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Caenorhabditis Elegans (caenorhabditis + elegan)
Kinds of Caenorhabditis Elegans Selected AbstractsEvolutionary origins of the purinergic signalling systemACTA PHYSIOLOGICA, Issue 4 2009G. Burnstock Abstract Purines appear to be the most primitive and widespread chemical messengers in the animal and plant kingdoms. The evidence for purinergic signalling in plants, invertebrates and lower vertebrates is reviewed. Much is based on pharmacological studies, but important recent studies have utilized the techniques of molecular biology and receptors have been cloned and characterized in primitive invertebrates, including the social amoeba Dictyostelium and the platyhelminth Schistosoma, as well as the green algae Ostreococcus, which resemble P2X receptors identified in mammals. This suggests that contrary to earlier speculations, P2X ion channel receptors appeared early in evolution, while G protein-coupled P1 and P2Y receptors were introduced either at the same time or perhaps even later. The absence of gene coding for P2X receptors in some animal groups [e.g. in some insects, roundworms (Caenorhabditis elegans) and the plant Arabidopsis] in contrast to the potent pharmacological actions of nucleotides in the same species, suggests that novel receptors are still to be discovered. [source] Caenorhabditis elegans expresses three functional profilins in a tissue-specific mannerCYTOSKELETON, Issue 1 2006D. Polet Abstract Profilins are actin binding proteins, which also interact with polyphosphoinositides and proline-rich ligands. On the basis of the genome sequence, three diverse profilin homologues (PFN) are predicted to exist in Caenorhabditis elegans. We show that all three isoforms PFN-1, PFN-2, and PFN-3 are expressed in vivo and biochemical studies indicate they bind actin and influence actin dynamics in a similar manner. In addition, they bind poly(L -proline) and phosphatidylinositol 4,5-bisphosphate micelles. PFN-1 is essential whereas PFN-2 and PFN-3 are nonessential. Immunostainings revealed different expression patterns for the profilin isoforms. In embryos, PFN-1 localizes in the cytoplasm and to the cell,cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in the neurons, the vulva, and the somatic gonad, PFN-2 in the intestinal wall, the spermatheca, and the pharynx, and PFN-3 localizes in a striking dot-like fashion in body wall muscle. Thus the model organism Caenorhabditis elegans expresses three profilin isoforms and is the first invertebrate animal with tissue-specific profilin expression. Cell Motil. Cytoskeleton, 2006.© 2005 Wiley-Liss, Inc. [source] GATA factors as key regulatory molecules in the development of Drosophila endodermDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2005Ryutaro Murakami Essential roles for GATA factors in the development of endoderm have been reported in various animals. A Drosophila GATA factor gene, serpent (srp, dGATAb, ABF), is expressed in the prospective endoderm, and loss of srp activity causes transformation of the prospective endoderm into ectodermal foregut and hindgut, indicating that srp acts as a selector gene to specify the developmental fate of the endoderm. While srp is expressed in the endoderm only during early stages, it activates a subsequent GATA factor gene, dGATAe, and the latter continues to be expressed specifically in the endoderm throughout life. dGATAe activates various functional genes in the differentiated endodermal midgut. An analogous mode of regulation has been reported in Caenorhabditis elegans, in which a pair of GATA genes, end-1/3, specifies endodermal fate, and a downstream pair of GATA genes, elt-2/7, activates genes in the differentiated endoderm. Functional homology of GATA genes in nature is apparently extendable to vertebrates, because endodermal GATA genes of C. elegans and Drosophila induce endoderm development in Xenopus ectoderm. These findings strongly imply evolutionary conservation of the roles of GATA factors in the endoderm across the protostomes and the deuterostomes. [source] Non-apoptotic cell death in Caenorhabditis elegansDEVELOPMENTAL DYNAMICS, Issue 5 2010Manolis Vlachos Abstract The simple nematode worm Caenorhabditis elegans has been instrumental in deciphering the molecular mechanisms underlying apoptosis. Beyond apoptosis, several paradigms of non-apoptotic cell death, either genetically or extrinsically triggered, have also been described in C. elegans. Remarkably, non-apoptotic cell death in worms and pathological cell death in humans share numerous key features and mechanistic aspects. Such commonalities suggest that similarly to apoptosis, non-apoptotic cell death mechanisms are also conserved, and render the worm a useful organism, in which to model and dissect human pathologies. Indeed, the genetic malleability and the sophisticated molecular tools available for C. elegans have contributed decisively to advance our understanding of non-apoptotic cell death. Here, we review the literature on the various types of non-apoptotic cell death in C. elegans and discuss the implications, relevant to pathological conditions in humans. Developmental Dynamics 239:1337,1351, 2010. © 2010 Wiley-Liss, Inc. [source] Spermatogenesis-defective (spe) mutants of the nematode Caenorhabditis elegans provide clues to solve the puzzle of male germline functions during reproductionDEVELOPMENTAL DYNAMICS, Issue 5 2010Hitoshi Nishimura Abstract In most species, each sex produces gametes, usually either sperm or oocytes, from its germline during gametogenesis. The sperm and oocyte subsequently fuse together during fertilization to create the next generation. This review focuses on spermatogenesis and the roles of sperm during fertilization in the nematode Caenorhabditis elegans, where suitable mutants are readily obtained. So far, 186 mutants defective in the C. elegans male germline functions have been isolated, and many of these mutations are alleles for one of the ,60 spermatogenesis-defective (spe) genes. Many cloned spe genes are expressed specifically in the male germline, where they play roles during spermatogenesis (spermatid production), spermiogenesis (spermatid activation into spermatozoa), and/or fertilization. Moreover, several spe genes are orthologs of mammalian genes, suggesting that the reproductive processes of the C. elegans and the mammalian male germlines might share common pathways at the molecular level. Developmental Dynamics 239:1502,1514, 2010. © 2010 Wiley-Liss, Inc. [source] PRP-17 and the pre-mRNA splicing pathway are preferentially required for the proliferation versus meiotic development decision and germline sex determination in Caenorhabditis elegansDEVELOPMENTAL DYNAMICS, Issue 5 2010Jessica Amrozowicz Kerins Abstract In C. elegans, the decision between germline stem cell proliferation and entry into meiosis is controlled by GLP-1 Notch signaling, which promotes proliferation through repression of the redundant GLD-1 and GLD-2 pathways that direct meiotic entry. We identify prp-17 as another gene functioning downstream of GLP-1 signaling that promotes meiotic entry, largely by acting on the GLD-1 pathway, and that also functions in female germline sex determination. PRP-17 is orthologous to the yeast and human pre-mRNA splicing factor PRP17/CDC40 and can rescue the temperature-sensitive lethality of yeast PRP17. This link to splicing led to an RNAi screen of predicted C. elegans splicing factors in sensitized genetic backgrounds. We found that many genes throughout the splicing cascade function in the proliferation/meiotic entry decision and germline sex determination indicating that splicing per se, rather than a novel function of a subset of splicing factors, is necessary for these processes. Developmental Dynamics 239:1555,1572, 2010. © 2010 Wiley-Liss, Inc. [source] Analysis of conserved residues in the ,pat-3 cytoplasmic tail reveals important functions of integrin in multiple tissuesDEVELOPMENTAL DYNAMICS, Issue 3 2010Xiaojian Xu Abstract Integrin cytoplasmic tails contain motifs that link extracellular information to cell behavior such as cell migration and contraction. To investigate the cell functions mediated by the conserved motifs, we created mutations in the Caenorhabditis elegans ,pat-3 cytoplasmic tail. The ,1D (799FK800), NPXY, tryptophan (784W), and threonine (797TT798) motifs were disrupted to identify their functions in vivo. Animals expressing integrins with disrupted NPXY motifs were viable, but displayed distal tip cell migration and ovulation defects. The conserved threonines were required for gonad migration and contraction as well as tail morphogenesis, whereas disruption of the ,1D and tryptophan motifs produced only mild defects. To abolish multiple conserved motifs, a ,1C-like variant, which results in a frameshift, was constructed. The ,pat-3(,1C) transgenic animals showed cold-sensitive larval arrests and defective muscle structure and gonad migration and contraction. Our study suggests that the conserved NPXY and TT motifs play important roles in the tissue-specific function of integrin. Developmental Dynamics 239:763,772, 2010. © 2010 Wiley-Liss, Inc. [source] Critical and sensitive periods for reversing the effects of mechanosensory deprivation on behavior, nervous system, and development in Caenorhabditis elegansDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2007Susan Rai Abstract In these studies the nematode Caenorhabditis elegans was used as a model to investigate ways to reverse the effects of mechanosensory deprivation on behavior and development. Rose et al. (J Neurosci 2005; 25:7159,7168) showed that worms reared in isolation responded significantly less to a mechanical tap stimulus, were significantly smaller, and expressed significantly lower levels of a postsynaptic glutamate receptor subunit on the command interneurons of the tap response circuit and a presynaptic vesicle marker in the tap sensory neurons compared with worms raised in groups. Here, brief mechanical stimulation at any time throughout development reversed the effects of isolation on the response to tap and on postsynaptic glutamate receptor expression on the command interneurons, suggesting there is no critical period for these measures. In contrast to the high level of plasticity in glutamate receptor subunit expression on the interneurons, low levels of stimulation only rescued vesicle expression in the tap sensory neurons early in development and progressively higher levels of stimulation were required as the worm developed, suggesting a sensitive period immediately after hatching, followed by a period of decreasing plasticity. Stimulation during the first three stages of larval development, but not later, rescued the effects of isolation on worm length, suggesting there is a critical period for this measure that ends in the third larval stage. These results indicate that different effects of early isolation required different amounts and/or timing of stimulation to be reversed. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Drosophila neuropeptide F mediates integration of chemosensory stimulation and conditioning of the nervous system by foodDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2001Ping Shen Abstract The conserved neuropeptide Y (NPY) signaling pathway has been strongly implicated in the stimulation of food uptake in vertebrates as well as in the regulation of food conditioned foraging behaviors of Caenorhabditis elegans. Using in situ RNA hybridization and immunocytochemistry, we report the neuronal network of Drosophila neuropeptide F (dNPF), a human NPY homologue, in the larval central nervous system and its food-dependent modifications. We provide indications that gustatory stimulation by sugar, but not its ingestion or metabolism, is sufficient to trigger long-term, dose-dependent alterations of the dNPF neuronal circuit through both dnpf activation and increased synaptic transmission. Our results strongly suggest that the dNPF neuronal circuit is an integral part of the sensory system that mediates food signaling, providing the neural basis for understanding how invertebrate NPY regulates food response. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 16,25, 2001 [source] Monogenic migraine syndromes highlight novel drug targetsDRUG DEVELOPMENT RESEARCH, Issue 7 2007J. Jay Gargus Abstract In the post-genomic era, the paradigm for drug discovery has changed, as every gene may become a potential target. Genetic diseases provide a special window into gene target selection. This approach is being applied to migraine making use of the genes and mutations causing familial hemiplegic migraine (FHM). FHM is caused by missense mutations in CACNA1A, altering a neuronal P/Q Ca2+ channel, in ATP1A2, altering ,2 Na,K-ATPase, and in SCN1A, altering a neuronal sodium channel. These genes provide insights into migraine pathogenesis that likely extend to other forms of migraine as well. Since the three FHM genes are only co-expressed in neurons, FHM is a neuronal, not a vascular, disease and because they all encode ion transport proteins, FHM is a neuronal channelopathy,meaning meta-stable neuronal hyperexcitability is the substrate of migraine, much as it is for genetic epilepsy syndromes. This similarity is reinforced, since different mutations of all three FHM genes can produce seizure syndromes. This has implications for drug discovery in that seizure medications already known to modulate the FHM channel mechanisms warrant more targeted development, and that drugs targeted to vascular headaches, such as the historically effective triptans, or experimental botulinum toxin, may well work by similar nonvascular mechanisms. Finally, in model neurogenetic systems such as Caenorhabditis elegans, the FHM genes also provide both a comprehensive means to discover all genes involved in their signaling pathway,genes potentially involved in common forms of the disease, and an in vivo whole animal means to screen rapidly for novel therapeutics. Drug Dev Res 68:432,440, 2007. © 2008 Wiley-Liss, Inc. [source] Experimental evolution of dispersal in spatiotemporally variable microcosmsECOLOGY LETTERS, Issue 10 2003Nicholas A. Friedenberg Abstract The world is an uncertain place. Individuals' fates vary from place to place and from time to time. Natural selection in unpredictable environments should favour individuals that hedge their bets by dispersing offspring. I confirm this basic prediction using Caenorhabditis elegans in experimental microcosms. My results agree with evolutionary models and correlations found previously between habitat stability and individual dispersal propensity in nature. However, I also find that environmental variation that triggers conditional dispersal behaviour may not impose selection on baseline dispersal rates. These findings imply that an increased rate of disturbance in natural systems has the potential to cause an evolutionary response in the life history of impacted organisms. [source] Novel DNA repair alkyltransferase from Caenorhabditis elegansENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2001Sreenivas Kanugula Abstract O6 -Alkylguanine DNA-alkyltransferase (AGT) is a widely distributed DNA repair protein that protects living organisms from endogenous and exogenous alkylation damage to DNA at the O6 -position of guanine. The search of the C. elegans genome database for an AGT protein revealed the presence of a protein (cAGT-2) with some similarity to known AGTs in addition to the easily recognized cAGT-1 protein. The predicted protein sequence of cAGT-2 contains the amino acid sequence ,ProCysHisPro, at the presumed active site of the protein, whereas all other known AGTs have ,ProCysHisArg,. A truncated version of the cAGT-2 protein was expressed in E. coli. This purified recombinant protein was able to repair O6 -methylguanine and O4 -methylthymine adducts in DNA in vitro and also reacted with the bulky benzyl adduct in O6 -benzylguanine. This fragment of cAGT-2 (104 amino acids) is the smallest protein possessing AGT activity yet described. The full-length cAGT-2 protein (274 amino acids) totally lacks the N-terminal domain present in all other known AGTs but has a long C-terminal extension that has significant homology to histone 1C. Expression of cAGT-2 in an E. coli strain lacking endogenous AGT activity provided modest but statistically significant resistance to the toxicity of N -methyl- N,-nitro- N -nitrosoguanidine, confirming that cAGT-2 is an alkyltransferase. Environ. Mol. Mutagen. 38:235,243, 2001. © 2001 Wiley-Liss, Inc. [source] Toxicity of manufactured zinc oxide nanoparticles in the nematode Caenorhabditis elegansENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2009Hongbo Ma Abstract Information describing the possible impacts of manufactured nanoparticles on human health and ecological receptors is limited. The objective of the present study was to evaluate the potential toxicological effects of manufactured zinc oxide nanoparticles (ZnO-NPs; 1.5 nm) compared to aqueous zinc chloride (ZnCl2) in the free-living nematode Caenorhabditis elegans. Toxicity of both types of Zn was investigated using the ecologically relevant endpoints of lethality, behavior, reproduction, and transgene expression in a mtl-2::GFP (gene encoding green fluorescence protein fused onto the metallothionein-2 gene promoter) transgenic strain of C. elegans. Zinc oxide nanoparticles showed no significant difference from ZnCl2 regarding either lethality or reproduction in C. elegans, as indicated by their median lethal concentrations (LC50s; p = 0.29, n = 3) and median effective concentrations (EC50s; Z = 0.835, p = 0.797). Also, no significant difference was found in EC50s for behavioral change between ZnO-NPs (635 mg Zn/L; 95% confidence interval [CI], 477,844 mg Zn/L) and ZnCl2 (546 mg Zn/L; 95% CI, 447,666 mg Zn/L) (Z = 0.907, p = 0.834). Zinc oxide nanoparticles induced transgene expression in the mtl-2::GFP transgenic C. elegans in a manner similar to that of ZnCl2, suggesting that intracellular biotransformation of the nanoparticles might have occurred or the nanoparticles have dissolved to Zn2+ to enact toxicity. These findings demonstrate that manufactured ZnO-NPs have toxicity to the nematode C. elegans similar to that of aqueous ZnCl2. [source] Toxicological assessment of chemicals using Caenorhabditis elegans and optical oxygen respirometryENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2009Katherine Schouest Abstract Oxygen consumption is indicative of an organism's metabolic state, whereby alterations in respiration rate can result from the presence of different stimuli. Here, we develop a novel approach based on quenched fluorescence oxygen sensing and respirometry method for toxicity screening assays using the nematode Caenorhabditis elegans. Previously, C. elegans was established as a useful model in soil and aquatic toxicology studies. For existing toxicology screening approaches with C. elegans, however, the endpoint is lethality. In addition, the assay time frame for the existing approaches is considerably longer than that for the approach described here. We present a sensitive, robust, high-throughput platform using standard laboratory equipment for toxicological studies by measuring respiration rate in C. elegans animals using a phosphorescent probe. [source] Transgenic strains of the nematode Caenorhabditis elegans as biomonitors of metal contaminationENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2000L. K. Cioci Abstract Transition metal contamination poses a serious environmental and human health threat. The bioavailability of transition metals in environmental samples can best be assessed with living organisms. A transgenic strain of the free-living soil nematode Caenorhabditis elegans has been engineered for monitoring the bioavailability of metals. A reporter transgene consisting of a fragment of the promoter from the C. elegans metallothionein-2 gene (mtl-2) that controls the transcription of a ,-galactosidase reporter (lacZ) has been integrated into the genome of this organism. By using these transgenic C. elegans, the toxicological response to metals in samples can be quickly measured with a simple histochemical staining assay. The C. elegans that contain the mtl-2:lacZ transgene provide a more sensitive assay of exposure to cadmium, mercury, zinc, and nickel than 24-h LC50 assays or those using nematodes with heat-shock protein,based reporter transgenes. This study demonstrates that C. elegans that contain mtl-2:lacZ transgenes can function as sensitive toxicological indicators of metals. [source] Synthesis and Hormonal Activity of the (25S)-Cholesten-26-oic Acids , Potent Ligands for the DAF-12 Receptor in Caenorhabditis elegansEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 22 2009René Martin Abstract Using a highly stereoselective Evans aldol reaction for the introduction of the stereogenic center at C-25, we describe an efficient synthesis of the orthogonally diprotected (25S)-26-hydroxycholesterol 11. In a few synthetic steps, this crucial intermediate 11 has been converted into the four (25S)-cholesten-26-oic acids 1,4, which have been obtained in 12,15 steps and 19,53,% overall yield based on commercially available 3,-hydroxychol-5-en-24-oic acid (5). Our biological studies of the compounds 1,4 reveal that (25S)-,7 -dafachronic acid (1) represents the most active steroidal ligand for the hormonal receptor DAF-12 in Caenorhabditis elegans. Moreover, the saturated (25S)-dafachronic acid (3) represents a new ligand for this receptor and the (25S)-steroidal acids are more active as compared to their corresponding (25R)-counterparts.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] THE FITNESS EFFECTS OF SPONTANEOUS MUTATIONS IN CAENORHABDITIS ELEGANSEVOLUTION, Issue 4 2000Larissa L. Vassilieva Abstract. Spontaneous mutation to mildly deleterious alleles has emerged as a potentially unifying component of a variety of observations in evolutionary genetics and molecular evolution. However, the biological significance of hypotheses based on mildly deleterious mutation depends critically on the rate at which new mutations arise and on their average effects. A long-term mutation-accumulation experiment with replicate lines of the nematode Caenorhabditis elegans maintained by single-progeny descent indicates that recurrent spontaneous mutation causes approximately 0.1% decline in fitness per generation, which is about an order of magnitude less than that suggested by previous studies with Drosophila. Two rather different approaches, Bateman-Mukai and maximum likelihood, suggest that this observation, along with the observed rate of increase in the variance of fitness among lines, is consistent with a genomic deleterious mutation rate for fitness of approximately 0.03 per generation and with an average homozygous effect of approximately 12%. The distribution of mutational effects for fitness appears to have a relatively low coefficient of variation, being no more extreme than expected for a negative exponential, and for one composite fitness measure (total progeny production) approaches constancy of effects. These results are derived from assays in a benign environment. At stressful temperatures, estimates of the genomic deleterious mutation rate (for genes expressed at such temperatures) is sixfold lower, whereas those for the average homozygous effect is approximately eightfold higher. Our results are reasonably compatible with existing estimates for flies, when one considers the differences between these species in the number of germ-line cell divisions per generation and the magnitude of transposable element activity. [source] A sensitized genetic background reveals evolution near the terminus of the Caenorhabditis germline sex determination pathwayEVOLUTION AND DEVELOPMENT, Issue 4 2009Robin Cook Hill SUMMARY Caenorhabditis elegans and Caenorhabditis briggsae are both self-fertile hermaphroditic nematodes that evolved independently from male/female ancestors. In C. elegans, FEM-1, FEM-2, and FEM-3 specify male fates by promoting proteolysis of the male-repressing transcription factor, TRA-1. Phenotypes of tra-1 and fem mutants are consistent with this simple linear model in the soma, but not in the germline. While both XX and XO tra-1(lf) mutants have functional male somas, they produce both sperm and oocytes. Further, all three tra-1; fem double mutants retain the expected male soma, but make only oocytes (the germline fem phenotype). Thus, a poorly characterized tra-1 activity is important for sustained male spermatogenesis, and the fem genes affect germline sexual fate independently of their role in regulating TRA-1. C. briggsae tra-1 mutants are phenotypically identical to their C. elegans counterparts, while the fem mutants differ in the germline: XX and XO C. elegans fem mutants are true females, but in C. briggsae they are self-fertile hermaphrodites. To further explore how C. briggsae hermaphrodites regulate germline sex, we analyzed Cb-tra-1/Cb-fem interactions. Cb-tra-1 is fully epistatic to Cb-fem-2 in the germline, unlike the orthologous C. elegans combination. In contrast, Cb-fem-3 shifts the Cb-tra-1(lf) germline phenotype to that of a nearly normal hermaphrodite in the context of a male somatic gonad. This suggests that Cb-fem-3 is epistatic to Cb-tra-1(lf) (as in C. elegans), and that the normal control of C. briggsae XX spermatogenesis targets Cb-tra-1 -independent factors downstream of Cb-fem-3. The effect of Cb-fem-3(lf) on Cb-tra-1(lf) is not mediated by change in the expression of Cb-fog-3, a likely direct germline target of Cb-tra-1. As Cb-fem-2 and Cb-fem-3 have identical single mutant phenotypes, Cb-tra-1 provides a sensitized background that reveals differences in how they promote male germline development. These results represent another way in which C. briggsae germline sex determination is incongruent with that of the outwardly similar C. elegans. [source] Characterization of the Hox cluster from the mosquito Anopheles gambiae (Diptera: culicidae)EVOLUTION AND DEVELOPMENT, Issue 6 2000Thomas P. Powers SUMMARY The Hox genes have been found to encode transcription factors, which specify the morphological identity of structures along the anteroposterior axis of animals ranging from worms to mice. The canonical set of nine genes is organized in a cluster in the genome of several protostomes and deuterostomes. However, within insects, whereas the Hox genes are organized in a single cluster in the beetle Tribolium castaneum, they are split into two separate groups in the flies Drosophila melanogaster and Drosophila virilis. The significance of a split Hox cluster is unknown and has been observed in only one organism outside the Drosophila lineage: the nematode Caenorhabditis elegans. We have cloned a majority of the Hox genes from the mosquito Anopheles gambiae (Diptera: Culicidae) and compared their genomic organization with that of Tribolium and Drosophila to determine if a split Hox cluster is found in dipterans aside from the Drosophilidae. We find that the Hox genes in Anopheles, as in Tribolium, are organized in a single cluster that spans a genomic region of at least 700 kb. This finding suggests that, within the insect genome, the partition of the Hox cluster may have evolved exclusively within the Drosophila lineage. The genomic structures of the resident genes, however, appear to be largely conserved between A. gambiae and D. melanogaster. [source] A Caenorhabditis elegans model of orotic aciduria reveals enlarged lysosome-related organelles in embryos lacking umps-1 functionFEBS JOURNAL, Issue 6 2010Steven Levitte Gut granules are cell type-specific lysosome-related organelles found within the intestinal cells of Caenorhabditis elegans. To investigate the regulation of lysosome-related organelle size, we screened for C. elegans mutants with substantially enlarged gut granules, identifying alleles of the vacuolar-type H+ -ATPase and uridine-5,-monophosphate synthase (UMPS)-1. UMPS-1 catalyzes the conversion of orotic acid to UMP; this comprises the two terminal steps in de novo pyrimidine biosynthesis. Mutations in the orthologous human gene UMPS result in the rare genetic disease orotic aciduria. The umps-1(,) mutation promoted the enlargement of gut granules to 250 times their normal size, whereas other endolysosomal organelles were not similarly affected. UMPS-1::green fluorescent protein was expressed in embryonic and adult intestinal cells, where it was cytoplasmically localized and not obviously associated with gut granules. Whereas the umps-1(,) mutant is viable, combination of umps-1(,) with mutations disrupting gut granule biogenesis resulted in synthetic lethality. The effects of mutations in pyr-1, which encodes the enzyme catalyzing the first three steps of de novo pyrimidine biosynthesis, did not phenotypically resemble those of umps-1(,); instead, the synthetic lethality and enlargement of gut granules exhibited by the umps-1(,) mutant was suppressed by pyr-1(,). In a search for factors that mediate the enlargement of gut granules in the umps-1(,) mutant, we identified WHT-2, an ABCG transporter previously implicated in gut granule function. Our data suggest that umps-1(,) leads to enlargement of gut granules through a build-up of orotic acid. WHT-2 possibly facilitates the increase in gut granule size of the umps-1(,) mutant by transporting orotic acid into the gut granule and promoting osmotically induced swelling of the compartment. [source] A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situFEBS JOURNAL, Issue 12 2008Fanjie Meng To measure mechanical stress in real time, we designed a fluorescence resonance energy transfer (FRET) cassette, denoted stFRET, which could be inserted into structural protein hosts. The probe was composed of a green fluorescence protein pair, Cerulean and Venus, linked with a stable ,-helix. We measured the FRET efficiency of the free cassette protein as a function of the length of the linker, the angles of the fluorophores, temperature and urea denaturation, and protease treatment. The linking helix was stable to 80 °C, unfolded in 8 m urea, and rapidly digested by proteases, but in all cases the fluorophores were unaffected. We modified the ,-helix linker by adding and subtracting residues to vary the angles and distance between the donor and acceptor, and assuming that the cassette was a rigid body, we calculated its geometry. We tested the strain sensitivity of stFRET by linking both ends to a rubber sheet subjected to equibiaxial stretch. FRET decreased proportionally to the substrate strain. The naked cassette expressed well in human embryonic kidney-293 cells and, surprisingly, was concentrated in the nucleus. However, when the cassette was located into host proteins such ,-actinin, nonerythrocyte spectrin and filamin A, the labeled hosts expressed well and distributed normally in cell lines such as 3T3, where they were stressed at the leading edge of migrating cells and relaxed at the trailing edge. When collagen-19 was labeled near its middle with stFRET, it expressed well in Caenorhabditis elegans, distributing similarly to hosts labeled with a terminal green fluorescent protein, and the worms behaved normally. [source] Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirementsFEBS JOURNAL, Issue 2 2006Hiromi Kochiwa Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA,RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg2+ ions, Caenorhabditis elegans has four RNase H1-related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1-related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene-specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg2+ ions, C. elegans has another enzyme that shows preference for Mn2+ ion as a cofactor. We characterized this Mn2+ -dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism. [source] Cloning of the guanylate kinase homologues AGK-1 and AGK-2 from Arabidopsis thaliana and characterization of AGK-1FEBS JOURNAL, Issue 2 2000Vinod Kumar Guanylate kinase is an essential enzyme for nucleotide metabolism, phosphorylating GMP to GDP or dGMP to dGDP. The low molecular mass cytosolic forms of guanylate kinase are implicated primarily in the regulation of the supply of guanine nucleotides to cell signalling pathways. The high molecular mass and membrane-associated forms of guanylate kinase homologues, notably found in neuronal tissues, are assigned roles in cell junction organization and transmembrane regulation. Here, we describe the first plant guanylate kinase-encoding genes, AGK1 and AGK2, from Arabidopsis thaliana. The nucleotide sequences of their genomic and cDNA clones predict proteins that carry N-terminal and C-terminal extensions of the guanylate kinase-like domain. The amino acid sequences of this domain share 46,52% identity with guanylate kinases from yeast, Escherichia coli, human, mouse and Caenorhabditis elegans. Arabidopsis guanylate kinases (AGKs) exhibit a high degree of conservation of active site residues and sequence motifs in common with other nucleoside monophosphate kinases, which suggests overall structural similarity of the plant proteins. Although bacterially expressed AGK-1 is enzymatically much less active than yeast guanylate kinase, its kinase domain is shown to complement yeast GUK1 recessive lethal mutations. AGKs are expressed ubiquitously in plant tissues with highest transcriptional activity detected in roots. The identification of AGKs provides new perspectives for understanding the role of guanylate kinases in plant cell signalling pathways. [source] Genome characterization, analysis of virulence and transformation of Microbacterium nematophilum, a coryneform pathogen of the nematode Caenorhabditis elegansFEMS MICROBIOLOGY LETTERS, Issue 2 2006Tatiana Akimkina Abstract A coryneform bacterium designated Microbacterium nematophilum has previously been reported to act as a pathogen for Caenorhabditis elegans. This bacterium is able to colonize the rectum of infected worms and cause localized swelling, constipation and slowed growth. Additional isolates and analysis of this bacterium are described here. Tests of pathogenicity on other Caenorhabditis nematodes show that M. nematophilum infection is lethal to most species in the genus, in contrast to its relatively mild effects on C. elegans. The size and geometry of the pathogen genome have been determined as a closed circular molecule of 2.85 Mb with high G+C content. Bacteria also harbor a 55 kb plasmid, pMN1, which is largely composed of a lysogenic bacteriophage genome. Mutagenesis experiments have yielded stable avirulent mutants of M. nematophilum. As a first step towards molecular genetic analysis, methods for low-efficiency transformation of M. nematophilum have been developed. [source] FLR-2, the glycoprotein hormone alpha subunit, is involved in the neural control of intestinal functions in Caenorhabditis elegansGENES TO CELLS, Issue 10 2009Akane Oishi The intestine plays an essential role in organism-wide regulatory networks in both vertebrates and invertebrates. In Caenorhabditis elegans, class 1 flr genes (flr-1, flr-3 and flr-4) act in the intestine and control growth rates and defecation cycle periods, while class 2 flr genes (flr-2, flr-5, flr-6 and flr-7) are characterized by mutations that suppress the slow growth of class 1 flr mutants. This study revealed that flr-2 gene controls antibacterial defense and intestinal color, confirming that flr-2 regulates intestinal functions. flr-2 encoded the only glycoprotein hormone alpha subunit in C. elegans and was expressed in certain neurons. Furthermore, FLR-2 bound to another secretory protein GHI-1, which belongs to a family of lipid- and lipopolysaccharide-binding proteins. A ghi-1 deletion mutation partially suppressed the short defecation cycle periods of class 1 flr mutants, and this effect was enhanced by flr-2 mutations. Thus, FLR-2 acts as a signaling molecule for the neural control of intestinal functions, which is achieved in a functional network involving class 1 and class 2 flr genes as well as ghi-1. These results are informative to studies of glycoprotein hormone signaling in higher animals. [source] [FeFe]-hydrogenase-like gene is involved in the regulation of sensitivity to oxygen in yeast and nematodeGENES TO CELLS, Issue 4 2009Michihiko Fujii Oxygen is essential for the life of aerobic organisms, but reactive oxygen species (ROS) derived from oxygen can be a threat for it. Many genes are involved in generation of ROS, but not much attention has been focused on the reactions from which ROS are generated. We therefore screened for mutants that showed an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans, and isolated a novel mutant, oxy-4(qa5001). This mutant showed an increased sensitivity to a high concentration of oxygen, and decreased longevity at 20 °C but not at 26 °C. The genetic analysis has revealed that oxy-4 had a causative mutation in an [FeFe]-hydrogenase-like gene (Y54H5A.4). In the yeast Saccharomyces cerevisiae, a deletion of NAR1, a possible homologue of oxy-4, also caused a similar increased sensitivity to oxygen. [FeFe]-hydrogenases are enzymes that catalyze both the formation and the splitting of molecular hydrogen, and function in anaerobic respiration in anaerobes. In contrast, [FeFe]-hydrogenase-like genes identified in aerobic eukaryotes do not generate hydrogen, and its functional roles are less understood. Our results suggested that [FeFe]-hydrogenase-like genes were involved in the regulation of sensitivity to oxygen in S. cerevisiae and C. elegans. [source] Non-receptor tyrosine kinase CSK-1 controls pharyngeal muscle organization in Caenorhabditis elegansGENES TO CELLS, Issue 3 2009Nozomu Takata C-terminal Src kinase (Csk) is a non-receptor type of tyrosine kinase, and serves as an essential negative regulator of Src family tyrosine kinases (SFKs) in vertebrates. However, analyses of Csk and SFKs from primitive animals suggest that the Csk-mediated mechanisms regulating SFK activity might diverge between evolutional branches, different tissues or SFK family members. We examined in vivo roles of CSK-1, a Caenorhabditis elegans orthologue of Csk, by generating animals lacking csk-1 function. Although some csk-1 mutants died during embryogenesis, the majority of mutants died during the first stage of larval development. In csk-1 mutants, the function of pharyngeal muscles, the major site of CSK-1 expression, was severely damaged. The pumping of pharyngeal grinder cells became arrhythmic, causing disabled feeding. Electron microscopy showed that pharyngeal muscle filaments were disorientated in the csk-1 mutants. These indicate that CSK-1 is crucial for proper organization of pharyngeal muscles. However, the growth arrest phenotype in csk-1 mutants could not be suppressed by src-1 and/or src-2 mutation, and SRC-1 was not significantly activated in the csk-1 mutants. These results suggest that CSK-1 has an essential function in organization of pharyngeal muscle filaments that does not require C. elegans SFKs. [source] Efficient production of monoclonal antibodies recognizing specific structures in Caenorhabditis elegans embryos using an antigen subtraction methodGENES TO CELLS, Issue 7 2008Kazumasa Takeda Monoclonal antibodies (mAbs) have been widely used to probe molecular components of specific cell types or cellular structures. We have developed a method to enrich antigens of low abundance in heterogeneous molecule mixtures by subtracting abundant antigens. The subtracted immunogen mixture is then used for immunization, which significantly increases the production of mAbs that exhibit specific staining patterns. By applying this "antigen subtraction" method to the embryonic extract of Caenorhabditis elegans, we have successfully isolated 35 mAbs that recognize specific structures, including P granules, muscles, the pharynx, and subsets of hypodermal cells; some of the mAbs revealed previously unreported cellular structures. This antigen subtraction approach can be used in various applications to produce mAbs against relatively scarce antigens in complex molecular mixtures. The mAbs will be useful tools for developmental and cell biological studies. [source] Caenorhabditis elegans DYF-11, an orthologue of mammalian Traf3ip1/MIP-T3, is required for sensory cilia formationGENES TO CELLS, Issue 1 2008Hirofumi Kunitomo Cilia and flagella play critical roles in cell motility, development and sensory perception in animals. Formation and maintenance of cilia require a conserved protein transport system called intraflagellar transport (IFT). Here, we show that Caenorhabditis elegans dyf-11 encodes an evolutionarily conserved protein required for cilium biogenesis. dyf-11 is expressed in most of the ciliated neurons and is regulated by DAF-19, a crucial transcription factor for ciliary genes in C. elegans. dyf-11 mutants exhibit stunted cilia, fluorescent dye-filling defects (Dyf) of sensory neurons, and abnormal chemotaxis (Che). Cell- and stage-specific rescue experiments indicated that DYF-11 is required for formation and maintenance of sensory cilia in cell-autonomous manner. Fluorescent protein-tagged DYF-11 localizes to cilia and moves antero- and retrogradely via IFT. Analysis of DYF-11 movement in bbs mutants further suggested that DYF-11 is likely associated with IFT complex B. Domain analysis using DYF-11 deletion constructs revealed that the coiled-coil region is required for proper localization and ciliogenesis. We further show that Traf3ip1/MIP-T3, the mammalian orthologue of DYF-11, localizes to cilia in the MDCK renal epithelial cells. [source] Dicer-related drh-3 gene functions in germ-line development by maintenance of chromosomal integrity in Caenorhabditis elegansGENES TO CELLS, Issue 9 2007Masaharu Nakamura In the course of systematic RNA interference (RNAi)-based screens with helicase-like genes in Caenorhabditis elegans, we have identified the drh-3(D2005.5) gene as a candidate gene for protection against X-ray irradiation. This gene encodes a novel RNA helicase-like protein that is similar to two nematode Dicer-related helicases (DRH). Here, we have showed the increased expression of drh-3 transcripts during maturation of larvae to adults, and characterized the phenotype of drh-3 -interferred nematodes using feeding RNAi method. RNAi-mediated depletion of the drh-3 transcripts caused embryonic lethality of F1 progeny and temperature-sensitive reproductive capacity but did not affect the nematode life span. F1 progeny from drh-3(RNAi) animals exhibited increased lethality after X-ray irradiation or exposure to camptothecin. In drh-3(RNAi) worms, aggregated chromosomes were observed in diakinesis oocyte nuclei. In developing early zygotic embryos from drh-3(RNAi) worms, abnormally segregated chromosomes were observed and embryonic development was largely arrested at the mid-stages of embryogenesis. Finally, examination of checkpoint responses in mitotic germ cells with regards to replication arrest by hydroxyurea and X-ray-induced DNA damage suggested that both checkpoints function normally under these genotoxic stress conditions. Taken together, these results indicate that the drh-3 gene is essential for the development of germ-lines by maintaining chromosomal integrity in C. elegans. 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