title: Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae.
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.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Fukunaga
authors.forename: Kamonchai
authors.initials: K
authors.lastname: Cha-Aim
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hirakawa
authors.forename: Ryota
authors.initials: R
authors.lastname: Sakai
authors.forename: Takao
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nakamura
authors.forename: Sanom
authors.initials: S
authors.lastname: Nonklang
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D004274:DNA, Recombinant / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci; D059765:Homologous Recombination; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D004274:DNA, Recombinant; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae