Au, Vinci et al. (2021) International Worm Meeting "Crispr-ing C. elegans genes conserved to humans"

Au, Vinci, Moerman, Donald, Rougvie, Ann, Edgley, Mark, Doell, Claudia, Vargas, Marcus, Palma, Wilber, Hutter, Harald, Park, Heenam, Knott, Julie, Sternberg, Paul, Fernando, Lisa, Juciute, Viktorija, Li-Leger, Erica, Flibotte, Stephane, Martin, Kiana

[International Worm Meeting, 2021]

We are continuing our community resource project to generate gene deletions of high value to those interested in human biology and disease. Our highly coordinated three-site knockout (KO) production strategy has branches in California, Minnesota, and British Columbia, with the Canadian branch transitioning from the University of British Columbia to Simon Fraser University upon the retirement of KO legend Don Moerman. We employ a dual-pipeline strategy to generate KOs, with the Minnesota and Canadian sites using a scheme based on the Calarco Lab's dual selection cassette method. It deletes all or most of the target gene, effectively eliminating function. In addition, it replaces the gene with a fluorescent reporter, allowing simple tracking of the allele in cases where the mutation results in homozygous lethality or sterility, or fails to cause a discernible phenotype, facilitating genetic manipulation of the alleles. Alternatively, the California site developed and uses a "STOP-IN" cassette that places stop codons in all three reading frames near the beginning of the coding region, eliminating activity. This method does not allow visual tracking of alleles but has the advantage in that it inserts a unique guide RNA recognition site, enabling reversion of the locus to wild-type allowing the phenotype to be reassessed. This feature is desired by some users, for example those performing metabolomics experiments requiring quantitation of phenotypes that are sensitive to strain background effects. Each gene edit is carefully confirmed, and validated strains are grossly phenotyped and promptly deposited into the Caenorhabditis Genetics Center (CGC) strain collection along with detailed strain information. To date, 1,040 of our KOs are available through the CGC. Going forward, we plan to target an additional 2500 C. elegans orthologs of human genes, prioritizing known or suspected human disease genes, druggable gene classes, as well as understudied genes that are conserved to humans but that have no actionable information. Finally, we will consider community requests to prioritize specific genes.

Claudia Walser et al. (2006) European Worm Meeting "Distinct Roles of the Pumilio and FBF Translational Repressors during C. elegans Vulval Development"

Gopal Battu, ALEX HAJNAL, Claudia Walser, Erika Frohli Hoier

[European Worm Meeting, 2006]

Claudia Walser, Gopal Battu, Erika Frhli Hoier and Alex Hajnal. Members of the conserved PUF (Pumilio and FBF repeat) protein family regulate various aspects of germ line development by selectively binding to the 3 untranslated region of their target mRNAs and repressing translation. FBF-1 and FBF-2 regulate the sperm/oocyte switch and mitosis versus meiosis decision by repressing fem-3 and gld-1 translation, respectively (Zhang et al., 1997; Crittenden et al., 2002). We found that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulval development.. Loss-of-function mutations in puf-8 cause excess vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway, and they suppress the Vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signaling. A PUF-8::GFP translational reporter is initially expressed in all six vulval precursor cells (the VPCs P3.p through P8.p) but gets restricted to the distal, uninduced (3) VPCs after vulval fate specification. Moreover, the fusion of the distal VPCs to the hypodermal syncytium is retarded in puf-8(lf) mutants. Thus, PUF-8 acts cell-autonomously to limit the temporal competence of the vulval precursor cells to respond to the extrinsic patterning signals. fbf-1 and fbf-2, on the other hand, function as redundant inhibitors of vulval cell fate specification in a distinct pathway that involves the repression of gld-1 translation. fbf-1 fbf-2 double mutants show ectopic 1 cell fate marker expression in adjacent VPCs. We conclude that the FBFs ensure that only one VPC (P6.p) is selected for the 1 cell fate.. Therefore, the PUF family translational repressors regulate various aspects of cell fate specification in the soma, and they may play a conserved role in modulating signal transduction during animal development.

Swenson SI et al. (1995) International C. elegans Meeting "THE STEROID RECEPTOR HOMOLOGUE GENE, nhr-1, IN C. ELEGANS AND C. BRIGGSAE"

Purac A, Eagen RM, Honda BM, Swenson SI, Davison PJ

[International C. elegans Meeting, 1995]

We are studying the nhr-1 (nuclear hormone receptor) gene, one of a number of steroid receptor homologues identified in C. elegans. Sequencing of the cDNA has shown interesting features for this putative "orphan" receptor, including an unusual sequence in the P box of the DNA binding domain, which might indicate a very different DNA binding specificity and function, and a very long 3' untranslated region. The nhr-1 gene has been mapped to the right arm of the X chromosome, very close to sup-10. We are now characterizing the genomic organization of nhr-1 in C. elegans. We have also initiated work with C. briggsae in order to identify conserved, functionally important regions. The presence of an apparent homologue in C. briggsae was of special interest to us, given the unusual properties of nhr-1 (different P box, the long 3'UT region), and the apparent viability of homozygous deficiencies in this region around sup-10 (C. Cummins and P. Anderson). The C. elegans nhr-1 gene appears to contain 12 exons spread over 9.5 kb, including several relatively large introns, 1.3 to 2.3 kb in size. Work to date with genomic clones from C. briggsae shows conserved exons which share approximately 81-84% homology at the nucleotide level with the nhr-1 gene in C. elegans. This conservation between species, in the absence of a lethal phenotype for the homozygous deficiency, suggests that the nhr-1 gene may nevertheless have an evolutionarily relevant function. We wish to thank Ann Sluder, Gary Ruvkun, Claudia Cummins and Dave Baillie et al. for their assistance, and Alan Coulson et al. for help with mapping clones. Supported by a Research Reorientation Associateship (NSERC Canada) to SS, and an NSERC Operating grant to BMH.

Sprunger SA et al. (1991) International C. elegans Meeting "Molecular Analysis of mlc-3, the C. elegans Alkali Myosin Light Chain Gene."

Anderson P, Sprunger SA

[International C. elegans Meeting, 1991]

Our goal is the molecular genetic study of the structure, function, and interaction of muscle proteins. To that end, we have undertaken the molecular analysis of mlc-3, which encodes the single C. elegans'alkali' (or 'essential') myosin light chain (MLC). Claudia Cummins cloned mlc-3 by homology to the Drosophila melanogaster alkali MLC. The deduced mlc-3 amino acid sequence is more than 40% identical to other vertebrate and invertebrate MLCs. mlc-3 is a single-copy gene. Unlike the regulatory MLCs, which are encoded by the mlc-1 and mlc-2two-gene family, all the alkali MLCs are produced from mlc-3, as indicated by the absence of additional mlc-3- hybridizing bands on very low stringency Southern blots. mlc-3 has two polyadenylation sites. Two distinct groups of mlc-3 transcripts are observed on Northern blots as two thick bands of roughly 1.05 and 0.75 kb. These two groups of mRNAs differ in size due to the utilization of two polyadenylation sites separated by 0.25 kb, as shown by cDNA sequences and by Northern blots with transcript-specific probes. mlc-3 has multiple transcription initiation sites. Use of these sites creates additional heterogeneity in mlc-3 transcript size. Primer- extension analysis suggests that mlc-3 mRNAs can initiate at any of six different sites clustered 50 to 100 bp upstream of the AUG start codon. These putative transcription initiation sites are similar to those found in mlc-1, unc-54, act-4, and the vit genes, with a concensus sequence of TCA(C/G)T. None of the mlc-3 transcripts appear to be trans-spliced. Physical mapping has placed mlc-3 on LGIII, between ced-4 and daf-4. We are currently using a PCR technique to isolate mutant animals that do not produce any functional mlc-3 gene product. Our method is described in detail in the abstract by Rushforth et al. in this volume.