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[
International C. elegans Meeting,
1999]
Nuclear hormone receptors (NHRs) are known to be essential for developmental processes in a wide variety of multicellular organisms, including C. elegans . One extensively studied example of the developmental role of NHRs is in the control of molting and metamorphosis in Drosophila . Molting and metamorphosis in Drosophila are regulated by the steroid hormone 20-OH ecdysone, hereafter referred to as ecdysone. The ecdysone signal is transduced by NHRs. Nematodes also undergo a series of molts during development. Molting from the third to the fourth larval stage of the filarial parasite Dirofilaria immitis (the causative agent of dog heartworm disease) can be prematurely induced in vitro by ecdysone, suggesting that ecdysone, or a related compound, may play a role in the development of this nematode. Putative homologs of the EcR and usp genes, which encode NHRs that form the functional ecdysone receptor in Drosophila , have been identified in D. immitis . The ecdysone signal in Drosophila is modulated by a number of transcription factors that are induced as a primary response to ecdysone. We have identified a putative D. immitis homolog of the Drosophila E75 ecdysone primary response gene. E75 encodes multiple isoforms and is essential for metamorphosis in Drosophila . The protein encoded by the D. immitis E75 homolog, dinhr-6, is 83% identical to E75A in the DNA binding domain. We cloned the 5' end of dinhr-6 and determined that the transcript uses the SL1 spliced leader. We are currently using a variety of techniques to clone the 3' end of the gene and have identified the complete DNA and ligand binding domains. Northern blot analysis suggests that dinhr-6 encodes multiple isoforms and is female-specific in adults. A putative E75 homolog,
nhr-85 , was recently identified in C. elegans (Ann Sluder, personal communication). We discuss the potential of using C. elegans to help determine the function of parasite proteins.
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Kohara Y, Tzellas N, Thierry-Mieg N, Jackson C, Temple GF, Hill DE, Vidal M, Lamesch PE, Thierry-Mieg D, Vandenhaute J, Brasch MA, Vaglio P, Doucette-Stamm L, Moore T, Hartley JL, Shin-i T, Lee H, Reboul J, Hitti J, Thierry-Mieg 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
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[
International Worm Meeting,
2007]
The Wnt signaling pathway is highly conserved throughout evolution and plays an integral part in both development and homeostasis of a diverse range of metazoan organisms. The pathway has been shown to regulate such processes as cell polarity, migration and fate determination. Mis-activation of the pathway has been implicated in both birth defects and disease in humans. We are interested in the role Wnt signaling plays in development, specifically in the regulation of cell fate determination. In C. elegans, homologs for most of the core vertebrate Wnt pathway components have been identified. However, only a handful of the worms Wnt target genes have been found. Wnt signaling is required for both embryonic and larval development. And our lab has shown that a beta-catenin dependent Wnt signaling pathway is involved in fate specification of the epithelial Vulva Precursor Cells (VPCs) and certain Ventral Cord Neurons (VCNs). The aim of my research is to identify Wnt pathway gene targets in the VPCs, VCNs and other cells. I used Affymetrix microarray analysis to identify differentially expressed genes in worms subjected to Wnt pathway wild-type activation vs. hyper-activation. The hyper-activated state was induced using a stable variant of the transcriptional activator BAR-1/beta-catenin. 117 up- and 72 down-regulated genes were identified (2-fold greater or lesser that wild-type expression levels). qRT-PCR analysis of these gene candidates, comparing expression levels under conditions of wild-type Wnt pathway activation to both pathway hyper- or hypo-activation, showed that 30% of the up-regulated microarray identified targets to be bona fide Wnt responsive genes. Of these, 18% are cuticular collagen genes and 9% are dauer related genes 41% are novel genes. I am currently characterizing the spatial and temporal expression patterns of these gene targets and analyzing them for altered expression when subjected to conditions of pathway hyper- or hypo-activation. In order to determine their biological function, future work will include analysis of phenotypic effects caused by RNA interference and/or mutation or deletion of these genes. Characterization of the Wnt responsive genes identified in this study will expand our knowledge of the genetic controls involved in cell fate determination.
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[
Biochim Biophys Acta,
2000]
Mammalian Elongin C is a 112-amino acid protein that binds to the von Hippel-Lindau (VHL) tumor suppressor and to Elongin A, the transcriptionally active subunit of the RNA polymerase II elongation factor, SIII. It is conserved in eukaryotic cells, as homologs have been identified in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans. The mammalian protein is thought to function as part of a ubiquitin targeting E3 ligase, yet the function in yeast has not been determined. In this report we examine the role of Elongin C in yeast and establish that yeast Elongin C may function in a mode distinct from its role as an E3 ligase. The RNA is expressed ubiquitously, albeit at low levels. Two hybrid analyses demonstrate that Elongin C in yeast interacts with a specific set of proteins that are involved in the stress response. This suggests a novel role for Elongin C and provides insights into additional potential functions in mammalian cells.
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Reboul J, Jackson C, Brasch MA, Vidal MI, Thierry-Mieg J, Temple GF, Matthews LR, Hartley JL, Vaglio P, Tzellas N, Moore T
[
European Worm Meeting,
2000]
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Two basic strategies have been developed for the characterization of (nearly) complete sets of proteins (the proteome). In the "bottom-up" approach, endogeneous proteins expressed in vivo are analyzed using high-throughput techniques such as mass spectrometry (MS) or matrix-laser desorption/ionization (MALDI). In the "top-down" approach, (nearly) complete sets of ORFs (ORFeomes) are expressed exogeneously to perform various protein function assays such as large-scale two-hybrid analysis, biochemical assays, protein production and purification for structural analyses, etc... The "top-down" approach relies upon the availability of ORFeomes cloned into various expression vectors (i.e., for each ORF: the sequence between the start and the stop codons, in the absence of 5 and 3 untranslated sequences and introns). To clone the C. elegansORFeome into various expression vectors, we use the Recombination Cloning technique GatewayTM . This system, first developed by Life Technologies Inc. (Hartley et al., submitted; Walhout et al., 2000, Science, 287, 166-122; Walhout et al., 2000, Methods in Enzymology, in press), allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro . The features of RC make it amenable to automation and 96-well (or 384-) plates settings, which is crucial for large-scale ORFeome cloning. So far we have cloned 1500 C. elegans ORFs. With our current throughput, 3,000 ORFs (15% of the ORFeome) should be cloned by the time of the European C. elegansmeeting and ~70% of the ORFeome should be cloned by the end of year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a description of the cloning quality, iv) how this resource will be made available to the community, and v) how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et al.). The C. elegansORFeome project is likely to illustrate how to undertake ORFeome cloning projects for more complex multicellular organisms.
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Vidal MI, Tzellas N, Moore T, Vaglio P, Thierry-Mieg D, Jackson C, Temple GF, Thierry-Mieg J, Brasch MA, Hartley JL, Reboul J
[
East Coast Worm Meeting,
2000]
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Most of protein-based approaches rely upon the availability of near complete set of open reading frames ("ORFeomes") cloned into various expression vectors (i.e., for each ORF: the sequence between the start and the stop codons, in the absence of 5 and 3 untranslated sequences and introns). To clone the C. elegans ORFeome into various expression vectors, we use a Recombination Cloning technique (RC) referred to as GatewayTM (Walhout et al., 2000, Science, 287, 166-122). RC allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro. In addition, RC is amenable to automation in 96-well (or 384-) plate settings, which is crucial for large-scale ORFeome cloning. So far we have cloned 2,000 C. elegans ORFs. At the current throughput (~400 ORFs/week), ~70% of the C. elegans ORFeome should be cloned by the end of the year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a description of the cloning quality, iv) how this resource will be made available to the community, and v) how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et al).
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Hartley JL, Moore T, Temple GF, Thierry-Mieg D, Vaglio P, Brasch MA, Tzellas N, Thierry-Mieg J, Reboul J, Jackson C, Vidal MI
[
West Coast Worm Meeting,
2000]
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein interaction mapping, protein localization mapping, and biochemical and structural genomics. Most of protein-based approaches rely upon the availability of near complete set of open reading frames ("ORFeomes") cloned into various expression vectors (i.e., for each ORF: the sequence between the start and the stop codons, in the absence of 5 and 3 untranslated sequences and introns). To clone the C. elegans ORFeome into various expression vectors, we use a Recombination Cloning technique (RC) referred to as GatewayTM (Walhout et al., 2000, Science, 287, 166-122). RC allows both the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific recombination in vitro. In addition, RC is amenable to automation in 96-well (or 384-) plate settings, which is crucial for large-scale ORFeome cloning. So far we have cloned 2,000 C. elegans ORFs. At the current throughput (~400 ORFs/week), ~70% of the C. elegans ORFeome should be cloned by the end of the year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a description of the cloning quality, iv) how this resource will be made available to the community, and v) how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et al).
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[
Development & Evolution Meeting,
2006]
Wnt signaling functions in multiple developmental processes in the model organism C. elegans. Homologs of β-catenin and TCF/LEF are BAR-1 and POP-1, respectively. POP-1 functions in endoderm induction, Q-neuroblast migration, P12 and vulva precursor cell fate specification, and T cell and somatic gonad precursor cell polarity. We are taking two approaches to identify Wnt target genes in these developmental processes. First, by performing microarray analysis on worms that have been induced to over-express key Wnt pathway components (BAR-1 and POP-1) we will reveal target genes that are up or down regulated by Wnt signaling (see poster by Belinda Jackson). Second, I am using a selection and amplification method with the DNA-binding domain of POP-1 to identify genomic binding sequences. Fragments bound by POP-1 utilizing this technique will be subjected to alignment and sequence comparison with the C. elegans genome to identify putative gene targets. Candidate target genes regulated by POP-1 will be verified by inserting the promoter of genes in question upstream of the
pes-10 basal promoter::GFP vector and injected into worms. Lines expressing GFP will be subjected to
pop-1 RNAi to identify sequences regulated by POP-1 in vivo. Additionally, reporter constructs will be made containing mutated POP-1 consensus binding sites to verify that the consensus site(s) are needed.
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[
BMC Genomics,
2007]
ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae
dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans
dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.
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[
West Coast Worm Meeting,
2002]
To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.