An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae
An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae
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DOI：
10.1002/yea.2932
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发表时间：
2012-12-01
期刊：
影响因子：
2.6
通讯作者：
Errede, Beverly
中科院分区：
文献类型：
--
作者：
Houser, John R.;Ford, Eintou;Errede, Beverly
时间转录程序的理想报告基因有很短的半衰期，这将它们的检测限制在其转录存在和翻译的窗口。为了满足单个活细胞转录报告分子的这一标准，我们采用泛素融合策略对N-末端规则进行可编程降解，以产生半衰期接近7min的N-降解型绿色荧光蛋白(GFP)。我们在这里使用的GFP变异体(命名为GFP*)具有良好的荧光亮度和成熟特性，这使得不稳定的报告程序非常适合于跟踪活细胞中基因表达的诱导和衰减动力学。这些属性通过它跟踪半乳糖和信息素诱导的酿酒酵母转录的能力得到了说明。我们进一步表明，使用短寿命N-degron GFP*报告基因的荧光测量准确地预测了典型信息素诱导的FUS1基因的瞬时mRNA谱。版权所有(C)2012 John Wiley&Sons，Ltd.
Ideal reporter genes for temporal transcription programmes have short half-lives that restrict their detection to the window in which their transcripts are present and translated. In an effort to meet this criterion for reporters of transcription in individual living cells, we adapted the ubiquitin fusion strategy for programmable N-end rule degradation to generate an N-degron version of green fluorescent protein (GFP) with a half-life of similar to 7?min. The GFP variant we used here (designated GFP*) has excellent fluorescence brightness and maturation properties, which make the destabilized reporter well suited for tracking the induction and attenuation kinetics of gene expression in living cells. These attributes are illustrated by its ability to track galactose- and pheromone-induced transcription in S. cerevisiae. We further show that the fluorescence measurements using the short-lived N-degron GFP* reporter gene accurately predict the transient mRNA profile of the prototypical pheromone-induced FUS1 gene. Copyright (c) 2012 John Wiley & Sons, Ltd.