Christopher W Brey et al. (2001) International C. elegans Meeting "ENHANCEMENT OF STEINERNEMA CARPOCAPSAE DESICCATION TOLERANCE BY GENETIC IMPROVEMENT"

Randy Gaugler, Christopher W Brey

[International C. elegans Meeting, 2001]

The entomopathogenic nematode (ENP) Steinernema carpocapsae , is an important biological control agent for a wide range of soil dwelling insect pests. However, the field efficacy of this ENP is limited by its sensitivity to high drought and salinity conditions. We report efforts to improve the desiccation tolerance of S. carpocapsae by transforming it with the trehalose-6-phosaphate synthase (tps1) and glycogen synthase(gsy1)genes. Trehalose-6-phosphate synthase and glycogen synthase are enzymes involved in the biosynthesis of trehalose, a disaccharide that accumulates to stabilize the lipid biomembranes in many organisms when in response to stress. To increase desiccation tolerance by genetic modification, we have cloned gene tps1 from yeast and C. elegans into expression vectors pJJ436 and pPD95.67, respectively. In addition, we also cloned gsy1 from Steinernema feltiae into expression vector pJJ436. Vector pJJ436 contained the Ce sq-1 promoter, whereas pPD95.67 contained the promoter of the tps1 Ce gene. All vectors contained the gfp transformation gene which was used as a selection marker. Vector constructions (yeast: pJYeTr.1; C. elegans : pP67CeTr.2; S. feltiae : pJSfTr.1) were microinjected independently into young S. carpocapsae females (48 h from infective juvenile stage). Injected females were mated with noninjected males for 2-4 days and progeny were screened for gfp expression. After selecting and retaining gfp expressing individuals for three generations, F3 progeny were tested for desiccation tolerance. We will present details of our methodology and results in our presentation.

Alison Slinskey Legg et al. (2007) International Worm Meeting "C. elegans research for high school teachers and students: The Gene Team."

Lew Jacobson, Alison Slinskey Legg

[International Worm Meeting, 2007]

The Gene Team is a 5-year program to explore and refine a novel mechanism for engaging high school teachers and high school students in biomedical research and inquiry-based learning, with a focus on the genetics of model organisms. The objectives are: (a) To improve the knowledge and process skills of high school biology teachers; (b) To introduce high school students to the practice of scientific research and develop their skills in scientific thinking; (c) To design and implement inquiry-based curricular modules using model organisms for high school biology classes. The core of the program is a 7-week summer session (40 hrs/week), during which participants in a single dedicated 1000 sq. ft. lab work in parallel on three distinct research projects involving bacteria, yeast and C. elegans. Nine participating research labs provide projects (3 each year), with the intention that the "products" (mutants, constructs, data) of the summer research are returned to the originating labs for further investigation. The novelty of the team concept is that the participants work as peers despite a wide diversity of age and experience: The 2006 Gene Team was composed of 3 high school teachers, 6 high-school students, 2 research-experienced undergraduates and 3 graduate students, under the daily supervision of a faculty member and a program staff member. Simultaneously, the teacher-participants work at the Curriculum Roundtable with a group of master teachers and University faculty to design and refine inquiry-based learning modules, targeted at state curricular standards for biology in grades 9-12. Modules may be classroom-based or lab-based, and we provide equipment, supplies and staff support for implementation during the school year. Modules are field-tested and iteratively refined through one academic year before wider dissemination. The Gene Team is supported by a Science Education Partnership Award (SEPA) from the National Center for Research Resources, NIH.

H Hwang et al. (1999) International C. elegans Meeting "Nucleotide-sugar biosynthesis and glycosylation are involved in vulval morphogenesis"

H Hwang, HR Horvitz

[International C. elegans Meeting, 1999]

Eight sqv ( sq uashed v ulva) genes ( sqv-1 to 8 ) have a mutant vulval morphogenetic phenotype that is defined by reduced separation between the anterior and posterior halves of the vulva in the L4 (1). SQV-3 and SQV-8 are glycosyltransferase-like proteins, and SQV-7 is similar to a putative nucleotide-sugar transporter (2). We have previously reported the cloning of sqv-1 and sqv-4 (3). Both SQV-1 and SQV-4 are similar to enzymes involved in nucleotide-sugar metabolism. These molecular identities suggest that glycosylation is important in shaping the vulva during development. We have now determined biochemically that SQV-4 is a UDP-glucose dehydrogenase. Recombinant SQV-4 expressed in E. coli can reduce NAD in the presence of UDP-glucose, suggesting that UPD-glucose is being converted to UDP-glucuronate. Like known UDP-glucose dehydrogenases, SQV-4 appears to act specifically on UDP-glucose, as it fails to reduce NAD using any of the many other nucleotide-sugars tested. Mutant recombinant SQV-4 derived from either of the two genetically identified mutant alleles does not show detectable activity. Preliminary antibody staining using a rabbit polyclonal antibody directed against a GST::SQV-4 indicates that SQV-4 is localized to the vulval cells and several other tissues, including seam cells. This result agrees with the expression of SQV-4::GFP fusion protein and supports a model in which SQV-4 acts in the vulval cells during vulval development. We also are trying to determine the functions of the other four cloned sqv genes using biochemical assays and heterologous complementation. Lastly, we are mapping and cloning the three remaining sqv genes. 1. Herman, T. et al. (1999). PNAS 96 : 968-973. 2. Herman, T. and Horvitz, H.R. (1999). PNAS 96 : 974-979. 3. Hwang, H. and Horvitz, H.R. (1998) East Coast C. elegans meeting, p. 103.

Isaac RE et al. (1995) International C. elegans Meeting "THE METABOLISM AND INACTIVATION OF AF1 AND PF1 BY NEMATODE NEPRILYSIN."

Coates D, Priestly RM, Sajid M, Isaac RE

[International C. elegans Meeting, 1995]

AF1 (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2) and PF1 (Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-NH2) have potent effects on the locomotory muscle of Ascaris suum. AF1 is inactivated by three neuropeptide-degrading enzymes found in crude plasma membranes of A.suum muscle. One of these enzymes is an endopeptidase which hydrolyses the Glu3-Phe4 bond of AF1 and which is inhibited by phosphoramidon (IC50, 0.7M), a potent inhibitor of mammalian neprilysin (neutral endopeptidase 24.11, enkephalinase). A phosphoramidon-sensitive endopeptidase is also present in membranes prepared from Caenorhabditis elegans and we are characterising the enzyme by biochemical and molecular approaches. The enzyme appears to be a membrane protein which can be solubilised with detergents but it does not partition into the detergent-rich layer formed during the temperature induced phase separation of Triton X-114 and therefore does not behave like a typical integral membrane protein. The enzyme attacks peptide bonds comprising the amino group of hydrophobic amino acids and hydrolyses the Asn4-Phe5, Phe5-Leu6 and the Arg7-Phe8 peptide bonds of the C.elegans peptide, PF1. Phe-X-Arg-Phe-NH2 is normally the core sequence for bioactivity and therefore cleavage of PF1 within this sequence will result in inactivation. The endopeptidase has a pH optimum of 7.8, and is inhibited by EDTA and 1,10 bis phenanthroline, indicating the involvement of a bivalent metal ion at the active site. Phosphoramidon, thiorphan, SCH 39370, SCH 32615 and SQ 28603 are inhibitors of mammalian neprilysin and all inhibit the C.elegans endopeptidase. The nematode enzyme is also inhibited by Co2+, Cu2+, Zn2+ and Fe2+ but not by Ca2+, Mg2+ and Mn2+. A putative neprilysin gene has been identified in the genome of C.elegans as part of the genome sequencing project and we are now screening for a Tc1 insertion induced knockout of this gene to determine the effect of neprilysin gene inactivation on nematode behaviour.

Sonnhammer ELL et al. (1998) European Worm Meeting "Bioinformatics driven functional genomics: a multifront approach to understand complex pathways with a "simple" little worm"

Vaz Gomes A, Sonnhammer ELL, Krause MW, Schrimpf S, Wahlestedt C

[European Worm Meeting, 1998]

C. elegans has been a useful model organism for developmental studies because of its ease of culture, simple anatomy, and available genetics. These attractive features are now be augmented by a wealth of molecular data from a multitude of nematode research labs as well as data provided by the completion of the full genome sequencing project. Already, the genomic information is allowing accurate detection of C.elegans orthologues to various genes relevant in human diseases or other conserved pathways (Mushegian et al., 1997). The time is right to take a bioinformatics driven approach to functional studies. The combination of the complete genomic sequence, bioinformatics, and an experimentally facile organism makes C. elegans an excellent system in which to dissect complex signaling pathways. A large body of signal-transduction pathways involving seven-transmembrane G-protein coupled receptors mediate responses to different types of chemicals like odorants, neurotransmitters, hormones, and also to more !physical! stimuli, like osmotic pressure, temperature, pressure, etc. Many members of these types of pathways have been studied in some detail in C. elegans while other potential candidates have so far only been identified !in silico! (Sonnhammer & Durbin, 1997). Clearly, the nematode has been, and will continue to be, helpful in identifying potential roles for these factors in regulating behaviour and responses to environmental cues, or in crucial developmental processes. The emergence of RNA-mediated interference (RNAi) (Fire et al., 1998) provides a powerful technique that will facilitate the identification of phenotypes associated with the elimination of single or combinations of multiple signalling factors. To improve biochemical analysis in the worm, we aim to apply the new generation of protein 2D-gels analysis systems. We expect this to become a powerful tool (e.g. Bini et al., 1997), for example revealing mutation effects and more accurate proteomics. This is especially important for genes encoding transcription factors, where changes of the expression pattern might be detected most easily at the protein level. For such studies the main requirement is non-lethality, health and fertility of the mutation, so that enough material can be obtained for the analysis. REFERENCES Bini, L., Heid, H., Liberatori, S., Geier, G., Pallini, V. & Zwilling, R. (1997) Electrophoresis18, 557-562 Fire, A., Xu, SQ., Montgomery, M.K., Kostas, S., Driver, S.E. & Mello, C. (1998) Nature 391, 806-811 Mushegian, A.R., Bassett, D.E.Jr, Boguski, M.S., Bork, P. & Koonin, E.V. (1997) PNAS 94, 5831-5836 Sonnhammer, E. & Durbin, R. (1997) Genomics 46, 200-216

Ho-Yon Hwang et al. (2001) International C. elegans Meeting "A PROTEOGLYCAN BIOSYNTHETIC PATHWAY INVOLVED IN C. elegans EMBRYOGENESIS AND VULVAL MORPHOGENESIS AND IN HUMAN AGING DISORDERS"

Bob Horvitz, Ho-Yon Hwang

[International C. elegans Meeting, 2001]

Mutations in eight sqv ( sq uashed v ulva) genes can result in several developmental abnormalities, including defective vulval invagination and maternal-effect lethality. We report the cloning of two sqv genes, sqv-2 and sqv-6 , and discuss their roles in a molecular genetic pathway defined by the six previously cloned sqv genes. We also report the characterization of sqv-5 and sqv-7 expression. The molecular identities of the eight sqv genes suggest that the molecular basis for the sqv defects lies in the disruption of the biosynthesis of glycosaminoglycans (GAGs) of the structure (Serine residue in the protein core)-Xylose-Galactose-Galactose-Glucuronic acid-(X-Glucuronic acid) n , where X is either N-acetylgalactosamine or N-acetylglucosamine. The biosynthesis of GAGs requires the synthesis of nucleotide sugars in the cytoplasm and the translocation of nucleotide sugars into the endoplasmic reticulum (ER) and/or Golgi, where polymerization of sugars is catalyzed by glycosyltransferases. SQV-4 is a UDP-glucose dehydrogenase, a key enzyme in UDP-Glucuronic acid synthesis. SQV-7 is an atypical multi-pass transmembrane protein that transports UDP-Glucuronic acid, UDP-N-acetylgalactosamine and UDP-Galactose from the cytoplasm into the ER and/or Golgi. Mammalian homologs of sqv-3 , sqv-6 and sqv-8 encode glycosyltransferases necessary for the biosynthesis of the GAG-protein linkage region of proteoglycans: SQV-6, SQV-3 and SQV-8 are similar to Xylosyltransferase, Galactosyltransferase I, and Glucuronyltransferase I, respectively. SQV-1 is a cytoplasmic protein with weak similarities to nucleotide-sugar modifying enzymes, and SQV-2 and SQV-5 are both type II transmembrane proteins and candidate glycosyltransferases. We postulate that sqv-1 , sqv-2 and sqv-5 are components of the same GAG biosynthesis pathway and that the GAGs are important for cell-cell or cell-matrix interactions in embryonic and vulval development. Preliminary antibody staining using rabbit polyclonal antibodies against SQV-4, SQV-5 and SQV-7 shows localization of these proteins consistent with their deduced functions. SQV-4 appears to localize broadly to the cytoplasm of many tissues, including the vulva, consistent with its function in the biosynthesis of UDP-glucuronic acid in the cytoplasm. SQV-5 shows punctate cytoplasmic localization in the vulva and germline, consistent with our model that SQV-5 functions as a glycosyltransferase in the Golgi. SQV-7 also shows punctate cytoplasmic localization in the hypodermal seam and gonadal distal tip cells, consistent with its function in transporting nucleotide sugars into the Golgi. We plan to characterize the subcellular localization of SQV-5 and SQV-7 further using immuno-electron microscopy. Mutations in the human homolog of sqv-3 are implicated as the cause of a progeroid variant of the connective-tissue disorder Ehlers-Danlos syndrome. All eight sqv genes have close human counterparts, suggesting that a common pathway for modifying important cell surface and/or extracellular GAGs is present in humans and in C. elegans . Defects in the human counterparts of other sqv genes therefore may be responsible for aging disorders and connective tissue diseases.