RT Fazzio et al. (1999) International C. elegans Meeting "vav Function in C. elegans"

MC Beckerle, RT Fazzio, AV Maricq

[International C. elegans Meeting, 1999]

The proto-oncoprotein Vav is a guanine nucleotide exchange factor (GEF) for members of the Rho/Rac family of monomeric GTPases. Vav homologs have been identified in humans, mice, rat and C. elegans . Extensive studies in mammalian tissue culture cells and chimeric mice have implicated Vav in signal transduction pathways that lead to the reorganization of the actin cytoskeleton. Vav comprises several functional domains that are highly conserved in known signaling molecules. At the amino acid level, the C. elegans homolog, VAV-1, shares 33% identity with the human VAV protein and displays each of the domains thought to be important for signaling. VAV-1 contains a calponin-homology (CH) domain and an acidic domain (AD). Selective disruption of these regions of the mammalian proteins results in oncogenic transformation. VAV-1 also contains a Dbl homology (DH) domain, two SH3 domains and one SH2 domain. In humans, the DH domain is responsible for the GEF activation of Rho, Rac-1 and Cdc-42, and the Src-homology regions are known to interact with identified signaling molecules. To determine the function of VAV-1, in vivo , we used a Tc1 transposon-based mutagenesis approach to generate a genomic disruption of the vav-1 locus. This resulted in the deletion of the carboxy-terminal 80% of VAV-1, including the DH, SH3 and SH2 domains. The putative null was outcrossed multiple times to remove the Tc1 background, and the region was sequenced to confirm the presence of a genomic deletion. Phenotypic characterization of this mutant indicated that the disruption of vav-1 results in larval lethality. Experiments designed to rescue the vav-1 phenotype and better define the developmental basis for lethality are currently in progress. Future experiments will focus on the identification of proteins that interact with VAV-1. To complement the functional analysis, VAV-1 expression was studied using several constructs that fused regions of the vav-1 gene to the reporter gene encoding Green Fluorescent Protein (GFP). Transgenic lines expressing these constructs indicated the presence of VAV-1 in several neurons, body wall muscle, gonad, and vulval cells. We are currently generating monoclonal antibodies to this protein to further characterize vav-1 expression.

Fazzio RT et al. (1998) West Coast Worm Meeting "INITIAL CHARACTERIZATION OF VAV-1: AN INTRACELLULAR SIGNAL TRANSDUCER IN C. ELEGANS"

Fazzio RT, Beckerle MC, Maricq AV

[West Coast Worm Meeting, 1998]

Central to the regulation of cellular proliferation and differentiation are the mechanisms by which external stimuli influence intracellular processes. Signal transduction at sites of membrane-substrate contact are mediated by transmembrane receptors, and several associated intracellular cytoskeletal proteins. The proto-oncogene encoded protein, Vav, is a multifunctional protein that has been shown to mediate signaling in several Rho/Rac pathways involved in cytoskeletal control. To gain a better understanding of the molecular mechanisms involved in such signaling, we have undertaken a genetic and molecular analysis of Vav in the nematode Caenorhabditis elegans. In mammals, Vav, and its family member, Vav-2, exhibit GEF activity for the Rac GTPase. Rac has been implicated in the control of cytoskeletal reorganization through the stimulation of actin polymerization at focal contacts and newly forming lamellipodia. In addition to GEF function, Vav contains a number of other structural motifs. Vav is potentially localized to the membrane through its pleckstrin-homology (PH) domain, where it could participate in a variety of protein-protein interactions. For example, Vav has been shown to bind to the focal adhesion scaffolding protein, zyxin, through its carboxy-terminal SH3 domain. Lastly, Vav contains an amino-terminal calponin-homology (CH) domain that could function in actin binding. Deletion of this domain results in oncogenesis, producing dense, non-refractile foci in NIH3T3 cells, and tumors in nude mice. Clearly, such a multifunctional protein will play key roles in the control of diverse intracellular signaling events. The study of Vav in C. elegans will contribute a novel approach to the understanding of such signaling mechanisms. Recently, the C. elegans genome sequencing project identified a vav homolog in the nematode that is located on linkage group III. We have screened a cDNA library provided by Robert Barstead to isolate a full-length cDNA encoding vav-1 in C. elegans. This vav-like protein (Vav-1) is highly homologous to mammalian Vav, containing an overall 55% identity with the murine protein. The predicted protein contains many of the structural motifs found in mammalian Vav, such as an amino terminal calponin homology (CH) domain, central GEF and PH domains, and a carboxy-terminal SH2 domain. To identify the cellular localization of this signaling protein, we constructed several fusions of vav-1 to a reporter gene encoding Green Fluorescent Protein (GFP). In transgenic strains that express these fusion proteins, we identified expression primarily in neurons and muscle. In order to determine the function of Vav-1, in vivo, we have taken two different approaches to disrupt Vav function: RNA-mediated interference, and Tc1-transposon based mutagenesis. Using a PCR-based strategy, we identified a strain containing a Tc1 transposon in exon 4 of the vav-1 gene. This strain is viable and exhibits no obvious phenotype on preliminary analysis. Rigorous phenotypic analysis of this strain, and strains derived from imprecise excision of the transposon, are currently in progress.

DM Madsen et al. (1999) International C. elegans Meeting "A Zyxin-like Molecule in C. elegans"

DM Madsen, RT Fazzio, MC Beckerle, AV Maricq

[International C. elegans Meeting, 1999]

Neuronal cells depend on cues from their environment to guide them to their correct targets. Focal adhesions are sites of contact between a cell and its substrate. These sites contain many specialized proteins that control and regulate cellular motility and shape. Amongst these is zyxin, a multifunctional LIM domain containing protein that colocalizes with cytoskeletal proteins. Zyxin may function to recruit components of the actin assembly machinery to specific sites in the cell and to stimulate spatially restricted actin polymerization. In mammalian cells, zyxin can shuttle between adhesion plaques and the nucleus, suggesting a mechanism by which changes in cell adhesion can trigger changes in gene expression and cellular differentiation. To gain a mechanistic understanding of signaling at adhesion plaques we have undertaken a study of a zyxin homologue in C. elegans . We isolated a cDNA that encodes a zyxin-like molecule in C. elegans . This gene, zyx-1 , has the characteristic features of zyxin, including a proline-rich N-terminal region believed to be important for protein-protein interactions, as well as 3 LIM domains, each with a canonical LIM consensus sequence. Using various fusions of zyx-1 to the reporter gene Green Fluorescent Protein (GFP), we observe expression in both neurons and muscle. We find localization to dense bodies and in the spermatheca. We are also generating antibodies to ZYX-1 to better define its subcellular localization. We anticipate that ZYX-1 functions as part of a protein complex that transduces substrate adhesion into changes in cytoskeleton function. To perturb the function of zyx-1 , we mapped its genomic structure and used a PCR-based strategy to identify two strains containing Tc1 transposable elements in the zyx-1 gene. Imprecise excision of the Tc1 elements led to deletion mutations that disrupt either the N-terminal or C-terminal regions. These strains are viable and phenotypic analysis is underway.

Fungtammasan A et al. (2016) Mol Biol Evol "Reverse Transcription Errors and RNA-DNA Differences at Short Tandem Repeats."

Fungtammasan A, Eckert K, Campos-Sanchez R, Makova KD, DeGiorgio M, Tomaszkiewicz M

[Mol Biol Evol, 2016]

Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to non-genetically encoded transcript variants, or RNA-DNA differences (RDDs). Such variation has been understudied, in part because its detection is obscured by reverse transcription (RT) and sequencing errors. It has only been evaluated for inter-transcript base substitution differences. Here we investigated transcript sequence variation for short tandem repeats (STRs). We developed the first maximum likelihood estimator (MLE) to infer RT error and RDD rates, taking next generation sequencing error rates into account. Using the MLE, we empirically evaluated RT error and RDD rates for STRs in a large-scale DNA and RNA replicated sequencing experiment conducted in a primate species. The RT error rates increased exponentially with STR length and were biased towards expansions. The RDD rates were approximately one order of magnitude lower than the RT error rates. The RT error rates estimated with the MLE from a primate data set were concordant with those estimated with an independent method, barcoded RNA sequencing, from a C. elegans data set. Our results have important implications for medical genomics, as STR allelic variation is associated with >40 diseases. STR non-allelic transcript variation can also contribute to disease phenotype. The MLE and empirical rates presented here can be used to evaluate the probability of disease-associated transcripts arising due to RDD.

Iyengar, Anusha et al. (2021) MicroPubl Biol

Iyengar, Anusha, Diamantakis, Stavros, Norris, Adam

[MicroPubl Biol, 2021]

C. elegans was the first animal to have its genome completely sequenced. In the decades since, the genome continues to be actively curated, annotated, and improved. Here we report the discovery of a new gene in a region of the genome that is currently not associated with any annotated gene or feature. We present RNA-seq and RT-PCR evidence that this gene is expressed at detectable levels, and that it is alternatively spliced. The new gene (Y97E10C.2) shares an operon with two upstream genes. We provide RNA-seq and RT-PCR evidence for a missing exon in the upstream gene T05B11.7, as well as an alternatively-spliced exon.

Kushibiki Y et al. (1996) Pathophysiology "Molecular cloning of oxygen-inducible genes in Caenorhabditis elegans by RT-PCR differential display."

Kushibiki Y, Yanase S, Nakazawa H, Ishii N

[Pathophysiology, 1996]

To investigate the genetic response to oxidative stress in eukaryotic cells, we have applied the method RT-PCR differential display to the small, free-living nematode Caenorhabditis elegans (C. elegans) which was cultured under atmospheric or high oxygen concentrations. As a result, a new gene was cloned whose expression increased at high oxygen concentration.

Zhang Y et al. (2012) PLoS One "Selection of reliable reference genes in Caenorhabditis elegans for analysis of nanotoxicity."

Pan X, Zhang Y, Chen D, Zhang B, Smith MA

[PLoS One, 2012]

Despite rapid development and application of a wide range of manufactured metal oxide nanoparticles (NPs), the understanding of potential risks of using NPs is less completed, especially at the molecular level. The nematode Caenorhabditis elegans (C.elegans) has been emerging as an environmental model to study the molecular mechanism of environmental contaminations, using standard genetic tools such as the real-time quantitative PCR (RT-qPCR). The most important factor that may affect the accuracy of RT-qPCR is to choose appropriate genes for normalization. In this study, we selected 13 reference gene candidates (act-1, cdc-42, pmp-3, eif-3.C, actin, act-2, csq-1, Y45F10D.4, tba-1, mdh-1, ama-1, F35G12.2, and rbd-1) to test their expression stability under different doses of nano-copper oxide (CuO 0, 1, 10, and 50 g/mL) using RT-qPCR. Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative Ct method, were employed to evaluate these 13 candidates expressions. As a result, tba-1, Y45F10D.4 and pmp-3 were the most reliable, which may be used as reference genes in future study of nanoparticle-induced genetic response using C.elegans.

Chauve L et al. (2020) J Vis Exp "High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology."

Andrews S, Casanueva O, Miska EA, Chauve L, Le Pen J, Biggins L, Hodge F, Todtenhaupt P

[J Vis Exp, 2020]

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named 'Worm-to-CT', allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

Lewis E et al. (1999) Mol Biochem Parasitol "cut-1-like genes are present in the filarial nematodes, Brugia pahangi and Brugia malayi, and, as in other nematodes, code for components of the cuticle."

Bazzicalupo P, Tetley L, Nunes CP, Lewis E, Hunter SJ, Devaney E

[Mol Biochem Parasitol, 1999]

A fragment of a cut-1 like gene from the filarial nematode Brugia pahangi (designated Bp-cut-1) was isolated by PCR from genomic DNA. The sequence was used to design primers for use in RT-PCR and resulted in the isolation of a cDNA fragment from larvae in the process of the L3-L4 moult. Screening of a B. malayi genomic library identified a single clone, Bm-cut-1. Using primers designed from the Brugia sequences, semi-quantitative RT-PCR was carried out on 11 different life cycle stages chosen to cover periods around the moult and inter-moult periods. This analysis demonstrated that the cut-1 mRNA was most abundant preceding the moult, consistent with its function as a cuticular protein. Immuno-gold electron microscopy using an affinity purified antiserum raised to the highly conserved region of Ascaris CUT-1 confirmed that the protein was restricted to a tight band in the median layer of the cuticle. Despite the fact that no transcripts could be detected in mature adult worms by RT-PCR, immuno-gold microscopy revealed staining of the microfilarial cuticle within the uterus of the adult female worm, suggesting that other cut-1-like genes are present in Brugia.

Y Kim et al. (1999) International C. elegans Meeting "cDNA cloning and functional analysis of the C. elegans dna topoisomerase IIIa gene"

Y Kim, H Koo

[International C. elegans Meeting, 1999]

Eukaryotic DNA topoisomerase III which is classified as type I DNA topoisomerase catalyzes the partial removal of negative DNA supercoils. The enzyme is required to maintain genome stability, and its null mutation blocks yeast sporulation and the embryogenesis of mice. cDNA clones of C. elegans DNA topoisomerase III a were obtained by performing RT-PCR using degenerate deoxynucleotides encoding amino acid sequences conserved in the human and yeast enzymes and then screening a C. elegans cDNA library using the RT-PCR product as a probe. The exact 5'-end cDNA clone was obtained by carrying out RT-PCR using a SL1 primer, which produced a 2.4 kb long cDNA sequence in combination with the clone from the cDNA library. C. elegans DNA topoisomerase III a polypeptide overexpressed in E. coli showed a catalytic activity of relaxing negatively supercoieled DNA in vitro . When the DNA topoisomerase III a expression in C. elegans was inhibited by microinjecting the double-stranded RNA, blockages in the oocyte formation and early embryogenesis were observed. The strong mRNA expression in the gonad coincided with the RNA interference phenotype. These results suggest that the DNA topoisomerase III a plays an important role in the gametogenesis and also embryogenesis. The specific reaction which the enzyme catalyzes in these developmental progressions remains to be explained.