Fox Jennifer E, Burow Matthew E, McLachlan John A, Miller Charles A
Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon 97403, USA.
Nat Protoc. 2008;3(4):637-45. doi: 10.1038/nprot.2008.33.
This is a general protocol for the identification of natural and xenobiotic ligands of metazoan nuclear receptors (NRs) expressed in yeast. Yeast engineered to express an NR and a response element-driven reporter gene provide a system to detect and quantify ligand-dependent transcriptional activity. Such assays allow researchers to measure different types of ligands and determine dose-dependent activation of NRs. This methodology can also be used to examine the components of signal transduction pathways when conducted with mutant or engineered yeast strains expressing additional proteins or having alternate DNA response elements. This assay typically takes 2-3 d to complete, but most of this time entails cell growth rather than 'hands on' time.
这是一份用于鉴定在酵母中表达的后生动物核受体(NRs)的天然和异源生物配体的通用方案。经过工程改造以表达NR和响应元件驱动的报告基因的酵母提供了一个检测和定量配体依赖性转录活性的系统。此类测定使研究人员能够测量不同类型的配体,并确定NRs的剂量依赖性激活。当与表达额外蛋白质或具有替代DNA响应元件的突变或工程酵母菌株一起进行时,该方法还可用于检查信号转导途径的组成部分。该测定通常需要2至3天完成,但大部分时间用于细胞生长而非实际操作时间。