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

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.

Utilizing Modular Biobanking Software in Different Types of Biobanking Activities.

Biobanking defines all activities linked to bioresource management-whether of human, animal, microbial, or environmental origin-which means that any biobank information management system should take into account the multistep life cycle of the samples: from acquisition, through preparation, storage, to distribution to the end users (medical or research teams). Different types of biobanks can use diverse approaches, making it difficult to find software that can handle all types of scenarios. Modul-Bio has developed MBioLIMS BioBanking®, a software dedicated to biobanking, as a modular solution so that our various clients can access the functionalities and scale in a system to match their needs. These projects range from biobanks setup and managed by academic institutions, hospitals, and private companies to small and large clinical trials across different countries, as well as to whole campus or organization solutions for multiple biorepositories. Each solution differs in size, requirements, and number of users, from small biobanks with a few members of staff accessing the software to large operations with multiple sites that can collect and ship samples to a centralized site. This article explores different projects that use Modul-Bio's software in a myriad of ways to manage the complete life cycle of biospecimens and associated data.

BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3.

Programmed destruction of regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism for controlling signalling pathways. Cullins are proteins that function as scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-Cul1-F-box) complex. The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to Cul1, and a member of the F-box family of proteins. F-box proteins bind Skp1 through the F-box motif, and substrates by means of carboxy-terminal protein interaction domains. Similarly, Cul2 and Cul5 interact with BC-box-containing specificity factors through the Skp1-like protein elongin C. Cul3 is required for embryonic development in mammals and Caenorhabditis elegans but its specificity module is unknown. Here we report the identification of a large family of BTB-domain proteins as substrate-specific adaptors for C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 and its genetic target MEI-1 (refs 12, 13) indicate that BTB proteins merge the functional properties of Skp1 and F-box proteins into a single polypeptide.

Combined functional genomic maps of the C. elegans DNA damage response.

Many human cancers originate from defects in the DNA damage response (DDR). Although much is known about this process, it is likely that additional DDR genes remain to be discovered. To identify such genes, we used a strategy that combines protein-protein interaction mapping and large-scale phenotypic analysis in Caenorhabditis elegans. Together, these approaches identified 12 worm DDR orthologs and 11 novel DDR genes. One of these is the putative ortholog of hBCL3, a gene frequently altered in chronic lymphocytic leukemia. Thus, the combination of functional genomic mapping approaches in model organisms may facilitate the identification and characterization of genes involved in cancer and, perhaps, other human diseases.

EDGEdb: a transcription factor-DNA interaction database for the analysis of C. elegans differential gene expression.

BACKGROUND: Transcription regulatory networks are composed of protein-DNA interactions between transcription factors and their target genes. A long-term goal in genome biology is to map protein-DNA interaction networks of all regulatory regions in a genome of interest. Both transcription factor -and gene-centered methods can be used to systematically identify such interactions. We use high-throughput yeast one-hybrid assays as a gene-centered method to identify protein-DNA interactions between regulatory sequences (e.g. gene promoters) and transcription factors in the nematode Caenorhabditis elegans. We have already mapped several hundred protein-DNA interactions and analyzed the transcriptional consequences of some by examining differential gene expression of targets in the presence or absence of an upstream regulator. The rapidly increasing amount of protein-DNA interaction data at a genome scale requires a database that facilitates efficient data storage, retrieval and integration. DESCRIPTION: Here, we report the implementation of a C. elegans differential gene expression database (EDGEdb). This database enables the storage and retrieval of protein-DNA interactions and other data that relate to differential gene expression. Specifically, EDGEdb contains: i) sequence information of regulatory elements, including gene promoters, ii) sequence information of all 934 predicted transcription factors, their DNA binding domains, and, where available, their dimerization partners and consensus DNA binding sites, iii) protein-DNA interactions between regulatory elements and transcription factors, and iv) expression patterns conferred by regulatory elements, and how such patterns are affected by interacting transcription factors. CONCLUSION: EDGEdb provides a protein-DNA -and protein-protein interaction resource for C. elegans transcription factors and a framework for similar databases for other organisms. The database is available at http://edgedb.umassmed.edu.

Integrating interactome, phenome, and transcriptome mapping data for the C. elegans germline.

By integrating functional genomic and proteomic mapping approaches, biological hypotheses should be formulated with increasing levels of confidence. For example, yeast interactome and transcriptome data can be correlated in biologically meaningful ways. Here, we combine interactome mapping data generated for a multicellular organism with data from both large-scale phenotypic analysis ("phenome mapping") and transcriptome profiling. First, we generated a two-hybrid interactome map of the Caenorhabditis elegans germline by using 600 transcripts enriched in this tissue. We compared this map to a phenome map of the germline obtained by RNA interference (RNAi) and to a transcriptome map obtained by clustering worm genes across 553 expression profiling experiments. In this dataset, we find that essential proteins have a tendency to interact with each other, that pairs of genes encoding interacting proteins tend to exhibit similar expression profiles, and that, for approximately 24% of germline interactions, both partners show overlapping embryonic lethal or high incidence of males RNAi phenotypes and similar expression profiles. We propose that these interactions are most likely to be relevant to germline biology. Similar integration of interactome, phenome, and transcriptome data should be possible for other biological processes in the nematode and for other organisms, including humans.

Functional cdc25C dual-specificity phosphatase is required for S-phase entry in human cells.

In view of the common regulatory mechanism that induces transcription of the mitotic phosphatase cdc25C and cyclin A at the beginning of S-phase, we investigated whether cdc25C was required for S-phase transit. Here, we show that in both nontransformed human fibroblasts and HeLa cells, cdc25C protein levels significantly increased concomitant with S-phase onset and cyclin A synthesis. Activity measurements on immunoprecipitates from synchronized HeLa cells revealed a sharp rise in cdc25C-associated phosphatase activity that coincided with S-phase. Microinjection of various antisense-cdc25C molecules led to inhibition of DNA synthesis in both HeLa cells and human fibroblasts. Furthermore, transfection of small interfering RNA directed against cdc25C specifically depleted cdc25C in HeLa cells without affecting cdc25A or cdc25B levels. Cdc25C RNA interference was also accompanied by S-phase inhibition. In cells depleted of cdc25C by antisense or siRNA, normal cell cycle progression could be re-established through microinjection of wild-type cdc25C protein but not inactive C377S mutant protein. Taken together, these results show that cdc25C not only plays a role at the G2/M transition but also in the modulation of DNA replication where its function is distinct from that of cdc25A.

WorfDB: the Caenorhabditis elegans ORFeome Database.

WorfDB (Worm ORFeome DataBase; http://worfdb.dfci.harvard.edu) was created to integrate and disseminate the data from the cloning of complete set of approximately 19 000 predicted protein-encoding Open Reading Frames (ORFs) of Caenorhabditis elegans (also referred to as the 'worm ORFeome'). WorfDB serves as a central data repository enabling the scientific community to search for availability and quality of cloned ORFs. So far, ORF sequence tags (OSTs) obtained for all individual clones have allowed exon structure corrections for approximately 3400 ORFs originally predicted by the C. elegans sequencing consortium. In addition, we now have OSTs for approximately 4300 predicted genes for which no ESTs were available. The database contains this OST information along with data pertinent to the cloning process. WorfDB could serve as a model database for other metazoan ORFeome cloning projects.

Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA.

The targeting of RNA for the design of novel anti-viral compounds represents an area of vast potential. We have used NMR and computational methods to model the interaction of a series of synthetic inhibitors of the in vitro RNA binding activities of a peptide derived from the transcriptional activator protein, Tat, from human immunodeficiency virus type 1. Inhibition has been measured through the monitering of fluorescence resonance energy transfer between fluorescently labeled peptide and RNA components. A series of compounds containing a bi-aryl heterocycle as one of the three substituents on a benzylic scaffold, induce a novel, inactive TAR conformation by stacking between base-pairs at the site of a three-base bulge within TAR. The development of this series resulted in an enhancement in potency (with Ki < 100 nM in an in vitro assay) and the removal of problematic guanidinium moieties. Ligands from this series can act as inhibitors of Tat-induced transcription in a cell-free system. This study validates the drug design strategy of using a ligand to target the RNA receptor in a non-functional conformation.

ICeE an interface for C. elegans experiments.

An increasing number of laboratories are using the COPAS Biosort™ to implement high-throughput approaches to tackle diverse biological problems. While providing a powerful tool for generating quantitative data, the utility of the Biosort is currently limited by the absence of resources for data management. We describe a simple electronic database designed to allow easy storage and retrieval of Biosort data for C. elegans, but that has a wide potential application for organizing electronic files and data sets. ICeE is an Open Source application. The code and accompanying documentation are freely available via the web at http://www.ciml.univ-mrs.fr/EWBANK_jonathan/software.html.

High-throughput expression of C. elegans proteins.

Proteome-scale studies of protein three-dimensional structures should provide valuable information for both investigating basic biology and developing therapeutics. Critical for these endeavors is the expression of recombinant proteins. We selected Caenorhabditis elegans as our model organism in a structural proteomics initiative because of the high quality of its genome sequence and the availability of its ORFeome, protein-encoding open reading frames (ORFs), in a flexible recombinational cloning format. We developed a robotic pipeline for recombinant protein expression, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. Using the pipeline, we have carried out heterologous protein expression experiments on 10,167 ORFs of C. elegans. With one expression vector and one Escherichia coli strain, protein expression was observed for 4854 ORFs, and 1536 were soluble. Bioinformatics analysis of the data indicates that protein hydrophobicity is a key determining factor for an ORF to yield a soluble expression product. This protein expression effort has investigated the largest number of genes in any organism to date. The pipeline described here is applicable to high-throughput expression of recombinant proteins for other species, both prokaryotic and eukaryotic, provided that ORFeome resources become available.

Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network.

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.

A map of the interactome network of the metazoan C. elegans.

To initiate studies on how protein-protein interaction (or "interactome") networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains approximately 5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.