Selection, mutants

Mutation. For industrial appHcations, mutations are induced by x-rays, uv irradiation or chemicals (iiitrosoguanidine, EMS, MMS, etc). Mutant selections based on amino acid or nucleotide base analogue resistance or treatment with Nystatin or 2-deoxyglucose to select auxotrophs or temperature-sensitive mutations are easily carried out. Examples of useful mutants are strains of Candida membranefaciens, which produce L-threonine Hansenu/a anomala, which produces tryptophan or strains of Candida lipolytica that produce citric acid. An auxotrophic mutant of S. cerevisiae that requires leucine for growth has been produced for use in wine fermentations (see also Wine). This yeast produces only minimal quantities of isoamyl alcohol, a fusel oil fraction derived from leucine by the Ehrlich reaction (10,11). A mutant strain of bakers yeast with cold-sensitive metaboHsm shows increased stabiUty and has been marketed in Japan for use in doughs stored in the refrigerator (12). 

The choice of the strain of microorganism is one of the important variables in the process. The strains to be used in manufacture are mutants of the original producer, which are chosen as the result of a planned program of mutant selection. Sometimes a spontaneous mutation occurs usually, it is induced by mutagenic agents or irradiation of various sorts. The choice of the best strain depends on its abiUty to produce large amounts of the proper antibiotic in a reasonable time from ingredients that are economically feasible (73). 

S. V. Gulnik, L. I. Suvorov, B. Liu, B. Yu, B. Anderson, H. Mitsuya, J. W. Erickson, Kinetic Characterization and Cross-Resistance Patterns of HIV-1 Protease Mutants Selected under Drug Pressure , Biochemisry 1995, 34, 9282-9287. 

A variety of mammalian cell culture systems can be used to detect mutations induced by chemical substances. The L5178Y mouse l)nnphoma line, measuring mutation at the TK locus, is preferred. TK is an important enz)une involved in DNA synthesis. Cells are exposed to the test substance at various concentrations, in the presence and absence of a metabolic activation system, for a suitable period of time, and then subcultured to assess cytotoxicity and to allow phenotypic expression prior to mutant selection. Cells deficient in TK because of a forward mutation are resistant to the cytotoxic effects of pyrimidine analogues (antimetabolites), such as trifluorothymidine (TFT). This is because the antimetabolites cannot be incorporated into cellular nucleotides and kill the cell through inhibition of cellular metabolism. After treatment, cells are grown in a medium containing TFT mutant cells can proliferate in the presence of TFT, whereas normal cells containing TK are killed. This allows the detection of an increase in mutant... 

Geosmin synthesis in channel catfish aquaculture systems, 322-333 biosynthesis, 328-329 occurrence in organisms, 324,325r odor quality, 13 synthesis, 322,323/ synthetic mutant selection, 330-333 Glucosyloxyethyl methacrylate polymerization, 224,225/ synthesis and toxicity, 222,224 Glucosyloxyethyl methacrylate polymers potential applications, 226r properties, 224-227... 

Gulnik SV, Suvorov LI, Liu B, Yu B, Anderson B, Mitsuya H, Erickson JW. Kinetic characterization and cross-resistance patters of HIV-1 protease mutants selected under in vitro drug pressure. Biochemistry 1995 34 9282-9287. 

Ping, L. H. and Lemon, S. M (1992) Antigenic structure of human hepatitis-A virus defined by analysis of escape mutants selected against murine monoclonal antibodies. J Virol 66,2208-2216... 

Two Wga mutants selected from CHO cells have been partially characterized by Briles et al. (12). One of the WgaK cell lines described by these authors is designated clone 1021 and it exhibits many properties similar to those of WgaRH cells. The other mutant (clone 13) possesses certain properties similar to WgaRVIH cells (for example, a very high degree of resistance to... 

Morrissey JH, Mack BG, Neuenschwander PR Comp PC. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation, Blood 1993 81 734-744. 

It is important to be aware of the criteria used by the authors of the studies cited in selecting their data, because similar requirements should apply to schemes to which uniform standards will be applied. Test protocols, test strains or cell lines, genetic loci, metabolic-activation requirements, mutant-selection protocols, and many other factors were evaluated before data were acceptable for publication. This kind of evaluation is absolutely necessary in constructing a relative-potency scale like the one we propose. Although not all GENE-TOX evaluations are complete, the Committee views the current GENE-TOX effort as a good source of protocol and data evaluation... 

It is not yet clear whether any rules for thermal adaptation are to be found in the qualitative features of the mutations discovered during directed evolution. The trends seen in Table V (preference for surface substitutions over buried substitutions, conservative over nonconservative, etc.) would most likely be seen in any pool of functional mutants selected from random mutant libraries. 

Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutant bacteria measurement and potential use of the mutant selection window./. Infect. Dis., 2002, 185, 561-565. 

The assay was described by Clive and co-workers (Clive et al. 1972) as a mutational assay system using the TK locus in mouse lymphoma cells. In the following years, he and his collaborators undertook a large-scale of investigation of the potential and optimal conduct of the assay. This included the use of the above mentioned TFT to select tk mutants, a comparison of the hypoxanthine guanine phosphoribosyl transferase (hprt) and tk loci, an analysis of the best expression time for tk mutant selection and a description of distinct large and small colony tk mutants. 

In these two examples, the principle of transition-state stabilization as a source of enzymatic catalysis is successfully exploited for the selection of active mutants. Selection with a TSA appears to be able to enrich libraries in variants with high catalytic activity. 

Martin F, Michel F, Zenklusen D, Muller B, Schumperli D. Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system. Nucleic Acids Res. 2000 28 1594-1603. 

It is possible that the virus mutant selected under the pressure of a certain drug is a mixture of two or more populations of virus. It has been found that the HSV-1 mutants arising under the selective pressure of HPMPC or HPMPA are not pure populations. Thus, plaque-purified virus must be prepared, and the drug-susceptibility profile of a few classes of compounds should be determined as well as the mutations in the drug-targeted viral gene that are associated with the resistant phenotype. 

Applicable to all assays that use X phage as shuttle vector (e.g., MutaTMMouse, and Big Blue mouse and rat), except gpt delta models. The XEGIO phage used as vector in the later model bears a mutation in the chiC gene, involved in positive mutant selection. 

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