| RRC ID |
22731
|
| 著者 |
Ishii J, Tanaka T, Matsumura S, Tatematsu K, Kuroda S, Ogino C, Fukuda H, Kondo A.
|
| タイトル |
Yeast-based fluorescence reporter assay of G protein-coupled receptor signalling for flow cytometric screening: FAR1-disruption recovers loss of episomal plasmid caused by signalling in yeast.
|
| ジャーナル |
J Biochem
|
| Abstract |
Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.
|
| 巻・号 |
143(5)
|
| ページ |
667-74
|
| 公開日 |
2008-5-1
|
| DOI |
10.1093/jb/mvn018
|
| PII |
mvn018
|
| PMID |
18281298
|
| MeSH |
Cell Cycle
Cell Cycle Proteins / genetics*
Cyclin-Dependent Kinase Inhibitor Proteins
Flow Cytometry*
Fluorescent Dyes / analysis
GTPase-Activating Proteins / genetics
Gene Deletion
Genes, Reporter*
Green Fluorescent Proteins / analysis
Green Fluorescent Proteins / genetics
Ligands
Plasmids / genetics
Receptors, G-Protein-Coupled / metabolism*
Repressor Proteins / genetics*
Saccharomyces cerevisiae / genetics*
Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins / genetics*
Signal Transduction*
|
| IF |
2.476
|
| 引用数 |
26
|
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| 酵母 |
BY4741 |
