Hawkins, E.G. et al. (2015) International Worm Meeting "Tolerance to a cholinergic activating effect of alcohol requires a specific function of the EAT-6 Na+/K+-ATPase."

Kondo, L.M., Davies, A.G., Hawkins, E.G., Martin, I., Bettinger, J.C.

[International Worm Meeting, 2015]

Alcohol (ethanol) produces activating and depressing behavioral effects in humans. We have investigated the basis for an activating effect of ethanol in C. elegans that produces body hypercontraction. Acute ethanol exposure induces a shortening of the animal that requires normal acetylcholine levels and a functional UNC-63 acetylcholine receptor (AChR) subunit. Elimination of the two receptors currently known to contain the UNC-63 subunit, the ACR-2R and the levamisole-sensitive AChR, do not alter sensitivity to ethanol. This suggests that UNC-63 may be acting in an unidentified receptor to mediate this effect of ethanol. AChRs in mammals have been implicated in mediating activating effects of ethanol, and genetic variation in AChR subunit-encoding genes has been associated with the development of alcoholism in humans. We are pursuing the tissue-specific requirements for unc-63 expression for its role in this ethanol effect.The hypercontracted animals show a recovery of pre-treatment body length despite continuous exposure to ethanol (and a gradual increase in internal ethanol concentration). This observation suggests that a physiological tolerance is developing to the drug. We identified the eat-6(eg200) mutation as a mutant that fails to develop tolerance to this ethanol effect. An examination of several existing alleles of eat-6 found that mutants with strong effects on pharyngeal pumping did not alter the response to ethanol-induced hypercontraction, whereas the ad601 allele, which resembles the eg200 mutant in having weak effects on pumping but strong effects on aldicarb sensitivity, does alter the maintenance of tolerance to ethanol. These data suggest that eg200 and ad601 alter a specific function of the Na+/K+-ATPase that is important for the development and/or maintenance of tolerance to ethanol for this cholinergic activating effect of ethanol. One possibility that we are testing, is that EAT-6 has a structural relationship with an ethanol-sensitive AChR, and that the mutations eg200 and ad601 prevent desensitization or compensatory trafficking of that receptor.

Katsura I et al. (1992) Worm Breeder's Gazette "More Mutants From Mishima"

Hishida R, Katsura I, Ishihara T, Kawakami M

[Worm Breeder's Gazette, 1992]

(1) Complex epistasis We have been studying fluoride-resistant mutants of C. elegans (Katsura, Kondo and Amano: the 1991 C. elegans Meeting Abstract p179 ).The mutants can be classified into two classes. Class 1 mutants are resistant to 400 mg/ml NaF and grow slowly even in the absence of NaF. Class 2 mutants are not completely resistant to 400 mg/ml NaF and grow normally in the absence of NaF. Class 1-class 2 double mutants are resistant to 400 mg/ml and grow normally in the absence and presence of NaF. Namely, class 1 mutations are epistatic to class 2 mutations concerning degree of fluoride-resistance but hypostatic concerning growth rate. We have reported flr-1 Xand flr-3 IVas class 1 genes and flr-2 Vas a class 2 gene. Here we report flr-4 Xas a new class 1 gene and flr-5 (LG not yet determined) and flr-6 Vas new class 2 genes. The latter two mutations were obtained as suppressors of the slow-growing phenotype of class 1 mutants. We have cloned a 3.6kb EcoRI fragment containing Tc1 that caused flr-3 (ut9 ),a spontaneous mutation isolated from a mut-5 strain. (2) Clr-1 phenotype lethals In the course of studying larval lethal mutations that affect gross morphology of worms we found that quite a few mutants have a phenotype similar to clr-1 mutants, that is, the intestine and the body wall are gradually detached during larval growth. We reported that one of them mapped in clr-1 ,and another in let-23 (Katsura, Kondo and Kawase: WBG 12(2) p108 ).After that we mapped one of them (ut58 )in let-341 .Two of them (ut103 and ut105 )were sent to Dr. J. Kimble's Lab and shown to be deletions that remove lag-2 gene (E. Lambie, D. Gao and J. Kimble, personal communication). Therefore, it has become more likely that the clr-1 phenotype is somehow related to signal transduction. We found a unique Tc1 containing DNA fragment (HindIII 2.7kb) in sma-1 let(ut102 )unc-761 sqt-3 ,where the mutation ut102 was isolated by K. Kondo from a mut-6 strain. We are also isolating mutants that have a phenotype of incompletely penetrant clr-1 lethals. Many of them are defective also in the shape or movement of the head. For instance, ut78 IVhas a notched head (it may be an allele of vab-2 ),and ut79 IIis defective in foraging movement. Since the worms that grow to adults are not so sick, it is possible that those mutants may be mutants of cell lineage and that only those worms that adopt a lethal cell lineage may die. Anyway, we hope they will be useful in elucidating the nature of clr-1 lethals.

Hoogeboom D et al. (2009) Biochim Biophys Acta "Should I stay or should I go: beta-catenin decides under stress."

Burgering BM, Hoogeboom D

[Biochim Biophys Acta, 2009]

Reactive oxygen species (ROS) are essential for efficient and proper execution of a large number of cellular processes including signalling induced by exogenous factors. However, ROS are highly reactive in nature and excessive or prolonged ROS formation can result in considerable damage to cellular constituents and is implicated in the onset of a large variety of diseases as well as in the process of ageing [reviewed in [1] T.M. Paravicini, R.M. Touyz, Redox signaling in hypertension, Cardiovasc. Res. 71 (2006) 247-258, [2] P. Chiarugi, From anchorage dependent proliferation to survival: lessons from redox signalling, IUBMB life 60 (2008) 301-307, [3] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, Int. J. Biochem. Cell Biol. 39 (2007) 44-84]. Management of ROS to prevent potential damage, yet enabling its signalling function is achieved through numerous enzyme systems e.g. peroxidases, superoxide dismutases etc. and small molecules e.g. glutathione that collectively form the cellular anti-oxidant system. The O-class of Forkhead box (FOXO) transcription factors regulates amongst others cellular resistance against oxidative stress [[4] Y. Honda, S. Honda, The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans, Faseb J. 13 (1999) 1385-1393]. In turn FOXOs themselves are regulated by ROS and cellular oxidative stress results in the activation of FOXOs [[5] M.A. Essers, S. Weijzen, A.M. de Vries-Smits, I. Saarloos, N.D. de Ruiter, J.L. Bos, B.M. Burgering, FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK, EMBO J. 23 (2004) 4802-4812]. A prominent feature of ROS-induced FOXO activation is ROS-induced binding of beta-catenin to FOXO [[6] M.A. Essers, L.M. de Vries-Smits, N. Barker, P.E. Polderman, B.M. Burgering, H.C. Korswagen, Functional interaction between beta-catenin and FOXO in oxidative stress signaling, Science (New York, NY) 308 (2005) 1181-1184, [7] M. Almeida, L. Han, M. Martin-Millan, C.A. O'Brien, S.C. Manolagas, Oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting beta-catenin from T cell factor- to forkhead box O-mediated transcription, J. Biol. Chem. 282 (2007) 27298-27305, [8] D. Hoogeboom, M.A. Essers, P.E. Polderman, E. Voets, L.M. Smits, B.M. Burgering, Interaction of FOXO with beta-catenin inhibits beta-catenin/T cell factor activity, J. Biol. Chem. 283 (2008) 9224-9230]. However, ROS affect many transcriptional programs besides that of FOXOs. Here, we discuss the recent progress in our understanding as to how ROS may regulate the interplay between some of the ROS-sensitive transcription factors through diverting beta-catenin binding to these transcription factors. We propose that beta-catenin acts as a key switch between the various ROS-sensitive transcription programs.

Li L et al. (1998) Mol Cell Biol "Differential expression of individual suppressor tRNA(Trp) gene gene family members in vitro and in vivo in the nematode Caenorhabditis elegans."

Kondo K, Li L, Honda BM, Linning RM

[Mol Cell Biol, 1998]

Eight different amber suppressor tRNA (suptRNA) mutations in the nematode Caenorhabditis elegans have been isolated; all are derived from members of the tRNA(Trp) gene family (K. Kondo, B. Makovec, R. H. Waterston, and J. Hodgkin, J. Mol. Biol. 215:7-19, 1990). Genetic assays of suppressor activity suggested that individual tRNA genes were differentially expressed, probably in a tissue- or developmental stage-specific manner. We have now examined the expression of representative members of this gene family both in vitro, using transcription in embryonic cell extracts, and in vivo, by assaying suppression of an amber-mutated lacZ reporter gene in animals carrying different suptRNA mutations. Individual wild-type tRNA(Trp) genes and their amber-suppressing counterparts appear to be transcribed and processed identically in vitro, suggesting that the behavior of suptRNAs should reflect wild-type tRNA expression. The levels of transcription of different suptRNA genes closely parallel the extent of genetic suppression in vivo. The results suggest that differential expression of tRNA genes is most likely at the transcriptional rather than the posttranscriptional level and that 5' flanking sequences play a role in vitro, and probably in vivo as well. Using suppression of a lacZ(Am) reporter gene as a more direct assay of suptRNA activity in individual cell types, we have again observed differential expression which correlates with genetic and in vitro transcription results. This provides a model system to more extensively study the basis for differential expression of this tRNA gene family.

Clark DV et al. (1986) Worm Breeder's Gazette "distribution of lethal mutations in LGIV (right)."

Baillie DL, Clark DV

[Worm Breeder's Gazette, 1986]

We have isolated lethal mutations in a screen to determine the distribution of essential genes on LGIV (right) in the region balanced by nT1. Previous work in the 1.5 m.u. sDf2 region has identified 20 essential genes (Rogalski, T. M. and Baillie, D. L., 1985, MGG 201:409-414). Sixteen of these lie within 1 m.u. to the left of unc- 22. unc-22(s7)unc-31(e169)/nT1(IV);+/nT1(V) hermaphrodites were exposed to .012M EMS for four hours and 3398 F1's were screened for the absence of Unc-22Unc-31 progeny. 303 lethal strains were isolated. Of the 283 strains analyzed so far, 164 lethals map to LGIV and these were retained. This data, together with data from a similar screen (Donati, L.M. et al, CSH C. 1985, p. 38), shows a frequency of lethal mutations recovered over nT1 of .078, with .044 on LGIV and .034 on LGV. The distances of LGIV lethals from unc-22 were also determined by two-factor mapping. Results show a cluster around unc 22. This parallels the distribution of visibles on the 1985 C. ese lethals are now being left- right positioned relative to unc-22 by employing a set of three deficiencies which define seven zones (sDf2, sDf21, and mDf7, which was kindly supplied by Teresa Rogalski). In the figure below, the height of the bars corresponds to the number of lethal alleles on LGIV which map in the 1 m.u. interval ending with the distance indicated. [See Figure 1]

O'Neil NJ et al. (1998) West Coast Worm Meeting "ANALYSIS OF ESSENTIAL GENES IN THE sDf125 REGION"

Vatcher GP, Baillie DL, Kuervers LM, O'Neil NJ

[West Coast Worm Meeting, 1998]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab has constructed a map of 112 essential genes isolated on LGIII left. We have divided the region between dpy-17 and dpy-19 into 12 zones that are defined by overlapping deficiencies and duplications. These zones were determined physically by PCR testing the deficiencies to determine endpoints. Into these zones, we have mapped the 112 essential gene mutations. One such zone (zone 7) is defined by sDf125 and sDp8. This region spans 380 000 base pairs and is completely covered by sequenced cosmids. Within this zone there are 101 predicted coding elements. To date, we have mapped 14 essential genes into this zone. We have used a systematic approach to rescue these essential genes with cosmid transgenics from our cosmid transgenic library. We have to date rescued 10 genes in this zone. These transgenic rescues map the genes to 40 kB (one cosmid) resolution as opposed to the 380 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of predicted coding elements which may be responsible for the gene function. The next step will be to subclone the cosmids with which we have rescued essential gene mutations. This will directly correlate the lethal or maternal effect lethal phenotype with the appropriate coding sequence. We have already begun the subcloning of the cosmids (see abstracts by G. P. Vatcher et al., L.M. Kuervers et al. this meeting).

Alicea, Bradly et al. (2015) International Worm Meeting "An experimental evolution model of C. Elegans adaptability."

Schroeder, Nathan, Androwski, Rebecca, Alicea, Bradly

[International Worm Meeting, 2015]

While the genetic architecture of C. elegans is well-studied, the adaptability of various strains and their reproductive fitness is less understood. To address this, an experimental-based model will allow for characterization of C. elegans adaptability amongst strains which may exhibit low reproductive potential and/or survivability under selection. Two experimental approaches representing within- and between-generation survivability (preconditioning and experimental evolution, respectively) will allow survivability to be assessed. Preconditioning [1] assesses adaptability within a generation using environmental selection, while experimental evolution [2] assesses adaptability via cross-generational artificial selection. For a given strain, populations grown from single founder individuals are repeatedly subject to genetic drift and then selected for fecundity (largest brood size). These results demonstrate how specific genotypes exhibit robustness in the face of adaptive constraints.Biological adaptability involves both survivability and reproductive fitness. Survivability is the degree to which a single founder worm can survive and produce viable clones (e.g. the number of generations constituting a single genealogy). Reproductive fitness is the mean population size for a given genealogy. In strains with high survivability, founder populations with high reproductive fitness should also generate subsequent founder populations with a high degree of survivability. Strains with high reproductive fitness but low overall progeny sizes may also exhibit high adaptability if selection results enhances adaptation to varying environmental conditions. While this model does not control for stochastic effects and demographic constraints [3], it does allow for genealogies with consistently high survivability [4].1. Calabrese, E.J. et.al, Tox App Pharm, 222(1), 122-128 (2007).2. Kawecki, T.J. et.al, Trends Ecol Evol, 27(10), 547-560 (2012).3. Szendro, I.G. et.al, PNAS, 110(2), 571-576 (2012).4. Wahl, L.M., Krakauer, D.C., Genetics, 156(3), 1437-1448 (2000).

Nakajima T et al. (2000) Japanese Worm Meeting "A possible candidate of the DAF-2 ligands: structure, expression, and physiological functions"

Kawano T, Kimura Y, Ito Y, Nakajima T, Ishiguro M, Takuwa K

[Japanese Worm Meeting, 2000]

The DAF-2 protein, a C. elegans homolog of an insulin/IGF-1 receptor, is involved in dauer formation, fat metabolism, and a long life span (1). In the previous meeting, we reported two distinct genes encoding insulin/IGF-1-like peptides designated Ceinsulin-1 and Ceinsulin-2 (2). In this meeting, we will make a short talk of the Ceinsulin-1 regarding its structure, expression, and physiological functions. The precursor protein of Ceinsulin-1 consists of 95 amino acid residues and seems to include a signal peptides, B and A chains, and a so-call C peptide that could be removed out through post-translational proteolysis like the preproinsulin. First, to detect the mature peptide, a polyclonal antibody against the putative B chain was prepared and used for analyzing C. elegans protein by Western blotting. The result showed that the mature Ceinsulin-1 should consist of one polypeptide like IGFs. However, the predicted tertiary structure of Ceinsulin-1 was more similar to the crystal structure of insulin than that of IGF-1, suggesting that the Ceinsulin-1 should be a hybrid molecule of insulin and IGF-1. Next, we analyzed expression pattern of the peptide at several developmental stages using RT-PCR and Western blotting. Interestingly, the transcriptional and translational patterns were not always coincident with each other, suggesting that the peptide biosynthesis could be regulated post-transcriptionally with some degrees. Details will be shown in the meeting. Finally, an RNA-i experiment was carried out to elicit physiological functions of the Ceinsulin-1. The RNAi led to reduction of the corresponding transcript and an extended life span. The inhibition of the Ceinsulin-1 biosynthesis exhibited no drastic effects on entry into and recovery from the dauer stage. Now, the effect on fat accumulation is examined. Anyhow, other insulin/IGF-1 peptides may be involved in the dauer formation and fat metabolism. References 1. Kimura, K.D., et al. (1997) Science 227, 942-946. 2. Kawano, T., et al. (1999) Peptide Science 1998: M. Kondo (Ed.) pp117-120.

Rogalski TM et al. (1991) International C. elegans Meeting "GENETIC APPROACHES TO C. ELEGANS RNA POLYMERASE III."

Golomb M, Watson N, Lancy ED, Rogalski TM, Riddle DL, Albert PS

[International C. elegans Meeting, 1991]

RNA polymerase III (Rpo m) transcribes genes encoding SS and transfer RNA's. This enzyme and its associated factors have been studied extensively in other systems, but Rpo m function has not been studied genetically in a metazoan. Developmental regulation of Rpo m activity in C. elegans has been suggested by the molecular genetic analysis of nonsense suppressors (Kondo et al., J. Mol. Biol. 215:7-19, 1990). The gene for the largest subunit of Rpo m, rpc-l, has been cloned (Bird and Riddle, Mol. Cell. Biol. 9:4119-4130, 1989) and positioned on the physical map between unc44 and unc-24 on chromosome IV. The availability of cloned genes and an in vitro transcription system (Honda et al., Nucl. Acid Res. 14B:869-881, 1986) provides potential tools for a detailed analysis of Rpo III structure and function using mutants, if appropriate mutants can be identified. Two approaches to genetic analysis of rpc-l have been employed. The first approach utilized resistance to the fungal toxin, ct-amanitin. A unique strain with an extremely amanitin-resistant Rpo II (Rogalski et al., Genetics 126:889-898, 1990) was used as a parent to select even higher levels of resistance. One such mutation was mapped on chromosome IV, and preliminary nuclear run-on assays indicate that Rpo III is abnormally resistant to amanitin, in appropriate recombinants derived from this mutant strain. The mutant Rpo III transcribes SS RNA in the presence of 200 g/ml amanitin, whereas wild-type Rpo m is inhibited by 25 g/ml. These results suggest that the resistance mutation affects Rpo III, and it may be within the rpc-l gene. A second approach to rpc-l genetics utilized a collection of EMSinduced lethal mutations generated by Denise Clark. We mapped fourteen of her lethal mutations to the interval between unc44 and unc-24, a region estimated to contain approximately 30 essential genes. These lethal mutations are now being assigned to complementation groups. Lethal derivatives of the putative amanitin-resistant rpc-l allele will also be sought, and selected lethal strains will be injected with the cloned rpc-l gene in an effort to identify the genetic correlate to rpc-l by DNA transformation. Genetic, phenotypic, and biochemical analysis of these mutants may provide a useful entree into structure- function relationships in this important enzyme.

Jha, Sweta et al. (2021) International Worm Meeting "UPS modulation and autophagy."

Jha, Sweta, Holmberg, Carina

[International Worm Meeting, 2021]

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Autophagy is responsible for degrading defective cellular organelles and long-lived proteins in the cytosol and is involved in cell growth, survival, development and death. UPS mostly degrades soluble and short-lived proteins in the nucleus and cytosol and affects various cellular processes. These two proteolytic systems are essential components of the cellular protein quality control system. Several studies have reported an interplay between the UPS and ALP, however it still remains largely unknown how these systems communicate in a multicellular organism. We have recently shown that downregulation of autophagy genes elicits tissue-specific effects on UPS function in C. elegans1. Here, we address how genetic or pharmacological modulation of the proteasome and the proteasome-associated DUBs affects autophagy. We use transgenic C. elegans expressing the autophagy reporters GFP::LGG-12 or mCherry::GFP::LGG-13 and analyze the accumulation of LGG-1 puncta and autophagic flux. Our preliminary data show that downregulation of the proteasome-associated DUBs ubh-4, usp-14 and rpn-11 as well as some proteasomal subunits affect the number of puncta positive for autophagosomes in hypodermal seam cells, intestinal cells as well as in pharynx. To nvestigate a conserved function of the UPS on autophagy in human cells, we are utilizing a HeLa cell line expressing the fluorescence probe GFP::LC3::RFP::LC3G4. A better understanding of the multilayered crosstalk between UPS and ALP in vivo may facilitate development of therapeutic options for various disorders linked to dysfunction in proteostasis. References Jha, S., Holmberg, C.I. Tissue-Specific Impact of Autophagy Genes on the Ubiquitin-Proteasome System in C. elegans. Cells 2020, 9, 1858. Melendez A, Talloczy Z, Seaman M, Eskelinen E-L, Hall DH, Levine B. Autophagy genes are essential for dauer develop-ment and life-span extension in C. elegans. Science 2003. 301:1387-91 Chang J.T., Kumsta C., Hellman A.B., Adams L.M., Hansen M. (2017). Spatiotemporal regulation of autophagy during caenorhabditis elegans aging. Elife. pii: e18459 Kaizuka T., Morishita H., Hama Y., Tsukamoto S., Matsui T., Toyota Y., Kodama A., Ishihara T., Mizushima T., Mizushima N. (2016). An autophagic flux probe that releases an internal control. Mol Cell. 64(4):835-849.