Contribution of the GTPase Domain to the Subcellular Localization of Dynamin in the Nematode Caenorhabditis elegans

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.

A common muscarinic pathway for diapause recovery in the distantly related nematode species Caenorhabditis elegans and Ancylostoma caninum

Converging TGF-β and insulin-like neuroendocrine signaling pathways regulate whether Caenorhabditis elegans develops reproductively or arrests at the dauer larval stage. We examined whether neurotransmitters act in the dauer entry or recovery pathways. Muscarinic agonists promote recovery from dauer arrest induced by pheromone as well as by mutations in the TGF-β pathway. Dauer recovery in these animals is inhibited by the muscarinic antagonist atropine. Muscarinic agonists do not induce dauer recovery of either daf-2 or age-1 mutant animals, which have defects in the insulin-like signaling pathway. These data suggest that a metabotropic acetylcholine signaling pathway activates an insulin-like signal during C. elegans dauer recovery. Analogous and perhaps homologous cholinergic regulation of mammalian insulin release by the autonomic nervous system has been noted. In the parasitic nematode Ancylostoma caninum, the dauer larval stage is the infective stage, and recovery to the reproductive stage normally is induced by host factors. Muscarinic agonists also induce and atropine potently inhibits in vitro recovery of A. caninum dauer arrest. We suggest that host or parasite insulin-like signals may regulate recovery of A. caninum and could be potential targets for antihelminthic drugs.

Does recombination improve selection on codon usage? Lessons from nematode and fly complete genomes

Understanding the factors responsible for variations in mutation patterns and selection efficacy along chromosomes is a prerequisite for deciphering genome sequences. Population genetics models predict a positive correlation between the efficacy of selection at a given locus and the local rate of recombination because of Hill–Robertson effects. Codon usage is considered one of the most striking examples that support this prediction at the molecular level. In a wide range of species including Caenorhabditis elegans and Drosophila melanogaster, codon usage is essentially shaped by selection acting for translational efficiency. Codon usage bias correlates positively with recombination rate in Drosophila, apparently supporting the hypothesis that selection on codon usage is improved by recombination. Here we present an exhaustive analysis of codon usage in C. elegans and D. melanogaster complete genomes. We show that in both genomes there is a positive correlation between recombination rate and the frequency of optimal codons. However, we demonstrate that in both species, this effect is due to a mutational bias toward G and C bases in regions of high recombination rate, possibly as a direct consequence of the recombination process. The correlation between codon usage bias and recombination rate in these species appears to be essentially determined by recombination-dependent mutational patterns, rather than selective effects. This result highlights that it is necessary to take into account the mutagenic effect of recombination to understand the evolutionary role and impact of recombination.

Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans

CHR3 (nhr-23, NF1F4), the homologue of Drosophila DHR3 and mammalian ROR/RZR/RevErbA nuclear hormone receptors, is important for proper epidermal development and molting in the nematode Caenorhabditis elegans. Disruption of CHR3 (nhr-23) function leads to developmental changes, including incomplete molting and a short, fat (dumpy) phenotype. Here, we studied the role of CHR3 during larval development by using expression assays and RNA-mediated interference. We show that the levels of expression of CHR3 (nhr-23) cycle during larval development and reduction of CHR3 function during each intermolt period result in defects at all subsequent molts. Assaying candidate gene expression in populations of animals treated with CHR3 (nhr-23) RNA-mediated interference has identified dpy-7 as a potential gene acting downstream of CHR3. These results define CHR3 as a critical regulator of all C. elegans molts and begin to define the molecular pathway for its function.

Characterization of LeMir, a Root-Knot Nematode-Induced Gene in Tomato with an Encoded Product Secreted from the Root1

A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet. This gene was therefore named LeMir (L. esculentum miraculin). Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz). LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root. Rapid induction of expression upon nematode infection is localized to root tips. In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis. The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies. Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding. LeMir is also expressed in tomato callus tissue. Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction. Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms.

Environmental induction and genetic control of surface antigen switching in the nematode Caenorhabditis elegans.

Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection. Surface antigen switching mechanisms are presently unknown. In a genetic study of surface antigen switching, we have used a monoclonal antibody, M37, that recognizes a surface antigen on the first larval stage of the free-living nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans can be induced to display the M37 antigen on a later larval stage by altering the growth conditions. Mutations that result in nonconditional display of this antigen on all four larval stages fall into two classes. One class defines the new gene srf-6 II. The other mutations are in previously identified dauer-constitutive genes involved in transducing environmental signals that modulate formation of the dauer larva, a developmentally arrested dispersal stage. Although surface antigen switching is affected by some of the genes that control dauer formation, these two process can be blocked separately by specific mutations or induced separately by environmental factors. Based on these results, the mechanisms of nematode surface antigen switching can now be investigated directly.

Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.

Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Phylogeny of mRNA capping enzymes

The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and Chlorella virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes. We performed a structure–function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping enzyme. The results also allowed us to identify the capping enzyme of Caenorhabditis elegans. The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5′ end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5′ end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3′ end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.

The genetics of caloric restriction in Caenorhabditis elegans

Low caloric intake (caloric restriction) can lengthen the life span of a wide range of animals and possibly even of humans. To understand better how caloric restriction lengthens life span, we used genetic methods and criteria to investigate its mechanism of action in the nematode Caenorhabditis elegans. Mutations in many genes (eat genes) result in partial starvation of the worm by disrupting the function of the pharynx, the feeding organ. We found that most eat mutations significantly lengthen life span (by up to 50%). In C. elegans, mutations in a number of other genes that can extend life span have been found. Two genetically distinct mechanisms of life span extension are known: a mechanism involving genes that regulate dauer formation (age-1, daf-2, daf-16, and daf-28) and a mechanism involving genes that affect the rate of development and behavior (clk-1, clk-2, clk-3, and gro-1). We find that the long life of eat-2 mutants does not require the activity of DAF-16 and that eat-2; daf-2 double mutants live even longer than extremely long-lived daf-2 mutants. These findings demonstrate that food restriction lengthens life span by a mechanism distinct from that of dauer-formation mutants. In contrast, we find that food restriction does not further increase the life span of long-lived clk-1 mutants, suggesting that clk-1 and caloric restriction affect similar processes.

The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells

The nematode Caenorhabditis elegans exhibits behavioral responses to many volatile odorants. Chemotaxis toward one such odorant, diacetyl (butanedione), requires the function of a seven-transmembrane receptor protein encoded by the odr-10 gene. To determine directly whether ODR-10 protein is an odorant receptor, it is necessary to express the protein in a heterologous system and show that it responds to diacetyl by activation of a G protein signaling pathway. Here we demonstrate that human cells expressing ODR-10 on their surfaces exhibit a transient elevation in intracellular Ca2+ levels after diacetyl application. Volatile compounds that differ from diacetyl only by the addition of a methyl group (2,3-pentanedione) or the absence of a keto group (butanone) are not ODR-10 agonists. Behavioral responses to these compounds are not dependent on odr-10 function, so ODR-10 specificity in human cells resembles in vivo specificity. The apparent affinity of ODR-10 for diacetyl observed in human cells is consistent with the diacetyl concentration ranges that allow efficient nematode chemotaxis. ODR-10 expressed in human cells also responds to two anionic compounds, pyruvate and citrate, which are metabolic precursors used for diacetyl production by certain bacterial species. Ca2+ elevation in response to ODR-10 activation is due to release from intracellular stores.

Genetically targeted cell disruption in Caenorhabditis elegans

The elimination of identified cells is a powerful tool for investigating development and system function. Here we report on genetically mediated cell disruption effected by the toxic Caenorhabditis elegans mec-4(d) allele. We found that ectopic expression of mec-4(d) in the nematode causes dysfunction of a wide range of nerve, muscle, and hypodermal cells. mec-4(d)-mediated toxicity is dependent on the activity of a second gene, mec-6, rendering cell disruption conditionally dependent on genetic background. We describe a set of mec-4(d) vectors that facilitate construction of cell-specific disruption reagents and note that genetic cell disruption can be used for functional analyses of specific neurons or neuronal classes, for confirmation of neuronal circuitry, for generation of nematode populations lacking defined classes of functional cells, and for genetic screens. We suggest that mec-4(d) and/or related genes may be effective general tools for cell inactivation that could be used toward similar purposes in higher organisms.

Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa

Identification of host factors that interact with pathogens is crucial to an understanding of infectious disease, but direct screening for host mutations to aid in this task is not feasible in mammals. The nematode Caenorhabditis elegans is a genetically tractable alternative for investigating the pathogenic bacterium Pseudomonas aeruginosa. A P. aeruginosa toxin, produced at high cell density under control of the quorum-sensing regulators LasR and RhlR, rapidly and lethally paralyzes C. elegans. Loss-of-function mutations in C. elegans egl-9, a gene required for normal egg laying, confer strong resistance to the paralysis. Thus, activation of EGL-9 or of a pathway that includes it may lead to the paralysis. The molecular identity of egl-9 was determined by transformation rescue and DNA sequencing. A mammalian homologue of EGL-9 is expressed in tissues in which exposure to P. aeruginosa could have clinical effects.

Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated Mr 26,558) has been cloned and expressed in Escherichia coli. The purified recombinant human protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. Database searches identified amino acid sequences from Saccharomyces cerevisiae, Drosophila, and the nematode Caenorhabditis elegans with significant identity to the deduced amino acid sequence of human eIF6, suggesting the presence of homologues of human eIF6 in these organisms.