Charlesworth AG et al. (2019) Cell "Next-Gen Learning: The C.elegans Approach."

Seroussi U, Claycomb JM, Charlesworth AG

[Cell, 2019]

In this issue, Moore etal. and Posner etal., provide evidence for how the activity of the nervous system in C.elegans results in gene expression changes in the germline to pass on parental experiences and learned behavior to their progeny.

Courtney Wilkins et al. (2005) International Worm Meeting "RNAi as an antiviral defense mechanism in C. elegans"

Ryan Dishongh, Steve Moore, Courtney Wilkins, Marie Chow, Khaled Machaca, Michael Whitt

[International Worm Meeting, 2005]

Although RNA interference has been postulated to be an antiviral defense mechanism in C. elegans, there has been little evidence demonstrating the effect of RNAi on viral infection in the worm. At the 2004 Midwest Worm Meeting, we described a model of viral infection in primary C. elegans cell culture using a mammalian virus, vesicular stomatitis virus (VSV), that results in production of viral antigens and RNAs in neuronal cells following exposure to virus. At that time, preliminary results suggested that RNAi inhibits VSV infection in these cells in the absence of exogenous inducers of RNAi. Subsequently, we have characterized VSV infection of cells with mutations in genes known to modulate the RNAi response. We show that VSV infection is potentiated in RNAi-deficient strains (rde-1 and rde-4), as evidenced by a greater percentage of infected cells as well as increased expression of viral proteins and RNA. In addition, loss of RNAi responses leads to a significant increase in viral titers, demonstrating production of infectious progeny virus. In contrast to the RNAi-deficient strains, mutations in genes that negatively regulate RNAi (eri-1 and rrf-3) lead to a decrease in the numbers of infected cells following exposure to VSV. To further elucidate the antiviral mechanism of RNAi, molecular characterization of the VSV replication process in these and other C. elegans strains with altered RNAi responses will be discussed.

Moore RS et al. (2021) STAR Protoc "Protocol for transgenerational learned pathogen avoidance behavior assays in Caenorhabditis elegans."

Moore RS, Murphy CT, Kaletsky R

[STAR Protoc, 2021]

Animal experiences, including learned behaviors, can be passed down to several generations of progeny in a phenomenon known as transgenerational epigenetic inheritance. Yet, little is known regarding the molecular mechanisms regulating physiologically relevant transgenerational memories. Here, we present a method for <i>Caenorhabditis elegans</i> in which worms learn to avoid the pathogen <i>Pseudomonas aeruginosa</i> (PA14). Unlike previous protocols, this training paradigm, either using PA14 lawns or through exposure to a PA14 small RNA (P11), induces memory in four generations of progeny. For complete details on the use and execution of this protocol, please refer to Moore etal. (2019) and Kaletsky etal. (2020).

Reboul J et al. (2001) Nat Genet "Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans."

Reboul J, Vandenhaute J, Tzellas N, Jackson C, Hartley JL, Lamesch PE, Vidal M, Brasch MA, Hill DE, Thierry-Mieg J, Thierry-Mieg N, Hitti J, Vaglio P, Thierry-Mieg D, Moore T, Shin-i T, Doucette-Stamm L, Temple GF, Lee H, Kohara Y

[Nat Genet, 2001]

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.AD - Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.FAU - Reboul, JAU - Reboul JFAU - Vaglio, PAU - Vaglio PFAU - Tzellas, NAU - Tzellas NFAU - Thierry-Mieg, NAU - Thierry-Mieg NFAU - Moore, TAU - Moore TFAU - Jackson, CAU - Jackson CFAU - Shin-i, TAU - Shin-i TFAU - Kohara, YAU - Kohara YFAU - Thierry-Mieg, DAU - Thierry-Mieg DFAU - Thierry-Mieg, JAU - Thierry-Mieg JFAU - Lee, HAU - Lee HFAU - Hitti, JAU - Hitti JFAU - Doucette-Stamm, LAU - Doucette-Stamm LFAU - Hartley, J LAU - Hartley JLFAU - Temple, G FAU - Temple GFFAU - Brasch, M AAU - Brasch MAFAU - Vandenhaute, JAU - Vandenhaute JFAU - Lamesch, P EAU - Lamesch PEFAU - Hill, D EAU - Hill DEFAU - Vidal, MAU - Vidal MLA - engID - R21 CA81658 A 01/CA/NCIID - RO1 HG01715-01/HG/NHGRIPT - Journal ArticleCY - United StatesTA - Nat GenetJID - 9216904SB - IM

Ouyang YB et al. (1998) J Biol Chem "Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans."

Moore KL, Ouyang YB

[J Biol Chem, 1998]

Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.

Joshua Snow et al. (2001) International C. elegans Meeting "Functional analysis of potential E2 ubiquitin conjugating enzymes using RNAi."

Joshua Snow, Rhonda Moore, Lynn Boyd, Claudia Morales

[International C. elegans Meeting, 2001]

We are investigating how genes predicted to be involved protein degradation effect embryogenesis in Caenorhabditis elegans . Within the cell, protein degradation is primarily accomplished through the ubiquitin-proteasome pathway. Studies in other systems show that E2 and E3 enzymes work in tandem to attach ubiquitin to a specific protein substrate, thereby condemning the substrate to degradation by the proteasome. We have identified 26 potential E2 genes within the completed genome of C. elegans . We are assessing the function of these genes through the use of RNAi-mediated interference (RNAi). E3 ligases are less conserved and more numerous than E2s. One class of E3 enzymes contains proteins with RING finger domains. We have previously identified 112 genes containing a RING finger in the C. elegans database. Four of the RING finger proteins were found to be required for embryogenesis (Moore, ECWM 2000, 154). By comparing E2 RNAi phenotypes with the RING finger mutant phenotypes, we hope to determine which E2 ubiquitin-conjugating enzymes partner with specific RING finger proteins. One of the four essential RING finger containing genes is par-2 , a gene involved in establishing anterior-posterior polarity in the embryo. PAR-2 protein is localized asymmetrically to the posterior cortex in embryos. In order to understand if protein degradation is involved in PAR-2 localization, we are using a transgenic strain expressing PAR-2:GFP to observe PAR-2 localization in E2 RNAi embryos.

Beisswanger R et al. (1998) Proc Natl Acad Sci U S A "Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2."

Ashman K, Kellner R, Thiele C, Huttner WB, Niehrs C, Corbeil D, Vannier C, Dohrmann U, Beisswanger R

[Proc Natl Acad Sci U S A, 1998]

Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [35S]sulfate transfer from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS); and (ii) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. &amp; Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896-2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an approximately 13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.

JC Bettinger et al. (1999) International C. elegans Meeting "Behavioral Effects of Exposure to Ethanol"

SL McIntire, JC Bettinger

[International C. elegans Meeting, 1999]

Exposure to ethanol interferes with complex behaviors in many model systems, but it has been difficult to correlate effects of ethanol on behavior with observations of its effects on specific molecular targets. Recently, studies of Drosophila demonstrated a link between ethanol sensitivity and a learning pathway 1 : A screen for mutations that cause flies to be hypersensitive to the effects of ethanol on postural control yielded an allele of amnesiac , a putative neuropeptide known to be involved in learning 2 . After exposure to ethanol, C. elegans display uncoordinated movement (characterized by a decreased amplitude of the sine waveform and lethargy), and decreased rate of pumping and egg-laying (SLM, unpublished observations). After several hours of exposure, worms develop an acute tolerance to ethanol, and recover to resemble untreated controls. We are interested in determining whether or not exposure to and development of tolerance to ethanol alter any of the more complex behaviors exhibited by worms, including chemotaxis. Our preliminary experiments on the effect of ethanol on chemotaxis suggest that brief or prolonged exposure to moderate concentrations of ethanol (100-200 mM) does not prevent chemotaxis to the volatile odorant benzaldehyde. With extended exposure, worms become insensitive to chemoattractants in a process termed adaptation. Worms that have adapted to a particular chemoattractant will not climb a gradient of that chemoattractant 3 . Given that worms are able to chemotax in the presence of ethanol, we can test the effect of ethanol on adaptation. We are determining whether or not exposure to low-to-moderate concentrations of ethanol interferes with the process of adaptation to benzaldehyde and other odorants. 1 Moore, M.S.; DeZazzo, J.; Luk, A.Y.; Tully, T.; Singh, C.M. and Heberlein, U. (1998). Cell 93: 997-1007. 2 Feany, M.B. and Quinn, W.G. (1995). Science 268: 869-873. 3 Colbert, H.A. and Bargmann, C.I. (1995). Neuron 14: 803-812.

Delia M O'Rourke et al. (2005) International Worm Meeting "Analysis of gene expression changes in C. elegans following infection with Microbacterium nematophilum."

Delia M O'Rourke, Richard Mott, Jonathan Hodgkin, Maria Demidova, Dilair Baban

[International Worm Meeting, 2005]

Microbacterium nematophilum is a bacterial pathogen of C. elegans (Hodgkin et al, 2000) that adheres to the rectum and post-anal region of the worm causing swelling of the underlying hypodermal tissue, mild constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype. C. elegans responds to this infection in part through activation of an ERK MAP kinase cascade which mediates tail swelling and prevents severe constipation (Nicholas and Hodgkin, 2004). To identify the downstream transcriptional changes that occur in C. elegans during this host/pathogen interaction we have conducted a genome wide analysis of gene expression using Affymetrix gene chips. We infected a synchronised population of larval C. elegans in liquid culture for 6hrs before harvesting the worms and extracting RNA. Our control sample was an identical experiment using an avirulent M. nematophilum generated in our laboratory by Tanya Akimkina and Steve Curnock. Analysis of data from triplicate microarray experiments has identified a number of statistically significant gene clusters whose expression changes upon infection. Clusters containing up-regulated genes are located on chromosomes IV and V and include C-type lectins, lysozyme-like proteins and proteins containing metridin-like ShK toxin and DUF141 domains. An RNAi feeding screen of these and other induced genes has identified a number that affect the Dar response. The microarray data suggest that although similar functional domains are found in proteins induced by both M. nematophilum and other pathogens, the response of C. elegans to M. nematophilum is specific. Hodgkin, J. Kuwabara, P. E. Corneliussen, B. Current Biology 200010 ;1615-1618 Nicholas, HR, Hodgkin, J. Current Biology 2004 14 ;1256-1261

Tenenhaus T et al. (1997) International C. elegans Meeting "GENETIC REQUIREMENTS FOR PIE-1 EXPRESSION AND RNA POLYMERASE II INHIBITION IN THE EMBRYONIC GERM LINEAGE OF C. ELEGANS"

Tenenhaus T, Seydoux GC

[International C. elegans Meeting, 1997]

The germline blastomeres (P1-P4) fail to transcribe many messenger RNAs expressed in somatic cells(1). This deficiency in mRNA transcription requires the germline factor PIE-1(1,2), and correlates with the absence of a particular phosphoepitope on RNA polymerase II (RNAP II-H5) (see abstract by Dunn and Seydoux). To begin to understand the genetic requirements for the repression of RNA polymerase II activity in the germ lineage, we have analysed pre-existing embryonic lethal mutations for the expression of PIE-1 and RNAP II-H5 by immunohistochemistry. We find that many mutations (par-1, par-2, par-3, par-4, par-6, cib-1, mes-1, emb-1, emb-6, emb-8, emb-16, emb-20, emb-21, emb-25, emb-27, emb-30, zyg-1, zyg-2, zyg-9) result in the complete or progressive loss of PIE-1 expression in the germ lineage. In all cases, these mutations cause inappropriate expression of RNAP II-H5 in the germ lineage, suggesting that PIE-1 is required in each germline blastomere to repress RNAP II activity. We have also found two mutations (mex-1, mex-3) that cause ectopic expression of PIE-1 in somatic blastomeres; preliminary results suggests that these mutations also affect RNAP II-H5 expression, suggesting that RNAP II-H5 expression is sensitive to variations in PIE-1 levels. Further analysis of these and other mutations will test the hypothesis that PIE-1 is necessary and sufficient to repress RNA polymerase II phosphorylation, and will identify loci required to restrict PIE-1-dependent repression of transcription to the germ lineage. We thank the CGC for strains and Craig Mello, Charlotte Schubert, Jim Priess and Steve Warren for antibodies. 1. Seydoux et al. (1996). Nature 382, 713-716. 2. Mello et al. (1996). Nature 382, 710-712.