De Stasio E et al. (1994) Worm Breeder's Gazette "Mutagenesis of C. elegans Using N-ethyl-N-nitrosourea."

De Stasio E, Stanislaus D, Lephoto C

[Worm Breeder's Gazette, 1994]

Mutagenesis of C. elegans using N-ethyl-N-nitrosourea Elizabeth De Stasio, Dinesh Stanislaus and Catherine Lephoto. Department of Biology, Lawrence University, Appleton, Wl 54911

Yan, Wenwei Richard et al. (2021) International Worm Meeting "Real-time volumetric whole-animal imaging at cellular resolution with SCAPE microscopy in NeuroPAL worms"

Yemini, Eviatar, Yan, Wenwei Richard, Hillman, Elizabeth M. C., Li, Wenzi, Hobert, Oliver

[International Worm Meeting, 2021]

Recording neural activity at single cell resolution during unrestrained behavior holds tremendous potential for investigating the C. elegans neural code on a global scale. As fluorescent calcium indicators and 3D microscopy speeds have improved, recording from 100's of cells in moving worms has become feasible. However, 2 problems remain 1) being able to extract robust information from individual cells in a moving worm, and 2) knowing the defined identity of each individual tracked cell. Recently, a novel C. elegans strain termed NeuroPAL was developed that labels individual neuronal identities via a stereotypical multi-color fluorescence map. To harness the power of this worm we developed a high-speed multispectral volumetric microscopy platform with sub-cellular resolution, optimized for the NeuroPAL worms. Our SCAPE microscopy-based approach uses a scanning oblique light sheet which provides low phototoxicity and optical sectioning capabilities in a convenient single-objective geometry compatible with common C. elegans sample mounting procedures. The system's multi-laser launch, spectral image splitter and high-speed intensified camera make it possible to rapidly acquire a fully 3D NeuroPAL image in under 0.3 s. These scans can be interspersed with dual channel imaging of GCaMP and RFP with 0.33 x 0.67 x 0.25 microm sampling density over a 310 x 210 microm FEP-covered agarose arena at 13 volumes per second. The resulting data suggests much simpler tracking of uniquely identifiable cells throughout the worm, and analysis of the cellular calcium dynamics during free behavior.

Goodwin EB et al. (1994) Worm Breeder's Gazette "The laf-1 Gene Functions in the 3'UTR-Mediated Translational Repression of the Sex Determination Gene, tra-2"

Kimble JE, Goodwin EB

[Worm Breeder's Gazette, 1994]

The laf-l Gene Functions in the 3'UTR-mediated Translational Repression of the Sex Determination Gene, tra-2. Elizabeth Goodwin# and Judith Kimble University of Wisconsin, Madison, WI. # Dept. of CMS Biology, Northwestern Univ. Medical School, Chicago Ill.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Silverman PH (1999) Science "C. elegans as a model."

Silverman PH

[Science, 1999]

Elizabeth Pennisi, in her excellent commentary "Worming secrets from the C. elegans" (News Focus, 11 Dec 1998, p.1972), states that "The first person to sense that the worm might take on such a prominent role in biology was molecular biologist Sydney Brenner." I am sure that Brenner would wish to acknowledge the role that Ellsworth C. Dougherty played in this matter. Dougherty originally described in 1949, "[a] new species of the free-living nematode genus Rhabditis of interest in comparative physiology and genetics".

Navarro IC et al. (2020) EMBO J "Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans."

Miska EA, Legrand C, Braukmann F, Jordan D, Navarro IC, Lyko F, Akay A, Price J, Helm M, Tuorto F, Kotter A, Hendrick AG

[EMBO J, 2020]

Methylation of carbon-5 of cytosines (m⁵ C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m⁵ C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m⁵ C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m⁵ C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C.elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m⁵ C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m⁵ C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m⁵ C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m⁵ C tRNA wobble methylation in the adaptation to higher temperatures.

Navarro IC et al. (2022) Wellcome Open Res "Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA."

Suen KM, Navarro IC, Balasubramanian S, Tanpure A, Lamond A, Miska EA, Bensaddek D

[Wellcome Open Res, 2022]

<b>Background:</b> Methylation of carbon-5 of cytosines (m ⁵C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield&#xa0;5-hydroxymethylcytidine (hm ⁵C), 5-formylcytidine (f ⁵C), 2&#xb4;-O-methyl-5-hydroxymethylcytidine (hm ⁵Cm) and 2&#xb4;-O-methyl-5-formylcytidine (f ⁵Cm).&#xa0;How m ⁵C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m ⁵C is required for <i>Caenorhabditis elegans</i> development and fertility under heat stress. m ⁵C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications. <b>Methods:</b> To contribute to the understanding of how m ⁵C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in <i>C. elegans</i>. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m ⁵C was validated using immunoblotting. <b>Results:</b> Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. <i>In silico</i> analysis for phenotype enrichments suggested that hm ⁵Cm unique readers are enriched in proteins involved in RNA processing, while readers for m ⁵C, hm ⁵C and f ⁵C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m ⁵C <i>in vitro</i>. Finally, sequence alignment analysis showed that several of the putative m ⁵C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. <b>Conclusions:</b> The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m ⁵C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m ⁵C in <i>C. elegans</i>.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.

Butler JA et al. (1994) Worm Breeder's Gazette "Looking For Extracellular Matrix Proteinases in C. elegans"

Kramer JM, Butler JA

[Worm Breeder's Gazette, 1994]

LOOKING FOR EXTRACELLULAR MATRIX PROTEINASES IN C. ELEGANS. James A. Butler and James M. Kramer, Northwestern University Medical School. Department of CMS Biology, Chicago IL 60611