Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Authors

  • Tomoaki Fukunaga,

    1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
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  • Kamonchai Cha-aim,

    1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
    Current affiliation:
    1. Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Wattana, Bangkok, Thailand
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  • Yuki Hirakawa,

    1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
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  • Ryota Sakai,

    1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
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  • Takao Kitagawa,

    1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
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  • Mikiko Nakamura,

    1. Innovation Centre, Yamaguchi University, Tokiwadai, Ube, Japan
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  • Sanom Nonklang,

    1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
    Current affiliation:
    1. Department of Biological Science, Faculty of Science, Ubonratchathani University, Warinchumrap, Ubonratchathani, Thailand
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  • Hisashi Hoshida,

    Corresponding author
    • Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
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  • Rinji Akada

    1. Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan
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Correspondence to: H. Hoshida, Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan.

E-mail: hoshida@yamaguchi-u.ac.jp

Abstract

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre-existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C-rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.

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