jones, Combining Site-Specific Chemical Modification with Site-Directed Mutagenesis: A Versatile Strategy To Move Beyond the Structural Limitations of the 20 Natural Amino Acids Side Chains in Protein Engineering , in In Vitro Mutagenesis Protocols , Methods in Molecular Biology, Humana Press, 2002, vol 182, pp55-65 ...
Weird problem in in vitro mutagenesis - posted in Molecular Biology: Hi, Iam doing invitro mutagenesis for a 5.2Kb plasmid using stratagene kit. I did not get any results with my plasmid, so checked the control in the kit. Unfortunately the control is not working. I have tried many times, but got no colonies. The conditions used were according to the instructions given by the manufacturer. 95 C 50 sec 95 C 50 seconds 55 C 1 min 68 C 5 minute x 18 times 68 C 5 minutes. I even tried addin...
For contributions to the development of methods within DNA-based chemistry [specifically, for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies ...
Kajohn Boonrod is the author of this article in the Journal of Visualized Experiments: Homemade Site Directed Mutagenesis of Whole Plasmids
Cysteine is the ubiquitous ligand of iron-sulfur clusters in proteins, although chemical models have indicated that functional groups other than thiolates can coordinate iron in iron-sulfur...
Plasmids ME1646 (S29, nucleotide 2555 A → G; amino acid 853 H → R), ME1647 (S41, nucleotide 2555 A → G; amino acid 853 H → R), and ME1648 (S55, nucleotides 2260 A → G, 2545 G → A; amino acids 754 N → D, 849 A → T) were sequenced between the DraI and HpaI sites to determine the mutations responsible for the mutant phenotypes. Site-directed mutagenesis was performed to generate the single (S29/S41) and double mutations (S55) using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as recommended. The primers used were: S29/S41 P126, 5′-CAG AAT TGC TGA TTT TCG TTT AGA TAA ACC ATT C, and P127, 5′-G AAT GGT TTA TCT AAA TCA CGA AAA TCA GCA ATT CTG; S55 (nucleotide 2260 A → G) P135, 5′-TTG TGA TTT ATA GGG ATG TGG GGA ATA TTG, and P136, 5′-CAA TAT TCC CCA CAT CCC TAT AAA TCA CAA; and S55 (nucleotide 2545 G → A) P132, 5′-TAT TCA AAT GCC AGA ATT ACT GAT TTT CAT GAT TTA G, and P133, 5′-C TAA ATC ATG AAA ATC AGT AAT TCT GGC ATT TGA ATA (the boldface ...
Mechano-growth factor (MGF), an alternative splicing variant of insulin-like growth factor-1 (IGF-1) gene, promotes cell proliferation and inhibits cell differentiation. It also plays an important role in tumor development. It is important to optimize the production process and achieve MGF protein because there is no commercial MGF protein available. In this study, the human MGF gene is cloned into pGEX-4T-1 and the recombinant human MGF (rhMGF) protein could be expressed in Rosetta (DE3) by isopropyl β-D-1-thiogalactopyranoside induction but not in BL21 (DE3). Mutation from rare codons to Escherichia coli preferred ones is performed. We obtain MGF(Mut54-56) and MGF(Mut-total) fragments through site-directed mutagenesis and overlapping PCR. Both pGEX-4T-1/MGF(Mut54-56)- and pGEX-4T-1/MGF(Mut-total)-transformed BL21 (DE3) can be induced to express rhMGF protein. To optimize the production technology, expression and purification of rhMGF are analyzed and compared in Rosetta (DE3) and BL21 (DE3). Results
Cited for: VARIANTS TRP-18; CYS-34; HIS-34; LEU-216; SER-286; SER-291; MET-299; CYS-376; GLY-447; ALA-449; VAL-461; SER-475; TRP-481; TYR-524; ARG-558; HIS-568; ARG-579; LYS-592; GLY-596; ALA-601; PHE-618; ASP-638; LEU-656; THR-672; HIS-689; LYS-692; PHE-705; ILE-924; GLN-986; MET-1016; ARG-1040; ALA-1082; LEU-1090; LEU-1098; TYR-1103; LYS-1107; TRP-1116; GLN-1193; MET-1251; SER-1293; PHE-1308; TRP-1512; ASN-1787; THR-1836; LYS-1901; CYS-1919; LEU-1951; GLN-1958; LEU-1962; MET-1968; GLN-1991; LEU-2004 AND ALA-2006; VARIANTS BRGDA1 GLN-18; LYS-70; ASN-84; SER-93; SER-94; GLN-104; TRP-104; LYS-109; GLN-121; TRP-121; GLU-126; PRO-136; MET-146; GLN-161; LYS-161; ASN-175; GLY-178; ARG-182; VAL-185; VAL-204; GLN-212; ILE-220; GLN-222; LEU-223; TRP-225; VAL-226; ILE-232; MET-240; LYS-270; GLN-276; ASP-278; CYS-282; ILE-300; PRO-315; ASN-320; ARG-325; LEU-336; ASP-351; VAL-351; ASN-356; CYS-367; HIS-367; LEU-367; LYS-369; GLY-374; HIS-376; ARG-386; GLU-386; ALA-396; LEU-396; LYS-439; GLY-501; HIS-526; ...
31150DNAArtificial Sequencesynthetic primer for the site-directed mutagenesis 1cctccccctg aacctgaaac aagatctgaa tgcaattgtt gttgttaacg 50251DNAArtificial Sequencesynthetic primer for the site-directed mutagenesis 2cgttaacaac aacaattgca ttcaggatct tgtttcaggt tcagggggag g 51316DNAArtificial Sequencesynthetic primer, RT-telo-4 3ctcatatatt cagtat 16415DNAArtificial Sequencesynthetic primer, RT-telo-3 4ctggacactc gctca 15515DNAArtificial Sequencesynthetic primer, RT-telo-2 5tcagccggac atgca 15615DNAArtificial Sequencesynthetic primer, RT-telo-1 6tcactcaggc ctcag 15717DNAArtificial Sequencesynthetic primer, RCR-telo-10 7gctggtgtct gctctcg 17825DNAArtificial Sequencesynthetic primer, RCR-telo-11 8ctgcagcagg aggatcttgt agatg 25921DNAArtificial Sequencesynthetic primer, RCR-telo-6 9gcaggtgaac agcctccaga c 211024DNAArtificial Sequencesynthetic primer, RCR-telo-R13 10cacaggctgc agagcagcgt ggag 241125DNAArtificial Sequencesynthetic primer, RCR-telo-F12 11gtcctacgtc cagtgccagg ggatc 251225DNAArtificial ...
For our British Journal of Pharmacology publication regarding the structural chemogenomics analysis of aminergic GPCRs (publication) we created a comprehensive overview of all site-directed mutagenesis studies performed against HRH1, HRH2, HRH3, HRH4, DRD3, ACM2, ACM3, ADRB1 and ADRB2 (aminergic GPCRs). In total 1420 reported single-point mutations were registered (including their effect on the binding affinity of reported ligands) at 128 individual B&W positions. ...
Cited for: VARIANTS EIEE6 PHE-17 DEL; THR-68; ASN-79; CYS-84; PRO-98; GLN-101; TRP-101; ARG-108; ASP-127; ARG-199; SER-227; THR-227; SER-232; ARG-233; VAL-342; ASP-343; TRP-351; SER-359; ARG-363; ARG-384; CYS-393; HIS-393; VAL-400; VAL-403; PHE-406; GLY-626; ASP-762; THR-785; ILE-812; ARG-842; 854-GLY-LEU-855 DEL; CYS-859; GLN-862; PRO-890; CYS-932; PRO-933; CYS-946; HIS-946; ARG-950; LYS-954; LYS-956; LEU-957; ILE-976; VAL-979; ARG-993; 999-ASN-LEU-1000 DELINS LEU-ILE-SER; LYS-1208; LYS-1221; PHE-1230; ASP-1238; ALA-1266; ASN-1288; VAL-1320; PRO-1326; GLY-1350; ARG-1358; PRO-1370; HIS-1378; THR-1378; ILE-1394; TYR-1396; SER-1417; PHE-1423; ALA-1429 DEL; VAL-1433; LYS-1450; SER-1451; LYS-1454; HIS-1462; LYS-1476; LYS-1503; GLY-1544; GLU-1586; ARG-1588; HIS-1592; PRO-1592; SER-1605; GLU-1637; THR-1638; CYS-1648; GLU-1653; PRO-1660; PRO-1667; LEU-1668; ILE-1672; THR-1673; THR-1683; ASP-1684; TRP-1688; ARG-1714; ASN-1763; ASN-1770; PHE-1770; THR-1770; THR-1780; VAL-1783; LYS-1787; PRO-1832; ...
Sodium, Electrophoresis, Escherichia, Escherichia Coli, Family, Flexibility, Inhibition, Muscle, Mutagenesis, PH, Polyacrylamide Gel Electrophoresis, Polymerization, Polymers, Report, Serpins, Site-directed Mutagenesis, Skeletal Muscle, Sodium Dodecyl Sulfate, Temperature
We have constructed highly constitutively active H3 receptors (MT6; A357K) by induced point mutagenesis in the C-terminal portion of the third intracellular loop of mouse H3 receptors. This substitution resulted in three major biochemical modifications of the H3 receptors: 1) increased constitutive activity leading to agonist-independent inhibition of adenylyl cyclase in cells; 2) moderately increased affinity for some agonists; and 3) reduced affinities for antagonist acting as inverse agonists.. Several mutagenesis studies indicated that the C-terminal portion of the i3 might act in concert with other structural domains to determine the selectivity and efficiency of G-protein-coupling receptor. For the adrenergic receptors, it has been shown that the C-terminal portion of the i3 plays a crucial role in G-protein-coupling receptor (ODowd et al., 1988; Cotecchia et al., 1990; Liggett et al., 1991). Ren et al. (1993) previously showed that the T373K mutation adjacent to TM6 causes dramatically ...
Mixed bases are used in primers to bind to templates that contain variability or a mixture of sequences at the primer binding sites. Mixed bases can also be used to create diversity in clone libraries and in site directed mutagenesis.. IDT offers random base oligos. Order them by using an upper case letter N or other IUPAC-IUB symbol (see table below). IDT offers two types of randomization, machine mix and hand mix. Machine mix bases are charged at the standard base price for the scale ordered. Hand mixing is done to provide custom ratios of the bases, and incurs an additional charge. When entering your sequence, the "Mixed Base" tab at the bottom of the page lists the IUB symbols and is where custom mix ratios need to be entered ...
Buy In Vitro Mutagenesis Protocols (Methods in Molecular Biology v. 57) by TROWER From WHSmith today! FREE delivery to store or FREE UK delivery on all ...
1ENA: Crystal structures of the binary Ca2+ and pdTp complexes and the ternary complex of the Asp21Glu mutant of staphylococcal nuclease. Implications for catalysis and ligand binding.
Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster ...
Dear Netters I am looking for the GUS gene from which the glycosylation sites have been removed by site directed mutagenesis. I know it is around but I dont have the reference. Sjef Smeekens Utrecht University email: wbtmcbw at cc.ruu.nl ...
Virtual plasmid consisting of wild-type human ubiquitin cloned between KpnI and NotI sites of pCDNA3.1(+)-Zeo. Used for site-directed mutagenesis tutorial.. 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT ...
Production of ser125-interleukin-2 by site directed mutagenesis / Seongman Kang; Ju Won Kwak; Jong Bum Kwon; Sung Wan Kim; In Young Lee; Sun Bok Lee; Hye-Young Yun; Kyung Soo Hahm; Moon Hi Han; Doe Sun Na , 1989 ...
Mutagenesis of the mouse Bhlhb4 gene. A: Gene targeting strategy showing partial restriction map of WT Bhlhb4 allele, the targeting vector, the targeted ES cell
Melet, A., Marques-Soares, C., Schoch, G. A., Macherey, A. C., Jaouen, M., Dansette, P. M., Sari, M. A., Johnson, E. F., Mansuy, D. Analysis of human cytochrome P4502C8 substrate specificity using a substrate pharmacophore and site-directed mutants Biochemistry 2004 43:15379-15392 DOI:10.1021/bi0489309 PMID:15581350 ...
Despite the extensive work being performed to understand cancer and carcinogenic properties of chemicals and other agents, there are still gaps in our ability t...
Enzymatic conversion of pectinaceous biomasses such as potato and sugar beet pulp at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, and increased substrate solubility and lowers the risk of contamination. Such high-temperature processing requires development of thermostable enzymes. Talaromyces stipitatus was found to secrete endo-1,4- β -galactanase when grown on sugar beet pectin as sole carbon source. The mature protein contained 353 AA and the MW was estimated to 36.5 kDa. It was subjected to codon optimization and pro- duced in Pichia pastoris in 2 l scale yielding 5.3 g. The optimal reaction condition for the endo-1,4- β -galactanase was determined to be 46 °C at pH 4.5 at which the specific activity was estimated to be 6.93 μ mol/min/mg enzyme with half-lives of 13 and 2 min at 55 and 60 °C, respectively. For enhancement of the half-life of TSGAL, nine single amino acid residues were selected for site-directed mutagenesis on the basis ...
Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica
Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella ...
To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele-characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding. ...
The metallo-β-lactamases (MBLs) are enzymes with the ability to hydrolyse the β-lactam antibiotics. The worldwide emergence of the antibiotic resistant MBLs poses an increasing clinical threat. The VIM enzymes are a growing family of carbapenemases with a wide geographic distribution in Europe, South America and the USA. The VIM-7, the first VIM enzyme to be discovered in the USA, is the most divergent member of the VIM-enzymes with the closest similarity to VIM-1 with a 77% amino acid identity. The VIM-7 has a conserved D120 in the active site, which, in catalysis, plays an important role. The Y224 residue present in the VIM-2, which currently is the most widespread MBL, is though to have an impact on the activity. Three site-directed mutations of the VIM-7, with a previously solved structure, were made; D120A, D120N and H224Y respectively. All three mutants and the VIM-7 wt were sequenced, and the mutants VIM-7 D120A and VIM-7 H224Y with the VIM-7 wt, containing an N-terminal hexahis-tag and ...
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I want to site-directed mutation on genome other than plasmid DNA. Anyone has protocol like this. I donnot want to introduce neo etc antibiotic maker. I need the clear genetic background which the mutation differ to the wild type with only the nucleo..
To understand how the protein and chromophore components of a light-sensing protein interact to create a light cycle, we performed time-resolved spectroscopy on site-directed mutants of photoactive yellow protein (PYP). Recently determined crystallographic structures of PYP in the ground and colorless I2 states allowed us to design mutants and to study their photosensing properties at the atomic level. We developed a system for rapid mutagenesis and heterologous bacterial expression for PYP apoprotein and generated holoprotein through formation of a covalent thioester linkage with the p-hydroxycinnamic acid chromophore as found in the native protein. Glu46, replaced by Gln, is buried in the active site and hydrogen bonds to the chromophores phenolate oxygen in the ground state. The Glu46Gln mutation shifted the ground state absorption maximum from 446 to 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds. Arg52, which separates the active site from ...
Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2-5-oligoadenylate (2-5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293-15299). Sequence comparison of this region among the different isozymes of 2-5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized
Those poor male bacteria! They have to contend with invading filamentous phage - something that Rogaine just cant cure! Well be talking more about male and female bacteria in a later lecture (sex in bacteria isnt quite the same concept as in eukaryotes).. What is significant here is that the virion of the filamentous phage (i.e. the viral particle) carries a single-strand of DNA - not a double helix. In the cell, this single-stranded genome (2.) is used as a template to synthesize a double-stranded replicative form (RF), which is essentially a plasmid (3.). The replicative form is used as a template to generate new single-stranded genomes (4.) that are packaged into virions (5.) to generate new phage. The cell doesnt die - it just grows more slowly and continues to secrete phage indefinitely.. The practical side of this story - if you use a cloning vector that is based on a filamentous bacteriophage (such as M13mp18 which is an engineered version of the phage M13) or merely contains an ...
It has been suggested [4] that these transport proteins have evolved from the duplication of an ancestral protein with six transmembrane regions, this hypothesis is based on the conservation of two G-R-[KR] motifs. The first one is located between the second and third transmembrane domains and the second one between transmembrane domains 8 and 9. We have developed two patterns to detect this family of proteins. The first pattern is based on the G-R-[KR] motif; but because this motif is too short to be specific to this family of proteins, we have derived a pattern from a larger region centered on the second copy of this motif. The second pattern is based on a number of conserved residues which are located at the end of the fourth transmembrane segment and in the short loop region between the fourth and fifth segments. Last update: April 2006 / Patterns revised. ...
Volume 35: Physical and Chemical Induced Mutagenesis Study for Identifying Lethality Dose in Chick Pea (|i|Cicer|/i| a|i|rietinum|/i| L.) Var. Co - 4
Gene of interest may include tag fusion for rapid purification and essay detection. Site-directed mutagenesis. N-terminal and C-terminal His-tag vectors.
Zidi-Yahiaoui N, Ripoche P, Le Van Kim C, et al. (2006). "Ammonium transport properties of HEK293 cells expressing RhCG mutants: preliminary analysis of structure/function by site-directed mutagenesis". Transfusion clinique et biologique : journal de la Société française de transfusion sanguine. 13 (1-2): 128-31. doi:10.1016/j.tracli.2006.02.025. PMID 16580862 ...
1. For mutagenesis bundled with gene synthesis, our Express Mutagenesis (sc1441) offers price starts at $99 per mutation and TAT is 5...
The key proposals were: Regarding new technologies, option 3 best supports the effectiveness of the legislative framework at this time. Under option 3 organisms modified using site-directed nucleases without templates to guide genome repair (i.e. SDN-1) would not be regulated as GMOs. Currently, if a template is used to guide genome repair (i.e. SDN-2 and SDN-3), the resulting organisms are GMOs, as are organisms modified using oligonucleotide-directed mutagenesis. These would continue to be regulated under this option ...
This proposal is to renew Environmental Carcinogenesis and Mutagenesis (T32 ES-09250-20). It is a fairly large training program due to an NIEHS-directed merger...
Gentaur molecular products has all kinds of products like :search , Sceti K.K. \ Arg_Arg_Leu_Ile_Glu_Asp_Ala_Glu_Tyr_Ala_ \ 4184-v for more molecular products just contact us
Yesterday Cindy presented the results of her Masters rotation project, which she carried out in our lab over the past couple of months. During the rotation, she worked with Lennart to develop and optimize a new gene excision protocol. Not all details can be revealed yet, but it looks like we can cut long sequences surprisingly early and efficiently!. As was also recognized in Cindys exam presentation, she completed a considerable amount of work. She also became a lively part of the lab - so that time passed too fast, again, and her next rotation period is coming up. We wish the best of luck and exciting results!. ...
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After my first few attempts at making models using 123d catch I noticed that the models I got back were a little melted and deformed. This along with an episode of...
山口沙織,丸本朋稔,二井偉暢,河野紘隆,寥紀元,永井陽子,岡田美智代,高橋淳,井上博之,佐々木えりか,藤井浩,岡野慎士,海老瀬速雄,佐藤哲也,須山幹太,岡野栄之,三浦由恵,谷憲三朗.初期化因子搭載レンチウイルスベクターにより誘導された未分化胚細胞腫様コモンマーモセット腫瘍の特性.第4回日本マーモセット研究会大会,愛知, ...
Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-Ag7, typically have a small, uncharged amino acid residue at position 57 of their β chain (β57); this results in the absence of a salt bridge between β57 and Argα76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Argα76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3β. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable ...
Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants ...
The coordination chemistry and electron-transfer properties of a single-site mutant of the mononuclear copper electron-transfer protein azurin from Pseudomonas aeruginosa have been studied. The active-site cysteine at position 112 was replaced by an aspartate (Cys112Asp) to assess directly the importance of this ligand to the structure-function properties of azurin. Although the mutant protein retains a high-affinity copper-binding active site, the absorption and EPR spectra of Cu[superscript II]Cys112Asp azurin are quite distinct from those of the wild-type protein and indicate the presence of a normal (type 2) copper center. A Cu[superscript II/I] reduction potential of 180 mV vs. NHE (pH 7.0) was obtained through a redox titration experiment with cytochrome c[subscript 551]. The Co[superscript II] derivative of Cys112Asp azurin was prepared and found to be amenable to paramagnetic NMR spectroscopy. In conjunction with electronic absorption data, the NMR data were used to generate a computer ...
Discussion. The novel αA-crystallin mutation G98R was identified and reported in an Indian family by Santhiya et al. [37]. G98R is the only mutation in αA-crystallin associated with cataract where a positively charged amino acid replaces the uncharged amino acid in the native protein. The other known mutations in αA-crystallin, such as R116C, R41C, and R21L have a substitution of a uncharged amino acid [30-32]. Therefore, we investigated the effect of the G98R mutation on the structure and function of αA-crystallin. Using a site-directed mutagenesis method, we constructed the αAG98R mutant and expressed it in E. coli cells. The mutant αA-crystallin protein was expressed but the recombinant protein formed inclusion bodies. The protein from the inclusion bodies was solubilized with urea and refolded. Interestingly, the urea-refolded mutant proteins stayed in soluble form. We observed that when the recombinant protein was extracted within 2 h of IPTG induction G98R was present in both ...
Plasmids used are in Table S1. To generate Fab1 mutants, pRS413 Fab1 was mutagenized by site-directed mutagenesis using the following primers (sequences in Table S3): S75A(FW1 and REV1), T183A(FW2 and REV2), T392A(FW3 and REV3), T526A(FW4 and REV4), T712A(FW5 and REV5), S729A(FW6 and REV6), S855A(FW7 and REV7), S1095A(FW8 and REV8), T1208A(FW9 and REV9), T1296A(FW10 and REV10), T1364A(FW11 and REV11), S1447A(FW12 and REV12), T1569A(FW13 and REV13), T1583A(FW14 and REV14), T1594A(FW15 and REV15), T1691A(FW16 and REV16), T1818A(FW17 and REV17), S1924A(FW18 and REV18), T1953A(FW19 and REV19), T1963A(FW20 and REV20), and S2166A(FW21 and REV21).. To generate HA6-fused Fab1 on the C terminus, pRS413 Fab1 including the 3′UTR was mutagenized for introducing NheI sites by site-directed mutagenesis using the primers FW22 and REV22. An HA6 fragment was amplified by PCR using the primers FW23 and REV23, digested by NheI and SpeI, and inserted the NheI sites of pRS416 Fab1-NheI.. To generate pRS413 ...
Altered sugar donor specificity and catalytic activity of pteridine glycosyltransferases by domain swapping or site-directed mutagenesis;kpubs;kpubs.org
Definition of mutagenesis in the Legal Dictionary - by Free online English dictionary and encyclopedia. What is mutagenesis? Meaning of mutagenesis as a legal term. What does mutagenesis mean in law?
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The identification of amino acid residues essential for function of the hematopoietic growth factor erythropoietin has been approached by several methods, including comparisons of related sequences, immunochemical approaches, mutagenesis and computer modeling. We have reported previously that mutations within amino acids 100-109 of erythropoietin can have profound effects on the hormones structure and/or activity and that Arg103 is especially important for function (Chern, Y., Chung, T. & Sytkowski, A.J. (1991) Eur. J: Biochem. 202, 225-229; Grodberg, J. Davis, K. L. & Sytkowski, A. J. (1993) Eur. J Biochem. (218, 597-601). We have now constructed a series of Arg 103 substitutions in order to determine the structural features of amino acid 103 required for biological activity. Each of the mutants was expressed and secreted efficiently by transfected COS1 cells. Mutants Arg103Asn, Arg103Gln, Arg103CGlu exhibited no biological activity. In contrast, Arg103His and Arg103Lys had specific activities equal
51728PRTHomo sapiens 1Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu1 5 10 15Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln20 25 30Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr35 40 45Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val50 55 60Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu65 70 75 80Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys85 90 95Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe100 105 110Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys115 120 125Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys130 135 140Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His145 150 155 160Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr165 170 175Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser180 185 190Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu195 200 205Val Glu Cys Met Thr Cys Asn Gly Glu ...
Ramesh, Sneha and Bharath, Srinivas MM and Chandra, Nagasuma R and Rao, Manchanahalli Satyanarayana R (2006) A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties. In: FEBS Letters, 580 (25). pp. 5999-6006. Bharath, Srinivas MM and Chandra, Nagasuma R and Rao, MRS (2003) Molecular modeling of the chromatosome particle. In: Nucleic-Acids-Research, 31 (14). pp. 4264-4274. Bharath, Srinivas MM and Ramesh, Sneha and Chandra, Nagasuma R and Rao, MRS (2002) Identification of a 34 Amino Acid Stretch within the C-Terminus of Histone H1 As the DNA-Condensing Domain by Site-Directed Mutagenesis. In: Biochemistry, 41 (24). 7617 -7627. Bharath, Srinivas MM and Chandra, Nagasuma R and Rao, MRS (2002) Prediction of an HMG-box fold in the C-terminal domain of histone H1: Insights into its role in DNA condensation. In: Proteins: Structure, Function, and Genetics, 49 (1). pp. 71-81. ...
Mutations occur when a DNA strand is damaged and the cells own repair mechanism mends the damage so that the end result is different from what it looked like to start with. The mutations thus arise through the organisms own system; regardless of whether it is a spontaneous mutation, a mutation with traditional methods, to which the EU court refers to having shown a history of safe use, or new improved methods of refinement. Since spontaneous mutations as well as mutagenesis through traditional methods have demonstrated safe use, it cannot be interpreted otherwise than that all mutagenesis involving a breach in the DNA strand and mutation induced through the organisms repair system do not cause any safety problems. It is not possible to distinguish in a crop the technique used for mutagenesis, and thus the exception in the GMO Directive must apply regardless of the mutagenesis technique used. In the judgment, it has been decided primarily to review recital 17 in combination with Annex IB of ...
Changes in Specific Amino Acid Residues and Na^+,K^+ -ATPase Activity in Cell Membranes of Rat Cerebral Cortex by Low Dose X-Irradiation : Changes in Specific Amino Acid Residues and Na^+,K^+ -ATPase Activity in Cell Membranes of Rat Cerebral Cortex by Low Dose X-Irradiation (1994 ...
First, the experiments were conducted with cells transformed with two different pPaB plasmids. These plasmids contained either the pBR322 origin (pMB1) or the p15A origin, which yielded a stable two-plasmid system with about 15-20 copies per plasmid and cell (medium-copy). Also, exactly the published riboregulator sequences were used in the the first place to have a positive control for functionality. This is important, because the original riboregulator sequence includes two forbidden restriction sites (EcoRI and XbaI). Second, the restriction sites were removed by two different approaches: a) multiple site-directed mutagenesis, b) blunting and ligation (Klenow and T4 DNA polymerase). Afterwards, the functionality of the construct was again tested to approve that no severe loss-of-function occurred due to the site-removal. In a third step, the construct was transferred into the pSB1C3 backbone (pMB1 origin, high copy number of 100-300) to be in line with the BioBrick Standard. Below, you can ...
van Tilbeurgh, H., J. Jenkins, M. Chiadmi, J. Janin, S. J. Wodak, N. T. Mrabet, and A. M. Lambeir, Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis., Biochemistry, vol. 31, issue 24, pp. 5467-71, 1992 Jun 23. ...
van Tilbeurgh, H., J. Jenkins, M. Chiadmi, J. Janin, S. J. Wodak, N. T. Mrabet, and A. M. Lambeir, Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis., Biochemistry, vol. 31, issue 24, pp. 5467-71, 1992 Jun 23. ...
CJ236 Electrocompetent Cells High efficiency cells to create uracil-containing DNA for site-directed mutagenesis Sole source of highly efficient Electrocompetent cells (1 × 109 cfu/µg) ung- and dut- mutations to generate DNA... ...
Site-directed mutagenesis of rodent liver bile acid CoA: amino acid N-acyltransferase (BAT) - this project involves a combination of molecular biology, enzymology and protein mass ...
CJ236 Electrocompetent Cells High efficiency cells to create uracil-containing DNA for site-directed mutagenesis Sole source of highly efficient Electrocompetent cells (1 × 109 cfu/µg) ung- and dut- mutations to generate DNA... ...
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L-Carnitine is an important amino acid that promotes cellular health and energy, aids in mental focus and concentration, and may support improved insu
L-Carnitine is an important amino acid that promotes cellular health and energy, aids in mental focus and concentration, and may support improved insu
International round-robin study on the Ames fluctuation test (pages 185-197). G. Reifferscheid, H.M. Maes, B. Allner, J. Badurova, S. Belkin, K. Bluhm, F. Brauer, J. Bressling, S. Domeneghetti, T. Elad, S. Flückiger-Isler, H.J. Grummt, R. Gürtler, A. Hecht, M.B. Heringa, H. Hollert, S. Huber, M. Kramer, A. Magdeburg, H.T. Ratte, R. Sauerborn-Klobucar, A. Sokolowski, P. Soldan, T. Smital, D. Stalter, P. Venier, Chr. Ziemann, J. Zipperle and S. Buchinger. Version of Record online: 4 JAN 2012 , DOI: 10.1002/em.21677. ...
Тезисы принимаются до 2019-05-01. Конференция проводится в Washington, США, с 2019-09-19 до 2019-09-23.
The protein encoded by this gene was identified by its RED repeat, a stretch of repeated arginine, glutamic acid and aspartic acid residues. The…
ASP Projects for $500 - $2500. Simply looking for an eCommerce site that matches musiciansfriend.com thats just as robust and powerful with feature...
سیانوباکترها یک گروه منحصر به فرد از باکتری-های فتواتوتروف هستند که برخی از آن‌ها به دلیل ویژگی-های ساختاری، تحمل قابل توجهی به تنش شوری نشان می-دهند. این موجودات نقش مهمی در محیط-های خاکی به ویژه در نواحی خشک و نیمه-خشک ایفا می-کنند. در مطالعه حاضر سیانوباکترهای خاک از مناطق بیابانی ایران جداسازی شده و سپس جدایه-های مقاوم به شرایط فوق شور شناسایی شدند. 40 نمونه خاک از پارک ملی کویر تهیه شد. نمونه-ها پس از کشت در محیط BG11 و ASN III (5/3، 5، 6 و 7 درصد کلرید سدیم) و انکوباسیون در شرایط مناسب دمایی و نور، جداسازی شده و با استفاده از کلیدهای مورفولوژیکی به طور اولیه و سپس با
By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM beta-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of ...
Alzheimers, Parkinsons and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including αBcrystallin, play a role in the prevention of protein deposition. A series of site-directed mutants of the human molecular chaperone, αB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of αB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of αB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein. Together, our results highlight the important role of the C-terminal region of αB-crystallin in regulating its
Staphylococcus aureus alpha-toxin is a membrane-damaging exoprotein that oligomerizes to form transmembrane pores. Chemical modification of histidines with diethylpyrocarbonate has been shown to reduce the hemolytic activity of alpha-toxin, suggesting that one or more of the histidine residues is important for toxin function. To individually assess the functional importance of each of the four histidine residues (residues 35, 48, 144, and 259), we used oligonucleotide-directed mutagenesis of the cloned alpha-toxin gene to replace each histidine with leucine. The mutant toxins were expressed in S. aureus and evaluated for hemolytic activity in vitro and for lethality in an intraperitoneal murine model. Substitution of histidine 35 with leucine produced a mutant toxin (H35L) without hemolytic or lethal activity. Mutant toxins H48L, H144L, and H259L exhibited 7, 16, and 46%, respectively, of the hemolytic activity of wild-type toxin. Immunoblotting of purified H35L toxin incubated with liposomal ...
NADPH: protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. Sequence comparisons have revealed that POR is a member of the short-chain alcohol dehydrogenase family of enzymes. A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site. This present study describes how site-directed mutagenesis has been used to change Tyr-189 to Phe and Lys-193 to Arg in the Synechocystis POR enzyme. The mutant enzymes were produced with a His tag in Escherichia coli and subsequently purified on a Ni2+-affinity column. The two mutations resulted in inactive enzymes, indicating that both residues are crucial for activity. The Kd value for NADPH binding to the K193R mutant was significantly higher than for the wild-type enzyme, suggesting that the affinity for NADPH has also been reduced. ...
Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly166), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (kcat/Km) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft (∼160 Å3), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (kcat/Km) (up to 5000 times) as a result of steric hindrance. ...
The Kv1 subfamily of voltage-gated potassium channels (Kv1.1-Kv1.6) is involved in repolarizing the membrane potential after an action potential, shaping the action potential, and neurotransmitter release. The ability to carry out these functions requires proper trafficking and localization to the cell surface. Kv1 subfamily members have very different trafficking and TGG patterns despite having very similar amino acid sequences. Both positive and negative amino acid trafficking determinants of these processes have been mapped to the outer pore-forming region, which differ in these channels. The two important amino acid determinants responsible for those differences have been identified by lab personnel. Using a site-directed mutagenesis approach to make targeted substitutions, I have defined the physical and chemical properties at those two key positions that affect the Kv1.4 potassium channels localization pattern and TGG. The mutations were characterized by their effect on levels of surface and
"The Development of Site-directed Mutagenesis by Michael Smith" (PDF). Journal of Biological Chemistry. 281 (39).. ... Site-directed mutagenesis[edit]. In 1971, Clyde Hutchison and Marshall Edgell showed that it is possible to produce mutants ... Clyde A. Hutchison III is an American biochemist and microbiologist notable for his research on site-directed mutagenesis and ... Hutchison later collaborated with Michael Smith and developed a more general method of site-directed mutagenesis using a mutant ...
Analysis by site-directed mutagenesis". J. Biol. Chem. 267 (26): 18407-18412. PMID 1526981. Guest JR, Russell GC (1991). " ...
Identification by site-directed mutagenesis". J. Biol. Chem. 275 (32): 24294-303. doi:10.1074/jbc.M002437200. PMID 10827082. ...
Identification by site-directed mutagenesis". J. Biol. Chem. 268 (6): 3980-5. PMID 8095045. GGT1 protein, human at the US ... Catalytic properties, subunit structure, and localization of the gamma-glutamyl binding site on the light subunit". J. Biol. ...
Analysis using site-directed mutagenesis". J. Biol. Chem. 265: 21027-21031. PMID 2250008. Kim, W.S.; Hatsuzawa, K.; Ishizuka, Y ...
MacDonald NJ, Freije JM, Stracke ML, Manrow RE, Steeg PS (1996). "Site-directed mutagenesis of nm23-H1. Mutation of proline 96 ... and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement". J. Biol. Chem. 277 (23): 20895- ...
Szasz J, Yaffe MB, Sternlicht H (1993). "Site-directed mutagenesis of alpha-tubulin. Reductive methylation studies of the Lys ... http://www.genetex.com/WebPage/Product/PromotionDetail.aspx?Type=Promotion&PromotionID=178 Venter JC (1993). "Identification of ...
Biochemical characterization and site-directed mutagenesis". The Journal of Biological Chemistry. 266 (7): 4315-21. PMID ...
This can be achieved by: site-directed mutagenesis employing either phage- or polymerase chain reaction (PCR)-mediated methods ... Storici, Francesca; Lewis, L. Kevin; Resnick, Michael A. (2001-08-01). "In vivo site-directed mutagenesis using ... Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases" Proceedings of the National Academy of ... many target sites, single genetic mutations; (ii) single target site, many genetic mutations; and (iii) many target sites, many ...
The Gibson Assembly method can also be used for site directed mutagenesis to incorporate site-specific mutations such as ... By using this site, you agree to the Terms of Use and Privacy Policy. Wikipedia® is a registered trademark of the Wikimedia ... No restriction site scar remains between two DNA fragments, but the region between the double strands and hanging ends is ...
The delitto perfetto technique is also simpler compared to other methods for in vivo site-directed mutagenesis. Other methods ... Delitto perfetto (Italian: [deˈlitto perˈfɛtto]) is a genetic technique for in vivo site-directed mutagenesis in yeast. This ... 2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol. 19(8):773-6. ... 2006) The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic ...
Site-directed mutagenesis of a critical lysine 262". The Journal of Biological Chemistry. 266 (35): 24031-7. PMID 1748675. ... The tertiary structure consists of a beta/alpha-barrel, with the coenzyme-binding site located at the carboxy-terminus end of ... identification of glycation sites". Biochemistry. 34 (4): 1433-8. doi:10.1021/bi00004a038. PMID 7827091. Robinson B, Hunsaker ... characterization of the active site pocket". Biochemistry. 34 (35): 11264-75. doi:10.1021/bi00035a036. PMID 7669785. Takahashi ...
Lounsbury KM, Schlegel B, Poncz M, Brass LF, Manning DR (1993). "Analysis of Gz alpha by site-directed mutagenesis. Sites and ... "Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin". Proc. ...
Another application of site directed mutagenesis is exchanging an amino acid residue far from the active site with a lysine ... Site-directed mutagenesis is a technique that has been around since the 1970s. The early days of research in this field yielded ... Site directed mutagenesis is a valuable technique that allows for the replacement of a single base in an oligonucleotide or ... Site-directed mutagenesis can be useful for many different reasons. A single base pair replacement, could change a codon, and ...
This is often investigated through site-directed mutagenesis. In addition the synthesis of a model complex can suggest the ... Defining the active site structure. A number of important active sites are still poorly defined. This includes the oxygen ... Understanding the active site function. The structure of some enzymes are very well characterized, however, the function of ... ". "Breslow Group Homepage". www.columbia.edu. Retrieved 2015-12-11. Breslow, Ronald (1995-03-01). "Biomimetic Chemistry and ...
2010). "Site-directed mutagenesis of the Anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological ... Through direct mutagenesis, the scientists exchanged the Asn9 for both Asp (Aspartic Acid) and Glu (Glutamic Acid). This change ... "ATP Drives Direct Photosynthetic Production of 1-butanol in Cyanobacteria". Proceedings of the National Academy of Sciences. ... The coenzymes bind to the active site of an enzyme to promote catalysis. By engineering cofactors and coenzymes, a naturally ...
1999). "Site-directed mutagenesis of restriction endonuclease HindIII". Biosci. Biotechnol. Biochem. 63 (10): 1703-7. doi: ... Despite the uncertainty concerning the structure-catalysis relationship of type II endonucleases, site-directed mutagenesis of ... As a result of the site-mutagenesis experiments previously outlined, it is thus proposed that Lys-125, Asp-123, and Asp-108 of ... Despite the lack of evidence suggesting an exact mechanism for the cleavage of DNA by HindIII, site-mutagenesis analysis ...
Site-directed mutagenesis Restriction digest DNA footprinting Kamvysselis, M. (2003). Computational molecular genomics: genes, ... Non-directed PCR-based mutagenesis can also be used; the parameters of the mutagenic PCR reaction can be adjusted to introduce ... The effects of protein interactions with each other as well as the binding sites can also be assayed in this way; candidate ... This is often necessary because one mutation cannot be guaranteed to inactivate a binding site. ...
... and site-directed mutagenesis of inhibitor-2". J. Biol. Chem. 269 (2): 944-54. PMID 8288648. Kakinoki Y, Somers J, Brautigan DL ... 2006). "Global, in vivo, and site-specific phosphorylation dynamics in signaling networks". Cell. 127 (3): 635-48. doi:10.1016/ ...
His cloning vectors were also used to develop the method for oligonucleotide site-directed mutagenesis. DNA cloning, shotgun ... sequencing and site-directed mutagenesis became widely used to sequence large DNA molecules like human chromosomes and to ... "Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis". Gene. 26 (1): 101-6. doi:10.1016/0378- ... "Messing's personal website at the Waksman Institute". Waksman.rutgers.edu. Retrieved 2010-11-10. "Jo Messing, Highly cited ...
Chueh PJ, Morré DM, Morré DJ (2002). "A site-directed mutagenesis analysis of tNOX functional domains". Biochim. Biophys. Acta ...
van Koppen CJ, Nathanson NM (1991). "Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the ...
Daubner SC, Lauriano C, Haycock JW, Fitzpatrick PF (Jun 1992). "Site-directed mutagenesis of serine 40 of rat tyrosine ... A direct pathogenetic role of tyrosine hydroxylase has also been suggested, as the enzyme is a source of H2O2 and other ... By using this site, you agree to the Terms of Use and Privacy Policy. Wikipedia® is a registered trademark of the Wikimedia ... Ramsey AJ, Hillas PJ, Fitzpatrick PF (Oct 1996). "Characterization of the active site iron in tyrosine hydroxylase. Redox ...
Stradal TB, Troxler H, Heizmann CW, Gimona M (2000). "Mapping the zinc ligands of S100A2 by site-directed mutagenesis". J. Biol ... 1998). "Repression of the candidate tumor suppressor gene S100A2 in breast cancer is mediated by site-specific hypermethylation ...
"Cloning and site-directed mutagenesis of human ADP-ribosylarginine hydrolase". J. Biol. Chem. 268 (24): 17837-43. PMID 8349667 ... although these proteins appear to have lost the presumed active site residues. ADP-ribosylarginine hydrolase ADP-ribosyl-( ...
Westheimer et al.'s proposal was further supported by site-directed mutagenesis studies. When Lys116 was mutated to cysteine or ... The active site, consisting of residues such as Phe27, Met97, and Tyr113, is mostly hydrophobic. However, the active site does ... Further research led to the isolation of an active site peptide sequence and identification of the active site lysine, Lys115, ... Arg29 is thought to play a role in substrate binding, while Glu76 is thought to play a role in the orienting the active site ...
By using this site, you agree to the Terms of Use and Privacy Policy. Wikipedia® is a registered trademark of the Wikimedia ... Crotty S, Cameron C, Andino R (2002). "Ribavirin's antiviral mechanism of action: lethal mutagenesis?". J. Mol. Med. 80 (2): 86 ... Infection typically occurs by direct or indirect exposure to animal excrement through the respiratory or gastrointestinal ... Treatment is directed at addressing dehydration and improving symptoms.[1] The antiviral medication, ribavirin may be useful ...
Nelson DR, Lawson JE, Klingenberg M, Douglas MG (April 1993). "Site-directed mutagenesis of the yeast mitochondrial ADP/ATP ... In this conformation, the inhibitor may bind to the ATP-binding site. Functional and structural roles for residues in the TMSs ... 2008) identified the substrate-binding sites and salt bridge networks that are important for transport. The symmetry analyses ... Kunji ER, Robinson AJ (2006-10-01). "The conserved substrate binding site of mitochondrial carriers". Biochimica et Biophysica ...
... WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de). Mon Aug 15 03:50:43 EST 2011 * ...
... philip hardwidge hardp3 at mayo.edu Fri Feb 23 09:16:01 EST 2001 *Previous message: help ... I would like undertaken site directed mutagenesis, but never did. : , : , Can somebody tell me if it is very difficult to do, ... Several papers from about 10 years ago offer facile PCR-based methods for making site-directed mutants. -Phil Hardwidge Mayo ... Previous message: help for site directed mutagenesis *Next message: help for site directed mutagenesis ...
Citations may include links to full-text content from PubMed Central and publisher web sites. ... Efficient site-directed mutagenesis using uracil-containing DNA.. Kunkel TA, Bebenek K, McClary J. ...
... site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours. The kit utilizes the robust Q5 Hot Start High- ... The Q5® Site-Directed Mutagenesis Kit enables rapid, ... The Q5 Site-Directed Mutagenesis Kit enables rapid, site- ... DNA Assembly, Cloning and Mutagenesis Kits. Applications: Site Directed Mutagenesis, Site Directed Mutagenesis. * Advantages ... Home DNA Assembly Cloning and Mutagenesis Kits Products Q5® Site-Directed Mutagenesis Kit ...
... any number of mutageneses. ExonBio provides the best service with double sequecing to make sure the success of the mutagenesis ... Site-directed Mutagenesis from Exon BioSystems,Any form, any size, ... Site-directed mutagenesis-Within 10 bases from Bio S&T. 2. Site-directed DNA Mutagenesis Service from Yorkshire Bioscience Ltd ... AMAP Multi Site-directed Mutagenesis Kit from MBL International. 4. GPS -M Mutagenesis System from New England Biolabs. 5. ...
PCR site-directed mutagenesisEdit. The limitation of restriction sites in cassette mutagenesis may be overcome using polymerase ... Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and ... Whole plasmid mutagenesisEdit. For plasmid manipulations, other site-directed mutagenesis techniques have been supplanted ... "SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites". BMC Bioinformatics. 14 ...
Nobel Lecture on Invention of Site-Directed Mutagenesis OpenWetWare Diagram summarizing site-directed mutagenesis. ... Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker Direct gene deletion and site-specific ... Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and ... "SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites". BMC Bioinformatics. 14 ...
Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ... Chen, Wei, 1965-. Site Directed Mutagenesis Of Dienelactone Hydrolase, thesis, December 1992; Denton, Texas. (digital.library. ... Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ... Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ...
firefly luciferase Luciola mingrelica site-directed mutagenesis cysteine residues polyhistidine tag kinetic parameters thermal ... firefly luciferase carrying C-terminal His6-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. ...
... ,Please email us with project details and we will respond within 24 hours ... Site-directed Mutagenesis from Exon BioSystems. 2. Site-directed DNA Mutagenesis Service from Yorkshire Bioscience Ltd. 3. AMAP ... Date:12/10/2018)... ... December 10, 2018 , ... ... Manufacturing Organization (CDMO) with sites in China and the United States ...
This system replaces the popular GeneTailor Site-Directed Mutagenesis System, and has been completel ... The GeneArt Site-Directed Mutagenesis System provides a state-of-the-art, simple, convenient, and highly efficient means to ... Site-Directed Mutagenesis System, and has been completely redesigned to be at the leading edge of commercial site-directed ... Optimized Mutagenesis Protocol. The GeneArt® Site-Directed Mutagenesis System has been optimized for efficiency and simplicity ...
... the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated ... This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for ... In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved ...
... identify enzyme active sites, and design novel proteins in drug discovery. ... Site-directed mutagenesis is a powerful research tool used to study protein function, ... Which site-directed mutagenesis system is right for you?. GeneArt Site-Directed Mutagenesis System GeneArt Site-Directed ... Multisite-directed mutagenesis efficiency. Figure indicates the multisite-directed mutagenesis efficiency of 3 sites of 1 bp or ...
... spectroscopic and site-directed mutagenesis study Dolores Linde, Rebecca Pogni, Marina Cañellas, Fátima Lucas, Victor Guallar, ... Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus Jing Cheng, Yuichi Hanada, ... Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase Fuyuan Jing, Marna D. ...
Puett D., Huang J., Xia H. (1994) Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ. In: ... Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ. ... Site-directed mutagensis of virtually any plasmid by eliminating a unique site. Anal Biochem 1992; 200: 81-8.PubMedCrossRef ... El-Deiry S, Kaetzel D, Kennedy G, Nilson J, Puett D. Site-directed muta-genesis of the human chorionic gonadotropin β-subunit: ...
Citations may include links to full-text content from PubMed Central and publisher web sites. ... Site-directed mutagenesis has been used to change amino acid residues of a recombinant Fc-hinge fragment derived from the ... Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis.. Kim JK1 ... The results show that the site of the IgG1 molecule that controls the catabolic rate (the catabolic site) is located at the ...
Problems with Stratagenes site directed mutagenesis kit - Primers, PCR, Mutagenesis (Jun/17/2010 ). Hi all. Ive been having ... problems with the Stratagenes site directed kit.. Ive been trying to troubleshoot, and think its my primers coz I ran the ... Im using a website: Northwestern university/ biotools that shows if your primers have any complementarity and unfortunately ... Im using a website: Northwestern university/ biotools that shows if your primers have any complementarity and unfortunately ...
... can save you both time and money over some of the common cloning methods used for mutagenesis. ... In vitro site-directed mutagenesis is a core technique in functional genomics studies. Until recently, labs performing site- ... Site-directed mutagenesis techniques have relied primarily on PCR and standard cloning methods. Read about some of the common ... Figure 1. Examples of site-directed mutagenesis by traditional PCR. Primers incorporating the desired base changes are used in ...
... 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 ...
... hypothesis that the segment between amino acid position 63 to 73 of the H-2Dd antigen forms a major allo-antigenic site, ... Introduction of H-2Dd determinants into the H-2Ld antigen by site directed mutagenesis. J. Exp. Med. (in press).Google Scholar ... Site Directed Mutagenesis Identifies Allo-Antigenic Epitopes of an H-2 Antigen Recognized by Antibodies and by Cytotoxic T- ... 1987) Site Directed Mutagenesis Identifies Allo-Antigenic Epitopes of an H-2 Antigen Recognized by Antibodies and by Cytotoxic ...
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Wikiomics:Site_Directed_Mutagenesis&oldid=396866" ...
Lidstrom:Site-Directed Mutagenesis. From OpenWetWare. Revision as of 06:44, 29 February 2012 by Janet B. Matsen. (talk , ... Retrieved from "https://openwetware.org/mediawiki/index.php?title=Lidstrom:Site-Directed_Mutagenesis&oldid=588129" ... Kunkel Mutagenesis. *Justin Siegel at the Baker lab is our local expert. The UW iGem team uses this method. ...
Problems with Stratagene Site Directed Mutagenesis - posted in Molecular Cloning: I would check your NZY+ broth by growing an E ... Problems with Stratagene Site Directed Mutagenesis. Started by yean_ny_nie, Apr 07 2009 09:14 PM ... instead of using the high price Strategene site-directed mutagenesis kit. I have gained about ~ 19 mutaed clones, and find it ... Forward mutagenesis primer 2.0 µl. Reverse mutagenesis primer 2.0 µl. dsDNA template (150 ng) 2.0 µl. pfu Ultra fusion II ...
... Gullesen, Jonathan ... positions in the sequences of specific C1 oxidizing cellulose-active AA10 proteins were targeted for site-directed mutagenesis ...
3 Designing Primers for Site-Directed Mutagenesis (2014). Biochemistry Laboratory Manual For Undergraduates An Inquiry-Based ... 4 Performing Site-Directed Mutagenesis. *5 Purifying Mutant Toxin Sensor DNA from Bacterial Cells and Evaluating its Quality ... 3 Designing Primers for Site-Directed Mutagenesis. * ... 3 Designing Primers for Site-Directed Mutagenesis. Gerczei, ...
  • I'm using a website: Northwestern university/ biotools that shows if your primers have any complementarity and unfortunately two of my forward primers happen to be complementary at a particular stretch. (protocol-online.org)
  • 2010). In the RF method, PCR primers are replaced with long dsDNA that has 5' ends containing homologous overlaps with the desired vector insertion site (Figure 2). (idtdna.com)
  • Unlike kits that rely on linear amplification, primers designed for the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits of exponential amplification are realized. (neb.com)
  • Previously in our laboratory, eight highly active OmpT variants were engineered with novel catalytic sites. (utexas.edu)
  • Site-directed mutagenesis of human myeloperoxidase: further identification of residues involved in catalytic activity and heme interaction. (ac.be)
  • Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes. (jimmunol.org)
  • 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)
  • Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. (aspetjournals.org)
  • Limited proteolysis of the pyruvate decarboxylase (E1, α 2 β 2 ) component of the pyruvate dehydrogenase (PDH) multi-enzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1α subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. (elsevier.com)
  • Lipoamide Dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. (growkudos.com)
  • Efficient site-directed mutagenesis using uracil-containing DNA. (nih.gov)
  • These involve (1) subcloning a promoterless sucCD fragment downstream of the lac promoter in M13mp10, and (2) precise splicing of the suc coding regions with the efficient atpE ribosome-binding site and expression from the thermoinducible lambda promoters in the pJLA503 vector. (portlandpress.com)
  • PickMutant™ is a reliable, robust and highly efficient PCR-based Mutagenesis Kit. (canvaxbiotech.com)
  • 5 PCFT has a low pH optimum that allows efficient transport of folates in the acid microclimate of the duodenum and jejunum, 6 the major sites of folate absorption, where this transporter is highly expressed. (bloodjournal.org)
  • Selected active site residues were mutated by site-directed mutagenesis to lysine, arginine, and histidine. (utexas.edu)
  • No activity was detected after the histidine residue at the phosphorylation site of the alpha-subunit was replaced by aspartate (His-246 alpha----Asp), but this alteration seemed to have a deleterious effect on the accumulation of the enzyme in cell-free supernatant extracts. (portlandpress.com)
  • Using non-conical amino acids (ncAAs), we probe an adjacent residue (Phe 93) to one of three key histidine residues involved in the active site. (oregonstate.edu)
  • Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidine-tyrosine, the Cu B and its ligands, and the active-site heme and its ligands. (pnas.org)
  • The K-channel leads from a glutamate residue at the cytoplasmic interface of subunits I and II to the cross-linked histidine-tyrosine cofactor in the active site (see ref. 6 for review). (pnas.org)
  • Since 2013, the development of the CRISPR /Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing of the genome , and mutagenesis may be performed in vivo with relative ease. (wikipedia.org)
  • We further demonstrate the ease with which mutagenesis can be achieved by deleting 13.5 kb from the Toledo genome using a PCR-based technique. (biomedsearch.com)
  • The site-directed mutagenesis using the plasmid vector to introduce the mutated DNA, a process which provides accurate and precise changes and manageable segments of the genome. (facmedicine.com)
  • The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure-function relationships of this important family of proteins. (frontiersin.org)
  • Following the initial characterization, site-directed mutagenesis of conserved cysteinyl residues was performed in order to gain further insight into the structure/function relationship of NifU. (vt.edu)
  • Bacterial sRNA's activate and repress translation by exposing or covering RBS (ribosome binding site) on mRNA. (mywordsolution.com)
  • Past site-directed mutagenesis experiments, combined with structural analysis, have identified 2 conserved proton channels critical for function within the A-family: the K- and D-channels ( 4 - 6 ). (pnas.org)
  • Ni H, Zeng S, Qin X, Sun X, Zhang S, Zhao X, Yu Z, Li L. Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic, Synergistic Lepidopteran-larvicidal and Nematicidal Activities. (ijbs.com)
  • Message Body (Your Name) thought you would like to see this page from the Biochemical Journal web site. (biochemj.org)
  • A final transformation of the mutated DNA into DH5α™ E.coli cells digests any methylated parental DNA, leaving behind only the intact unmethylated mutagenesis reaction product. (thermofisher.com)
  • The present study examined the roles of several residues surrounding the active site of OmpT while attempting to use rational design to modulate fine specificity enough to create a novel protease that prefers phosphotyrosine containing substrates relative to sulfotyrosine or unmodified tyrosine residues. (utexas.edu)
  • Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. (nus.edu.sg)