Julie-Anne Fritz et al. (2003) International Worm Meeting "Assessing the functions of succinate dehydrogenase and fumarate reductase by RNA interference in Caenorhabditis elegans."

Julie-Anne Fritz, Carolyn Behm

[International Worm Meeting, 2003]

Respiratory Complex II, or succinate dehydrogenase/fumarate reductase (SDH/FR), of the mitochondrial electron transport chain catalyses either oxidation of succinate under aerobic conditions or reduction of fumarate under microaerobic conditions. The Complex is composed of four polypeptides, Fp, Ip, cybL and cybS. Fp and Ip form the catalytic site while the cyb subunits catalyse electron transport. We chose the Fp subunit for RNA interference (RNAi) studies as it is expected to be a critical component in determining the catalytic function of Complex II. While C. elegans normally lives aerobically, with SDH activity predominating, worms can also survive microaerobic conditions, excreting metabolic end-products indicative of FR activity. We aim to assess which Fp subunit is responsible for FR activity. Three genes encode putative Fp subunits in C. elegans. We silenced the expression of each gene, individually and in combination, using the RNAi feeding method. No obvious phenotype was evident for any gene silenced individually, but simultaneous knockdown of the two closely-related Fp genes (C03G5.1 and C34B2.7) that have high homology to SDH/FR Fp subunits from nematode parasites, induced an 80% decrease in fecundity. The two genes are thus functionally redundant and important under aerobiosis. We also tested worm viability in the presence of sodium azide, an inhibitor of respiratory Complex IV. To date we have observed that silencing expression of the third putative SDH/FR Fp gene (F48E8.3) induced an increased trend in mortality. Further work is planned to identify which gene encodes the Fp subunit catalysing FR activity in the nematode.

Hirani, Nisha et al. (2011) International Worm Meeting "Operon transcriptional complexity: dissection via recombineered fosmid-based reporters."

Sturzenbaum, Stephen, Dolphin, Colin, Hirani, Nisha

[International Worm Meeting, 2011]

Operons are common in prokaryotic genomes but, except for Nematoda, are uncommon in eukaryotes. Nematoda "operons" comprise gene clusters with short, approx. 100bp, intergenic regions. A discrete nascent transcript, transcribed from a major upstream promoter, undergoes trans-splicing and 5' addition of one of two possible spliced-leader sequences, followed by cis-splicing and finally translation of mature mRNA. Operons are common in C. elegans and approx. 15% of genes are clustered into operons. The significance of Nematoda operons is unclear although evidence suggests they may have arisen as a consequence of genome evolution and compaction - the "easy come, slow go" scenario. The classical view of operon transcription is of an upstream promoter generating a single "operon" transcript, however evidence suggests operon gene transcription maybe more complex. For example, microarray data indicates that correlated operon gene expression is weaker with increasing intergenic distance suggesting expression is influenced by intergenic sequence-located elements. Further evidence comes from comparison of expression data between fluorescent protein (FP) transcriptional reporters driven either by the upstream "promoter" or intergenic "internal promoter" sequences. These two reporter types generated different expression patterns indicating the presence of regulatory elements within the operon. As an alternative to these transcriptional reporters dissection of operon-clustered gene expression could be investigated using translational reporters generated directly from genomic clones. As they contain 5' and 3' flanking and intronic sequences such genomic clone-based reporters are more likely to contain all, or many, of the regulatory elements associated with the gene of interest (GOI). Furthermore, these reporters are ideal for analyzing operon transcriptional complexity as the size of the fosmid insert (approx. 40kb) easily accommodates an intact operon. Tagging each operon gene with a different FP would permit operon gene expression analyses within the genomic context of that operon. We have used counter-selection recombineering to insert seamlessly commercially-synthesized, C. elegans-codon-optimized FP genes into the 3' ends of genes within a number of different 3-gene operons each contained within a different fosmid clone. The suitability of these FP genes, as reporters for multiple tagging has been validated by both in vivo and in vitro experiments. Operon targets, with confirmed gene structures and preliminary expression pattern data available from published literature, have been identified and the corresponding, multi-gene-tagged fosmid-based reporters created in readiness for expression pattern analyses.

Harumi Ichimiya et al. (2001) International C. elegans Meeting "RNAi-Mediated Embryonic Lethality by Three Genes Encoding Complex II Subunits of the Electron Transport Chain."

Phil Hartman, Nao Ishii, Harumi Ichimiya

[International C. elegans Meeting, 2001]

The electron transport system consists of four complexes that alternatively accept and donate electrons, generating ATP in the process. We have demonstrated previously that the mev-1 gene encodes one (Cyt-1) of the four subunits (Ip, Cyt-1, Cyt-S and Fp) of complex II. mev-1 mutants are hypersenstive to oxidative stress and age precociously, particularly at elevated oxygen concentrations. To more systematically explore the consequences of complex II inactivation, we performed RNAi, specifically the soaking method, directed against three of the four subunits of complex II. Embryonic lethality was the predominant result in each case, with hatching reduced by 87%, 75% and 98.3% after functional inactivation of Ip, Cyt-1, and Cyt-S, respectively . The survivors were healthy and not hypersensitive to hyperoxia. The inviable zygotes arrested at various times throughout embryogenesis in proportions specific to the subunit inactivated. For example, Cyt-S inactivation yielded almost 70% inviable zygotes that arrested immediately after fertilization (either prior to nuclear fusion or at the one-cell stage) whereas as almost 60% of Cyt-1-treated embryos reached at least the late morula stage. This embryonic lethality is not surprising given the energy demands throughout embryogenesis.

Natalya Morsci et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "The Role of Kinesin-Like Protein 6 in Cilia Structure and Function"

Natalya Morsci, Maureen Barr

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

The kinesin-3 family member klp-6 is required for male sensory behaviors and localization of the polycystin LOV-1 receptor/PKD-2 channel complex in C. elegans (Peden and Barr, 2005). In humans, mutated polycystins cause autosomal dominant polycystic kidney disease. Understanding the molecular motor KLP-6 may shed light on the etiology of human ciliary diseases. We aim to elucidate the role of KLP-6 in ciliary morphogenesis, understand the mechanisms by which KLP-6 controls sensation and polycystin localization, and identify KLP-6 cargo and regulatory proteins. Cilia are built and maintained by intraflagellar transport (IFT). We will test the hypothesis that klp-6 acts with the IFT machinery to sculpt male-specific ciliated neurons. We will use electron microscopy to identify any ultrastructural defects in klp-6 mutants and also examine genetic interactions between klp-6 and IFT components. Domain architecture of a given kinesin determines its cellular localization and function. To identify innate domains necessary for KLP-6 localization, auto-regulation, motility, and function in polarized cells like sensory neurons, we will use a transgenic approach to express various FP-tagged KLP-6 fragments in wild-type and klp-6 mutant backgrounds. The identity of KLP-6 regulators and cargo is unknown. We will screen a male-enriched yeast two-hybrid library for proteins that interact with KLP-6. We will also test for direct interactions between KLP-6 and its proposed cargo proteins LOV-1 and PKD-2. Combined, these studies will address the functions of the microtubule-based motor KLP-6.

Volkers, R. J. M. et al. (2013) International Worm Meeting "Gene-environment interactions drive genomic and transcriptomic diversity in wild Caenorhabditis elegans populations."

Chen, W., Schulenburg, H., van Hellenberg hubar, C. J., Coopman, R., Sterken, M. G., Kammenga, J. E., Volkers, R. J. M., Braekman, B. P., Snoek, L. B.

[International Worm Meeting, 2013]

RJMV and LBS contributed equally to this work. Background: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research in the nematode C. elegans has been instrumental for unravelling many genotype-phenotype relations. But almost all studies, including forward and reverse genetic screens, are dominated by investigations in one canonical single strain. In order to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map, it is important to study these relations in wild populations. Results: We used multiple wild strains freshly isolated from local habitats to investigate the gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness and behavioural traits. These showed a direct link with the original habitat of the strains. The separation between the isolation sites was prevalent on all chromosomes, chr. V contributing the most. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Moreover, we show that genomic and transcriptomic diversity was driven by genes involved in gene-environment interactions, like c-type lectins and fbox genes. Conclusions: Importantly, where wild C. elegans strains show a broad range of genotype-phenotype relations the widely studied canonical genotype N2 covers only a diminutive part of the myriad of genotype-phenotype relations which are present in the wild. Funding: RJMV and LBS: NWO-ALW (project 855.01.151) NEMADAPT, RC and BPB: ESF-EEFG (09-EuroEEFG-FP-002 / G.0998.10N) NEMADAPT. JEK: ERASysbio-plus ZonMW project GRAPPLE (project nr. 90201066). MGS: Graduate School Production Ecology & Resource Conservation. WC and HS: NEMADAPT (DFG grant SCHU 1415/11-1).

Peder Zipperlen et al. (2003) International Worm Meeting "An allele specific PCR based approach to map mutations to subchromosomal regions"

Peder Zipperlen, ALEX HAJNAL

[International Worm Meeting, 2003]

The availability of a high-density SNP map between C. elegans varieties Bristol and Hawaii allows for the rapid mapping of mutations. Numerous different assays exist to determine the molecular nature of a SNP, such as restriction fragment length polymorphism (RFLP), fluorescence polarisation (FP) or microsequencing. Common to all approaches is that the region containing the SNP is first amplified in a PCR reaction and then in a second step, the PCR fragment is analysed using different approaches like the ones mentioned above. This two step procedure is labour intense and, depending on the chosen SNP assay, requires special equipment and, hence, can be costly. Therefore, we set out to develop an allele specific PCR based approach that would allow us to determine the molecular nature of a SNP already in the initial PCR. All our assays are designed such that there is amplification only if the Hawaii allele is present whereas there is no amplification from a Bristol allele. Results obtained so far by using a set of six SNP assays, one for the centre of each chromosome, are encouraging. This set allows us to link mutations to one of the six chromosomes by testing 14 F2 single worm lysates in a total of 84 PCR reactions only. Together with 2x6 positive and negative controls on Bristol and Hawaii lysates gives a total of 96 reactions. Currently, we are developing two additional assays for each chromosomal arm, resulting in a total of five assays/chromosome. This set will allow us to map mutations to subchromosomal regions by performing an estimated additional 200-300 PCR reactions. At the meeting, we will report on our progress in developing those additional assays and their use in mapping mutations.

Donavan TS Cheng et al. (2007) International Worm Meeting "A NON-ALIGNMENT GIBBS-SAMPLING BASED METHOD FOR SEQUENCE ELEMENT DETECTION USING PHYLOGENETIC CONSERVATION."

Donavan TS Cheng, Michael A Beer

[International Worm Meeting, 2007]

Current attempts to infer regulatory networks from genome-wide expression data require motif detection algorithms to identify transcription factor (TF) binding sites in the regulatory regions of co-expressed genes. These algorithms typically use either over-represention or phylogenetic conservation to predict TF binding sites. While these approaches have been successful in yeast [1,2,5], in larger genomes motif detection is significantly more challenging, and will likely require comparative genomics. Several recent algorithms have begun to combine these two sources of information to predict functional sites more accurately. [1,2] However, many rely on an initial global alignment of orthologous regulatory regions as a preprocessing step. Unfortunately, TFs generally recognize sites that are short and degenerate, and global alignment may be overly restrictive for the detection of such sites. We have developed a non-alignment based Gibbs sampling [3,4] algorithm that searches for over-represented sequence elements, conserved across orthologous regulatory regions in related species. Motif composition is represented probabilistically by a position weight matrix and a posterior probability score is used to assess the significance of the motif found. The algorithm employs a stochastic search to converge to optima and unlike other alignment-based techniques, does not require a prior estimate of the evolutionary relationship between species sampled. We compare the performance of our algorithm on synthetic data to that of existing algorithms that employ either over-representation, phylogenetic conservation, or both. We further validate our motif predictions on in vivo binding data in yeast. [5] We present novel motif predictions from sets of coexpressed genes C. elegans and the related sequenced nematode species C. briggsae, and C. remanei. 1. Siddharthan R, Siggia ED, van Nimwegen E. PLOS Comp. Biol. 1, e67 (2005). 2. Sinha S, Blanchette M, Tompa M. BMC Bioinf. 5, 170 (2004). 3. Roth FP, Hughes JD, Estep PW, Church, GM. Nat. Biotech. 16, 939-945 (1998). 4. Neuwald AF, Liu JS, Lawrence CE. Prot. Sci. 4, 1618-1632 (1995). 5. Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, et al. Nature 431, 99-104 (2004).

Daniel C. Koboldt et al. (2007) International Worm Meeting "High-throughput identification of structural variations from C. briggsae sequence traces."

Julia E. Staisch, Raymond D. Miller, Daniel C. Koboldt

[International Worm Meeting, 2007]

Recent reports of widespread copy number differences including large insertion/deletions (indels) have demonstrated that this variation affects such substantial portions of the human genome that it is likely to play a significant role in human disease. C. elegans strains also harbor large indels (Maydan, J.S. et al., 2007, Genome Res 17:337). While comparative genome hybridization and SNP genotyping technologies have been adapted to detect structural variants, doing so at the nucleotide level remains a challenge. We have developed a method that allows for the high-throughput identification of structural variation from sequence trace data. We tested the method using sequence traces from C. briggsae strain HK104, which has a SNP on average every 115 bp when compared to the canonical strain (AF16). Each set of sequence traces was used to create a BLAST database against which the reference genome was aligned using WU-BLAST. The BLAST results were parsed and filtered to identify reads that appear to span an indel. Alignment positions and orientations were analyzed to characterize the size and precise molecular boundaries of the apparent indels. From a database of 13,632 sequencing traces from strain HK104 we identified 399 candidate indel events of 50 - 1,000 bp, 62 indels of 1-10 kbp, and a number of larger indels. We chose an initial test set of two indels for each of the six chromosomes, and designed PCR assays for them. Nine of the 12 PCR assays were successful under uniform conditions and each of the nine reflected the predicted indel. We also designed and successfully used three FP-TDI genotyping assays for the indels. Additional candidates are being tested. Our method is both efficient and sensitive. It has shown that large indels are common in C. briggsae. The large indels are very useful as tools for mapping studies and they raise the question about their effects on gene expression.

Clark SG et al. (1996) West Coast Worm Meeting "ANALYSIS OF LONGITUDINAL CELL MIGRATION AND AXON GUIDANCE"

Clark SG, Bargmann CI

[West Coast Worm Meeting, 1996]

The assembly of the C. elegans nervous system requires the directed migration of cells and growth cones along the anteroposterior body axis. To understand the mechanisms that guide these migrations, we initiated a genetic and molecular approach to identify molecules required for longitudinal cell migration and axon outgrowth. Transgenic strains expressing the green fluorescent protein (GFP) under cell-type-specific promoters were generated to observe particular neurons and their axonal processes in living animals. For example, AVA, AVB, AVD and PVC were examined using a glr-1::GFP strain and CAN was visualized using a ceh-23::GFP strain. In collaboration with B. Wightman and G. Garriga, we found that vab-8 is needed to guide most posteriorly-directed cell and growth cone migrations. To find additional guidance genes, we sought mutants with phenotypes similar to those of vab-8 animals. From screening the progeny of 7,000 mutagenized glr-1::GFP or ceh-23::GFP F1 animals, we recovered 19 uncoordinated and withered tail mutants. These mutations represent 12 genes, including epi-1, mig-2, mig-10, mig-11, unc-33, unc-34, unc-35, unc-44, unc-73 and vab-8. Some of these genes were known to act in cell migration and axon outgrowth; others were less well understood. For example, in addition to blocking the growth of the AVA, AVB and AVD axons, we found that mutations in mig-2, unc-34 and unc(ky190) caused these axons to extend inappropriately along the dorsal or lateral nerve cords, indicating that these genes are required for axon outgrowth and pathfinding. Mutations in epi-1, which encodes a laminin A subunit (K. Joh & E. Hedgecock, pers. comm.) caused the CAN axons to branch inappropriately and stray from their normal trajectory, suggesting a role for laminin in maintaining normal axon morphology, fasciculation and directed growth. The unc-35 mutant isolated in our screen, ky196, has an uncoordinated phenotype like that of the canonical unc-35 allele, e259. However, unlike e259 mutants, many ky196 animals have notched heads and protruding vulvae, are sterile or die as larvae. ky196 mutants have defects in the organization of dense bodies and other phenotypes characteristic of Pat mutants (B. Barstead, pers. comm.; our observations). These phenotypes are similar to those conferred by mutations in an alpha integrin gene (P. Baum, D. Perry & G. Garriga, pers. comm.), suggesting that unc-35 might act in an integrin-mediated process. We have begun the molecular characterization of unc-35 and mig-11, which, like vab-8, appears to act in the guidance of posteriorly-directed cell and growth cone migrations.

Gu GQ et al. (1997) International C. elegans Meeting "Characterization of mec-15 and mec-18"

Gu GQ, Chalfie M

[International C. elegans Meeting, 1997]

At least twelve genes (mec-1, 2, 4, 5, 6, 7, 9, 10, 12, 14, 15, and mec-18) are needed for mechanotransduction process in the C. elegans touch receptor neurons. From molecular genetic data, we proposed that these genes encode different components of a touch apparatus. This touch apparatus includes a mechanically-gated degenerin channel (formed by MEC-4, 10 and, possibly, MEC-6), extracellular matrix (MEC-5, 9 and, perhaps, MEC-1), cyotskeletal components (MEC-7,12), a molecule that couples the channel and microtubules (MEC-2) and several channel regulators (MEG-14, 15, and MEG-18). This putative touch apparatus enables the mechanical stimuli to directly open the degenerin channel. To understand better the regulation of this touch apparatus, we started to characterize mec-15 and mec-18, two genes that may encode modifiers of the apparatus. We found that mec-7 and mec-12 loss of function mutations dominantly suppressed the mec-15 Mec phenotype in an allele-specific manner. These results lead us to believe that mec-15 product physically interacts with the microtubule structure. mec-15 maps about 0.2 mu to the right of the polymorphism stP98. Cosmids spanning ~0.6 mu around this region were injected into mec-15 mutants, but none have rescued the mutant phenotype. We are planning to use phosmids that lie within the cosmic gaps to try the rescue of mec-15 phenotype. Previous genetic analysis showed that mec-18 mutations enhanced the mec-10(gof)-induced degenerations, implying that MEC-18 might be a negative regulator of the putative degenerin channel (one model is that it could resensitize the touch apparatus). We mapped mec-18 between the polymorphism sy8 (W. Katz) and a deletion associated with dpy-23 (e840, P. Baum). One cosmid in this region, C52B9 fully rescued the mec-18 phenotype. A 3.5 Kb fragment from C52B9 also rescues. The sequence of the genomic DNA and cDNA showed that this rescuing fragment encodes a novel protein with an AMP-binding motif. Expression study using lac-z and gfp reporters showed that this putative protein was produced by the six touch neurons. These results suggested that the putative AMP binding protein may participate in the mechanosensation process. Unfortunately, we have yet to found mutations in the five known mec-18 alleles throughout the rescuing fragment. At this point, we are not sure whether the rescuing fragment contains the mec-18 locus or it is another gene whose product interacts with mec-18 when it is over-expressed.