Schein JE et al. (1997) International C. elegans Meeting "POSITIONING OF ESSENTIAL GENES IN THE lin-3 - let-60 INTERVAL"

Franz NW, Rose AM, Janke DL, Bilkhu N, Baillie DL, Schein JE

[International C. elegans Meeting, 1997]

The lin-3 - let-60 interval of the right arm of chromosome IV contains thirteen essential gene mutations and 22 overlapping sequenced cosmids. The Sanger Center has nearly finished sequencing the entire interval. Analysis of cosmid sequences shows over one hundred potential coding regions. Of the coding regions with proposed protein function, more than 50% have strong homology to human genes. Most notably, the putative genes include three phosphatases, four protein kinases, a kinesin, and a cluster of histones. C. elegans strains carrying sequenced cosmids (in extrachromosomal arrays) have been constructed (see abstract for D. Janke et al., this meeting) and will be used in rescue experiments involving the essential genes in this interval. lin-3 and let-60(ras), the two genes bracketing this interval, were previously placed on the physical map by others. One of the remaining eleven essential genes has been already positioned to a cosmid: let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido). By the time of the meeting, we anticipate that several of the remaining mutations will have been positioned to cosmids. This work is being funded by a grant from the Medical Research Council to Ann Rose and David Baillie and by a grant from the Natural Sciences & Engineering Research Council to David Baillie.

Dao, Chris et al. (2017) International Worm Meeting "Development of a high-statistics, three dimensional imaging system for C. elegans under magnetic and chemical stimuli."

Arisaka, Katushi, David, Angela, Vincent, Nitin, Thatcher, Joseph, Chow, Aaron, Wang, Charles, Dao, Chris, Du, Angela, Alberto, Jasmine, Li, Lina

[International Worm Meeting, 2017]

In contrast to previous behavioral experiments limited to two dimensional surfaces, we extended the study of C. elegans into three dimensional environments to observe the resulting unconstrained motion. While research has been initiated towards single worm, high magnification tracking, this has limited practicality in terms of gathering a large number of statistics with bulk behavior of many worms simultaneously. Incorporating stimuli allows for a new, more thorough examination of motion and environmental interaction, extending inquiry beyond simple free motion. Previously performed, two dimensional results suggest hypotheses, which may serve as limiting-case projections of greater trends in three dimensional space. A novel, cost effective system was developed to image C elegans' behavior over a period of 30 to 60 minutes using time lapse acquisition. The setup consists of three, perpendicularly oriented DSLR cameras, synchronized via the open source, MIT developed DigiCam control software and illuminated with lowly-invasive, conical, red LED light sources. Samples consisted of N2 type worms embedded in the center of 2% porcine gelatin (4 to 5 cm) quartz cuvettes. Each camera had an affixed aperture to elongate its depth of field, promoting focus throughout the volume of the cubes. In addition to free motion studies, three sets of Helmholtz coils (with axes coinciding with the cameras' optical axes) were utilized to generate a uniform magnetic field at the location of the sample. NaCl concentration gradients throughout the gelatin were also simulated and created for three dimensional chemotaxis experiments. Numerous free motion trials have been conducted alongside magnetic field experiments of field magnitude ranging from 0 Gauss (cancelling the geomagnetic field) to 10 Gauss. Using this data, analysis software tools were developed in MATLAB in order to extract the center of mass trajectory of worms navigating the gelatin, enabling further quantification of motional trends such as the worms' velocities and distributions of initial and final positions. While preliminary results for free motion (with chemical attractants) appear to reflect standard biased random motion, positive verification of magnetic field trials remains elusive.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

O'Neil NJ et al. (1996) West Coast Worm Meeting "SYSTEMATIC HIGH RESOLUTION MAPPING OF ESSENTIAL GENES IN THE sDf125 REGION BY TRANSGENIC RESCUE."

Kuervers LM, Baillie DL, Vatcher GP, O'Neil NJ

[West Coast Worm Meeting, 1996]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab is constructing a map of essential genes on LGIII left. We have divided the left end of the central cluster into manageable regions which are defined by overlapping deficiencies and duplications. These regions are determined physically by PCR testing the deficiencies to determine endpoints (see N.Franz et al. abstract, this meeting). Into these regions, we have mapped a set of EMS induced lethals and maternal effect lethals. (see abstracts by H. Stewart et al. and G. Vatcher and D. Baillie, this meeting). One such region is defined by sDf125 and sDp8. This region spans 340 000 base pairs and is completely covered by nine cosmids. Within this region there are 75 putative genes predicted by Genefinder. We have used a sytematic approach to rescue essential genes within the sDf125 region with transgenic cosmids from the library created by J. Schein, D. Janke and The Ha as part of a CGAT grant to Ann Rose and David Baillie (see abstracts by J.Schein et al. and D. Janke et al., this meeting) To date this method has proven very successful. At least 8 genes have been completely rescued in this region by this approach. These transgenic rescues map these genes to 40 kB (one cosmid) resolution as opposed to the 340 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of ORFs which may be responsible for the gene function. The next step will be the subcloning of cosmids with which we have rescued essential genes. This will directly corellate the lethal or maternal effect lethal phenotype to the appropriate ORF. We have already begun the subcloning of cosmid C05D11 (see G.Vatcher and D.Baillie abstract, this meeting). This project is funded by a CGAT grant to Ann Rose and David Baillie.

O'Neil NJ et al. (1997) International C. elegans Meeting "ANALYSIS OF THE sDf127 REGION BY TRANSGENIC RESCUE."

O'Neil NJ, Vatcher GP, Kuervers LM, Baillie DL

[International C. elegans Meeting, 1997]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab is constructing a map of essential genes on LGIII left. We have divided the region between dpy-17 and unc-36 into manageable regions that are defined by overlapping deficiencies and duplications. These regions are determined physically by PCR testing the deficiencies to determine endpoints. Into these regions, we have mapped a set of EMS induced lethal and maternal effect lethal mutations (See abstract H.Stewart et al., this meeting). One such region is defined by sDf127 and sDp8. This region spans 340 000 base pairs and is completely covered by ten cosmids. Within this region there are 75 putative genes predicted by Genefinder. To date, we have mapped 14 essential genes into this region. We have used a sytematic approach to rescue these essential genes with cosmid transgenics from the library created as part of a CGAT grant to Ann Rose and David Baillie (See abstract by D. Janke et al., this meeting) To date this method has proven very successful. At least 8 genes have been completely rescued in this region utilizing this approach. These transgenic rescues map these genes to 40 kB (one cosmid) as opposed to the 340 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of coding sequences which may be responsible for the gene function. The next step will be to subclone the cosmids with which we have rescued essential gene mutations. This will directly corellate the lethal or maternal effect lethal phenotype to the appropriate coding sequence. We have already begun the subcloning of the cosmids C05D11 (see abstract by G.Vatcher et al., this meeting), C31H11 and C16A3. This project is funded by a grant from the Medical Research Council of Canada to Ann Rose and David Baillie.

David R. Bell et al. (2006) European Worm Meeting "The Function of Latrophilin in C. elegans"

S. van der Poel, P.N.R. Usherwood, D. Kendall, J. Davy, A. Ademola, I.R. Duce, D. de Pomerai, David R. Bell, I. R. Mellor

[European Worm Meeting, 2006]

David R. Bell, A. Ademola, J. Davy, S. van der Poel, D. Kendall, P.N.R. Usherwood, I.R. Duce, I. R. Mellor and D. de Pomerai. Black Widow spider venom (BWSV) contains latrotoxins that induce catastrophic neurotransmitter release in mammals, and is known to bind with high affinity to three neural proteins in mammals, including latrophilin. We have investigated C. elegans as a model system for studying the function of these proteins, and their role in regulating neurotransmitter release by latrotoxins.. We have shown that latrophilin is required for the lethality of BWSV in C. elegans by RNAi experiments. We now present data on the characterisation of null mutants of the C. elegans latrophilin, lat-1, illustrating the function of this gene in C. elegans, and show that lat-1 nulls are resistant to emodepside. The expression of latrophilin-GFP constructs is examined, and compared with the phenotypes seen in null mutants. Genetic interactions between endogenous signaling pathways and the lat-1 nulls have been examined by the construction of double mutant worms, revealing novel facets of lat-1 function.

Victor L. Jensen et al. (2006) European Worm Meeting "A Potential DAF-16 Target Involved in Longevity"

David L. Baillie, Victor L. Jensen, Donald L. Riddle

[European Worm Meeting, 2006]

Victor L. Jensen1, David L. Baillie2, and Donald L. Riddle3. Genes differing in steady-state RNA levels between N2 and daf-2 adults and N2 dauer larvae were identified using Serial Analysis of Gene Expression (SAGE) databases. One gene, a putative transcription factor, was isolated from a screen for genes involved in longevity. The outcrossed knockout allele was shown to have extended longevity, ~3% Him and ~2% Vab phenotypes. The 1 kb of genomic sequence 5? of the confirmed transcription start site contains several possible DAF-16 binding sites. By using a series of truncated promoter sequences to drive GFP expression, possible in vivo activity for these DAF-16 binding sites was observed. One consensus site DBE (DAF-16 Binding Element) may act to repress transcription. This is in agreement with the SAGE data which shows down regulation of this transcription factor in the daf-2 background. In daf-2 mutants DAF-16 is activated and is required for extended longevity. Transcriptional targets and the DNA binding motif for the transcription factor will be elucidated, and its role in longevity will be characterized.

Materi WP et al. (1998) West Coast Worm Meeting "THE ROLE OF UNC-119 IN AXONOGENESIS IN C. ELEGANS"

Pilgrim DB, Maduro MF, Materi WP

[West Coast Worm Meeting, 1998]

UNC-119 is the first example of a novel class of protein found in both the developing and adult nervous systems of the nematode, the fly, the zebrafish (see poster by Angela Manning) and in mammals. While UNC-119 is found throughout the C. elegans nervous system, beginning around the 60-cell stage, the human and rat homologs (HRG4 and RRG4, respectively) have only been found in retinal tissue, beginning shortly after birth. The fly homolog (DmUnc-119) is expressed throughout the larval central nervous system. In the worm, four unc-119 null alleles have been characterized in addition to the wild type. Apart from the canonical nearly paralyzed, uncoordinated phenotype, these mutants also exhibit constitutive feeding behavior even in the absence of food and are unable to enter into the dauer larva stage when subjected to overcrowding and starvation. Both the behavioral and morphological phenotypes suggest that UNC-119 plays a role in growth cone guidance in the developing nervous system. An UNC-119::GFP fusion protein is found in the cytoplasm of all cells of the nervous system. In an unc-119 mutant the ventral nerve cord (VNC) is defasciculated and the lateral commisures, which normally project from the VNC dorsolaterally to join the dorsal nerve cord have an abnormal, highly branched structure. We have integrated GFP constructs, driven by promoters specific to subsets of neurons, into both wild-type and unc-119 mutant strains and compared neural structures at high magnification using confocal microscopy. The projections of many neurons are unaffected by the unc-119 mutation while abnormally long axons, abnormally branched axons and defasciculated axons are associated with neurons affected by the mutation. We have also conducted a yeast 2-hybrid screen using an unc-119 bait construct and have found several positive interactions that are selective for UNC-119 versus a SNF-4 bait. These preliminary results are presently being confirmed using the human homolog, HRG4, as bait in a 2-hybrid experiment. Co-immunoprecipitation experiments are also underway.

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.

O'Connell KF et al. (1997) International C. elegans Meeting "NEW MUTATIONS AFFECTING THE GENERATION OF ZYGOTIC POLARITY"

White JG, Badrinath AS, O'Connell KF, Maxwell KN

[International C. elegans Meeting, 1997]

Division of the C. elegans zygote is asymmetric, producing daughter cells that differ in size and developmental potential. To produce distinct daughter cell fates, the zygote must be polarized prior to division such that a different complement of cell fate determinants is inherited by each of the daughters. It is currently thought that the unfertilized egg possesses no inherent polarity and that polarity is established only after fertilization. A number of par genes have been described that are required for partitioning of developmental instructions during the early cleavages and it is thought that the products of these genes interact directly or indirectly with microfilament and microtubule systems to establish polarity and cleavage orientation. Here we describe two new mutations that affect zygotic polarity, both identified in screens for conditional mutations producing a sterile uncoordinated phenotype. oj29 embryos lack functional centrosomes and a number of indicators of embryonic polarity; membrane ruffling and pronuclear migration are aberrant and the posterior-directed cytoplasmic streaming seen in wild-type embryos is missing. stu-4(e2406) isolated by David Livingstone appears to affect the actin cytoskeleton; embryos are defective in polar body formation, pronuclear migration, pseudocleavage and cytokinesis. In addition the first mitotic spindle is often misaligned. We are currently analyzing these mutants for defects in other aspects of asymmetry such as the segregation of P granules. In addition we are screening a small bank of emb mutations, produced in the sterile unc screen, for similar phenotypes.