title: Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.
abstract: The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Nobutada
authors.initials: N
authors.lastname: Murakami
authors.forename: Ayako
authors.initials: A
authors.lastname: Suzuki
authors.forename: Ryoko
authors.initials: R
authors.lastname: Tamura
authors.forename: Jun
authors.initials: J
authors.lastname: Asakawa
authors.forename: Babiker M A
authors.initials: BM
authors.lastname: Abdel-Banat
authors.forename: Sanom
authors.initials: S
authors.lastname: Nonklang
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nakamura
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D007716:Kluyveromyces / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012641:Selection, Genetic; D014170:Transformation, Genetic
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae
keywords: Kluyveromyces marxianus; URA3; functional marker selection; non-homologous end joining; plasmid; recombinant DNA