The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Narendra Wajapeyee - One of the best experts on this subject based on the ideXlab platform.
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Methods for In Vitro Mutagenesis
CSH Protocols, 2019Co-Authors: Matteo Forloni, Narendra WajapeyeeAbstract:: Several different methodologies for Mutagenesis have been developed to introduce mutations at predetermined sites or regions within mammalian genes. These methods of in vitro Mutagenesis have had a transforming effect on the understanding of functions of protein, transcription regulatory elements, and noncoding RNAs and are now integral to molecular biology investigations. In this introduction, we summarize the most commonly used experimental approaches for Mutagenesis and their major applications.
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Random Scanning Mutagenesis.
CSH Protocols, 2019Co-Authors: Matteo Forloni, Narendra WajapeyeeAbstract:: In vitro oligonucleotide and polymerase chain reaction (PCR)-based Mutagenesis is generally used for altering the nucleotide sequence of genes to study their functional importance and the products they encode. A thorough approach to this problem is to systematically change each successive amino acid residue in the protein to alanine (i.e., alanine-scanning Mutagenesis) or to a limited number of alternative amino acids. Although these strategies can provide useful information, it is sometimes desirable to test a broader spectrum of amino acid changes at the targeted positions. "Random scanning Mutagenesis" was developed to examine the functional importance of individual amino acid residues in the conserved structural motif of human immunodeficiency virus (HIV) reverse transcriptase, and this protocol is adapted from that method. This strategy is an oligonucleotide-based method for generating all 19 possible replacements at individual amino acid sites within a protein.
Susan M Rosenberg - One of the best experts on this subject based on the ideXlab platform.
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Stress-induced β-lactam antibiotic resistance mutation and sequences of stationary-phase mutations in the Escherichia coli chromosome
Journal of Bacteriology, 2009Co-Authors: Joseph F. Petrosino, Rodrigo S. Galhardo, Liza D. Morales, Susan M RosenbergAbstract:In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations are a common route to clinically relevant beta-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial beta-lactam resistance mutations using the well-developed genetics of E. coli by integrating enterobacterial ampRC genes into the E. coli chromosome. Like the cells of other enterobacteria, E. coli cells acquire beta-lactam resistance by ampD mutation. Here we show that starvation and stress responses provoke ampD beta-lactam resistance Mutagenesis. When starved on lactose medium, Lac(-) strains used in Mutagenesis studies accumulate ampD beta-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this Mutagenesis, in agreement with the results obtained for lac reversion in these cells. Surprisingly, the stress-induced ampD mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike lac Mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the ampD system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced Mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced Mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.
Matteo Forloni - One of the best experts on this subject based on the ideXlab platform.
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Methods for In Vitro Mutagenesis
CSH Protocols, 2019Co-Authors: Matteo Forloni, Narendra WajapeyeeAbstract:: Several different methodologies for Mutagenesis have been developed to introduce mutations at predetermined sites or regions within mammalian genes. These methods of in vitro Mutagenesis have had a transforming effect on the understanding of functions of protein, transcription regulatory elements, and noncoding RNAs and are now integral to molecular biology investigations. In this introduction, we summarize the most commonly used experimental approaches for Mutagenesis and their major applications.
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Random Scanning Mutagenesis.
CSH Protocols, 2019Co-Authors: Matteo Forloni, Narendra WajapeyeeAbstract:: In vitro oligonucleotide and polymerase chain reaction (PCR)-based Mutagenesis is generally used for altering the nucleotide sequence of genes to study their functional importance and the products they encode. A thorough approach to this problem is to systematically change each successive amino acid residue in the protein to alanine (i.e., alanine-scanning Mutagenesis) or to a limited number of alternative amino acids. Although these strategies can provide useful information, it is sometimes desirable to test a broader spectrum of amino acid changes at the targeted positions. "Random scanning Mutagenesis" was developed to examine the functional importance of individual amino acid residues in the conserved structural motif of human immunodeficiency virus (HIV) reverse transcriptase, and this protocol is adapted from that method. This strategy is an oligonucleotide-based method for generating all 19 possible replacements at individual amino acid sites within a protein.
James H Naismith - One of the best experts on this subject based on the ideXlab platform.
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an efficient one step site directed deletion insertion single and multiple site plasmid Mutagenesis protocol
BMC Biotechnology, 2008Co-Authors: James H NaismithAbstract:Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed Mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. We have developed a site-directed plasmid Mutagenesis protocol that preserved the simple one step procedure of the QuikChange™ site-directed Mutagenesis but enhanced its efficiency and extended its capability for multi-site Mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site Mutagenesis. Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange™. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange™) but increased the overall success rates.
Claudio Gualerzi - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis of the cyanobacterium Spirulina platensis by UV and nitrosoguanidine treatment
FEMS Microbiology Letters, 1991Co-Authors: Luisa Lanfaloni, M Trinei, M. Russo, Claudio GualerziAbstract:The production of Spirulina platensis cells resistant to 8-azaguanine or beta-(2-thienyl)-DL-alanine following Mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation is described. The conditions for the Mutagenesis were determined by monitoring cell viability and the appearance of the two types of mutants as a function of the stage of growth of the tricomes and the length and the conditions of the treatment. The optimal conditions for UV and MNNG Mutagenesis were found to be 1-3 min irradiation and 30 min incubation with 50 micrograms MNNG/ml of tricomes derived from cultures entering stationary phase sonicated for 10 s and 5 s respectively. Under these conditions beta-(2-thienyl)-DL-alanine-resistant mutants appeared at a frequency greater than or equal to 10(-4) and greater than or equal to 10(-5) following UV- and MNNG-Mutagenesis, respectively. Mutants resistant to 8-azaguanine were found at a frequency approx. 10(-5) only after MNNG Mutagenesis. A few chlorate-resistant mutants were also obtained following UV treatment.
