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Genetic aspects of targeted insertion mutagenesis in yeasts

Genetic aspects of targeted insertion mutagenesis in yeasts,10.1016/j.femsre.2003.10.002,Fems Microbiology Reviews,U Klinner,B Schäfer

Genetic aspects of targeted insertion mutagenesis in yeasts   (Citations: 8)
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Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to “non-conventional” yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.
Journal: Fems Microbiology Reviews - FEMS MICROBIOL REV , vol. 28, no. 2, pp. 201-223, 2004
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    • ...In contrast to S. cerevisiae, the predominant pathway for DNA integration in most other yeast species is NHEJ, especially when short homologous sequences are used for the targeting (Klinner and Schafer 2004; Kegel et al. 2006; Maassen et al. 2008)...
    • ...using as short as 30–45 bp Xanks (Manivasakam et al. 1995), whereas as a general rule most other yeast species require much longer homologous tracts varying from 100 bp to several kb [reviewed in (Klinner and Schafer 2004)]...
    • ...For this type of targeting the so called “ends-in” vectors are most commonly used (two-step gene disruption) (Klinner and Schafer 2004), which are derived from circular integrating plasmids that are linearized to promote more eYcient integration (Fig. 1b)...
    • ...To enhance HR and targeting eYciency we used long homologous regions of TLC1 sequences Xanking the KanMX cassette (»1 kb) (Klinner and Schafer 2004)...
    • ...In most other yeast species ends-in constructs are more eYcient as gene targeting tools than ends-out constructs (Klinner and Schafer 2004)...
    • ...The two pathways of homologous and ectopic integration may be competing for the same incoming vector DNA, and the mechanism being the most successful may depend on various diVerent internal and external factors (Klinner & Schafer 2004)...
    • ...NHEJ is the predominant DNA integration pathway in most non-Saccharomyces species analyzed, like the pre-WGD species K. lactis and P. pastoris (Klinner and Schafer 2004; Kegel et al. 2006; Maassen et al. 2008)...

    Eimantas Astromskaset al. Ends-in vs. ends-out targeted insertion mutagenesis in Saccharomyces c...

    • ...In S. cerevisiae insertion is easily directed to specific loci by incorporating the cognate homologous sequence into the vector or construction of interest in order to promote homologous recombination between chromosomal and vector sequences (Klinner and Schafer 2004) The frequency of homologous recombination would depend on several factor, including the length of the homologous regions, topology of the transforming DNA (linear or ...

    Eduardo Cebolleroet al. Transgenic wine yeast technology comes of age: is it time for transgen...

    • ...This fact hampers our use of the far-easier PCR-based molecular construction reported in several yeast strains, where constructs with homologous regions of only about 50 bp are applicable (7, 11, 13)...

    Takaaki Satoet al. Improved and Versatile Transformation System Allowing Multiple Genetic...

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