title: Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae.
abstract: A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multi-cloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.
authors.affiliation: Department of Biological Sciences, SCEN601, University of Arkansas, Fayetteville, AR 72701, USA. dmcnabb@uark.edu
authors.forename: David S
authors.initials: DS
authors.lastname: McNabb
authors.forename: Robin
authors.initials: R
authors.lastname: Reed
authors.forename: Robert A
authors.initials: RA
authors.lastname: Marciniak
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D048888:Fireflies / Q000201:enzymology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism; D049410:Luciferases, Renilla / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D048790:Renilla / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D020798:Two-Hybrid System Techniques
chemical_list: C014059:CYC1 protein, S cerevisiae; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly