Hackett Elizabeth A, Esch R Keith, Maleri Seth, Errede Beverly
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
Yeast. 2006 Apr 15;23(5):333-49. doi: 10.1002/yea.1358.
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.
利用N端规则降解途径的“可编程”特性和泛素融合策略，创建了一系列不稳定的青色荧光蛋白（CFP），用作转录报告基因。本报告中表征的N-降解决定子CFP报告基因的半衰期约为75、50和5分钟，但对N-降解决定子信号序列的进一步修饰可以很容易地在该范围内产生更多变体。这些不稳定的CFP报告基因已被构建到方便的质粒构建体中，其特征是能够从选择的上游激活序列表达，并便于它们靶向整合到第五条染色体的URA3-TIM9基因间区域。作为半乳糖和信息素诱导转录的报告基因，这些报告基因在活细胞中作为基因表达的时间指标的优点和局限性得到了体现。我们描述的质粒设计以及该策略在理论上可行的不同稳定性范围，使得N-降解决定子CFP报告基因很容易适用于各种应用。
