title: A family of destabilized cyan fluorescent proteins as transcriptional reporters in S. cerevisiae.
abstract: The 'programmable' features of the N-end rule degradation pathway and a ubiquitin fusion strategy were exploited to create a family of destabilized cyan fluorescent proteins (CFP) to be used as transcriptional reporters. The N-degron CFP reporters characterized in this report have half-lives of approximately 75, 50 and 5 min, but further modification of the N-degron signal sequences could readily generate additional variants within this range. These destabilized CFP reporters have been engineered into convenient plasmid constructs with features to enable their expression from upstream activating sequences of choice and to facilitate their targeted integration to the URA3-TIM9 intergenic region of chromosome V. The advantages and limitations of these reporters as temporal indicators of gene expression in living cells are illustrated by their application as reporters of galactose- and pheromone-induced transcription. The plasmid design we describe and the range of different stabilities that are theoretically feasible with this strategy make the N-degron CFP reporters easily adapted to a variety of applications.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Hackett
authors.forename: R Keith
authors.initials: RK
authors.lastname: Esch
authors.forename: Seth
authors.initials: S
authors.lastname: Maleri
authors.forename: Beverly
authors.initials: B
authors.lastname: Errede
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D015825:Chromosomes, Fungal / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005686:Galactokinase / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter / Q000235:genetics* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics* / Q000502:physiology
chemical_list: C485182:Cyan Fluorescent Protein; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005686:Galactokinase