Stepek, Gillian et al. (2015) International Worm Meeting "Nematode moulting enzymes as potential drug targets."

Houston, Doug, Walkinshaw, Malcolm, McCormack, Gillian, Page, Antony, Stepek, Gillian

[International Worm Meeting, 2015]

The nematode cuticle is a collagenous extracellular matrix that is synthesised repeatedly during development via the moulting process. The enzymology of cuticle collagen biosynthesis and more importantly the enzymology of cuticle ecdysis and moulting represent potential novel targets for parasitic nematode control. Using C. elegans we have identified and characterized key moulting enzymes and have focused on the nematode astacin (NAS) metalloproteases. As an example, mutation of the enzyme-encoding genes NAS-36 and NAS-37 reveals an essential developmental role for these enzymes in C. elegans moulting. We have identified the orthologs in the diverse parasitic nematodes Brugia malayi and Haemonchus contortus that are able to rescue the moulting phenotypes associated with nas-36/ -37.Using in silico-modelling we have screened available chemical libraries and have tested several hundred potential inhibitors using in vivo and in vitro assays in C. elegans, Brugia malayi and Haemonchus contortus.The screening process will be described and the main finding of this study will be presented.

Jamie White et al. (2008) C.elegans Neuronal Development Meeting "Deconstructing the neural circuitry for sex-specific pheromone responses"

Nadja Schaefer, Eliott Davidson, Erik Jorgensen, Jamie White

[C.elegans Neuronal Development Meeting, 2008]

C.elegans hermaphrodites release pheromones that elicit sex-specific behaviors: males are attracted to the pheromones and hermaphrodites avoid them. We are interested in the differences in neural circuitry that account for sex-specific responses to common pheromone signals. Sex-specific pheromone responses depends only on the sexual identity of the nervous system, shown by experimentally swapping the sexual identity of the nervous system, but not other tissues. Specifically, expression of the masculinizing factor FEM-3 using the pan-neuronal rab-3 promoter (Prab-3::fem-3), results in an animal with the physical appearance of a hermaphrodite, but the pheromone response of a male (attraction behavior). Conversely, expression of a truncated tra-2 gene (encoding the TRA-2ic fragment, generously provided by Bill Mowrey and Doug Portman prior to publication), or the e1575 allele of the tra-1 gene, both feminizing factors, results in an animal with the physical appearance of a male, but the pheromone response of a hermaphrodite (avoidance behavior). Presumably, the entire nervous system does not have to be sexualized; for example, it is unlikely that motor neurons must be male to generate male behavior. Accordingly, we are switching the sexual identity of subsets of neurons. Masculinizing either the sensory neurons (using Posm-5) or, independently, a set of interneurons (using Pglr-1) fails to induce male behavior in animals that are otherwise hermaphrodites, suggesting that both sensory and interneurons must be sexualized to generate the male-specific pheromone response (some Posm-5 and Pglr-1 lines generously provided by Bill Mowrey and Doug Portman). Similar experiments are underway to feminize either sensory neurons or interneurons. When during development do neural circuits acquire the sex-dependent characteristics that impart sex-specific pheromone responses? To address this, we are switching the sexual identity of the nervous system at different times during development using the FLP-ON system (Davis et. al. 2008 PMID 18369447). Additionally, the FLP-ON system allows us to investigate whether the C. elegans nervous system can be reprogrammed during adulthood. The sex-specificity of pheromone-response behaviors can be reversibly switched in adult flies (Grosjean et. al. 2008 PMID 18066061) and adult mice (Kimchi et. al. 2007 PMID 17676034); it may turn out that the C. elegans nervous system is similarly plastic.

Taylor, Remington et al. (2021) International Worm Meeting "Snip-SNP mapping of a trembler C. elegans mutant strain with undergraduate students"

Pham, Kelvin, Henderson, Jacklyn, Soebianto, Sarita, Mathai, Kristina, Taylor, Remington, Nguyen, Gina, LaForce, Michael, Simmons, Dr. Alexandra

[International Worm Meeting, 2021]

Caenorhabditis elegans is a valued model organism that is easy to maintain in the lab. We obtained a mutant strain of C. elegans that exhibits constant involuntary muscle spasms, a phenotype that has not been described before in the literature (MCW230, curtesy of Dr Wang, BCM, Houston, TX). The 'trembler' mutant strain has a nre-1(hd20) lin-15b(hd126) genetic background. We use snip-SNP mapping to seek the causal mutant gene responsible for the trembling phenotype in the MCW230 strain. MCW230 worms were backcrossed with wild-type N2 worms to eliminate the double mutant background (nre-1, lin-15b). For snip-SNP gross mapping, we crossed our mutant with CB4856 worms. DNA was extracted from 43 F2 individuals resulting from the MCW230 x CB4856 cross. The F2 samples were studied to identify the presence of specific single nucleotide polymorphisms at 18 genomic locations (3 per chromosome) and assess how the alleles segregate relative to the trembler phenotype. We completed genotyping at all genomic locations and our data supports the idea that the trembler phenotype seems to be linked to one of the markers on chromosome III. Our work is an example of how research using C. elegans can be done in the undergraduate environment with basic laboratory equipment.

Hamill, Danielle R. et al. (2009) International Worm Meeting "Analysis of a spindle assembly mutant in C. elegans."

Hamill, Danielle R., Everett, Molly, Bowerman, Bruce, Price, Meredith, McNeeley, Marie, Mazhar, Sahar, Gearica, Amy

[International Worm Meeting, 2009]

Cell division is a highly conserved and complex process that must be carefully orchestrated. or452ts was isolated in a screen for temperature sensitive embryonic lethal cell division mutants. In or452ts mutant embryos, bipolar spindles often fail to form. In these mutants DNA localizes around microtubules emanating from a single, often misshapen, centrosome. Staining with centriole markers suggests that a centriole duplication defect may underlie the spindle assembly defect. Using live cell imaging and fluorescence transgenes (including GFP::gamma tubulin), we have noticed centrosome streaking and splitting that is not seen in wild-type embryos. We also have observed the formation of asymmetric spindles. Towards the goal of identifying the gene that is mutated in or452ts, we have done meiotic mapping using phenotypic markers and snip-SNPs. We have narrowed the genetic location down to about 0.5 cM around +2.5 on LGIII. This region contains less than 500 kbp of DNA, and there are about 40 predicted genes in this region. In collaboration with Doug Turnbull and Eric Johnson (University of Oregon), we are sequencing the DNA from this region to try to identify the mutation responsible for the or452ts mutant phenotype. We believe that characterization of this mutant, and identification of the gene responsible, will provide new insights into the process of mitotic spindle assembly and cell division.

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.

G Lochnit et al. (2004) European Worm Meeting "FIRST IDENTIFICATION OF A PHOSPHORYLCHOLINE-SUBSTITUTED PROTEIN FROM CAENORHABDITIS ELEGANS ISOLATION AND CHARACTERIZATION OF THE ASPARTYL PROTEASE ASP-6."

R Geyer, G Lochnit, B Henkel, J Grabitzki

[European Worm Meeting, 2004]

Caenorhabditis elegans has been found to be a good model system for parasitic nematodes, drug screening and developmental studies. Structural analyses have revealed nematode specific glycosphingolipid structures of the arthro-series, carrying, in part, phosphorylcholine (PC) substituents. PC is a widespread antigenic epitope of pathogens like parasitic nematodes and has also been detected on N-glycans of this model organism (1, 2). The PC modification seems to play an important role in nematodes development, fertility and survival within the host. With the exception of ES-62 from Achanthocheilonema viteae no protein carrying this epitope has been yet identified and further characterized (3). In the axenic medium of C. elegans culture we detected a single protein with an apparent molecular mass of 40 kDa which reacted with the PC-specific antibody TEPC-15. The protein was purified by anion-exchange chromatography and 2D-gel electrophoresis. After in-gel digestion with trypsin, the protein was identified by MALDI-TOF-MS peptide finger print and nanoLC-ESI-MS/MS microsequencing as the aspartyl protease ASP-6. RNAi experiments confirmed is assignment. Lectin analysis of the purified ASP-6 protein revealed the presence of the GlcNAc-residues by binding of WGA, whereas Con A showed no binding, indicating the absence of terminal mannosyl residues. Treatment of the protein with PNGase A and PNGase F abolished the binding of WGA, but not that of TEPC-15. This might be an indication for a PC-epitope distinct from those described so far. References: 1. Lochnit, G. et al. (2000) Biol. Chem. 381: 839-47 2. Houston, K., et al. (2002) Mol. Biochem. Parasitol. 123: 55-66 3. Harnett, W., et al. (2003) Curr.Protein Pept. Sci. 4: 59-71