Zygotic genome activation (ZGA) is a critical event in early embryonic development, and thousands of genes are involved in this delicate and sophisticated biological process. To date, however, only a handful of these genes have revealed their core functions in this special process, and therefore the roles of other genes still remain unclear. In the present study, we used previously published transcriptome profiling to identify potential key genes (candidate genes) in minor ZGA and major ZGA in both human and mouse specimens, and further identified the conserved genes across species. Our results showed that 887 and 760 genes, respectively, were thought to be specific to human and mouse in major ZGA, and the other 135 genes were considered to be orthologous genes. Moreover, the conserved genes were most enriched in rRNA processing in the nucleus and cytosol, ribonucleoprotein complex biogenesis, ribonucleoprotein complex assembly and ribosome large subunit biogenesis. The findings of this first comprehensive identification and characterization of candidate genes in minor and major ZGA provide relevant insights for future studies on ZGA.

The effect of kiwi fruit at two dietary levels on the adaptation of intestinal fermentation over time in the growing pig was studied. A semi-synthetic fibre-free diet and two semi-synthetic diets containing kiwi fruit as a model fibre source (133 or 266 g/kg (DM basis); 28 or 48 g fibre/kg) were formulated and the diets contained titanium dioxide as an indigestible marker. A total of fourteen ileal cannulated pigs (41 kg body weight) were fed the fibre-free diet for 7 d followed by either the low or high kiwi fruit-containing diets (n 7/diet) for a further 44 d. Ileal digesta and faeces were collected at five times throughout the study. Ileal digesta were fermented (in vitro) with a standard pooled human faecal inoculum, while fresh pig faeces were used as inocula to ferment in vitro a standard purified fibre. Observations were normalised for diet DM intake using the marker. The 16S ribosomal RNA gene copy number of ileal and total faecal bacteria were high for the high-kiwi fruit level diet (P<0·05). The ileal bacteria tended to decrease over time (P<0·1), while the faecal bacteria increased (P<0·05), at the same rate for both diets. The amounts of crude protein and insoluble dietary fibre entering the hindgut changed over time similarly for both diets, whereas for starch it changed only for the low kiwi fruit-containing diet (P<0·05). Changes over time were also observed for the predicted hindgut valeric acid production and butyric acid absorption (P<0·05). In conclusion, adaptational changes over time of some characteristics of intestinal fermentation depended on the dietary level of kiwi fruit.

In the present work, we carried out a comparative molecular study of Stenoponia tripectinata tripectinata isolated from Mus musculus from the Canary Islands, Spain. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and 18S ribosomal RNA partial gene and cytochrome c-oxidase 1 (cox1) mitochondrial DNA partial gene sequences of this subspecies were determined to clarify the taxonomic status of this subspecies and to assess inter-population variation and inter-specific sequence differences. In addition, we have carried out a comparative phylogenetic study with other species of fleas using Bayesian, Maximum Parsimony, Maximum Likelihood and Neighbor-Joining analysis. A geographical signal was detected between the cox1 partial gene sequences of S. t. tripectinata isolated from M. musculus from different islands and those isolated from Apodemus sylvaticus from the Iberian Peninsula. Our results assess the monophyletic origin of Stenoponiinae and a different genetic lineage from Ctenophthalmidae. Thus, the elevation of subfamily Stenoponiinae to family level (Stenoponiidae) is suggested.

The purpose of the present study was to compare digestibility of grass hay, faecal and plasma volatile fatty acid (VFA) concentrations, and faecal bacterial abundance in overweight and moderate-condition mares. Five overweight adult mixed-breed mares and five adult mixed-breed mares in moderate condition were housed individually and limit-fed orchard grass (Dactylis glomerata) hay at 20 g/kg body weight (as fed) daily for 14 d. Forage DM and fibre digestibility were determined using AOAC methods; digestible energy was measured using bomb calorimetry; plasma and faecal VFA concentrations were determined by use of GC and MS; faecal Firmicutes, Bacteroidetes, Fibrobacter succinogenes, Ruminococcus flavefaciens and total bacteria abundance was determined by quantitative real-time PCR using previously designed phylum-specific 16S ribosomal RNA gene primers. No differences in hay digestibility, faecal VFA concentrations or faecal bacterial abundance were detected between overweight and moderate-condition mares. Mean plasma acetate concentrations were higher (P = 0·03) in overweight (1·55 (range 1·43–1·65) mmol/l) v. moderate-condition (1·39 (range 1·22–1·47) mmol/l) mares. We conclude that the higher plasma acetate in overweight mares should be further investigated as a potential link between gut microbes and obesity in horses.

Although clam populations in France are known to be infected with protozoans of the genus Perkinsus, no molecular characterization was previously performed on these parasites. Considering that several members of this genus have been associated with mortalities of molluscs worldwide, a study was undertaken in order to characterize these parasites in France. For that purpose, clams, Ruditapes philippinarum and R. decussatus, collected from different production areas and found to be infected with Perkinsus sp. in thioglycolate culture medium, were selected for PCR-RFLP tests and sequencing. Perkinsus olseni was detected in all the investigated areas and results also suggested the presence of P. chesapeaki in Leucate, a lagoon on the Mediterranean coast and in Bonne Anse in Charente Maritime, on the Atlantic coast. Clonal cultures from both detected species were produced in order to describe and compare in vitro stages. Differences in size between both Perkinsus spp. were noticed especially for schizonts and zoosporangia. Lastly, in situ hybridization tests allowed confirmation of the presence of both species in the same R. decussatus population and even in same clams. This is the first detection of P. chesapeaki in Ruditapes species and outside North America, which questions its introduction into Europe.

Monocystis sp. are sporocyst-forming apicomplexan parasites common in seminal vesicles of the earthworm Lumbricus terrestris where they may account for temporary castration. This study describes the internal transcribed spacer (ITS) region of the ribosomal cistron of Monocystis sp. This region, including ITS-1, the 5·8S ribosomal RNA gene, and ITS-2, was PCR amplified, cloned, and sequenced for Monocystis sp. isolated from the seminal vesicles of several wild-caught L. terrestris. Our analysis revealed substantial polymorphisms, also within single host organisms, indicating intra-host diversity of parasites. These genetic markers are the first that allow distinction of Monocystis sp. genotypes, opening new avenues for the study of parasite diversity within and between hosts.

An entire 18S rRNA gene sequence from Schistosoma spindale (1990 bases) and partial 18S rRNA gene sequences from S. haematobium (1950 bases) and S. japonicum (1648 bases) have been determined. Together with the previously published sequence for the S. mansoni 18S rRNA gene, these data encompass the 4 recognized Schistosoma species groups. Although Schistosoma 18S rRNA genes are highly conserved, the sequences permit a preliminary molecular phylogeny to be established for the genus. This identifies S. haematobium and S. spindale as sister taxa in a clade with S. mansoni. S. japonicum does not appear to be closely related to this clade. Much of the observed variation occurs within a ‘hypervariable’ stretch of the gene corresponding to part of the V4 region of 18S rRNA. Despite this variation, the 3 new sequences fit models of 18S rRNA secondary structure predicted from the S. mansoni sequence.

The sequences reported in this paper have been submitted to the EMBL Database under accession numbers Z11976 (S. haematobium), Z11590 (S. japonicum) and Z11979 (S. spindale).

Since 1993, strains of Entamoeba histolytica sensn lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. were established for the first time. Single microbes were successfully isolated from a mixture of micro-organisms obtained from caecal contents of turkeys, using a micromanipulation approach. The cloned parasites were propagated in vitro and maintained through continuous passages multiplying to high numbers. Identification of the protists was done by morphological investigation identifying various forms of each parasite. PCR and partial sequencing of the small subunit rRNA were used to confirm clonality and to determine the relationship of the cloned parasites with known protozoan parasites. The clonal cultures established by this technique will be useful to gain more insight into the biological repertoire of the organisms. In addition, refined infection experiments in different poultry species can now be performed to elucidate the pathological pathways of the respective protozoa.

The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

In a manner similar to many other groups of organisms, the tapeworm order Proteocephalidea poses a difficult phylogenetic problem if treated on the basis of single-gene analysis. Since the biogeography and host distribution of proteocephalideans make these tapeworms a potentially interesting model for evolutionary and co-evolutionary studies, we tried to resolve their phylogenetic relationships by applying a multi-gene approach. The ITS2 sequences and V4 hypervariable loop of 18S rRNA were obtained for 43 and 35 proteocephalidean taxa, respectively, and combined with other sequences available in the GenBank. The phylogenetic analysis of the combined DNA set was confronted with characters derived from ITS2 secondary structures. Using this approach, a species-rich Neotropical lineage of proteocephalideans could be reliably resolved. The phylogenetic relationships within this group show a high degree of phylogeny-independent host distribution. The reconstruction of ITS2 secondary structure revealed a universal 4-domain arrangement, which is conserved across a wide range of Neodermata. Several motifs of the secondary structure could be mapped to the phylogenetic tree as possible clade synapomorphies.

Freshwater snails belonging to the genus Biomphalaria are intermediate hosts of the trematode Schistosoma mansoni in the Neotropical region and Africa. In Brazil, one subspecies and ten species of Biomphalaria have been identified: B. glabrata, B. tenagophila, B. straminea, B. occidentalis, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and B.t. guaibensis. However, only the first three species are found naturally infected with S. mansoni. The classical identification of these planorbids is based on comparison of morphological characteristics of the shell and male and female reproductive organs, which is greatly complicated by the extensive intra-specific variation. Several molecular techniques have been used in studies on the identification, genetic structure as well as phylogenetic relationships between these groups of organisms. Using the randomly amplified polymorphic DNAs (RAPD) analysis we demonstrated that B. glabrata exhibits a remarkable degree of intra-specific polymorphism. Thus, the genetics of the snail host may be more important to the epidemiology of schistosomiasis than those of the parasite itself. Using the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) in intra-populational and intra-specific studies we have demonstrated that snails belonging to the B. straminea complex (B. straminea, B. kuhniana and B. intermedia) clearly presented higher heterogeneity. Using the low stringency polymerase chain reaction (LS-PCR) technique we were able to separate B. glabrata from B. tenagophila and B. tenagophila from B. occidentalis. To separate all Brazilian Biomphalaria species we used the restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer region (ITS) of the DNA gene. The method also proved to be efficient for the specific identification of DNA extracted from snail eggs. Recently we have sequenced the ITS2 region for phylogenetic studies of all Biomphalaria snails from Brazil.

Saccharomyces cerevisiae contains three nonessential genes (NGL1, NGL2, and NGL3) that encode proteins containing a domain with similarity to a Mg2+-dependent endonuclease motif present in the mRNA deadenylase Ccr4p. We have investigated a possible role of these proteins in rRNA processing, because for many of the pre-rRNA processing steps, the identity of the responsible nuclease remains elusive. Analysis of RNA isolated from cells in which the NGL2 gene has been inactivated (ngl2Δ) demonstrates that correct 3′-end formation of 5.8S rRNA at site E is strictly dependent on Ngl2p. No role in pre-rRNA processing could be assigned to Ngl1p and Ngl3p. The 3′-extended 5.8S rRNA formed in the ngl2Δ mutant is slightly shorter than the 6S precursor previously shown to accumulate upon combined deletion of the 3′ → 5′ exonuclease-encoding REX1 and REX2 genes or upon depletion of the exosomal subunits Rrp40p or Rrp45p. Thus, our data add a further component to the set of nucleases required for correct 3′-end formation of yeast 5.8S rRNA.

The validity of Gyrodactylus thymalli has been questioned, based on its morphological and genetic resemblance to G. salaris. This taxonomic problem has practical implications regarding correct diagnosis of G. salaris, which has proved to be highly pathogenic to stocks of wild Norwegian Atlantic salmon. The host specificity and pathogenicity of G. salaris and G. thymalli were experimentally tested on salmon and grayling. Both parasite species were able to infect, live and reproduce on both fish species. G. salaris was highly pathogenic for the experimental stock of salmon, while grayling mounted an effective response against this parasite. Both fish species responded to an infection with G. thymalli. The results did not support the hypothesis of conspecificity between G. thymalli and G. salaris. The ribosomal RNA gene intergenic spacer of both species was sequenced. Variation in sequence was lower than expected for different species. Variation in the sequences of tandemly repeated elements was found and may prove useful in distinguishing G. salaris and G. thymalli.

A new RNA structural motif consisting of two double helices closely packed via minor grooves is found in many places in the ribosome structure. The packing requires that a GU base pair in one helix be packed against a Watson–Crick pair in the other helix. Two such motifs mediate the interaction of the P- and E-tRNA with the large ribosomal subunit. Analysis of the particular positions of these two motifs in view of the available data on occupancy of tRNA-binding sites and structural changes in the ribosome during the elongation cycle suggests a distinct role for each motif in tRNA translocation.

Seven European laboratories co-operated in a joint project (FAIR CT97-3035) to develop, refine and apply molecular methods towards facilitating elucidation of the complex composition of the human intestinal microflora and to devise robust methodologies for monitoring the gut flora in response to diet. An extensive database of 16S rRNA sequences for tracking intestinal bacteria was generated by sequencing the 16S rRNA genes of new faecal isolates and of clones obtained by amplification with polymerase chain reaction (PCR) on faecal DNA from subjects belonging to different age groups. The analyses indicated that the number of different species (diversity) present in the human gut increased with age. The sequence information generated, provided the basis for design of 16S rRNA-directed oligonucleotide probes to specifically detect bacteria at various levels of phylogenetic hierarchy. The probes were tested for their specificity and used in whole-cell and dot-blot hybridisations. The applicability of the developed methods was demonstrated in several studies and the major outcomes are described.

The publication of the crystal structures of the ribosome offers an opportunity to retrospectively evaluate the information content of hundreds of qualitative biochemical and biophysical studies of these structures. We assessed the correspondence between more than 2,500 experimental proximity measurements and the distances observed in the ribosomal crystals. Although detailed experimental procedures and protocols are unique in almost each analyzed paper, the data can be grouped into subsets with similar patterns and analyzed in an integrative fashion. We found that, for crosslinking, footprinting, and cleavage data, the corresponding distances observed in crystal structures generally did not exceed the maximum values expected (from the estimated length of the agent and maximal anticipated deviations from the conformations found in crystals). However, the distribution of distances had heavier tails than those typically assumed when building three-dimensional models, and the fraction of incompatible distances was greater than expected. Some of these incompatibilities can be attributed to the experimental methods used. In addition, the accuracy of these procedures appears to be sensitive to the different reactivities, flexibilities, and interactions among the components. These findings demonstrate the necessity of a very careful analysis of data used for structural modeling and consideration of all possible parameters that could potentially influence the quality of measurements. We conclude that experimental proximity measurements can provide useful distance information for structural modeling, but with a broad distribution of inferred distance ranges. We also conclude that development of automated modeling approaches would benefit from better annotations of experimental data for detection and interpretation of their significance.

The only eukaryotes found in highly acidic environments (pH 0·5–3) with elevated temperatures (up to 56 °C) are three species of unicellular red algae: Cyanidioschyzon merolae, Cyanidium caldarium and Galdieria sulphuraria. These habitats are scattered all over the world and are usually very small. Because all three species are strictly acidophilic and will not tolerate desiccation, distribution by wind or water seems very unlikely. The populations in the various habitats might have been isolated for very long times, providing a unique opportunity to observe a significant degree of independent evolution under strong selective pressure. We investigated the biogeography of 18 isolates of these red algae by comparison of partial sequences of the 18S rRNA. A gene tree based on 18S rRNA assigns 15 strains to one branch with high bootstrap values. These isolates share the feature of facultative heterotrophy, whether or not they were originally placed in the genus Galdieria. The remaining three strains – Galdieria maxima, C. merolae and C. caldarium – form a sister clade to this group. The exact position of these two groups in relation to other red algae remains unresolved. The evolutionary distance between individual Galdieria strains is high, indicating that within this genus several races have developed significantly altered 18S sequences. Our comparison of 18S sequences from thermo-acidophilic red algae indicates that even within a seemingly homogeneous group of eukaryotic organisms the limits of phylogenetic analysis may be reached.

Domain V of Escherichia coli 23 S rRNA (residues 2023–2630) was replaced by that from Staphylococcus aureus, thereby introducing 132 changes in the rRNA sequence. The resulting ribosomal mutant was unable to support cell growth. The mutant was rescued, however, by restoring an interaction between domains IV and V (residues 1782 and 2586). Although the importance of this interaction, U/U in E. coli, C/C in S. aureus, is therefore demonstrated, it cannot be the only tertiary interaction important for ribosomal function as the rescued hybrid grew more slowly than the wild type. Additionally, although the single-site mutations U1782C and U2586C in E. coli are viable, the double mutant is lethal.

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598–600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C Gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along Gram-negative and Gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.