Universal Clone Resources for Drosophila Proteomics

描述(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识别和组装每个生物的完整蛋白质集。在伯克利果蝇基因组计划(BDGP)中，我们正在积极地生成一个全面的cDNA资源--果蝇基因集合(DGC)，其中将包含约14,000个注释蛋白质编码基因中的每个基因的至少一个cDNA。我们的cdna收集极大地加快了建立所有果蝇基因的全面转录图谱的进程，并被用于确定胚胎中的基因表达模式。目前，9,020个cDNA克隆代表三分之二的当前注释的基因组成了DGC Gold Collection，这是一组没有无义和错义突变的全长cDNA。我们已经使用略多于一半的Gold cDNA克隆构建了两个通用供体克隆，一个带有天然终止密码子，另一个没有天然终止密码子。我们建议通过DGC Gold Set中现有的和预期的额外克隆来扩展这些集合，这些集合现在是并将继续成为高质量黑腹果蝇开放阅读框架(ORF)的主要来源，这些ORF很容易被转移到许多不同的表达载体中。此外，我们计划使用供体克隆来制作三组表达克隆。其中两个是为组织培养细胞设计的，一个是用于蛋白质纯化研究的表位标记的，另一个是未标记的用于功能获得筛选，以补充正在进行的RNAi筛选。第三个目的是生产带有表位标记蛋白的转基因果蝇，用于体内研究内源复合体的表达、定位和纯化。所有研究人员都可以不受限制地获得这些克隆。 相关性：我们的目标是更详细地了解果蝇基因组编码的一整套蛋白质，并为研究界提供功能基因组学和蛋白质组学的资源。这些克隆资源将被用来加深我们对高等真核生物中保守基因、途径和细胞分化的理解，对改善人类健康具有广泛的意义。

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.