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Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry
Author(s) -
Mateus Carolina,
Avery Simon V.
Publication year - 2000
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/1097-0061(200010)16:14<1313::aid-yea626>3.0.co;2-o
Subject(s) - biology , nocodazole , green fluorescent protein , saccharomyces cerevisiae , microbiology and biotechnology , cell cycle , gene , flow cytometry , reporter gene , yeast , gene expression , genetics , cell , cytoskeleton
Green fluorescent protein (GFP) has many advantages as a reporter molecule, but its stability makes it unsuitable for monitoring dynamic changes in gene expression, among other applications. Destabilized GFPs have been developed for bacterial and mammalian systems to counter this problem. Here, we extend such advances to the yeast model. We fused the PEST‐rich 178 carboxyl‐terminal residues of the G 1 cyclin Cln2 to the C terminus of yEGFP3 (a yeast‐ and FACS‐optimized GFP variant), creating yEGFP3‐Cln2 PEST . We tested the hybrid protein after integrating modules harbouring the yEGFP3 or yEGFP3–CLN2 PEST ORFs into the Saccharomyces cerevisiae genome. yEGFP3– Cln2 PEST had a markedly shorter half‐life ( t ½ ) than yEGFP3; inhibition of protein synthesis with cycloheximide lead to a rapid decline in GFP content and fluorescence ( t ½ ∼30 min) in cells expressing yEGFP3–Cln2 PEST , whereas these parameters were quite stable in yEGFP3‐ expressing cells ( t ½ ∼7 h). We placed yEGFP3–CLN2 PEST under the control of the CUP1 promoter, which is induced only transiently by copper. This transience was readily discernible with yEGFP3–Cln2 PEST , whereas yEGFP3 reported only on CUP1 switch‐on, albeit more slowly than yEGFP3–Cln2 PEST . Cell cycle‐regulated transcriptional activation/inactivation of the CLN2 promoter was also discernible with yEGFP3– Cln2 PEST , using cultures that were previously synchronized with nocodazole. In comparison to CLN2 , expression from the ACT1 promoter was stable after release from nocodazole. We also applied a novel flow‐cytometric technique for cell cycle analysis with asynchronous cultures. The marked periodicities of CLN2 and CLB2 (mitotic cyclin) transcription were readily evident from cellular yEGFP3–Cln2 PEST levels with this non‐perturbing approach. The results represent the first reported successful destabilization of a yeast–GFP. This new construct expands the range of GFP applications open to yeast workers. Copyright © 2000 John Wiley & Sons, Ltd.