C. elegans ORFeome version 3.1: Increasing the coverage of ORFeome resources with improved gene predictions

Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Genome Research (Impact Factor: 14.63). 11/2004; 14(10B):2064-9. DOI: 10.1101/gr.2496804
Source: PubMed


The first version of the Caenorhabditis elegans ORFeome cloning project, based on release WS9 of Wormbase (August 1999), provided experimental verifications for approximately 55% of predicted protein-encoding open reading frames (ORFs). The remaining 45% of predicted ORFs could not be cloned, possibly as a result of mispredicted gene boundaries. Since the release of WS9, gene predictions have improved continuously. To test the accuracy of evolving predictions, we attempted to PCR-amplify from a highly representative worm cDNA library and Gateway-clone approximately 4200 ORFs missed earlier and for which new predictions are available in WS100 (May 2003). In this set we successfully cloned 63% of ORFs with supporting experimental data ("touched" ORFs), and 42% of ORFs with no supporting experimental evidence ("untouched" ORFs). Approximately 2000 full-length ORFs were cloned in-frame, 13% of which were corrected in their exon/intron structure relative to WS100 predictions. In total, approximately 12,500 C. elegans ORFs are now available as Gateway Entry clones for various reverse proteomics (ORFeome v3.1). This work illustrates why the cloning of a complete C. elegans ORFeome, and likely the ORFeomes of other multicellular organisms, needs to be an iterative process that requires multiple rounds of experimental validation together with gradually improving gene predictions.

Download full-text


Available from: Stuart Milstein,
  • Source
    • "All bait plasmids were transformed into Mav203 (Li et al, 2004) and Y8930 (Yu et al, 2008) yeast strains. C. elegans AD-ORFeome v1.1 (Reboul et al, 2003) and v3.1 (Lamesch et al, 2004), which contain about 13 000 AD-ORFs in total, were transformed into MaV103 (Li et al, 2004) and Y8800 (same genotype as Y8930 except opposite mating type) in this study. ORFeome Y2H screening was performed using a mating method, as previously described (Rual et al, 2005), with different yeast strain combinations, that is, Y8930 Â Y8800, MaV203 Â MaV103, Y8930 Â MaV103, and MaV203 Â Y8800. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.
    Molecular Systems Biology 04/2013; 9(1):652. DOI:10.1038/msb.2013.9 · 10.87 Impact Factor
  • Source
    • "Beyond the improved gene expression and delivery of multigene expressing vectors, the most powerful aspect of RecWay assembly is that it uses widely available modular entry vector components. There have been numerous reports using Gateway cloning to assemble modular ORFeome cDNA libraries in entry vectors from various organisms using the BP Clonase reaction (40–44). These libraries can then be used to produce cDNA expression libraries en masse using the LR Clonase reaction, which can then be used to perform forward genetic screens. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Studying complex biological processes such as cancer development, stem cell induction and transdifferentiation requires the modulation of multiple genes or pathways at one time in a single cell. Herein, we describe straightforward methods for rapid and efficient assembly of bacterial marker free multigene cassettes containing up to six complementary DNAs/short hairpin RNAs. We have termed this method RecWay assembly, as it makes use of both Cre recombinase and the commercially available Gateway cloning system. Further, because RecWay assembly uses truly modular components, it allows for the generation of randomly assembled multigene vector libraries. These multigene vectors are integratable, and later excisable, using the highly efficient piggyBac (PB) DNA transposon system. Moreover, we have dramatically improved the expression of stably integrated multigene vectors by incorporation of insulator elements to prevent promoter interference seen with multigene vectors. We demonstrate that insulated multigene PB transposons can stably integrate and faithfully express up to five fluorescent proteins and the puromycin-thymidine kinase resistance gene in vitro, with up to 70-fold higher gene expression compared with analogous uninsulated vectors. RecWay assembly of multigene transposon vectors allows for widely applicable modelling of highly complex biological processes and can be easily performed by other research laboratories.
    Nucleic Acids Research 02/2013; 41(8). DOI:10.1093/nar/gkt115 · 9.11 Impact Factor
  • Source
    • "Finally, we create an ORFeome-based molecular toolset of mutant rab ORFs to be used for rab gene function studies in C. elegans. The C. elegans ORFeome resource was created in 2003 [11] and is a partially-verified, Gateway-compatible library of ORFs representing more than 63% of the proteome [12]. Our molecular toolset includes 44 full-length, sequence-verified, dominant-negative (DN) and constitutive-active (CA) mutant rab ORFs. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).
    PLoS ONE 11/2012; 7(11):e49387. DOI:10.1371/journal.pone.0049387 · 3.23 Impact Factor
Show more