The mitochondrial ribosomal RNA genes of the nematodes Caenorhabditis elegans and Ascaris suum: Consensus secondary-structure models and conserved nucleotide sets for phylogenetic analysis

The small- and large-subunit mitochondrial ribosomal RNA genes (mt-s-rRNA and mt-1-rRNA) of the nematode worms Caenorhabditis elegans and Ascaris suum encode the smallest rRNAs so far reported for metazoa. These size reductions correlate with the previously described, smaller, structurally anomalous mt-tRNAs of C. elegans and A. suum. Using primer extension analysis, the 5 end nucleotides of the mt-s-rRNA and mt-1-rRNA genes were determined to be adjacent to the 3 end nucleotides of the tRNA^Glu and tRNA^His genes, respectively. Detailed, consensus secondary-structure models were constructed for the mt-s-rRNA genes and the 3 64% of mt-1-rRNA genes of the two nematodes. The mt-s-rRNA secondary-structure model bears a remarkable resemblance to the previously defined universal core structure of E. coli 16S rRNA: most of the nucleotides that have been classified as variable or semiconserved in the E. coli model appear to have been eliminated from the C. elegans and A. suum sequences. Also, the secondary structure model constructed for the 3 64% of the mt-1-rRNA is similar to the corresponding portion of the previously defined E. coli 23S rRNA core secondary structure. The proposed C. elegans/A. suum mt-s-rRNA and mt-1-rRNA models include all of the secondary-structure element-forming sequences that in E. coli rRNAs contain nucleotides important for A-site and P-site (but not E-site) interactions with tRNAs. Sets of apparently homologous sequences within the mt-s-rRNA and mt-1-rRNA core structures, derived by alignment of the C. elegans and A. suum mt-rRNAs to the corresponding mt-rRNAs of other eukaryotes, and E. coli rRNAs were used in maximum-likelihood analyses. The patterns of divergence of metazoan phyla obtained show considerable agreement with the most prevalent metazoan divergence patterns derived from more classical, morphological, and developmental data.Correspondence to: D.R. Wolstenholme     

Caenorhabditis elegans and Ascaris suum: inhibition of isocitrate lyase by itaconate

The largest forms of isocitrate lyase from Caenorhabditis elegans and Ascaris suum of 543,000 and 549,000 daltons, respectively, can be purified from three- to five-fold in excellent yield by pelleting from extracts at 160,000g for 4 hr. Isocitrate lyase in the pellet is much more stable toward proteolysis. Itaconate which both inhibits isocitrate lyase and suppresses the level of this enzyme in bacteria inhibits the partially purified isocitrate lyase from both C. elegans and A. suum. The inhibition is noncompetitive with respect to d_s-isocitrate at one itaconate concentration. The K_i values at 30 C, pH 7.7, are 19 and 7.3 μM for the enzyme from C. elegans and A. suum, respectively. Itaconate inhibits the growth of C. elegans in random axenic as well as monoxenic cultures. At a concentration of 10 mM, itaconate is more effective in the inhibition of random axenic cultures than is oxalate, maleate, or succinate. At 60 mM itaconate, reproduction of C. elegans larvae is completely abolished.     

Caenorhabditis elegans and Ascaris suum: fragmentation of isocitrate lyase in crude extracts

It is well known that proteolysis often occurs after rupture of metazoan cells. Thus proteins isolated from extracts may not be representative of their native cellular counterparts. In the present research, extensive proteolysis was observed in crude extracts of the freeliving soil nematode Caenorhabditis elegans and the parasitic nematode Ascaris suum. Phenylmethylsulfonyl fluoride (PMSF) reduced the loss in activity of isocitrate lyase (EC 4.1.3.1), fumarase (EC 4.2.1.2), and citrate synthase (EC 4.1.3.7) in extracts of C. elegans but had little or no effect upon loss of malate synthase (EC 4.1.3.2). Catalase (EC 1.11.1.6) was stable. The loss of isocitrate lyase and citrate synthase was less pronounced in extracts of 22-day-old embryos of A. suum. Catalase decayed in these extracts. The addition of PMSF reduced the loss in all three of these activities. Fumarase was stable. The number of active fragments of isocitrate lyase recovered after filtration on Sephadex G-200 increased with the length of storage of crude extracts in the absence of PMSF at 4 C. Even in the presence of PMSF five activity peaks were observed after storage of extracts of C. elegans at 4 C for 72 hr. The molecular weights of active species ranged between 549,000 and 128,000 for isocitrate lyase in extracts of either C. elegans or A. suum. The 549,000- and 214,000-dalton species of isocitrate lyase from A. suum were much more labile at 50 C than the 543,000- and 195,000-dalton species from C. elegans.     

Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.     

Preparation of biologically active Ascaris suum mitochondrial tRNAMet with a TV-replacement loop by ligation of chemically synthesized RNA fragments.

Ascaris suum mitochondrial tRNA Met lacking the entire T stem was prepared by enzymatic ligation of two chemically synthesized RNA fragments. The synthetic tRNA could be charged with methionine by A.suum mitochondrial extract, although the charging activity was considerably low compared with that of the native tRNA, probably due to lack of modification. Enzymatic probing of the synthetic tRNA showed a very similar digestion pattern to that of the native tRNA Met, which has already been concluded to take an L-shape-like structure [Watanabe et al. (1994) J. Biol. Chem., 269, 22902-22906]. These results suggest that the synthetic tRNA possesses almost the same conformation as the native one, irrespective of the presence or absence of modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too much difficulty in the amounts necessary for physicochemical studies such as NMR spectroscopy.     

Effects of plumbagin on development of the parasitic nematodes Haemonchus contortus and Ascaris suum.

1. Plumbagin (5-hydroxy,2-methyl-1,4-napthoquinone) inhibited the motility and survival of Haemonchus contortus first-stage larvae (L1) with an ED50 of 1 microgram/ml, but was less effective in preventing the development of H. contortus to infective third-stage larvae in a faecal slurry assay. 2. Of the structural analogs tested, plumbagin was the most potent in preventing development of L1 followed in decreasing order of potency by 1,4-naphthoquinone, 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone. 3. Plumbagin had a biphasic effect on development of the fourth-stage Ascaris suum larvae that caused an increase in growth at low concentrations but was lethal at higher doses. 4. Plumbagin and 1,2-napthoquinone partially inhibited embryonation of A. suum eggs.     

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.     

A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum: MOLECULAR MODELING AND FUNCTIONAL PROPERTIES*

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O₂ with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.     

Effects of plumbagin on development of the parasitic nematodes Haemonchus contortus and Ascaris suum

1. Plumbagin (5-hydroxy,2-methyl-l,4-napthoquinone) inhibited the motility and survival of Haemonchus contonus first-stage larvae (L1) with an ed₅₀ of 1 μg/ml, but was less effective in preventing the development of H. contortus to infective third-stage larvae in a faecal slurry assay.2. Of the structural analogs tested, plumbagin was the most potent in preventing development of L1 followed in decreasing order of potency by 1,4-napthoquinone, 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone.3. Plumbagin had a biphasic effect on development of the fourth-stage Ascaris suum larvae that caused an increase in growth at low concentrations but was lethal at higher doses.4. Plumbagin and 1,2-napthoquinone partially inhibited embryonation of A. suum eggs.     

Transposable elements, mutational correlations, and population divergence in Caenorhabditis elegans

Transposable element activity is thought to be responsible for a large portion of all mutations, but its influence on the evolution of populations has not been well studied. Using mutation accumulation experiments with the nematode Caenorhabditis elegans, we investigated the impact of transposable element activity on the production of mutational variances and covariances. The experiments involved the use of two mutator strains (RNAi-deficient mutants) that are characterized by high levels of germline transposition, as well as the Bristol N2 strain, which lacks germline transposition. We found that transposition led to an increase in mutational heritabilities, as well as to the intensification of correlation patterns observed in the absence of transposition. No mutational trade-offs were detected and mutations generally had a deleterious effect on components of fitness. We also tested whether the pattern of mutational covariation could be used to predict observed patterns of population divergence in this species. Using 15 natural populations, we found that population divergence of C. elegans in multivariate phenotypic space occurred in directions only partially concordant with mutation, and thus other evolutionary factors, such as natural selection and genetic drift, must be acting to produce divergence within this species. Our results suggest that mutations induced by mobile elements in C. elegans are similar to other spontaneous mutations with respect to their contribution to the microevolution of quantitative traits.     

Detection of deletions in the mitochondrial genome of Caenorhabditis elegans.

We have examined an aging population of Caenorhabditis elegans via a PCR assay to determine if deletions in the mitochondrial genome occur in the nematode. We detected eight such deletions, identified the breakpoints of four of these, and discovered direct repeats of 4-8 base pairs at the site of all four deletions. Six of the eight repeats involved in the deletions are located in or immediately adjacent to tRNAs. Without a biochemical bias, the probability of direct repeats being present at all four breakpoints was 4 x 10(-6).     

Rhodoquinone reaction site of mitochondrial complex I, in parasitic helminth, Ascaris suum

The components and organization of the respiratory chain in helminth mitochondria vary remarkably depending upon the stage of the life cycle. Mitochondrial complex I in the parasitic helminth Ascaris suum uses ubiquinone-9 (UQ(9)) and rhodoquinone-9 (RQ(9)) under aerobic and anaerobic conditions, respectively. In this study, we investigated structural features of the quinone reduction site of A. suum complex I using a series of quinazoline-type inhibitors and also by the kinetic analysis of rhodoquinone-2 (RQ(2)) and ubiquinone-2 (UQ(2)) reduction. Structure-activity profiles of the inhibition by quinazolines were comparable, but not completely identical, between NADH-RQ(2) and NADH-UQ(2) oxidoreductase activities. However, the inhibitory mechanism of quinazolines was competitive and partially competitive against RQ(2) and UQ(2), respectively. The pH profiles of both activities differed remarkably; NADH-RQ(2) oxidoreductase activity showed an optimum pH at 7.6, whereas NADH-UQ(2) oxidoreductase activity showed two optima pH at 6.4 and 7.2. Our results indicate that although A. suum complex I uses both RQ(2) and UQ(2) as an electron acceptor, the manner of reaction (or binding) of the two quinones differs.     

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.     

Divergent structures of Caenorhabditis elegans cytochrome P450 genes suggest the frequent loss and gain of introns during the evolution of nematodes

The Caenorhabditis elegans genome contains more than 60 cytochrome P450 (CYP) genes. The exon-intron organizations of all of the available and potentially active C. elegans CYP genes were inferred by a newly developed program for predicting protein-coding exons based on the alignment of a genomic DNA sequence and a protein profile. From the predicted amino acid sequences, all of the C. elegans CYP genes except one were classified into three groups, which were closely related to the mammalian drug-metabolizing P450 gene families CYP2, CYP3, and CYP4. The gene structures were strikingly divergent within each group; 20, 10, and 5 unique gene organizations were identified among 40, 18, and 5 genes in the CYP2-, CYP3-, and CYP4-related groups, respectively. The degrees of divergence in gene organization were strongly correlated with those in the amino acid sequences of encoding proteins, and the minimum rate of change in an intron insertion site was estimated to be about 90 times less frequent than amino acid substitutions. Parsimonious analyses suggested that frequent loss and gain of introns has occurred during the evolution of CYP genes in each group after the divergence of nematodes, arthropods, and deuterostomia. Few, if any, incidents of intron sliding were evident, and a model that did not allow intron insertions was highly inconsistent with the observations. All of these findings are explained better by the intron-late view than by the intron-early view.     

Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b5 in parasitic adaptation

We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b₅, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b₅ and the canonical cytochrome b₅ proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b₅-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b₅ in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b₅, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b₅ precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b₅ and highlight the crucial function of this A. suum adult-specific secretory cytochrome b₅ in parasitic adaptation.