Search results for " Site-Directed"
showing 10 items of 132 documents
Transmembrane beta-barrel of staphylococcal alpha-toxin forms in sensitive but not in resistant cells.
1997
Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity coul…
SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion
1999
The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well a…
Identification of sequences in the human peptide transporter subunit TAP1 required for transporter associated with antigen processing (TAP) function
2001
The heterodimeric peptide transporter associated with antigen processing (TAP) consisting of the subunits TAP1 and TAP2 mediates the transport of cytosolic peptides into the lumen of the endoplasmic reticulum (ER). In order to accurately define domains required for peptide transporter function, a molecular approach based on the construction of a panel of human TAP1 mutants and their expression in TAP1(-/-) cells was employed. The characteristics and biological activity of the various TAP1 mutants were determined, and compared to that of wild-type TAP1 and TAP1(-/-) control cells. All mutant TAP1 proteins were localized in the ER and were capable of forming complexes with the TAP2 subunit. H…
Homemade Site Directed Mutagenesis of Whole Plasmids
2009
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensi…
The cost of replication fidelity in an RNA virus
2005
It is often argued that high mutation rates are advantageous for RNA viruses, because they confer elevated rates of adaptation. However, there is no direct evidence showing a positive correlation between mutation and adaptation rates among RNA viruses. Moreover, theoretical work does not argue in favor of this prediction. We used a series of vesicular stomatitis virus clones harboring single amino acid substitutions in the RNA polymerase to demonstrate that changes inducing enhanced fidelity paid a fitness cost, but that there was no positive correlation between mutation an adaptation rates. We demonstrate that the observed mutation rate in vesicular stomatitis virus can be explained by a t…
Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies
2010
The fitness effects of mutations are central to evolution, yet have begun to be characterized in detail only recently. Site-directed mutagenesis is a powerful tool for achieving this goal, which is particularly suited for viruses because of their small genomes. Here, I discuss the evolutionary relevance of mutational fitness effects and critically review previous site-directed mutagenesis studies. The effects of single-nucleotide substitutions are standardized and compared for five RNA or single-stranded DNA viruses infecting bacteria, plants or animals. All viruses examined show very low tolerance to mutation when compared with cellular organisms. Moreover, for non-lethal mutations, the me…
Role for calnexin and N-linked glycosylation in the assembly and secretion of hepatitis B virus middle envelope protein particles.
1998
ABSTRACT Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is required for efficient secretion of M empty envelope particles. Moreover, this glycan mediates specific association of M with the chaperone calnexin. Conversely, the N glycan, common to all three envelope proteins, is involved neither in calnexin binding nor in subviral particle release. As proper folding and trafficking of M need the assistance of the chaperone, the glycan-dependent association of M with calnexin may thus play a crucial role in the assembly …
The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its…
1996
Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786–5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been …
Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis.
2003
The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g. substitution of Glu368 by Asp. Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enz…
Mutational analysis of the cysteine residues in the hepatitis B virus small envelope protein.
1993
The small envelope protein of hepatitis B virus is the major component of the viral coat and is also secreted from cells as a 20-nm subviral particle, even in the absence of other viral proteins. Such empty envelope particles are composed of approximately 100 copies of this polypeptide and host-derived lipids and are stabilized by extensive intermolecular disulfide cross-linking. To study the contribution of disulfide bonds to assembly and secretion of the viral envelope, single and multiple mutants involving all 14 cysteines in HepG2 and COS-7 cells were analyzed. Of the six cysteines located outside the region carrying the surface antigen, Cys-48, Cys-65, and Cys-69 were each found to be …