Gene expression analysis using strains constructed by NHEJ-mediated one-step promoter cloning in the yeast Kluyveromyces marxianus

• Published in: FEMS yeast research 2015-09, Vol.15 (6), p.fov059
• Main Authors: Suzuki, Ayako, Fujii, Hiroshi, Hoshida, Hisashi, Akada, Rinji
• Format: Article
• Language: English
• Subjects: Artificial Gene Fusion; Cell culture; Cloning, Molecular - methods; Culture Media - chemistry; Deoxyribonucleic acid; DNA; DNA microarrays; Fluorometry - methods; Gene expression; Gene Expression Profiling - methods; Gene Expression Regulation, Fungal; Genes, Reporter; Genetic Vectors; Glycolysis; Kluyveromyces - genetics; Kluyveromyces marxianus; Luminescent Proteins - analysis; Luminescent Proteins - genetics; N-Terminus; Non-homologous end joining; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Promoters; Recombination, Genetic; Red Fluorescent Protein; Reporter gene; Selection, Genetic; Transformation, Genetic; Uracil
• ISSN: 1567-1356; 1567-1364; 1567-1364
• DOI: 10.1093/femsyr/fov059

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. One-step promoter cloning method was developed in the yeast Kluyveromyces marxianus to analyze multiple promoter activities and expression analysis of 36 promoters were performed in a real-time manner.