-
[
Neuron,
2001]
Weighing in at about 5 ug, with 302 neurons and 5000 synapses, C. elegans is unlikely to prove theorems, write poetry, or challenge Mike Tyson. Still, remarkable behavioral complexity is packed into this tiny worm.
-
[
Science,
2000]
Protein interaction mapping using large-scale two-hybrid analysis has been proposed as a way to functionally annotate large numbers of uncharacterized proteins predicted by complete genome sequences. This approach was examined in Caenorhabditis elegans, starting with 27 proteins involved in vulval development. The resulting map reveals both known and new potential interactions and provides a functional annotation for approximately 100 uncharacterized gene products. A protein interaction mapping project is now feasible for C. elegans on a genome-wide scale and should contribute to the understanding of molecular mechanisms in this organism and in human diseases.AD - Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA.FAU - Walhout, A JAU - Walhout AJFAU - Sordella, RAU - Sordella RFAU - Lu, XAU - Lu XFAU - Hartley, J LAU - Hartley JLFAU - Temple, G FAU - Temple GFFAU - Brasch, M AAU - Brasch MAFAU - Thierry-Mieg, NAU - Thierry-Mieg NFAU - Vidal, MAU - Vidal MLA - engID - 1 R21 CA81658 A 01/CA/NCIID - 1 RO1 HG01715-01/HG/NHGRIPT - Journal ArticleCY - UNITED STATESTA - ScienceJID - 0404511RN - 0 (Genetic Vectors)RN - 0 (Helminth Proteins)RN - 0 (LIN-35 protein)RN - 0 (LIN-53 protein)RN - 0 (Repressor Proteins)RN - 0 (Retinoblastoma Protein)SB - IM
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[
International Worm Meeting,
2003]
It has been argued that, to globally understand development, it will be crucial to generate databases describing where (in what cell) and when (at what stage of development) each protein is expressed. One possible approach is to construct transgenic C. elegans strains carrying reporter genes, such as that encoding the Green Fluorescent Protein (GFP), driven by particular promoters and examine expression patterns throughout development. Such studies have already been successfully carried out with relatively small subsets of genes, this project aims to address conceptual and technical issues that will allow us to apply this approach to a genome wide scale. We propose to clone the majority of worm promoters into a novel Gateway compatible vector. The availability of a comprehensive collection of cloned worm promoters (promoterome) will be very useful to the scientific community. We use an innovative Gateway technology with which we will transfer cloned promoters into various destination vectors. Each promoter can then be used to detect expression of reporter genes, or to drive expression of their cognate ORFs, fused to either GFP, or any other Tag-encoding sequence. These can be used to examine sub-cellular protein localization, for complex purifications or in genetic rescue experiments. To increase the throughput of generating transgenic worms to a genome scale, we will adapt the recently developed ballistic transformation protocol (Praitis, 2001) This method allows genomic integration of low copy plasmids which may allow more physiological regulation of the transgenes than extrachromosomal arrays. We will then use a semi-automated 96 well confocal microscopic platform (Atto Bioscience) for rapid imaging and analysis of multiple independent transgenic strains. All the data generated will be made available to the community on a freely accessible database. To illustrate usage of Multisite Gateway to generate promoter::ORF::GFP fusion proteins. We created a construct in which a Histone2B (B0035.8) is expressed from the
myo-2 promoter, which drives expression in the muscle cells of the pharynx. We then used the microparticle bombardment technique to integrate this construct into the C. elegans genome. As expected, the
pmyo-2::H2B::GFP construct expressed in the muscle cells of the pharynx, and the H2B::GFP fusion localized to the nucleus.
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[
Worm Breeder's Gazette,
1993]
It would be useful if worm labs had access to each other's strain lists. For instance, one lab may have put away in the freezer as uninteresting a mutation that is just what a second lab wants. Unfortunately, there have always been practical difficulties to sharing strain lists: they change frequently, and there is no common format. A new network program called Gopher (see contribution from Mike Cherry on previous page of this WBG issue, which discusses the use of Gopher in connection with accessing the ACEDB database) may be the solution to these problems. Gopher sends an inquiry over the Internet directly to the source, without the user having to know about any messy details. The reply is current, and comes back as text, so that format problems don't arise. Other kinds of information (e.g., pictures) can also be made available. To test the feasibility of Gopher for data sharing, we have set up Gopher service at the CGC (elegans.cbs.umn.edu; IP address 134.84.210.1) and the Avery lab (eatworms.swmed utexas.edu; IP address 129.112.11.21). By gopher to either of these addresses you can get the CGC bibliography, strain list, WBG subscriber directory, recent WBG tables of contents, Avery lab strain lists, pictures of mutants, manuscripts in press, and (thanks to Mike Cherry at Massachusetts General Hospital) access to ACEDB information. Gopher is available for Macintosh, IBM-PC, Unix, and Xwindows by anonymous ftp from boombox.micro.umn.edu (134.84.132.2). If you don't know how to get it, send e-mail to leon@eatworms.swmed.utexas.edu and we will try to help. Also, we would like to urge other labs to make data available by Gopher. If you do, send us e-mail so you can be included in the CGC menu. If you would like to make your strain lists available but don't know how, send e-mail and we'll see if we can help with that.
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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
-
[
J Exp Zool,
1999]
A memorable workshop, focused on causal mechanisms in metazoan evolution and sponsored by NASA, was held in early June 1998, at MBL. The workshop was organized by Mike Levine and Eric H. Davidson, and it included the PI and associates from 12 different laboratories, a total of about 30 people. Each laboratory had about two and one half hours in which to represent its recent research and cast up its current ideas for an often intense discussion. In the following we have tried to enunciate some of the major themes that emerged, and to reflect on their implications. The opinions voiced are our own. We would like to tender apologies over those contributions we have not been able to include, but this is not, strictly speaking, a meeting review. Rather we have focused on those topics that bear more directly on evolutionary mechanisms, and have therefore slighted some presentations (including some of our own), that were oriented mainly toward developmental processes. J. Exp. Zool. (Mol. Dev. Evol. ) 285:104-115, 1999.
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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.
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Troy Moore, Jerome Reboul, Monica Martinez, Mike Brasch, Alban Chesneau, Marc Vidal, David E Hill, Jean-Francois Rual, Lynn Doucette-Stamm, Hongmei Lee, Philippe Vaglio, Nicolas Bertin, Laurent Jacotot, Philippe Lamesch, Jim Hartley, Christopher Armstrong
[
West 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.
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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.
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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.