Takuji Yoshimura et al. (2002) Japanese Worm Meeting "Analysis of a cross-reacting protein to anti-ryanodine-receptor antibody of Caenorhabditis elegans"

Hiroaki Kagawa, Takuji Yoshimura, Tomoyo Hamada

[Japanese Worm Meeting, 2002]

Antibody has specific reactivity to protein and is useful to identify the protein in vitro and in vivo as a prob. Antibody has sometimes cross-reactivity to unexpected proteins. The molecular mechanism of the antibody cross-reactivity is not well understood. Polyclonal antibody R16, which was raised against a fusion protein composed of -galactosidase and KH30 domain of the ryanodine receptor in C. elegans (CeRyR), reacted not only to CeRyR but also other C. elegans proteins. Cross-reacted bands are not derived from the CeRyR because these were also detected in the total protein extract from a null mutant of the CeRyR. As homologous protein to the KH30 domain of the CeRyR was not obtained by the worm-pep database search, proteins reactive with R16 could not share the same sequence but conformational epitope as KH30 domain. In this study, the clone reactive with R16 was obtained by screening C. elegans cDNA expression library. The predicted amino acid sequence of the cDNA clone of H02I12.1 had little homology to the KH30 region. To demonstrate the reason why the H02I12.1 product cross-reacted with R16, we applied the epitope mapping to identify the cross-reactive region with R16. We will present the conformational epitope to the KH30 domain. The result may provide a clue to the mechanism of the cross-reactivity by antibody. This approach could have the potential to classify proteins in the genome wide database.

Martyn Quinn et al. (2001) International C. elegans Meeting "The structural role and interactions of the DPY-7 cuticular collagen in the exoskeleton of Caenorhabditis elegans."

Martyn Quinn, Iain Johnstone

[International C. elegans Meeting, 2001]

The C.elegans cuticle (exoskeleton) is a multi-layered extracellular matrix consisting predominantly of small collagen-like proteins that are extensively cross-linked. The cuticle is synthesised by the underlying hypodermis 5 times throughout the lifecycle of the animal. Previous investigations have indicated that the cuticular collagen DPY-7 has an important structural role within the complex architecture of the exoskeleton. Using immuno-fluorescence we have shown that this collagen locates to circumferential stripes within the cuticle. There is one DPY-7 containing stripe per cuticular annulus. We are utilizing a range of techniques (including immuno-electron microscopy and scanning electron microscopy) to further elucidate the precise localisation of this protein within the cuticular matrix. Using a mono-clonal antibody specific for DPY-7, we can detect the protein on western blots from cuticle extracts. The DPY-7 detected is much larger than monomer. Cuticle collagens are crosslinked by non-reducible bonds during formation of the cuticle. The various high molecular weight species we detect will be cross-linked forms of DPY-7. By a combination of genetic and immuno-fluorescent means we have shown that DPY-7 physically interacts with at least three other cuticle collagens. We are interested to determine if the cross-linking of DPY-7 we detect by westerns is with the same collagen molecules we know by other means DPY-7 interacts with. We are attempting to purify the cross-linked species with the intention of identifying cross-linked partners.

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

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

[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.

Arata, Yukinobu et al. (2021) International Worm Meeting "Insulin signaling underlies a heavy-tailed temporal organization in C. elegans episodic swimming"

Jurica, Peter, Kimura, Hiroshi, Arata, Yukinobu, Ikeda, Yusaku, Kiyono, Ken, Sako, Yasushi, Shiga, Itsuki

[International Worm Meeting, 2021]

Episodic bouts of animal behaviors are not periodic, but temporally organized in a scale-free manner with long memory in many animal species including human. It is known that depression in human is associated with defects in the scale-free temporal organization of behaviors and impacts on social fitness, and accordingly exacerbates disease state. C. elegans crawls in on agar plate and swim in a solution while switching between actively-moving state and inactive state, which is referred to as episodic behavior. We have shown that the residence time in each state in episodic swimming has a scale-free or heavy-tailed property with long memory. Here, we studied how the residence times between two states are cross-correlated by using detrending moving-average cross-correlation analysis (DMCA) to activity time series obtained from a long-term culture of C. elegans. We found that residence times in the active and inactive states are correlated with each other over the time scale from minutes to hours, or at the ultradian time scale. This temporal cross-correlation disappeared when cultured in M9 buffer without food bacteria, and restored when cultured M9 buffer only with glucose. Additionally, the temporal cross-correlation disappeared in the mutant animals of daf-2 gene, which encodes the membrane receptor for insulin growth factor signal and in the mutant of daf-16, which encodes a downstream transcriptional repressor FOXO. Therefore, we conclude that the temporal cross-correlation is achieved by the insulin signaling. Glucose metabolism disorders and behavioral disorders are known to be closely related in human depression, raising the possibility that the temporal organization by insulin signaling found in C. elegans is conserved in humans.

SW Jin et al. (1999) International C. elegans Meeting "A diet of double-stranded RNA allows simple male/female genetics"

D Salinsky, SW Jin, RE Ellis, PJ Chen

[International C. elegans Meeting, 1999]

Although male/hermaphrodite genetics has given us many blessings, it also creates one major problem — distinguishing between cross-progeny and self-progeny. Traditionally, this problem has been solved in one of three ways: (1) using a marker mutation to distinguish between self- and cross-progeny, (2) using mutations to create true females, which produce only cross-progeny, or (3) using hermaphrodites that have been purged of sperm. The first two methods require extra crosses, and occasionally create problems because of the effects of the marker mutation on the phenotype being studied. The final method requires careful handling of worms to identify purged hermaphrodites, which usually yield few progeny. We have found that double-stranded RNA homologous to fog-1 creates female worms with high efficiency. Almost 100% of the progeny of worms injected with a 1 mg/ml solution of fog-1 dsRNA develop as females. To simplify administration of the double-stranded RNA, we developed a procedure for raising large numbers of larvae in a 1 microl drop. This method yields female animals rapidly, easily, and with high efficiency; these females can be used for any desired cross. At our poster we will provide information and materials for production of fog-1 dsRNA, and for producing female worms by feeding this dsRNA to larvae.

Yang Wang et al. (2006) East Asia C. elegans Meeting "Establishment of learning choice model using heavy metals and identification of genetic loci affecting the learning choice in C. elegans"

Yang Wang, Yaou Hu, Dayong Wang

[East Asia C. elegans Meeting, 2006]

We explored these 12 heavy metals to establish the learning choice model in worms. In this model, worms were tested for their ability to cross different metal barriers to reach the attractive odorant. Without attractive odorant, nearly all the indexes for worms having ability to cross the metal barriers were below the 50%. Using these 12 metals, we established two types of learning choice models. In the first type of learning choice model, the indexes for worms to cross the Cu and Hg barriers were still below the 50%. In the second type of learning choice model, the indexes for worms to cross the barriers of other ten metals were above 50%. These learning choice models suggest that worms may have different response or sensitivity to different heavy metal and may take different choice in dilemma.Further, we recently used these models to screen the genetic loci and related mutants affecting the leaning choice and attempt to elucidate the molecular mechanism of leaning choice in C. elegans. Mutation of 21 genes screened out had abnormal learning choice in Cu, Hg and Zn-mediated models. Among these identified genes, 9 genes were found to be involved in the regulation of ASE, ASH, ASI, AWA and AWC sensory neuron function. In addition, mutations of seven miRNAs were found to be associated with the learning choice regulation. Five genetic loci affecting the synaptic function were also identified in our models.Among these identified genetic loci, we are recently elucidating the roles of dopamine receptors in learning choice regulation. We found that gain of the antagonistic functions of dopamine receptors in the learning choice might through the regulation of series of sensory neuron functions.

D Zorio et al. (1999) International C. elegans Meeting "Identification of the 3' splice site by both subunits of U2AF"

D Zorio, T Blumenthal

[International C. elegans Meeting, 1999]

How are introns and outrons recognized in C. elegans ? The 5' splice sites on introns are identified by base pairing with U1 snRNA, as they are in other animals. However, C. elegans introns lack both the polypyrimidine tract and the branchpoint consensus sequences found in other animals. Instead, they have a highly conserved 3' splice site octamer, U 4 CAG/R, that is required for both intron and outron recognition. In other animals, the polypyrimidine tract is bound by the two-subunit splicing factor, U2AF, but it has never been determined how the invariant AG at the 3' splice site is recognized. Here we report a probable solution to this problem for C. elegans . We have previously shown that C. elegans has both U2AF subunits and both are required for viability. We also showed that the large subunit gene contains an alternatively spliced exon containing multiple copies of the U 4 CAG/R consensus. We proposed that these octamers in the alternative exon serve as sites for autoregulation of U2AF levels by alternative splicing. To test whether U2AF does in fact bind to this consensus, we performed in vitro cross-linking experiments with crude embryo extracts from transgenic worms that overexpress both U2AF subunits in response to heat shock (with the small subunit epitope tagged). We then used immunoprecipitation to identify U2AF-cross-linked bands. We found that both subunits cross-link to an RNA oligonucleotide containing the complete consensus (U 4 CAG/G), but that only the large subunit cross-links to an RNA containing the 5 pyrimidines, but lacking the AG (U 4 CGAA). Furthermore, the presence of the AG-less RNA causes dissociation of the two subunits from each other. An RNA lacking 3 of the pyrimidines, but retaining the remainder of the consensus (UA 3 CAG/G), does not cross-link to either subunit. Our results suggest that U2AF small subunit may be responsible for recognition of the invariant AG at the 3' splice site, and that simultaneous recognition of the short polypyrimidine tract by U2AF large subunit and the AG/R by U2AF small subunit may be the key event in 3' splice site identification.

Deng, L. et al. (2017) International Worm Meeting "The role of cross-inhibition in C. elegans locomotion."

Haspel, G., Deng, L., Doyle, C., Marfil, V.

[International Worm Meeting, 2017]

To achieve translocation, many locomotor systems produce alternation of antagonist muscle, including axial bending and limb alternation. Cross-inhibition is believed to be key to antagonistic alternation so that when a muscle is activated by the neural circuit, cross-inhibitory elements inhibit a counteracting muscle. Nineteen GABAergic motoneurons in C. elegans have been suggested to provide cross inhibition, based on their morphology and inhibitory effect on muscles. They each receive synaptic input from excitatory motoneurons and provide inhibition to its opposing motoneurons and muscles. However, the activity of D motoneurons during locomotion has not been reported. The locomotion behavior of mutants that are deficient in GABA transmission has been described and their phenotype was generally termed Shrinker. While their forward locomotion was described as normal, shrinker mutants contract their anterior section instead of undulating backward in response to noxious stimuli. However, we show that the deficiencies are not related to the direction of locomotion but rather to its speed and that while cross-inhibition is not necessary for slow antagonistic alternation, it plays a crucial role in fast alternation. We use a combination of behavior analysis, optogenetics, and calcium imaging to clarify the role of the inhibitory D motoneurons. GABA transmission mutants moved at lower frequency and slower translocation speed in both forward and backward directions. Moreover, we found that both forward and backward undulation frequencies of wild type animals each fall into slow and fast distributions, and that the frequencies of mutants overlap with the low frequency distribution of wild type. When mutant animals were exposed to noxious stimuli that would have induced fast forward or backward undulation in wild type animals, they contracted their posterior or anterior sections, respectively. Similar changes in locomotion behavior occurred when D motoneurons were acutely inactivated with optogenetics. We recorded the neuronal activity of D motoneurons, as well as the activity of excitatory motoneurons and muscle cells, during restricted locomotion using microfluidic channels that mimic natural shape of natural undulation so that all elements can be correlated in the same framework of undulatory phase. We manipulate undulation frequency by changing the ambient viscosity. We hypothesize that cross inhibition sharpens the coordination of fast antagonistic alternation.

Andrew M Spence (2001) International C. elegans Meeting "Maternal-effect germline silencing of the sex-determining gene fem-1"

Andrew M Spence

[International C. elegans Meeting, 2001]

The fem-1 gene is required for male development of germline and somatic tissues in C. elegans . Animals lacking fem-1 activity develop as cross-fertile females regardless of their X chromosome dose. Following a cross to wild-type males, most fem-1 mutants produce phenotypically wild-type XO male and XX hermaphrodite heterozygous progeny. In contrast, females homozygous for any of three deficiencies that remove all or part of fem-1 produce cross-progeny of both sexes that exhibit germline feminization. The penetrance of this effect varies from 20% to over 90%, depending upon the deficiency, and it dramatically increases in the second generation following a backcross to females homozygous for the deficiency. Feminization appears to result from maternal absence of some product of the fem-1 locus rather than a zygotic interaction involving the deficiency chromosome, because the paternally disomic progeny of a cross to deficiency homozygotes exhibit germline feminization. It is unlikely that maternal loss of FEM-1 protein is responsible for the effect, because alleles carrying point mutations that prevent production of FEM-1 complement the deficiencies with respect to their maternal effect on the germline. Genetic evidence suggests that the affected animals fail to express fem-1 at normal levels in the germline, which could account for their phenotype. I suggest that a fem-1 transcript is maternally required to license fem-1 for expression in the germline. A similar requirement might exist for the germline expression of other genes, but it would be difficult to detect in the absence of deficiency alleles that allow homozygotes to develop into fertile adults. I am investigating the relationship between mechanisms involved in maternal-effect silencing of fem-1 and other silencing phenomena in C. elegans .

Lamelza, Piero et al. (2013) International Worm Meeting "If we have children together, will they be less fit? Hybrid incompatibilities in Caenorhabditis species."

Lamelza, Piero, Cattin, Jerome, Ailion, Michael, Wilson, Vanessa, Topalidou, Irini

[International Worm Meeting, 2013]

Speciation often occurs by the accumulation of genetic incompatibilities between populations. We are studying the genetic basis of hybrid incompatibility in several Caenorhabditis species. In C. elegans, hybrid incompatibility between the Bristol and Hawaiian strains is mediated by the peel-1/zeel-1 genetic element. peel-1 encodes a sperm-expressed toxin and zeel-1 encodes its zygotically-expressed antidote. We are interested in determining the cellular mechanism of PEEL-1 toxicity. To identify other factors required for PEEL-1 toxicity, we conducted a screen for suppressors of ectopic PEEL-1 driven by a heat-shock promoter. Thus far, we have isolated five suppressors. The strongest suppressor fully suppresses PEEL-1 toxicity and is not allelic to the heat-shock transcription factor gene hsf-1. We have also expressed GFP-tagged PEEL-1 under a galactose-inducible promoter in yeast. Preliminary data suggest that PEEL-1 strongly inhibits growth of the yeast when induced. We have also begun a search for hybrid incompatibility genes in other Caenorhabditis species. We performed crosses between two recently isolated Caenorhabditis species, C. sp. 17 and C. sp. 29. When C. sp. 17 males were crossed to C. sp. 29 females, all F1 progeny arrested as embryos. However, when C. sp. 29 males were crossed to C. sp. 17 females, a very small percentage of F1 progeny did not arrest as embryos, but instead grew to the adult stage. Thus, as with peel-1, a parental effect contributes to the hybrid incompatibility. Almost all of the viable hybrids were female, indicating that the cross obeys Haldane's rule. The few F1 hybrid males are sterile. F1 hybrid females did not give viable progeny when crossed to C. sp. 29 males, but gave large numbers of viable F2 progeny and some arrested embryos when crossed to C. sp. 17 males. Among the surviving F2, there are more females than males, indicating that this cross also obeys Haldane's rule. There is significant variability in the relative proportion of males, females, and arrested embryos in the F2 generation from cross to cross, suggesting that particular combinations of genes in the rare surviving F1 females determine the proportions seen in the F2 crosses.