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[
East Coast Worm Meeting,
2002]
Six lethal mutations near the
unc-29 locus on chromosome I of Caenorhabditis elegans have been isolated previously. Among these mutants, one particular mutant (
jh1) showed embryonic lethal phenotype that shows specific defects in posterior body morphology during early development. Following the outcrosses, three-factor crosses were conducted to place the
jh1 mutation on a precise genetic map near the
unc-29 locus on chromosome I using
dpy-5(
e61),
dpy-5(
e61)
unc-13(e-1091) as genetic markers. The results of three factor crosses have placed the
jh1 mutation in the region between 0.42 and 0.64 map unit right from the center of chromosome I, which corresponds to approximately a 300 Kb region in the physical map. Along with the results of three-factor crosses, cosmid rescue experiments were performed by microinjection of 8 cosmids, which are located within the genetically mapped position. Among 8 cosmids, only the cosmid B0414 decreased the embryonic lethality in the subsequent generations, suggesting that genes in the cosmid B0414 could rescue the mutation. We are currently injecting subclones of this cosmid the gene that rescues the lethal mutations.
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[
International Worm Meeting,
2015]
The sequencing of the genome of Caenorhabditis elegans remains one of the milestones of modern biology, and this genome sequence is the essential backdrop to a vast body of work on this key model organism. "Nothing in biology makes sense except in the light of evolution" (Dobzhansky) and thus it is clear that complete understanding of C. elegans will only be achieved when it is placed in an evolutionary context. While several additional Caenorhabditis genomes have been published or made available, a recent surge in the number of available species in culture makes the determination of the genomes of all the species in the genus a timely and rewarding project.We have initiated the Caenorhabditis Genomes Project. From material supplied by collaborators we have so far generated raw Illumina short-insert data for sixteen species. Where possible we have also generated mixed stage stranded RNASeq data for annotation. The data are being made publicly available as early as possible (warts-and-all) through a dedicated genome website at htttp://caenorhabditis.bio.ed.ac.uk, and completed genomes and annotations will be deposited in WormBase as mature assemblies emerge. We welcome additional collaborators to the CGP, whether to assemble new genomes or to delve into the evolutionary history of favourite gene sets and systems.Species sequenced thus far in Edinburgh: Caenorhabditis afra, Caenorhabditis castelli, Caenorhabditis doughertyi, Caenorhabditis guadeloupensis, Caenorhabditis macrosperma, Caenorhabditis nouraguensis, Caenorhabditis plicata, Caenorhabditis virilis, Caenorhabditis wallacei, Caenorhabditis sp. 1, Caenorhabditis sp. 5, Caenorhabditis sp. 21, Caenorhabditis sp. 26, Caenorhabditis sp. 31, Caenorhabditis sp. 32, Caenorhabditis sp. 38, Caenorhabditis sp. 39, Caenorhabditis sp. 40, Caenorhabditis sp. 43.[Samples have been supplied by Aurelien Richaud, Marie-Anne Felix, Christian Braendle, Michael Alion, Piero Lamelza].
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[
International C. elegans Meeting,
1997]
We are studying nematode surface antigen switching, defined as the restriction of specific surface molecules to a particular time or developmental stage. This device may help parasitic nematodes to avoid host immune responses during infections. In wild-type C. elegans grown under standard conditions, monoclonal antibodies M37 and M38 recognize epitopes on the L1 surface. Mutations that result in display of these markers on all larval stages include those in a new gene,
srf-6 II, and temperature-sensitive mutations in dauer-constitutive (daf-c) genes. Some daf-c genes encode transmembrane ser/thr receptor kinases for TGF-b-like ligands. We have attempted to identify the
srf-6 gene by marker rescue. Srf-6 mutants transformed with either a mixture of three cloned cosmid DNAs or a single cosmid from the candidate interval showed significant rescue. The rescuing cosmid contains two interesting coding sequences. One shows similarity to a cDNA that encodes the major larval surface protein of the parasite Toxocara canis; the other shows similarity to ser/thr kinases. The proximity of these genes suggests that their expression may be coregulated. To further localize
srf-6, we are performing marker rescue experiments with portions of the rescuing cosmid and attempting to identify mutant-correlated DNA polymorphisms.
-
[
International C. elegans Meeting,
1991]
The C. elegans physical map has been assembled with a combination of cosmid and yeast artificial chromosome (YAC) clones. In the first phase, cosmid clone overlaps were detected using a high resolution fingerprinting technique. This produced a map with more than 700 contigs. Next, YACs were used to link together the multiple cosmid contigs via hybridization of the YACs to colony grids of representative cosmids. By the time this approach reached its practical limits, the map had been reduced to about 170 contigs. The current phase has involved detecting overlaps between protruding YAC clones at the ends of existing contigs, and also between small cosmid contigs and YACs. End sequence from the clones was obtained by using flanking vector primers either on total yeast genomic DNA for YACs or miniprep DNA for cosmids. PCR was then used to get unique hybridization probes. Hybridizing YACs were checked by PCR to confirm overlaps. From these efforts, the map now contains less than 90 contigs. Through the supporting evidence of genetic and physical information from other sources, more than 85 Mb of the assembled DNA has been assigned to chromosomes in 35 large contigs. Since C. elegans genes are preferentially located in the centers of chromosomes and the continuity of the map is greatest in these locations, all but a few coding sequences are now covered by contigs. The results of the physical map effort are available to the community in three forms: The database can be accessed either via network or modem from several locations throughout the world; a colony grid of 958 YACs, genomically ordered at the time of selection, has been prepared in a postcard sized array and replicas have been distributed to interested labs; YACs and cosmid clones covering any region are available upon request.
-
[
International C. elegans Meeting,
1997]
The early embryonic cell lineage of Pellioditis marina, a marine rhabditid with relatively short developing time (9hrs at 25!C) was traced using a 4D-microscope. Although the general pattern of cell division is congruent with the lineage described for Caenorhabditis elegans by Sulston and Co-workers, striking differences can be observed concerning migrations, timing of divisions and cell deaths. The AB, MS and C lineage of Pellioditis marina differ from those of Caenorhabditis elegans both in the occurence of additional cell deaths as well as in the abscence of certain cell deaths. Additionaly, Caap does not divide in accordance with the characteristic period for the rest of the C-lineage. In contrast with Caenorhabditis elegans, the E founder cell in Pellioditis marina undergoes a migration before gastrulation and divides into Ea and Ep only after E has entered the interior of the embryo. D and P4 divide in a similar way as in Caenorhabditis elegans.
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Hay, Megan, Smith, Mark, Emerson, Soren, Snyder, Nicole, Blauch, David, El Bejjani, Rachid, Granger, Ricky
[
International Worm Meeting,
2021]
DESPITE THE TOXICITY AND ADDICTIVE LIABILITY ASSOCIATED WITH COCAINE ABUSE, ITS MODE OF ACTION IS NOT COMPLETELY UNDERSTOOD, AND EFFECTIVE PHARMACOTHERAPEUTIC INTERVENTIONS REMAIN ELUSIVE. THE CHOLINERGIC EFFECTS OF COCAINE ON ACETYLCHOLINE RECEPTORS, SYNTHETIC ENZYMES, AND DEGRADATIVE ENZYMES HAVE BEEN THE FOCUS OF RELATIVELY LITTLE EMPIRICAL INVESTIGATION. DUE TO ITS GENETIC TRACTABILITY AND ANATOMICAL SIMPLICITY, THE EGG LAYING CIRCUIT OF THE HERMAPHRODITIC NEMATODE, CAENORHABDITIS ELEGANS, IS A POWERFUL MODEL SYSTEM TO PRECISELY EXAMINE THE GENETIC AND MOLECULAR TARGETS OF COCAINE IN VIVO. HERE, WE REPORT A NOVEL COCAINE-INDUCED BEHAVIORAL PHENOTYPE IN CAENORHABDITIS ELEGANS, COCAINE-STIMULATED EGG LAYING. IN ADDITION, WE PRESENT THE RESULTS OF AN IN VIVO CANDIDATE SUPPRESSION SCREEN OF SYNTHETIC ENZYMES, RECEPTORS, DEGRADATIVE ENZYMES, AND DOWNSTREAM COMPONENTS OF THE INTRACELLULAR SIGNALING CASCADES OF THE MAIN NEUROTRANSMITTER SYSTEMS THAT CONTROL CAENORHABDITIS ELEGANS EGG LAYING. OUR RESULTS SHOW THAT COCAINE-STIMULATED EGG LAYING IS DEPENDENT ON ACETYLCHOLINE SYNTHESIS AND SYNAPTIC RELEASE, FUNCTIONAL NICOTINIC ACETYLCHOLINE RECEPTORS, AND THE CAENORHABDITIS ELEGANS ACETYLCHOLINESTERASES.
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[
International C. elegans Meeting,
1995]
We have analyzed genetic mosaic animals using a procedure in which mosaic animals are generated by spontaneous somatic loss of an extrachromosomal array that is constructed by microinjection of cosmid DNA. We identified a cosmid clone that rescues the mutant phenotype of the cell-autonomous mosaic marker,
ncl-1. With the identification of this cosmid clone, any cloned gene can be linked to
ncl-1 on an extrachromosomal array, and the resulting array can be used efficiently in mosaic analysis experiments to determine in which cell or cells a gene acts. We used this modified method for mosaic analysis to show that
lin-31, a gene that is a member of the HNF-3 transcription factor famility and specifies cell fates during vulval induction, acts cell-autonomously in the target cells of a signaling pathway. We also used this method of mosaic analysis to show that the
unc-29 gene, which encodes a subunit of the acetylcholine receptor, acts in the body muscle cells.
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[
International Worm Meeting,
2007]
I found that males from the gonochoristic species Caenorhabditis sp. 4 (PB2801) and Caenorhabditis remanei (PB4641) induced behavioral passivity in con- and heterospecific virgin females during the spicule insertion step of mating. Males from the hermaphroditic species Caenorhabditis briggsae (AF16, VT847, PB826) also induced behavioral passivity in Caenorhabditis sp. 4 and C. remanei females, but not in their own conspecific hermaphrodites. Caenorhabditis elegans males (N2, CB4855, CB4856) did not induce mating-induced behavioral passivity in con- or heterospecific mates. I used C. elegans male spicule insertion behavior as a reference to understand how gonochoristic males induce behavioral changes in their mates. Through comparative laser ablation- and behavioral analyses between C. elegans and C. remanei, I found that gonochoristic males required the SPC neurons, the p.c.s. neurons and the somatic gonad to produce a soporific factor that immobilizes virgin females and stimulates their vulval slit to widen during copulation. C. elegans and C. briggsae hermaphrodites and non-virgin gonochoristic females were not affected by this factor. At present, I am using the C. elegans hermaphrodite anatomy and molecular biology as a reference to explore potential cells and molecules in gonochoristic females that respond to the male soporific factor. I found that the uterine vulval cells are required for gonochoristic females to be sedated by males. In C. elegans, the UV1 cells are believed to have secretory properties; thus I plan to over-express and RNAi-knock down C. remanei and species 4 genes that in C. elegans are expressed in the UV1 cells.
-
[
International Worm Meeting,
2005]
The availability of genomic clones with 35 to 40kb inserts has been a critical resource to the C. elegans community. Fingerprinted and ordered cosmid clones harboring such inserts were the backbone of the sequencing project. Further utility was found when individual research labs were trying to map mutants. Complementing a mutant by injecting a strain with overlapping cosmid clones eventually led to mutation identity. This approach is still widely used and is an important tool for mutational analysis. Many of the cosmid libraries are now 20 years old and there are reports of some of them losing viability. This persuaded us to consider building a new DNA library with total coverage of the genome. We also felt it was best to make a 40kb clone insert library rather than rely on YAC or BAC clone libraries. The large insert clones are not trivial to manipulate and do not in many cases give you the resolution that is required for many experiments. With these considerations in mind we have chosen to make a fosmid rather than a cosmid library. The cosmid libraries have been excellent workhorses but many clones within the libraries have been prone to rearrangements. By using a fosmid vector (pCC1FOS from Epicentre) that is maintained at low copy number until induced we hope that the occurrence of such rearrangements will be reduced. The use of fosmids as backbones allow for the maintenance of large pieces of DNA (around 40kB) in limited number (1-5) per bacterial host. Up to this point, such a resource was unavailable for C. elegans We have created an N2 fosmid library and are currently mapping clones to the genome. We are mapping all fosmid clones to the C. elegans genome by pair-wise alignment of fosmid end-reads. Initial analysis of the library quality shows that the average insert size is ~35 kb and 86% of the clones have paired end-reads with correct relative orientation. Currently, with over 6,100 clones sequenced we have obtained approximately 2X clone coverage of the genome. From this, we estimate that we will require ~16,000 clones to obtain our goal of 5X coverage of the genome. This resource was created for public domain use and is available to interested researchers.
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Franz, Carl J., Wang, David, Felix, Marie-Anne, Frezal, Lise, Renshaw, Hilary, Jiang, Yanfang
[
International Worm Meeting,
2013]
Orsay, Santeuil and Le Blanc viruses were recently discovered, enabling for the first time the study of virus-host interactions using a natural pathogen in the well-established model organism Caenorhabditis elegans and its relative Caenorhabditis briggsae. All three viruses share less than 50% amino acid identity and are most closely related to nodaviruses, which are positive sense RNA viruses with bipartite genomes. Comparison of their complete genomes demonstrated unique coding and noncoding features absent in known nodaviruses. Le Blanc virus, similar to Santeuil virus, was capable of infecting wild C. briggsae isolates but not the AF16 C. briggsae laboratory reference strain nor any tested C. elegans strains. We characterized the tissue tropism of infection in Caenorhabditis nematodes by all three viruses. Using immunofluorescence assays targeting viral proteins, as well as in situ hybridization, we demonstrated that viral proteins and RNAs localized primarily to intestinal cells in larval stage Caenorhabditis nematodes. The viral proteins could be detected in one to six of the 20 intestinal cells present in Caenorhabditis nematodes. In Orsay virus-infected C. elegans, viral proteins could be detected as early as six hours post infection. Furthermore, the RNA-dependent RNA polymerase and capsid proteins of Orsay virus exhibited different subcellular localization patterns from each other. Collectively, these observations broaden our understanding of viral infection in Caenorhabditis nematodes.