Chemical sensitivity in Caenorhabditis elegans.

The small nematode Caenorhabditis elegans lives in the soil, where mechanical, thermal and most of all chemical stimuli strongly influence its behavior. Here we briefly review how chemical sensitivity is organized at the cellular and molecular level in this organism. C. elegans has less than 40 chemosensory neurons. With few exceptions each neuron senses more than one substance and each substance is sensed by more than one neuron. At the molecular level, as in other organisms, also in C. elegans, seven transmembrane G-protein-coupled receptors (GPCRs), heterotrimeric G proteins, cyclic nucleotidegated ion channels, TRP channels and Ca++ play crucial roles in chemical sensitivity. An unusual feature, possibly due to C. elegans's strong dependence on chemical cues, is the very large number of GPCR chemoreceptor genes (1300-1700) coded in its genome. Genetic approaches have also allowed the identification of new molecules involved in chemical sensitivity that would not have been discovered otherwise. In addition to the basic factors involved in primary signalling, the studies in C. elegans have revealed a network of regulatory pathways and molecules suggesting that fine modulation of the responsiveness of neurons is important, possibly to allow worms to negotiate a continuously changing environment. The experimental versatility of C. elegans has made it possible, in many cases, to determine precisely in which neuron a given molecule or pathway is required and for which biological response. This type of information can contribute to the general field of sensory signalling because it provides correlations between the biochemical properties of molecules and their cellular functions and between these and the in vivo behavioral responses of the animal.

Nematode chitin synthases: gene structure, expression and function in Caenorhabditis elegans and the plant parasitic nematode Meloidogyne artiellia.

Although the presence of chitin in nematodes is well documented little is known about its synthesis in this phyletic group. The recently completed genome sequence of Caenorhabditis elegans predicts two sequences with homology to chitin synthases (chitin-UDP acetyl-glucosaminyl transferase; EC 2.4.1.16). We show that these genes are differentially expressed in a pattern that may reflect different functional roles. One gene is expressed predominantly in the adult hermaphrodite (the main egg-producing stage in the nematode) and later larval stages, which is consistent with a role in production of chitin for the eggshell. The other gene, however, is expressed in the cells that form the pharynx, and only in the period directly preceding a moult. These data suggest that the product of this gene is involved in synthesis of the feeding apparatus, which is replaced during each moult. We have also isolated a full-length genomic sequence of a chitin synthase orthologue from the plant parasitic nematode Meloidogyne artiellia. The single gene present in M. artiellia shows an expression pattern that is consistent with a role for the protein in production of the eggshell.

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling.

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

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.

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.

Immuno-cross-reactivity of CUT-1 and cuticlin epitopes between ascaris lumbricoides, Caenorhabditis elegans, and Heterorhabditis.

Cuticlin is the insoluble residue of nematode cuticle. It has been proved that cuticlin and CUT-1-like epitopes are conserved between the free-living Caenorhabditis elegans and the entomopathogenic nematode Heterorhabditis sp. The cloning of a cut-1 homologous gene from the animal intestinal parasite Ascaris lumbricoides has allowed us to extend the study of immuno-cross-reactivity at the ultrastructural level to this important species. Antibodies against recombinant CUT-1 protein and against cuticlin from Ascaris as well as from C. elegans were used for immuno-labeling ultrathin sections of high-pressure cryoprocessed worms. All the antisera used showed the same specific pattern of localization on sections of C. elegans of Heterorhabditis dauer larvae, and of Ascaris larvae in mature eggs. It was also shown that sera raised against the cuticlin residue contain anti-CUT-1 antibodies. CUT-1-like proteins are thus possibly important components in the immune response of hosts to invading nematodes. The results presented support the use of C. elegans as a model for the study of vertebrate parasitic nematodes.

Structural analysis of ASCUT-1, a protein component of the cuticle of the parasitic nematode Ascaris lumbricoides.

CUT-1 from the intestinal parasitic nematode Ascaris lumbricoides is a protein component of the insoluble residue of the cuticle, cuticlin. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The structure and the thermal stability of recombinant CUT-1 from A. lumbricoides (ASCUT-1) were investigated by Fourier-transform infrared (FT-IR) and CD spectroscopy. The data revealed that the secondary structure of the protein at 20 degrees C, both as insoluble inclusion bodies or in soluble form, contains about 50% beta structure, 14% alpha-helix and 25% turns. A tendency of A. lumbricoides CUT-1 to form aggregates was documented by FT-IR spectroscopy which showed also that the addition of SDS disrupts these interactions. Near-ultraviolet CD spectra confirmed these data and suggested that phenylalanine residues are probably involved in intermolecular hydrophobic interactions responsible for the tendency of the protein to aggregate. Near-ultraviolet spectra showed also that part of the cysteine residues forms disulphide bridges responsible for the tertiary architecture of the protein. Finally, FT-IR and CD data revealed that ASCUT-1 is very stable at high temperatures. This stability and the tendency of ASCUT-1 to form aggregates suggest that these properties may be important for a protein which is a component of a particularly resistant extracellular matrix such as the nematode cuticle.

cut-1-like genes of Ascaris lumbricoides.

Three genomic fragments homologous to cut-1 of Caenorhabditis elegans (C. elegans) have been identified in the intestinal parasitic nematode Ascaris lumbricoides (A. lumbricoides). Two of these fragments identify one region of the A. lumbricoides genome; they are separated by 8-9 kb and have opposite orientation, with the direction of transcription converging toward the center of the region. The third gene, which has been studied more completely, is in a different region of the genome separated from the first one by not less than 12-15 kb. The complete genomic sequence of this third gene has been determined. cDNA overlapping clones were obtained from adult A. lumbricoides RNA via the rapid amplification of cDNA ends (RACE) procedure [Frohman et al., 1988. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002] and sequenced. The mature mRNA of this gene, which we have named ascut-1, is trans-spliced to the spliced leader sequence of nematodes (SL1) [Krause, M., Hirsh, D., 1987. A trans-spliced leader sequence on actin mRNA in C. elegans. Cell 49, 753-761]. The mRNA is 1684 nt long plus the poly(A) tail and contains four exons with a 138 nt untranslated 5' leader and a 388 nt untranslated 3' tail. Conceptual translation of the coding sequence shows a protein of 385 aa with a signal peptide of 16 aa. The protein shows very high homology with CECUT-1, the product of the C. elegans gene cut-1 and with other cuticlin proteins of nematodes. A 262 amino acids region which is strongly conserved between these proteins seems to identify a group of proteins, so far restricted to nematodes, for which the name CUT-1-like is proposed.

Assembly of nematode cuticle: role of hydrophobic interactions in CUT-2 cross-linking.

CUT-2 is a component of cuticlin, the highly cross-linked, insoluble residue of the cuticle of the nematode Caenorhabditis elegans. A recombinant fragment of CUT-2, produced in E. coli, can be cross-linked in vitro by horse radish peroxidase via dityrosine formation to give large molecular species [1]. In this paper it is shown that the formation of CUT-2 polymers is greatly favoured over that of CUT-2 oligomers as no low molecular weight intermediates, dimers or trimers can be detected even when the cross-linking reaction is slowed or interrupted before completion. This suggests that recombinant CUT-2 forms large non-covalent complexes that are the only competent substrate for cross-linking. The inhibition of cross-linking by urea and the behavior of recombinant CUT-2 in size-exclusion chromatography under a variety of conditions suggest that hydrophobic interactions are important in the formation and stabilization of these complexes. The complexes are excellent substrates for cross-linking but react poorly with free tyrosine. In contrast, a soluble recombinant CUT-2 is a poor substrate for cross-linking but can efficiently react with free tyrosine.

Immuno-gold-labelling of CUT-1, CUT-2 and cuticlin epitopes in Caenorhabditis elegans and Heterorhabditis sp. processed by high pressure freezing and freeze-substitution.

CUT-1 and CUT-2 are two distinct protein components of cuticlin, the insoluble residue of the cuticles of nematodes. In previous experiments of gold-immuno-labelling on sections of chemically fixed Caenorhabditis elegans, CUT-1 and CUT-2 epitopes were specifically lost. Cryo-immobilization of C. elegans under high pressure followed by freeze-substitution, however, resulted in a good preservation of these antigenic sites and of the ultrastructure of the worms. The entomopathogenic nematode Heterorhabditis sp. processed by the same cryopreparation protocol has shown a strong reactivity with anti-sera raised against CUT-1, CUT-2 and against the whole cuticlin residue of C. elegans. The localization of these epitopes was conserved across the two species.

Ultrastructural immuno-localization of CUT-1 and CUT-2 antigenic sites in the cuticles of the nematode Caenorhabditis elegans.

CUT-1 and CUT-2 are two distinct proteins found in cuticlin, the insoluble residue of the cuticles of the nematode Caenorhabditis elegans. They are the products of genes which have been previously characterized molecularly. These proteins have been expressed as recombinant in Escherichia coli and specific antisera have been raised against them. The experiments reported here regard their ultrastructural immuno-gold localization either on purified cuticles or on whole worms of various stages of development of Caenorhabditis elegans. A location in the cortical layer of the isolated cuticles is common to all stages, whereas there is a dauer specific location in the fibrous ribbon underneath the alae. These localizations are compared with immuno-labelling obtained using a serum raised against the whole cuticlin residue. CUT-1 and CUT-2 epitopes are easily and specifically lost during conventional chemical fixation.

The role of dityrosine formation in the crosslinking of CUT-2, the product of a second cuticlin gene of Caenorhabditis elegans.

A second cuticlin gene, cut-2, of the nematode Caenorhabditis elegans, has been isolated and its genomic and cDNA sequences determined. The gene codes for a component of cuticlin, the insoluble residue of nematode cuticles. Conceptual translation of cut-2 reveals a 231-amino acid secreted protein which, like CUT-1, begins with a putative signal peptide of 16 residues. The central part of the protein consists of 13 repetitions of a short hydrophobic motif, which is often degenerated with substitutions and deletions. Parts of this motif are present also in CUT-1 (Caenorhabditis elegans) as well as in several protein components of the larval cuticle and of the eggshell layers of various insects (Locusta migratoria, Ceratitis capitata and Drosophila species). These sequence similarities are related to the similar functions of these proteins: they are all components of extracellular insoluble protective layers. Immunolocalisation and transcription analysis suggest that CUT-2 contributes to the cuticles of all larval stages and that it is not stage-specific. Analysis by reverse transcriptase-PCR suggests that it is not stage-specific. Analysis by reverse transcriptase-PCR suggests that transcription is not continuous throughout larval development but occurs in peaks which precede the moults. Dityrosine has been detected in the cuticle of nematodes and of insects; formation of dityrosine bridges may be one of the cross-linking mechanisms contributing to the insolubility of cuticlins. Recombinant, soluble CUT-2 is shown to be an excellent substrate for an in vitro cross-linking reaction, catalysed by horseradish peroxidase in the presence of H2O2, which results in the formation of insoluble, high-molecular weight CUT-2 and of dityrosine.

Amphid defective mutant of Caenorhabditis elegans.

Studies are reported on a chemoreception mutant which arose in a mutator strain. The mutant sensory neurons do not stain with fluoresceine isothiocyanate (Dyf phenotype), hence the name, dyf-1, given to the gene it identifies. The gene maps on LGI, 0.4 map units from dpy-5 on the unc-11 side. The response of mutant worms to various repellents has been studied and shown to be partially altered. Other chemoreception based behaviors are less affected. The cilia of the sensory neurons of the amphid are shorter than normal and the primary defect may be in the capacity of the sheath cells to secrete the matrix material that fills the space between cilia in the amphid channel. Progress toward the molecular cloning of the gene is also reported. Relevant results from other laboratories are briefly reviewed.

cut-1 a Caenorhabditis elegans gene coding for a dauer-specific noncollagenous component of the cuticle.

We have molecularly identified a new gene of Caenorhabditis elegans that codes for a component of the cuticle. The gene has been physically mapped on LGII near the locus sqt-1. The structure and the sequence of the gene have been determined and antisera have been raised against parts of the protein produced as fusions in Escherichia coli. By transcription analysis, and by the use of specific antisera, we have determined that this gene is expressed specifically during dauer larva formation. In extracts of worms completing the dauer transformation the product of this gene migrates in sodium dodecyl sulfate acrylamide gels with an apparent molecular mass of 40 kDa. By immunofluorescence we have determined that it is a component of the cuticles of dauer larvae. It forms a ribbon approximately 2 microns wide running along the lateral lines underneath the alae. Once it is assembled in the cuticle the protein becomes insoluble even in the presence of strong detergents and reducing agents in a manner that is similar to that described for the noncollagenous, insoluble residue of nematode cuticles called cuticlins; therefore, we have named the gene cut-1 for cuticlin 1. cut-1 represents the first gene for a noncollagenous component of C. elegans cuticle that has been characterized molecularly.

Structure, evolution and properties of a novel repetitive DNA family in Caenorhabditis elegans.

We have identified a moderately repeated DNA sequence in Caenorhabditis elegans present at least at twenty different locations in the genome. Elements of this intermingled repetitive DNA family are made up of tandem subreapeats whose smaller unit is ten base pairs long. The occurrence of single base changes between units is reminiscent of mammalian satellite DNA. Sequence analysis has shown that the consensus of these repeats is identical to the consensus of the heat-shock element (HSE) common to all eukaryotes (C--GAA--TTC--G). This consensus in our sequences is repeated in tandem with an overlap of four bases (C--GAA--TTC--GAA--TTC...). We studied in detail one cloned element of the family and we were unable to detect transcription in the flanking regions either under normal growth or after heat induction. Nevertheless a 242 bp sequences out of this same element was sufficient, when located on a multicopy plasmid in Saccharomyces cerevisiae, to drive transcription from a downstream gene under heat shock conditions.

A two-year study of enteric infections associated with diarrhoeal diseases in children in urban Somalia.

A hospital-based systematic sample of 1667 children with severe diarrhoeal disease was studied in Mogadishu, Somalia, throughout 1983 and 1984. One or more enteric pathogens were found in 61% of the patients. Rotavirus (25%), enterotoxigenic Escherichia coli (11%), Shigella spp. (9%), Aeromonas hydrophila (9%), Giardia lamblia trophozoites (8%), Campylobacter jejuni (8%), and Vibrio cholerae non-O1 (6%) were the most frequently identified pathogens. Age-specific detection rates of enteric pathogens and helminths, seasonal patterns, and relationship of some specific infections with feeding status and main clinical features have been defined for all the sample examined.

Beta-glucuronidase mutants of the nematode Caenorhabditis elegans.

Using a screening procedure that is based on a histochemical stain for the enzyme beta-glucuronidase, we have isolated several mutants of the nematode Caenorhabditis elegans affected in beta-glucuronidase activity. All of the mutations fall into one complementation group and identify a new gene, gus-1, which has been mapped on the right arm of linkage group I (LG I), 1.1 map units to the left of unc-54. The mutants have no visible phenotype, and their viabilities and fertilities are unaffected. Linked revertants of two of the mutations have been isolated. They restore enzyme activity to almost wild-type levels; the beta-glucuronidase that one of the revertants produces differs from that of the wild type. We propose that gus-1 is the structural locus for beta-glucuronidase.

Segregation of developmental potential in early embryos of Caenorhabditis elegans.

We have followed the appearance of differentiation markers in cleavage-inhibited and uninhibited early blastomeres of C. elegans and have compared the cleavage patterns of blastomeres in partial and complete embryos. The results indicate that at least some primary differentiation of embryonic cells is determined by internal factors that segregate in early cleavages, whereas patterns of cleavage are dictated by both internally segregating determinants and external cues.

Plasmids and transposable elements in Salmonella wien.

The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.

Particulate DNA polymerase from the cytoplasm of Xenopus laevis oocytes.

A DNA polymerase has been partially purified and characterized from Xenopus laevis stage 6 oocytes. The enzyme is present only in the cytoplasm and has been shown to be able to copy Poly(A) x oligo(dT), to be sensitive to N-ethylmaleimide, and to sediment faster than 4 S in high salt glycerol gradient. The enzyme can be extracted from particulate material which has a density in sucrose gradient ranging from 1.200 to 1.225 g/cc. This particulate material is identified by its ability to use Poly(A) x oligo(dT) as template in an exogenous DNA polymerase reaction and by its endogenous DNA synthesizing capacity.