A novel mode of ecdysozoan growth in Caenorhabditis elegans

Whereas growth in many ecdysozoa is associated with only molting, larval growth in nematodes, specifically Caenorhabditis elegans, is thought to be continuous and exponential. However, this has never been closely investigated. Here we report several detailed studies of growth in wild-type and dwarf C. elegans strains. We find that apparent exponential growth between hatching and adulthood comprises a series of linear phases, one per larval stage, with the linear growth rate increasing at successive molts. Although most structures grow continuously, the buccal cavity does not; instead, it grows saltationally at molts, like arthropod structures. We speculate that these saltational changes in mouth size permit changes in growth rate and that molting exists in nematodes to facilitate rapid growth. We study the cellular basis of this growth in the hypodermis. At each larval stage, lateral seam cells produce daughters that fuse with hyp7, a syncytium covering most of the worm. We find that seam cells and fusing daughter cells obtain larger sizes in successive molts. The total seam cell volume remains constant relative to the size of the worm. However, fusing daughter cells contributes only a very small amount directly to hypodermal growth, suggesting that most hyp7 growth must be intrinsic. Thus, dwarfism mutations studied principally act via adult syncytial growth, with cell size being near normal in both dbl-1 and dpy-2 mutant worms. We speculate that the main function of seam cell proliferation may be to supply the hypodermis with additional genomes for the purpose of growth.     

Actin gene family of Caenorhabditis elegans

Four actin genes have been isolated from Caenorhabditis elegans that account for all of the major actin hybridization to total genomic DNA. Actin genes I, II and III are clustered within a 12 X 10(3) base region; gene IV is unlinked to the others. All four genes have been sequenced from at least nucleotide -109 to +250. Genes I and III are identical for the first 307 coding nucleotides. Genes I and II differ in 14 positions within the first 250 coding nucleotides; one difference substitutes an aspartic acid for a glutamic acid at codon 5. Genes I and IV differ in 18 positions within the first 259 coding nucleotides without causing any amino acid differences. Genes I, II and III have introns after the first nucleotide of codon 64 and gene IV has an intron between codons 19 and 20. The four nucleotide sequences thus far define two different amino acid sequences. Both of the amino acid sequences resemble vertebrate cytoplasmic actin more than vertebrate muscle actin. A DNA polymorphism between the Bristol and Bergerac strains has been used as a phenotypic marker in genetic crosses to map the cluster of actin genes within a 2% recombination interval on linkage group V between unc-23 and sma-1 in order to begin a molecular genetic analysis of the actin loci.     

Ribosomal DNA of Caenorhabditis elegans

We have characterized the organization of the genes coding for 18 S, 5·8 S and 26 S ribosomal RNAs in the nematode Caenorhabditis elegans. These ribosomal genes, present in about 55 copies per haploid genome, alternate in a repeating tandem array. The repeating unit is only 7000 base-pairs, containing a non-transcribed spacer of no more than 1000 base-pairs. Most of the repeating units have identical restriction maps, but one repeat contains a deletion of 2900 base-pairs, which eliminates all or part of the 18 S coding region. We have found no difference in the major ribosomal DNA restriction endonuclease cleavage patterns between two interbreeding strains of C. elegans, but found differences between C. elegans and the closely related Caenorhabditis briggsae.     

A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans

The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors.     

Identification of a novel gene family involved in osmotic stress response in Caenorhabditis elegans

Organisms exposed to the damaging effects of high osmolarity accumulate solutes to increase cytoplasmic osmolarity. Yeast accumulates glycerol in response to osmotic stress, activated primarily by MAP kinase Hog1 signaling. A pathway regulated by protein kinase C (PKC1) also responds to changes in osmolarity and cell wall integrity. C. elegans accumulates glycerol when exposed to high osmolarity, but the molecular pathways responsible for this are not well understood. We report the identification of two genes, osm-7 and osm-11, which are related members of a novel gene family. Mutations in either gene lead to high internal levels of glycerol and cause an osmotic resistance phenotype (Osr). These mutants also have an altered defecation rhythm (Dec). Mutations in cuticle collagen genes dpy-2, dpy-7, and dpy-10 cause a similar Osr Dec phenotype. osm-7 is expressed in the hypodermis and may be secreted. We hypothesize that osm-7 and osm-11 interact with the cuticle, and disruption of the cuticle causes activation of signaling pathways that increase glycerol production. The phenotypes of osm-7 are not suppressed by mutations in MAP kinase or PKC pathways, suggesting that C. elegans uses signaling pathways different from yeast to mount a response to osmotic stress.     

The astacin protein family in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.     

A novel calmodulin-like gene from the nematode Caenorhabditis elegans

A novel gene from the nematode Caenorhabditis elegans was isolated by hybridization with a human calmodulin complementary DNA probe. This gene, cal-1, is present at one copy per haploid genome. In-situ hybridization of the cloned gene to metaphase chromosomes allowed us to assign it to the nematode linkage group IV. The polypeptide predicted from the sequence of this gene displays structural features of both calmodulin and troponin C.     

Prediction of structured non-coding RNAs in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae

We present a survey for non-coding RNAs and other structured RNA motifs in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae using the RNAz program. This approach explicitly evaluates comparative sequence information to detect stabilizing selection acting on RNA secondary structure. We detect 3,672 structured RNA motifs, of which only 678 are known non-translated RNAs (ncRNAs) or clear homologs of known C. elegans ncRNAs. Most of these signals are located in introns or at a distance from known protein-coding genes. With an estimated false positive rate of about 50% and a sensitivity on the order of 50%, we estimate that the nematode genomes contain between 3,000 and 4,000 RNAs with evolutionary conserved secondary structures. Only a small fraction of these belongs to the known RNA classes, including tRNAs, snoRNAs, snRNAs, or microRNAs. A relatively small class of ncRNA candidates is associated with previously observed RNA-specific upstream elements.     

A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity

PUF proteins comprise a highly conserved family of sequence-specific RNA binding proteins that regulate target mRNAs via binding directly to their 3'UTRs. The Caenorhabditis elegans genome encodes several PUF proteins, which cluster into four groups based on sequence similarity; all share amino acids that interact with the RNA in the cocrystal of human Pumilio with RNA. Members of the FBF and the PUF-8/9 groups bind different but related RNA sequences. We focus here on the binding specificity of representatives of a third cluster, comprising PUF-5, -6, and -7. We performed in vivo selection experiments using the yeast three-hybrid system to identify RNA sequences that bind PUF-5 and PUF-6, and we confirmed binding to optimal sites in vitro. The consensus sequences derived from the screens are similar for PUF-5 and PUF-6 but differ from those of the FBF or PUF-8/-9 groups. Similarly, neither PUF-5 nor PUF-6 bind the recognition sites preferred by the other clusters. Mutagenesis studies confirmed the unique RNA specificity of PUF-5/-6. Using the PUF-5 consensus derived from our experiments, we searched a database of C. elegans 3'UTRs to identify potential targets of PUF-5, several of which indeed bind PUF-5. Therefore the consensus has predictive value and provides a route to finding genuine targets of these proteins.     

A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans

For many organisms the ability to transduce light into cellular signals is crucial for survival. Light stimulates DNA repair and metabolism changes in bacteria, avoidance responses in single-cell organisms, attraction responses in plants, and both visual and nonvisual perception in animals. Despite these widely differing responses, in all of nature there are only six known families of proteins that can transduce light. Although the roundworm Caenorhabditis elegans has none of the known light transduction systems, we show here that C. elegans strongly accelerates its locomotion in response to blue or shorter wavelengths of light, with maximal responsiveness to ultraviolet light. Our data suggest that C. elegans uses this light response to escape the lethal doses of sunlight that permeate its habitat. Short-wavelength light drives locomotion by bypassing two critical signals, cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG), that neurons use to shape and control behaviors. C. elegans mutants lacking these signals are paralyzed and unresponsive to harsh physical stimuli in ambient light, but short-wavelength light rapidly rescues their paralysis and restores normal levels of coordinated locomotion. This light response is mediated by LITE-1, a novel ultraviolet light receptor that acts in neurons and is a member of the invertebrate Gustatory receptor (Gr) family. Heterologous expression of the receptor in muscle cells is sufficient to confer light responsiveness on cells that are normally unresponsive to light. Our results reveal a novel molecular solution for ultraviolet light detection and an unusual sensory modality in C. elegans that is unlike any previously described light response in any organism.     

The HSP70 multigene family of Caenorhabditis elegans

1. The heat shock response of the nematode Caenorhabditis elegans has been characterized. 2. There are at least nine genes in the hsp70 multigene family of C. elegans. 3. Five of the hsp70 genes have been characterized and assigned to one of at least three hsp70 gene subfamilies. One of the subfamilies consists of an hsp70 protein that has the potential to be translocated into the endoplasmic reticulum and another subfamily consists of a protein that has the potential to be translocated into the mitochondria. 4. The C. elegans hsp70 multigene family has several unique characteristics including introns in the heat inducible hsp70 genes, at least one trans-spliced hsp70 mRNA and two grp78 related genes, one of which is highly heat inducible. 5. The identification and characterization of C. elegans hsp70 multigene family is the basis for a genetic characterization of the regulation and function of a gene family during the development of a multicellular eukaryote.     

Extrachromosomal DNA transformation of Caenorhabditis elegans.

DNA was introduced into the germ line of the nematode Caenorhabditis elegans by microinjection. Approximately 10% of the injected worms gave rise to transformed progeny. Upon injection, supercoiled molecules formed a high-molecular-weight array predominantly composed of tandem repeats of the injected sequence. Injected linear molecules formed both tandem and inverted repeats as if they had ligated to each other. No worm DNA sequences were required in the injected plasmid for the formation of these high-molecular-weight arrays. Surprisingly, these high-molecular-weight arrays were extrachromosomal and heritable. On average 50% of the progeny of a transformed hermaphrodite still carried the exogenous sequences. In situ hybridization experiments demonstrated that approximately half of the transformed animals carried foreign DNA in all of their cells; the remainder were mosaic animals in which some cells contained the exogenous sequences while others carried no detectable foreign DNA. The presence of mosaic and nonmosaic nematodes in transformed populations may permit detailed analysis of the expression and function of C. elegans genes.     

Identification of a novel chondroitin hydrolase in Caenorhabditis elegans

Hyaluronidases have been postulated to be the enzyme acting at the initial step of chondroitin sulfate (CS) catabolism in vivo. Since chondroitin (Chn) but not hyaluronic acid (HA) has been detected in Caenorhabditis elegans, the nematode is a good model for elucidating the mechanism of the degradation of CS/Chn in vivo. Here we cloned the homolog of human hyaluronidase in C. elegans, T22C8.2. The Chn-degrading activity in vitro was first demonstrated when it was expressed in COS-7 cells. The enzyme cleaved preferentially Chn. CS-A and CS-C were also depolymerized but to lesser extents, and HA was hardly degraded. In order of preference, the substrates ranked Chn > CS-A > CS-C > HA. The products of the degradation of Chn by the enzyme were characterized by anion-exchange high performance liquid chromatography and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The structure of the major component in the digest was determined as GlcUAbeta1-3GalNAcbeta1-4GlcUAbeta1-3GalNAc, where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively, indicating that this enzyme is a Chn hydrolase, an endo-beta-galactosaminidase specific for Chn. Investigation of the effects of pH on the activity revealed the optimum pH of Chn hydrolase to be 6.0. Since Chn in C. elegans has been demonstrated to play critical roles in cell division, Chn hydrolase possibly regulates the function of Chn in vivo. This is the first demonstration of a Chn hydrolase in an animal.     

DNA glycosylase activities in the nematode,Caenorhabditis elegans

DNA glycosylases acting upon uracil- or 3-methyl-adenine-containing DNA have been detected in the sonic extracts of the nematode, Caenorhabditis elegans. 4 types of the asynchronously-growing worms, embryos obtained from gravid hermaphrodites, aseptically-hatched larvae, or dauer larvae. Uracil-DNA glcosylase activity was found in all 4 types of the extracts, and the activity was highest in the embryonic extract. In contrast, 3-methyladenine-DNA glycosylase activity was undetectable in the embryonic extract, while an equal level of activity was found in the other 3 types of the extracts. The results substantiate the ubiquity of base-excision repair in various organisms, and suggest that some of the repair functions may be developmentally regulated in multicellular animals.