Crook, Helen Marie et al. (2009) International Worm Meeting "An RNAi screen for regulators of phase II detoxification genes."

Crook, Helen Marie, Olahova, Monika, Veal, Elizabeth A., Blackwell, T. Keith

[International Worm Meeting, 2009]

The phase II detoxification system is an important cellular defence against the toxic effects of free radicals and xenobiotics which cause oxidative tissue damage that has been implicated in many diseases and in ageing. Thus, in response to stress, the expression of many phase II detoxification enzyme genes is co-ordinately induced. The antioxidant glutathione is utilized as an important component of many phase II detoxification reactions.g-glutamine cysteine synthetase, encoded by gcs-1, catalyses the rate-limiting step in glutathione biosynthesis. Thus gcs-1 is amongst the phase II detoxification genes that are transcriptionally activated in response to stress. Conserved bZIP transcription factors, including Nrf-2 in mammals and SKN-1 in C. elegans, are required for regulation of phase II detoxification genes, including gcs-1. Several signaling pathways including the PMK-1/p38 MAPK signaling pathway, act to regulate levels of active SKN-1 which is important for both stress resistance and ageing [1, 2]. However, in the absence of the conserved 2-Cys Peroxiredoxin PRDX-2, expression of phase II detoxification genes such as gcs-1 is also activated by alternative SKN-1-independent mechanisms [3]. Moreover, activation by these SKN-1-independent mechanism/s is apparently sufficient to increase resistance to arsenite stress. To determine the mechanism/s by which PRDX-2 inhibits phase II detoxification gene expression, we are undertaking RNAi screens to identify genes required for expression of a stress-inducible Pgcs-1::GFP reporter in prdx-2 mutant worms. Phosphatases play key roles in the regulation of signal transduction and are intrinsically sensitive to oxidative stress. Hence, we have begun by conducting a screen of RNAi targeting C. elegans phosphatase-encoding genes. Here we will present the data obtained in this screen. It is expected that characterisation of genes identified by these screens will uncover new mechanisms for regulating phase II detoxification gene expression and provide insight into the important contributions made by PRDX-2 and phase II detoxification systems to stress resistance and longevity [2, 3].

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.

Todd N Wylie et al. (2001) International C. elegans Meeting "NEMATODE.NET, a Tool for Navigating Sequences from Parasitic and Free-living Nematodes"

Todd N Wylie, John C Martin, Robert H Waterston, James P McCarter, Sandra W Clifton

[International C. elegans Meeting, 2001]

Two web sites have been established to allow easier access to nematode sequences from species other than C. elegans and C. briggsae ; WWW.NEMATODE.NET is maintained by the Genome Sequencing Center (GSC) at Washington University in collaboration with North Carolina State University, and WWW.NEMATODES.ORG is maintained by Mark Blaxter's lab at the University of Edinburgh. Useful features being built for NEMATODE.NET include the following - 1) Searches: All nematode expressed sequence tags (ESTs) generated at the GSC, currently 32,000 from 10 species, and NemaGene clusters built from these ESTs, are available for BLAST and text searching. Searches can be directed by species, library, or nematode clade in a way that is not possible using the NCBI EST database dbEST. 2) FTP: All EST project data can be downloaded for local analysis including FASTA files and sequence trace image files. 3) Trace Viewer: Fluorescent trace representations for each EST can be viewed. Traces can sometimes provide additional sequence information not included in the EST due to quality value cut-offs. 4) Project Updates: Information is available about libraries in construction and sequencing in progress as the project expands toward 235,000 ESTs. 5) Clone Requests: Details on clone availability and ordering procedure are provided. 6) Links: The site includes an up-to-date set of 300 links to information on human, animal, and plant parasitic nematodes. Further plans for NEMATODE.NET include linking of ESTs to their closest C. elegans homologues by DAS third-party curation of Wormbase. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.

Schein JE et al. (1996) West Coast Worm Meeting "CONSTRUCTION AND UTILIZATION OF THE CHROMOSOME IV COSMID TRANSGENIC LIBRARY"

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

[West Coast Worm Meeting, 1996]

Construction of a chromosome IV cosmid transgenic library of C. elegans strains carrying sequenced cosmids in extrachromosomal arrays has begun as part of our labs' collaboration with the Genome Sequencing Consortium (see abstract by D. Janke et al.). There are 20 chromosome IV cosmids available in transgenic strains at the time of this writing, and we estimate a total of 40-50 will be available to the community by the end of July. In order to enhance the utility of this library, we are also using these strains to localize approximately 50 essential gene mutations, previously identified in the Baillie lab, to cosmids by way of transgenic rescue experiments (for results of similar efforts on chromosomes III and V, see abstracts by Nigel O'Neil et al., Helen Stewart et al., and Brad Barbazuk et al.). Correlation of the genetic and physical maps in this manner will facilitate the selection of candidate cosmids for use in further rescue experiments, and thereby increase the speed with which the connection between genetic mutations and DNA sequence is made. Currently these rescue experiments are focused in the sDf2 region, in particular in the lin-3 - let-60 interval, which contains 13 essential gene mutations and 21 overlapping cosmids. The two genes bracketing this interval, lin-3 and let-60(ras), were previously placed on the physical map by others. To date, two additional essential genes have been positioned to cosmids on the physical map; let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido) and let-64. It is anticipated that by the time of the meeting several additional mutations will have been positioned to cosmids. This work is being funded by a grant from the Canadian Genome and Associated Technologies Program to Ann Rose and David Baillie.

Birgit Gerisch et al. (2006) European Worm Meeting "RNAi Screen for Genes that Regulate Stress Resistance and Life Span of C. elegans"

Adam Antebi, Marie-Laure Yaspo, Birgit Gerisch, Hans Lehrach

[European Worm Meeting, 2006]

Birgit Gerisch1, Adam Antebi2, Marie-Laure Yaspo1 and Hans Lehrach1. Oxidative stress appears to play major role in life span regulation. Long-lived mutants of daf-2/insulin-like receptor (insulin/IGF-1 signaling) also show increased resistance to environmental stress, such as high temperature and reactive oxygen species. Moreover most known long-lived mutants tested so far are resistant to various environmental stress. We are using this correlation between longevity and stress resistance to identify genes that are involved in the process of life span regulation, and screened 275 RNAis corresponding to phosphatases for oxidative and heat stress resistance. Positive RNAis were tested in aging experiments. Out of the 275 RNAis 15 result in strong resistance to oxidative stress, 3 in resistance to heat stress, and 5 in oxidative and heat stress resistance. At the moment these RNAis are being tested in aging experiments. The most interesting candidate we have obtained up to now is an evolutionary conserved phosphatase involved in cell division, which results in resistance to heat stress and shows a strong life span extension. Further characterization and assignment of the identified genes to known pathways should yield insights into cellular processes involved in longevity and stress resistance.

Verster, Adrian et al. (2009) International Worm Meeting "Examining Changes in Gene Function in Caenorhabditis nematodes using RNAi libraries."

Felix, Marie-Anne, Verster, Adrian, Mckay, Sheldon, Ramani, Arun

[International Worm Meeting, 2009]

Gene functions change throughout evolutionary history and understanding how this occurs is a key goal of molecular evolutionary biology. To date researchers have systematically looked at changes in gene function by comparing loss of function phenotypes in orthologs from different model organisms, such as S. cerevisiae and C. elegans. However, since there has been such a large evolutionary divergence between these organisms their body plans are so different and many phenotypes are impossible to compare. It is much more practical to compare phenotypes from organisms with similar body plans and ecological niches. Here we use Caenorhabditis nematodes to systematically look at changes in gene function through evolution because loss of function phenotypes map easily between species. In order to address this problem we are building an RNAi library for C. briggsae to look for changes in gene phenotype from those that were observed in C. elegans. Genome scale RNAi has been successfully used in C. elegans and thus we are going to use it in C. briggsae. In order to do this we take advantage of the sid-2 transgenic C. briggsae line (gratefully contributed by Marie-Anne Felix''s group) which has been shown to uptake RNAi by feeding. We will present preliminary screening data from the C. briggsae RNAi library and showcase the changes in gene function we have found so far.

Hartman PS et al. (1991) International C. elegans Meeting "SOME RAD HAPPENINGS FROM FORT WORTH."

DeWilde D, Reddy J, Hartman PS

[International C. elegans Meeting, 1991]

1. UV radiation mutagenesis in C. elegans: Helen Stewart and David Baillie have noted that many UV radiation-induced mutations are chromosomal rearrangements rather than point mutations. We have begun to extend this observation by isolating UV radiation-induced mutations in fem-3 and unc-22. As suggested to us by Judith Kimble, and as will be described at the meeting,fem-3 is particularly well suited for these studies. Mutations have been selected at low and high radiation fluences, as well as in both rad(+) and rad(-) genetic backgrounds. These are currently the subject of both genetic and molecular analyses, with the ultimate hope of elucidating specific sequence contexts which are misrepaired into deficiencies. 2. Cloning a rad gene: We have isolated two allelic mutations in the high-hopper strain TR679 which confer hypersensitivity to methyl methanesulfonate and UV radiation. They map in between unc-36 and dpy-17 on linkage group III. Each mutation was crossed 12 times into an N2 background, several of which including forced recombinations in between the mutations and either unc-36 or dpy-17. Southern blots were performed, using Tcl as a probe (kindly provided by Phil Anderson) and revealed one novel Tc l- containing fragment in one mutant and three in the other. We are currently cloning these. Since both mutants are hypersensitive only during embryogenesis, we look forward to performing Northern blot analyses in order to determine if this developmental regulation of DNA is exercised at the transcriptional level. 3. The rad mutants are not hypersensitive to 8-methoxypsoralen (8MOP) phototreatment. Extending the observations of Fujita and coworkers (Photochem. Photobiol. 39, 831), survival of three staged populations N2 and four rad mutants was determined after 8-MOP phototreatment. As opposed to their responses after exposure to other DNA-damaging agents, none of the four were hypersensitive. Split-dose experiments indicated that DNA-DNA crosslinks were primarily responsible for lethality. Little if any crosslink repair was observed in N2 using two different assays, explaining why the rad mutants were not hypersensitive to 8-MOP inactivation.

Artan, Murat et al. (2021) International Worm Meeting "Interactome analysis of C. elegans synapses by TurboID-based proximity labeling"

Skehel, Mark, Nicolas, Armel, Barratt, Stephen, de Bono, Mario, Artan, Murat, Begum, Farida, Flynn, Sean M.

[International Worm Meeting, 2021]

Characterizing the proteome of subcellular structures and the interactome of specific proteins provides entry points to probe molecular function. Several methods for screening protein-protein interactions (PPIs) have proven useful, including affinity purification and two hybrid screens. However, these methods have limitations, including poor detection of transient or weak PPIs, high false positives, and difficulty detecting PPIs in specific cell types or subcellular compartments. Many of these limitations can be overcome using proximity-dependent protein labeling (PL). We optimize a PL protocol for C. elegans using TurboID, a recently improved E. coli promiscuous biotin ligase. We analyse the proteome of individual tissues including the nervous system, intestine, hypodermis and muscle, and, to demonstrate single cell resolution, of the pair of AFD neurons. We observe enrichment of tissue-specific proteins, and identify both known and previously unknown AFD-specific proteins. To characterize the interactome of a specific protein, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. ELKS-1-TurboID highlights both characterized and previously uncharacterized synaptic proteins. We use our data to define non-specific contaminants that should help interpretation of C. elegans PL data. TurboID proximity labeling provides a powerful tool to investigate C. elegans biology. Research support: This work was supported by an Advanced ERC Grant (269058 ACMO) and a Wellcome Investigator Award (209504/Z/17/Z) to MdB and an ISTplus Fellowship to MA (Marie Sklodowska-Curie agreement No 754411).

David Johnson et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Regulators of Mitochondrial Morphology Affect Intracellular pH"

David Johnson, Keith Nehrke

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Within living cells mitochondria constantly change shape and size through the processes of fission and fusion. Mitochondrial dynamics are important for numerous cellular processes such as apoptosis, energy production, and cell signaling, and mitochondrial morphology is tightly regulated. Misregulation of mitochondrial dynamics in humans results in diseases such as Charcot Marie Tooth syndrome and autosomal dominant optic atrophy. Specifically, mitochondrial fusion in C. elegans is regulated by the mitochondria associated GTPases encoded by the genes fzo-1 and eat-3. Loss-of-function mutations in either gene have been shown to cause high levels of mitochondrial fragmentation in the muscle cells of C. elegans (Kanazawa et al., 2008 Feb 29;4(2)). Here, using a combination of RNAi and deletion mutants, we show that the loss of either fzo-1 or eat-3 is sufficient to reduce intracellular pH (pHi) in both muscle and intestinal cells. We also show that these genes are epistatic to one another and that they function cell autonomously to maintain pH homeostasis. Furthermore, failure to rescue using dichloroacetate suggests that the reduction in pHi is not simply a result of lactic acidosis. Finally, since intestinal pHi oscillates during defecation (Pfeiffer et al., Curr Biol. 2008 Feb 26;18(4):297-302), we have examined how morphology regulates dynamic changes in pHi in live worms. These results implicate mitochondrial morphology as a possible regulator of pHi and provide new insight into the pathology of human diseases caused by the altered mitochondrial dynamics in humans.

Stewart HI et al. (1996) West Coast Worm Meeting "Recessive Lethal Mutations and Deficiency Analysis in Caenorhabditis elegans on LGIII(left) and Balanced by sDp3"

Vatcher GP, Ha TT, Janke DL, Gilchrist EJ, Stewart HI, Baillie DL

[West Coast Worm Meeting, 1996]

We have isolated 202 new, EMS induced, recessive lethal mutations on the left arm of LGIII. The free duplication sDp3 (III;f) was used to rescue recessive mutations linked to dpy-17 (recovery rate 1.9%). Of these 202 mutations, 109 have been characterized and analysis of the remaining mutations is in progress. A subset, of 62 recessive lethals, are also marked with ncl-1 and will facilitate the mosaic analysis of these lethal mutations. We have identified 82 new essential genes let-700 to let-790. Of these new essential genes, 13 have multiple alleles, thus we estimated that we have only a 28% saturation of the area for essential genes. As there is about 4.8 Mb of DNA, in the interval between unc-93 and dpy-19, we estimate that there will be approximately 293 essential genes, in this region. We intend to generate another set of recessive lethal mutations to facilitate saturation of this area for essential genes. Both gamma rays and ultraviolet light were used to generate a set of overlapping deficiencies. We have shown that duplications can be used to rescue large deletions such as sDf121, sDf125, sDf127 and sDf130, as well as smaller deficiencies. This indicates that there are no "holes" or lethal mutations along the length of sDp3, in the areas uncovered by these large deficiencies. We have shown that duplications, such as sDp3, are valid and useful as balancer systems and have utilized deficiency mapping to resolve the genetic mapping of essential genes. Selected visible mutations have also been complementation tested with our deficiencies. PCR analysis was utilized, with these deficiencies, to correlate the genetic and physical maps and to further define the end points of these deficiencies ( See poster ; Norm Franz et al, this session). As well, work has been progressing on the rescue of essential genes (See poster; Helen Stewart et al, this session), utilizing transgenic strains constructed in this laboratory (see presentation by Diana Janke et al, this session). This work has been funded by a grant from NIH (National Institute of Health), U.S.A. to Ann. Rose; D. Riddle and D.L. Baillie.