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Understanding of the C. elegans homeobox gene cluster has been significantly expanded since the genes egl-5 and lin-39 have been shown to correspond to homeobox genes in the cluster. Genes of the homeobox cluster not only function as regionally restricted homeotic genes along the anterior-posterior body axis, but also control cell migrations within the affected body regions.
Why is there variation in the virulence of infectious diseases? Virulence can have substantial effects on the genetic contribution of both host and pathogen to future generations. Understanding it therefore requires explanation not only in terms of cellular and molecular mechanisms, but also in evolutionary terms: what is the nature of the selection acting on genes responsible for virulence?
Research carried out in mammalian epithelial cell systems over the past 25 years has delineated pathways and sorting signals involved in polarized delivery of plasma membrane proteins. Recently some progress has been made in the identification of mechanisms underlying this polarized trafficking and in the visualization of trafficking routes in live cells. A promising area of research is the study of trafficking functions of novel polarity genes identified in Drosophila and Caenorhabditis elegans.
Quantitative trait locus (QTL) mapping provides a means to discover and roughly position regions of the genome that harbor genes responsible for natural variation in a complex trait. QTL mapping has been utilized extensively in the pursuit of genes contributing to longevity, chiefly in two animal models, the nematode Caenorhabditis elegans and the dipteran insect Drosophila melanogaster. Research on both species has demonstrated that a relatively small set of loci accounts for most of their genetic variance in lifespan. QTL mapping complements the discovery of longevity genes by mutagenesis screens, because the two procedures are predicted to unveil overlapping but distinct types of genes. We argue that information gained from animal models, even invertebrates, can greatly facilitate the process of gene identification and testing of homologous genes in humans.
A previous study in nematodes suggested that developmental constraint reduces the duplicability of genes involved in early development. Recent large-scale gene expression studies of fly development enabled us to conduct a more detailed study of this issue. We found that the average duplicability of genes involved in embryonic development is indeed lower than that of genes involved in larval development but not significantly lower than that of genes involved in later stages of development. Moreover, in both flies and nematodes genes with multiple expression peaks do not seem to have a lower duplicability than do genes with a single expression peak. Thus, although developmental constraint does appear to reduce gene duplicability, the effect seems weak or at best moderate.
A key requirement for several theories involving the evolution of sex and sexual selection is a specificity between host and parasite genotypes, i.e. the resistance of particular host genotypes to particular parasite genotypes and the infectivity of particular parasite genotypes for particular host genotypes. Determining the scope and nature of any such specificity is also of applied relevance, since any specificity for different parasite genotypes to infect particular host genotypes may affect the level of protection afforded by vaccination, the efficacy of selective breeding of livestock for parasite resistance and the long-term evolution of parasite populations in response to these control measures. Whereas we have some evidence for the role of specificity between host and pathogen genotypes in viral and bacterial infections, its role in macroparasitic infections is seldom considered. The first empirical test of this specificity for a vertebrate-nematode system is provided here using clonal lines of parasite and inbred and congenic strains of rat that differ either across the genome or only at the major histocompatibility complex. Although significant differences between the resistance of host genotypes to infection and between the fitness of different parasite genotypes are found, there is no evidence for an interaction between host and parasite genotypes. It is concluded that a specificity between host and parasite genotypes is unlikely in this system.
We have previously identified 60 predicted ABC transporter genes in the Caenorhabditis elegans genome and classified them into eight groups. As an initial step towards understanding how these putative ABC genes work in worms, we generated promoter-fluorescent protein fusions for the entire family to address when and where these genes are turned on in vivo. Both Aequoria green fluorescent protein (GFP) and Discosoma red fluorescent protein (RFP) were used as reporters in our transgenic assay. Observable expression is more frequently seen from fusions to genes in subfamilies B, C, D and E than those in subfamilies A and G. Sixteen worm ABC genes are found in tandem duplications, forming two four-gene clusters and four two-gene clusters. Fifteen out of the 16 duplicated gene promoters drove different or partially overlapping expression patterns, suggesting active functions for these duplicated genes. Furthermore, our results suggest that an internal promoter can cause differential expression of genes within an operon. Finally, our observations suggest that it is possible for coding sequences to function as a regulatory region for a neighbouring gene.
Small heat shock proteins are induced by various stresses. We here report the differential hypoxia responses of the hsp-16 genes in the nematode. The hsp-16.1 and hsp-16.2 genes in Caenorhabditis elegans responded to hypoxia, while hsp-16.41 and hsp-16.48, which share the promoter regions with hsp-16.1 and hsp-16.2, respectively, did not. For comparative genomic analysis, we identified ten hsp-16 genes in the nematode C.briggsae from the genome database. The comparison of the promoter sequences revealed a new conserved sequence block, CAC(A/T)CT, that was required for the orientation-dependent hypoxia response, but not for other stress responses such as heat or ethanol. We propose a working model for the orientation-dependent promoter usage between two genes sharing the promoter region. We also discuss a possible application of the hypoxia-inducible promoter for conditional gene expression.
DNA from Onchocerca volvulus from Oaxaca and Chiapas, Mexico were used as templates to amplify members of the O-150 Onchocerca specific repeat sequence family. The resulting PCR amplicons all hybridized with OVS2, an oligonucleotide that has been previously shown to recognize amplicons derived from O. volvulus with 100% sensitivity. However, when PCR products amplified from the O. volvulus specific plasmid pOVS134 were used as a probe, most samples did not hybridize. Similarly, when PCR products amplified from DNA isolated from adult O. volvulus from Oaxaca were used as a probe, amplicons from adult worms from both Oaxaca and Chiapas were recognized, but PCR products from infected black flies from Chiapas were not recognized. Amplicons derived from an adult worm from Chiapas hybridized with PCR products produced from adult parasites from both Oaxaca and Chiapas and to PCR products derived from the DNA of infected black flies from Chiapas. These data, when taken together, suggest that differences exist among the repeat sequence populations of parasites from Oaxaca and Chiapas in Mexico, suggesting that the O-150 repeat sequence family may be a useful tool for biogeographic studies of O. volvulus in the Americas.
We have isolated and expressed a cDNA from the parasitic nematode Trichinella spiralis encoding a novel secreted nucleotidase which catalyses the hydrolysis of nucleoside 5'-diphosphates and 5'-monophosphates, but not 5'-triphosphates. The full length cDNA encodes a protein of 550 amino acids with an N-terminal signal peptide, but lacking a C-terminal signature sequence for addition of a glycosyl phosphatidylinositol (GPI) anchor. Expression in Pichia pastoris resulted in the secretion of an active enzyme with the catalytic properties of both a Mg2+-dependent diphosphohydrolase/apyrase and a 5'-nucleotidase. The protein sequence is homologous to 5'-nucleotidases from a wide variety of organisms but contains no sequences specifically conserved in apyrases, suggesting that it is a representative of a new class of secreted nucleotidase. The enzyme was essentially monospecific for AMP among the nucleoside 5'-monophosphates and catalysed the hydrolysis of nucleoside 5'-diphosphates in the order of UDP >> ADP. The diphosphatase activity was dependent on the presence of magnesium ions and a reducing agent, while the 5'-nucleotidase activity was enhanced by these additions. Kinetic analyses indicated that the enzyme exhibits allosteric behaviour. Determination of the number of active sites suggested that catalysis of the two different reactions occurs at the same active site. The data are discussed in terms of regulation of host purinergic signalling during infection.
Collagens and proteins with collagen-like domains form large superfamilies in various species, and the numbers of known family members are increasing constantly. Vertebrates have at least 27 collagen types with 42 distinct polypeptide chains, >20 additional proteins with collagen-like domains and approximately 20 isoenzymes of various collagen-modifying enzymes. Caenorhabditis elegans has approximately 175 cuticle collagen polypeptides and two basement membrane collagens. Drosophila melanogaster has far fewer collagens than many other species but has approximately 20 polypeptides similar to the catalytic subunits of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. More than 1300 mutations have so far been characterized in 23 of the 42 human collagen genes in various diseases, and many mouse models and C. elegans mutants are also available to analyse the collagen gene family and their modifying enzymes.
The Caenorhabditis elegans genome encodes 284 nuclear receptor (NR) genes. Among these 284 NR genes are 15 genes conserved among the Metazoa. Here, we analyze the expression and function of eight heretofore uncharacterized conserved C. elegans NR genes. Reporter gene analysis demonstrates that these genes have distinct expression patterns and that a majority of the C. elegans cell types express a conserved NR gene. RNA interference with NR gene function resulted in visible phenotypes for three of the genes, revealing functions in various processes during postembryonic development. Five of the conserved NR genes are orthologs of NR genes that function during molting and metamorphosis in insects. Functional studies confirm a role for most of these 'ecdysone cascade' NR orthologs during the continuous growth and dauer molts. Transcript levels for these genes fluctuate in a reiterated pattern during the molting cycles, reminiscent of the expression hierarchy observed in the insect ecdysone response. Together, these analyses provide a foundation for further dissecting the role of NRs in nematode development as well as for evaluating conservation of NR functions among the Metazoa.
Sex determination in C. elegans is controlled by the TRA-1 zinc finger protein, a Ci/GLI homolog that promotes female cell fates throughout the body. The regulatory hierarchy that controls TRA-1 is well established, but the downstream effectors that establish sexual dimorphism during larval development remain largely unknown. Here, we describe the use of cDNA microarrays to identify sex-enriched transcripts expressed during three stages of C. elegans larval development. By excluding previously identified germline-enriched transcripts, we focused on somatic sexual development. This approach identified a large number of sex-enriched transcripts that are good candidates to encode regulators of somatic sexual development. We found little overlap between genes with sex-enriched expression in early versus late larval development, indicating that distinct sexual regulatory programs operate at these times. Genes with sex-enriched expression are found throughout the genome, with no strong bias between autosomes and X chromosomes. Reporter gene analysis revealed that these genes are expressed in highly specific patterns in a variety of sexually dimorphic cells. We searched for TRA-1 consensus DNA binding sites near genes with sex-enriched expression, and found that most strongly sex-enriched mRNAs are likely to be regulated indirectly by TRA-1. These results suggest that TRA-1 controls sexual dimorphism through a small number of intermediary regulators rather than by acting directly on the full constellation of genes involved in sex-specific differentiation.
Mutations in MCOLN1, which encodes the protein h-mucolipin-1, result in the lysosomal storage disease Mucolipidosis Type IV. Studies on CUP-5, the human orthologue of h-mucolipin-1 in Caenorhabditis elegans, have shown that these proteins are required for lysosome biogenesis. We show here that the lethality in cup-5 mutant worms is due to two defects, starvation of embryonic cells and general developmental defects. Starvation leads to apoptosis through a CED-3-mediated pathway. We also show that providing worms with a lipid-soluble metabolite partially rescues the embryonic lethality but has no effect on the developmental defects, the major cause of the lethality. These results indicate that supplementing the metabolic deficiency of Mucolipidosis Type IV patients mat not be sufficient to alleviate the symptoms due to tissue degeneration.
Toxocara canis is an ascarid nematode parasite of canids. Larvae infect a wide range of accidental hosts including humans, in whom they are the aetiologic agent of visceral and ocular Larva migrans. The labile surface coat of T. canis larvae consists of a family of mucin glycoproteins termed TES-120, for which the cDNAs have recently been cloned. In this paper, we describe the identification of a novel cDNA (Tc-muc-5) encoding an apomucin by expression screening of a cDNA library with antiserum raised to T. canis excretory/secretory products, and compare the predicted Tc-MUC-5 protein with those of other T. canis mucins (Tc-MUC-1-Tc-MUC-4) that include the TES-120 surface coat glycoproteins. Tc-MUC-5 has both a larger open reading frame and a more divergent sequence than the other T. canis mucins. It contains a putative signal peptide followed by two six-cysteine (SXC) domains, an extended threonine-rich central mucin core domain and two C-terminal SXC domains. Amino acid composition analysis of secreted TES-120 glycoproteins revealed a distinct lack of lysine residues; while this finding is in agreement with the primary sequences of Tc-MUC-1-Tc-MUC-4, Tc-MUC-5 is conspicuous by its relative abundance of lysines (6.7%), suggesting that this protein is not part of the TES-120 family of surface coat proteins.
The heterochronic gene lin-28 is a regulator of developmental timing in the nematode Caenorhabditis elegans. It must be expressed in the first larval stage and downregulated by the second stage for normal development. This downregulation is mediated in part by lin-4, a 21-nt microRNA. If downregulation fails due to a mutation in a short sequence in the lin-28 3' UTR that is complementary to lin-4, then a variety of somatic cell lineages fail to progress normally in development. Here, we report that Lin-28 homologues exist in diverse animals, including Drosophila, Xenopus, mouse, and human. These homologues are characterized by the LIN-28 protein's unusual pairing of RNA-binding motifs: a cold shock domain (CSD) and a pair of retroviral-type CCHC zinc knuckles. Conservation of LIN-28 proteins shows them to be distinct from the other conserved family of CSD-containing proteins of animals, the Y-box proteins. Importantly, the LIN-28 proteins of Drosophila, Xenopus, and mouse each appear to be expressed and downregulated during development, consistent with a conserved role for this regulator of developmental timing. In addition, the extremely long 3' UTRs of mouse and human Lin-28 genes show extensive regions of sequence identity that contain sites complementary to the mammalian homologues of C. elegans lin-4 and let-7 microRNAs, suggesting that microRNA regulation is a conserved feature of the Lin-28 gene in diverse animals.
MicroRNAs (miRNAs) are a large family of small regulatory RNAs that are poorly understood. The let-7 miRNA regulates the timing of the developmental switch from larval to adult cell fates during Caenorhabditis elegans development. Expression of let-7 RNA is temporally regulated, with robust expression in the fourth larval and adult stages. Here, we show that, like let-7 RNA, a transcriptional fusion of the let-7 promoter to gfp is temporally regulated, indicating that let-7 is transcriptionally controlled. Temporal upregulation of let-7 transcription requires an enhancer element, the temporal regulatory element (TRE), situated about 1200 base pairs upstream of the start of the mature let-7 RNA. The TRE is both necessary and sufficient for this temporal upregulation. A TRE binding factor (TREB) is able to bind to the TRE, and a 22-base pair inverted repeat within the TRE is necessary and sufficient for this binding. We also find that the nuclear hormone receptor DAF-12 and the RNA binding protein LIN-28 are both required for the correct timing of let-7 RNA and let-7::gfp expression. We speculate that these heterochronic genes regulate let-7 expression through its TRE.
We wish to understand how the trajectories of the twenty pharyngeal neurons of C. elegans are established. In this study we focused on the two bilateral M2 pharyngeal motorneurons, which each have their cell body located in the posterior bulb and send one axon through the isthmus and into the metacorpus. We used a GFP reporter to visualize these neurons in cell-autonomous and cell-non-autonomous axon guidance mutant backgrounds, as well as other mutant classes. Our main findings are: 1). Mutants with impaired growth cone functions, such as unc-6, unc-51, unc-73 and sax-3, often exhibit abnormal terminations and inappropriate trajectories at the distal ends of the M2 axons, i.e. within the metacorpus; and 2). Growth cone function mutants never exhibit abnormalities in the proximal part of the M2 neuron trajectories, i.e. between the cell body and the metacorpus. Our results suggest that the proximal and distal trajectories are established using distinct mechanisms, including a growth cone-independent process to establish the proximal trajectory. We isolated five novel mutants in a screen for worms exhibiting abnormal morphology of the M2 neurons. These mutants define a new gene class designated mnm (M neuron morphology abnormal).