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Results for “Ribosome Profiling”

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PubMed2003

Genomic diversity and relatedness of bifidobacteria isolated from a porcine cecum.

This study initially involved the isolation of a number of bifidobacteria from either the lumen or the epithelium of a porcine cecum. A total of 160 isolates were selected at random on MRS plates containing cysteine hydrochloride (0.5 g/liter) and mupirocin (50 mg/liter). All were identified as bifidobacteria based on fructose-6-phosphate phosphoketolase activity. Following genomic digestion with the restriction enzyme XbaI and pulsed-field gel electrophoresis (PFGE), the isolates produced 15 distinct macro-restriction patterns. Several of the PFGE patterns differed by only 1, 2, or 3 DNA fragments and were grouped as related patterns into seven PFGE types, termed A through G. The related patterns appeared to show genomic plasticity within the isolates arising from chromosomal mutations or possibly horizontal transfer of plasmids. The relative frequency of each PFGE type was maintained within each cecal sample, with PFGE type E representing approximately 50% of the isolates. Randomly amplified polymorphic DNA PCR, cell morphology, whole-cell protein profiling, 16S ribosomal DNA sequencing, and DNA-DNA hybridization were used to determine if the seven apparently unrelated PFGE types represented genetically distinct isolates. Four groups were identified: PFGE types A, C/D/G, B/E, and F, and these appeared to represent Bifidobacterium minimum, Bifidobacterium pseudolongum subsp. pseudolongum, and Bifidobacterium pseudolongum subsp. globosum and two new species, respectively. The data demonstrate the presence of considerable genomic diversity within a relatively simple bifidobacteria population, consisting of 15 distinct strains representing four groups, which was maintained throughout the porcine cecal contents and epithelial layer.

Animals↗

PubMed1994

Characterization of Leptospira isolates from serovar hardjo by ribotyping, arbitrarily primed PCR, and mapped restriction site polymorphisms.

Leptospira serovar hardjo isolates of the hardjoprajitno and hardjobovis genotypes were characterized by ribotyping, arbitrarily primed PCR (AP-PCR) fingerprinting, and the study of mapped restriction site polymorphisms (MRSPs) in rrs and rrl genes. After restriction of chromosomal DNA with BglII, EcoRI, or HindIII, each genotype was individualized with a distinct ribotype. The fingerprints produced by AP-PCR with seven primers clearly separated the two groups; primers KF and RSP produced species-specific products which assigned hardjoprajitno and hardjobovis isolates to the species L. interrogans sensu stricto and L. borgpetersenii, respectively. Furthermore, AP-PCR fingerprints gave evidence of a considerable genomic heterogeneity at the strain level among the hardjobovis group. Conversely, the hardjoprajitno group was homogeneous. MRSP profiles in ribosomal genes indicated that hardjoprajitno and hardjobovis isolates belonged to L. interrogans MRSP group B and L. borgpetersenii group C, respectively. AP-PCR and determination of MRSPs in ribosomal genes proved to be quick and reliable methods for typing Leptospira strains and for studying intraspecific population structures.

Animals↗

PubMed1980

Fine structure of the parathyroid glands in baboons, Papio hamadryas in response to experimental hypercorticoidism.

Female baboons maintained under laboratory conditions were subjected to a series of 10 weekly injections (4 mg/kg body weight) of the synthetic glucocorticoid, triamcinolone hexacetonide. In response to the treatment, serum immunoreactive parathyroid hormone (PTH) levels were raised, though blood calcium levels remained within normal physiological limits. Light and electron-microscopic studies were made on the parathyroid glands at the end of the experimental period. The baboon parathyroid glands were composed of 'light' and 'dark' forms of the chief cells in varying ratios from gland to gland even within a single animal. Glucocorticoid-induced parathyroid hyperactivity as measured by circulating PTH levels was not accompanied by cellular hypertrophy, though there was an increase in the relative number of 'light' cells. At the ultrastructural level, after treatment, many of the 'light' cells were found to contain more free ribosomes, larger profiles of granular endoplasmic reticulum and had better developed mitochondria. Interdigitations between adjacent chief cells were more complex in treated glands. Apart from these features, chief cells of treated glands were basically similar to those of untreated controls. Our study showed that functional parathyroid hyperactivity in baboons is not necessarily accompanied by significant ultrastructural changes in chief cells.

Animals↗

PubMed1993

Ultrastructural similarity between bat and human mast cell secretory granules.

Mast cells in the tongue of the bat (Artibeus lituratus) show a well-developed Golgi area and abundant mitochondria in the granule-free perinuclear cytoplasm. Rough endoplasmic reticulum profiles, free ribosomes, mitochondria, bundles of filaments and a great number of secretory granules are found throughout the remaining cytoplasm. The granules, of various shapes and sizes, are simple containing an electron-dense, homogeneous matrix, coarse particles or cylindrical scrolls, or combinations (cylindrical scrolls with either electron-dense, homogeneous matrix or coarse particle contents). Up to now, scroll-containing granules have been considered to be a unique feature of human mast cells.

Animals↗

PubMed2005

Immunohistochemical analysis of Ki-67, PCNA and S6K1/2 expression in human breast cancer.

AIM: To assess the correlation between the expression profiles of ribosomal protein S6 kinase (S6K1/2), Ki-67 nuclear antigen (Ki-67) and proliferating cell nuclear antigen (PCNA) in human breast adenocarcinomas. METHODS: The expression pattern of S6K1/2, Ki-67 and PCNA has been investigated by immunohistochemical analysis of formalin fixed paraffin embedded sections of 40 human breast adenocarcinomas. Analysis was performed using specific monoclonal and polyclonal antibodies directed against S6K2, Ki-67 and PCNA followed by semi-quantitative measurements. RESULTS: Positive correlation between nuclear staining for S6K2 and PCNA (p < or = 0.01) or Ki-67 (p < or = 0.01) in breast cancer cells has been detected. No significant correlation in nuclear staining was observed between S6K1 and PCNA or Ki-67. In addition, we have detected an increase in S6K2 expression in the nuclei of cancer cells localized predominantly in peripheral areas of the tumor contacting with normal tissue. CONCLUSION: The accumulation of S6K2, but not S6K1, in the nuclei of cancer cells and the correlation with the expression of PCNA and Ki-67 suggest the involvement of S6K2 in the regulation of malignant growth.

Biomarkers, Tumor↗

PubMed1982

The ultrastructure of the centroacinar cells within the pancreas of the starling (Sturnus vulgaris).

Tissues from the dorsal lobe of the pancreas of 8 starlings (Sturnus vulgaris) were examined electron microscopically using conventionally prepared samples. Centroacinar cells were distinguished from exocrine pancreas cells by differences in size, shape, distribution, position and zymogen granule content. The centroacinar cells closely resembled ductal cells and were separated from them only by position. Both electron-lucent and electron-dense forms of centroacinar cells were observed. Centroacinar cells show a prominent single nucleus, often displaying at least one nucleolus, surrounded by cytoplasm in which lies a spectrum of both primary and secondary lysosomes, mitochondria which appear to have some tubulovesicular cristae amongst the predominating transverse cristae, a sparse para- or supranuclear Golgi apparatus, and variable populations of ribosomes and profiles of both rough and smooth endoplasmic reticulum. Cell surface features include microvilli, 'blebs' and the occasional single cilium. Centroacinar cells are believed to be involved in electrolyte secretion although, from a study of their ultrastructure, the absence of beta cytomembranes with numerous mitochondria in their neighbourhood, would appear to contra-indicate the maintenance of an osmotic gradient between the extracellular fluid and any secretory product.

Animals↗

PubMed1983

Radioactive puromycin formation as an assay for the proportion of active ribosomes in mammalian cells: correlation with polysome profiles under different physiological conditions.

We have established optimal conditions for the in vitro formation of peptidyl-[3H] puromycin by mammalian ribosomes. The growth conditions of cultured Ehrlich ascites tumor cells were manipulated to produce changes in the polysome profiles. The correlation between polysome content and peptidyl-[3H] puromycin formation was linear and excellent when different cell densities were compared. The percentage of ribosomes actively engaged in protein synthesis, calculated from the number of 3H-peptide bonds formed, was similar in rapidly growing Ehrlich cells (47%) and in young rat gastrocnemius muscle (44%). Starvation resulted in a 50% reduction in the number of puromycin-reactive ribosomes in rat gastrocnemius.

Animals↗

PubMed1967

Ribosomes and ribosomal protein from Neurospora crassa. I. Physical, chemical, and immunochemical properties.

Ribosomes from Neurospora crassa, initially characterized by ultracentrifugal and immunochemical analyses, have been used to prepare ribosomal protein for physical, chemical, and immunochemical study. The acrylamide gel disc electrophoretic profiles of Neurospora ribosomal protein exhibit a degree of heterogeneity comparable to what has been observed in other systems. Only by chemical modification or by aggregation of the protein do alterations in the profile become apparent. Disulfide-bond formation appears to play a role in the aggregation of ribosomal protein to complexes of S(20,w) = 200. The aggregation can be prevented by alkylation of -SH groups, and protein treated in this fashion has a subunit molecular weight of about 20,000 as determined by equilibrium centrifugation. Finger-printing of tryptic peptides indicates that more than one unique sequence of amino acids must be present in ribosomal protein, although gross primary structural heterogeneity is questioned. Antigenic heterogeneity is much less apparent; only a few precipitin bands are resolved by immunodiffusion tests, although complete reactivity of total ribosomal protein is suggested by quantitative precipitin analysis. The antigenically active ribosomal protein components appear to reside in at least two fractions; one is removed readily from the ribosome by CsC1 treatment. Ribosomal protein of N. crassa possesses antigenic determinants present in E. coli ribosomal protein as judged by spur formation in immunodiffusion tests.

Amino Acids↗

PubMed2002

Microbial community analysis of thermophilic contact oxidation process by using ribosomal RNA approaches and the quinone profile method.

Microbial community structure of a lab scale thermophilic aerobic wastewater treatment reactor was analyzed by a combination of culture-independent methods. Quinone profile method provides for chemical analysis of respiratory quinone molecular species, which corresponds to bacterial groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA partial sequences (PCR-DGGE) clarifies community changes at species level, as DGGE can separate DNA fragments of different sequences. Certain phvlogenetic groups of bacterial cells can be labeled by fluorescence in situ hybridization (FISH). Quinone profile showed a predominant presence of MK-7. PCR-DGGE revealed that constituents of the community were unchanged during the stable phase. FISH demonstrated the existence of the relatives of Bacillus lentus and B. thermocloacae in considerable proportions. The community was mainly composed of Bacillaceae, and obligate thermophilic and mesophilic Bacillus appeared in spite of the temperature fluctuation from 35 degrees C to 60 degrees C. The combination of these culture-independent methods revealed the community precisely enough to evaluate the reactor performance.

Bacillaceae↗

PubMed1967

Decomposition of ribosomal particles in Escherichia coli treated with mitomycin C.

Exposure of cells of Escherichia coli to mitomycin C (5 mug/ml) resulted in a marked change in the sedimentation profiles of the cell-free extracts, indicating a specific decomposition of ribosomal particles. When the extracts were prepared in the presence of 0.01 m Mg(++) and analyzed by sucrose density gradient centrifugations, the 100S fraction disappeared rapidly from the treated cells. The 70S ribosomes were also degraded, but more slowly, with a concomitant accumulation of a fraction having a sedimentation coefficient of about 50S. However, decomposition of the 70S ribosomes was preceded by an almost complete loss of the 50S ribosomal subunits, as revealed by sedimentation analyses in the presence of 10(-4)m Mg(++). Synthesis of the ribosomes in the treated cells was also suppressed, being demonstrated by a lower incorporation of uracil-2-(14)C into the ribosomal fractions. However, the change in the ribosomal profile in the treated cells apparently resulted from the decomposition of pre-existing ribosomes, rather than from the inhibition of the net synthesis of ribosomes. Sedimentation analyses and chromatography of the nucleic acids extracted from the treated cells indicated extensive but delayed degradation of the ribosomal ribonucleic acid (RNA), but not of the soluble RNA or deoxyribonucleic acid fractions. Altered structure of the ribosomes in the treated cells was also indicated by their lower melting temperature, broadened thermal profile, higher electrophoretic mobility, and extreme sensitivity to ribonuclease treatment, compared with normal ribosomes. The synthesis of messenger RNA was inhibited progressively with time in the treated cells.

Carbon Isotopes↗

PubMed1971

Characterization of mitochondrial ribosomes from yeast.

A method for the preparation of mitochondrial ribosomes from yeast is presented. Mitochondrial ribosomes differ from their cytoplasmic counterpart in nucleotide composition, electrophoretic mobility of a number of their respective ribosomal proteins, and thermal denaturation profiles. The mitochondrial and cytoplasmic ribosomes have identical sedimentation coefficients (80 S) and dissociate into 60S and 40S subunits. These findings demonstrate that, in yeast, mitochondrial ribosomes are distinct from cytoplasmic ribosomes.

Cytoplasm↗

PubMed1993

Characterization of unrelated strains of Staphylococcus schleiferi by using ribosomal DNA fingerprinting, DNA restriction patterns, and plasmid profiles.

The molecular characteristics of 31 unrelated strains of Staphylococcus schleiferi isolated from 13 hospitals between 1973 and 1991 were determined by ribosomal DNA fingerprinting by using a digoxigenin-labeled DNA probe, genomic DNA restriction patterns, and plasmid profiles. Only six strains harbored one or two plasmids. DNA restriction analysis, which was carried out with five endonucleases (EcoRI, HindIII, PstI, PvuII, and ClaI), did not allow us to discriminate between isolates. Ribotyping with HindIII, ClaI, or EcoRI enzymes generated six, seven, and nine distinct patterns, respectively. With the combination ClaI-EcoRI, 13 ribotypes were obtained among the 31 strains, suggesting a relative heterogeneity within the species. Moreover, all strains shared two or three common bands, according to the endonuclease used, which were relatively specific for S. schleiferi in comparison with the ribosomal banding patterns described for other coagulase-negative staphylococci. These results illustrate that ribotyping can be used for the epidemiological investigation of S. schleiferi isolates and possibly for taxonomic analysis in this species.

DNA Fingerprinting↗

PubMed1968

Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus.

1. Spermine and spermidine were the main polyamines detectable in Bacillus stearothermophilus. 2. When grown at 65 degrees B. stearothermophilus contained lower concentrations of polyamines per mg. of RNA than when grown at 45 degrees or at 55 degrees . 3. Ribosomes isolated from B. stearothermophilus in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride contained sufficient polyamines to neutralize between 4% and 9% of their RNA phosphorus. 4. Removal of polyamines from the ribosomes by dialysis against m-potassium chloride did not appreciably alter the hypochromicity or thermal denaturation profiles of the ribosomes when measured in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride, though it did cause a loss of ribosome particles sedimenting at greater than 78s. 5. When ribosomes were dialysed against acridine orange solutions acridine orange bound to the ribosomes and did not displace spermine, but when a mixture of ribosomal RNA and spermine was dialysed against acridine orange the acridine orange displaced the spermine. It is concluded that polyamines in the ribosomes are less accessible for displacement by acridine orange than when polyamines are bound to ribosomal RNA.

Acridines↗

PubMed1980

Denaturation mapping of the ribosomal DNA of Saccharomyces cerevisiae.

The thermal melting profile of purified Saccharomyces cerevisiae ribosomal DNA (rDNA) is biphasic indicating considerable intramolecular heterogeneity in base composition. The first phase of the transition, about 20% of the total hyperchromic shift, has a Tm of 80.6 degrees C and the second phase has a Tm of 87.3 degrees C, corresponding to GC contents of 28 and 44%, respectively. The Tm of the nonribosomal nuclear DNA, called alpha DNA, is 85.7 degrees C. This heterogeneity in GC distribution in the rDNA is also reflected in its denaturation map. A denaturation map of the 5.6 X 10(6) dalton rDNA SmaI restruction fragment, which represents monomer units of the rDNA, shows that specific regions of the repeating unit denature more readily than the remainder and apparently have a significantly higher AT content. By aligning the rDNA denaturation map with the restriction endonuclease map, we have been able to determine that the AT-rich segments are localized in the transcribed and nontranscribed spacer regions of the rDNA repeating unit. Buoyant density determinations of individual rDNA restriction fragments corroborate the locations of AT-rich regions. A denaturation map of the tandem repeating units in higher molecular weight rDNA has also been constructed and compared with the map of the SmaI fragment. The results show that the repeating units are uniform in size, that they are not separated by large heterogeneous regions, and that they are arranged in head-to-tail array.

Base Composition↗

PubMed2006

Comparison of amplified ribosomal DNA restriction analysis, peptidoglycan hydrolase and biochemical profiles for rapid dairy propionibacteria species identification.

Species of dairy propionibacteria are used as cheese-ripening cultures as well as probiotics. However, no rapid identification methods are currently available. With this in mind, the present study compared three methods, (i) carbohydrate fermentation, (ii) ARDRA (amplified ribosomal DNA restriction analysis) and (iii) peptidoglycan hydrolase (PGH) activity profiles to improve the identification of Propionibacterium thoenii, Propionibacterium jensenii, Propionibacterium acidipropionici and Propionibacterium microaerophilum. The species Propionibacterium freudenreichii and Propionibacterium cyclohexanicum have previously been shown to be easily distinguishable from the other species. Principal component analysis of the carbohydrate fermentation profiles of 113 P. thoenii, P. jensenii, P. acidipropionici and P. microaerophilum strains correctly classified 85% of the strains based on the fermentation of seven carbohydrates. Regarding PGH profiles, optimized conditions of PGH-renaturing SDS-PAGE were applied to 34 of the strains. The PGH profiles of P. acidipropionici and P. microaerophilum were indistinguishable from one another, but were easily distinguished from P. jensenii and P. thoenii. However, four strains exhibited atypical profiles. Hence, in general, the PGH profiles were shown to be conserved within a species, with some exceptions. Four endonucleases were tested for ARDRA and the four species differentiated by combining the profiles obtained with MspI and HaeIII. P. freudenreichii and P. cyclohexanicum profiles were also performed but showed wide differences. Consequently, ARDRA was shown to be the most appropriate method for rapidly distinguishing strains of propionibacteria. Carbohydrate fermentation and peptidoglycan hydrolase activity profiles are useful as complementary identification tools, since about 15% of the 34 strains tested showed atypical profiles.

Bacterial Typing Techniques↗

PubMed2004

Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet).

The composition of microbial communities on and within seeds may effect their storage and field performance, whether they are indigenous or applied as biocontrol agents. In this study, we have explored the usefulness of profiling small subunit ribosomal (SSR) gene fragments for studying the microflora associated with seeds. DNA was amplified by the polymerase chain reaction (PCR) and the amplicons separated using denaturing gradient gel electrophoresis (DGGE). Primers targeting eukaryotic SSRs were used to investigate fungal communities, and primers targeting bacterial SSRs were employed to study the eubacterial microflora. As a case study, we attempted to profile the fungi and bacteria associated with seeds of Beta vulgaris (sugar beet) to permit an insight into the varying field performance of several well-characterised commercial seed lots. Serious interference with the microbial signals was observed from the plant's own nuclear 18S rRNA genes and chloroplast 16S rRNA genes using standard PCR conditions and DNA extracted from whole seeds as template. Hot-start and touchdown PCR made no appreciable improvement to these signals. Seed imbibition and dissection into operculum and fruit wall and true seed prior to DNA extraction improved signal recovery in the fruit fraction. With primer modification, bacteria and fungi were detected in an excess of plant DNA of 100:1 and 10:1, respectively. With this method, microbial communities on seeds could be profiled, however, it is likely that targeted depletion of plant rDNA targets will be a necessary extra step before this approach can be used to screen seeds routinely.

Beta vulgaris↗

PubMed2003

The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli.

RbfA, a 30S ribosome-binding factor, is a multicopy suppressor of a cold-sensitive C23U mutation of the 16S rRNA and is required for efficient processing of the 16S rRNA. At 37 degrees C, DeltarbfA cells show accumulation of ribosomal subunits and 16S rRNA precursor with a significantly reduced polysome profile in comparison with wild-type cells. RbfA is also a cold-shock protein essential for Escherichia coli cells to adapt to low temperature. In this study, we examined its association with the ribosome and its role in 16S rRNA processing and ribosome profiles at low temperature. In wild-type cells, following cold shock at 15 degrees C, the amount of free RbfA remained largely stable, while that of its 30S subunit-associated form became several times greater than that at 37 degrees C and a larger fraction of total 30S subunits was detected to be RbfA-containing. In DeltarbfA cells, the pre-16S rRNA amount increased after cold shock with a concomitant reduction of the mature 16S rRNA amount and the formation of polysomes was further reduced. A closer examination revealed that 30S ribosomal subunits of DeltarbfA cells at low temperature contained primarily pre-16S rRNA and little mature 16S rRNA. Our results indicate that the cold sensitivity of DeltarbfA cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature. Importantly, when the C-terminal 25 residue sequence was deleted, the resulting RbfADelta25 lost the abilities to stably associate with the 30S subunit and to suppress the dominant-negative, cold-sensitive phenotype of the C23U mutation in 16S rRNA but was able to suppress the 16S rRNA processing defect and the cold-sensitive phenotype of the DeltarbfA cells, suggesting that RbfA may interact with the 30S ribosome at more than one site or function in more than one fashion in assisting the 16S rRNA maturation at low temperature.

Cell Division↗

PubMed2006

A new approach for separating low-molecular-weight RNA molecules by staircase electrophoresis in non-sequencing gels.

Low-molecular-weight (LMW) RNA profiles, which include ribosomal and transfer RNA molecules with similar small sizes, are molecular signatures of microorganisms with a great potential in microbial identification. The greatest resolution of these profiles was achieved by staircase electrophoresis in sequencing gels. Nevertheless, this technique is difficult to use because it takes 7 h, the gels have large sizes and it is necessary to heat the system and to recycle the buffer to maintain the denaturing conditions and avoid smile effects. Most available sequencing slabs have no internal temperature control or homogenizing devices, which by contrast are present in some newly designed non-sequencing slabs. Nevertheless, these slabs present two important problems for separating LMW RNA molecules, the size of gels is only 20 cm (instead of 40 cm) and the maximum voltage that can be reached is only 840 V (instead 2400 V). Staircase electrophoresis follows a model in which the external polarization is incrementally modified with a constant time step value. In the present work, we experimentally confirmed that by reducing the time step and increasing the total number of steps a suitable resolution is achieved. Under these conditions, despite the smaller size of the gels and the lower values of the electric field, the intensity reaches higher values than in sequencing gels and the LMW RNA profiles are correctly separated in 5 h. The resolution of these profiles obtained in non-sequencing gels is similar to that obtained in sequencing ones facilitating the analysis of large populations of microorganisms in any laboratory.

Bacteria↗