The present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.0 μg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 μg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription.

Neospora caninum and N. caninum-like organisms are cyst-forming coccidian parasites known to cause neuromuscular disorders in dogs and abortion in cattle. In this article we report on the use of the polymerase chain reaction (PCR) for the detection of DNA from N. caninum. After determining the sequence of the internal transcribed spacer 1 (ITSl) of N. caninum and Toxoplasma gondii, and part of the sequences for & species of Sarcocystis, we designed a primer set for the amplification of a 279-base-pair fragment of ITSl from N. caninum. The PCR system made possible the specific detection of 5 N. caninum organisms and no amplification was observed from any of the other cyst-forming coccidia tested, including the closely related T. gondii. Furthermore, we were also able to demonstrate the presence of N. caninum in brain and lung tissue samples from experimentally infected mice. Our data also link the 5-8S rRNA gene for T. gondii and N. caninum to the 16S-like rRNA gene, within the rDNA unit.

Nondenatured and denatured total RNA from the tapeworm Diphyllobothrium dendriticum (Cestoda) was analysed by agarose gel electrophoresis. It was found that the large subunit ribosomal RNA (lrRNA) is 28.0S and the small subunit ribosomal RNA (srRNA) is 19.5S. Following denaturation the 28.0S rRNA was disrupted into a 19.5S subfragment and a 20.7S subfragment due to the presence of a centrally located hidden break. By hybridization of Northern blot membranes with oligonucleotide probes specific for the 5′- and 3′-ends of the lrRNA respectively, we have shown that the 19.5S sub-fragment is from the 5′-end (the α-subfragment) and the 20.7S subfragment from the 3′-end (the β-subfragment) of the 28.0S rRNA of D. dendriticum.

rRNA and a heterologous cloned rDNA probe have been used to detect the rRNA genes of Eimeria species which infe the chicken, and has allowed the isolation and preliminary characterization of cloned rDNA sequences from a genomic DNA library of Eimeria tenella. It is demonstrated that rRNA and rDNA probes can be used to identify individual Eimeria species by the restriction fragment patterns detected after Southern hybridization. In addition, studies have shown that the large and small subunit rRNAs are expressed throughout sporulation.

The ribosomal DNA (rDNA) unit of the liver fluke Opisthorchis viverrini has been cloned and characterized. The results demonstrated that the total length of this unit is approximately 13 kb, containing 4·2 kb of large subunit (LSU) rDNA, 2·0 kb of small subunit (SSU) rDNA, 1·0 kb of transcribed spacer DNA and 5·8 kb of non-transcribed + external transcribed spacer DNA. Examination of the non-transcribed spacer region between different rDNA units showed variation in the restriction sites rather than in the length. Judging from hybridization of the rDNA plasmid to the restriction endonuclease digest of genomic DNA, rDNA units represent 6·1% of the total genomic DNA. At the RNA level, the LSU rRNA of O. viverrini and Fasciola gigantica contained a hidden break. The molecule consisted of two fragments co-migrating with a SSU rRNA, when electrophoresis was carried out under denaturing conditions. Ribosomal RNA sequence comparison has been previously used to determine phylogenetic classification of parasitic organisms. The sequence of 381 nucleotides at the 5′ terminus of the LSU rRNA gene was determined and compared with those from species previously reported by other investigators. Phylogenetic classification of O. viverrini, as determined by rRNA gene sequence comparison, is comparable with the conventional taxonomic classification.

Neospora caninum is an apicomplexan, protozoan parasite, which causes severe disease in dogs and cattle. It has previously been isolated only in the United States. A 5-week-old Boxer pup with a progressive hindlimb paresis was diagnosed as suffering from neosporosis on the basis of clinical signs and the presence of anti-Neospora antibodies in it, 2 litter-mates and its darn. Despite treatment with sulphonamides, the pup was euthanased 3 days later. The diagnosis of neosporosis was confirmed by immunohistochemical examination of muscle and CNS tissue sections from the pup. Parasites were isolated into Vero cell culture from the cerebrum, and confirmed as Neospora caninum by immunofluorescence with specific antibody, tachyzoite ultrastructure and 16S-like ribosomal RNA sequences. This isolate (designated NC-Liverpool) has been continuously passaged every 7–10 days. Its growth characteristics, ultrastructure and antigenic profile, as revealed by immunoblotting, have revealed no major differences from the American NC-1 isolate. Furthermore, no difference was seen when comparing the sequences of 16S-like ribosomal RNA and the ITS1 region of the two isolates.

Cultivation-independent analyses were carried out to compare the bacterial community structure found in the rhizospheres of a transplastomic tobacco plant carrying the antibiotic resistance marker-gene aadA and its non-engineered parental line. PCR- and reverse transcriptase PCR-amplifications of 16S rRNA and their corresponding genes were carried out with primers targeting the domain Bacteria. The diversity of PCR-products amplified from total nucleic acids extracted from rhizospheres of 10-week-old plants, which had been grown in potting soil in the greenhouse, was visualized by genetic profiling using the single-strand conformation polymorphism (SSCP) technique. The SSCP profiles generated from DNA extracted with two different protocols, one including total RNA and the other only DNA, did not show any differences. The SSCP profiles amplified from RNA and DNA were also highly similar to each other, indicating that the dominant bacteria detected were metabolically active. High similarities were seen between the SSCP profiles from the transplastomic and the non-engineered plants, except for a single band that consistently occurred with samples from the non-engineered plants (six replicates), but not, or only weakly, with their engineered counterparts. DNA sequencing and database analysis revealed that the partial rRNA gene matched to a Flavobacterium sp. Other bands of the SSCP-profiles, related to Burkholderia and Bordetella were variable between individual plants but not affected by the transplastomic modification. Thus, the transplastomic modification caused a relative decline of a specific Flavobacterium population but not of other bacteria. Further studies including additional tobacco cultivars, soils and conditions of cultivation would be desirable, to elucidate the ecological importance of this difference.

In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling. All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene. L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene. This interaction results in the formation of a pseudoknot. Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro. The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene. By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions. Therefore, these two regions define two L20-binding sites in the operator. Using mutations and deletions of rpmI’-‘lacZ fusions, we show that both sites are essential for repression in vivo. However L20 can bind to each site independently in vitro. One site is similar to the L20-binding site on 23S rRNA. Here we propose that L20 recognizes its mRNA and its rRNA in similar way.

RNase MRP is a ribonucleoprotein enzyme involved in processing precursor rRNA in eukaryotes. To facilitate our structure–function analysis of RNase MRP from Saccharomyces cerevisiae, we have determined the likely secondary structure of the RNA component by a phylogenetic approach in which we sequenced all or part of the RNase MRP RNAs from 17 additional species of the Saccharomycetaceae family. The structure deduced from these sequences contains the helices previously suggested to be common to the RNA subunit of RNase MRP and the related RNA subunit of RNase P, an enzyme cleaving tRNA precursors. However, outside this common region, the structure of RNase MRP RNA determined here differs from a previously proposed universal structure for RNase MRPs. Chemical and enzymatic structure probing analyses were consistent with our revised secondary structure. Comparison of all known RNase MRP RNA sequences revealed three regions with highly conserved nucleotides. Two of these regions are part of a helix implicated in RNA catalysis in RNase P, suggesting that RNase MRP may cleave rRNA using a similar catalytic mechanism.

The universally conserved A2451 of 23S rRNA has been proposed to participate directly in the catalysis of peptide bond formation in the ribosomal peptidyl transferase center. An unusually high, near neutral, pKa of A2451 is a prerequisite for its action as a general acid–base catalyst. Increased reactivity of A2451 to dimethylsulfate (DMS) at pH 8.5 compared to pH 6.5 was taken as evidence that the pKa of this nucleotide falls within this pH range. Structural data suggested that the interaction between A2451 and G2447 in the ribosome is responsible for A2451 pKa perturbation. In contrast to expectation, our studies did not show pH dependence of A2451 dimethylsulfate modification in ribosomes of Thermus aquaticus and Mycobacterium smegmatis. Other rRNA regions, however, showed major alterations in DMS reactivity at pH 8.5 compared to pH 6.5, suggesting that conformational rearrangements in the structure of the large ribosomal subunit may occur upon the pH shift. The G2447U mutant of M. smegmatis was viable, indicating that the G2447-A2451 interaction is not critical for the ribosome function. We concluded that the proposed unusual pKa of A2451, if existing, may not be crucial for the ribosome activity and that the previously reported pH-dependent alterations in the DMS modification of A2451 do not necessarily reveal an unusual pKa of this nucleotide.

The U3 small nucleolar ribonucleoprotein (snoRNP) is composed of a small nucleolar RNA (snoRNA) and at least 10 proteins. The U3 snoRNA base pairs with the pre-rRNA to carry out the A0, A1, and A2 processing reactions that lead to the release of the 18S rRNA from the nascent pre-rRNA transcript. The yeast U3 snoRNA can be divided into a short 5′ domain (nt 1–39) and a larger 3′ domain (73 to the 3′ end) separated by a stretch of nucleotides called the hinge region (nt 40–72). The sequences required for pre-rRNA base pairing are found in the 5′ domain and hinge region whereas the 3′ domain is largely covered with proteins. Mpp10p, one of the protein components unique to the U3 snoRNP, plays a role in processing at the A1 and A2 sites. Because of its critical role in U3 snoRNP function, we determined which sequences in the U3 snoRNA are required for Mpp10p association. Unlike fibrillarin and all the previous U3 snoRNP components studied in this manner, sequences in the 3′ domain are not sufficient for Mpp10p association. Instead, a conserved sequence element in the U3 snoRNA hinge region is required, placing Mpp10p near the 5′ domain that carries out the pre-rRNA base-pairing interactions in the functional center of the U3 snoRNP.

The Escherichia coli DEAD protein DbpA is an RNA-specific ATPase that is activated by a 153-nt fragment within domain V of 23S rRNA. A series of RNA subfragments and sequence changes were used to identify the recognition elements of this RNA–protein interaction. Reducing the size of the fully active 153-nt RNA yields compromised substrates in which both RNA and ATP binding are weakened considerably without affecting the maximal rate of ATP hydrolysis. All RNAs that stimulate ATPase activity contain hairpin 92 of 23S rRNA, which is known to interact with the 3′ end of tRNAs in the ribosomal A-site. RNAs with base mutations within this hairpin fail to activate ATP hydrolysis, suggesting that it is a critical recognition element for DbpA. Although the isolated hairpin fails to activate DbpA, RNAs with an extension of approximately 15 nt on either the 5′ or 3′ side of hairpin 92 elicit full ATPase activity. These results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension. A model relating the RNA binding and ATPase activities of DbpA is presented.

Self-splicing of the Tetrahymena pre-rRNA is inhibited by a conserved rRNA hairpin P(−1) upstream of the 5′ splice site. P(−1) inhibits self-splicing by competing with formation of the P1 splice site helix. Here we show that the P(−1) hairpin also enhances dissociation of the spliced products, which was monitored by native gel electrophoresis. Mutations that stabilize the rRNA hairpin increase the rate of dissociation approximately 10-fold, from 0.5 min−1 for the wild-type RNA to ∼4 min−1 at 30 °C. Conversely, mutations or oligonucleotides that inhibit refolding of the exons and that stabilize the P1 helix decrease the rate of product release. The results suggest that refolding of products can be used to stimulate the turnover of ribozyme-catalyzed reactions. In the pre-rRNA, this conformational change helps shift the equilibrium of self-splicing toward the mature rRNA.

Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5′ terminus of 16S rRNA, in the 300 region of its 5′ domain, and in the 1070 region of its 3′-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-Å map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein–protein and protein–RNA crosslinking, chemical and enzymatic footprinting, and genetics.

Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus. The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way junctions at both ends composed of portions of helices 20, 21, and 23. Addition of the remaining portion of helix 23 to this construct results in the minimal site for S11. Surprisingly, almost half of the central domain (helices 24, 25, and 26) is dispensable for binding the central domain proteins. Thus, at least two classes of RNA elements can be identified in ribosomal RNA. A protein-binding core element (such as helices 20, 21, 22, and 23) is required for the association of ribosomal proteins, whereas secondary binding elements (such as helices 24, 25, and 26) associate only with the preformed core RNP complex. Apparently, there may be a hierarchy of ribosomal RNA elements similar to the hierarchy of primary, secondary, and tertiary binding ribosomal proteins.

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5′ end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

In two recent articles by Nitta et al. (1998a, 1998b), evidence was presented that in vitro-transcribed protein-free 23S rRNA of Escherichia coli (and its individual domains) can catalyze peptide bond formation. The implications of these studies are considerable because demonstration of catalytic activity associated with protein-free 23S rRNA would be fundamental to our understanding of the mechanism and evolution of protein synthesis (Noller et al., 1992; Khaitovich et al., 1999).

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.

Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth. Processing requires numerous factors which act in trans as well as RNA sequence/structural elements which function in cis. To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast. Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3′ end of 5.8S rRNA and the 5′ end of 25S. Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem. Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency. This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates. This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP. Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing.

The sequences for the entire intergenic spacer (IGS) and 3′ end region of the large subunit (28S) ribosomal RNA gene are presented for Schistosoma intercalatum, S. haematobium and S. mansoni. The IGS was found to have at least 2 size formats for S. intercalatum and S. haematobium and the region for all 3 species contains numerous repeats and evidence of recombination. An A+T rich sequence found in S. intercalatum and S. haematobium is described and its possible function and origins are discussed. The amplification of this region by means of the polymerase chain reaction can be used to discriminate clearly between the 3 species involved in this study. The putative end of the 28S gene is identified.