Tat-mediated protein delivery in living Caenorhabditis elegans

The Tat protein from HIV-1 fused with heterologous proteins traverses biological membranes in a transcellular process called: protein transduction. This has already been successfully exploited in various biological models, but never in the nematode worm Caenorhabditis elegans. TAT-eGFP or GST-eGFP proteins were fed to C. elegans worms, which resulted in the specific localization of Tat-eGFP to epithelial intestinal cells. This system represents an efficient tool for transcellular transduction in C. elegans intestinal cells. Indeed, this approach avoids the use of tedious purification steps to purify the TAT fusion proteins and allows for rapid analyses of the transduced proteins. In addition, it may represent an efficient tool to functionally analyze the mechanisms of protein transduction as well as to complement RNAi/KO in the epithelial intestinal system. To sum up, the advantage of this technology is to combine the potential of bacterial expression system and the Tat-mediated transduction technique in living worm.     

Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.     

Genetic and fine structure analysis of unc-26(IV) and adjacent regions in Caenorhabditis elegans

Summary The genetic organization of unc-26(IV) and adjacent regions was studied in Caenorhabditis elegans. We constructed a fine structure genetic map of unc-26(IV), a gene that affects locomotion and pharyngeal muscle movement but not muscle structure. Eleven alleles were positioned relative to each other recombinationally and were classified according to phenotypic severity. The unc-26 gene spans at least 0.026 map units, which is exceptionally large for a C. elegans gene. All but one allele, e205, are amorphic alleles. Interestingly, e205 is hypomorphic but also suppressible by the amber suppressor sup-7. Nineteen lethal mutations in the unc-26 region were isolated and characterized. The unc-26 region is subdivided into four zones by five deficiency breakpoints. These mutations fall into 15 complementation groups. The stages of development affected by these mutations were determined.     

Tests for parental imprinting in the nematode Caenorhabditis elegans

Summary The mutation him-6(e1423) leads to generalized chromosomal nondisjunction during meiosis in oogenesis and spermatogenesis of C. elegans. As a result, gametes nullisomic or disomic for each of the six chromosomes occur at appreciable frequency. Crosses utilizing marked him-6 strains were used to generate and identify exceptional euploid progeny which had received both homologues of a marked autosome either from the male parent or from the female parent. Examples of all ten possible exceptions were identified and found to be viable and fertile. These results (together with previous data for the X chromosome) indicate that major chromosomal imprinting effects do not occur during gametogenesis in this organism.     

Analysis of the flt-1 Gene Encoding a ECM Protein, Flectin, in Caenorhabditis elegans

Flectin is a new type of extracellular matrix protein and its function was suggested to provide a micro-environment of great elasticity. The C. elegans genome database revealed the presence of a flectin homologue, flt-1, which shows approximately 40% similarity (20% identity) to chick flectin. Here we propose a new gene structure for the flt-1 based on our experiments and the partial cDNA clones obtained from Y. Kohara and further suggest that the previous gene prediction is incorrect. FLT-1 is shown to be expressed in various neurons, hypodermal cells, distal tip cells and vulva epithelial cells. Immunostaining results with anti-FLT-1 antibody, further confirm the FLT-1 expression in vulva epithelial cells. The lipophilic dye, DiI, was used to identify the head neurons expressing GFP and results indicated that none of the head neurons expressing GFP are the 6 chemosensory neurons. In order to determine the function of flt-1 gene, RNA-mediated interference (RNAi) experiments were conducted.     

A novel zinc-carboxypeptidase SURO-1 regulates cuticle formation and body morphogenesis in Caenorhabditis elegans

Cuticle formation and molting are critical for the development of Caenorhabditis elegans. To understand cuticle formation more clearly, we screened for suppressors in transgenic worms that expressed dominant ROL-6 collagen proteins. The suro-1 mutant, which is mild dumpy, exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in three suro-1 mutants, and found that suro-1 (ORF R11A5.7) encodes a putative zinc-carboxypeptidase homologue. The expression of this enzyme in the hypodermis and the genetic interactions between this enzyme and other collagen-modifying enzyme mutants suggest a regulatory role in collagen processing and cuticle organization for this novel carboxypeptidase. These findings aid our understanding of cuticle formation during worm development.     

The major gut esterase locus in the nematode Caenorhabditis elegans

Summary Mutations in the major gut esterase of the nematode Caenorhabditis elegans have been induced by ethylmethane sulfonate and detected by isoelectric focusing. The gut esterase locus, denoted ges-1, maps less than 0.3 map units to the right of the unc-60 locus, at the left end of chromosome V.     

The Caenorhabditis elegans EGL-26 protein mediates vulval cell morphogenesis.

In screens for Caenorhabditis elegans mutants defective in vulval morphogenesis, we isolated multiple mutants in which the uterus and the vulva fail to make a functional connection, resulting in an egg-laying defective phenotype. Two of these connection of gonad defective (Cog) mutants carry alleles of the egl-26 gene. We demonstrate that vulval lineages in egl-26 mutant animals are normal, but one vulval cell, vulF, adopts an abnormal morphology. This results in formation of an abnormally thick layer of vulval tissue at the apex of the vulva and a physical blockage of the exit to the vulva from the uterus. egl-26 was cloned and is predicted to encode a novel protein. Mosaic analysis indicates that egl-26 activity is required in the primary vulval lineage for vulF morphogenesis. Expression of a functional translational fusion of EGL-26 to GFP was observed within the primary vulval lineage only in vulE, which neighbors vulF. EGL-26 is localized at the apical edge of the vulE cell. It is thus possible that vulE acts to instruct morphological changes in the neighboring cell, vulF, in an interaction mediated by EGL-26.     

Abundance,Distribution, and Mutation Rates of Homopolymeric Nucleotide Runs in the Genome of Caenorhabditis elegans

Homopolymeric nucleotide runs, also called mononucleotide microsatellites, are a ubiquitous, dominant, and mutagenic feature of eukaryotic genomes. A clear understanding of the forces that shape patterns of homopolymer evolution, however, is lacking. We provide a focused investigation of the abundance, chromosomal distribution, and mutation spectra of the four strand-specific homopolymer types (A, T, G, C) 8 bp in the genome of Caenorhabditis elegans. A and T homopolymers vastly outnumber G and C HPs, and the run-length distributions of A and T homopolymers differ significantly from G and C homopolymers. A scanning window analysis of homopolymer chromosomal distribution reveals distinct clusters of homopolymer density in autosome arms that are regions of high recombination in C. elegans. Dramatic biases are detected among closely spaced homopolymers; for instance, we observe 994 A homopolymers immediately followed by a T homopolymer (5 to 3) and only 8 instances of T homopolymers directly followed by an A homopolymer. Empirical homopolymer mutation assays in a set of C. elegans mutation-accumulation lines reveal an 20-fold higher mutation rate for G and C homopolymers compared to A and T homopolymers. Nuclear A and T homopolymers are also found to mutate 100-fold more slowly than mitochondrial A and T homopolymers. This integrative approach yields a total nuclear genome-wide homopolymer mutation rate estimate of 1.6 mutations per genome per generation.Novel sequences are deposited in GenBank under accession numbers AY219759–AY219789.     

Circadian stress tolerance in adult Caenorhabditis elegans

Circadian rhythms control several behaviors through neural networks, hormones and gene expression. One of these outputs in invertebrates, vertebrates and plants is the stress resistance behavior. In this work, we studied the circadian variation in abiotic stress resistance of adult C. elegans as well as the genetic mechanisms that underlie such behavior. Measuring the stress resistance by tap response behavior we found a rhythm in response to osmotic (NaCl LC(50) = 340 mM) and oxidative (H(2)O(2) LC(50) = 50 mM) shocks, with a minimum at ZT0 (i.e., lights off) and ZT12 (lights on), respectively. In addition, the expression of glutathione peroxidase (C11E4.1) and glycerol-3-phosphate dehydrogenase (gpdh-1) (genes related to the control of stress responses) also showed a circadian fluctuation in basal levels with a peak at night. Moreover, in the mutant osr-1 (AM1 strain), a negative regulator of the gpdh-1 pathway, the osmotic resistance rhythms were masked at 350 mM but reappeared when the strain was treated with a higher NaCl concentration. This work demonstrates for the first time that in the adult nematode, C. elegans stress responses vary daily, and provides evidence of an underlying rhythmic gene expression that governs these behaviors.     

ABC transporters and RNAi in Caenorhabditis elegans

RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants, we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles for some transporter genes in endogenous RNAi-related processes.     

Using Caenorhabditis elegans for functional analysis of genes of parasitic nematodes

Information on the functional genomics of Caenorhabditis elegans has increased significantly in the last few years with the development of RNA interference. In parasitic nematodes, RNA interference has shown some success in gene knockdown but optimisation of this technique will be required before it can be adopted as a reliable functional genomics tool. Comparative studies in C. elegans remain an appropriate alternative for studying the function and regulation of some parasite genes and will be extremely useful for fully exploiting the increasing parasite genome sequence data becoming available.     

The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans

Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1 in ASJ neurons during aging, which in turn triggers TRX-1-dependent mechanisms to extend adult lifespan in the worm.     

Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.     

A new transmembrane 4 superfamily molecule in the nematode,Caenorhabditis elegans

Transmembrane 4 superfamily (TM4SF) molecules are predominantly mammalian cell surface glycoproteins that are thought to transduce signals mediating cell development, activation, and motility. Analysis of the Genpept sequence database reveals YKK8, a novel member of the TM4SF in the nematode,Caenorhabditis elegans. YKK8 is a putative 27.4-kDa protein encoded by a gene on chromosome III of theC. elegans genome (Wilson et al. [1994]Nature 368:32–38). The assignment of YKK8 to the TM4SF is justified by three criteria: statistical comparison of protein sequences, conserved TM4SF protein sequence motifs, and conserved TM4SF intron/exon boundaries in the genomic sequence. The discovery of a TM4SF molecule in the nematode extends this superfamily to a more primitive branch of the phylogenetic tree and suggests a fundamental role for TM4SF molecules in biology. Correspondence to: M.G. Tomlinson     

<Emphasis Type="Italic">Caenorhabditis elegans gcs-1</Emphasis> Confers Resistance to Arsenic-Induced Oxidative Stress

Gamma-glutamylcysteine synthetase (γ-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. The amount of metal ion required to kill half of the population of worms falls in the order of wild-type/As(V)>gcs-1/As(V)> wild-type/As(III)>gcs-1/As(III). gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Pretreatment with GSH significantly raised the survival rate of gcs-1 mutant worms compared to As(III)- or As(V)-treated worms alone. These results indicate that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress.     

Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.     

A mutation in CHN-1/CHIP suppresses muscle degeneration in Caenorhabditis elegans

Duchenne muscular dystrophy (DMD) is one of the most severe X-linked, inherited diseases of childhood, characterized by progressive muscle wasting and weakness as the consequence of mutations in the dystrophin gene. The protein encoded by dystrophin is a huge cytosolic protein that links the intracellular F-actin filaments to the members of the dystrophin-glycoprotein-complex (DGC). Dystrophin deficiency results in the absence or reduction of complex components that are degraded through an unknown pathway. We show here that muscle degeneration in a Caenorhabditis elegans DMD model is efficiently reduced by downregulation of chn-1, encoding the homologue of the human E3/E4 ubiquitylation enzyme CHIP. A deletion mutant of chn-1 delays the cell death of body-wall muscle cells and improves the motility of animals carrying mutations in dystrophin and MyoD. Elimination of chn-1 function in the musculature, but not in the nervous system, is sufficient for this effect, and can be phenocopied by proteasome inhibitor treatment. This suggests a critical role of CHIP/CHN-1-mediated ubiquitylation in the control of muscle wasting and degeneration and identifies a potential new drug target for the treatment of this disease.