Suraj Nath, Nisa et al. (2015) International Worm Meeting "Identifying the metabolic pathways involved in phosphine resistance in alh-6(wr3) mutant ."

R Ebert, Paul, Suraj Nath, Nisa

[International Worm Meeting, 2015]

Pest insects of stored grain have developed high level resistance to the fumigant phosphine, which threatens the grain industry. Since there are no suitable alternative fumigants to replace phosphine it is essential to understand the mechanisms of phosphine resistance to increase the effectiveness of resistance management. We have created phosphine resistance mutations in C. elegans that identify two genes, one of which is alh-6. The alh-6 gene encodes delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) that is involved in proline catabolism. In humans, alh-6 mutation causes a condition called hyperprolinemia II (HP II) that is associated with an increase in free amino acid proline. In our alh-6(wr3) mutant the amount of free proline is nearly six times that in the wild type strain (N2). As proline is a stress protectant we have also tested the ability of the strain to withstand osmotic stress and UV radiation, but we find that its resistance toward NaCl and UV radiation does not differ significantly from the N2 control. To determine whether proline accumulation is responsible for phosphine resistance, the effect of epigenetic suppression of proline oxidase on phosphine toxicity will be tested. We will also investigate the level of proline accumulation in response to phosphine exposure as well as the effect of P5C on phosphine resistance. While the alh-6(wr3) mutation causes an increase in lifespan under well-fed conditions, it did not influence survival under starvation.

Ayyadevara S et al. (1995) International C. elegans Meeting "MAPPING OF LONGEVITY DETERMINING GENES IN C. ELEGANS"

Thaden JJ, Shmookler Reis RJ, Ayyadevara S, Ebert RHA

[International C. elegans Meeting, 1995]

Absence of inbreeding depression, short mean life span and reduced generation time make C. elegans an excellent model system for studying genes that determine longevity. Recombinant inbred strains have been used in a number of organisms to map genes of aging. Recombinant inbred (RI) lines were generated from a cross between a low-Tc1-copy strain (Cl2a) and a high-copy strain (Bergerac-BO). A similar cross (Ebert et al; Genetics 1993) was made between Bristol N2 and Bergerac-BO for the same purpose, and the genotypes were analysed by using sequence-tagged strain specific markers. As aging is polygenic and any two strains will be polymorphic at only some of the relevent loci the present cross is aimed at mapping additional genes that determine longevity. Bergerac-BO and Cl2a worms were crossed for seven generations to accumulate recombinations, followed by five generations of selffertilizations to achieve homozygsoity. Analysed genotypes of young and old worms from a synchronously aging population, will be presented.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Duke A, Duerr JS, McManus JR, Rand JB, Moulder GL, Kohn RE, Barstead RJ

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, H H Rees, R A Clegg, M J Fisher, M Tabish

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zelmanovich, V., Livshits, L., Zuckerman, B., Smith, Y., Gross, E., Abergel, R., Romero, L., Abergel, Z.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Rubin CS, Angelo RG

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Balaji, Vishnu et al. (2021) International Worm Meeting "Ubiquitin-Dependent Dimer-Monomer Switch Defines Substrate Specificity and Processivity of the E3 Ligase CHIP"

Lorenz, Robin, Balaji, Vishnu, Loewe, Thomas, Mueller, Leonie, Hoppe, Thorsten, Pokryzwa, Wojciech

[International Worm Meeting, 2021]

Proteostasis is achieved by quality control pathways that support the generation of correctly folded proteins, prevent protein misfolding and remove toxic proteins. The quality control E3 ligase CHIP ubiquitylates damaged proteins consigned by chaperone partners for disposal through the endo-lysosomal pathway, proteasomal degradation, or autophagy. Additionally, CHIP has been reported to modulate essential signaling pathways by specifically delivering a myriad of native proteins to destined fates homeostasis (Paul I and Ghosh M.K., 2015). We aimed at understanding the substrate specificity and processivity through a "structure to function" approach, by examining the modeled 3D structure of the C. elegans ortholog of CHIP, CHN-1, based on the reported structure of murine CHIP (Zhang M et al., 2005). Using C. elegans and mammalian cells as model system and with various genetic and biochemical analyses, we demonstrate that monomeric CHN-1/CHIP has preserved ubiquitylation activity and promotes longevity via the DAF-2 Insulin-like signaling pathway (Tawo R et al., 2017). Our data reveal that the CHN-1/CHIP autoubiquitylation and its chaperone binding interplay modulates the alteration between monomer and dimer (Balaji V and Hoppe T., 2020). Together, the conserved dimer-monomer transition provides a molecular switch regulating CHN-1/CHIP activity in response to proteotoxic stress and aging.

Consortium Wormbase (2000) Worm Breeder's Gazette "Update on the C. elegans database WormBase"

Consortium Wormbase

[Worm Breeder's Gazette, 2000]

WormBase (www.wormbase.org) is an international consortium of biologists and computer scientists dedicated to providing the research community with accurate, current, accessible information concerning the genetics, genomics and biology of C. elegans and some related nematodes. WormBase builds upon the existing ACeDB database of the C. elegans genome by providing curation from the literature, an expanded range of content and a user friendly web interface. The team that developed and maintained ACeDB (Richard Durbin, Jean Thierry-Mieg) remains an important part of WormBase. Lincoln Stein and colleagues at Cold Spring Harbor are leading the effort to develop the user interface, including visualization tools for the genome and genetic map. Teams at Sanger Centre (led by Richard Durbin) and the Genome Sequencing Center at Washington University, St. Louis (led by John Spieth) continue to curate the genomic sequence. Jean and Danielle Thierry-Mieg at NCBI spearhead importation of large-scale data sets from other projects. Paul Sternberg and colleagues at Caltech will curate new data including cell function in development, behavior and physiology, gene expression at a cellular level; and gene interactions. Paul Sternberg assumes overall responsibility for WormBase, and is delighted to hear feedback of any sort. WormBase has recently received major funding from the National Human Genome Research Institute at the US National Institutes of Health, and also receives support from the National Library of Medicine/NCBI and the British Medical Research Council. WormBase is an expansion of existing efforts, and as such continues to need you help and feedback. Even with the increased scope and funding, all past contributors to ACeDB remain involved. The Caenorhabditis Genetics Center (Jonathan Hodgkin and Sylvia Martinelli) collaborate with WormBase to curate the genetic map and related topics. Ian Hope and colleagues continue to supply expression data to WormBase. Leon Avery will continue his superb website and serves as one advisor to WormBase. While the major means of access to WormBase is via the world wide web, downloadable versions of WormBase as well as the acedb software engine will continue to be available.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Smolikove, Sarit, Williams, Cameron, Koury, Emily, Hicks, Tara

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.

Korswagen HC et al. (1995) International C. elegans Meeting "PHYSICAL MAPPING OF Tc1 SEQUENCE-TAGGED SITES IN THREE Tc1-POLYMORPHIC STRAINS"

Thaden JJ, Plasterk RHA, Korswagen HC, Shmookler Reis RJ

[International C. elegans Meeting, 1995]

Williams et al. [(1992) Genetics 131, 609] have described a genetic mapping system based on polymorphic sequence-tagged sites (STS). It is an alternative to classical two- or three-factor crosses, and can also be used to map polygenic traits [Ebert et al. (1993) Genetics 135,1003; Ayyudevara et al. poster]. One weakness has been that fewer than 80 C. elegans polymorphisms had been sequenced and mapped. This has changed. By shotgun sequencing, Korswagen et al. [WBG 13(5),90] have generated over 800 sequences adjoining Tc1 insertions in strains RW7000, KR1787, and CB4000. At this writing, 35 Tc1 alleles map to sequenced genome regions. This number will slowly increase as sequencing proceeds. We are mapping Tc1 alleles by YAC grid annealing to pooled DNA probes. Pooling is via the "matrix addressing" strategy [Zwaal et al. (1993) PNAS 90, 7431]; DNAs are arranged in 3-D arrays and pooled along each plane. An allele thus contributes to three orthogonal probe pools. A YAC or adjacent YACs labeled by only those three pools will in principle map the allele. In preliminary work, we identified and set aside alleles that align with repetitive DNA elements or anneal to total genomic DNA. We are evaluating probe mixtures of increasing complexity, to optimize 3-D array size.