Performing Yeast One-Hybrid Library Screens.
Performing Yeast One-Hybrid Library Screens.
复制标题
进行酵母单杂交库筛选。
DOI：
10.1101/pdb.prot088955
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发表时间：
2016
影响因子：
--
通讯作者：
Walhout,AlberthaJM
中科院分区：
文献类型：
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作者：
FuxmanBass,JuanI;Reece-Hoyes,JohnS;Walhout,AlberthaJM
酵母单杂交（Y1H）测定用于确定哪些转录因子（TF）“猎物”分子可以结合用作“诱饵”的感兴趣的DNA片段。Y1H试验涉及将编码tf的质粒引入酵母“诱饵菌株”中，其中感兴趣的DNA片段整合在一个或多个报告基因的上游，这些报告基因的激活表明TF-DNA相互作用已经发生。这些质粒将每个TF表达为与酵母TF Gal4的激活域（AD）融合的杂交蛋白（因此称为“单杂交”）。AD片段激活报告基因表达，即使与之融合的TF通常起抑制作用。在这里，我们描述了如何对克隆到pPC86质粒中的cDNA片段库进行Y1H筛选，该质粒表达由cDNA编码的蛋白质作为AD融合。该方法假设有商业上可用的文库或使用从感兴趣的组织中提取的mRNA在内部生成的文库。我们还假设用户可以接触到一种酵母诱饵菌株，该菌株拥有两种不同的聚合体——his3（营养不良标记）和LacZ（将无色X-gal变成蓝色化合物的比色标记）上游的DNA片段。简单地说，筛选包括将AD-cDNA文库转化为酵母诱饵菌株，鉴定显示两个报告者激活的菌落，在新鲜生长的诱饵菌株中重新测试相互作用，并对cDNA插入进行测序以确定相互作用的TF。
Yeast one-hybrid (Y1H) assays are used to identify which transcription factor (TF)“prey” molecules can bind a DNA fragment of interest that is used as “bait”. Y1H assays involve introducing plasmids that encode TFs into a yeast “bait strain” in which the DNA fragment of interest is integrated upstream of one or more reporters, and activation of these reporters indicates that a TF–DNA interaction has occurred. These plasmids express each TF as a hybrid protein (hence the “one-hybrid” name) fused to the activation domain (AD) of the yeast TF Gal4. The AD moiety activates reporter expression even if the TF to which it is fused typically functions as a repressor. Here, we describe how to perform a Y1H screen of a library of cDNA fragments cloned into a pPC86 plasmid expressing the protein encoded by the cDNA as an AD fusion. The method assumes availability of either commercially available libraries or libraries generated in house using mRNA extracted from a tissue of interest. We also assume that users have access to a yeast bait strain that possesses the DNA fragment of interest integrated upstream of two different reporters—HIS3, an auxotrophic marker, and LacZ, a colorimetric marker that changes colorless X-gal into a blue compound. Briefly, the screen involves transforming the AD-cDNA library into the yeast bait strain, identifying colonies that show activation of both reporters, retesting the interaction in a freshly grown bait strain, and sequencing the cDNA insert to identify the interacting TF.