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Results for “Ribosome Profiling”

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PubMed1990

[The pineal ultrastructure of the Mongolian ground squirrel, Citellus dauricus].

The distal part of pineal gland of the Mongolian ground squirrel was ultrastructurally studied. The gland was composed of low electron-dense parenchymal cells, among which glial cells, pigment cells, blood vessels and neural elements were occasionally interspersed. The pinealocytes contained numerous mitochondria, lysosomes, microtubules, microfilaments, Golgi apparatus and free ribosomes, as well as less prominent profiles of rough- and smooth-surfaced endoplasmic reticula and some cilia, centrioles, synaptic ribbons and few subsurface cisterns. Some pinealocytes were vacuolated. The content of the vacuoles released into the extracellular space by exocytosis could be observed. The gap junctions between pinealocytes were also observed. Of particular interest was that many mitochondria "fused together" and formed gap junction-like structure in about five percent pinealocytes. The pigment cell has a amorphous nucleus which contains many aggregated chromatin, its cell membrane has a few microvilli projecting into a central lumen, these features may indicated that this kind of cell differs either from the pinealocyte or astrocyte. There are axo-axonic synapses or axo-dendritic synapses between neuron processes or between neuron processes and pinealocytes.

Animals↗

PubMed1982

The fine structure of normal lymphocyte subpopulations--a study with monoclonal antibodies and the immunogold technique.

The ultrastructural characteristics of normal lymphocyte subpopulations, identified by monoclonal antibodies and visualized by a colloidal gold labelled anti-mouse IgG were analysed. Our study demonstrates: (1) the major T lymphocyte subsets (OKT4+ and OKT8+) have distinct ultrastructural morphology. The majority of OKT4+ cells have a high nuclear/cytoplasmic ratio (N/C) and few cytoplasmic organelles whilst most OKT8+ cells have a low N/C ratio and numerous organelles, namely a well developed Golgi apparatus, lysosomal granules and parallel tubular arrays (PTA); (2) a unique subtype with irregular nuclear outline that resembles Sézary cells was seen in 5-10% of OKT4+ lymphocytes; (3) OKM1, a reagent that reacts with monocytes and granulocytes, is positive in a small lymphocyte subset which appears to be negative with the OKT reagents and is morphologically identical to OKT8+ cells; (4) 'hand-mirror' cells were only seen labelled with OKT8 and OKM1; (5) B lymphocytes labelled with FMC4 (anti-IA) could be distinguished from OKT3+ lymphocytes by having numerous profiles of endoplasmic reticulum (ER) and ribosomes; these were particularly prominent in lymphoplasmacytoid cells. Morphological similarities between normal T lymphocyte subsets and T neoplasias of the same membrane phenotype suggest that these disorders arise from specific T cell types present in normal peripheral blood or from common precursors.

Antibodies, Anti-Idiotypic↗

PubMed1980

[Ultrastruct characteristics of neurons and synapses in the central cortex during circulatory hypoxia].

During short-term (3.7 and 15 min) circulatory hypoxia (ischemia) pronounced ultrastructural changes, having an obvious focal character, were observed in the rabbit cerebral cortex. In some regions, the extracellular space was widened; in the neurons, changes are seen both in the nucleus (appearance of polymorphic vacuoles and deep invaginations of nuclear envelope, aggregation of chromatin) and cytoplasmic organelles (mitochondrial swelling, swelling of profiles of endoplasmic reticulum, dislocation of ribosomes). Most striking reactions were observable in synapses - the number of synaptic vesicles was sharply reduced, its location in the axon terminal changed, the dense material associated with pre- and postsynaptic membranes was enlarged, mitochondria were swollen. The degree of the above alterations was proportional to the duration of ischemia.

Animals↗

PubMed1976

Segregation of Col Ib and drd7 into minicells.

The wild-type plasmid ColIb and its mutant drd7 derepressed in conjugation were transferred to Escherichia coli K12 P678-54 which produces minicells. Fertility functions of drd7 remained derepressed in the new host. P678-54drd7 transmitted the plasmid at a high frequency (28.6%) and it was effectively lysed by the phage If1. Significant amounts of 3H-DNA segregated from P67854Col+ into minicells dependent upon the presence of the plasmid. The depressed plasmid segregated more effectively into minicells than the wild-type plasmid. ColIb segregated into 2% whereas ColIbdrd7 into 8.4% of minicells. The difference in the frequency of segregation of the wild-type and the derepressed plasmid indicated different cell membrane attachment sites of each plasmid studied. Mini-drd7 were able to transfer the plasmid to E. coli Row at a low frequency (0.1%). Minicells carrying either of plasmid were capable to synthesize RNA and protein. RNA and protein synthesis were plasmid specific and the precursors were not incorporated into minicells without plasmids. Rifampin and chloramphenicol inhibited RNA and protein synthesis in minicells, respectively. The more effective incorporation of 3H-uridine or 14C-leucine into minicells harboring drd7 than ColIb resulted presumably from the high efficiency of the segregation of drd7 into minicells. Polyacrylamide gel electrophoresis of 3H-RNA has shown that plasmids in minicells were able to code low molecular RNA of 4s. No 16 or 23 ribosomal RNA was found in the profiles of de novo synthesized RNA in minicells.

Bacterial Proteins↗

PubMed1987

Effects of ribosome dissociation on the structure of the ribosome-associated 5.8S RNA.

Diethyl pyrocarbonate reactivity and thermal denaturation were used to probe potential ribosomal interactions between tRNA and the small 5.8S and 5S rRNAs. Puromycin, an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, was observed to increase the accessibility of the 5.8S rRNA, including the highly conserved GAACp sequences. EDTA which releases both tRNA and the 5S rRNA-protein complex resulted in an even greater accessibility in the 5.8S rRNA. The thermal dissociation of whole ribosomes resulted in the release of all three RNAs, with a striking similarity in the denaturation profiles. These results strongly suggest an interdependence in the ribosome-associated structures of the small rRNAs and provide in situ evidence for the various 5S rRNA, 5.8S rRNA, and tRNA containing ribonucleoprotein complexes previously reconstituted through affinity chromatography.

Base Sequence↗

PubMed2005

Characterization of the Bacillus subtilis GTPase YloQ and its role in ribosome function.

We present an analysis of the cellular phenotype and biochemical activity of a conserved bacterial GTPase of unknown function (YloQ and YjeQ in Bacillus subtilis and Escherichia coli respectively) using a collection of antibiotics of diverse mechanisms and chemical classes. We created a yloQ deletion strain, which exhibited a slow growth phenotype and formed chains of filamentous cells. Additionally, we constructed a conditional mutant in yloQ, where growth was dependent on inducible expression from a complementing copy of the gene. In phenotypic studies, depletion of yloQ sensitized cells to antibiotics that bind at the peptide channel or peptidyl transferase centre, providing the first chemical genetic evidence linking this GTPase to ribosome function. Additional experiments using these small-molecule probes in vitro revealed that aminoglycoside antibiotics severely affected a previously characterized ribosome-associated GTPase activity of purified, recombinant YjeQ from E. coli. None of the antibiotics tested competed with YjeQ for binding to 30 or 70 S ribosomes. A closer examination of YloQ depletion revealed that the polyribosome profiles were altered and that decreased expression of YloQ led to the accumulation of ribosomal subunits at the expense of intact 70 S ribosomes. The present study provides the first evidence showing that YloQ/YjeQ may be involved in several areas of cellular metabolism, including cell division and ribosome function.

Anti-Bacterial Agents↗

PubMed2004

Organization, differential expression and methylation of rDNA in artificial Solanum allopolyploids.

Uniparental activity of ribosomal RNA genes (rDNA) in interspecific hybrids is known as nucleolar dominance (ND). To see if difference in rDNA intergenic spacers (IGS) might be correlated with ND, we have used artificial Solanum allopolyploids and back-crossed lines. Combining fluorescence in situ hybridization and quantification of the level of the rRNA precursor by real-time PCR, we demonstrated that an expression hierarchy exists: In leaves, roots, and petals of the respective allopolyploids, rDNA of S lycopersicum (tomato) dominates over rDNA of S. tuberosum (potato), whereas rDNA of S. tuberosum dominates over that of the wild species S. bulbocastanum . Also in a monosomic addition line carrying only one NOR-bearing chromosome of tomato in a potato background the dominance effect was maintained. These results demonstrate that there is possible correlation between transcriptional dominance and number of conservative elements downstream of the transcription start in the Solanum rDNA. In anthers and callus tissues under-dominant rDNA was slightly (S. lycopersicum/S. tuberosum) or strongly (S. tuberosum/S. bulbocastanum) expressed indicating developmental modulation of ND. In leaves and petals, repression of the respective parental rDNA correlated with cytosine methylation at certain sites conserved in the IGS, whereas activation of under-dominant rDNA in anthers and callus tissues was not accompanied by considerable changes of the methylation pattern.

Base Sequence↗

PubMed2006

Seasonal influences on fungal community structure in unimproved and improved upland grassland soils.

Seasonal and management influences on the fungal community structure of two upland grassland soils were investigated. An upland site containing both unimproved floristically diverse (U4a) and improved mesotrophic (MG7b) grassland types was selected. Samples from both grassland types were taken at five times in one year. Soil fungal community structure was assessed using fungal automated ribosomal intergenic spacer analysis (ARISA), a DNA-profiling approach. A grassland management regime was found to strongly affect fungal community structure, with fungal ARISA profiles from unimproved and improved grassland soils differing significantly. The number of fungal ribotypes found was higher in unimproved than improved grassland soils, providing evidence that improvement may reduce the suitability of upland soil as a habitat for specific groups of fungi. Seasonal influences on fungal community structure were also noted, with samples taken in autumn (October) more correlated with change in ribotype profiles than samples from other seasons. However, seasonal variation did not obscure the measurement of differences in the fungal community structure that were due to agricultural improvement, with canonical correspondence analysis indicating grassland type had a stronger influence on fungal profiles than did season.

Analysis of Variance↗

PubMed1977

Iodination-deiodination. A radiochemical method for detection of structure and changes in structure in RNA.

Bound iodine is released from radioiodinated nucleotides in polymers exposed to sodium bisulfite. The rate of bisulfite-catalyzed deiodination of pyrimidines can be controlled both by change of temperature of pH and is also dependent on the molecular association of the nucleotide. The rate of release of iodine from iodocytidine in polycytidylate is greater than the rate of elimination from RNA. Experiments testing the influence of base-pairing of the iodopyrimidines in synthetic polynucleotides showed that pairing of the substituted nucleotide protected the iodine bond. The rates of bisulfite-catalyzed deiodination of several radioiodinated RNAs were measured. The action of bisulfite on all single stranded RNAs tested was multiphasic consisting of a rapid early deiodination reaction supplanted by a slower phase which was followed by reacceleration of release. The release of iodine from double stranded RNA and DNA-RNA duplexes was retarded in comparison with the release from ribosomal and messenger RNA fractions. The deiodination profiles of single and double stranded RNA suggested that the intermediate stage iodine release is governed by melting of paired zones of low stability. Late release may result from destablization of the molecule through the addition of bisulfite to the pyrimidine ring or deamination. The effect of several substances expected to complex with polynucleotides was tested. Acridine orange and ethidium bromide increased loss of iodine from ribosomal RNA but slightly decreased elimination from double stranded viral RNA. A basic protein fraction isolated from ribosomal particles accelerated the deiodination of ribosomal RNA. While the destabilization caused by this protein fraction was greater than that caused by an equal amount of albumin, as tested the effect was non-specific. The results show that a change in sensitivity to chemical deiodination may folow the interaction of small amounts of protein with polynucleotides.

Animals↗

PubMed2002

Characterisation of the first Australian isolate of Neospora caninum from cattle.

OBJECTIVE: To isolate Neospora caninum from a congenitally infected calf. PROCEDURE: A calf was obtained from a N. caninum infected dam maintained in a dairy herd of Holstein-Friesian cattle located on the south coast of NSW near Nowra. The calf was euthanased and samples collected for serology and pathology. Samples of brain and spinal cord of the calf were homogenised and injected into immunocompromised mice in an attempt to recover protozoa by in vivo culture. Sequential passage of brain homogenate through IFNgammaPKO mice was performed and tissue culture flasks were inoculated with brain homogenate. Parasites were identified by electron microscopy and DNA sequencing. The antigen profile of the isolate was analysed using Western blotting. Pathogenicity was examined in BALB/c mice and transmission of the parasite during pregnancy was examined in Qs mice. RESULTS: The calf was seropositive for N. caninum and histopathological examination of sections of cerebrum identified lesions consistent with a very mild infection with N. caninum. The parasites isolated using tissue culture were identified as N. caninum, based on the sequence of the ribosomal DNA and electron microscopy. The antigen profile of the new isolate was similar to that of the NC-Liverpool isolate, but quite different from that of Toxoplasma gondii. In BALB/c mice inoculated with the new isolate, severe clinical signs developed in only three of ten infected mice, compared with six of ten mice infected with NC-Liverpool. Mild to moderate nonsuppurative encephalitis was observed in BALB/c mice infected with the new isolate, compared with mice infected with NC-Liverpool, that developed severe nonsuppurative encephalitis. Transplacental transmission of the isolate arising from an acute infection during pregnancy occurred in about 87% of pups. CONCLUSION: This is the first isolation of bovine Neospora caninum in Australia. This isolate, called NC-Nowra, appears to be a less virulent form and may prove to be a suitable candidate for vaccine development.

Abortion, Veterinary↗

PubMed1981

Analytical fractionation of human liver microsomal fractions: localization of cholesterol and of the enzymes relevant to its metabolism.

1. The submicrosomal distribution of three enzymes concerned in cholesterol metabolism, and of free and esterified cholesterol, was determined in human liver by analytical isopycnic centrifugation on sucrose gradients. 2. The distribution profile and median density of acyl-CoA:cholesterol O-acyltransferase was similar to that of RNA, showing that this enzyme is confined largely to the ribosome-rich membranes of the endoplasmic reticulum. The distribution profiles and median densities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7 alpha-mono-oxygenase showed that both enzymes are confined to the smooth, ribosome-poor, endoplasmic reticulum. 3. Most of the free cholesterol in the microsomal preparations was present in smooth membranes from the Golgi apparatus and in vesicles from plasma-membrane fragments. The distribution of esterified cholesterol was multimodal and extended throughout the whole gradient.

Adult↗

PubMed2005

Uncovering the enzymatic pKa of the ribosomal peptidyl transferase reaction utilizing a fluorinated puromycin derivative.

The ribosome-catalyzed peptidyl transferase reaction displays a complex pH profile resulting from two functional groups whose deprotonation is important for the reaction, one within the A-site substrate and a second unidentified group thought to reside in the rRNA peptidyl transferase center. Here we report the synthesis and activity of the beta,beta-difluorophenylalanyl derivative of puromycin, an A-site substrate. The fluorine atoms reduce the pK(a) of the nucleophilic alpha-amino group (<5.0) such that it is deprotonated at all pHs amenable to ribosomal analysis (pH 5.2-9.5). In the 50S modified fragment assay, this substrate reacts substantially faster than puromycin at neutral or acidic pH. The reaction follows a simplified pH profile that is dependent only upon deprotonation of a titratable group within the ribosomal active site. This feature will simplify characterization of the peptidyl transferase reaction mechanism. On the basis of the reaction efficiency of the doubly fluorinated substrate compared to the unfluorinated derivative, the Bronsted coefficient for the nucleophile is estimated to be substantially smaller than that reported for uncatalyzed aminolysis reactions, which has important mechanistic implications for the peptidyl transferase reaction.

Catalysis↗

PubMed1978

Partial purification and evidence for multiple molecular forms of the scrapie agent.

A procedure for the partial purification of the scrapie agent from mouse spleen was developed based on its sedimentation profile. Differential centrifugation and detergent treatment with sodium deoxycholate yielded a fraction designated "P5" which was enriched for scrapie infectivity approximately 20-fold with respect to cellular protein. The P5 fraction was devoid of cellular membranes but heavily contaminated with ribosomes as judged by electron microscopy. On centrifugation of the fraction P5 to near equilibrium in a sucrose gradient scrapie infectivity was distributed over a range of densities from 1.08 to 1.30 g/cm3. Parallel rate-zonal analysis showed that the infectivity was distributed over a range of particle sizes with s20.w values from approximately 40 S to greater than 500 S. Incubation of P5 at 37 or 80 degrees C, under conditions that disrupt ribosomes, dramatically altered the rate-zonal gradient profile of the agent. Under these conditions, the agent sedimented as particles with s20.w greater than 500 S. The apparent heterogeneity of the scrapie agent with respect to both size and density and its ability to shift from one form to another suggest that the agent may contain hydrophobic domains on its surface.

Animals↗

PubMed2001

Primary structure and transcription patterns of RPL36, a ribosomal protein-encoding gene of the mycoparasitic fungus, Trichoderma hamatum.

We report the isolation and expression profiles of a single-copy gene from the mycoparasitic fungus Trichoderma hamatum encoding a 60 S cytoplasmic ribosomal protein. The gene, named RPL36, was cloned through its nutrient-mediated expression, using mRNA differential screening. Its predicted ORF, interrupted by two introns, encoded a 105-aa polypeptide. The deduced rpL36 protein showed high overall homologies with other L36-type ribosomal proteins isolated from yeast, rat and human. Analysis of the promoter region of RPL36 revealed the presence of two ribosomal protein gene (RPG) boxes and a T-rich region known to be involved in the regulation of most ribosomal protein genes. Expression of RPL36 was tightly regulated by carbon and nitrogen availability. The mRNA levels of this gene decreased upon exposure of the mycelium to different stresses, whereas the addition of cycloheximide resulted in a super-induction. Levels of RPL36 transcripts also increased during mycoparasitic interaction between T. hamatum and Botrytis cinerea.

Amino Acid Sequence↗

PubMed2005

Evaluation of telithromycin against Streptococcus pneumoniae with ribosomal mutations utilizing in vitro time-kill methodology.

In a recent study, our in vivo data suggested that clinically achievable levels of telithromycin are more effective than azithromycin against selected Streptococcus pneumoniae isolates with ribosomal mutations in 23S rRNA gene alleles and L22 region mutations. In the current study, we attempt to investigate further the antibacterial activity of telithromycin against these isolates to better delineate the disparity between isolates based on allelic differences. Four isolates of S. pneumoniae with ribosomal mutations were tested using in vitro time-kill methodology. Isolates were exposed to telithromycin at concentrations of 0.5-8 x the minimum inhibitory concentration (MIC). At these exposures, telithromycin demonstrated concentration-dependent killing for three of the four isolates. Against the fourth isolate, telithromycin affected only a 1 log decrease in colony-forming units/mL despite exposures of 8 x MIC. These data demonstrate the in vitro killing profile of telithromycin against S. pneumoniae isolates with ribosomal and L22 mutations. Whilst telithromycin did not demonstrate bactericidal activity against all isolates in these time-kill studies, the in vivo human-simulated exposures did result in a high degree of bacterial kill. Full evaluation of the potential utility of new antimicrobial agents against these emerging genotypic profiles requires both in vitro and in vivo assessments.

Ketolides↗

PubMed1991

Leishmania: genus identification based on a specific sequence of the 18S ribosomal RNA sequence.

The analysis of PvuII restriction patterns of Leishmania spp. and Trypanosoma spp. genomic DNA showed genus distinctive profiles. A specific PvuII site was detected in the 5' domain of 18S ribosomal DNA of Leishmania. A 20-mer oligonucleotide encompassing this PvuII region was synthesized. This sequence, when utilized as probe, on short exposures of dot tests, detected 10(3) whole promastigotes of all Leishmania species analyzed but did not hybridize with T. cruzi or human nucleic acids. Two other oligonucleotides were synthesized to be used as primers for amplification through polymerase chain reaction of the 18S ribosomal DNA region containing the PvuII site. The probes described may be useful for the detection of Leishmania spp. under clinical and epidemiological trials.

Animals↗

PubMedDate not supplied

Anatomic evidence suggestive of dendrodendritic synapses in the opossum basilar pons.

In Golgi-prepared material, opossum basilar pontine intrinsic neurons measured less than 22 microns and gave origin to only two or three primary dendrites which gradually tapered in diameter, branched infrequently and exhibited few spines or protrusions. Characteristically, such neurons gave rise to several axon-like processes which might take origin from proximal or distal dendrites as well as the soma. Morphologically similar neurons have been observed in several other regions throughout the CNS where they have been shown to be a source of presynaptic dendritic elements. That opossum basilar pontine intrinsic neurons might also give rise to presynaptic dendrites was supported by the following electron microscopic observations. Profiles containing a small cluster of pleomorphic vesicles, occasional ribosomes and numerous microtubules formed synaptic active sites in which the membrane densities were intermediate between Gray's type I and type II. Such presumed presynaptic dendritic profiles were observed to participate in serial synaptic arrangements in which they were always postsynaptic to a round vesicle bouton and presynaptic to another dendritic element. The above features are compatible with electron microscopic descriptions of presynaptic dendrites in other CNS regions and thus suggest that basilar pontine intrinsic neurons may represent a source of presynaptic dendritic elements.

Animals↗

PubMed2000

PCR-restriction fragment length polymorphism analysis for identification of Bacteroides spp. and characterization of nitroimidazole resistance genes.

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroides infections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novel nim gene exhibited 75% DNA sequence similarity with nimB. These rapid, accurate, and inexpensive methods should enable improved identification of Bacteroides spp. and the detection of MTZ resistance determinants.

Anti-Bacterial Agents↗