Zeigler, Mark T et al. (2011) International Worm Meeting "Exploring the role of RNP granules in regulating RNA stability."

Schisa, Jennifer, Zeigler, Mark T, Rice, Breanna

[International Worm Meeting, 2011]

RNA-binding proteins function in a diversity of ways to regulate RNA metabolism, including RNA translation, stability, and subcellular localization. Increasingly, it has become clear that many of these regulatory processes occur in compartmentalized regions of the cytoplasm, including RNP (ribonucleoprotein) structures such as stress granules and P bodies (processing bodies). We have been investigating RNPs that are induced in C. elegans oocytes in response to extended meiotic arrest or stress. Based on their composition, the hypothesis for the function of the RNP granules is to regulate mRNA stability and/or translation during periods of extended meiotic arrest or stress. The Schisa lab has identified 59 genes as required for RNP granule assembly in an RNAi screen. To investigate the role of RNP granules in regulating mRNA stability, qRT-PCR is being performed. We are currently comparing levels of pos-1 mRNA, an mRNA at high levels in RNP granules, in arrested fog-2 oocytes with levels in arrested fog-2 oocytes after RNAi inhibits assembly of RNP granules. The goal is to determine whether failure to assemble RNP granules affects RNA stability. We are using pgk-1 as our control RNA. We are planning to assay several mRNAs known to localize to RNP granules in arrested oocytes, and have a large number of RNAi targets to use to interfere with RNP granule assembly. We hope that this approach will address part of our hypothesis for the function of RNP granules in meiotically-arrested oocytes.

Jeff Harford (2004) East Coast Worm Meeting "High Throughput TILLING, Ecotilling, Genotyping, and Sequencing Instrumentation"

Jeff Harford

[East Coast Worm Meeting, 2004]

Targeting Induced Local Lesions In Genomes, or TILLING R , is a novel high throughput technology for reverse genetics. TILLING uses chemical mutagenesis to yield a traditional allelic series of point mutations for virtually all genes. TILLING is of particular value for essential genes where sublethal alleles are required for phenotypic analysis. TILLING has become an established technique on many model organisms, including C. Elegans, Arabidopsis, Zebrafish, Maize, Rice, and others. A variation on TILLING is called Ecotilling, in which high throughput SNP discovery is performed by locating natural variations throughout the genome. LI-COR Biosciences is a manufacturer of DNA Analysis Instrumentation for high throughput TILLING and Ecotilling, as well as for DNA Sequencing, AFLP R , and Microsatellite analysis. LI-COR also manufactures instrumentation for infrared fluorescent protein imaging, offering superior quantification over standard chemiluminescence. Stop by our booth to pick up the latest literature and publications on our products. TILLING is a registered trademark of Anawah, Inc. AFLP is a registered trademark of KeyGene, N.V.

Kipreos ET et al. (1995) International C. elegans Meeting "lin-19 and lin-23: Novel Regulators of the Cell Cycle"

Hedgecock EM, Kipreos ET, He WW

[International C. elegans Meeting, 1995]

We are interested in the regulation of cell proliferation during development. We have been characterizing two mutants, lin-19 and lin-23, that exhibit hyperplasia of multiple tissues. When maternal product is present, lin-19 or lin-23 embryos develop normally. In lin-23 mutants, all larval blast cells, with the exception of germ cells, produce extra cells. lin-19 mutants develop normally through the L1 stage, but all later larval blast cells produce extra cells. In the absence of maternal product, lin-19 and lin-23 embryos arrest with many cells but no overt morphogenesis. Both lin-23 and lin-19 are expressed at their highest levels in embryos, with appreciable expression during larval stages, and little expression in adults. A lin-23 transcriptional lacZ expression construct is transiently expressed in dividing tissues during larval development, consistent with a role in regulating the cell cycle. Sequence homology suggests that LIN-19 and LIN-23 work to modulate the cell cycle machinery by targeting key cell cycle regulators for destruction. LIN-19 is 28% identical to the S. cerevisiae cell cycle protein CDC53 (M. Goebl, pers. comm.). LIN-23 is homologous to the yeast protein CDC4. CDC53 and CDC4 interact with CDC34, a ubiquitin- conjugating enzyme, to target both positive and negative cell cycle regulators (G1 cyclin and cyclin kinase inhibitors) for degradation. lin-19 is a member of a new multi-gene family in higher eukaryotes, with at least 5 human, 4 C. elegans, 1 rice, and 2 Arabidopsis members. We envision that this gene family evolved to target specific classes of cell cycle regulators, to effect either positive or negative control of the cell cycle.

Sugimoto A et al. (1995) International C. elegans Meeting "DAD-1, A CONSERVED PROGRAMMED CELL DEATH SUPPRESSOR IN C. ELEGANS AND VERTEBRATES"

Halberg R, Nakashima T, Nishimoto T, Rothman JH, Hozak RR, Sugimoto A

[International C. elegans Meeting, 1995]

Multicellular organisms have a mechanism to eliminate unnecessary or harmful cells during development and tissue maintenance. Recent studies suggest that this process of programmed cell death is molecularly conserved throughout the animal kingdom. We are analyzing a new cell death inhibitor, dad-1, that functions in both C. elegans and vertebrates. The dad-1 gene, which encodes a novel protein, was originally identified in a mutant hamster cell line (tsBN7) that undergoes apoptosis at restrictive temperature. We identified a dad-1 homologue in C. elegans whose predicted product is >60% identical to vertebrate DAD-1. From the sequence databases, we also identified cDNA sequences encoding dad-1-like proteins from two plant species, Arabidopsis thaliana and the rice Oryza sativa. To test whether dad-1 is sufficient to prevent cell death, we generated worms transgenic for dad-1 genes expressed under control of a heat-shock promoter. Heat-shock-induced expression of either human or C. elegans dad-1 reduced the number of cell corpses present at comma stage. Extra cells were observed in the anterior pharynx of heat-shocked animals, confirming that cell deaths were indeed prevented. The C. elegans dad-1 gene was found to rescue mutant tsBN7 hamster cells from apoptotic death as efficiently as vertebrate dad-1 genes. Thus, dad-1 is a functionally conserved cell death suppressor that appears to be necessary (in cultured hamster cells) and sufficient (in C. elegans) to promote cell survival. In attempt to isolate loss-of-function mutants of Ce-dad-1, we have isolated multiple Tc1 insertions in the gene and are characterizing the expression pattern of the gene.

Negishi, Takefumi et al. (2021) International Worm Meeting "The auxin-inducible degron 2 (AID2) system provides sharp degradation control with low ligand concentrations and works at all developmental stages of C. elegans"

Kanemaki, Masato, Tanaka, Yuka, Negishi, Takefumi, Hayashi, Ken-ichiro

[International Worm Meeting, 2021]

In cases where genetic mutants are not readily available, RNA interference has been used for many years for loss-of-function analyses. Recently, the genome-editing technology has made it possible to fuse a degron tag to endogenous proteins of interest, allowing us to induce their degradation for loss-of-function analyses . The auxin-inducible degron (AID) system is one of the major degron-based technologies that has been used in many research communities, including the C. elegans filed. The pair of a 44-amino acid degron tag (namely AID*) and the TIR1 E3 ligase subunit derived from Arabidopsis thaliana (AtTIR1) has been used in studies using C. elegans. While the AID system is getting popular, two major drawbacks have been reported: 1) leaky degradation of AID-fused proteins in the absence of auxin and 2) the requirement for a high concentration of auxin. Recently, we developed an improved version, namely AID2, that worked in yeast, mammalian cells, and mice. In the AID2 system, we employed a TIR1 mutant derived from rice (OsTIR1(F74G)) and a new ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA). In this study, we applied AID2 to C. elegans and compared it with the original AID system. To this end, we inserted a transgene encoding AtTIR1(F79G) into the genome. First, we found that strain expressing AtTIR1(F79G) showed no significant basal degradation. Then, we found 5-Ph-IAA for AID2 induced rapid target degradation with a concentration 1,300 times lower than IAA for AID. Moreover, we developed a modified 5-Ph-IAA that has better permeability. Using this new ligand, we showed that AID2 worked effectively in the embryos surrounded by the eggshell. Our results indicate that the AID2 system is a better option for loss-of-function analyses of C. elegans in the future.

Hill, Robin L. (2011) International Worm Meeting "Characterizing wild nematode isolates in an undergraduate research lab."

Hill, Robin L.

[International Worm Meeting, 2011]

Small Public Liberal Arts Colleges pose multiple challenges, particularly in teaching laboratory courses. Resources are limited and the majority of students have no lab experience. In addition, faculty struggle to maintain a full-time active research program due to heavy teaching loads. A significant percentage of students expressing an interest in the sciences also express a desire for graduate or professional studies. As many of these programs desire "hands-on" scientific experience, providing research opportunities could be vital to their future success. To address these issues, a full semester research-based project was developed around isolating and identifying wild nematode isolates. An overview of nematode phylogeny and population genetics studies is coupled with students working in small groups to develop a research project, isolate nematodes and characterize the isolates using morphological and molecular analysis. Results are presented in a format similar to graduate lab meetings. The molecular techniques utilized during the pilot project included species-specific PCR (glp-1) (1) as well as sequencing of the 18S ribosomal RNA gene (2). I adapted universal rice primers (URP) as a fingerprint assay (3). Morphological analysis included buccal (lips, buccal tube, pharynx) and tail (shape, papillae and spicule structure) morphology as well as mating system (gonochoristic vs. hermaphroditic). Strains from the CGC have been used for comparison (both morphological and molecular) to wild isolates. Mating crosses with C.elegans and C.briggsae males have been performed with hermaphroditic species. Males from gonochoristic strains can be employed for gonochoristic isolates. Hermaphroditic isolates were obtained from leaf litter in wooded settings, open grasslands and under a rotting pumpkin. Species-specific PCR as well as mating crosses reject identification as C.elegans or C.briggsae. Sequencing of the 18S RNA gene is ongoing. Gonorchorisitc isolates were obtained from both household and grass cutting compost. Morphological analysis based on tail morphology suggests the genus Rhabditis, subgenus Rhabditella and Cephalopoides. Student feedback was uniformly positive. Isolates were easily obtained and the self-directed nature of the projects deepened their understanding of lab methods and research design. The range of analysis methods allows the course to be adapted to resource availability, therefore providing a method of research experience while enhancing knowledge of the model species. (1) Barriere, A., and Felix M.A. (2005). Curr. Biol. 15,1176-184. (2) Floyd, R. et al. (2002). Mol Ecol. 11, 839-850. (3) Kang, H. W. et al. (2002). Mol. Cells. 2, 281-287.