Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

Carta L (1998) East Coast Worm Meeting "TOXICITY OF 4 SERRATIA MARCESCENS BACTERIAL STRAINS TOWARD C. ELGANS N2, PGP-3 AND OTHER NEMATODES IS INVERSELY RELATED TO PIGMENT"

Carta L

[East Coast Worm Meeting, 1998]

We are testing a number of biocontrol bacteria (eg. Burkholderia, Pseudomonas and Stenotrophomonas) for toxicity to bacterial feeding nematodes including C. elegans N2 and pgp-3. Serratia marcescens in particular had a measurable effect on number of eggs over 24 hours and relative motility in microtiter wells. Serratia marcescens strains with increasing amounts of red pigmentation (BF1-5@ (albino), D1, N4-5, NIMA) were tested for relative motility to C. elegans N2, pgp3, Oscheius myriophila DF5020, Panagrellus redivivus PS 1163, Mesorhabditis sp. PS1170 and Zeldia punctata PS 1153. Toxicity directly increases with decreasing amount of pigment in Caenorhabditis, Oscheius and Panagrellus. The order of relative toxicity is reversed for the 2 highly pigmented strains and the 2 least pigmented strains (2143 vs. 1234) in Mesorhabditis and Zeldia. These nematodes are phylogenetically distant from C. elegans. Bacterial strains were provided by Phyllis Martin and Dan Roberts, USDA.

Chung, George et al. (2015) International Worm Meeting "The molecular identification of a gene which controls the distribution of meiotic recombination events in Caenorhabditis elegans."

Sanchez-Pulido, Luis, Petalcorin, Mark, Chu, Jeffrey, Chung, George, Boulton, Simon, Rose, Ann, Ponting, Chris, Yanowitz, Judith, Kessler, Zebulin, O'Neil, Nigel, Martin, Julie

[International Worm Meeting, 2015]

The placement of meiotic crossover events is regulated such that crossover events are not distributed randomly along the chromosome. In Caenorhabditis elegans, meiotic crossover placement favours chromosome arms over chromosome centres, a bias abolished by mutations in the rec-1 gene. This mutant phenotype has precluded positional cloning efforts for 30 years. Here, we describe the identity of rec-1, which was uncovered by whole-genome sequencing and confirmed by new alleles engineered with MosSCI and CRISPR-Cas9. The rec-1 gene encodes a previously uncharacterized protein, which possesses putative phosphorylation motifs as well as weak homology to the meiotic protein HIM-5. We demonstrate that the REC-1 protein is a substrate for CDK phosphorylation in vitro and in C. elegans extracts. This phosphorylation was abolished in a REC-1(8A) mutant lacking the 8 CDK phosphorylation sites. Moreover, the rec-1(8A) and rec-1(8E) (phosphor-mimetic) mutant transgenes did not rescue the rec-1 defect. Given the homology shared between REC-1 and HIM-5, we also examined their genetic relationship. Strikingly, we found that in contrast to the single mutants, the rec-1; him-5 double mutant was defective for the formation of meiotic double-strand breaks (DSBs), which was rescued by artificially introducing DSBs using ionizing radiation. Thus, we establish that REC-1 plays an indispensable role in the distribution of meiotic crossovers, which is controlled by CDK phosphorylation. Furthermore, we uncover an unappreciated redundancy in the mechanisms required to generate meiotic DSBs involving REC-1 and HIM-5.

Aurlie Blanc et al. (2005) International Worm Meeting "A French functional genomics platform."

Daniel Wong, Jerome Reboul, Jonathan Ewbank, Aurlie Blanc, Yohann Duverger

[International Worm Meeting, 2005]

For the last two years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates 500 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community.This French functional genomics platform has been made possible through funding from the National Genopole<sym02> network, Marseille-Nice genopole<sym02>, the CNRS and collaboration with Union Biometrica.

Yohann Duverger et al. (2007) International Worm Meeting "A French functional genomics platform."

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Sarah Scaglione, Daniel Wong

[International Worm Meeting, 2007]

For the last four years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates up to 1000 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented. This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community. This French functional genomics platform has been made possible through funding from the National Genopole&#174; network, Marseille-Nice genopole&#174;, INSERM, the CNRS and support from Union Biometrica.

Gogonea CB et al. (1997) International C. elegans Meeting "EXPRESSION OF THE REPORTER GENE MEC-12::LACZ IN LOCOMOTORY AND CHEMOSENSORY MUTANTS OF C. ELEGANS"

Siddiqui ZK, Siddiqui SS, Gogonea CB

[International C. elegans Meeting, 1997]

Mutations in close to 100 genes affect locomotion. These mutants show uncoordinated locomotion, ranging from slow movement, poor backing, colier, kinkier, and paralyzed phenes (J. Hodgkin, 1997: Appendix in C. elegans II). Previously it was shown that mutants in at least 25 genes harbor defects in the axonal growth and process placement of the six touch receptor neurons (Siddiqui, 1990). A reporter gene mec-12::lacZ has been shown to express in the set of six touch receptor neurons (Fukushige et al., 1997). So far, a variety of cellular defects in the process placement, axonal guidance and axonal growth of the touch cells and the PVR in 15 genes (unc-4, unc-5,unc-30, unc-33, unc-44, unc-51, unc-53, unc-55, unc-61, unc-71, unc-76, unc-83, unc-86, and unc-119) have been observed. Defects include ectopic branching of axons, fore- shortened processes, misplaced cell body positions, extra commissures, and a lack of staining. In addition, we have examined the reporter gene expression in the background of various mechano- sensory mutations (Chalfie and Au, 1989). These results will be summarized. We thank the Caenorhabdtis Genetics Center, and Martin Chalfie for various mutant strains.

May Tran et al. (2005) International Worm Meeting "RNAi knockout of the mitochondrial carrier protein ucp-4: effects on metabolism"

May Tran, Craig LaMunyon, Aidyl Gonzalez-Serricchio

[International Worm Meeting, 2005]

We are investigating the phenotype of ucp-4 RNAi knockout worms. Mitochondrial uncoupling protein (UCP) function in humans has been linked to obesity. UCPs are mitochondrial carrier proteins, and human UCP-1, -2, and -3 are protonophores, transporting protons from the intermembrane space to the matrix, bypassing ATP synthase, and thereby raising resting metabolic rate in response to certain cues. The function of human UCP-4 is not clear, but it may be a mitochondrial fatty-acid transporter, and it is a homolog to the C. elegans ucp-4 (K07B1.3). Initial RNAi experiments by Dr. Julie Ahringers lab deemed knockout worms to be wild-type. However, we wondered if the RNAi phenotype might be a subtle alteration of metabolic activity. We obtained Dr. Ahringers RNAi feeding clone from the UK HGMP Resource Centre and measured egg laying and defecation rates. Compared to control worms fed HT115 bacteria containing the empty feeding vector, ucp-4 RNAi fed young adults that were exposed beginning as L1 larvae laid eggs at a significantly slower rate, although preliminary data on defecation rate suggests no difference between these treatments. If the trends in these data hold, it would suggest that ucp-4 RNAi knockout worms are processing food at the same rate, but converting that energy into offspring at a reduced rate.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Martin Gutternigg et al. (2006) European Worm Meeting "Glycosidases of Caenorhabditis elegans involved in N-glycan processing"

Martin Gutternigg, Matthias Hackl, Ute Stemmer, Iain B. H. Wilson, Petra Geier, Gunter Lochnit, Ramona Ranftl, Katharina Paschinger, Verena Jantsch, Dorothea Lubich

[European Worm Meeting, 2006]

Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.

Vallin E. et al. (2006) European Worm Meeting "Molecular Characterization of MOS Transposon-Tag Strains Produced by the NemaGENETAG Project"

Vallin E., Granger L., Segalat L., Martin E.

[European Worm Meeting, 2006]

Vallin E., Granger L., Martin E. & Segalat L.. A collection of Caenorhabditis elegans insertion mutants is being created as part of the NemaGENETAG project. The aim is to produce characterized transposon-tagged mutants for the European and international scientific community. We use the Drosophila mariner element Mos-1, which has been shown to be active in C.elegans. The J. Ewbank laboratory (Marseille) generates and provides us with Mos-positive strains, which we molecular characterize. The localization of the insertion is determined by inverse PCR and sequencing. The sequence is compared with the C. elegans genome sequence to determine the insertion sites using the blast program. At the beginning of 2006, we have characterized approximately 2000 strains. Strains are currently analyzed sequentially with enzymes Hae III and Mbo I. Approximately 70% of the strains received from Marseille give a workable PCR band in these conditions. The percentage of PCR bands that give an unambiguous genome localization is approximately 66%. These strains are frozen in triplicate in both -80C freezer and liquid nitrogen. In case of request or for quality control, strains are thawed and PCR-tested with a Mos primer and an insertion-specific primer to check the presence on the insertion. On 30 strains tested in recovery tests, 28 were positive. These results prove that the insertions are stable and can be easily recovered from frozen samples. The aim of the project is to generate and provide strains for the worm community. The list of the insertions can be found on a database available on the web.