title: Gene expression analysis using strains constructed by NHEJ-mediated one-step promoter cloning in the yeast Kluyveromyces marxianus.
abstract: Gene expression analysis provides valuable information to evaluate cellular state. Unlike quantitative mRNA analysis techniques like reverse-transcription PCR and microarray, expression analysis using a reporter gene has not been commonly used for multiple-gene analysis, probably due to the difficulty in preparing multiple reporter-gene constructs. To circumvent this problem, we developed a novel one-step reporter-gene construction system mediated by non-homologous end joining (NHEJ) in the yeast Kluyveromyces marxianus. As a selectable reporter gene, the ScURA3 selection marker was fused in frame with a red fluorescent gene yEmRFP (ScURA3:yEmRFP). The N-terminally truncated ScURA3:yEmRFP fragment was prepared by PCR. Promoter sequences were also prepared by PCR using primers containing the sequence of the deleted ScURA3 N-terminus to attach at their 3(') ends. The two DNA fragments were used for the transformation of a ura3(-) strain of K. marxianus, in which two DNA fragments are randomly joined and integrated into the chromosome through NHEJ. Only the correctly aligned fragments produced transformants on uracil-deficient medium and expressed red fluorescence under the control of the introduced promoters. A total of 36 gene promoters involved in glycolysis and other pathways were analyzed. Fluorescence measurements of these strains allowed real-time gene expression analysis in different culture conditions.
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Suzuki
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujii
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan Research Center for Thermotolerant Microbial Resources, Yamaguchi University, Yamaguchi 753-8315, Japan Yamaguchi University Biomedical Engineering Center, Ube 755-8611, Japan hoshida@yamaguchi-u.ac.jp.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.affiliation: Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan Research Center for Thermotolerant Microbial Resources, Yamaguchi University, Yamaguchi 753-8315, Japan Yamaguchi University Biomedical Engineering Center, Ube 755-8611, Japan.
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D019677:Artificial Gene Fusion; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D005470:Fluorometry / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors; D007716:Kluyveromyces / Q000235:genetics*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014170:Transformation, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D003470:Culture Media; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: Kluyveromyces marxianus; glycolysis; metabolism; non-homologous end joining (NHEJ); reporter analysis; xylose