Universal Clone Resources for Drosophila Proteomics

描述（由申请人提供）：随着来自各种生物体的大量基因组序列，基因组学的下一个主要挑战是鉴定和组装每个生物体的完整蛋白质集。在伯克利果蝇基因组计划（BDGP）中，我们正在积极生成一个全面的cDNA资源，果蝇基因收集（DGC）将包含约14,000个注释蛋白质编码基因中的每个基因至少一个cDNA。我们的cDNA收集极大地加速了所有果蝇基因的全面转录图谱的进展，并被用于确定胚胎中的基因表达模式。目前，9020个cDNA克隆代表了三分之二的当前注释基因，组成了DGC金集合，这是一组全长cDNA，没有无义和错义突变。我们用一半多一点的Gold cDNA克隆构建了两个通用供体克隆集合，一个带有和一个不带有天然终止密码子。我们建议用DGC Gold系列中现有的和预期的其他克隆来扩展这些集合，这些集合将继续成为高质量果蝇开放阅读框（orf）的主要来源，这些orf很容易转移到许多不同的表达载体中。此外，我们计划利用供体克隆制作三组表达克隆。其中两个设计用于组织培养细胞，一个表位标记用于蛋白质纯化研究，另一个未标记用于功能获得筛选，以补充正在进行的RNAi筛选。第三个目的是培育带有表位标记蛋白的转基因果蝇，用于体内研究内源性复合物的表达、定位和纯化。所有研究人员都可以无限制地使用克隆技术。

DESCRIPTION (provided by applicant): With the vast amount of genomic sequence from a wide variety of organisms, the next major challenge in genomics is to identify and assemble complete protein sets for each. At the Berkeley Drosophila Genome Project (BDGP) we are actively generating a comprehensive cDNA resource, the Drosophila Gene Collection (DGC) that will contain at least one cDNA for each of the ~14,000 annotated protein-coding genes. Our cDNA collection has greatly accelerated progress towards a comprehensive transcript map of all Drosophila genes and is being used to determine gene expression patterns in the embryo. Presently, 9,020 cDNA clones representing two-thirds of the currently annotated genes comprise the DGC Gold Collection, which is a set of full-length cDNAs that are free of nonsense and missense mutations. We have used a little over half of the Gold cDNA clones to construct two collections of universal donor clones, one with and one without the native stop codon. We propose to extend these collections with the existing and anticipated additional clones in the DGC Gold set these collections are and will continue to be the primary source of high quality Drosophila melanogaster open reading frames (ORFs) that are easily transferred into a number of different expression vectors. Further, we plan to use the donor clones to make three sets of expression clones. Two are designed for use in tissue culture cells, one epitope-tagged for protein purification studies and the other untagged for gain-of-function screens to complement ongoing RNAi screens. The third is designed to produce transgenic flies with epitope-tagged proteins for in vivo studies of expression, localization and purification of endogenous complexes. The clones are available without restrictions to all researchers. RELEVANCE: Our goals are to obtain a more detailed understanding of the complete set of proteins that are encoded by the Drosophila genome and to provide resources for functional genomics and proteomics to the research community. These clone resources will be used to further our understanding of conserved genes, pathways and cellular differentiation in higher eukaryotes with broad implications for improvements in human health.