Sequence-specific cleavage of small-subunit (SSU) rRNA with oligonucleotides and RNase H: a rapid and simple approach to SSU rRNA-based quantitative detection of microorganisms

A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a "scissor probe") that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.     

Sequence-Specific Cleavage of RNA Using Macrocyclic Lanthanide Complexes Conjugated to Oligonucleotides: A Structure Activity Study

Abstract

A series of lanthanide complexes were synthesized and conjugated to an oligodeoxynucleotide. Sequence-specific cleavage of a complementary RNA by the conjugates was performed and results are discussed in terms of structure of the complexes.     

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.     

Identification of Species and Sources of Cryptosporidium Oocysts in Storm Waters with a Small-Subunit rRNA-Based Diagnostic and Genotyping Tool

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of Cryptosporidium oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of Cryptosporidium parasites in environmental samples.     

Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.     

Conjugates of RNase P-Guiding Oligonucleotides with Oligo(N-Methylpyrrole) as Prospective Antibacterial Agents

Russian Journal of Bioorganic Chemistry - The conjugates of RNase P guiding oligo(2'-O-methylribo)- and oligodeoxyribonucleotides (EGS oligonucleotides) with oligo(N-methylpyrrole) have been...     

Phylogenetic Analysis of Bacterial Communities Associated with Leaves of the Seagrass Halophila stipulacea by a Culture-Independent Small-Subunit rRNA Gene Approach

Abstract The phylogenetic diversity of the bacterial community associated with leaves of the marine plant Halophila stipulacea in the northern Gulf of Elat was examined by 16S rRNA gene (rDNA) sequence analyses of a clone library. For 59 clones corresponding to 51 ARDRA (amplified rDNA restriction analysis) groups, the sequence of ∼1 kb was determined, and the fraction of the corresponding ARDRA groups of the leaf library was calculated. The class Proteobacteria was represented by 62.6% of the clone sequences. Most sequences originated from members of the γ-subclass (27.3%), affiliated with members of the genera Pseudomonas, Vibrio, Marinomonas, Oceanospirillum, and other marine groups. Affiliation to the α-subclass was determined for 24.2% of the sequences. They were related to the genera Hyphomonas, Roseobacter, Ruegeria, and Rhizobiaceae. Several α-proteobacterial sequences were distantly related to known sequences. Only 4% of the clone sequences were related to β-Proteobacteria. Additionally, 7.1% of the sequences possibly belonged to the class Proteobacteria, but branched deeply from known subclasses. Several sequences were affiliated to members of the orders Verrucomicrobiales and Planctomycetales, the Holophaga/Acidobacterium phylum, and chloroplasts of marine diatoms. Received: 20 March 1999; Accepted: 13 August 1999; Online Publication: 2 March 2000     

Cleavage of a 23S rRNA pseudoknot by phenanthroline-Cu(II)

Studying the intricate folding of rRNA within the ribosome remains a complex problem. Phenanthroline-Cu(II) complexes cleave phosphodiester bonds in rRNA in specific regions, apparently especially where the rRNA structure is constrained in some fashion. We have introduced phenanthroline-copper complexes into 50S Escherichia coli ribosomal subunits and shown specific cleavages in the regions containing nucleotides 60-66 and 87-100. This specificity of cleavage is reduced when the ribosome is heated to 80 degrees C and reduced to background when the ribosomal proteins are extracted and the cleavage repeated on protein-free 23S rRNA. It has been suggested that nucleotides 60-66 and 87-95 in E.coli 23S rRNA are involved in a putative pseudoknot structure, which is supported by covariance data. The paired cleavages of nearly equal intensity of these two regions, when in the ribosome, may further support the existence of a pseudoknot structure in the 100 region of 23S rRNA.     

Radial Immunodiffusion: a Simple and Rapid Method for Detection of Marek''s Disease Antigen(s)

A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek's disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek's disease.     

Radial Immunodiffusion: a Simple and Rapid Method for Detection of Marek's Disease Antigen(s)

A qualitative radial immunodiffusion technique is described which detects antigen(s) in feathers from live or dead chickens infected with Marek''s disease herpesvirus. Antiserum, which is incorporated into a support medium, reacts with antigen(s) in the feather tip producing a radial precipitin ring. Antigen(s) was detected in 93.3% of experimentally inoculated chickens 21 days postinoculation and in 100% of infected birds subsequently tested through 6 weeks. No antigen was detectable in the feathers of uninoculated control chickens. The technique is simple and rapid to perform. Positive tests could be detected after 1 to 2 hours of incubation. Antigen detection by the radial immunodiffusion test correlated well with other criteria of infection. This technique should have application as a laboratory research tool and as an adjunct for a rapid flock diagnosis of Marek''s disease.     

SSU rRNA reveals a sequential increase in shell complexity among the euglyphid testate amoebae (Rhizaria: Euglyphida)

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus. Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae. The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae).     

Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene

Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.     

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.     

Simple,Rapid and Inexpensive Quantitative Fluorescent PCR Method for Detection of Microdeletion and Microduplication Syndromes

Because of economic limitations, the cost-effective diagnosis of patients affected with rare microdeletion or microduplication syndromes is a challenge in developing countries. Here we report a sensitive, rapid, and affordable detection method that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR). Our procedure is based on the finding of genomic regions with high homology to segments of the critical microdeletion/microduplication region. PCR amplification of both using the same primer pair, establishes competitive kinetics and relative quantification of amplicons, as happens in microsatellite-based Quantitative Fluorescence PCR. We used patients with two common microdeletion syndromes, the Williams-Beuren syndrome (7q11.23 microdeletion) and the 22q11.2 microdeletion syndromes and discovered that MQF-PCR could detect both with 100% sensitivity and 100% specificity. Additionally, we demonstrated that the same principle could be reliably used for detection of microduplication syndromes, by using patients with the Lubs (MECP2 duplication) syndrome and the 17q11.2 microduplication involving the NF1 gene. We propose that MQF-PCR is a useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes, ideally suited for use in developing countries, but having general applicability as well.     

Evolutionary relationships of Spirurina (Nematoda: Chromadorea: Rhabditida) with special emphasis on dracunculoid nematodes inferred from SSU rRNA gene sequences

The analysis of 26 new small subunit rRNA sequences obtained from helminths that primarily parasitize fishes sampled from five continents provided well-supported trees, allowing us to study the phylogenetic relationships among spirurid nematodes. The analyses have shown that Dracunculoidea is a paraphyletic taxon and Anguillicolidae and Gnathostomatidae constitute the basal branch of the suborder Spirurina. The genera Philometra and Philometroides appear to be paraphyletic, while on the higher taxonomic level, good correlation between the morphology-based system and molecular data was observed. Neither co-evolution of the studied helminths with their hosts, nor phylogeographic pattern, are apparent in our dataset.     

A Simple and Rapid Quantitative Method of Detection of the Common Achondroplasia Mutation: Analysis in Mismatch Repair Deficient Cells

Achondroplasia is the most common form of dwarfism and has an incidence of approximately 1/7500. In more than 97% of cases, it is caused by a recurrent point mutation, a G to A substitution at nucleotide position 1138 (G1138A) of the fibroblast growth factor receptor 3 gene. Although this is an autosomal dominant condition, more than 90% of all mutations occur sporadically making this one of the most mutagenic sites in the human genome. The reasons for the high spontaneous G1138A mutation rate are not known. This investigation was performed by developing a simple and rapid semi-quantitative allele specific PCR based assay capable of reliably detecting more than 25 mutant G1138A copies in a pool of 300 000 wild type molecules. Using this assay, the G1138A mutation frequency was measured in cell lines deficient in mismatch repair (LoVo, SW48) and comparing it with controls. No differences were found in the frequency of this point mutation between the mismatch repair deficient and wild type cell lines.__________From Genetika, Vol. 41, No. 8, 2005, pp. 1137–1141.Original English Text Copyright © 2005 by Grewal.This article was submitted by the author in English.     

Neoparamoeba spp. and their eukaryotic endosymbionts similar to Perkinsela amoebae (Hollande, 1980): coevolution demonstrated by SSU rRNA gene phylogenies

The molecular phylogeny of Neoparamoeba spp. based on SSU rDNA was updated by including new sequences of strains isolated from an invertebrate and an alga. In total, 59 sequences of strains representating N. pemaquidensis, N. branchiphila, N. aestuarina and N. perurans were analysed. Sequences of SSU rDNA of eukaryotic endosymbionts (Perkinsela amoebae-like organisms) were prepared from 34 samples of genomic DNA of strain-representatives of N. pemaquidensis, N. branchiphila and N. aestuarina. Comparison of phylograms reconstructed from corresponding SSU rDNA sequences of host amoebae and their symbionts revealed a high level of congruence, which argues very strongly for coevolution of these two eukaryotic organisms.     

Trefusiidae Are a Subtaxon of Marine Enoplida (Nematoda): Evidence from Primary Structure of Hairpin 35 and 48 Loops of SSU rRNA Gene

A rare nucleotide substitution was found in the evolutionarily conserved loop of hairpin 35 of the 18S rRNA gene of marine free-living nematode, Trefusia zostericola(Nematoda: Enoplida). The same substitution was found in all the marine Enoplida studied but not in other nematodes. Such a molecular synapomorphy indicates that marine enoplids are more closely related to T. zostericolathan to freshwater Triplonchida. Maximum parsimony, neighbor-joining, and maximum likelihood analyses of complete nucleotide sequences of the gene, with the heterogeneity of nucleotide sites in evolution rates taken into account, support this conclusion. Hence, the hypothesis of particular primitiveness of Trefusiidae among nematodes should be rejected. Phylogenies based on molecular data support the morphological reduction of metanemes in Trefusiidae. Alongside with the unique change in hairpin 35 loop among marine Enoplida (including T. zostericola), hairpin 48 is also modified by a rare transversion which could be found among Mesorhabditoidea nematodes, in related genera Pelodera, Mesorhabditis, Teratorhabditis, Parasitorhabditis, Crustorhabditis, and Distolabrellus, and in 11 orders of Rhodophyta. Rare mutations in hairpins 35 and 48 tend to be fixed correlatively in evolution and could be found in all the Acanthocephala species. X-Ray data show that these regions (H31 and H43, in alternative nomenclature) are spatially brought together in native ribosomes. The nature and distribution of molecular autoapomorphies in phylogenetic trees of high-rank taxa are discussed.     

Synthesis and Antibody Mediated Detection of 2,4-Dinitrophenyl (DNP) Labelled Oligonucleotides

Abstract

Different DNP phosphoramidites based on non-nucleoside and nucleoside backbone molecules are developed and used in the multiple labelling of oligonucleotides during the solid phase synthesis. It is demonstrated that the antibody mediated detection of DNP labelled oligonucleotides is comparable to that of digoxigenin, biotin and fluorescein.