Jerome Reboul et al. (2002) European Worm Meeting "The C. elegans orfeome cloning project : version 1.0"

Christopher Armstrong, Monica Martinez, Philippe Vaglio, Mike Brasch, Laurent Jacotot, Hongmei Lee, Troy Moore, Lynn Doucette-Stamm, Alban Chesneau, Marc Vidal, Jim Hartley, David E Hill, Nicolas Bertin, Philippe Lamesch, Jerome Reboul, Jean-Francois Rual

[European Worm Meeting, 2002]

In addition to the draft of the human genome sequence, the genome sequences of an increasing number of model organisms are now available. This sequence information is expected to revolutionize the way biological questions can be addressed. Molecular mechanisms should now be approachable on a more global scale in the context of (nearly) complete sets of genes, rather than by analyzing genes individually. However most protein-encoding open reading frames (ORFs) predicted from these sequencing projects have remained completely uncharacterized at the functional level. For example, out of 19,000 ORFs predicted from the C. elegans genome sequence, the function of approximately 1,200 has been experimentally characterized during the last 30 years. Functional genomics and proteomics address this limitation through the simultaneous annotation of large numbers of predicted ORFs. Despite the urgent need for large- scale functional annotation projects, functional genomics approaches have remained relatively undeveloped in multicellular organisms, primarily because of the lack of suitable methods to clone large numbers of protein-encoding ORFs into many different expression vectors. Indeed, most strategies developed in these projects are based upon the expression of large numbers of proteins in exogenous settings and in fusion with relevant tags. In order to facilitate these different proteome-wide projects, a complete set of ORFs (or ORFeome) will need to be cloned multiple times into many different expression vectors for each model organism of interest. To achieve this goal, one solution is to clone an ORFeome of interest once and for all in a "resource" vector allowing a convenient transfer to various expression vectors. To clone the C. elegans ORFeome into various expression vectors, we use a recombination cloning technique referred to as Gateway. This technique allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro. We have now finished the first part of the C. elegans ORFeome project which was to attempt to clone the ~19,000 predicted ORFs. We will present the success rate in cloning of the ORFs and the overall quality of the ORFeome to date. We will also describe how the ORFeome was used as a new approach to construct a ~100% normalized yeast two-hybrid library. Finally we will show how we could transfer thousands of ORFs from the resource clones into a dozen different expression vectors for uses in large-scale functional genomic and proteomic projects such as gene inactivation by RNAi, protein interaction mapping by yeast two-hybrid, protein production for structural genomics etc.

Jerome Reboul et al. (2002) Mid-west Worm Meeting "THE C. ELEGANS ORFEOME CLONING PROJECT : VERSION 1.0"

Marc Vidal, Laurent Jacotot, David E Hill, Jim Hartley, Christopher Armstrong, Troy Moore, Philippe Vaglio, Jerome Reboul, Monica Martinez, Lynn Doucette-Stamm, Mike Brasch, Nicolas Bertin, Hongmei Lee, Jean-Francois Rual, Philippe Lamesch

[Mid-west Worm Meeting, 2002]

In addition to the draft of the human genome sequence, the genome sequences of an increasing number of model organisms are now available. This sequence information is expected to revolutionize the way biological questions can be addressed. Molecular mechanisms should now be approachable on a more global scale in the context of (nearly) complete sets of genes, rather than by analyzing genes individually. However most protein-encoding open reading frames (ORFs) predicted from these sequencing projects have remained completely uncharacterized at the functional level. For example, out of 19,000 ORFs predicted from the C. elegans genome sequence, the function of approximately 1,200 has been experimentally characterized during the last 30 years. Functional genomics and proteomics address this limitation through the simultaneous annotation of large numbers of predicted ORFs. Despite the urgent need for large-scale functional annotation projects, functional genomics approaches have remained relatively undeveloped in multicellular organisms, primarily because of the lack of suitable methods to clone large numbers of protein-encoding ORFs into many different expression vectors. Indeed, most strategies developed in these projects are based upon the expression of large numbers of proteins in exogenous settings and in fusion with relevant tags. In order to facilitate these different proteome-wide projects, a complete set of ORFs (or "ORFeome") will need to be cloned multiple times into many different expression vectors for each model organism of interest. To achieve this goal, one solution is to clone an ORFeome of interest once and for all in a "resource" vector allowing a convenient transfer to various expression vectors. To clone the C. elegans ORFeome into various expression vectors, we use a recombination cloning technique referred to as Gateway. This technique allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro. We have now finished the first part of the C. elegans ORFeome project which was to attempt to clone the ~19,000 predicted ORFs. We will present the success rate in cloning of the ORFs and the overall quality of the ORFeome to date. We will also describe how the ORFeome was used as a new approach to construct a ~100% normalized yeast two-hybrid library. Finally we will show how we could transfer thousands of ORFs from the resource clones into a dozen different expression vectors for uses in large-scale functional genomic and proteomic projects such as gene inactivation by RNAi, protein interaction mapping by yeast two-hybrid, protein production for structural genomics etc.

Jerome Reboul et al. (2002) East Coast Worm Meeting "THE C. ELEGANS ORFEOME CLONING PROJECT : VERSION 1.0"

Marc Vidal, Troy Moore, Alban Chesneau, Lynn Doucette-Stamm, Monica Martinez, Philippe Vaglio, Mike Brasch, Jean-Francois Rual, Philippe Lamesch, Christopher Armstrong, Hongmei Lee, David E Hill, Jerome Reboul, Jim Hartley, Nicolas Bertin, Laurent Jacotot

[East Coast Worm Meeting, 2002]

In addition to the draft of the human genome sequence, the genome sequences of an increasing number of model organisms are now available. This sequence information is expected to revolutionize the way biological questions can be addressed. Molecular mechanisms should now be approachable on a more global scale in the context of (nearly) complete sets of genes, rather than by analyzing genes individually. However most protein-encoding open reading frames (ORFs) predicted from these sequencing projects have remained completely uncharacterized at the functional level. For example, out of 19,000 ORFs predicted from the C. elegans genome sequence, the function of approximately 1,200 has been experimentally characterized during the last 30 years. Functional genomics and proteomics address this limitation through the simultaneous annotation of large numbers of predicted ORFs. Despite the urgent need for large-scale functional annotation projects, functional genomics approaches have remained relatively undeveloped in multicellular organisms, primarily because of the lack of suitable methods to clone large numbers of protein-encoding ORFs into many different expression vectors. Indeed, most strategies developed in these projects are based upon the expression of large numbers of proteins in exogenous settings and in fusion with relevant tags. In order to facilitate these different proteome-wide projects, a complete set of ORFs (or "ORFeome") will need to be cloned multiple times into many different expression vectors for each model organism of interest. To achieve this goal, one solution is to clone an ORFeome of interest once and for all in a "resource" vector allowing a convenient transfer to various expression vectors. To clone the C. elegans ORFeome into various expression vectors, we use a recombination cloning technique referred to as Gateway. This technique allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro. We have now finished the first part of the C. elegans ORFeome project which was to attempt to clone the ~19,000 predicted ORFs. We will present the success rate in cloning of the ORFs and the overall quality of the ORFeome to date. We will also describe how the ORFeome was used as a new approach to construct a ~100% normalized yeast two-hybrid library. Finally we will show how we could transfer thousands of ORFs from the resource clones into a dozen different expression vectors for uses in large-scale functional genomic and proteomic projects such as gene inactivation by RNAi, protein interaction mapping by yeast two-hybrid, protein production for structural genomics etc.

Jerome Reboul et al. (2001) International C. elegans Meeting "Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans"

Marc Vidal, James L Hartley, Philippe E Lamesch, Yuji KOHARA, Tadasu SHIN-I, Philippe Vaglio, David E Hill, Jerome Reboul, Jean Vandenhaute, Gary F Temple, Jean Thierry-Mieg, Jean Francois Rual, Michael A Brasch, Lynn Doucette-Stamm, Cindy Jackson, Troy Moore, Joseph Hitti, Nicolas Thierry-Mieg, Danielle Thierry-Mieg, Hongmei Lee

[International C. elegans Meeting, 2001]

The genome sequences of Caenorhabditis elegans , Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than D. melanogaster . Second, intron/exon predictions need to be experimentally tested. Third, complete sets of open reading frames (ORFs), or ORFeomes, need to be cloned into various expression vectors. To simultaneously address these issues, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning, and then sequenced to generate ORF sequence tags (OSTs), as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a different structure from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans . Hence we suggest that gene counts based primarily upon ESTs may underestimate the number of genes, in human and in other organisms.

Mecenas, Desirea G. et al. (2015) International Worm Meeting "The Dynamics of Alternative Polyadenylation Usage in C. elegans Germ Cell Development."

Piano, Fabio, West, Sean M., Polanowska, Jola, Reboul, Jerome, Gutwein, Michelle, Gunsalus, Kristin C., Mecenas, Desirea G., Bian, Wenting

[International Worm Meeting, 2015]

Proper spatio-temporal control of gene activity is vital for C. elegans germline development and maintenance and is determined primarily by regulatory elements within 3'UTRs (Merritt et al., Curr Biol 2008). Because almost half of protein-coding genes in the genome are subject to alternative polyadenylation (Mangone et al., Science 2010; Jan et al., Nature 2011), we are investigating whether the regulatory potential of genes during germline development is controlled by alternative 3'UTR isoform expression. We have established a Low Input 3'-End Sequencing (LITE-Seq) method to simultaneously identify and quantify mRNA transcript abundance and 3'UTR isoforms from small RNA samples, and we have applied it to investigate differences in transcripts and 3'UTR isoforms expressed in oocyte- and sperm-producing germline and in three distinct developmental stages within the hermaphrodite germline (mitosis, early meiosis, and developing oocytes). We observe on a global level that 3'UTRs in sperm-producing germline tend to be shorter than those expressed in oocyte producing germline, and that 3'UTRs become progressively longer as germ cell nuclei proliferate, enter meiosis, and differentiate into oocytes. We have identified numerous transcripts whose abundance and/or 3'UTR isoforms differ in a sex- or developmental stage-dependent manner. We also detect examples of 3'UTR isoform switching between sexes or developmental stages, including for some genes whose total transcript abundance is similar. To test the idea that individual transcripts may be subject to differential post-transcriptional regulation by selective expression and/or degradation of alternative 3'UTR isoforms at different developmental times, we developed an in vivo assay that reports on the translational regulatory potential of alternative 3'UTR isoforms in the germline. The reporter construct enables the cloning of two distinct 3'UTR isoforms into a Gateway-compatible, two-color reporter system in which each fluorophore is subject to translational regulation by a single 3'UTR isoform. Using this reporter system, we found that protein expression for several genes identified above is altered in a 3'UTR isoform-dependent manner, and that protein levels vary in different developmental contexts. Future work to identify cis-regulatory elements within the variable regions of 3'UTRs will enable us to assay their relative contributions to specific spatio-temporal expression patterns of known developmental regulators in the germline and to ascertain their functional significance in different developmental processes.

Cipriani, Patricia G. et al. (2013) International Worm Meeting "A genome-wide network of genetic interactions in embryonic development."

Erickson, Katherine, Cipriani, Patricia G., White, Amelia, Piano, Fabio, Gunsalus, Kristin, Kao, Huey-Ling, Reboul, Jerome, Munarriz, Eliana, Lucas, Jessica, Chatterjee, Indrani

[International Worm Meeting, 2013]

The phenotypes manifested by genetic alleles are influenced by the genetic background in which they reside. Yet, we still have a very limited understanding of how genetic interactions (GIs) influence animal development. The goal of our project is to use genome-wide screens to identify all enhancing and suppressing GIs for a set of strains harboring temperature sensitive (ts) mutations in 24 essential embryonic genes. We have completed over three million primary GI assays and secondary screening of putative suppressors, and we have archived in a database all experimental metadata and images, along with quantitative scoring results from an automated phenotypic scoring algorithm we developed (DevStaR). DevStaR combines computer vision and machine learning methods to count different developmental stages in mixed populations of animals. Using these results we have developed a quantitative phenotypic "GI score" based on the multiplicative model of independence: if the effects of perturbing two genes are independent, then their combined effects should not deviate from the product of their individual effects. GI scores for individual experimental replicates correlate positively with semi-quantitative manual estimates of interaction strength. Using manual inspection as a reference, we devised criteria to combine GI scores across replicates that reliably detect suppressing interactions. We then generated final interaction scores that reflect both strength and reproducibility, which we used to define ~800 high-confidence and ~750 intermediate-confidence suppressing interactions. Based on comparisons with manual scoring, we estimate the false discovery rates in these two sets as 2% and 10%, respectively. The resulting GI network provides the first genome-wide map of suppressing genetic interactions for the embryo based on quantitative phenotypic analysis of viability.

Anita G Fernandez et al. (2003) International Worm Meeting "Toward an RNAi-based phenotypic map in C. elegans"

Marc Vidal, Hsiao-Lan Liang, Kris Gunsalus, Caroline Tang, Ling-Shiang Chuang, Fabio Piano, Nancy Ying, Anita G Fernandez

[International Worm Meeting, 2003]

We are using C. elegans as a model to develop a "systems biology" approach to early embryogenesis. As a step in building a comprehensive map of the events that drive early development, we have been systematically characterizing RNAi phenotypes in the early embryo and have developed a digital representation of time-lapse DIC phenotypes composed of 47 discrete phenotypic characters. We have used this encoding system to generate and test hypotheses about the biological functions of unknown genes and are now beginning to combine our results with other functional genomics data to investigate additional correlations. The general phenotypic trends we observe (e.g. lethality level) are largely, but not completely, in agreement with other large-scale analyses. Based on comparisons of results from several studies, we conclude that the most promising strategy for eliciting potent gene-specific RNAi effects is to treat animals with dsRNA corresponding to a single spliced gene product. To pursue the next phase of high-content RNAi analysis, we have therefore chosen to use cloned gene products represented in the ORFeome (Reboul et al., 2003, in press). We will present our progress toward building a phenotypic map of the early C. elegans embryo.

Reboul J et al. (2001) Nat Genet "Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans."

Doucette-Stamm L, Lamesch PE, Reboul J, Temple GF, Hartley JL, Brasch MA, Hill DE, Vaglio P, Thierry-Mieg N, Shin-i T, Lee H, Moore T, Vandenhaute J, Kohara Y, Vidal M, Jackson C, Thierry-Mieg J, Tzellas N, Thierry-Mieg D, Hitti J

[Nat Genet, 2001]

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.AD - Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.FAU - Reboul, JAU - Reboul JFAU - Vaglio, PAU - Vaglio PFAU - Tzellas, NAU - Tzellas NFAU - Thierry-Mieg, NAU - Thierry-Mieg NFAU - Moore, TAU - Moore TFAU - Jackson, CAU - Jackson CFAU - Shin-i, TAU - Shin-i TFAU - Kohara, YAU - Kohara YFAU - Thierry-Mieg, DAU - Thierry-Mieg DFAU - Thierry-Mieg, JAU - Thierry-Mieg JFAU - Lee, HAU - Lee HFAU - Hitti, JAU - Hitti JFAU - Doucette-Stamm, LAU - Doucette-Stamm LFAU - Hartley, J LAU - Hartley JLFAU - Temple, G FAU - Temple GFFAU - Brasch, M AAU - Brasch MAFAU - Vandenhaute, JAU - Vandenhaute JFAU - Lamesch, P EAU - Lamesch PEFAU - Hill, D EAU - Hill DEFAU - Vidal, MAU - Vidal MLA - engID - R21 CA81658 A 01/CA/NCIID - RO1 HG01715-01/HG/NHGRIPT - Journal ArticleCY - United StatesTA - Nat GenetJID - 9216904SB - IM

Katja Ziegler et al. (2007) International Worm Meeting "A Sodium:Neurotransmitter symporter Family protein required for the induction of antimicrobial peptides in C.elegans."

Jonathan Ewbank, Nathalie Pujol, Katja Ziegler

[International Worm Meeting, 2007]

C. elegans responds to infection with the nematophagous fungus Drechmeria coniospora by up-regulating the expression of antimicrobial peptides including certain NLPs (neuropeptide-like proteins) and CNCs (Caenorhabditis bacteriocins) (1). We have generated a reporter strain containing the promoter of the antimicrobial peptide gene, nlp-29, fused to gfp. This results in a strain in which green fluorescence is highly induced after infection with D. coniospora. Coupled with the Union Biometrica BioSort, this provides us with a valuable tool to study infection-dependent induction in a qualitative and quantitative fashion. Using this strain and a direct visual screen following EMS mutagenesis, we identified 5 Nipi (no induction of peptide after Drechmeria infection) mutants (see abstracts by Pujol et al., Zugasti et al.). This study describes the isolation, characterization and SNP mapping of nipi-2. The mutant was found to carry a mutation in a conserved residue of a member of the sodium-dependent neurotransmitter transporter family in C. elegans. We are currently further characterizing this SNF protein and investigating its role in the induction of nlp-29 that follows infection. Progress will be reported at the meeting. 1.Couillault, C., Pujol, N., Reboul, J., Sabatier, L., Guichou, J. F., Kohara, Y. & Ewbank, J. J. (2004) Nat Immunol 5, 488-494. .

Coux O (2003) Biochem Soc Trans "An interaction map of proteasome subunits."

Coux O

[Biochem Soc Trans, 2003]

Despite the central role of the 26 S proteasome in eukaryotic cells, many facets of its structural organization and functioning are still poorly understood. To learn more about the interactions between its different subunits, as well as its possible functional partners in cells, we performed, with Marc Vidal's laboratory (Dana-Farber Cancer Institute, Boston, MA, U.S.A.), a systematic two-hybrid analysis using Caenorhaditis elegans 26 S proteasome subunits as baits (Davy, Bello, Thierry-Mieg, Vaglio, Hitti, Doucette-Stamm, Thierry-Mieg, Reboul, Boulton, Walhout et al. (2001) EMBO Rep. 2, 821-828). A pair-wise matrix of all subunit combinations allowed us to detect numerous possible intra-complex interactions, among which some had already been reported by others and eight were novel. Interestingly, four new interactions were detected between two ATPases of the 19 S regulatory complex and three alpha-subunits of the 20 S proteolytic core. Possibly, these interactions participate in the association of these two complexes to form the 26 S proteasome. Proteasome subunit sequences were also used to screen a cDNA library to identify new interactors of the complex. Among the interactors found, most (58) have no clear connection to the proteasome, and could be either substrates or potential cofactors of this complex. Few interactors (7) could be directly or indirectly linked to proteolysis. The others (12) interacted with more than one proteasome subunit, forming 'interaction clusters' of