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PubMed2004

mRNA sequences for Haemonchus contortus intestinal cathepsin B-like cysteine proteases display an extreme in abundance and diversity compared with other adult mammalian parasitic nematodes.

Cathepsin B-like cysteine protease (cbl) genes produce the most abundant mRNAs ( approximately 16%) detected in the adult female intestine of the parasitic nematode Haemonchus contortus. CBL enzymes appear to digest host proteins and are vaccine candidates for immune control of H. contortus and potentially other parasitic nematodes. Hence, it is important to quantify the extent of diversity of H. contortuscbl genes. Here, expressed sequence tags (ESTs) were used to assess both the size and diversity of the H. contortuscbl gene family. Contig analysis of 686 cbl ESTs from a USA isolate resolved 123 clusters. ESTs were grouped into discrete sets and analyzed using an additive model. Discovery of new cbl clusters increased with each set and reached a terminal rate of about 1 per 10 ESTs. The extreme diversity was unique to cbls relative to other genes investigated and was ascribed to specific cbl clades. Sixty percent of cbl clusters from a UK isolate were shared with those identified in the USA isolate, suggesting conservation of cbl gene repertoires across regions, although minor to moderate geographic variation cannot be excluded. Sequence comparisons also suggested high potential for antigenic diversity among CBL proteins, which is relevant to vaccine strategies. Compared to other parasitic nematodes of mammals, the extreme abundance and diversity of intestinal cbl transcripts appear to be relative specializations for H. contortus. Therefore, adaptations related to nutrient acquisition may vary markedly among these parasitic nematodes.

Amino Acid Sequence↗
PubMed1998

Secondary structure of some elements of 18S rRNA suggests that strongylid and a part of rhabditid nematodes are monophyletic.

Analysis of the secondary structure of 18S rRNA molecules in nematodes revealed some new traits in the secondary structure peculiar to their hairpin 17. Some of them are characteristic of all the nematodes, whereas others are characteristic exclusively of the order Rhabditida. The loss of a nucleotide pair in the highly conservative region of hairpin 17 distinguishes 18S rRNA of the Strongylida and some species of the Rhabditida from other nematodes and, moreover, from all other organisms. Hence, it is possible to regard the Strongylida and a part of the Rhabditida including Caenorhabditis elegans as a new monophyletic taxon.

Animals↗
PubMed1996

cul-1 is required for cell cycle exit in C. elegans and identifies a novel gene family.

The gene cul-1 (formerly lin-19) is a negative regulator of the cell cycle in C. elegans. Null mutations cause hyperplasia of all tissues. cul-1 is required for developmentally programmed transitions from the G1 phase of the cell cycle to the GO phase or the apoptotic pathway. Moreover, the mutant phenotype suggests that G1-to-S phase progression is accelerated, overriding mechanisms for mitotic arrest and producing abnormally small cells. Significantly, diverse aspects of cell fate and differentiation are unaffected in cul-1 mutants. cul-1 represents a conserved family of genes, designated cullins, with at least five members in nematodes, six in humans, and three in budding yeast.

Amino Acid Sequence↗
PubMed2000

Mitochondrial genetic code in cestodes.

The flatworm mitochondrial genetic code, which has been used for all species of the Platyhelminthes, is mainly characterized by AUA codon for isoleucine, AAA codon for asparagine and UAA codon for tyrosine. In eight species of cestodes (Echinococcus multilocularis, Echinococcus granlosus, Taenia solium Taenia saginata, Taenia hydatigena, Taenia crassiceps, Hymenolepis nama and Mesocestoides corti), the cytochrome c oxidase subunit I (COI) genes were partially sequenced to verify this genetic code. Comparison of the COI-encoding nucleotide sequences with those of human, sea urchin, fruit fly, nematode and yeast indicated that the assignments of AUA and AAA codons are adequate for cestodes. In addition, the nucleotide sequences of ATPase subunit 6 (ATP6) gene and its flanking region were compared to examine initiation and stop codons. In the related species of T. solium and T. saginata, the deduced amino acid sequences of ATP6 were homogeneous; however, the conversion of initiation codon AUG into GUG was observed in T. saginata. We also found the similar conversion in T. crassiceps. The C-terminal sequences of putative ATP6 proteins were highly conserved among the eight species and the stop codon UAG was altered to UAA in all Taenia species. The features of the gene-junctional region between NADH dehydrogenase subunit 4 (ND4) and glutamine tRNA (tRNAGln) genes also supported that UAA serves as a stop codon. Based on these results, we propose that the flatworm mitochondrial code should be modified for cestodes, particularly, in an initiating methionine codon (GUG) and a terminating codon (UAA).

Animals↗
PubMed1997

Characterization of a schistosome T cell-stimulating antigen (Sm10) associated with protective immunity in humans.

Parasite antigens that are strong T cell immunogens represent potential candidates for vaccines against pathogens susceptible to T cell-mediated immunity. We have previously shown that chromatographic fractions of schistosomula extracts contain components that are major T cell immunogen(s) in natural schistosome infections in humans and might contribute to the induction of human protective immunity against this parasite. In the present study, we report on the molecular cloning and on the biochemical characterization of the active components of these fractions. The screening of a schistosomula cDNA expression library with antibodies raised against the fractions allowed the cloning of a cDNA that hybridized to a 0.56-kb mRNA of schistosomula and adult worms. This cDNA contains an open reading frame of 267 base pairs (bp) which encodes a 10-kDa polypeptide. The analysis of the cDNA sequence revealed 70% homology with the sequences of previously reported proteins of unknown function. The native molecules in the active fractions were analyzed by mass spectrometry after additional purification by reverse phase high-performance liquid chromatography (HPLC). This procedure revealed two components in the fractions of molecular mass 10383 +/- 2 Da and 10401 +/- 9 Da. Both polypeptides stimulated immune T cells and yielded tryptic peptides whose sequences matched the sequence of the cloned molecule. These two polypeptides probably correspond to different post-translationally modified forms of the polypeptide encoded by the cloned cDNA.

Amino Acid Sequence↗
PubMed2002

On the systematic position of Ophiosacculus Macy, 1935 (digenea: Lecithodendriidae), with the erection of the Ophiosacculinae n. subfam.

The phylogenetic relationships and systematic position of the digenean genus Ophiosacculus Macy, 1935 has been controversial and opinions of different authors on its systematic position and content are contradictory. Molecular analysis based on the partial sequences of the large subunit ribosomal DNA gene of the type and only valid species of the genus, Ophiosacculus mehelyi (Mödlinger, 1930), as well as previously published sequences of members of several families of Plagiorchiata (including the Allassogonoporidae, Lecithodendriidae and Pleurogenidae as potential relatives of Ophiosacculus) has shown that Ophiosacculus forms a clade with the typical representatives of the Lecithodendriidae from bats. Ophiosacculus is basal to the cluster containing Lecithodendrium, Prosthodendrium and Pycnoporus and has quite pronounced differences in the sequenced fragment compared to these genera. Based on the results of the molecular study, morphological characteristics of Ophiosacculus (in particular, possession of a seminal vesicle lying freely in parenchyma) and the fact that the type-specimen of Gyrabascus brevigastrus Macy, 1835 (type-species of the monotypic genus Gyrabascus and type-genus of the subfamily Gyrabascinae) belongs to Allassogonoporus, a new subfamily, the Ophiosacculinae, with Ophiosacculus as the type-genus, is established within the Lecithodendriidae. Molecular study did not support a close phylogenetic relationship between Allassogonoporus and Ophiosacculus, although several authors previously allocated both these genera to the Allassogonoporidae. Morphological study revealed the position of the genital pore in O. mehelyi to be at the posterior margin of the ventral sucker. An amended diagnosis of Ophiosacculus and a diagnosis of Ophiosacculinae n. subfam. are given.

Animals↗
PubMed1994

The Caenorhabditis elegans muscle-affecting gene unc-87 encodes a novel thin filament-associated protein.

Mutations in the unc-87 gene of Caenorhabditis elegans affect the structure and function of bodywall muscle, resulting in variable paralysis. We cloned the unc-87 gene by taking advantage of a transposon-induced allele of unc-87 and the correspondence of the genetic and physical maps in C. elegans. A genomic clone was isolated that alleviates the mutant phenotype when introduced into unc-87 mutants. Sequence analysis of a corresponding cDNA clone predicts a 357-amino acid, 40-kD protein that is similar to portions of the vertebrate smooth muscle proteins calponin and SM22 alpha, the Drosophila muscle protein mp20, the deduced product of the C. elegans cDNA cm7g3, and the rat neuronal protein np25. Analysis of the genomic sequence and of various transcripts represented in a cDNA library suggest that unc-87 mRNAs are subject to alternative splicing. Immunohistochemistry of wildtype and mutant animals with antibodies to an unc-87 fusion protein indicates that the gene product is localized to the I-band of bodywall muscle. Studies of the UNC-87 protein in other muscle mutants suggest that the unc-87 gene product associates with thin filaments, in a manner that does not depend on the presence of the thin filament protein tropomyosin.

Actin Cytoskeleton↗
PubMed2004

Translation repression by GLD-1 protects its mRNA targets from nonsense-mediated mRNA decay in C. elegans.

Previously, we identified multiple in vivo mRNA targets of the maxi-KH/STAR domain protein GLD-1 by their ability to interact with GLD-1 in cytoplasmic extracts and, for all targets tested thus far, GLD-1 functions as a translational repressor. However, here we show that GLD-1 stabilizes the mRNAs of two targets, gna-2 (T23G11.2) and Y75B12B.1. gna-2 mRNA has two upstream open reading frames (uORF), resulting in two premature stop codons. We found that gna-2 mRNA is a naturally occurring mRNA target of nonsense-mediated mRNA decay (NMD) and that the binding of GLD-1 protects gna-2 mRNA from NMD, likely by repressing translation of the uORFs. Therefore, gna-2 mRNA comes under two posttranscriptional controls: (1) translation regulation by a specific translational repressor, GLD-1; and (2) uORF elicited regulation, mainly through NMD. As a result, these two posttranscriptional controls together provide precise temporal and spatial control of gene expression. Consistent with this novel mode of regulation, when GLD-1 mRNA targets acquire premature stop codon mutations, GLD-1 protects them from NMD. Analysis of several mRNA targets containing premature stop codons suggests that in translation repression, GLD-1 either represses ribosome assembly on the target mRNA, or subsequent ribosome elongation to the premature stop codon.

3' Untranslated Regions↗
PubMed1995

The Caenorhabditis elegans spe-26 gene is necessary to form spermatids and encodes a protein similar to the actin-associated proteins kelch and scruin.

Six independent mutations in the Caenorhabditis elegans spe-26 gene cause sterility in males and hermaphrodites by disrupting spermatogenesis. Spermatocytes in mutants with the most severe alleles fail to complete meiosis and do not form haploid spermatids. Instead, these spermatocytes arrest with missegregated chromosomes and mislocalized actin filaments, endoplasmic reticulum and ribosomes. In spite of this arrest some of the nuclei and the organelles that normally transport sperm-specific components to the spermatid mature as if they were in spermatids. The spe-26 gene is expressed throughout the testis in both spermatogonial cells and spermatocytes. It encodes a 570-amino-acid polypeptide, which contains five tandem repeat motifs, each of approximately 50 amino acids. These repeats are similar in sequence to repeats in the Drosophila kelch protein, in the invertebrate sperm protein scruin that cross-links actin filaments, as well as in the mouse and pox virus proteins. The functional importance of these repeat motifs is shown by the fact that five of the spe-26 mutations are in the tandem repeats, and one of the most severe mutations is a substitution in a highly conserved glycine. These results suggest that spe-26 encodes a cytoskeletal protein, perhaps actin binding, which is necessary to segregate the cellular components that form haploid spermatids.

Actins↗
PubMed1999

SMG-2 is a phosphorylated protein required for mRNA surveillance in Caenorhabditis elegans and related to Upf1p of yeast.

mRNAs that contain premature stop codons are selectively degraded in all eukaryotes tested, a phenomenon termed "nonsense-mediated mRNA decay" (NMD) or "mRNA surveillance." NMD may function to eliminate aberrant mRNAs so that they are not translated, because such mRNAs might encode deleterious polypeptide fragments. In both yeasts and nematodes, NMD is a nonessential system. Mutations affecting three yeast UPF genes or seven nematode smg genes eliminate NMD. We report here the molecular analysis of smg-2 of Caenorhabditis elegans. smg-2 is homologous to UPF1 of yeast and to RENT1 (also called HUPF1), a human gene likely involved in NMD. The striking conservation of SMG-2, Upf1p, and RENT1/HUPF1 in both sequence and function suggests that NMD is an ancient system, predating the divergence of most eukaryotes. Despite similarities in the sequences of SMG-2 and Upf1p, expression of Upf1p in C. elegans does not rescue smg-2 mutants. We have prepared anti-SMG-2 polyclonal antibodies and identified SMG-2 on Western blots. SMG-2 is phosphorylated, and mutations of the six other smg genes influence the state of SMG-2 phosphorylation. In smg-1, smg-3, and smg-4 mutants, phosphorylation of SMG-2 was not detected. In smg-5, smg-6, and smg-7 mutants, a phosphorylated isoform of SMG-2 accumulated to abnormally high levels. In smg-2(r866) and smg-2(r895) mutants, which harbor single amino acid substitutions of the SMG-2 nucleotide binding site, phosphorylated SMG-2 accumulated to abnormally high levels, similar to those observed in smg-5, smg-6, and smg-7 mutants. We discuss these results with regard to the in vivo functions of SMG-2 and NMD.

Alleles↗
PubMed1999

Contributions to the phylogeny of the Cyclophyllidea (Cestoda) inferred from mitochondrial 12S rDNA.

A 314-bp fragment of the mitochondrial 12S rRNA gene from 21 cestodes species of eight families was synthesized by PCR with specially designed primers. These allowed amplification of parasite DNA without concomitant synthesis of host DNA. Phylogenetic trees were inferred from the sequence data using three methods (maximum parsimony, maximum likelihood, and Fitch-Margoliash). At the major nodes all three trees were similar. For the first time the genus Mesocestoides could be arranged into the Cyclophyllidea and a narrow relationship between the Mesocestoididae, Taeniidae, Hymenolepididae, Anoplocephalidae, and Dipylidiidae was shown. Members of the families Catenotaeniidae and a cluster of two families (Hymenolepididae and Dilepididae) form two monophyletic groups which derive prior to the remaining families of this phylogenetic study. A third and a fourth clear monophyletic group were formed by the Taeniidae and by the Mesocestoididae. A high degree of variation within the examined 304-bp fragment was observed between two isolates of Taenia taeniaeformis, supporting often discussed genetic heterogeneity within this species. In contrast, only one nucleotide exchange was found in 23 isolates of Echinococcus multilocularis of various geographic origin, indicating that this species is genetically homogenous.

Animals↗
PubMed1996

Cloning and characterization of cDNAs encoding putative glutamate transporters from Caenorhabditis elegans and Onchocerca volvulus.

We report the identification and partial characterization of cDNAs encoding for putative glutamate transporters from the free-living nematode Caenorhabditis elegans and the filarial parasite Onchocerca volvulus. Glutamate transporters can be used as reliable markers for identifying cells and neurons that synaptically release glutamate and aspartate. An amplified PCR fragment containing a highly conserved amino acid heptamer found in all vertebrate glutamate transporters was used to screen a C. elegans cDNA library. Two full-length cDNA sequences from C. elegans were deduced from the isolated cDNA clones and RT-PCR products with the splice leader. The two C. elegans cDNA sequences differ by only 97 nucleotides at the 5' end. The C. elegans glutamate transporter gene glt-1 spans at least 2.9 kb of chromosomal DNA and possesses nine exons and eight introns. Primers directed to the CeGlt cDNA were used with O. volvulus first-strand cDNA to amplify and isolate the O. volvulus cDNA homolog. The C. elegans and O. volvulus glutamate transporters are 98% identical over 492 amino acids to each other and 52 to 58% identical to the mammalian glutamate transporters. Antibodies generated against partial coding regions of the C. elegans glutamate transporter recognized a protein of approximately 66 kDa in C. elegans and O. volvulus protein extracts.

ATP-Binding Cassette Transporters↗
PubMed1998

Common secondary structures for the second internal transcribed spacer pre-rRNA of two subfamilies of trichostrongylid nematodes.

Sequences of the second internal transcribed spacer ribosomal DNA for the parasitic trichostrongylid nematodes Trichostrongylus probolurus, Trichostrongylus rugatus and Camelostrongylus mentulatus were compared with previously published sequences for five other species within the genus Trichostrongylus. The secondary structures of the second internal transcribed spacer pre-rRNA for these nematodes were predicted using an energy minimisation method. The results indicate that a common secondary structure of the second internal transcribed spacer of these nematodes is maintained despite distinct differences in primary sequence between species. Sequence differences among Trichostrongylus species ranged from 1.3 to 7.6%, but each species differed by 22-26% in sequence when compared with C. mentulatus which belongs to a different subfamily.

Animals↗
PubMed1998

Candidate adaptor protein CED-6 promotes the engulfment of apoptotic cells in C. elegans.

The rapid engulfment (phagocytosis) of cells undergoing programmed cell death (apoptosis) is a fundamental biological process that is not well understood. Here we report the cloning and functional characterization of ced-6, a gene specifically required for the engulfment of apoptotic cells in the nematode C. elegans. The CED-6 protein contains a phosphotyrosine binding domain at its N terminus and a proline/serine-rich region in its C-terminal half. Genetic mosaic analysis demonstrates that ced-6 acts within engulfing cells. We also show that ced-6 can promote the engulfment of cells at both early and late stages of apoptosis. Our data suggest that CED-6 is an adaptor molecule acting in a signal transduction pathway that specifically mediates the recognition and engulfment of apoptotic cells.

Amino Acid Sequence↗
PubMed1999

Gender-specific gene expression in Brugia malayi.

Brugia malayi is a mosquito-borne filarial nematode that causes lymphatic filariasis and elephantiasis in humans. The purpose of this study was to identify and characterize genes that are expressed differentially in male and female B. malayi in hopes of gaining new insight into the reproductive biology of the parasite. Two approaches were used. A 5' differential display PCR (splice leader differential display PCR, SL DD-PCR) was performed by PCR with splice leader and random primers on cDNA templates, and electronic subtraction was performed on expressed sequence tag (EST) cluster databases developed by the Filarial Genome Project (FGP). Gender-specific expression of candidate clones was confirmed by RT-PCR for six of 22 (27%) clones identified by DD and in seven of 15 (47%) clones identified by electronic subtraction. One clone was identified by both methods. Several female-specific clones had homology to known nematode genes that encode a fatty acid binding protein, a high mobility group protein, an eggshell protein, a glutamate-gated ion channel, and a collagen. However, most of the clones have no significant homology to known genes or proteins in computer databases. This project has confirmed the value of SL DD-PCR and electronic subtraction for analysis of gene expression in filariae. These two complimentary techniques may be generally applicable to the study of gender-specific (and by analogy stage specific) gene expression in other nematodes.

Animals↗
PubMed2002

Expression profiles of the essential intermediate filament (IF) protein A2 and the IF protein C2 in the nematode Caenorhabditis elegans.

The multigene family of intermediate filament (IF) proteins in Caenorhabditis elegans covers 11 members of which four (A1-3, B1) are essential for development. Suppression of a fifth gene (C2) results in a dumpy phenotype. Expression patterns of three essential genes (A1, A3, B1) were already reported. To begin to analyze the two remaining RNAi phenotypes we followed the expression of the A2 and C2 proteins. Expression of A2 mRNA starts in larval stage L1 and continues in the adult. Transgenic A2 promoter/gfp larvae strongly display GFP in the main body hypodermis but not in seam cells. This pattern and the muscle displacement/paralysis induced by RNAi silencing are consistent with the role of this protein in keeping the correct hypodermis/muscle relationship during development. IF protein C2 occurs in the cytoplasm and desmosomes of intestinal cells and in pharynx desmosomes. Expression of C2 starts in the late embryo and persists in all further stages.

Animals↗
PubMed2003

The origin of the triploid in Paragonimus westermani on the basis of variable regions in the mitochondrial DNA.

Triploid, parthenogenetic forms of the lungfluke, Paragonimus westermani, occur in Japan, Korea and China. The origin(s) of triploidy has been debated over the years. Sequences of two regions in the mitochondrial DNA, i.e. partial lrRNA (16S), and a portion of the non-coding region, were obtained from natural populations of P. westermani. All triploid individuals (Japan, Korea, China) and a single tetraploid individual (China) had identical sequences in the 16S region studied. Some sequence variation was observed among diploids, with those from Taiwan being distinct from the remainder. Both neighbour joining and parsimony trees using the 16S region placed diploid individuals from southwestern Japan close to the triploids and the tetraploid. The fragment amplified from the mitochondrial non-coding region showed dimorphism. One form (type A) consisted of 239 bp comprising two identical tracts of 70 bp separated by a tract of 93 bp. The second form (Type B) consisted of only a single 70 bp tract. All diploid individuals from Taiwan, China and Korea possessed type A, while those from Japan were polymorphic; individuals from Oita and Hyogo had type B, those from Chiba had type A, but both types were found in Mie. On the other hand, all of the triploid individuals and two tetraploid individuals possessed type B. Both the form present in the non-coding region and the 16S sequence suggest an affinity between a south-eastern group of diploid populations in Japan and the triploid form. A possible mechanism responsible for the origin of the triploid is discussed.

Animals↗