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• W2280852983 endingPage "fov059" @default.
• W2280852983 startingPage "fov059" @default.
• W2280852983 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." @default.
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• W2280852983 date "2015-06-30" @default.
• W2280852983 modified "2023-10-16" @default.
• W2280852983 title "Gene expression analysis using strains constructed by NHEJ-mediated one-step promoter cloning in the yeast<i>Kluyveromyces marxianus</i>" @default.
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• W2280852983 doi "https://doi.org/10.1093/femsyr/fov059" @default.
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