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At least 19 recordsLinked to original sources
PubMed2004

RNA interference in protozoan parasites.

RNA interference or RNAi is defined as the mechanism through which gene-specific, double-stranded RNA (dsRNA) triggers degradation of homologous transcripts. Besides providing an invaluable tool to downregulate gene expression in a variety of organisms, it is now evident that RNAi extends its tentacles into both the nucleus and the cytoplasm and is involved in a variety of gene silencing phenomena. Here we review the current status of RNAi in protozoan parasites that cause diseases of considerable medical and veterinary importance throughout Africa, Asia and the Americas. RNAi was first discovered in Trypanosoma brucei, a species of the family Trypanosomatidae, and it rapidly became the method of choice to downregulate gene expression in these organisms. At the same time, mechanistic studies exposed a role for RNAi in the control of retroposon transcript abundance. Whereas RNAi is also present in T. congolense, other members of the same family of organisms, namely T. cruzi and Leishmania major, are RNAi-negative. In apicomplexan parasites, there is experimental evidence for RNAi in Plasmodium, but this is not supported by their genetic make up. In contrast, the genome of Toxoplasma gondii harbours gene candidates with convincing similarity to 'classical' RNAi genes. Thus, as previously shown in fungi, protozoan parasites are genetically heterogeneous as far as the RNAi pathway is concerned. Finally, database mining predicts that Entamoeba histolytica and Giardia intestinalis have an RNAi pathway and the presence of RNAi genes in Giardia supports the view that gene silencing by dsRNA appeared very early during evolution of the eukaryotic lineage.

Animals↗
PubMed2006

Structure-function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins.

RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name "triphosphate tunnel metalloenzyme," is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5'-end processing.

Acid Anhydride Hydrolases↗
PubMed2006

Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes.

We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected approximately 4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to alpha-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene.

Amanitins↗
PubMed2005

Microsporidia: emerging ocular pathogens.

Microsporidia are eukaryotic, spore forming obligate intracellular parasites, first recognized over 100 years ago. Microsporidia are becoming increasingly recognized as infectious pathogens causing intestinal, ocular, sinus, pulmonary, muscular and renal diseases, in both immunocompetent and immunosuppressed patients. Ocular microsporidiosis, though uncommon, could be isolated or part of systemic infections. It occurs mainly in two forms: keratoconjunctivitis form, mostly seen in immunocompromised individuals; stromal keratitis form seen in immunocompetent individuals. Recent reports indicate increasing number of cases of ocular microsporidiosis in immunocompetent individuals. The ocular cases present as superficial keratitis in AIDS patients, and these differ in presentation and clinical course from the cases seen in immunocompetent individuals which mainly appear to be as deep stromal keratitis. For most patients with infectious diseases, microbiological isolation and identification techniques offer the most rapid and specific determination of the etiologic agent, however this does not hold true for microsporidia, which are obligate intracellular parasites requiring cell culture systems for growth. Therefore, the diagnosis of microsporidiosis currently depends on morphological demonstration of the organisms themselves, either in scrapings or tissues. Although the diagnosis of microsporidiosis and identification of microsporidia by light microscopy have greatly improved during the last few years, species differentiation by these techniques is usually impossible and electron microscopy may be necessary. Immuno fluorescent-staining techniques have been developed for species differentiation of microsporidia, but the antibodies used in these procedures are available only at research laboratories at present. During the last 10 years, molecular techniques have been developed for the detection and species differentiation of microsporidia.

Americas↗
PubMed2006

Ultrastructural and molecular characterization of Haplosporidium montforti n. sp., parasite of the European abalone Haliotis tuberculata.

A new member of the parasitic phylum Haplosporidia, which was found infecting the connective tissue, gill, digestive gland, and foot muscle of Haliotis tuberculata imported from Ireland and experimentally grown in Galicia (NW Spain), is described. Scanning electron microscopy, transmission electron microscopy, and molecular characterization of the small subunit ribosomal RNA (SSU rRNA) gene were carried out to confirm the description of this species. The ultrastructural morphology of the spores and their surrounding ornaments attached to the spore wall was described from light, scanning, and transmission electron microscopy observations. Systemic infection with uninucleated and multinucleated plasmodia containing spherical nuclei was observed among several sporocysts containing the different spore maturation stages. The spores were spherical to slightly ellipsoidal (2.42 +/- 0.5 x 2.31 +/- 0.6 microm). The apical zone of the spore wall was modified into a complex opercular system covering a circular orifice that measured about 0.5 microm in diameter. The operculum was connected to the spore wall by a hinge. The spore wall was about 110 nm thick, with 4 filaments (20-28 microm long). The filaments were composed of the same material that formed the wall. The cross-sections through the base of these filaments showed T-like and X-like sections. Internally, the uninucleated endosporoplasm contained typical haplosporidian structures, such as, haplosporosomes, a spherulosome, and mitochondria with vesicular cristae. The SSU rRNA gene sequence was different from previously reported haplosporidian SSU rRNA gene sequences, corroborating morphological data that this was an undescribed species. Based on differences from previously described haplosporidians in ultrastructural characteristics of the spore and SSU rRNA gene sequence, we describe the abalone haplosporidian as Haplosporidium montforti n. sp.

Animals↗
PubMed2001

Genotyping Cryptosporidium parvum by single-strand conformation polymorphism analysis of ribosomal and heat shock gene regions.

Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.

Animals↗
PubMed1995

Relative orientation of RNA helices in a group 1 ribozyme determined by helix extension electron microscopy.

The relative orientation of helical elements in a folded RNA molecule provides key information about its three-dimensional architecture. We have developed a method that involves extending peripheral helices of an RNA, mounting for electron microscopy in the absence of protein and measuring interhelical angles. As a control, extended anticodon and acceptor stems of tRNA(Phe) were found to form a 92 +/- 20 degrees angle, consistent with the X-ray structure. Single, double and triple extensions (50-80 bp) of helical elements P2.1, P6b and P8 of the Tetrahymena group I ribozyme did not alter its catalytic activity. The measured angle between P6b and P8 is consistent with the Michel-Westhof structural model, while the P2.1-P6b and P2.1-P8 angles allow P2.1 to be positioned in the model. The angle distributions of the ribozyme are broader than those of the tRNA, which may reflect the dynamics of the RNA. Helix extension allows low-resolution electron microscopy to provide much higher resolution information about the disposition of helical elements in RNA. It should be applicable to diverse RNAs and ribonucleoprotein complexes.

Animals↗
PubMed1995

Genetic code deviations in the ciliates: evidence for multiple and independent events.

In several species of ciliates, the universal stop codons UAA and UAG are translated into glutamine, while in the euplotids, the glutamine codon usage is normal, but UGA appears to be translated as cysteine. Because the emerging position of this monophyletic group in the eukaryotic lineage is relatively late, this deviant genetic code represents a derived state of the universal code. The question is therefore raised as to how these changes arose within the evolutionary pathways of the phylum. Here, we have investigated the presence of stop codons in alpha tubulin and/or phosphoglycerate kinase gene coding sequences from diverse species of ciliates scattered over the phylogenetic tree constructed from 28S rRNA sequences. In our data set, when deviations occur they correspond to in frame UAA and UAG coding for glutamine. By combining these new data with those previously reported, we show that (i) utilization of UAA and UAG codons occurs to different extents between, but also within, the different classes of ciliates and (ii) the resulting phylogenetic pattern of deviations from the universal code cannot be accounted for by a scenario involving a single transition to the unusual code. Thus, contrary to expectations, deviations from the universal genetic code have arisen independently several times within the phylum.

Animals↗
PubMed1996

Localization of the newly initiated and processed ribosomal primary transcripts during the mitotic cycle in Physarum polycephalum.

We analyzed the fate of the rRNA released as a result of the prophasic disintegration of the nucleolus, during the "closed" mitosis of the naturally synchronous plasmodium of Physarum polycephalum. Using a probe complementary to the mature 19S and 26S rRNA, we previously showed that the nucleolus-derived rRNAs are stable in mitosis, mainly associated with numerous fibrillar nucleolar remnants (Pierron and Puvion-Dutilleul, 1993, Exp. Cell Res. 208, 509-517). However, a significant fraction of these mitotic rRNA precursors were also found in more diffuse nuclear, granular zones. In this paper, we trace pre-rRNA molecules in the early stages of their maturation by using probes derived from the 5' external transcribed spacer (5'-ETS), upstream and downstream of the processing site located 1.7 kb from the transcription initiation site. In agreement with a previous S1-mapping study (Blum et al., 1986, Nucleic Acids Res., 10, 4121-4133), we observed that the rRNA transcripts complementary to the 5'-ETS portion downstream of the processing site are stable in mitosis, and we demonstrate that they are specifically associated with the border of the fibrillar nucleolar remnants. On the other hand, the pre-rRNA chains containing the upstream portion of the 5'-ETS become undetectable in mitosis, marking the cessation of rDNA transcription. Since the reappearance of these nascent pre-rRNA molecules signals the resumption of the transcriptional activity, we determined that a burst of rRNA synthesis is taking place within the prenucleolar bodies, immediately after the separation of the daughter nuclei. Taken together, our results illustrate the persistence of the nucleolar components of Physarum, within nucleolar remnants that, although transcriptionally inactive during mitosis, function as independent entities very early in interphase.

Animals↗
PubMed1995

Plasmodium: genus-conserved primers for species identification and quantitation.

Stable RNAs have regions of primary sequence that are nearly identical in every member of the Plasmodium genus and not found in the host or in other common pathogens. Several "genus-conserved" sequences, which flank hypervariable regions, were identified within the small subunit ribosomal RNA of Plasmodium species. Primers based on these conserved sequences permit amplification of species- or possibly even strain-specific sequences from samples of unknown composition. As an example of this approach, sequences from the four human malaria species were successfully recovered from Giemsa-stained blood smears, including two different sequences for Plasmodium ovale (of 91.5% similarity). This type of information is useful for epidemiological and phylogenetic analysis of any malaria species. We show that amplification of rRNA-derived sequences behaves in a competitive fashion during the cycles of polymerase amplification and therefore target sequences from Plasmodium species are amplified in proportion to their abundance in the sample. There are several implications of this finding. (1) The proportion of different products resulting from amplification from samples with mixed infections is closely related to the proportion of infecting species. (2) Direct quantitation of parasite nucleic acids within a sample can be derived when known amounts of competitor RNA are added to the RT/PCR reaction. (3) Amplification of rRNA sequences, using genus-specific primers, allows one to monitor the development of the parasite in the mosquito.

Animals↗
PubMed1999

Carbon regulation of ribosomal genes in Neurospora crassa occurs by a mechanism which does not require Cre-1, the homologue of the Aspergillus carbon catabolite repressor, CreA.

Transcription of the ribosomal protein and 40S rRNA genes is coordinately regulated during steady state growth and carbon shifts in Neurospora crassa. Recognition sequences for the Aspergillus nidulans carbon catabolite repressor, CreA, overlap transcriptional elements of a 40S rRNA gene and the crp-2 ribosomal protein gene. They also occur in similar locations in the promoters of several other ribosomal protein genes. Substitutions encompassing the -74 and -167 CreA consensus sequences in the crp-2 promoter result in a decrease in transcription. A cDNA encoding the N. crassa homologue of CreA was cloned and designated Cre-1. The Cre-1 protein is 45% identical to CreA from A. nidulans. Cre-1 protein produced in Escherichia coli binds to the CreA sites in the promoters of the 40S rRNA and crp-2 genes. An amino acid change from histidine (92) to threonine changed the Cre-1 binding specificity from (5'G/CC/TGGG/AG3') to (5'G/CC/TGGCG3'). Base substitutions in the Cre-1 binding sites of the crp-2 promoter disrupted binding of wildtype Cre-1 in vitro but had no effect on transcription during steady state growth or carbon shifts, indicating that regulation of ribosomal genes by carbon source is not mediated by Cre-1, but via different proteins binding the Cre-1 sites and the Dde boxes.

Amino Acid Sequence↗
PubMed1996

Antibiotic-induced oligomerisation of group I intron RNA.

Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecular capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to react in trans instead of in cis. The phage T4-derived td intron uses the same sites for oligomerisation as for circularisation. Gel- retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA-RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derived td intron with the Tetrahymena rRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. The td intron attacks the Tetrahymena intron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA-RNA interactions in a pre-protein era.

Animals↗
PubMed2000

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs.

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.

Animals↗
PubMed2001

Trypanosoma brucei 5'ETS A'-cleavage is directed by 3'-adjacent sequences, but not two U3 snoRNA-binding elements, which are all required for subsequent pre-small subunit rRNA processing events.

Trypanosoma brucei pre-rRNA processing commences by cleavage near the 5' end of 5.8 S sequences. The 5' external transcribed spacer (5'ETS) is removed from pre-small subunit (SSU) rRNAs by sequential cleavages at internal A' and A0 sites, and A1 at the 5' end of SSU rRNA. The A' and A0 sites positionally resemble the U3 small nucleolar RNA-dependent, primary pre-rRNA cleavages of vertebrates and yeast, respectively. Uniquely in T. brucei, two U3-crosslinkable 5'ETS sites are essential for SSU rRNA production: site1b is novel in its 3' location to the A' site, and site3 lies upstream of A0 in a position analogous to the yeast U3-binding site. Here, in vivo analysis of mutated 5'ETS sequences shows that sequences 5' to the A' site are not needed for A' cleavage or SSU rRNA production. A' cleavage is linked to, but is not sufficient to trigger, downstream pre-SSU rRNA processing events. These events require an intact 11 nt sequence, 3'-adjacent to A', which directs efficient and accurate A' cleavage. Neither the A' nearby site1b nor the site3 U3-binding elements affect A' processing, yet each is required for A0 and A1 cleavage, and SSU rRNA production. The same U3 3' hinge bases evidently bind a core element, UGUu/gGGU, within site1a and site3; the U3-site1b interaction is less reliant on base-pairing than the U3-site3 interaction. As yeast U3 5' hinge bases pair to 5'ETS sequences, it is clear that distinct U3 hinge regions can interact at both novel and related 5'ETS sites to promote 3'-proximal 5'ETS processing events in diverse organisms. The T. brucei data fit a model wherein processing factors assemble at the 5'ETS site1a to affect A' cleavage and stabilize a U3-site1b complex, which may work in concert with the downstream U3-site3 complex to assist processing events leading to ribosomal SSU production.

Animals↗
PubMedDate not supplied

Queuosine modification of tRNA: a case for convergent evolution.

Queuosine is a hypermodified nucleoside found in position 34, the anticodon wobble position, of four tRNA species. This modification is distributed with near uniformity across all life forms found on this planet. Yet the molecular mechanisms involved with accomplishing this ubiquitous posttranscriptional modification of tRNA are dramatically different between prokaryotic and eukaryotic organisms, which suggests that these were formed by convergent evolution of a fundamental life process essential to nearly all life forms. This minireview describes the differences between these modification systems and points to a new direction for developing research on the molecular function queuosine-modified tRNA in diverse species.

Animals↗
PubMed1995

Phylogenetic relationships among karyorelictids and heterotrichs inferred from small subunit rRNA sequences: resolution at the base of the ciliate tree.

Ciliate protozoa are among the most diverse and complex cells that have been described. Ciliates are characterized by nuclear dimorphism, possessing a macronucleus and a micronucleus which share the same cytoplasm. An understanding of the evolution of ciliate diversity depends upon knowledge of their phylogeny. In this study we attempted to resolve some of the relationships at the base of the ciliate tree by determining the phylogenetic position of a sample of heterotrich and hypothesized primitive karyorelictid ciliates. Karyorelictids are considered primitive because they possess a "simple" form of nuclear dualism whereby the macronucleus does not divide once it has differentiated from a micronucleus. We micromanipulated cells of two heterotrichs, Spirostomum ambiguum and Gruberia sp., and two karyorelictids, Loxodes magnus and Tracheloraphis sp., and amplified their small subunit (SSU) rDNA using PCR. The primary structure of the SSU rDNA was determined for each species and used to infer their positions in the ciliate phylogenetic tree. The results indicate, with strong support, that the aerobic heterotrichs and the karyorelictids sampled constitute a monophyletic group. The most parsimonious interpretation of the form of nuclear dimorphism in karyorelictids is that it is derived from the general condition as found in its sister group the aerobic heterotrichs. The two anaerobic heterotrichs, Metopus contortus and Metopus palaeformis, comprise a distinct clade, so that the subclass Heterotrichia, as currently conceived, is not a monophyletic group. The complex mouth architecture which characterizes all heterotrichs must be reassessed in light of this finding.

Animals↗