Universal Clone Resources for Drosophila Proteomics

University Of Calif-Lawrenc Berkeley Lab, Berkeley CA

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Abstract

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.