Katic, I. et al. (2019) International Worm Meeting "A reagent toolkit for tagging genes with compound tags."

Towbin, B., Katic, I., Gro beta hans, H.

[International Worm Meeting, 2019]

Precise editing using CRISPR/Cas9-induced DNA breaks and subsequent homology-directed repair (HDR) have become a routine part of C. elegans research. Emerging consensus in the field is that single-stranded oligonucleotides work as highest-efficiency HDR templates for creating small inserts or sequence changes, as well as for generating defined deletions. Several recent methods have achieved increased efficiency of precise insertions of larger fragments (Farboud et al., 2019; Paix et al., 2016; Dokshin, Ghanta et al., 2018), using double-stranded or partially single stranded templates and ribonucleoprotein complex delivery of CRISPR-Cas9. To support the increased use of longer endogenous tagging by CRISPR/Cas9 gene editing in C. elegans, we present a set of plasmids that can serve as templates for generating linear double- or partially single-stranded HDR templates that are selection marker independent. The plasmids each contain a fluorescent protein coding region, as well as affinity tags and/or the auxin-dependent degron domain (Zhang et al., 2015). We have used this kit routinely for >40 tagging experiments with a 98% success rate. The kit can easily be expanded to encompass additional fluorescent proteins or other simple tags. Farboud, B., A. F. Severson, and B. Meyer. 2019 Strategies for Efficient Genome Editing Using CRISPR-Cas9. Genetics 211: 431-457. https://doi.org/10.1534/genetics.118.301775 Paix, A., H. Schmidt, and G. Seydoux. 2016 Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs. Nucleic Acid Res. doi: 10.1093/nar/gkw502 Dokshin, G. A., K. S. Ghanta, K. M. Piscopo, and C. C. Mello, 2018 Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans. Genetics 210: 781-787. https://doi.org/10.1534/ge- netics.118.301532 Zhang, L., J. D. Ward, Z. Cheng, and A. F. Dernburg, 2015. The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Development 142: 4374-4384. doi: 10.1242/dev.129635

Jimenez G et al. (1997) Genes Dev "Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed."

Paroush Z, Ish-Horowicz D, Jimenez G

[Genes Dev, 1997]

Relatively little is known about the molecular mechanisms involved in transcriptional repression, despite its importance in development and differentiation. Recent evidence suggests that some transcriptional repressors act by way of adaptor molecules known as corepressors. Here, we use in vivo functional assays to test whether different repressor activities are mediated by the Groucho (Gro) corepressor in the Drosophila embryo. Previously, Gro was proposed to mediate repression by the Hairy-related family of basic helix-loop-helix proteins. Our results indicate not only that repression by Hairy requires Gro, but that a repressor domain from the Engrailed (En) homeodomain protein is also Gro dependent. The latter result correlates with an ability of this En domain to bind to Gro in vitro. In contrast, repressor regions from the Even-skipped, Snail, Kruppel, and Knirps transcription factors are effective in the absence of Gro. These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En.

Lemieux J et al. (2001) Genetics "Regulation of physiological rates in Caenorhabditis elegans by a tRNA-modifying enzyme in the mitochondria."

Lemieux J, Barnes T, Bussiere F, Hekimi S, Meng Y, Ubach A, Lakowski B, Webb A

[Genetics, 2001]

We show that the phenotype associated with gro-1(e2400) comprises the whole suite of features that characterize the phenotype of the clk mutants in Caenorhabditis elegans, including deregulated developmental, behavioral, and reproductive rates, as well as increased life span and a maternal effect. We cloned gro-1 and found that it encodes a highly conserved cellular enzyme, isopentenylpyroptiosphate:tRNA transferase (IPT), which modifies a subset of tRNTAs. In yeast, two forms of the enzyme are produced by alternative translation initiation, oric of which is mitochondrial. In the gro-1 transcript there are also two possible initiator ATGs, between which there is a sequence predicted to encode a mitochondrial localization signal. A functional GRO-1::GFP fusion protein is localized diffusely throughout the cytoplasm and nucleus. A GRO-1:GFP initiated from the first methionine is localized exclusively to the mitochondria and rescues the mutant phenotype. In contrast, a protein initiated from the second methionine is localized diffusely throughout the cell and does not rescue the mutant phenotype. As oxygen consumption and ATP concentration have been reported to be unaffected in gro-1 mutants, our observations suggest that GRO-1 acts in mitochondria and regulates global physiology by unknown mechanisms.

Moorthy S et al. (2000) J Cell Biol "Caenorhabditis elegans beta-G spectrin is dispensable for establishment of epithelial polarity, but essential for muscular and neuronal function."

Bennett V, Chen LS, Moorthy S

[J Cell Biol, 2000]

The Caenorhabditis elegans genome encodes one alpha spectrin subunit, a beta spectrin subunit (beta-G), and a beta-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans alpha spectrin is reproduced by tandem depletion of both beta-G and beta-H spectrins. We propose that alpha spectrin combines with the beta-G and beta-H subunits to form alpha/beta-G and alpha/beta-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode beta-G spectrin and vertebrate beta spectrins exhibit three striking parallels including: (1) beta spectrins are associated with the sites of cell-cell contact in epithelial tissues; (2) the highest levels of beta-G spectrin occur in the nervous system; and (3) beta spectrin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode beta-G spectrin associates with plasma membranes at sites of cell-cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode beta-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, beta-G spectrin is not associated with internal membranes and depletion of beta-G spectrin was not associated with any detectable defects in secretion. Instead beta-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans beta-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.

Guenette S et al. (1992) Mol Biochem Parasitol "Identification of a novel Brugia pahangi beta-tubulin gene (beta 2) and a 22-nucleotide spliced leader sequence on beta 1-tubulin mRNA."

Matlashewski G, Prichard RK, Guenette S

[Mol Biochem Parasitol, 1992]

We have examined the expression of beta-tubulin genes in the parasitic nematode, Brugia pahangi. A genomic library was constructed and screened by hybridization with a Haemonchus contortus beta-tubulin cDNA fragment which recognizes several B. pahangi beta-tubulin sequences, including sequences which correspond to the previously characterized beta 1-tubulin gene. The B. pahangi beta 2-tubulin gene was isolated by selecting clones which hybridize to the H. contortus beta-tubulin gene but which do not hybridize to the beta 1-tubulin gene. A partial sequence of the beta 2-tubulin gene confirms that it codes for a distinct beta-tubulin. Southern hybridization analyses show that the beta 2-tubulin sequence exists as a single copy gene within the B. pahangi genome. Expression of the beta 2-tubulin gene is developmentally regulated and the message is found predominantly in adult male worms, whereas the beta 1-tubulin gene is expressed in microfilariae and approximately equal levels of the transcript are found in male and female adult worms. During mRNA maturation the beta 1-tubulin mRNA of microfilariae and adult worms acquires a trans-spliced leader identical to the SL1 of Caenorhabditis elegans.

Ewbank JJ et al. (1995) International C. elegans Meeting "TIME FOR SOME RESULTS: MOLECULAR CHARACTERISATION OF THE CLK-1 LOCUS."

Hekimi S, Ewbank JJ, Barnes TM, Lakowski B

[International C. elegans Meeting, 1995]

Mutation in the clk-1 locus disrupts the normal temporal control of a wide range of developmental and behavioral events; see Wong et al., Genetics 139:1247-1259 (1995). Perhaps fittingly for clk-1, progress towards the gene's cloning has been slow (wm93p124; ecwm94p77). Injection of cosmid pools spanning the candidate region has failed to produce rescue. RFLP mapping has been hampered by an apparent paucity of polymorphisms between N2, RC301 and RW7000 strains in the region. The strategy of refining the likely position of clk-1 by locating the left- and right-flanking genes dpy-17 and lon-1 has also proved problematic. Extensive Southern analyses have failed to reveal any allele-specific polymorphisms (thanks to Cathy Savage for lon-1 strains). Further, for lon-1, the usual co-injectable marker pRF4 on its own gives phenotypic rescue (wild-type length rollers; presumed to be a indirect effect), while arrays covering the supposed location of dpy-17 produce a dominant Dpy effect, potentially confounding detection of any rescue. A 32P non-complementation screen has generated new alleles of clk-1, dpy-17 and lon-1. These will be used in another round of Southern analyses in the hope of uncovering allele-specific polymorphisms. One positive lead that we are pursuing involves gro-1(e2400); see wbg11#5p60; ecmw92p5. The gro-1 locus lies close to clk-1; by scoring Sma non Dpy in the six factor cross dpy-17(e164)gro- 1(e2400)sma-4(e729)/ unc-79(e1030)clk-1(e2519)lon-1(e185) it has been positioned 0.03 cM (approx. 15 kb) to the left of clk-1. Two spontaneous mutants (bli-1(qm49) II and dpy-7(qm63) V) have been recovered from gro-1 strains, implicating the activity of a transposon which may be directly associated with the gro-1 mutation. As dpy-7 has been cloned, this may give us an indirect route to speed the cloning of both gro-1 and clk-1. The progress towards identifying clk-1, gro-1, dpy-17 and lon-1 will be reported.

Quarato P et al. (2021) STAR Protoc "Global Run-On sequencing to measure nascent transcription in C.elegans."

Quarato P, Cecere G

[STAR Protoc, 2021]

Global Run-On sequencing (GRO-seq) is one of the most sensitive techniques to detect nascent transcription from RNA polymerase (Pol) at a genome-wide level. The protocol incorporates labeled ribonucleotides into nascent RNAs from Pol I, II, and III. We have adapted the GRO-seq protocol to the nematode <i>Caenorhabditis elegans</i> to measure transcription from embryos and adult worms. Here, we provide a detailed overview of the protocol highlighting the critical steps for generating successful libraries. For complete details on the use and execution of this protocol, please refer to Quarato et al. (2021).

Bedrood S et al. (2009) Biochemistry "Annexin A5 directly interacts with amyloidogenic proteins and reduces their toxicity."

Jayasinghe S, Langen R, Erbel S, Butler PC, Chen M, Sieburth D, Ritzel RA, Bedrood S

[Biochemistry, 2009]

Protein misfolding is a central mechanism for the development of neurodegenerative diseases and type 2 diabetes mellitus. The accumulation of misfolded alpha-synuclein protein inclusions in the Lewy bodies of Parkinson's disease is thought to play a key role in pathogenesis and disease progression. Similarly, the misfolding of the beta-cell hormone human islet amyloid polypeptide (h-IAPP) into toxic oligomers plays a central role in the induction of beta-cell apoptosis in the context of type 2 diabetes. In this study, we show that annexin A5 plays a role in interacting with and reducing the toxicity of the amyloidogenic proteins, h-IAPP and alpha-synuclein. We find that annexin A5 is coexpressed in human beta-cells and that exogenous annexin A5 reduces the level of h-IAPP-induced apoptosis in human islets by approximately 50% and in rodent beta-cells by approximately 90%. Experiments with transgenic expression of alpha-synuclein in Caenorhabditis elegans show that annexin A5 reduces alpha-synuclein inclusions in vivo. Using thioflavin T fluorescence, electron microscopy, and electron paramagnetic resonance, we provide evidence that substoichiometric amounts of annexin A5 inhibit h-IAPP and alpha-synuclein misfolding and fibril formation. We conclude that annexin A5 might act as a molecular safeguard against the formation of toxic amyloid aggregates.

Thomas JH et al. (1994) Worm Breeder's Gazette "lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein"

Thomas JH, Horvitz HR

[Worm Breeder's Gazette, 1994]

lin-36, a Class B Synthetic Multivulva Gene, Encodes a Novel Protein Jeffrey H. Thomas and H. Robert Horvitz, HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA

Lemieux JY et al. (1997) International C. elegans Meeting "TOWARDS THE MOLECULAR CHARACTERIZATION OF THE BIOLOGICAL TIMING GENE gro-1"

Lakowski B, Barnes TM, Lemieux JY, Hekimi S

[International C. elegans Meeting, 1997]

In a screen for C. elegans viable maternal-effect mutants, a number of novel phenotypic classes were defined (1). One of these classes is defined by the three clk genes, mutations in which result in a mean lengthening of development and life span, and an increase in the periods of rhythmic behaviors such as defecation, swimming and pumping (1). We have found that a fourth gene, gro-1, also shows the Clk phenotype (2,3). gro-1 was first recovered by Jonathan Hodgkin as a spontaneous segregant from a wild strain (personal communication). Of the four genes, only clk-1 has been thoroughly studied; a comprehensive phenotypic analysis was carried out by Wong et. al. (2) and the molecular cloning by Ewbank et. al. (4). The molecular characterization of each of the Clk genes will aid in understanding the Clk phenotype and ultimately the processes in which these genes are involved. We have mapped gro-1 to the interval between dpy-17 and clk-1 on LGIII. Injection of individual cosmids spanning this region identified three overlapping cosmids which were capable of rescue. Using the available genomic sequence, we tested individual predicted genes from the common rescuing region. This led to the identification of a single gene necessary and sufficient for rescue. The subsequent sequencing of the gene from the gro-1 mutant strain identified the e2400 lesion, confirming the gene assignment. Studies are now underway to determine the expression pattern of GRO-1. Details will be presented at the meeting. 1. Hekimi, S. et. al. Genetics 141: 1351-1364. 2. Wong, A. et. al. Genetics 139: 1247-1259. 3. Lakowski, B. and S. Hekimi. Science. 272: 1010-1013. 4. Ewbank, J. J. et. al. Science 275: 980-983.