SEARCH · PubMed Health

Results for “Ribosome Profiling”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources

PubMed1998

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences.

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24S alpha ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24S alpha rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage 1 was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite.

Animals↗

PubMed2003

Investigation of MALDI-TOF and FT-MS techniques for analysis of Escherichia coli whole cells.

Recently, it has been demonstrated that bacteria can be characterized using whole cells and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, identification of specific bacterial proteins usually requires analysis of cellular fractions or purified extracts. Here, the first application of Fourier transform mass spectrometry (FTMS) to analysis of bacterial proteins directly from whole cells is reported. It is shown that accurate mass MALDI-FTMS can be used to characterize specific ribosomal proteins directly from Escherichia coli cells. High-accuracy mass measurements and high-resolution isotope profile data confirm posttranslational modifications proposed previously on the basis of low-resolution mass measurements. Seven ribosomal proteins from E. coli whole cells were observed with errors of less than 27 ppm. This was accomplished directly from whole cells without fractionation, concentration, or overt overexpression of characteristic cellular proteins. MALDI-FTMS also provided information regarding E. coli lipids in the low-mass region. Although ions with m/z values below 1000 have been observed by FTMS of whole cells, this represents the first report of detection of ions in the 5000 to 10,000 m/z range by MALDI-FTMS using whole cells.

Escherichia coli↗

PubMed2004

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

Ribosome biogenesis requires >100 nonribosomal proteins, which are associated with different preribosomal particles. The substrates, the interacting partners, and the timing of action of most of these proteins are largely unknown. To elucidate the functional environment of the putative ATP-dependent RNA helicase Dbp6p from Saccharomyces cerevisiae, which is required for 60S ribosomal subunit assembly, we have previously performed a synthetic lethal screen and thereby revealed a genetic interaction network between Dbp6p, Rpl3p, Nop8p, and the novel Rsa3p. In this report, we extended the characterization of this functional network by performing a synthetic lethal screen with the rsa3 null allele. This screen identified the so far uncharacterized Npa1p (YKL014C). Polysome profile analysis indicates that there is a deficit of 60S ribosomal subunits and an accumulation of halfmer polysomes in the slowly growing npa1-1 mutant. Northern blotting and primer extension analysis shows that the npa1-1 mutation negatively affects processing of all 27S pre-rRNAs and the normal accumulation of both mature 25S and 5.8S rRNAs. In addition, 27SA(2) pre-rRNA is prematurely cleaved at site C(2). Moreover, GFP-tagged Npa1p localizes predominantly to the nucleolus and sediments with large complexes in sucrose gradients, which most likely correspond to pre-60S ribosomal particles. We conclude that Npa1p is required for ribosome biogenesis and operates in the same functional environment of Rsa3p and Dbp6p during early maturation of 60S ribosomal subunits.

Base Sequence↗

PubMed1984

Biosynthesis of carbonic anhydrase in Chlamydomonas reinhardtii during adaptation to low CO(2).

The unicellular green alga Chlamydomonas reinhardtii synthesizes carbonic anhydrase in response to low levels of CO(2) (i.e., air levels of CO(2)). This enzyme, localized predominantly in the periplasmic space of the alga (or associated with the cell wall), is an important component of the machinery required for the active accumulation of inorganic carbon by C. reinhardtii and the saturation of ribulose-1,5-bisphosphate carboxylase at low extracellular carbon concentrations. We have begun to examine the synthesis and compartmentalization of carbonic anhydrase in C. reinhardtii. The monomeric species associated with carbonic anhydrase activity is synthesized as a precursor on 80S cytoplasmic ribosomes. This precursor can be detected immunologically in the profiles of translation products when a reticulocyte lysate, cell-free system is primed with poly(A)-RNA from either air-grown C. reinhardtii or cells shifted from growth on 5% CO(2) to air for 12 hr. It is not synthesized when the in vitro system is primed with poly(A)-RNA from CO(2)-grown algae. Since translatable RNA for the polypeptide responsible for carbonic anhydrase activity was only present in cells that experienced low levels of CO(2), the adaptation process either involves the regulation of transcription of the carbonic anhydrase gene (and perhaps other genes involved in adaptation) or the post-transcriptional processing of the messenger RNA. Furthermore, the appearance of the mature polypeptide associated with carbonic anhydrase activity in the periplasmic space of C. reinhardtii is inhibited by tunicamycin, an antibiotic that prevents core glycosylation of polypeptides on the endoplasmic reticulum. Together, these results suggest that the biosynthesis of this extracellular algal enzyme involves the translation of mRNA for the carbonic anhydrase monomer on ribosomes bound to the endoplasmic reticulum, the cleavage of a signal sequence during transport of the nascent polypeptide into the lumen of the endoplasmic reticulum, and subsequent glycosylation events prior to export across the plasmalemma.

Journal Article↗

PubMed1996

Polyribosome dynamics: size-distribution as a function of attachment, translocation and release of ribosomes.

Assembly of polyribosomes is the ubiquitous manifestation of the activation of translational machinery in cells. This is accompanied by a concomitant appearance of mRNAs either from an activation of pre-existing ribo-nucleoprotein particles (RNP) or from newly synthesized transcripts. The size-distributions of polyribosomes as inferred from their sedimentation profiles are essentially defined by various kinetic parameters responsible for continuous flux of ribosomes due to their recycling between the "free" and the "bound" states. In the theoretical analysis presented here the time-evolution of polyribosomes and their sustenance in the steady state is considered as a Markovian process with the states of mRNAs defined as the number of ribosomes attached. Expressions for the elements of one-step transition matrix as a function of kinetic parameters related to the initiation, translocation and the release of ribosomes and the size of mRNA are derived. The latter is shown to be relevant only in so far as it sets an upper limit to the size of polyribosomes. Implications of transient arrest of ribosomes at the initiation site as envisaged in the signal hypothesis for the synthesis of secretory proteins and the putative role of receptor-mediated post-translational transport of proteins have been examined. Results of computer simulations on model systems have focussed attention on the diverse types of profiles that may appear under varying physiological conditions.

Animals↗

PubMed1983

Insulin regulation of fat cell ribosomes, protein synthesis, and lipoprotein lipase.

Ribosomes of high purity were isolated from fat cells by discontinuous sucrose gradient centrifugation. Approximately 23% of the ribosomes were membrane bound. A reproducible fraction of ribosomes was recovered as polysomes capable of incorporating [3H]leucine into peptides in a cell-free system. Insulin (0.1-1.0 mU/ml) produced an increase in polysomal activity. Linear sucrose gradient profiles also revealed an increase in the ratio of polysomes to total ribosomes. Coincident with this effect, insulin increased the lipoprotein lipase activity fraction inhibitable by cycloheximide (0.01 mg/ml), and the immunotitratable enzyme activity. Insulin also enhanced the incorporation of labeled amino acids into adipose tissue immunoprecipitable lipoprotein lipase and total fat cell proteins. Cordycepin (0.1 mg/ml) or alpha-amanitin (10 micrograms/ml) partially inhibited insulin effects on ribosomes and protein synthesis but not on lipoprotein lipase. EGTA (1mM) prevented all of the insulin effects, whereas the calcium ionophore A-23187 (2 microM) augmented the hormone actions. The ionophore alone partially mimicked the insulin effects. In adipocytes, insulin increased the size of the protein-synthesizing polysomal pool by a mechanism which, in part, requires nuclear mRNA processing. Insulin, however, increased lipoprotein lipase synthesis independent of nuclear events. Calcium ions may be important for the expression of these insulin effects.

Adipose Tissue↗

PubMed2005

A microarray data-based semi-kinetic method for predicting quantitative dynamics of genetic networks.

BACKGROUND: Elucidating the dynamic behaviour of genetic regulatory networks is one of the most significant challenges in systems biology. However, conventional quantitative predictions have been limited to small networks because publicly available transcriptome data has not been extensively applied to dynamic simulation. RESULTS: We present a microarray data-based semi-kinetic (MASK) method which facilitates the prediction of regulatory dynamics of genetic networks composed of recurrently appearing network motifs with reasonable accuracy. The MASK method allows the determination of model parameters representing the contribution of regulators to transcription rate from time-series microarray data. Using a virtual regulatory network and a Saccharomyces cerevisiae ribosomal protein gene module, we confirmed that a MASK model can predict expression profiles for various conditions as accurately as a conventional kinetic model. CONCLUSION: We have demonstrated the MASK method for the construction of dynamic simulation models of genetic networks from time-series microarray data, initial mRNA copy number and first-order degradation constants of mRNA. The quantitative accuracy of the MASK models has been confirmed, and the results indicated that this method enables the prediction of quantitative dynamics in genetic networks composed of commonly used network motifs, which cover considerable fraction of the whole network.

Amino Acid Motifs↗

PubMed1985

Evolutionary conservation of ribosomal protein mRNA sequences: application for expansion of corresponding cDNA and gene libraries.

Cloned cDNAs, containing ribosomal protein sequences from mouse (five cDNAs) or Xenopus laevis (six cDNAs), were used to estimate the evolutionary conservation, from insects to mammals, of the corresponding mRNA sequences. Northern blot analysis reveals a variable degree of homology between these sequences in different eukaryotes. Thus, among the ribosomal protein cDNA clones utilized, some exhibit complete, others partial, and a few no interphyla cross-hybridization. Melting profile analysis was employed to quantitate this homology. It is proposed that for expansion of eukaryotic ribosomal cDNA and gene libraries, one can exploit the interspecies homology of the corresponding sequences. However, the diverse evolutionary conservation of individual ribosomal protein gene sequences should be taken into account.

Animals↗

PubMed2002

Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis.

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H'), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from beta-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.

Bacteria↗

PubMed1993

Automated ribosomal DNA fingerprinting by capillary electrophoresis of PCR products.

Capillary electrophoresis (CE) provides a rapid and automated technique for the analysis of subnanogram amounts of DNA fragments generated by the polymerase chain reaction (PCR). Here we describe the implementation of size-selective CE for DNA profiling and restriction fragment length polymorphism analysis of amplified polymorphic spacers of ribosomal DNA from fungi. Separations of unpurified and isopropanol-precipitated PCR-amplified DNA fragments in the size range of 20-1600 base pairs were achieved in less than 20 min with the use of hydroxypropyl methylcellulose as a sieving medium. The amplified internal transcribed spacer (ITS) and intergenic spacer (IGS) of RNA genes could be sized by coelectrophoresing a standard size ladder mixed with every sample, thereby eliminating errors in size estimation. This, together with the strictly controlled conditions of CE, permit the discrimination of amplified rDNA fragments differing only a few base pairs in length. Inter- and intraspecific variation in the size and number of restriction sites of the amplified rDNA spacers from the ectomycorrhizal basidiomycetes Laccaria laccata and Laccaria bicolor was observed and most strains could thus be reliably genotyped by PCR-CE. Multiple amplified IGS fragments of heterogeneous size were detected in several strains. This polymorphism is due to the occurrence of 5S rDNA subrepeats (i.e., multiple annealing of primer) within IGS. With CE, in contrast to slab gel electrophoresis, run times are short, the capillary can be reused, and full automation is feasible. Data acquisition and analysis are computer-controlled, which facilitates the locus identification and reduces error especially when large numbers of PCR products must be analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoanalysis↗

PubMed2006

Alu RNP and Alu RNA regulate translation initiation in vitro.

Alu elements are the most abundant repetitive elements in the human genome; they emerged from the signal recognition particle RNA gene and are composed of two related but distinct monomers (left and right arms). Alu RNAs transcribed from these elements are present at low levels at normal cell growth but various stress conditions increase their abundance. Alu RNAs are known to bind the cognate proteins SRP9/14. We purified synthetic Alu RNP, composed of Alu RNA in complex with SRP9/14, and investigated the effects of Alu RNPs and naked Alu RNA on protein translation. We found that the dimeric Alu RNP and the monomeric left and right Alu RNPs have a general dose-dependent inhibitory effect on protein translation. In the absence of SRP9/14, Alu RNA has a stimulatory effect on all reporter mRNAs. The unstable structure of sRight RNA suggests that the differential activities of Alu RNP and Alu RNA may be explained by conformational changes in the RNA. We demonstrate that Alu RNPs and Alu RNAs do not stably associate with ribosomes during translation and, based on the analysis of polysome profiles and synchronized translation, we show that Alu RNP and Alu RNA regulate translation at the level of initiation.

Alu Elements↗

PubMed2003

Moderate degradation does not preclude microarray analysis of small amounts of RNA.

Gene expression analysis by microarrays using small amounts of RNA is becoming more and more popular against the background of advances and increasing importance of small-sample acquisition methods like laser microdissection techniques. The quality of RNA preparations from such samples constitutes a frequent issue in this context. The aim of this study was to assess the impact of different extents of RNA degradation on the expression profile of the samples. We induced RNA degradation in human tumor and healthy tissue samples by endogeneous ribonucleases. Next, we amplified 20 ng total RNA degraded to different extents by two rounds of in vitro transcription and analyzed them using Affymetrix oligonucleotide microarrays. Expression differences for some genes were independently confirmed by real-time quantitative PCR. Our results suggest that gene expression profiles obtained from partially degraded RNA samples with still visible ribosomal bands exhibit a high degree of similarity compared to intact samples and that RNA samples of suboptimal quality might therefore still lead to meaningful results if used carefully.

Carcinoma, Renal Cell↗

PubMed1977

Entry of mRNA into polyribosomes during recovery from starvation in mouse sarcoma 180 cells.

Starvation in mouse sarcoma 180 cells leads to a block in initiation of protein synthesis which results in the conversion of polysomes to 80-S ribosomes. The mRNA is released as nucleoprotein particles. Addition of nutrients to the starved cell causes the rapid uptake of the mRNA into large polysomal structures. The ribosomes enter these structures more gradually, without much effect on the sedimentation profile of the labeled mRNA. The results indicate that the stored mRNA in starved cells is readily available for uptake into polysomes. They also suggest that the mRNA may be binding to components other than ribosomes during the initial stages of polysome formation.

Animals↗

PubMed1991

Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients.

Long-term cultures of melanocytes were established from 14 subjects with vitiligo and from five normal controls and analyzed ultrastructurally. Cultured melanocytes from 78.6% of the vitiligo patients demonstrated abnormalities that consisted of 1) dilation of the rough endoplasmic reticulum (RER), 2) circular RER profiles, and/or 3) membrane bound compartments of melanosomes. Cultured melanocytes from control subjects were predominantly normal with only one of the normal cultures demonstrating minimal circular RER profiles. The three unique abnormal structures in cultured vitiligo melanocytes were not always concomitantly expressed and could not be associated with any specific clinical feature of vitiligo. Quantitative analysis of the RER demonstrated that the profiles of dilated RER in cultured vitiligo melanocytes expressed a significant 1.5-2.8-times increase in mean cisternal area over cultured control melanocytes (i.e., 5.41-9.92 microns 2 versus 3.53 microns 2, respectively). The cisterna of the dilated RER profiles frequently contained floccular material that appeared to originate from the ribosomes, an indication that the floccular material may be translation products. The dilation of RER in melanocytes from the same patient persisted through repeated subculturing for up to 14.75 months. Epidermal melanocytes in biopsied skin from a patient whose cultured melanocytes were aberrant also demonstrated dilated and circular RER profiles. These results demonstrate that melanocytes from most vitiligo patients express an innate defect when cultured. Although this defect does not appear to be cytotoxic in vitro, this abnormality may be the primary defect that elicits melanocyte destruction in vivo.

Adolescent↗

PubMed1997

Ultrastructural changes in the deep cortical pyramidal cells of infant rats with inherited hydrocephalus and the effect of shunt treatment.

Pathological changes in the cortical gray matter in infantile hydrocephalus vary with the age at onset and may not be reversible with shunt treatment. We have used electron microscopy to investigate the sequence of pathological change and the effect of shunt treatment on layer VI pyramidal cells from infant H-Tx rats with inherited early-onset hydrocephalus. Tissue was prepared from the frontal and visual cortex of control and hydrocephalic rats at 4, 11, and 21 days after birth, together with 21-day rats previously treated with ventriculosubcutaneous shunts at 4-5 or 10-11 days after birth. Both cortical regions gave similar results but the effects were more severe in the visual cortex. In the early stages of hydrocephalus, the pyramidal cells were in clusters with fewer mature dendrites and less cytoplasmic organization than those in control rats, and some neuronal processes were vacuolated. In intermediate hydrocephalus the changes were more severe, with vacuolated cytoplasm, fewer cytoplasmic organelles, frequent swollen processes, and infrequent synapses. In advanced hydrocephalus at 21 days, many neurons showed degenerative changes, with edematous Golgi and dilated endoplasmic reticulum, distorted mitochondria, and single ribosomes. The neuropil contained many spongy areas with distended profiles. Shunt treatment prevented most of the changes if carried out at 4 days. Shunt treatment at 11 days also gave a dramatic recovery at the cellular level, but there were more immature pyramidal cells and edematous processes in the neuropil than in the 4-day-treated rats. The changes in hydrocephalus are consistent with progressive neuronal damage, which is largely prevented by early shunt treatment.

Animals↗

PubMed1996

The gene distribution of the human genome.

Linear correlations exist between the GC levels of third codon positions (GC3) of individual human genes and the GC levels of long genomic sequences and DNA molecules (50-100 kb in size) embedding the genes. These linear relationships allow the positioning of the GC3 histogram of cDNA sequences from the databases relative to the CsCl profile of human DNA. In turn, this allows an estimate of the relative concentrations of genes in genomic regions of different GC content. An estimate obtained by using current sequence data and Gaussian decompositions of the GC3 histogram and of the CsCl profile indicates that the GC-richest (non-ribosomal) component of the human genome is at least 17 times as gene-rich as the GC-poor regions. Moreover, our results suggest that the most recent physical maps of the human genome consisting of overlapping YACs cover less than 50% of the genes.

Chromosome Mapping↗

PubMed1988

The ultrastructure of the glandular epithelium in the timed endometrial biopsy.

This paper reports the electron microscopic appearance of the uterine glandular epithelium in a group of 25 women of known fertility who underwent endometrial biopsies at known times after the luteinizing hormone (LH) surge between LH + 2 and LH + 6. The use of these well-timed endometrial biopsies enables one to gain a clearer insight into the cellular dynamics of the glandular cells, between ovulation and the middle of the luteal phase. Under the influence of progesterone, the cells are transformed from relatively inactive cells full of free ribosomes to very active polarized cells, containing giant mitochondrial profiles, intracellular deposits of glycogen/glycoprotein-rich material and a complex intranuclear channel system. Intimate associations between organelles develop, the most notable being that between a semi-rough endoplasmic reticulum and the mitochondria. By taking biopsies that are accurately timed, relative to the LH surge, from a group of women of known fertility, the sequences of changes in ultrastructure are established with considerably more precision than was previously available.

Adult↗

PubMed2000

Molecular analysis of Shigella sonnei isolated from three well-documented outbreaks in school children.

Fifty-eight isolates of Shigella sonnei from three outbreaks in school children and eight control isolates from epidemiologically unrelated sporadic clinical infections in Taiwan were compared by antibiotic susceptibility testing and molecular typing. Antibiotic susceptibility testing showed that all strains except one sporadic isolate were multi-resistant. Ribotyping after restriction endonuclease digestion with SalI, PvuII and HindII generated the same ribosomal pattern in 65 of the 66 isolates. Plasmid profile analysis and pulsed-field gel electrophoresis (PFGE) produced eight and nine distinct patterns, respectively, and were in agreement with the epidemiological relationship of the outbreak strains. Nevertheless, some of the sporadic isolates could be discriminated only by a combination of these two methods. This study showed that plasmid profiling in combination with PFGE may be superior to ribotyping in molecular epidemiological investigations of S. sonnei.

Anti-Bacterial Agents↗