Gookin Timothy E, Bendtsen Jannick D
Department of Biology, Mueller Laboratory, The Pennsylvania State University, University Park, PA, USA.
Methods Mol Biol. 2013;1043:1-12. doi: 10.1007/978-1-62703-532-3_1.
Genomic sequencing has provided a vast resource for identifying interesting genes, but often an exact "gene-of-interest" is unknown and is only described as putatively present in a genome by an observed phenotype, or by the known presence of a conserved signaling cascade, such as that facilitated by the heterotrimeric G-protein. The low sequence similarity of G protein-coupled receptors (GPCRs) and the absence of a known ligand with an associated high-throughput screening system in plants hampers their identification by simple BLAST queries or brute force experimental assays. Combinatorial bioinformatic analysis is useful in that it can reduce a large pool of possible candidates to a number manageable by medium or even low-throughput methods. Here we describe a method for the bioinformatic identification of candidate GPCRs from whole proteomes and their subsequent in vivo analysis for G-protein coupling using a membrane based yeast two-hybrid variant (Gookin et al., Genome Biol 9:R120, 2008). Rather than present the bioinformatic process in a format requiring scripts or computer programming knowledge, we describe procedures here in a simple, biologist-friendly outline that only utilizes the basic syntax of regular expressions.
基因组测序为鉴定有趣的基因提供了丰富的资源，但通常一个确切的“感兴趣的基因”是未知的，仅通过观察到的表型，或通过已知存在的保守信号级联反应（如由异源三聚体G蛋白介导的信号级联反应）来推测其存在于基因组中。G蛋白偶联受体（GPCRs）的序列相似性较低，且植物中缺乏与之相关的高通量筛选系统的已知配体，这使得通过简单的BLAST查询或强力实验分析来鉴定它们变得困难。组合生物信息学分析很有用，因为它可以将大量可能的候选基因减少到通过中等通量甚至低通量方法可处理的数量。在这里，我们描述了一种从全蛋白质组中生物信息学鉴定候选GPCRs的方法，以及随后使用基于膜的酵母双杂交变体对其进行G蛋白偶联的体内分析（Gookin等人，《基因组生物学》9:R120，2008）。我们不是以需要脚本或计算机编程知识的格式呈现生物信息学过程，而是以一种简单、对生物学家友好的大纲形式描述程序，该大纲仅利用正则表达式的基本语法。
