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
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.
Sequence Homology, Amino Acid
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.
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.
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 Structure, Secondary
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.
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.
Genetic Complementation Test
Recombinant Fusion Proteins
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.
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.
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
Promoter Regions, Genetic
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.
Gene Expression Regulation, Bacterial
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.
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.
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.
Sequence Analysis, DNA
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)
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.
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
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.
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.
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.
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.
Sequence Homology, Nucleic Acid
Protein Structure, Quaternary
Nucleic Acid Conformation
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
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.
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.
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 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).
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 184.108.40.206.
Gene Expression Regulation
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.
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.
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)
Magnetic Resonance Spectroscopy
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)
Structural Homology, Protein
Membrane Transport Proteins
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).
Protein Interaction Domains and Motifs
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.
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.
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)
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.
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.
Hydrophobic and Hydrophilic Interactions
Bacterial Outer Membrane Proteins
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)
GENETAILOR SITE DIRECTED MUTAGENESIS PDF
GENETAILOR SITE DIRECTED MUTAGENESIS PDF - Site-specific mutagenesis techniques, also known as site-directed mutagenesis .. GeneTailor Site-Directed mutagenesis system (Invitrogen. I have designed
Analysis by Site-Directed Mutagenesis of Important Residues Invoved in the Assembly of Escherichia-Coli Heart-Labile NALYSIS BY...
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) ...
Mutagenesis Studies on the Membrane Anchoring Properties of Human CD2 - Enlighten: Theses
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 ...
Understanding the role of the essential Asp251 in cytochrome P450cam using site-directed mutagenesis, crystallography, and...
Fingerprint Dive into the research topics of Understanding the role of the essential Asp251 in cytochrome P450cam using site-directed mutagenesis, crystallography, and kinetic solvent isotope effect. Together they form a unique fingerprint. ...
Site-directed Mutagenesis Libraries - Creative Biostructure
Creative Biostructure develops comprehensive protein evolution and engineering platform to generate gene diversification by site-directed mutagenesis libraries.
Describe how you would use site-directed mutagenesis to, Ask an Expert
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
Gene site saturated mutagenesis
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, ...
Mutation of the ATP-Binding Pocket of SSA1 Indicates That a Functional Interaction Between Ssa1p and Ydj1p Is Required for Post...
Site-directed mutagenesis, cloning, strains, and media: Four mutant alleles of SSA1, ssa1-101 (K69Q), ssa1-102 (G198D), ssa1-103 (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 ...
Site-directed mutagenesis - experimental considerations | NEB
Site-drected mutagenesis considerations include: primer design, modification and purity, template removal, ligation, transformation, and product evaluation.
Lidstrom:Site-Directed Mutagenesis - OpenWetWare
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 ...
Wikiomics:Site Directed Mutagenesis - OpenWetWare
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. ...
SDM-Assist software to design site-directed mutagenesis primers introducing silent restriction sites
The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.
Site directed mutagenesis
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? ...
Site Directed Mutagenesis - 100% Präzision
Ortsgerichtete Mutagenese: Punktmutationen, Aminosäure-Austausche und Insertionen einführen. Bis zu 150 bp. Kurze Lieferzeit. Absolute Vertraulichkeit.
Mutagenesis: Site-specific - eLS - El-Gewely - Wiley Online Library
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. ...
Ergebnisse durchsuchen | Europäische Behörde für
Deadline: 4 June 2020 EFSAs GMO Panel has launched an open consultation on its draft scientific opinion on the applicability of the EFSA opinion on site-directed nucleases type 3 (SDN-3) for the safety assessment of plants developed using site-dire .... ...
Altmetric - Site-Directed Mutagenesis Study on the Thermal Stability of a Chimeric PQQ Glucose Dehydrogenase and Its Structural...
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 ...
Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not...
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 ...
Site-directed mutagenesis of the active site of diacylglycerol kinase α: calcium and phosphatidylserine stimulate enzyme...
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 ...
Site-Directed Mutagenesis - Cloning and Mutagenesis - GENEWIZ
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.. ...
Plasmid-based one-pot saturation mutagenesis<...
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. | [email protected]
Defining fibronectins cell adhesion synergy site by site-directed mutagenesis.s profile, publications, research topics, and co-authors
OneClick: A Program for Designing Focused Mutagenesis Experiments
OneClick is a user-friendly web-based program, developed specifically for quick-and-easy design of focused mutagenesis experiments (|em|e.g|/em|., site-directed mutagenesis and saturation mutagenesis). Written in Perl and developed into a web application using CGI programming, OneClick offers a step-by-step experimental design, from mutagenic primer design to analysis of a mutant library. Upon input of a DNA sequence encoding the protein of interest, OneClick designs the mutagenic primers according to user input, |em|e.g|/em|., amino acid position to mutate, type of amino acid substitutions (|em|e.g|/em|., substitution to a group of amino acids with similar chemical property) and type of mutagenic primers. OneClick has incorporated an extensive range of commercially available plasmids and DNA polymerases suitable for focused mutagenesis. Therefore, OneClick also provides information on PCR mixture preparation, thermal cycling condition, expected size of PCR product and agar plate to use during bacterial
A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study |...
The drug binding also produces significant displacement of several segments of the backbone. There is a large movement of the segment between Val107 and Leu113 (including the mutated residue Ala109), apparently because the segment between Gln104 and Gln108 becomes helical as a result of drug binding. Some of the side chain atoms appear to become displaced by nearly 3 Å upon drug binding. In addition, the segment between Gly861 and Gly870, just preceding TM helix 8, shows a slight backbone displacement and side chain movement up to 3 Å.. Similar interactions often involving the same set of residues appear to occur with the binding of R6G and Et in our model (not shown). Surprisingly, no acidic residue that would neutralize the positive charges of these dyes was found within 6 Å of the ligand (except Asp566, which is close to the benzoic acid moiety of R6G but more than 11 Å away from its amine nitrogens), although the partially negative π-electron cloud of the phenyl ring of Phe664 is about ...
Homemade Site Directed Mutagenesis of Whole Plasmids | Protocol
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we...
Structure Cluster - 1RQL: Crystal Structure of Phosponoacetaldehyde Hydrolase Complexed with Magnesium and the...
1RQL: X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis
Mutagenesis | BaseClear B.V.
Site-directed mutagenesis of a sequence to make specific, targeted changes to double stranded DNA. Based on optimised protocols, BaseGene is able to create mutations in virtually every DNA fragment. This is a valuable tool for studying DNA or protein structure and function. Site-directed or random mutagenesis can be applied to any DNA-fragment cloned into a plasmid vector. We can help you to introduce a range of mutations, varying from point mutations to deletions or insertions.. ...
Indian Patents. 247720:PROCESS FOR GENERATING AND DETECTING RECOMBINANT DNA SEQUENCES IN PROKARYOTES
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 ...
Q5 Site-Directed Mutagenesis Kit - Lab-JOT® Ltd. Sp.z o.o. Sp.k.
Site Directed Mutagenesis with Pfu Turbo
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol ...
mutagenesis expert come in~ - Molecular Cloning - BioForum
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...
Faculty Profile of J. Bryan Jones
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: post #1
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 ...
Protocols and Video Articles Authored by Kajohn Boonrod
Kajohn Boonrod is the author of this article in the Journal of Visualized Experiments: Homemade Site Directed Mutagenesis of Whole Plasmids
Team:Warsaw/Calendar-Stage1/14 September 2010 - 2010.igem.org
Directed mutagenesis was preformed on pSB-MinC in order to remove an additional PstI site. Two primers were used: MinCmutF (TAATTGCAGTCGCGCCGC) and MinCmutR (GTCGAAAACGCTTTGACCG). PCR was run with Yellow Pfu Polimerase (Eurex) in a standard reaction mix ...
The coordination sphere of iron-sulfur clusters: lessons from site-directed mutagenesis experiments | SpringerLink
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...
Identification of Critical Amino Acid Residues in Human Immuno...
Identification of Critical Amino Acid Residues in Human Immunodeficiency Virus Type 1 IN Required for Efficient Proviral DNA Formation at Steps prior to Integra
The structure of tumour necrosis factor--implications for biological function. - CAMS Oxford Institute
The three-dimensional structure of TNF has been determined at 0.29 nm using the technique of X-ray crystallography. Published data on site-directed mutagenesis and antibody binding may now be assessed in the light of the structure, thus the links between structure and function for TNF may be addressed. TNF is a compact trimer composed of three identical subunits of 157 amino acids. The main-chain topology for a single subunit is essentially a beta-sandwich structure formed by two anti-parallel beta-pleated sheets. This mainchain fold corresponds to the jelly roll motif observed in viral coat proteins such as VP1, VP2 and VP3 of rhinovirus, or the hemagglutinin molecule of influenza. TNF is the first non-viral protein to contain this motif. The subunits associate tightly about a threefold axis interacting through a simple edge-to-face packing of the beta-sandwich to form the solid, conical shaped trimer. A large number of the residues conserved between the amino acid sequences of TNF and lymphotoxin
SPO14 Separation-of-Function Mutations Define Unique Roles for Phospholipase D in Secretion and Cellular Differentiation in...
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 ...
Examination of alternate codon bias solutions for expression and purification of recombinant mechano-growth factor in...
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
UniProtKB/SwissProt variant VAR 026378
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; ...
TRANSIENT IMMORTALIZATION - Patent application
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 ...
Updated mutation overview - VU Medicinal Chemistry - Tools & Downloads
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. ...
UniProtKB/SwissProt variant VAR 073451
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; ...
JoVE Author Search: Br%C3%A9maud L
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 ...
Tryptophan scanning mutagenesis reveals distortions in the helical structure of the δM4 transmembrane domain of the Torpedo...
Caballero-Rivera, D., Cruz-Nieves, O.A., Oyola-Cintrón, J., Torres-Nunez, D.A., Otero-Cruz, J.D., Lasalde-Dominicci, J.A. (2012) Channels (Austin) 2012 Mar-Apr 6(2):111-23 ...
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 ...
In Vitro Mutagenesis Protocols (Methods... | WHSmith Books
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 ...
RCSB PDB - 1ENA: CRYSTAL STRUCTURES OF THE BINARY CA2+ AND PDTP COMPLEXES AND THE TERNARY COMPLEX OF THE ASP 21->GLU...
1ENA: Crystal structures of the binary Ca2+ and pdTp complexes and the ternary complex of the Asp21-->Glu 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 ...
pCDNA3.1-human-ub-wt | mBIOnet
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 ...
KRIBB: Search Results
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 ...
An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol - Nuffield...
Mutagenesis of the mouse Bhlhb4 gene. A: Gene targeting | Open-i
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 ...
Tools to Study Mammalian Mutagenesis - Stephen Dertinger
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...
Acid Resource | Learn About, Share and Discuss Acid At Popflock.com
Get Acid essential facts. View Videos or join the Acid discussion. Add Acid to your PopFlock.com topic list or share. Acid at popflock.com
Clyde A. Hutchison III
"The Development of Site-directed Mutagenesis by Michael Smith" (PDF). Journal of Biological Chemistry. 281 (39).. ... Site-directed mutagenesis. 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 ...
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. CS1 maint: discouraged parameter (link) Breslow, Ronald ( ...
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. ...
"Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis". The Journal of Cell Biology. 149 (2): 521-7. ...
Rouhier N, Gelhaye E, Jacquot JP (2002). "Exploring the active site of plant glutaredoxin by site-directed mutagenesis". FEBS ...
Zheng L, Baumann U, Reymond JL (August 2004). "An efficient one-step site-directed and site-saturation mutagenesis protocol". ... Gene site saturation mutagenesis (GSSM) is mutagenesis technique of one or more codons in a gene to create a library of ... "Gene site saturation mutagenesis: a comprehensive mutagenesis approach". Methods in Enzymology. 388 (1): 3-11. doi:10.1016/ ... Li A, Acevedo-Rocha CG, Reetz MT (July 2018). "Boosting the efficiency of site-saturation mutagenesis for a difficult-to- ...
"Attenuation of chicken anemia virus by site-directed mutagenesis of VP2". Journal of General Virology. 88 (8): 2168-2175. doi: ...
T7 DNA polymerase
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA ... This polymerase has various applications in site-directed mutagenesis as well as a high-fidelity enzyme suitable for PCR. It ... Bebenek K, Kunkel TA (July 1989). "The use of native T7 DNA polymerase for site-directed mutagenesis". Nucleic Acids Research. ... for oligonucleotide site-directed mutagenesis". Nucleic Acids Research. 17 (8): 3314. doi:10.1093/nar/17.8.3314. PMC 317753. ...
Identification of donor and acceptor binding sites by site-directed mutagenesis". J. Biol. Chem. 274 (36): 25608-12. doi: ... 1998). "Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell ...
For more information on rational design see site-directed mutagenesis. In directed evolution, random mutagenesis, e.g. by error ... This method utilizes user defined site directed mutagenesis at single or multiple sites simultaneously. OSCARR is an acronym ... Whole plasmid single PCR is also referred to as site directed mutagenesis (SDM). SDM products are subjected to Dpn endonuclease ... In general, this has the advantage of being inexpensive and technically easy, since site-directed mutagenesis methods are well- ...
Today phagemids are still useful for generating templates for site-directed mutagenesis. Detailed characterisation of the ...
Dang T, Prestwich GD (August 2000). "Site-directed mutagenesis of squalene-hopene cyclase: altered substrate specificity and ... Active site engineering resulted in loss of the enzyme's ability to form hopanoids, but enabled Brønsted acid catalysis for the ... Aromatic residues in the active site form several unfavorable carbocations on the substrate which are quenched by a rapid ... Researchers indicate this could be due to direct regulation of transport proteins by hopanoids or indirectly by altering ...
Analysis of the redox state of the flavin cofactor by site-directed mutagenesis". The Journal of Biological Chemistry. 283 (6 ... Biskup T, Schleicher E, Okafuji A, Link G, Hitomi K, Getzoff ED, Weber S (2009). "Direct observation of a photoinduced radical ... This indicates that opsin-less sponge eyes utilize cryptochrome, along with other proteins, to direct or act in eye-mediated ... The resulting change in protein conformation could lead to phosphorylation of previously inaccessible phosphorylation sites on ...
See genetic code.) Site-directed mutagenesis selectively introduces mutations that change the structure of a protein. The ... Some examples of genetic methods include conceptual translation, Site-directed mutagenesis, using a fusion protein, and ...
Computer modeling and site-directed mutagenesis". Mol. Pharmacol. 69 (3): 788-795. doi:10.1124/mol.105.018200. PMID 16354762. ... This has a direct effect on the peripheral nervous system (PNS). Batrachotoxin in the PNS produces increased permeability ( ... suggesting a common site of action. Due to these similarities, treatment for batrachotoxin poisoning might best be modeled ...
Quantitative structure-activity relationship
Protein-protein interactions can be quantitatively analyzed for structural variations resulted from site-directed mutagenesis. ... The molecular fragments could be substituents at various substitution sites in congeneric set of molecules or could be on the ... A comprehensive web resource for QSAR modelers Chemoinformatics Tools, Drug Theoretics and Cheminformatics Laboratory ... "Structure-Activity Correlation for Relative Chain Initiation to Propagation Rates in Single-Site Olefin Polymerization ...
In molecular biology, alanine scanning is a site-directed mutagenesis technique used to determine the contribution of a ... This is usually accomplished by site-directed mutagenesis or randomly by creating a PCR library. Furthermore, computational ... Then shotgun scanning method which combines the concepts of alanine scanning mutagenesis and binomial mutagenesis with phage ... Simonsen SM, Sando L, Rosengren KJ, Wang CK, Colgrave ML, Daly NL, Craik DJ (April 2008). "Alanine scanning mutagenesis of the ...
... by site-directed mutagenesis". Virology. 187 (2): 654-62. doi:10.1016/0042-6822(92)90468-5. PMID 1546460. Wang MB, Bian XY, Wu ... Parallel to agriculture-directed studies, more basic scientific research elucidated many of viroids' physical, chemical, and ...
... of an important cysteine residue in human glutamate-cysteine ligase catalytic subunit by site-directed mutagenesis". Biochem. J ... identification of the major transcriptional start site in HT29 cells". Biochim. Biophys. Acta. 1446 (1-2): 47-56. doi:10.1016/ ...
David S. Cafiso
Site-directed spin labeling is a powerful methodology that combines site-directed mutagenesis with and EPR spectroscopy. Dr. ... Cafiso's laboratory is directed at studying membranes and peripheral and integral membrane proteins. One area of investigation ... https://web.archive.org/web/20101115062638/http://ernst.chem.virginia.edu/dsclab/. ...
β-glucosidase / galactosidase in which the nucleophile glutamate 358 was mutated to an alanine by site directed mutagenesis. ... The leaving group is displaced by an alcohol of the acceptor sugar aided by the active site general base amino acid of the ... The first was that a change of the active site nucleophile of a glycosidase from a carboxylate to another amino acid resulted ... Mutation of the active site nucleophile to a non-nucleophilic amino acid prevents the formation of a covalent intermediate. An ...
... common patterns revealed by site-directed mutagenesis studies". Philosophical Transactions of the Royal Society of London B: ... Retrieved 2013-11-01.CS1 maint: archived copy as title (link) "Cluster home page". engineering.purdue.edu. Mächler, Martin (25 ...
A site-directed mutagenesis study". Environ. Health Perspect. 106 (2): 85-92. doi:10.1289/ehp.9810685. PMC 1533021. PMID ... 8-DHF and various other reported small-molecule TrkB agonists might not actually be direct agonists of the TrkB and might be ...
Site-directed mutagenesis is a technique often employed to create knock-in and knock-out models that express missense mRNAs. ... When the APC mRNA was analyzed in the mutational spectrum, it showed 3 sites where the frequency of substitutions were high. ...
Site-directed mutagenesis studies to the carbohydrate recognition domain removes glycan binding but does not prevent ... They can also hinder adhesion by binding to two glycans on the same cell, which blocks the integrin binding site. Galectin-8 is ... "TRIMs and Galectins Globally Cooperate and TRIM16 and Galectin-3 Co-direct Autophagy in Endomembrane Damage Homeostasis". ... "TRIM proteins regulate autophagy and can target autophagic substrates by direct recognition". Developmental Cell. 30 (4): 394- ...
She used site directed mutagenesis to probe the role of specific amino acid residues in the function of the enzyme. Rao also ... R, Rao; J, Pagan; Ae, Senior (1988-11-05). "Directed Mutagenesis of the Strongly Conserved Lysine 175 in the Proposed ... "Rao Lab Homepage". www.bs.jhmi.edu. Retrieved 2020-05-21. "Rajini Rao - Publications". neurotree.org. Retrieved 2020-05-21. R, ... In her graduate work, Rao discovered that three catalytic sites in the ATPase enzyme needed to interact to achieve maximal ...
Protein fold class
... site-directed mutagenesis, or Artificial gene synthesis. Protein superfamily SCOP database CATH database FSSP database Hubbard ...
Cold Spring Harbor Laboratory
Nociceptinski receptor - Vikipedija, slobodna enciklopedija
... evidence from a site-directed mutagenesis study of the ORL1 receptor transmembrane-binding domain.". Mol. Pharmacol. 57 (3): ...
List of atheists in science and technology
S100A6 - ويكيبيديا
Sudo T، Hidaka H (1999). "Characterization of the calcyclin (S100A6) binding site of annexin XI-A by site-directed mutagenesis ... Kordowska J، Stafford WF، Wang CL (1998). "Ca2+ and Zn2+ bind to different sites and induce different conformational changes in ...
نوبل انعام برائے کیمیا وصول کنندگان کی فہرست - آزاد دائرۃ المعارف، ویکیپیڈیا
... site-directed mutagenesis and its development for protein studies" 1994 George A. Olah امریکا / ہنگری "for his contribution ... As of 26 October 2008, the nobelprize.org website page for the 2008 award gives Shimomura's country as "USA". However, the ... "for their outstanding achievements in developing direct methods for the determination of crystal structures" ...
Methods commonly used to study protein structure and function include immunohistochemistry, site-directed mutagenesis, X-ray ... Through another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence ... [email protected]Home (Stanford University). *Protein Databank in Europe (see also PDBeQuips, short articles and tutorials on ... Tutorials and educational websites. *"An Introduction to Proteins" from HOPES (Huntington's Disease Outreach Project for ...
வேதியியலுக்கான நோபல் பரிசு பெற்றவர்கள் - தமிழ் விக்கிப்பீடியா
... site-directed mutagenesis and its development for protein studies" 1994 George A. Olah United States / Hungary "for his ... A. The form and spelling of the names in the name column is according to nobelprize.org, the official website of the Nobel ... C. The citation for each award is quoted (not always in full) from nobelprize.org, the official website of the Nobel Foundation ... B. The information in the country column is according to nobelprize.org, the official website of the Nobel Foundation. This ...
"Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human ... A novel Sp1-binding site occurs close to the major transcription start site (position - 65); a GC-rich core region including ... Ishii S, Noguchi M, Miyano M, Matsumoto T, Noma M (1992). "Mutagenesis studies on the amino acid residues involved in the iron- ... An Adenosine triphosphate (ATP) binding site; ATP is crucial for ALOX5's metabolic activity ...
Cykl Calvina, wolna encyklopedia
... nucleotide response and increased Rubisco activation activity of Arabidopsis rubisco activase by site-directed mutagenesis.. „ ...
5-HT4受體 - 维基百科，自由的百科全书
Computational model of the complex between GR113808 and the 5-HT4 receptor guided by site-directed mutagenesis and the crystal ...
Nuclear magnetic resonance spectroscopy of proteins
Green fluorescent protein
Lista över nobelpristagare i kemi
... site-directed mutagenesis and its development for protein studies". *^ "The Nobel Prize in Chemistry 1994". Nobelprize.org. ... https://web.archive.org/web/20070202025710/http://nobelprize.org/nobel_prizes/nobelprize_facts.html. Läst 7 oktober 2008.. ... Web. (9 oktober 2013). "Pressmeddelande: Nobelpriset i kemi 2013". Pressmeddelande. Läst 9 oktober 2013. ... https://web.archive.org/web/20080731233358/http://nobelprize.org/nobel_prizes/amounts.html. Läst 7 oktober 2008.. ...
Functional magnetic resonance imaging
Քաղցկեղ - Վիքիպեդիա՝ ազատ հանրագիտարան
DNA damage, homology-directed repair, and DNA methylation»։ PLoS Genetics 3 (7): e110։ July 2007։ PMC 1913100։ PMID 17616978։ ... SEER Stat Fact Sheets: All Cancer Sites»։ National Cancer Institute։ Արխիվացված օրիգինալից 26 September 2010-ին։ Վերցված է 18 ... Mutator pathways unleashed by epigenetic silencing in human cancer»։ Mutagenesis 22 (4): 247-53։ July 2007։ PMID 17412712։ doi: ...
Site-directed mutagenesis - Wikipedia
Nobel Lecture on Invention of Site-Directed Mutagenesis OpenWetWare Diagram summarizing site-directed mutagenesis. ... Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker Direct gene deletion and site-specific ... Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and ... "SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites". BMC Bioinformatics. 14 ...
Site directed mutagenesis
help for site directed mutagenesis
... philip hardwidge hardp3 at mayo.edu Fri Feb 23 09:16:01 EST 2001 *Previous message: help ... I would like undertaken site directed mutagenesis, but never did. : , : , Can somebody tell me if it is very difficult to do, ... Several papers from about 10 years ago offer facile PCR-based methods for making site-directed mutants. -Phil Hardwidge Mayo ... Previous message: help for site directed mutagenesis *Next message: help for site directed mutagenesis ...
... directed mutagenesis (SDM) aims to introduceprecise alterations in any coding or noncoding deoxyribonucleic acid (DNA) sequence ... directed mutagenesis. (b) SLiCE‐mediated PCR‐based site‐directed mutagenesis (SLiP site‐directed mutagenesis). Reprinted from ... All site‐directed alterations requiring site‐directed mutagenesis technique are done at the DNA level. ... El‐Gewely MR (1991) Oligonucleotide and multisite directed mutagenesis. In: El‐Gewely MR (ed.) Site Directed Mutagenesis and ...
Efficient site-directed mutagenesis using uracil-containing DNA. - PubMed - NCBI
Q5® Site-Directed Mutagenesis Kit | NEB
... 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 ...
Site-directed mutagenesis - experimental considerations | NEB
Site-directed Mutagenesis from Exon BioSystems
... 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. ...
Site-directed mutagenesis - Wikipedia
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 ...
Site Directed Mutagenesis Of Dienelactone Hydrolase - Digital Library
Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ... Chen, Wei, 1965-. Site Directed Mutagenesis Of Dienelactone Hydrolase, thesis, December 1992; Denton, Texas. (digital.library. ... Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ... Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. ...
Site-directed mutagenesis of cysteine residues of Luciola mingrelica firefly luciferase | SpringerLink
Enabling Fast, Efficient, and High Fidelity Site-Directed Mutagenesis through Continuous Innovation
A site-directed mutagenesis method can introduce point mutations, amino acid substitutions, deletions and small insertions in ... Synthetic Biology as the Next Step in Site-Directed Mutagenesis. The efficiency and ease-of-use of site-directed mutagenesis ... Home Resources Enabling Fast, Efficient, and High Fidelity Site-Directed Mutagenesis through Continuous Innovation ... Conventional techniques to perform site-directed mutagenesis at five sites (along with accompanying miniprep and sequencing ...
Site-directed mutagenesis-Within 10 bases from Bio S&T
... ,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 ...
GeneArt Site-Directed Mutagenesis System - Thermo Fisher Scientific
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 ...
SDM-Assist software to design site-directed mutagenesis primers introducing 'silent' restriction sites
The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on ... SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites BMC Bioinformatics. 2013 ... Background: Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure- ... Conclusions: The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the ...
Efficient multi-site-directed mutagenesis directly from genomic template.
... the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated ... This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for ... In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved ...
Site-Directed Mutagenesis | Thermo Fisher Scientific - CA
... 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 ...
site-directed mutagenesis | Biochemical Journal
... 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. ...
Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ | SpringerLink
Puett D., Huang J., Xia H. (1994) Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ. In: ... Delineation of Subunit and Receptor Contact Sites by Site-Directed Mutagenesis of hCGβ. ... Site-directed mutagensis of virtually any plasmid by eliminating a unique site. Anal Biochem 1992; 200: 81-8.PubMedCrossRef ... El-Deiry S, Kaetzel D, Kennedy G, Nilson J, Puett D. Site-directed muta-genesis of the human chorionic gonadotropin β-subunit: ...
Problems with Stratagene Site Directed Mutagenesis -> Attachments -...
Homemade Site Directed Mutagenesis of Whole Plasmids | Protocol
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. ... Site directed mutagenesis is a mutagenesis-method which provides a fast way to mutate a gene carried by a plasmid. The whole ... The site directed mutagenesis of whole plasmids explained in this video is a mutagenesis method which allows you to alter a ... In this tutorial we demonstrated performing a site directed mutagenesis by using a homemade mutagenesis kit. The benefit of ...
Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis. -...
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 Stratagene's site directed mutagenesis kit - Molecular Cloning
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 ...
Site-directed mutagenesis-improvements to established methods
... 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 ...
Spinach carbonic anhydrase: investigation of the zinc-binding ligands by site-directed mutagenesis, elemental analysis, and...
To investigate the nature of the zinc-binding site in spinach carbonic anhydrase, we targeted potential zinc ligands for ... Spinach carbonic anhydrase: investigation of the zinc-binding ligands by site-directed mutagenesis, elemental analysis, and ... To investigate the nature of the zinc-binding site in spinach carbonic anhydrase, we targeted potential zinc ligands for ... suggest that spinach carbonic anhydrase utilizes a Cys-His-Cys-H2O ligand scheme to bind the zinc ion at the active site. ...
IJMS | Free Full-Text | Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed...
... site-directed mutagenesis uricase; exon replacement/restoration; site-directed mutagenesis ... After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity ... Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis. Guangrong Xie †. ... In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis ...
Site-directed mutagenesis - OpenWetWare
An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004 Aug 10;32(14):e115. DOI: ... Site-directed mutagenesis can be used to change particular base pairs in a piece of DNA. There are a number of methods for ... Stratagene QuikChange Site-directed Mutagenesis Kit. Publications. * Zheng L, Baumann U, and Reymond JL. ... If you have problems with this procedure, you can try Round-the-horn site-directed mutagenesis which uses PCR to amplify the ...
Site Directed Mutagenesis Identifies Allo-Antigenic Epitopes of an H-2 Antigen Recognized by Antibodies and by Cytotoxic T...
... 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 ...
Wikiomics:Site Directed Mutagenesis - OpenWetWare
ResiduesPlasmidMutationPrimersSubstrateGeneTailor Site-Directed MutagenesProteinCloningMutationsSaturation mutagenesisGeneMutantsPlasmidsGenomeReagentsOligonucleotidePolymeraseEfficiency of mutagenesisVitro mutagenesisDeletionsOligonucleotidesAmino acidsMutantHigh-efficiencyEnzymeGeneArtEfficientMethodsSynthesisProtocolPrimerResidueCatalyticReactionSubstitutionsStratagenePutativeHomologyExperimentsMutagenicHistidineSequenceSyntheticOverlapEngland BiolabsSpecificity1985CatalysisEnzymesCharacterizationBiochemicalFragments
- Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis. (nih.gov)
- Site-directed mutagenesis has been used to change amino acid residues of a recombinant Fc-hinge fragment derived from the murine immunoglobulin (Ig)G1 molecule, and the effects of these mutations on the pharmacokinetics of the Fc-hinge fragment have been determined. (nih.gov)
- Highly conserved positions in the sequences of specific C1 oxidizing cellulose-active AA10 proteins were targeted for site-directed mutagenesis, and the C4 oxidizing activity of a MaLPMO10B variant, carrying two mutated residues, was almost completely lost. (bibsys.no)
- Kinetic consequences of site-directed substitutions of these residues are reported. (unboundmedicine.com)
- The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. (rcsb.org)
- Veit M., Kretzschmar E., Kuroda K., Garten W, Schmidt M.F.G., Klenk H.D., Rott R.. Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin. (springer.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)
- Selected active site residues were mutated by site-directed mutagenesis to lysine, arginine, and histidine. (utexas.edu)
- An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. (aspetjournals.org)
- Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. (aspetjournals.org)
- Ten unique 2B6 SRS residues were mutated to the corresponding residues in 2B1, and two additional mutants, L363V and V477I, were produced to test sites known to be important in 2B1. (aspetjournals.org)
- Site-directed mutagenesis of potential CoA binding-site residues indicated that Trp-43 beta and His-50 beta are essential residues in the beta-subunit, whereas Cys-47 beta could be replaced by serine without inactivating the enzyme. (portlandpress.com)
- We have used site-directed mutagenesis to examine the relative importance of the five residues in determining sensitivity of this strain to paclitaxel. (pubmedcentralcanada.ca)
- The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. (plantphysiol.org)
- These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. (plantphysiol.org)
- The closed structure is maintained by electrostatic and hydrophobic interactions between residues located in the active site and the substrate. (plantphysiol.org)
- 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)
- 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. (asm.org)
- It is speculated that domains 2 and 3 contain two regions as potential candidates for the reactive site of cysteine proteases : Gln-Val-Val-Ala-Gly (QVVAG) located at approximately two-thirds of the distance from the N-terminus of the domains and Gly located at 44 amino acid residues upstream from the QVVAG region. (nii.ac.jp)
- The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. (diva-portal.org)
- This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. (inserm.fr)
- In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. (ijbs.com)
- We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli . (ijbs.com)
- Mutational analysis of structurally-guided active site residues identified those involved in binding and catalysis. (concordia.ca)
- 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. (rupress.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)
- To determine whether any of the proposed ligand binding sites was responsible for TLR3 ligand recognition, we mutated residues around the two sulfate binding sites and at the positively charged patches. (pnas.org)
- Here we demonstrate that ligand binding depends on residues in a region encompassing the LRR20 sulfate binding site and extending to the glycan-free surface, thus providing the first indication of how TLRs interact with their cognate ligands. (pnas.org)
- Site-directed mutagenesis of human myeloperoxidase: further identification of residues involved in catalytic activity and heme interaction. (ac.be)
- The Q5 ® Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours. (neb.com)
- In order to ensure the success of the SDM reaction, the final plasmid should be isolated from a single E. coli clone and the site of mutation should be sequenced in both directions. (neb.com)
- Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His6-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis. (springer.com)
- Today's conventional site-directed mutagenesis methods, first introduced in 1995 (see Strategies 9(1): 3-4 under "Resources"), typically utilize a three-step, one-day method to introduce point mutations, amino acid substitutions, deletions, and small insertions in virtually any double-stranded plasmid template with high rates of efficiency. (genengnews.com)
- Multiple base mutagenesis is common, and we tested a 12 base substitution, insertion and deletion using a pUC19 plasmid. (thermofisher.com)
- Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. (jove.com)
- The site directed mutagenesis of whole plasmids explained in this video is a mutagenesis method which allows you to alter a cloned target gene by substitution, deletion or insertion of a few bases directly into a plasmid. (jove.com)
- Thereafter you can use this plasmid as template for a series of mutations in which this restriction site will be deleted by insertion of the new mutagenic primers. (jove.com)
- Purify template plasmid DNA from a dam + Escherichia coli strain (to ensure that all GATC sites are methylated for later digestion with DpnI). (openwetware.org)
- Cassette mutagenesis technique needs not involve primer extension using DNA polymerase, in which a fragment of DNA containing the mutation gene of interest and restriction enzyme sites is synthesized, and then inserted into a plasmid. (creative-biostructure.com)
- The most commonly used method for site-directed mutagenesis is the QuikChange ® method (Stratagene, La Jolla, CA, USA), which employs mutagenic plasmid amplification to introduce single site-directed mutations. (biomedcentral.com)
- Plasmid construction and luciferase reporter assays Data for genetailor site genetialor mutagenesis system gathered from related PubMed articles. (emailfinder.mobi)
- Stratagenes QuikChange SiteDirected Mutagenesis Kit allows site specific mutation in virtually any doublestranded plasmid, thus eliminating the need for Stratagenes QuikChange II sitedirected mutagenesis kit allows site specific mutation in virtually any doublestranded plasmid, thus eliminating the need for subcloning and for ssDNA rescue. (project-a-phone.com)
- Once the vector is set up with flanking restriction sites, all manipulations (i.e., mutagenesis, sequencing, expression) can be performed in the same plasmid. (wikipedia.org)
- These methods of mutagenesis, however, are limited by the kind of mutation they can achieve, and they are not as specific as later site-directed mutagenesis methods. (wikipedia.org)
- This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. (wikipedia.org)
- A large number of methods are available to effect site-directed mutagenesis, although most of them have rarely been used in laboratories since the early 2000s, as newer techniques allow for simpler and easier ways of introducing site-specific mutation into genes. (wikipedia.org)
- Site-directed mutagenesis is one of the most important techniques in laboratory for introducing a mutation into a DNA sequence. (wikipedia.org)
- For screening purposes it is practical to insert or delete a restriction site with your mutation. (jove.com)
- Because of the degeneration of the genetic code there are many possibilities to insert a restriction site with your desired mutation. (jove.com)
- The enzyme finder tool will tell you which restriction sites can be introduced parallel to your desired mutation. (jove.com)
- If you can't find any possible or practical restriction site by this way, you may insert any new restriction site without concerning the genetic code at the site of mutation. (jove.com)
- Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. (jimmunol.org)
- Publications] H.Nakaura: 'Functional changes in tropchin T by a splice cloror site mutation that causes hypertrophic cardiomyopathy' Am.J.Physiol.in press. (nii.ac.jp)
- The principle of site-directed mutagenesis is that a mismatched oligonucleotide is extended, incorporating the 'mutation' into a strand of DNA that can be cloned. (escience.ws)
- In detail the procedure employs two mutagenic oligonucleotide primers, one primer contains the desired mutation and the second contains a mutation with a unique, nonessential restriction site. (graduateway.com)
- Any downstream, effector interactions required for coupling Ca 2+ -bound synaptotagmin to vesicle fusion should cause as severe an effect as mutation of the C 2 B Ca 2+ -binding site itself. (jneurosci.org)
- All these mutation could be obtained by PCR using a FastPANGEA™ High Fidelity DNA Polymeras e and well-designed Mutagenesis primers. (canvaxbiotech.com)
- It means that we can direct the mutation according to our interests. (biochempages.com)
- But we can also commercially available site-directed mutagenesis kits to introduce a mutation. (biochempages.com)
- To ensure the validity of our analyses, we used site-directed mutagenesis to create positive controls containing the mutation. (ahajournals.org)
- In contrast, NEBaseChanger , a free online primer design tool, has been developed not only to aid in the design of primers for the Q5 Site-Directed Mutagenesis Kit , but also to provide an annealing temperature for Q5 that accounts for mismatched nucleotides. (neb.com)
- For some workflows, primers must be synthesized with a 5-prime phosphate to enable a downstream intramolecular ligation reaction (this is not required for the Q5 Site-Directed Mutagenesis Kit . (neb.com)
- We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest. (nih.gov)
- 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)
- The two primers are annealed to circular single-stranded DNA and direct synthesis of a new second strand containing both primers. (graduateway.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)
- Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. (unboundmedicine.com)
- In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. (frontiersin.org)
- Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. (aspetjournals.org)
- The group-to-group alignments of CYP2 sequences by Gotoh (1992) proposed the presence of six substrate recognition sites (SRSs) within the P-450 2 family. (aspetjournals.org)
- 1, 2] Haloperoxidases (hemeand vanadium-containing) catalyze the formation of hypohalous acids,[3, 4] which diffuse out of the active site and then react with substrate. (docme.ru)
- Active site of PrnA showing the binding of the substrate tryptophan. (docme.ru)
- Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding. (inserm.fr)
GeneTailor Site-Directed Mutagenes4
- This system replaces the popular GeneTailor™ Site-Directed Mutagenesis System, and has been completely redesigned to be at the leading edge of commercial site-directed mutagenesis products currently available on the market. (thermofisher.com)
- vitro mutagenesis system or the GeneTailor site-directed mutagenesis system can. (emailfinder.mobi)
- Thermo Fisher genetailor site directed mutagenesis system Bioz Stars score: ABM from strain Ecklund 17B] at amino acid level. (emailfinder.mobi)
- The Tbx3 mutant construct was generated using the GeneTailor Site-Directed Mutagenesis System Invitrogen according to the manuals and was confirmed by sequencing. (emailfinder.mobi)
- Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering. (wikipedia.org)
- These techniques are revolutionising our understanding of the genetic and molecular mechanisms, protein structure-function relationship, protein-protein interaction and binding sites in any biological system. (els.net)
- Mutagenesis experiments allow researchers to modulate protein activity and characterize structure- function relationships, which enriches our understanding of basic cellular processes and disease mechanisms and fuels discoveries in new therapies for complex diseases such as cancer. (genengnews.com)
- The results show that the site of the IgG1 molecule that controls the catabolic rate (the 'catabolic site') is located at the CH2-CH3 domain interface and overlaps with the Staphylococcal protein A binding site. (nih.gov)
- Furthermore, if you want to alter the sequence at more than one site (e.g., changing several codons at one site, and adding a protein tag to the resulting mutant), you will need to perform multiple rounds of mutagenesis, and the cost of reagents and time will rise accordingly. (idtdna.com)
- It occurs as a multiprotein complex ( 40 , 41 , 50 ) with the outer membrane channel TolC ( 9 , 15 ) and a periplasmic linker protein, AcrA ( 17 ) (the structure of an AcrA homolog, MexA, was solved recently [ 2 , 11 ]), and this complex structure allows the direct export of drugs to the external medium ( 28 ). (asm.org)
- Bos E.C.W., Heijnen L., Spaan W.J.M. (1995) Site Directed Mutagenesis of the Murine Coronavirus Spike Protein. (springer.com)
- The Escherichia coli multidrug efflux pump protein AcrB has recently been cocrystallized with various substrates, suggesting that there is a phenylalanine-rich binding site around F178 and F615. (uzh.ch)
- 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. (caltech.edu)
- 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. (caltech.edu)
- Structural and functional determinants of human plasma phospholipid transfer protein activity as revealed by site-directed mutagenesis of charged amino acids. (semanticscholar.org)
- Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants. (iupui.edu)
- The site-directed mutagenesis library offers a great technology for protein function and active center studies. (creative-biostructure.com)
- Site-directed mutagenesis (SDM), also termed site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique that is an important research tool for for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering. (creative-biostructure.com)
- To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli ( E. coli ) with the plasmids obtained. (biomedcentral.com)
- Site-directed and saturation mutagenesis are widely used to prepare mutant genes for studying a protein function in basic research and for preparation of engineered proteins for industrial and pharmaceutical applications. (biomedcentral.com)
- Hutchison later developed methods for "complete mutagenesis" in which each residue in a protein is individually altered. (wikipedia.org)
- In vitro sitedirected mutagenesis is an invaluable technique for characterizing the dynamic, complex relationships between protein structure and function, for studying gene b The QuikChange II SiteDirected Mutagenesis Kit (Catalog# ) contains enough reagents for 10 total reactions, which includes 5 control reactions. (project-a-phone.com)
- QuikChange SiteDirected Mutagenesis Kit 3 INTRODUCTION In vitro sitedirected mutagenesis is an invaluable technique for studying protein structurefunction relationships and gene expression, and for In vitro sitedirected mutagenesis is an invaluable technique for studying protein structure function relationships and for identifying intramolecular regions or amino acids, both of which may mediate gene expression and vector modification. (project-a-phone.com)
- Site-Directed Mutagenesis is an important research tool for protein engineering and structure/function studies. (blueskybioservices.com)
- When studying protein functions, cassette mutagenesis can allow a scientist to change individual amino acids by introducing different codons or omitting codons. (wikipedia.org)
- Site-directed mutagenesis techniques have relied primarily on PCR and standard cloning methods. (idtdna.com)
- Read about some of the common cloning methods used for mutagenesis and how double-stranded DNA fragments (gBlocks Gene Fragments) can save you both time and money. (idtdna.com)
- Until recently, labs performing site-directed mutagenesis relied primarily on PCR and standard cloning methods. (idtdna.com)
- PCR, and restriction cloning), and new methods (e.g., isothermal assembly) of mutagenesis, and are becoming a standard reagent in these techniques because they eliminate some of the time-consuming steps needed to produce both a wild-type sequence and any derivative variants. (idtdna.com)
- Now, for a reasonable cost, researchers can design all the requisite sequences for their experiments, order them online, and receive them ready for direct use or cloning. (idtdna.com)
- An example of the direct application of dsDNA to PCR mutagenesis is the restriction-free (RF) cloning method described by Unger T, et al. (idtdna.com)
- Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique. (biomedsearch.com)
- Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. (biomedcentral.com)
- This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis. (biomedcentral.com)
- It is not surprising that some better funded and less patient labs resorted to have the mutant gene completely synthesised at great expense or in a less extreme version known as cassette mutagenesis, partially synthesised with suitable cloning sites and inserted into the vector by ligation. (blogspot.com)
- Site-directed mutagenesis is one of the most important laboratory techniques for creating DNA libraries by introducing mutations into DNA sequences. (wikipedia.org)
- Early attempts at mutagenesis using radiation or chemical mutagens were non-site-specific, generating random mutations. (wikipedia.org)
- A selection system to distinguish and select the molecules with the desired site‐directed mutations from the rest of the molecules has to be planned. (els.net)
- Engineered site‐directed mutations are heritable modifications. (els.net)
- Quick Tips - Can I make multiple mutations with the Q5 site-directed mutagenesis kit? (neb.com)
- In contrast, site-directed methods may employ a linear amplification approach (only parental strands are copied) in addition to high-fidelity Pfu polymerases, to minimize the number of clones with undesired mutations. (genengnews.com)
- In addition to the improved site-directed mutagenesis method described above, a newer approach was developed with the ability to create mutations at multiple sites simultaneously. (genengnews.com)
- While error-prone PCR-based methods are typically limited to two simultaneous mutational sites, by employing a unique multi-enzyme polymerase blend, it is possible to introduce mutations at up to five sites simultaneously. (genengnews.com)
- The GeneArt® Site-Directed Mutagenesis System provides a state-of-the-art, simple, convenient, and highly efficient means to generate mutations on a target construct in vitro in less than three hours. (thermofisher.com)
- To test our previous hypothesis that the segment between amino acid position 63 to 73 of the H-2D d antigen forms a major allo-antigenic site, mutations were introduced into the H-2L d gene in a sequential fashion, which replaced the codons for amino acid position 63, 65, 66, 70 and 73 of the H-2L d antigen with those of the H-2D d antigen. (springer.com)
- 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. (genewiz.com)
- To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. (jimmunol.org)
- Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact. (biomedcentral.com)
- MUPAC could efficiently introduce various mutations at multiple specific sites within a gene. (biomedcentral.com)
- These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR3 toward the C terminus and suggest a model for dsRNA binding and TLR3 activation. (pnas.org)
- Site-directed mutagenesis is a procedure for the introduction of mutations in a target DNA sequence. (biochempages.com)
- Using the site-directed mutagenesis, it is possible to introduce three different kinds of mutations into the target DNA. (biochempages.com)
- Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. (biomedcentral.com)
- Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity. (biomedcentral.com)
- To facilitate the preparation of mutant genes, various methods of multiple site-directed and saturation mutagenesis have been developed. (biomedcentral.com)
- Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. (wikipedia.org)
- There are numerous methods for achieving site-directed mutagenesis, but with decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis. (wikipedia.org)
- The gene thus copied contains the mutated site, and is then introduced into a host cell in a vector and cloned. (wikipedia.org)
- Site-directed mutagenesis is the method of choice for altering a gene or vector sequence at a selected location. (genengnews.com)
- As was mentioned above, dsDNA gene fragments are compatible with familiar PCR mutagenesis methods, but they also offer some interesting advantages. (idtdna.com)
- Here we describe the comprehensive methodology used previously for the identification and functional characterization of MYC-responsive elements in the integrin α1 subunit (ITGA1) gene using a combination of in silico analysis, site-directed mutagenesis, and chromatin immunoprecipitation. (springer.com)
- Site-directed mutagenesis of the alpha-toxin gene of Staphylococcus aureus: role of histidines in toxin activity in vitro and in a murine model. (asm.org)
- Identification of an immunodominant region on the I-A beta chain using site-directed mutagenesis and DNA-mediated gene transfer. (rupress.org)
- Creative Biostructure can adapt different strategies to perform in vitro site-directed mutagenesis, such as transplacement "pop-in pop-out", direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker (or using using long homologous regions), and i n vivo site-directed mutagenesis with synthetic oligonucleotides. (creative-biostructure.com)
- Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. (elsevier.com)
- Targeted mutagenesis and gene targeting using sequence specific nucleases (SSNs) are powerful strategies used to accelerate molecular breeding of crops. (nature.com)
- Site directed mutagenesis (SDM) can help reveal the secrets of gene regulation. (eurofinsgenomics.eu)
- Cassette mutagenesis is a type of site-directed mutagenesis that uses a short, double-stranded oligonucleotide sequence (gene cassette) to replace a fragment of target DNA. (wikipedia.org)
- Since restriction enzymes are used, for this method to be useful, the restriction sites flanking the target DNA has to be unique in the gene/vector system so that the gene cassette can be inserted with specificity. (wikipedia.org)
- The length of the sequence flanked by the restriction sites is also a limiting factor due to the use of synthetic gene cassettes. (wikipedia.org)
- Several papers from about 10 years ago offer facile PCR-based methods for making site-directed mutants. (bio.net)
- Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. (unt.edu)
- Creating mutants with the GeneArt® Site-Directed Mutagenesis System relies on the inherent properties of DNA methylase, high fidelity DNA polymerase, recombination enzymes, and the native McrBC endonuclease of E. coli . (thermofisher.com)
- A series of mutants bearing single amino acid substitutions often encountered in the catalase/peroxidase, KatG, from isoniazid-resistant isolates of Mycobacterium tuberculosis has been produced by site-directed mutagenesis. (epfl.ch)
- Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. (biomedcentral.com)
- Strikingly, this method can generate mutants at close to 100% efficiency, however, is limited by the availability of suitable restriction sites flanking the site that is to be mutated. (creative-biostructure.com)
- Site-directed mutants of human myeloperoxidase. (ac.be)
- The high efficiency of the mutagenesis means mutants can be screened directly by sequencing. (wikipedia.org)
- The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most plasmids, the mutagenesis reaction is complete in less than two hours. (neb.com)
- Figure indicates the multisite-directed mutagenesis efficiency of 3 sites of 1 bp or 3 bp each in 5, 10, and 14 kb plasmids. (thermofisher.com)
- This site directed mutagenesis protocol works best with plasmids up to 10kb. (jove.com)
- 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)
- To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. (nature.com)
- In addition, optimized protocols and reagents reduced the length of the PCR and DpnI selection steps, providing up to a three-fold reduction in overall mutagenesis turn-around time. (genengnews.com)
- Each GeneArt® Site-Directed Mutagenesis System includes enough reagents for 16 reactions and a control: DNA methylase, S-adenosylmethionine, enzyme mix and buffer, enhancer, 0.5M EDTA, sterile water, pUC19WHITE mutagenesis control, and One Shot® MAX Efficiency DH5α™-T1R chemically competent cells. (thermofisher.com)
- We have available many products not yet on the website, so please enquire with us for any reagents you cannot find online. (focusbio.com.au)
- b The QuikChange II SiteDirected Mutagenesis Kit (Catalog# ) contains enough reagents for 10 total reactions, which includes 5 control reactions. (project-a-phone.com)
- a The QuikChange SiteDirected Mutagenesis Kits contain enough reagents for 5 control reactions. (project-a-phone.com)
-   Hutchison later collaborated with Michael Smith and developed a more general method of site-directed mutagenesis using a mutant oligonucleotide primer and DNA polymerase . (wikipedia.org)
- It is also called as site-specific mutagenesis or oligonucleotide-directed mutagenesis. (graduateway.com)
- Site-directed mutagenesis can be grouped generally into three categories of which oligonucleotide-directed mutagenesis is by far the most commonly used method. (graduateway.com)
- Hutchison later produced with his collaborator Michael Smith in 1978 a more flexible approach to site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA polymerase. (wikipedia.org)
- Continuing improvements to Pfu polymerase have led to enhancements, permitting protocols with higher fidelity, longer length-capability, multi-site targeting, and significantly faster time-to-completion. (genengnews.com)
- Basically, PCR-based mutagenesis involves the use of normal PCR using Taq-Polymerase and other necessary components. (biochempages.com)
- Unlike many site directed mutagenesis methods, cassette mutagenesis also does not involve primer extension by DNA polymerase. (wikipedia.org)
Efficiency of mutagenesis2
- In vitro mutagenesis using dut − ung − genetic selection method. (els.net)
- If you require substitutions, insertions or deletions at more than one target site, our SDM-Multi Service is the perfect choice. (eurofinsgenomics.eu)
- The results of these alterations are reflected in the encoded amino acids sequence of the proteins or in any targeted binding site in the DNA sequence. (els.net)
- To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. (diva-portal.org)
- We determined phospholipid transfer activity and HDL binding ability in wild-type PLTP and in 16 PLTP variants created by replacing 12 charged amino acids by site-directed mutagenesis. (semanticscholar.org)
- If you have problems with this procedure, you can try 'Round-the-horn site-directed mutagenesis which uses PCR to amplify the desired mutant product. (openwetware.org)
- This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product. (elsevier.com)
- Putative iron-binding sites of phenylalanine hydroxylase were studied by mutating either histidine 284 or 289 to serine and expressing these mutant enzymes (PAH-H284S and PAH-H289S) in Sf9 cells. (elsevier.com)
- Although this method demonstrated high efficiency for complete multiple mutagenesis, synthesis of phosphorothioate nucleotides is necessary to generate 3′-overhanging DNA fragments. (biomedcentral.com)
- My favorite thing about PickMutant™ Site-directed Mutagenesis Kit is its high efficiency and how easy is get the results, even for a beginner like me! (canvaxbiotech.com)
- 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)
- 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. (biochemsoctrans.org)
- Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 220.127.116.11). (inserm.fr)
- This methods can generate a larger DNA fragment to cover two convenient restriction enzyme sites. (creative-biostructure.com)
- A restriction enzyme called DpnI also acts on the same site and specifically digest the unmethylated strand. (blogspot.com)
- Identify enzyme active sites. (canvaxbiotech.com)
- In Directed Enzyme Evolution: Advances and Applications (pp. 201-227). (canvaxbiotech.com)
- The GeneArt® Site-Directed Mutagenesis System has been optimized for efficiency and simplicity. (thermofisher.com)
- An overview of the workflow for GeneArt Site-directed Mutagenesis. (thermofisher.com)
- Here, I am going to explain a simplified protocol for the site-directed mutagenesis that uses GENEART ® Site-Directed Mutagenesis System. (biochempages.com)
- GENEART ® Site-Directed Mutagenesis System comes with components necessary for mutagenesis reaction, recombination as well as transformation. (biochempages.com)
- Adachi Y and Fukuhara C (2012) TA strategy for rapid and efficient site‐directed mutagenesis. (els.net)
- Efficient site-directed mutagenesis using uracil-containing DNA. (nih.gov)
- Efficient multi-site-directed mutagenesis directly from genomic template. (biomedsearch.com)
- 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)
- Site-directed mutagenesis methods such as the QuikChange method have a number of advantages over PCR-based approaches. (genengnews.com)
- While the site-directed approach described above represent a marked improvement over PCR-based methods, within the last five years, new methods were introduced to address the demand for faster workflows and increased productivity. (genengnews.com)
- The efficiency and ease-of-use of site-directed mutagenesis methods have facilitated engineering of promoter and coding regions of numerous genes. (genengnews.com)
- Arguably, the most significant improvement to mutagenesis methods is the commercial availability of long, synthetic, double-stranded (ds), custom DNA fragments. (idtdna.com)
- Incomplete primer synthesis can lead to errors at the mutagenesis site. (neb.com)
- a contrast to the site-directed method's >80% efficiency and three-step one-day protocol. (genengnews.com)
- See my 'Round-the-horn site-directed mutagenesis protocol to get around these limitations. (openwetware.org)
- Your questions will be directed to the authors of the protocol. (bio-protocol.org)
- 5 In addition, the QuikChange Site Directed Mutagenesis Protocol Oligos For oligodesign, you can follow the protocol in the QuikChange Manual, which basically says to design a The Stratagene protocol species that the oligos need to be PAGE puried, although most people use cartridge Manual: QuikChange SiteDirected Mutagenesis Kit 2: 53 PM QuikChange SiteDirected Mutagenesis Kit INSTRUCTION MANUAL Please contact your local distributor. (project-a-phone.com)
- Schematic of the mutagenesis protocol. (iisc.ernet.in)
- Mutagenesis is carried out with one single stranded template (usually M13) by annealing a synthetic primer in which defined changes can be incorporated. (graduateway.com)
- We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. (asm.org)
- 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)
- The pyridoxal-phosphate (PLP) coenzyme resides at the bottom of the active site and forms a Schiff base with a Lys residue. (plantphysiol.org)
- A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site. (biochemsoctrans.org)
- One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. (inserm.fr)
- 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)
- To investigate the nature of the zinc-binding site in spinach carbonic anhydrase, we targeted potential zinc ligands for mutagenesis and examined the resulting enzymes for catalytic activity and stoichiometric zinc binding. (nih.gov)
- After site-directed mutagenesis, H 1-2 P 3 H 4 P 5-6 H 7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. (mdpi.com)
- Previously in our laboratory, eight highly active OmpT variants were engineered with novel catalytic sites. (utexas.edu)
- 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)
- 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)
- Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. (asm.org)
- The approach described here is adapted from the Stratagene site-directed mutagenesis kit, the manual can be found here . (openwetware.org)
- Site-directed mutagenesis was performed to create single amino acid changes in the putative ATP-binding site of rubisco activase. (illinois.edu)
- Gibbs, BS, Wojchowski, D & Benkovic, SJ 1993, ' Expression of rat liver phenylalanine hydroxylase in insect cells and site-directed mutagenesis of putative non-heme iron-binding sites ', Journal of Biological Chemistry , vol. 268, no. 11, pp. 8046-8052. (elsevier.com)
- 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)
- 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)
- Site-directed mutagenesis method was first benefited from recombinant DNA technology in 1970s, when isolated genes were exposed to conditions such as chemical agents or nucleotide analogs to localize their mutagenic effects. (graduateway.com)
- 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)
- Site‐directed mutagenesis (SDM) aims to introduce precise alterations in any coding or noncoding deoxyribonucleic acid (DNA) sequence, usually in vitro . (els.net)
- in one site or in multisite in the same DNA sequence. (els.net)
- In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated templates, such as genomic sequence or complementary DNA. (biomedsearch.com)
- This step removes the target sequence and everything between the restriction sites. (wikipedia.org)
- To use this method, the sequence of the target sequence and nearby restriction sites must be known. (wikipedia.org)
- Clyde A. Hutchison III is an American biochemist and microbiologist notable for his research on site-directed mutagenesis and synthetic biology . (wikipedia.org)
- a) Overlap extension method for site‐directed mutagenesis. (els.net)
- The website of the New England Biolabs provides a tool to find such an appropriate restriction site. (jove.com)
- Since the glycoprotein hormones contain a common α-subunit, and since both subunits are believed to participate in receptor binding (6, 7), interesting questions and models can be developed regarding subunit contact sites and hormone receptor contact sites, particularly those responsible for conferring specificity. (springer.com)
- 1985. T-cell antigenic sites tend to be amphipathic structures. (springer.com)
- The protons required for catalysis and translocation are derived from the cytoplasmic side of the membrane and are transferred to the active site via proton-conducting channels composed of internal water molecules orientated by conserved polar amino acid side chains. (pnas.org)
- The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. (jimmunol.org)
- Our design approach was to mimic the most selective phosphoryl-specific enzymes and binding proteins by increasing positive charge around the active site. (utexas.edu)
- DOI: 10.1002/anie.201007896 Enzymatic Halogenation Changing the Regioselectivity of the Tryptophan 7-Halogenase PrnA by Site-Directed Mutagenesis** Alexander Lang, Stefan Polnick, Tristan Nicke, Peter William, Eugenio P. Patallo, James H. Naismith, and Karl-Heinz van Pe* Dedicated to Professor Franz Lingens on the occasion of his 85th birthday For many years, haloperoxidases were the only type of halogenating enzymes known. (docme.ru)
- 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)
- Message Body (Your Name) thought you would like to see this page from the Biochemical Journal web site. (biochemj.org)
- This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for combination, deletion or insertion of DNA fragments. (biomedsearch.com)