RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.
RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.
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DOI：
10.1016/bs.mie.2016.02.003
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发表时间：
2016
影响因子：
--
通讯作者：
Grayhack EJ
中科院分区：
文献类型：
--
作者：
Brule CE;Dean KM;Grayhack EJ
鉴定和分析调控基因表达的序列是至关重要的，因为受调控的基因表达是生物学的基础。RNA-ID是发现和研究酿酒酵母中调控序列的有效且灵敏的方法，其使用基于荧光的测定来检测单个细胞中相对于RFP对照的GFP。推定的调控序列可以插入超折叠GFP融合蛋白的框内或上游，其表达与RFP的表达一样由双向GAL 1，10启动子驱动。在这一章中，我们描述的方法来确定和研究在RNA-ID系统中的顺式调控序列，解释的特点和变化的RNA-ID报告，以及该系统的一些应用。我们详细描述了分析单个调控序列的方法，从构建单个GFP变体到通过流式细胞术测定变体，以及同时筛选不同菌株文库所需的修改。我们还描述了随后的分析调控序列。
The identification and analysis of sequences that regulate gene expression is critical because regulated gene expression underlies biology. RNA-ID is an efficient and sensitive method to discover and investigate regulatory sequences in the yeast Saccharomyces cerevisiae, using fluorescence-based assays to detect GFP relative to an RFP control in individual cells. Putative regulatory sequences can be inserted either in-frame or upstream of a superfolder GFP fusion protein whose expression, like that of RFP, is driven by the bidirectional GAL1,10 promoter. In this chapter, we describe the methodology to identify and study cis-regulatory sequences in the RNA-ID system, explaining features and variations of the RNA-ID reporter, as well as some applications of this system. We describe in detail the methods to analyze a single regulatory sequence, from construction of a single GFP variant to assay of variants by flow cytometry, as well as modifications required to screen libraries of different strains simultaneously. We also describe subsequent analyses of regulatory sequences.
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