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      Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ

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          Abstract

          A novel mutagenesis technique using error-prone DNA polymerase δ ( polδ), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδ MKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδ MKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis.

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          Amino acid substitution matrices from protein blocks.

          Methods for alignment of protein sequences typically measure similarity by using a substitution matrix with scores for all possible exchanges of one amino acid with another. The most widely used matrices are based on the Dayhoff model of evolutionary rates. Using a different approach, we have derived substitution matrices from about 2000 blocks of aligned sequence segments characterizing more than 500 groups of related proteins. This led to marked improvements in alignments and in searches using queries from each of the groups.
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            A genome-wide view of the spectrum of spontaneous mutations in yeast.

            The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.
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              New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.

              We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.
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                Author and article information

                Journal
                Int J Evol Biol
                Int J Evol Biol
                IJEB
                International Journal of Evolutionary Biology
                Hindawi Publishing Corporation
                2090-8032
                2090-052X
                2012
                22 May 2012
                : 2012
                : 860797
                Affiliations
                1Genome Research Center, NODAI Research Institute, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
                2Neo-Morgan Laboratory Inc., 907 Nogawa, Miyamae-ku, Kawasaki, Kanagawa 216-0001, Japan
                3Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
                Author notes
                *Hirofumi Yoshikawa: hiyoshik@ 123456nodai.ac.jp

                Academic Editor: Hiromi Nishida

                Article
                10.1155/2012/860797
                3364565
                22675654
                30771c30-035e-4a90-a88a-620b557740d8
                Copyright © 2012 Yuh Shiwa et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 9 February 2012
                : 20 March 2012
                Categories
                Research Article

                Evolutionary Biology
                Evolutionary Biology

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