Mutations/mutagenesis

Abu-Shakra, A. Zeiger, E. (1990) Effects of Salmonella genotypes and testing protocols on H2O2-induced mutation. Mutagenesis, 5, 469-473... 

Dolle, M. E., Martus, H. J., Gossen, J. A., Boerrigter, M. E., and Vijg, J. (1996). Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations. Mutagenesis 11, 111-118. 

Fig. 9. Mutagenesis by a synthetic oligonucleotide of a cloned sequence available in single-stranded form (a) single-stranded M13 template containing uracil (U) residues (b) double-stranded product, uracil residues are not mutagenic (c) strong selection for M13 phages containing mutation of interest (23).

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

The specific role of each amino acid residue for the function of the protein can be tested by making specific mutations of the residue in question and examining the properties of the mutant protein. By combining in this way functional studies in solution, site-directed mutagenesis by recombinant DNA techniques, and three-dimensional structure determination, we are now in a position to gain fresh insights into the way protein molecules work. 

The disruption of C1C-2 in mice leads to male infertility, blindness, and leukodystrophy, and was attributed to defective extracellular ion homeostasis in narrow clefts. C1C-2 yields currents that slowly activate upon hyperpolarization. It is also activated by cell swelling and by extracellular acidification. Structural determinants that are essential for these types of activation were identified by mutagenesis. There is a report that C1C-2 might be mutated in human epilepsy, but this has not been confirmed in fiuther studies. 

The newest molecular addition to Cl- channels are bestrophins [5]. There are four different bestrophin isoforms in humans. Mutations in Bestl cause Best macular dystrophy, hence their name. All four isoforms induce chloride currents when expressed heterologously. Although they show a dependence on intracellular calcium, their biophysical properties differ from Ca-activated chloride currents typically observed in native cells. Mutagenesis experiments changed the activation of currents and induced slight changes in ion selectivity, lending support to the hypothesis that bestrophins themselves are Cl- channels. 

In molecular pharmacology research an indirect proof of a structural model is possible by functional examinations, e.g., by molecular biological experiments. Well-selected site directed mutagenesis and their functional characterization allows confirmation or rejection of a molecular protein model. The process is organized as an iterative procedure, where the biological answer of suggested mutations is used to refine the model. The iteration continues until the model... 

Systematic experimental and theoretical studies of this kind are helpful in performing in vitro evolution of enantioselectivity. Nevertheless, several questions are not fully answered. Are remote mutations more important than those close to the active site, or is the opposite true Is it more effective to allow randomization all over the enzyme rather than focusing on the region around the active site (or vice versa) To be sure, when applying epPCR or any other mutagenesis method that more or less... 

The data in Figure 2.12 are the results of initial mutagenesis experiments, which does not yet constitute directed evolution. An evolutionary process was subsequently induced by combining the mutations of two improved mutants of the first round [46]. Thereby new mutants were obtained, which show an increase of activity relative to the WT by more than 2 orders of magnitude. Although enantioselectivity was not the... 

Following several cycles of mutagenesis using the E. coli XLl-Red mutator strain and transformation of the plasmid library into E. coli, a total of about 150 000 bacterial colonies were assayed for activity using a colorimetric prescreen [100]. The best mutant Asn336Ser showed a 47-fold increase in activity and a 5.8-fold enhancement... 

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

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