25 Papers found (0.333 seconds)

Yuichi Nishi et al. (2005) International Worm Meeting "Regulation of OMA-1 degradation by the DYRK kinase, MBK-2 in newly fertilized embryos"

[

International Worm Meeting,

2005]

Oocyte maturation and fertilization are very dramatic transitions in which dormant oocytes are converted into rapidly dividing, differentiating embryos. Although this transition is clearly important for successful initiation of embryogenesis, its mechanism is poorly understood. We believe that OMA-1 and OMA-2 play important roles in the regulation of the egg-to-embryo transition and that timely degradation of OMA-1/2 is critical for this transition. oma-1 and oma-2 are close homologues that are redundantly required for oocyte maturation as well as embryogenesis. They encode CCCH Tis-11 like zinc finger proteins that are predicted to regulate RNA targets. OMA-1/2 are expressed in oocytes and are rapidly degraded in newly fertilized embryos. This timely degradation appears to be important for proper embryogenesis since the oma-1 (zu405) gain-of-function mutation, which causes a delay in OMA-1 degradation, results in embryonic lethality. Although our preliminary results suggest that OMA-1/2 are degraded through the ubiquitin proteasome pathway, the precise mechanism remains unclear. It has been shown previously that degradation of OMA-1, and several other maternal proteins, requires the MBK-2 kinase, a protein that also coordinates the egg-to-embryo transition.We show that OMA-1 is a direct target of MBK-2. We show that MBK-2 phosphorylates OMA-1 in vitro predominantly on T239, immediately next to the amino acid mutated in the oma-1(zu405) gain-of-function mutant. The amino acid change of the zu405 mutation prevents phosphorylation of OMA-1 at T239. Using an antibody recognizing OMA-1 phosphorylated at T239, we show that OMA-1 is phosphorylated in vivo at T239 during the very short developmental time window immediately after MBK-2 activation and until OMA-1 degradation. The in vivo phosphorylation of OMA-1 at T239 depends on MBK-2 kinase activity and is not observed in the zu405 mutant embryos. In addition, we show that an OMA-1 GFP reporter in which T239 is mutated to alanine fails to degrade properly, demonstrating that MBK-2 phosphorylation of OMA-1 at T239 promotes its degradation.In conclusion, our data suggest that OMA-1 is phosphorylated by MBK-2 at T239 shortly before its degradation and that this phosphorylation promotes OMA-1 degradation at the egg-to-embryo transition.

David Karow et al. (2001) International C. elegans Meeting "Guanylate Cyclase Beta 2 Homologues in C. elegans"

David Morton, Ronald E Ellis, Jennifer Chang, Scott Nicholls, Michael Marletta, Martin Hudson, David Karow

[

International C. elegans Meeting,

2001]

Soluble guanylate cyclases (sGCs) catalyze the conversion of GTP to cGMP. In mammals, the most well-studied sGC is a heme-containing, heterodimer, composed of a 1 and b 1 subunits. Nitric oxide (NO) binds to the heme, which is ligated to the b 1 subunit, and stimulates the activity of the enzyme 400-fold. A b 1 homologue, b 2, has been cloned from rat and human kidney cDNA libraries. This subunit appears to be expressed in the kidney collecting duct cells, but its heme binding status, sensitivity to NO, and intracellular localization are unknown. We want to elucidate the structure and function of this protein. Analysis of the C. elegans genome predicted seven putative sGC b subunits, including five b 2 homologues, ( gcy-32 , gcy-34 , gcy-35 , gcy-36 , and gcy-37 ). We cloned these five homologues, by RT-PCR and RACE, and found that each of them contains the residues necessary for GTP binding, catalysis and heme binding. These findings suggest that the b 2 homologues in C. elegans are receptors in a cGMP-dependent signaling system. Our data are consistent with this hypothesis. Preliminary characterization of the bacterially-expressed N-terminus from gcy-35 suggests that it can bind heme. Promoter::green fluorescent protein (GFP) fusion studies localize all of the b 2 homologues to four candidate sensory neurons (URXL, URXR, AQR and PQR). These neurons are all connected to the pseudocoelom, and might play a role in fluid homeostasis. This possibility is consistent with data demonstrating that their mammalian homologue, b 2, is expressed in the kidney. We are now using transgenes to express Egl-1 in these four candidate neurons, so that we can eliminate them and elucidate their functions. Does NO activate b 2 and its homologues? Analysis of the C. elegans genome did not reveal an open reading frame for nitric oxide synthase (NOS). Furthermore, we have identified GC activity in C. elegans lysates and shown that this activity is NO-insensitive. These results suggest that b 2 and its homologues define a novel class of NO-insensitive cyclases. We are using biochemical techniques to examine heme-binding and ligand specificity in the b 2 homologues.

Meghann E Mabon et al. (2005) International Worm Meeting "Genetic Screen to Identify Loci Required for Hypoxia Sensitivity"

Barbara A Scott, Meghann E Mabon, C Michael Crowder

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International Worm Meeting,

2005]

Cell death induced by low oxygen (hypoxia) has been observed in C. elegans. We have previously shown that the daf-2/daf-16 pathway regulates C. elegans cellular oxygen requirements1. This result suggests that other genes could also be involved in the regulation of hypoxia sensitivity. Therefore we are conducting an F2 EMS screen in N2 to identify mutations that result in viable animals following prolonged hypoxic insult.We will discuss current work in progress in addition to the strategy and caveats of this approach.1.Scott, B.A., Avidan, M.S. and C.M. Crowder. Regulation of Hypoxic Death in C. elegans by the Insulin/IGF Receptor Homolog DAF-2. Science, 2002 (296): 2388-91.

Buechner M et al. (2000) Japanese Worm Meeting "A faciogenital dysplasia gene homologue regulates the formation of organ structure in C. elegans"

Hisamoto N, Nishiwaki K, Matsumoto K, Suzuki N, Buechner M, Hall DH

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Japanese Worm Meeting,

2000]

The molecular controls governing the formation of organ structure in the development are less understood. The Caenorhabditis elegans excretory system, like the renal organs of other animals, is believed to regulate osmoregulation and water elimination1. The excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in the excretory canal. Here we report that exc-5 encodes a protein homologous to guanine nucleotide exchange factors (GEFs). EXC-5 contains, in order, a Dbl/Plecstrin homology (DH/PH) domain, a cysteine-rich FYVE domain and a second PH element. This motif architecture is similar to that of FGD1 (ref. 2), which is responsible for faciogenital dysplasia or Aarskog-Scott syndrome. Ultrastructural results suggest that exc-5 is required for the proper placement of cytoskeleton at the apical epithelial surface. Furthermore, EXC-5 associates with KEL-2, a protein similar to the actin-associated protein Drosophila Kelch3,4. We speculate that EXC-5 controls structural organization of the excretory canal by linking between the actin cytoskeleton and a Rho family GTPase. References: 1. Nelson, F. K., Albert, T. S. & Riddle, D. L. Fine structure of the Caenorhabditis elegans secretory-excretory system. J. Ultrastruct. Res. 82, 156-171 (1983) 2. Broeks, A., Janssen, H. W., Calafat, J. & Plasterk, R. K. A P-glycoprotein protects Caenor ha bditis elegans against natural toxins. EMBO J. 14, 1858-1866 (1995) 3. Pasteris, N. G. et al. Isolation and characterization of the faciogenital dysplasia (Aarskog-scott syndrome) gene: a putative Rho/Rac guanine nucleotide exchange factor. Cell 79, 669-678 (1994) 4. Chitwood, M. B. & Chitwood, B. G. The excretory system. In Introduction to Nematology:(eds. Chitwood, B. G. & Chitwood, M. B.; University Park Press, Baltimore) 126-135 (1974)

Meghann E Mabon et al. (2004) West Coast Worm Meeting "Studying Hypoxia Using C. elegans Microarrays"

Meghann E Mabon, C Michael Crowder

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West Coast Worm Meeting,

2004]

When cellular oxygen requirements are not met, cells eventually die. These requirements vary widely by cell type and organism. We have previously shown that the daf-2/daf-16 pathway regulates C. elegans cellular oxygen requirements 1 . Specifically, daf-2 (rf) mutants are resistant to cell death induced by low oxygen (hypoxia). The hypoxia resistance of daf-2 (rf) requires the DAF-16 forkhead transcription factor. To identify candidate genes regulating oxygen requirements, presumably some of which are DAF-16 targets, we are using whole-genome C. elegans microarrays. Preliminary data from N2 identifies a large number of genes whose expression is altered by hypoxia. The role of genes implicated via microarrays will be examined by RNAi and genetic analysis. Current work in progress will be presented. 1 Scott, B.A., Avidan, M.S. and C.M. Crowder. Regulation of Hypoxic Death in C. elegans by the Insulin/IGF Receptor Homolog DAF-2. Science, 2002 (296): 2388-91.

Norio Suzuki et al. (2001) International C. elegans Meeting "A putative GDP-GTP exchange factor is required for development of the excretory cell"

Naoki Hisamoto, David H Hall, Kunihiro Matsumoto, Matthew Buechner, Kiyoji Nishiwaki, Norio Suzuki

[

International C. elegans Meeting,

2001]

The molecular controls governing the formation of organ structure during development are not well understood. The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the hypodermis and mutations in the exc-5 gene cause tubulocystic defects at the distal tips of the canals. Here we report that exc-5 encodes a protein homologous to guanine nucleotide exchange factors (GEFs) and contains, in order, a Dbl/Pleckstrin homology (DH/PH) domain, a cysteine-rich FYVE domain and a second PH element. This motif architecture is similar to that of FGD1, which is responsible for faciogenital dysplasia or Aarskog-Scott syndrome 1 . Ultrastructural analysis suggests that EXC-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 interacts genetically with mig-2 encoding a Rho GTPase. Overexpression of exc-5 rescues the apical defect but causes defects at the basolateral surface of the excretory cell. These results suggest that EXC-5 controls the structural organization of epithelia by regulating Rho family GTPase activity. 1 Pasteris, N. G. et al. , Cell, 79, 669-678(1994)

James P McCarter et al. (2001) International C. elegans Meeting "Genes from Other"

Michael Dante, James P McCarter, John C Martin, Deana Pape, Brandi Chiapelli, Todd N Wylie, Robert H Waterston, Sandra W Clifton

[

International C. elegans Meeting,

2001]

To build upon knowledge gained from the genome of C. elegans , we have begun generating Expressed Sequence Tags (ESTs) from parasitic (and free-living) nematodes. This project will generate >225,000 5' ESTs from 14 species by 2003. Additionally, the Sanger Centre and Edinburgh Univ. will complete 80,000 ESTs from 7 species. Through these combined efforts, we anticipate the identification of >80,000 new nematode genes. At the GSC, approximately 35,000 ESTs have been generated to date including sequences from Ancylostoma caninum, Heterodera glycines, Meloidogyne incognita and javanica, Parastrongyloides trichosuri, Pristionchus pacificus, Strongyloides stercoralis and ratti, Trichinella spiralis, and Zeldia punctata . We will report on our progress in sequence analysis, including the creation of the NemaGene gene index for each species by EST clustering and consensus sequence generation, identification of common and rare transcripts, and identification of genes with orthologues in C. elegans and other nematodes. All sequences are publicly available at www.ncbi.nlm.nih.gov/dbEST. NemaGene sequences and project details are available at WWW.NEMATODE.NET. We would like to thank collaborators who have provided materials and ideas for this project including Prema Arasu, David Bird, Rick Davis, Warwick Grant, John Hawdon, Doug Jasmer, Andrew Kloek, Thomas Nutman, Charlie Opperman, Alan Scott, Ralf Sommer, and Mark Viney. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.

Rosoff ML et al. (1991) International C. elegans Meeting "MOLECULAR CHARACTERIZATION OF THE NEUROPEPTIDE GENE ENCODING FLRFamide."

Li C, Rosoff ML

[

International C. elegans Meeting,

1991]

Single neurons can express multiple neurotransmitters and in addition, can switch their transmitter phenotype during development. We are interested in identifying what factors may be involved in determining transmitter specificity. Approximately thirty neurons in C. elegans stain with an antibody specific for the RFamide moiety of the neuropeptide FMRFamide [see WBG 9(3):110]. Screening of Stuart Kim's cDNA library with degenerate oligonucleotides against FMRFGK(R) yielded a 659 bp hybridizing clone whose putative translation product encodes eight potential neuropeptides. Seven of these neuropeptides share the same carboxy terminal -PNFLRFamide structure. Primer extension analysis has shown that this cDNA is not full length. Since screening of the MIT and MRC cosmid libraries yielded no hybridizing clones, we screened a lambda 1059 genomic library ( courtesy of Scott Emmons), and isolated thirteen hybridizing clones. By restriction mapping, the clones were separated into four overlapping classes. A hybridizing 3 kb EcoRI fragment was subcloned and sequenced from two separate isolates. The clones contain approximately 300bp upstream of the start of the cDNA, the entire cDNA, and a poly adenylation signal. Constructs are being made to analyze the promoter region using the beta-galactosidase gene as a reporter in microinjection studies. Concurrently, we have begun the purification of FMRFamide-like peptides from worm extracts. Radioimmunoassays show the presence of FMRFamidelike peptides, and further characterization of the extracts by reverse phase HPLC reveals at least five peaks of FMRFamide-like immunoreactivity.

Brendan Galvin et al. (2001) International C. elegans Meeting "A ced-4 SUPPRESSION SCREEN TO IDENTIFY NEW GENES INVOLVED IN PROGRAMMED CELL DEATH"

Brendan Galvin, Bob Horvitz, Scott Cameron

[

International C. elegans Meeting,

2001]

Many genes involved in programmed cell death in C. elegans have been identified by screening for mutations that allow survival of cells that are normally destined to die. The opposite approach, screening for mutations that result in an increase in the number of cell deaths, has been comparatively unexplored, in part because of the lack of efficient methods to identify such mutations. A lin-11::gfp reporter (generated by Scott Cameron) expresses in the Pn.aap cells of the ventral cord. In wild-type animals, the six Pn.aap cells of the P3-P8 lineages survive, differentiate into VC neurons, and express lin-11::gfp . In strong cell-death defective mutants, all 12 Pn.aap cells survive and express GFP. In the lin-11::gfp reporter strain the amount of cell death can be quantified by scoring the number of fluorescent nuclei in the ventral cord using a fluorescence-equipped dissecting microscope. Using the lin-11::gfp reporter, I have performed a screen for suppressors of a partial loss-of-function ced-4 mutant by looking for mutants with a reduction in the number of GFP-positive Pn.aap cells. To date approximately 40,500 mutagenized genomes have been screened; 5,000 were screened clonally and 35,500 non-clonally. Two strong suppressors were obtained that reduce the number of GFP-positive Pn.aap cells. The first suppressor, identified in the clonal screen, is recessive and is recessively sterile. The second suppressor, identified in the non-clonal screen, is dominant and is recessively sterile. Further characterization of these mutants will be described.

David Hall et al. (2008) Development & Evolution Meeting "What's New on WormAtlas and WormImage?"

Tylon Stephney, Chris Crocker, Zeynep Altun, David Hall, Carolyn Norris, Laura Herndon

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Development & Evolution Meeting,

2008]

The pages of WormAtlas are getting a fresh look and organization.These changes will start from the front page and then be implemented throughout the Handbook and many other portions of the website.Here we will explain the principles of the new organization, and show you how to find your favorite features.This is the first major revamping of WormAtlas since its launch in 2002.We hope you will find the site simpler to navigate and we expect it will be more intuitive for beginners.As much as possible, these changes should not disrupt any previous weblinks you have established to your favorite pages.Inside the WormAtlas website, there will be several major changes. First will be an improved adult hermaphrodite handbook; it will include several completely revised chapters and a new one covering the nervous system. Second will be the launch of a handbook for anatomy of the worm embryo.Third will be the addition of more data to Slidable Worm.Lastly, we will be adding many new Neuron pages for the male nervous system in order to highlight new synaptic patterns emerging from the Wired Worm project conducted together with Scott Emmons. The WormImage website is expanding steadily.It now presents much more mutant data, particularly for genes affecting the nervous system.As before, we are relying heavily on MRC datasets, but we will continue to add more from the Riddle and Hall lab files.We encourage more laboratories to share your own best archival TEM and SEM images for this purpose.We are very grateful to many labs that have already contributed ideas, advice and experimental results that are featured on these websites.This work is supported by NIH RR12596.

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