C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression

Abstract

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

“…However, the mRNA of F08F1.3 (expected size: 650 base pairs) is not present in WT adult worms as demonstrated in Figure 5D showing a reverse transcription-PCR analysis on total RNA of WT worms. In accordance, reports by others also suggest that F08F1.3 may not be a functional gene: i) no Expressed Sequence Tag clones have been isolated for this gene (www.wormbase.org), ii) the ORFeome project was not able to amplify cDNA for this gene (Reboul et al, 2003) and (iii) RNAi of this gene causes no detectable phenotype (Kamath et al, 2003). Together, these observations strongly suggest that the deletion in tth-1 (gk43) specifically affects expression of tth-1.…”

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

“…Davis et al (2008) provide a similar Gateway strategy, but with different Gateway acceptor sites. If the starting sequences are in pDONR221 (Invitrogen), our destination vectors are most useful; if the starting sequences are from the promoterome (Dupuy et al 2004) or ORFeome (Reboul et al 2003) reagents, the Davis et al (2008) Gateway destination vectors may be more convenient.…”

“…Additional general information regarding useful strains and constructs: Davis et al (2008) introduce useful constructs that are compatible with promoterome (Dupuy et al 2004) and ORFeome (Reboul et al 2003) reagents; we expand the set of useful FLP-out technology reagents by introducing a variety of additional plasmids for constructing specific FLP recombinase and FLP-out target vectors of choice by both traditional methods and by Gateway cloning methods. The general strategies presented below were used to build plasmids used in this study.…”

“…Finally, to generate FLP-out targets in the form [promoter-of-interest,reporter,RNAi-inducing hairpin-of-interest], we provide a triple Gateway destination vector that simultaneously accepts a promoter of interest and, using Wormgate technology (Johnson et al 2005), generates an RNAi hairpin from an ORFeome (Reboul et al 2003) entry clone.…”

Microarrays to characterize protein interactions on a whole‐proteome scale

“…Nevertheless, various efforts are currently underway to generate nonredundant collections of genes suitable for use as the starting material for generating, among other things, whole-proteome arrays. These efforts range from the Caenorhabditis elegans ORFeome project [14] to the various public efforts to generate human genes collections, such as the Unigene set, which is a collection generated from a human fetal brain cDNA library [15], the Full-length Expression (FLEX) Gene repository under development at the Harvard Institute of Proteomics [16,17], the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) cDNA collection that is based at the Lawrence Livermore National Laboratory [18] and the associated sequence verified Mammalian Gene Collection (MGC) full-length clone collection [19,20].…”