89 Works

Worm corpses affect quantification of dauer recovery

Carolaing Gabaldon & Andrea Calixto
Diapause entry and recovery are behavioral adaptations that ensure the survival to environmental changes of animals capable of this type of developmental arrest. Caenorhabditis elegans enters diapause by forming the dauer larvae and at recovery resume post dauer L3 fate (Karp and Ambros, 2012). The precise quantification of dauer exit is required to estimate: post-dauer life span (Klass and Hirsh, 1976); dauer recovery ability after food replenishment (Cassada and Russell, 1975); to investigate components of bacterial...

GFP::PCN-1 does not reliably mark S phase in C. elegans adult germline progenitor zone cells

Tokiko Furuta & Swathi Arur
PCNA (proliferating cell nuclear antigen) is the DNA polymerase processivity factor that loads onto the chromatin during S phase of the cell-cycle (Brauchle et al. 2003). Thus, nuclear localization of PCNA (PCN-1 in C. elegans) is used as a marker for the S phase of the cell cycle (Brauchle et al. 2003). GFP::PCN-1 has been shown to label S phase in C. elegans embryo when driven through the germline and embryonic promoter pie-1 (Brauchle et...

nu444 is a novel allele of pkc-1 in C. elegans

Han Wang & Derek Sieburth
Here, we report nu444 as a novel allele of the gene pkc-1 that encodes the protein kinase C-1 in C. elegans. The nu444 allele was originally isolated from a forward genetic screen for mutants that suppressed the “Hic” (Hypersensitivity to Inhibitors of Cholinesterase) phenotype of dgk-1(nu62) mutants, which had increased acetylcholine release at the neuromuscular junction (Sieburth et al., 2007). In this screen, several genes that are important for neuropeptide secretion were recovered, including pkc-1(nu448)...

Establishment of a CRISPR/Cas9-based strategy for inducible protein dimerization

Jeffrey Zielich, Sriyash Mangal, Esther Zanin & Eric J. Lambie
We modified Norris’s mCherry-tag repair donor vector by fusing a C. elegans codon-optimized fkbp12 sequence 3’ to mCherry (Mangal et al. 2018) and re-establishing the critical NotI site for 3’ homology region insertion (Figure 1 A; pEL226). This vector can be used to C-terminally tag any GOI by following the protocol of Norris et al. (2015), including subsequent Cre-mediated excision of the dual marker selection cassette (Figure 1 B). As proof of principle we C-terminally...

oxi-1 and fshr-1 are required for neuromuscular signaling under normal and oxidative stress conditions in C. elegans

Barry Wei & Jennifer R Kowalski
Reactive oxygen species (ROS) contribute to neuronal degeneration by readily reacting with cellular components, consequently breaking down cellular integrity. Excess ROS often leads to oxidative stress, which results from destabilization of the organism's ability to control the balance between antioxidants and free radicals (Chandra et al. 2015). The ubiquitin-proteasome system helps to regulate oxidative stress and overall damage to cellular components by forming chains of ubiquitin polypeptides on cellular proteins; these chains then serve as...

acdh-1 transcripts are expressed in the intestine

Dustin Updike
​Stellaris smFISH probes targeting acdh-1, a short-chain acyl-CoA dehydrogenase, are shown in red (Cal Fluor 610). DAPI/blue marks embryonic nuclei, and PGL-1::GFP shows the corresponding location of P granules surrounding germ cell nuclei (arrowheads, green). During embryogenesis (A), acdh-1 expression begins in the E cells (arrow, red). Expression continues in the developing intestine, shown in red, throughout embryogenesis. Intestinal expression of acdh-1 persists through larval development in the L1 (B) and L2 (C) stages. These...

Genetic mapping of EgfrL.3.1 in Drosophila melanogaster

Joyce Stamm, Gnanada S Joshi, MA Anderson, Katie Bussing, Colton Houchin, Amber C Elinsky, Jacob T Flyte, Nadine Husseini, Dominika Jarosz, Chelsea L Johnson, Abby F Johnson, Christina E Jones, Taj P Kooner, Daniel Myhre, Thomas N Rafaill, Sarah Sayed, Kirby W Swan, Jonathan Toma & Jacob D Kagey

Reduced pharyngeal pumping rates observed in tph-1 mutants using microfluidic electropharyngeogram (EPG) recordings

Terra Hiebert, Adela Chicas-Cruz & Kathryn McCormick
In Caenorhabditis elegans, serotonin (5-HT) activates and controls pharyngeal pumping in response to food (Horvitz et al., 1982; Sze et al., 2000; Song and Avery 2012). Tryptophan hydroxylase, the enzyme required for serotonin biosynthesis, is encoded by the gene tph-1. Worms with a tph-1 deletion mutation exhibit phenotypes associated with a lack of serotonin-signaling, including reduced pharyngeal pumping (Sze et al., 2000; Avery and Horvitz 1990; Song and Avery 2012). We used a microfluidic electropharyngeogram...

Copulation defective mutants of C. elegans

Yvonne M. Hajdu-Cronin, Katharine S. Liu, Leslie Barber, Helen M. Chamberlin, William Boorstein & Paul W. Sternberg
​Description To identify genes involved in male copulatory behavior, we carried out an F2 clonal screen in a him-5 mutant background. We identified 20 mutations that affect male mating behavior without causing gross defects in morphology. ​ Male mating in C. elegans comprises at least five steps (Liu and Sternberg, 1995). (l) The male responds to the hermaphrodite by backing his tail along the length of the hermaphrodite, (2) he turns over or under her...

Novel deletion alleles of a C. elegans gene C38D4.9, named as tm4476 and tm4561

Sawako Yoshina, Sayaka Hori, Yuji Suehiro & Shohei Mitani
We report tm4476 and tm4561 as novel alleles of C38D4.9 gene that is an ortholog of human METTL5 (methyltransferase like 5)1. Human METTL5 function has not been reported. The alleles were isolated from the comprehensive screening of gene deletions generated by TMP/UV2. In the screening, both the alleles were detected by nested PCR using the following primer sets, 5’-CCGCCTATATCATGGCGCTT-3’ and 5’-GCTGGTAGTTGCCACCGCAT-3’ for first round PCR and 5’-ACTATCGAAGCCGCGCTTCA-3’ and 5’-AACCCCAACTCCGTCCCATT-3’ for second round PCR. By Sanger...

Mapping results for a set of cGAL effectors and drivers

​Sophie J Walton, Han Wang, Jonathan Lui & Paul W Sternberg
Recently, the GAL4-UAS system (cGAL) has been adapted for use in C. elegans for control of gene expression across 15°C - 25°C (Wang et al., 2017). In order to create a desired gene expression pattern, one crosses a transgenic strain containing a driver construct with another strain containing an effector gene. Here we mapped several cGAL driver and effector integrations. We first crossed each of the cGAL driver and effector strains with N2 males, picked...

SPAS-1 expression in neurons and vulva during L4 stage

George Brown & Rachid El Bejjani
spas-1 is the worm homolog of the human spastic paraplegia gene, spastin. Previous work in C. elegans has shown that SPAS-1 disassembles microtubules in an ATP-dependent manner (Matsushita-Ishiodori et al, 2007). Another study shows that SPAS-1 plays a role in synapse remodeling in motor neurons (Kurup et al, 2015). Here we describe the expression of SPAS-1 in L4 hermaphrodite larvae using the transcriptional reporter, axrEx8 [Pspas-1:mCh]: A) Low magnification (10X objective) image of an entire...

UNC-42 function is required for the ectopic expression of ASH markers in mls-2 mutant animals

Jordan F. Wood & Denise M. Ferkey
The HMX/NKS homeodomain transcription factor MLS-2 is required to initiate the expression of the AWC terminal selector ceh-36, and as such the AWCs of mls-2 loss-of-function mutant animals fail to express downstream AWC-specific terminal differentiation genes (Kim et al., 2010).  Interestingly, loss of mls-2 function was also shown to result in ectopic expression of ASH markers (sra-6p::gfp and osm-10p::gfp) in at least one neuron in a large percentage of mls-2 mutant animals (Kim et al., 2010).  The cells that ectopically express...

Simplified detection of a point mutation in C. elegans using tetra-primer ARMS-PCR

Matthew T Sullenberger & Eleanor M Maine
Single nucleotide polymorphisms (SNPs) can be difficult to detect using traditional PCR and gel electrophoresis, especially when no enzyme restriction sites overlap the SNP. Although alleles can be identified through amplification with flanking primers and sequencing, this adds time and cost, and sequencing is typically outsourced. Tetra-primer Amplification-Refractory Mutation System (ARMS)-PCR allows SNPs to be distinguished through a 3' primer mismatch at the SNP site for one allele, and an additional mismatch 1 - 3...

An isoform-specific allele of the sax-7 locus

Dylan Rahe, Ines Carrera, Filip Cosmanescu & Oliver Hobert

Mutations in KCNQ potassium channels cause pharyngeal pumping defects in C. elegans

Yoanne Clovis, Alexis Webb, Carl Turner & Bill Roberts
Pumps were stimulated with 10mM 5HT in M9 recorded as electropharyngeograms (EPGs) for 2 minutes in a NemaMetrix ScreenChip, and analyzed using NemAnalysis software (NemaMetrix). The null mutant strains kqt-1(aw3) and kqt-3 (aw1) were kindly donated by Dr. Aguan Wei (Wei et al., 2002) A) Pump frequency in kqt-1(aw3) animals was significantly lower than in N2s, while kqt-3(aw1) worms showed an increase in pump frequency (*p<0.05; ***p<0.01; 1-tailed Mann-Whitney U-test; n = 21-23 worms in...

The autophagy gene product BEC-1 supports normal aging and neurodevelopment in Caenorhabditis elegans I

Nicholas Ashley & Andrea M. Holgado
Macroautophagy (hereon referred as Autophagy) is a cellular housekeeping mechanism that uses a double membrane to target and engulf cell products for the formation of autophagosomes. These double membrane organelles then fuse to lysosomes where cell products are degraded and recycled (Nakamura and Yoshimori, 2018). Reports show that autophagy plays an important role in pathogen defense, development, starvation adaptations, and aging (Mizushima et al., 2008). Identifying molecular mechanisms responsible for autophagy in mammalian cells has...

SapTrap assembly of repair templates for Cas9-triggered homologous recombination with a self-excising cassette

Daniel J. Dickinson, Mark M. Slabodnick, Alicia H. Chen & Bob Goldstein
The advent of CRISPR/Cas9 technology in C. elegans has enabled an unprecedented level of control over this organism’s genome, which is facilitating research in a wide variety of fields. Several different experimental approaches exist for CRISPR in this organism (Dickinson and Goldstein 2016). One particularly powerful approach uses a drug selection with a Self-Excising Cassette (SEC) to isolate the desired genetically-modified animals following Cas9-triggered homologous recombination (Dickinson et al. 2015). The most labor-intensive step in...

New allele of C. elegans gene spch-3 (T27A3.4), called xc2

Nicholas R. Munoz, Dana T. Byrd & Diana Chu
We created a new mutant allele, named xc2, of the gene spch-3 (T27A3.4). spch-3 encodes one of three C. elegans small, highly basic proteins that resemble invertebrate protamines and associate with sperm meiotic chromosomes (Chu et al., 2006). The alleles were isolated from gene mutations generated by Non-Homologous End Joining (NHEJ) mediated repair of Cas9-generated breaks (Dickinson et al., 2013; Ran et al., 2013). The alleles were discovered using PCR with the following primers 5’-...

Loss of eif-2alpha phosphorylation on S49 (mammalian S51) associated with the integrated stress response hastens development in C. elegans​

Jarod Rollins, Noah Lind & Aric N. Rogers
The gene eif-2alpha (Y37E3.10) in C. elegans encodes for the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). The eIF2 trimer is required for delivering charged tRNAMet to the ribosome in a reaction requiring the hydrolysis of GTP to GDP (Sokabe et al., 2012). The phosphorylation of eif2-alpha on its 49th serine residue (Nukazuka et al., 2008) prevents replenishing GDP with GTP and thus reduces translation initiation by preventing delivery of tRNAMet. The phosphorylation...

The autophagy gene product BEC-1 supports normal aging and neurodevelopment in Caenorhabditis elegans: Integration

Nicholas Ashley & Andrea M. Holgado
Aging can be defined by many different physiological pathways that emphasize changes in a variety of cells and tissues. Disease and death are often results of aging (Viña et al., 2013). However, specific mechanisms in different cell types offer multiple hypotheses regarding the definition of aging (Viña et al., 2007). Although some researchers target specific aspects of physiological processes as major contributors to aging, such as free radical theory (Harman, 2003), inflammation theory (Chung et...

GABA Receptor mutant gbb-1 accelerates morphological aging of GABA neurons in Caenorhabditis elegans

I. Dhillon, A. Momin & I. Chin-Sang
The aging of an organism is heterogeneous, that is to say, tissues and organs may age at unique rates compared to overall organism longevity. Inhibition of the insulin/insulin-like growth factor signaling (IIS), such as the daf-2 IIS receptor increases Caenorhabditis elegans longevity. The daf-2 pathway plays a key role in the regulation of metabolism and stress responses and as such, impacts the longevity of the organism (Uno and Nishida 2016). Evidence suggests that an increase...

daf-2 modulates regeneration of mechanosensory neurons I

Zehra C Abay, Michelle Yu-Ying Wong & Brent Neumann
Caenorhabditis elegans (C. elegans) possess the ability to spontaneously regenerate injured axons via a highly efficient mechanism known as axonal fusion (Ghosh-Roy, et al., 2010; Neumann et al., 2011; Neumann et al., 2015; Abay et al., 2017). Following laser axotomy, regrowth from the proximal axon segment (still attached to the cell body) reconnects and fuses with its separated distal segment (Fig. 1A). We recently demonstrated that the level of axonal fusion increases with age (Abay...

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