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Transplantation of the GAL regulon into G-protein signaling circuitry in yeast

Author:
Ryo, Shintaro, Ishii, Jun, Iguchi, Yusuke, Fukuda, Nobuo, Kondo, Akihiko
Source:
Analytical biochemistry 2012 v.424 no.1 pp. 27-31
ISSN:
0003-2697
Subject:
G-protein coupled receptors, G-proteins, agonists, flow cytometry, galactose, gene induction, genetically engineered microorganisms, green fluorescent protein, ligands, regulator genes, regulon, reporter genes, screening, signal transduction, structural genes, transcription (genetics), yeasts
Abstract:
Here we present a successful transplantation of the GAL genetic regulatory circuitry into the G-protein signaling pathway in yeast. The GAL regulon represents a strictly regulated transcriptional mechanism that we have transplanted into yeast to create a highly robust induction system to assist the detection of on–off switching in G-protein signaling. In our system, we engineered yeast to drive the positive GAL regulatory gene in response to agonist-promoted G-protein signaling and to induce transcription of a green fluorescent protein (GFP) reporter gene under the control of the GAL structural gene promoter. Consequently, in response to agonist stimulation of G-protein-coupled receptors (GPCRs), the engineered yeast achieved more than a 150-fold increase in reporter intensity in up to 98% of cells, as determined by flow cytometric sorting. Surprisingly, agonist-stimulated induction of the GFP reporter gene was higher than that by galactose. Our approach to boost reporter gene induction could be applicable in establishing more efficient yeast-based flow cytometric screening systems for agonistic ligands for heterogeneous GPCRs.
Agid:
830719