Mutation-based resistance

Point mutation tests have been developed also for cultured mammalian cells (de Marini et al, 1989). These tests are based on the mutational resistance to otherwise cytotoxic agents (i.e. TKor HPRT mutations, conferring resistance to trifluorothymidine and 6-thioguanine, respectively). Compared to the Ames test and other bacterial assays they are, however, more laborious and time consuming. 

Fig. 8. The region of E. coli 16S rRNA involved in the response to monosubstituted and disubstituted 2-deoxy-streptamine compounds. Mutations conferring resistance to antibiotics of these classes are included within the boxed region most of them destroy the terminal G.C base pair of the long imperfect stem.

Biotypes with target-site-based resistance to ACCase inhibitors were also selected in wild oat species [Avena fatua, A. sterilis). The resistance patterns were found to be variable. For example, the resistance factors for ACCase from the Canadian A. fatua biotype UMl were 105 for sethoxydim, 10 for tralkoxydim, and 10 for didofop and fenoxaprop, whereas for the Aoena fatua biotype UM33 from Canada the ratios were 10.5 for fenoxaprop, 1.2 for diclofop, 5 for sethoxydim and 1.7 for tralkoxydim. It was proposed that this was due to different point mutations, each being assodated with a characteristic resistance pattern [37]. Another reason could be the frequency of homozygote and heterozygote resistant and susceptible plants within a tested population. 

Mechanisms of resistance to ACC-inhibiting herbicides can be divided into two categories ACC-related and metabolism-based. Metabolism-based resistance is well described and reviewed in the literature [35, 36]. In most cases, resistance is due to alteration of the target enzyme, making it less sensitive to inhibition, as reviewed by Devine [37] and by Delye [38] the latter gives an overview on homomeric plastidic ACC isoforms with altered sensitivities to AOPPs and CHDs or both [38]. Furthermore, the identification of mutations involved in altered sensitivity was achieved recently (Table 9.2) [38]. 

The induction of OU resistance occurs by a mutation that decreases the binding of OU to the Na /K ATPase but leaves the enzyme functional. As expected for such a restricted mutation, OU resistance is induced at a frequency that is approximately 1% of that for 6TG resistance. We hypothesize that nearly all mutations to OU resistance will be of the base-substitution type. Thus, we conjecture that the ratio of mutation to OU resistance and mutation to 6TG resistance will indicate the relative importance of base substitution to the overall mutation caused by a chemical. The ratio of OU /6TG for the alkylating agent EMS is 6 X 10", while the ratio for the polycyclic hydrocarbon FA is approximately 4 X 10 . This observation supports the hypothesis that OU is induced only by base substitution. A better test of this hypothesis would compare the mutagenicity of base analogues, such as BUdR, and frameshift mutagens, such as ICR-191. 

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). 

Other examples include rifampin resistance due to mutations in the ipoB gene encoding the (3-subunit of RNA polymerase, or oxazolidinone resistance due to a G2576T mutation in the gene for the 23 S rRNA as central part of the 50S large ribosomal subunit. Macrolide resistance is based upon the alteration of nucleotide A2058 by a point mutation. 

Melby TE, Despirito M, Demasi RA, HeUek G, Thommes JA, Greenberg ML, Graham N (2007b) Association between specific enfuvirtide resistance mutations and CD4 cell response during enfuvirtide-based therapy. Aids 21 2537-2539... 

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