Gu, Wei et al. (2011) International Worm Meeting "Purification and characterization of C. elegans cytoplasmic malate dehydrogenase."

Spengler, Justin W., Walstrom, Katherine M., Gu, Wei, Lindsay, Penelope L.

[International Worm Meeting, 2011]

Malate dehydrogenase (MDH) is the last enzyme in the citric acid cycle. MDH catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate. Since the forward reaction is energetically unfavorable, the reverse reaction is usually studied. Eukaryotes have two versions of this enzyme, one that is imported into the mitochondria and one that remains in the cytoplasm. The cytoplasmic MDH in C. elegans (F46E10.10) was originally mis-classified as lactate dehydrogenase. Hold and Riddle (Mech. Aging Dev. 124, 779, 2003) realized that the amino acids in the active site were compatible with malate binding rather than lactate. We named this enzyme MDH-1 and renamed the mitochondrial enzyme (F20H11.3) MDH-2 so that the naming convention corresponded to that used for other eukaryotes. We overexpressed MDH-1 in E. coli and removed the chitin binding domain tag used for the purification. In kinetic assays with the purified enzyme, MDH-1 had malate dehydrogenase activity that followed Michaelis-Menten kinetics. For the reverse reaction, we determined that the KM for oxaloacetate was 37 mM, and the KM for NADH was 70 mM. Our gel filtration results indicated that MDH-1 was active as a dimer, which is similar to the quaternary structure of most MDH enzymes (Minarik, P. et al., Gen. Physiol. Biophys. 21, 257, 2002). We are in the process of purifying endogenous MDH-1 from worms to compare its activity to our recombinant enzyme.

Halic M et al. (2009) Mol Cell "22G-RNAs in transposon silencing and centromere function."

Moazed D, Halic M

[Mol Cell, 2009]

Three recent papers (Gu et al., 2009; Claycomb et al., 2009; van Wolfswinkel et al., 2009) provide evidence that links a new class of small RNAs and Argonaute-associated complexes to centromere function and genome surveillance.

Hall DH et al. (1994) Worm Breeder's Gazette "Cytology of Degenerin-Induced Cell Death in the PVM Neuron"

Driscoll MA, Chalfie M, Gu GQ, Hall DH, Gong L

[Worm Breeder's Gazette, 1994]

Cytology of degenerin-induced cell death in the PVM neuron David H. Hall, Guoqiang Gu+, Lei Gong#, Monica Driscoll#, and Martin Chalfie+, * Dept. Neuroscience, Albert Einstein College of Medicine, Bronx, N.Y. 10461 + Dept. Biological Sciences, Columbia University, New York, N.Y. 10027 # Dept. Molecular Biology and Biochemistry, Rutgers University, Piscataway, N.J. 08855

Bhattacharya S et al. (2013) J Agric Food Chem "Bioactive components from flowers of Sambucus nigra L. increase glucose uptake in primary porcine myotube cultures and reduce fat accumulation in Caenorhabditis elegans."

Oksbjerg N, Bhattacharya S, Christensen LP, Young JF, Christensen KB, Kristiansen K, Faergeman NJ, Grevsen K, Olsen LC

[J Agric Food Chem, 2013]

Obesity and insulin resistance in skeletal muscles are major features of type 2 diabetes. In the present study, we examined the potential of Sambucus nigra flower (elderflowers) extracts to stimulate glucose uptake (GU) in primary porcine myotubes and reduce fat accumulation (FAc) in Caenorhabditis elegans. Bioassay guided chromatographic fractionations of extracts and fractions resulted in the identification of naringenin and 5-O- caffeoylquinic acid exhibiting a significant increase in GU. In addition, phenolic compounds related to those found in elderflowers were also tested, and among these, kaempferol, ferulic acid, p-coumaric acid, and caffeic acid increased GU significantly. FAc was significantly reduced in C. elegans, when treated with elderflower extracts, their fractions and the metabolites naringenin, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, quercetin-3-O-5-acetylglycoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, and isorhamnetin-3-O-glucoside and the related phenolic compounds kaempferol and ferulic acid. The study indicates that elderflower extracts contain bioactive compounds capable of modulating glucose and lipid metabolism, suitable for nutraceutical and pharmaceutical applications.

Suzuki JM et al. (2022) PLoS Genet "A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5' splice site identity."

Cartwright-Acar CH, Osterhoudt K, Katzman S, Suzuki JM, Gomez DR, Zahler AM

[PLoS Genet, 2022]

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5' GU and ending with 3' AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and "custody" of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5' splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5' splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5' splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5' splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.

Crittenden, Sarah L. et al. (2013) International Worm Meeting "Progress on developing the Q system to study GSCs and their control."

Crittenden, Sarah L., Kimble, Judith, Mohanty, Ipsita

[International Worm Meeting, 2013]

The Q system (Potter et al., 2010) permits inducible gene expression in C. elegans (Wei et al., 2012) and is similar in principal to the widely used GAL4-UAS system. The QF transcriptional activator directs transcription via an upstream activating sequence (QUAS). But in addition, another protein, QS, can inhibit QF activation and QS repression can be relieved by a small molecule, quinic acid or QA. QA is non-toxic and can be fed to worms (Wei et al., 2012). This inducible system has the potential to control target genes in both space and time. A chemically inducible method to control gene expression in specific cells at specific times would be tremendously useful for analyses of germline stem cells (GSCs) and their niche, the somatic distal tip cell (DTC). To this end, we are generating mosSCI insertion transgenes that rely on DTC-specific and GSC-specific regulatory sequences to drive QF expression. We are also generating a QUAS driven nuclear GFP (fused to H2B). Once we have the QF/QUAS pair working well for spatial regulation, we will add the QS/QA pair for temporal regulation. Preliminary experiments are promising and results will be shared at the meeting.

Driesschaert, Brecht et al. (2022) MicroPubl Biol

Driesschaert, Brecht, Temmerman, Liesbet

[MicroPubl Biol, 2022]

The Q system is a genetic tool developed to deliver spatiotemporal control over gene expression (Giles et al. 1991; Potter et al. 2010; Wei et al. 2012). Although it has already been adapted for use in C. elegans by Wei et al. in 2012, to date, the Q system has not been applied extensively in this nematode. In the relatively few available reports, it is mainly used to constitutively restrict gene expression in a spatial manner (e.g. Schild et al. 2014; Schild and Glauser 2015; Jee et al. 2016; Tolstenkov et al. 2018; Chiyoda et al. 2021), while but a handful of studies also explore the temporal aspect of the system (Matus et al. 2015; Yuan et al. 2016; Cottee et al. 2017; Hoang and Miller 2017). We aimed to apply this tool in the C. elegans nervous system to gain both spatial and temporal control over expression of a gene encoding a reporter protein that is targeted to the secretory pathway. Despite our efforts, we here report that in our hands, the Q system is not suitable for application in the neurons due to a lack of dynamic range.

Clovis Y et al. (2016) MicroPubl Biol "Mutations in KCNQ potassium channels cause pharyngeal pumping defects in C. elegans."

Roberts B, Webb A, Clovis Y, Turner C

[MicroPubl Biol, 2016]

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 each strain). B) Microfluidic EPG recordings show that pumping pattern in kqt-1(aw3) mutants is arrhythmic, with frequent drops in frequency. C) Overlay of first 50 pumps of recordings show that pump duration is higher in kqt-1(aw3) and kqt-3(aw1) animals than in N2s. Pumps are showed aligned on E spikes, which occur when the pharynx is fully contracted. D) Duration histogram illustrating the probability of occurrence of inter-pump interval (E to E duration) for each mutant strain. Histograms were binned to 4 ms width and normalized to reach an area underneath the curve equal to 1 (duration 100% likelihood to occur). In kqt-1(aw3)) animals, the time between two pumps is significantly increased compared to N2s (p < 0.01).

Ni, J. et al. (2017) International Worm Meeting "The triggering mechanism of C. elegans germline nuclear RNAi at the native targets."

Ni, J., Gu, S., Kalinava, N.

[International Worm Meeting, 2017]

We wish to understand the molecular mechanisms that distinguish "self" and "non-self" DNA in the germline genome of C. elegans. Nuclear RNAi refers to a set of small RNA-guided chromatin-based gene-silencing pathways (e.g., heterochromatin formation and transcriptional silencing), and plays an essential role in genome surveillance in yeast, plants, and animals. We previously identified a large set of genomic loci that are targeted by germline nuclear RNAi in C. elegans [1,2]. The triggering mechanisms at these native targets appear to be highly complex and are poorly understood. We are using two different approaches to resolve this gap. (1) We are identifying aberrant features associated with germline nuclear RNAi-targeted transcripts. To this end, we are using an unbiased RNA-seq approach to characterize both polyA and non-polyA transcripts in both wild type and mutant animals. In addition, subcellular localization of the native target transcripts are being examined using single-molecule FISH and RNA-seq combined with biochemical fractionation. (2) We are using CRISPR-mediated genome editing to test whether the silencing responses at native targets are indeed triggered by the aberrant features that are identified in (1). To date, we have found that "self" and "non-self" transcripts differ in RNA-pocessing, as well as subcellular localization. By using CRISPR, we have identified cis-regulatory elements that are required for the silencing response. Reference: 1. Ni JZ, Chen E, Gu SG. BMC Genomics. 2014. PMID: 25534009 2. Ni JZ, Kalinava N, Chen E, Huang A, Trinh T, Gu SG. Epigenetics Chromatin. 2016. PMID: 26779286

Huang CG et al. (2006) Mol Biotechnol "Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening."

Lamitina T, Agre P, Strange K, Huang CG

[Mol Biotechnol, 2006]

The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.