A family of destabilized cyan fluorescent proteins as transcriptional reporters in S. cerevisiae.
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.
Hackett
Elizabeth A
EA
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
Esch
R Keith
RK
Maleri
Seth
S
Errede
Beverly
B
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
C485182:Cyan Fluorescent Protein; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005686:Galactokinase