Ribosomal RNA Fragments Research Articles

Changes in transcript abundance relating to colony collapse disorder in honey bees ( Apis mellifera )

Colony collapse disorder (CCD) is a mysterious disappearance of honey bees that has beset beekeepers in the United States since late 2006. Pathogens and other environmental stresses, including pesticides, have been linked to CCD, but a causal relationship has not yet been demonstrated. Because the gut acts as a primary interface between the honey bee and its environment as a site of entry for pathogens and toxins, we used whole-genome microarrays to compare gene expression between guts of bees from CCD colonies originating on both the east and west coasts of the United States and guts of bees from healthy colonies sampled before the emergence of CCD. Considerable variation in gene expression was associated with the geographical origin of bees, but a consensus list of 65 transcripts was identified as potential markers for CCD status. Overall, elevated expression of pesticide response genes was not observed. Genes involved in immune response showed no clear trend in expression pattern despite the increased prevalence of viruses and other pathogens in CCD colonies. Microarray analysis revealed unusual ribosomal RNA fragments that were conspicuously more abundant in the guts of CCD bees. The presence of these fragments may be a possible consequence of picorna-like viral infection, including deformed wing virus and Israeli acute paralysis virus, and may be related to arrested translation. Ribosomal fragment abundance and presence of multiple viruses may prove to be useful diagnostic markers for colonies afflicted with CCD.

Evaluation for Helicobacter pylori in Meckel’s Diverticulum by Using Real-Time PCR

Although Helicobacter pylori (H. pylori) has been identified in heterotopic gastric mucosa of Meckel's diverticulum, controversial results are reported in the pertinent literature. The aim of this study was to evaluate for the presence of H. pylori histologically using hematoxylin-eosin and Toluidine Blue in Meckel's diverticulum and by real-time TaqMan polymerase chain reaction (PCR) in those with heterotopic gastric mucosa. The study included 21 consecutive patients who had undergone resection of Meckel's diverticulum at our hospital between 1995 and 2007. The paraffin-embedded tissues were retrieved and reviewed for the presence of histological abnormalities and H. pylori-like organisms and for the presence or absence of heterotopic mucosa. H. pylori was sought in those cases that contained heterotopic gastric mucosa using real-time TaqMan PCR to amplify a fragment of the 23S ribosomal RNA (rRNA) gene of H. pylori. Upon histological examination, heterotopic gastric mucosa was found to be present in 12 cases. H. pylori was not identified in any of the sections examined. A genomic PCR product was also not obtained in real-time PCR study. We have confirmed that colonization of H. pylori, if it occurs at all, is exceedingly rare in heterotopic gastric mucosa of Meckel's diverticulum.

A model of catheter‐associated urinary tract infection initiated by bacterial contamination of the catheter tip

To examine the microbial colonization of urinary catheters that have been used by patients, to model catheter colonization in vitro and thus provide information about the way bacteria gain access to the bladder during catheterization. Microbial growth patterns from patients' indwelling catheters and from catheters used in an in vitro model of the catheterized urinary tract were compared. Catheters were cut into short segments, microorganisms from the inside and outside of each segment of the catheters were removed by sonication, and viable bacteria counted. DNA was extracted from selected patient catheter isolates and the DNA fragment of 16S ribosomal RNA was amplified by polymerase chain reaction and confirmed by DNA sequencing. The DNA sequences from the isolates obtained from different catheter sections and from urine in the same patient were compared. After 1 day of catheterization there was significant bacterial growth on the outside of all the segments of patient catheters; there was significant growth on the inside of all segments by 4 days. Higher viable counts and a wider spectrum of genera were found on the outside than on the inside of these catheters. The same strains of bacteria, as determined by >or=98% similarity of the 16S ribosomal DNA sequence, were found on the outside and inside of catheters and in the urine. In the in vitro model, when the distal urethra was inoculated before inserting the catheter, the viable counts after incubation were variable along the outside of the catheter and the inside counts were uniformly high. By contrast, there was a different pattern after inoculating the inside of the distal end of the catheter, leading to an ascending biofilm on the inside. A smaller inoculum delayed but did not prevent infection in the model. The present results are consistent with the hypothesis that contamination of the tip of the catheter while it is being inserted is a possible means by which bacteria gain access to the bladder.

Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition

The Tetrahymena thermophila ribosomal DNA (rDNA) replicon contains dispersed cis-acting replication determinants, including reiterated type I elements that associate with sequence-specific, single-stranded binding factors, TIF1 through TIF4. Here, we show that TIF4, previously implicated in cell cycle-controlled DNA replication and rDNA gene amplification, is the T. thermophila origin recognition complex (TtORC). We further demonstrate that TtORC contains an integral RNA subunit that participates in rDNA origin recognition. Remarkably, this RNA, designated 26T, spans the terminal 282 nts of 26S ribosomal RNA. 26T RNA exhibits extensive complementarity to the type I element T-rich strand and binds the rDNA origin in vivo. Mutations that disrupt predicted interactions between 26T RNA and its complementary rDNA target change the in vitro binding specificity of ORC and diminish in vivo rDNA origin utilization. These findings reveal a role for ribosomal RNA in chromosome biology and define a new mechanism for targeting ORC to replication initiation sites.

Genetic characterization of Cryptosporidium species from humans in Spain

Several species of Cryptosporidium have been associated with infection. Cryptosporidium parvum and Cryptosporidium hominis are the main agents of cryptosporidiosis in humans. Stool samples from 108 Cryptosporidium-infected patients were submitted to PCR-RFLP analysis for a 553-bp fragment of Cryptosporidium oocyst wall protein (COWP) gene and an 826–864 bp fragment of the small-subunit ribosomal RNA (SSU-rRNA) gene. Ninety-two patients were immunocompetent children and 16 were HIV-infected adults. C. hominis was detected in 69 patients (59 immunocompetent and 10 HIV-infected); C. parvum, in 34 patients (28 immunocompetent and 6 HIV-infected); and C. meleagridis and C. felis in one patient each (both immunocompetent children). Three samples yielded negative results. C. parvum was significantly more frequent in children from rural areas than in those of urban residence ( p = 0.010). As far as we know, this is the first surveillance study about the molecular characterization of Cryptosporidium in humans performed in Spain. The finding of zoonotic species infecting humans calls for further research on this subject.

Correlation of the structure and conformational changes of selected fragments of plant small ribosomal RNA within the steps of polypeptide chain elongation

The interaction and conformational relationships between rRNAs and ribosomal proteins are responsible for ribosome activity. We tested seven different deoxyoligonucleotides complementary to the selected, highly conserved sequences of 18S rRNAs important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site on the ribosomes converted either to pre- or to post-translocational states (with or without pre-hybridized oligonucleotides). We found a correlation between the level of oligomer hybridization and the inhibition of AA-tRNA binding. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis.

Trisaccharide mimetics of the aminoglycoside antibiotic neomycin

A highly convergent approach for the chemical synthesis of eight structurally related trisaccharides that contain 3 to 5 amino groups has been described. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been employed to determine the dissociation constants (Kd) for the binding of the trisaccharides to a prototypical fragment of 16S ribosomal RNA. A compound that contained a 4,6-dideoxy-4-amino-beta-D-glucopyranoside moiety at C-3 displayed binding in the low micromolar range. It was found that small structural changes of the saccharides resulted in large differences in affinity. The described structure-activity relationship is expected to be valuable for the development of novel antibiotics that target rRNA.

Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan)

An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N + Nylon and FTA ® membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 °C for 10 min) of the Hybond N + Nylon discs in a buffered solution improved the detection, but for FTA ® membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.

SYSTEMATIC REVISION OF PATELLOIDA PYGMAEA (DUNKER, 1860) (GASTROPODA: LOTTIIDAE), WITH A DESCRIPTION OF A NEW SPECIES

Patelloida pygmaea (Dunker) and its closely allied species, P. heroldi (Dunker) and P. conulus (Dunker) have caused nomenclatural confusion because of their variable shell morphology and distinctive habitats. According to current nomenclature, these species of Patelloida have been synonymized and treated as one species with two ecological forms. Patelloida pygmaea lives on the shell of Crassostrea gigas (Ostreidae), P. pygmaea form heroldi occurs on intertidal rocks on sheltered shores and P. pygmaea form conulus is found on the shell of Batillaria multiformis (Batillariidae). Their taxonomic relationships and possible species status are, however, unclear. Using two mitochondrial genes (fragments of COI and 16S ribosomal RNA; total 1192 sites), we analysed 88 specimens of P. pygmaea, P. pygmaea form heroldi and P. pygmaea form conulus from 37 localities in East Asia. In the resulting molecular phylogenetic trees, the specimens of Patelloida fall into four clades with high bootstrap probabilities; these clades correspond taxonomically to P. pygmaea, P. conulus, P. heroldi and a fourth previously unrecognized taxon, Patelloida ryukyuensis n. sp., described here. A minimum-spanning network for 29 unique mitochondrial COI haplotypes obtained from 45 specimens in the same bay in central Japan form three distinct clusters, consisting of P. pygmaea, P. conulus and P. heroldi, respectively. This suggests that reproductive isolation has been established between each group. A detailed examination of radular and shell morphologies of the four taxa clarifies the morphological distinction between these species. The four species form a rather young clade in the genus Patelloida that diverged during the Pliocene. They provide an example of habitat segregation in a restricted marine environment.

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

Ribosomal protein L11 has two domains: the C-terminal domain (L11-C76) binds rRNA, whereas the N-terminal domain (L11-NTD) may variously interact with elongation factor G, the antibiotic thiostrepton, and rRNA. To begin to quantitate these interactions, L11 from Bacillus stearothermophilus has been overexpressed and its properties compared with those of L11-C76 alone in a fluorescence assay for protein-rRNA binding. The assay relies on 2'-amino-butyryl-pyrene-uridine incorporated in a 58-nucleotide rRNA fragment, which gives approximately 15-fold enhancement when L11 or L11-C76 is bound. Although the pyrene tag weakens protein binding, unbiased protein-RNA association constants were obtained in competition experiments with untagged RNA. It was found that (i) intact B. stearothermophilus L11 binds rRNA with K approximately 1.2 x 10(9) m(-1) in buffers with 0.2 m KCl, about 100-fold tighter than Escherichia coli L11; (ii) the N-terminal domain makes a small, salt-dependent contribution to the overall L11-RNA binding affinity (approximately 8-fold enhancement at 0.2 m KCl), (iii) L11 stimulates thiostrepton binding by 2.3 +/- 0.6 x 10(3)-fold, predicting an overall thiostrepton affinity for the ribosome of approximately 10(9) m(-1), and (iv) the yeast homolog of L11 shows no stimulation of thiostrepton binding. The latter observation resolves the question of why eukaryotes are insensitive to the antibiotic. These measurements also show that it is plausible for thiostrepton to compete directly with EF-G.GDP for binding to the L11-RNA complex, and provide a quantitative basis for further studies of L11 function and thiostrepton mechanism.

Risk of infection with Cryptosporidium parvum and Cryptosporidium hominis in dairy cattle in the New York City watershed

To determine the risk posed by Cryptosporidium parvum and Cryptosporidium hominis from dairy cattle in the New York City watershed (NYCW). Samples from cattle at risk for shedding Cryptosporidium organisms on randomly selected dairy farms in the NYCW. Feces were collected for 4 years from calves at risk for infection on 37 dairies. Oocysts were detected by use of centrifugation concentration-flotation microscopy. The DNA was directly isolated from fecal samples and used to amplify fragments of the small subunit ribosomal RNA and thrombospondin-related adhesion protein C-2 genes by use of nested polymerase chain reaction assays. Small subunit ribosomal RNA fragments were restriction digested by the enzyme Vspl and thrombospondin-related adhesion protein C-2 fragments were digested by Eco91l to distinguish between C hominis (formerly known as genotype 1) and C parvum (formerly known as genotype 2). Of 437 fecal samples examined, 214 contained oocysts. Amplicons were generated for 200 samples. We can be certain, with 95% confidence, that cattle in the NYCW did not harbor C hominis. Cryptosporidium infections in cattle are under examination because of the potential contamination of public waters by manure. Although cattle may be the source of zoonotic infection via C parvum, they pose little risk for C hominis (the strain commonly isolated from humans in waterborne outbreaks of disease). Other sources of oocysts should be considered when investigating outbreaks attributable to contaminated urban drinking water because cattle pose only a small risk via shedding of C hominis.

Zoonotic Genotype of Giardia intestinalis Detected in a Ferret

Giardia intestinalis has been found in a variety of mammals, including humans, and consists of host-specific and zoonotic genotypes. There has been only 1 study of G. intestinalis infection in weasels, but the genotype of its isolate remains unclear. In this study, we report the isolation of Giardia in a ferret exhibited at a pet shop. The isolate was analyzed genetically to validate the possibility of zoonotic transmission. Giardia diagnostic fragments of the small subunit ribosomal RNA, beta-giardin, and glutamate dehydrogenase genes were amplified from the ferret isolate and sequenced to reveal the phylogenetic relationships between it and other Giardia species or genotypes of G. intestinalis reported previously. The results showed that the ferret isolate represented the genetic group A-I in assemblage A, which could be a causative agent of human giardiasis.

Disaccharide mimetics of the aminoglycoside antibiotic neamine.

A highly convergent approach has been employed for the facile synthesis of a library of 24 disaccharides that are alpha(1-3), beta(1-3), alpha(1-4), or beta(1-4) linked and contain 2-4 amino groups. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to determine dissociation constant (Kd) values for the binding of the disaccharides to a prototypical fragment of 16S ribosomal RNA. Several derivatives bound with affinities similar to that of neamine. Structure-activity relationships have revealed the substitution pattern that is important for high-affinity binding. The compounds described here are unique lead compounds for the design of novel aminoglycoside antibiotics.

Pneumocystis jiroveci genotypes in the Spanish population.

This study describes the genotype distribution of Pneumocystis jiroveci in 79 respiratory samples obtained from 15 patients with acquired immunodeficiency syndrome (AIDS) with P. jiroveci pneumonia and 64 human immunodeficiency virus-negative subjects with different chronic pulmonary diseases. The genotyping was based in analysis of 2 independent genetic loci: the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) fragment (assessed by direct sequencing) and the gene for dihydropteroate synthase (DHPS; assessed by restriction fragment-length polymorphism). The mt LSU rRNA analysis revealed the presence of 3 different polymorphisms for both populations. The major genotype, 85C/248C, was found to be significantly higher in patients with AIDS and P. jiroveci pneumonia than in patients with pulmonary disease. The rate of genotypes 85A/248C and 85T/248C was similar in both groups. The analysis of DHPS genotypes assesses the prevalence of its 4 possible genotypes, with 35.5% of genotypes related to sulfa resistance. The data suggest a common source of infection between both groups.

First record of Cryptosporidium infection in a raccoon dog ( Nyctereutes procyonoides viverrinus)

Cryptosporidium species have been found in more than 150 species of mammals, but there has been no report in raccoon dogs. Here we found the Cryptosporidium organism in a raccoon dog, Nyctereutes procyonoides viverrinus, and identified this isolate using PCR-based diagnostic methods. Cryptosporidium diagnostic fragments of the 18S ribosomal RNA, Cryptosporidium oocyst wall protein and 70-kDa heat shock protein genes were amplified from the isolate and sequenced to reveal the phylogenetic relationships between it and other Cryptosporidium species or genotypes reported previously. The results showed that the raccoon dog isolate represented the C. parvum cattle genotype which could be a causative agent in human cryptosporidiosis.

PHYLOGENY AND HISTORICAL BIOGEOGRAPHY OF LIMPETS OF THE ORDER PATELLOGASTROPODA BASED ON MITOCHONDRIAL DNA SEQUENCES

Using new and previously published sequences of two mitochondrial genes (fragments of 12S and 16S ribosomal RNA; total 700 sites), we constructed a molecular phylogeny for 86 extant species, covering a major part of the order Patellogastropoda. There were 35 lottiid, one acmaeid, five nacellid and two patellid species from the western and northern Pacific; and 34 patellid, six nacellid and three lottiid species from the Atlantic, southern Africa, Antarctica and Australia. Emarginula foveolata fujitai (Fissurellidae) was used as the outgroup. In the resulting phylogenetic trees, the species fall into two major clades with high bootstrap support, designated here as (A) a clade of southern Tethyan origin consisting of superfamily Patelloidea and (B) a clade of tropical Tethyan origin consisting of the Acmaeoidea. Clades A and B were further divided into three and six subclades, respectively, which correspond with geographical distributions of species in the following genus or genera: (A1) northeastern Atlantic (Patella); (A2) southern Africa and Australasia (Scutellastra, Cymbula and Helcion); (A3) Antarctic, western Pacific, Australasia (Nacella and Cellana); (B1) western to northwestern Pacific (Patelloida); (B2) northern Pacific and northeastern Atlantic (Lottia); (B3) northern Pacific (Lottia and Yayoiacmea); (B4) northwestern Pacific (Nipponacmea); (B5) northern Pacific (Acmaea and Niveotectura) and (B6) northeastern Atlantic (Tectura). Approximate divergence times were estimated using geological events and the fossil record to determine a reference date. Divergence of the two major clades likely occurred as far back as the early Cretaceous. The phylogeny also suggests that nine principal geographic clades were formed during the late Mesozoic to early Cenozoic in association with the disruption of Pangea, which gave rise to new oceans and seaways.

Molecular characterization of a Cryptosporidium isolate from a banded mongoose Mungos mungo.

Cryptosporidium spp. has been found in more than 150 species of mammals, but there has been no report in mongooses. In this study, we report the isolation of Cryptosporidium sp. in a banded mongoose Mungos mungo, which was brought from Tanzania to Japan; the isolate was analyzed genetically to validate the occurrence of a new, host-adapted genotype. Cryptosporidium diagnostic fragments of 18S ribosomal RNA and 70-kDa heat shock protein genes were amplified from this isolate and compared with the other Cryptosporidium species and genotypes reported previously. Analyses showed that the mongoose isolate represents a new genotype, closely related to that of bears.

Development of PCR-based methods for detection of Sphaerothecum destruens in fish tissues

Single-round and nested polymerase chain reaction (PCR) tests were developed for amplification of a 434 bp fragment of the small subunit ribosomal RNA (18S rRNA) gene from Sphaerothecum destruens, previously known as the rosette agent, an intracellular parasite of salmonid fishes. Both tests have successfully amplified S. destruens-specific DNA from different isolates of S. destruens but not from related organisms. The limits of detection using the nested PCR test were 1 pg for purified S. destruens genomic DNA and 0.1 fg for plasmid DNA. We conducted 2 experimental transmission studies, consisting of injection or waterborne exposure of juvenile winter-run Chinook salmon Oncorhynchus tshawytscha to spore stages of the parasite. In the injection study, parasite DNA was detected in 100% of kidney samples from exposed fish (n = 83) at 1 and 3 mo post-exposure using nested PCR, versus 98% using microscopic analysis of Gram-stained impression smears made from the kidney. Following waterborne exposure, fish were sampled over the course of a year. From each fish, samples of gill, liver, posterior intestine and kidney were analyzed. S. destruens-specific DNA was detected most often in gill and kidney over the course of the experiment, and 71% (64/90) of the exposed fish were identified as positive for S. destruens using the nested PCR test, versus 16% (14/90) using microscopic analysis of Gram-stained kidney smears. Natural infections in captive broodstock of adult winter-run Chinook salmon, originally diagnosed by examination of Gram-stained kidney smears, were confirmed using the nested PCR test in all fish examined (15/15). Further, the nested test amplified parasite-specific DNA from other tissues in these fish with varying frequencies. This report introduces the first DNA-based detection method for S. destruens, to be used alone as a diagnostic tool or in conjunction with histologic tests for confirmatory identification of the parasite.

Detecting avian malaria: an improved polymerase chain reaction diagnostic.

We describe a polymerase chain reaction (PCR) assay that detects avian malarial infection across divergent host species and parasite lineages representing both Plasmodium spp. and Haemoproteus spp. The assay is based on nucleotide primers designed to amplify a 286-bp fragment of ribosomal RNA (rRNA) coding sequence within the 6-kb mitochondrial DNA malaria genome. The rRNA malarial assay outperformed other published PCR diagnostic methods for detecting avian infections. Our data demonstrate that the assay is sensitive to as few as 10(-5) infected erythrocytes in peripheral blood. Results of avian population surveys conducted with the rRNA assay suggest that prevalences of malarial infection are higher than previously documented, and that studies based on microscopic examination of blood smears may substantially underestimate the extent of parasitism by these apicomplexans. Nonetheless, because these and other published primers miss small numbers of infections detected by other methods, including inspection of smears, no assay now available for avian malaria is universally reliable.

Molecular phylogenetic relationship between two symbiotic photo-oxygenic prokaryotes, Prochloron sp. and Synechocystis trididemni

Synechocystis trididemni is a photo-oxygenic prokaryotic symbiont in some didemnid ascidians. Although the cells are pink and contain phycoerythrin, they morphologically resemble those of another symbiont of other didemnids, Prochloron didemni, which has no phycobilin pigments but has both chlorophyll a and chlorophyll b. The cytological similarities suggest that they might have evolved from a common ancestor. We examined this hypothesis by comparing nucleotide sequences in gene fragments of the 16S ribosomal RNA (16S rRNA) gene and in the large subunit of Rubisco gene (rbcL). ‘Blast’ search of GenBank showed that the most similar sequences to the 16S rRNA gene and rbcL of S. trididemni were those of Prochloron sp. The percent nucleotide and amino acid identities of the 16S rRNA genes, rbcL and the deduced Rubisco amino acid sequences between Prochloron sp. and S. trididemni were 94%, 90% and 99%, respectively. In trees based on the 16S rRNA gene and rbcL sequences, S. trididemni formed a clade with Prochloron. These data thus support the hypothesis that Prochloron sp. and S. trididemni arose from a common cyanobacterial ancestor and that the ancestor of Prochloron sp. somehow acquired the ability to synthesize chlorophyll b, as well as chlorophyll a, and subsequently lost the ability to synthesize phycobiliproteins.