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Results for “Ribosome Profiling”

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PubMed1998

Isolation and characterization of phenol-degrading denitrifying bacteria.

Phenol is a man-made as well as a naturally occurring aromatic compound and an important intermediate in the biodegradation of natural and industrial aromatic compounds. Whereas many microorganisms that are capable of aerobic phenol degradation have been isolated, only a few phenol-degrading anaerobic organisms have been described to date. In this study, three novel nitrate-reducing microorganisms that are capable of using phenol as a sole source of carbon were isolated and characterized. Phenol-degrading denitrifying pure cultures were obtained by enrichment culture from anaerobic sediments obtained from three different geographic locations, the East River in New York, N.Y., a Florida orange grove, and a rain forest in Costa Rica. The three strains were shown to be different from each other based on physiologic and metabolic properties. Even though analysis of membrane fatty acids did not result in identification of the organisms, the fatty acid profiles were found to be similar to those of Azoarcus species. Sequence analysis of 16S ribosomal DNA also indicated that the phenol-degrading isolates were closely related to members of the genus Azoarcus. The results of this study add three new members to the genus Azoarcus, which previously comprised only nitrogen-fixing species associated with plant roots and denitrifying toluene degraders.

Biodegradation, Environmental↗

PubMed2002

Nerve growth factor selectively regulates expression of transcripts encoding ribosomal proteins.

BACKGROUND: NGF exerts a variety of actions including promotion of neuronal differentiation and survival. The PC12 rat pheochromocytoma cell line has proved valuable for studying how NGF works and has revealed that the NGF mechanism includes regulation of gene expression. Accordingly, we used SAGE (Serial Analysis of Gene Expression) to compare levels of specific transcripts in PC12 cells before and after long-term NGF exposure. Of the approximately 22,000 transcripts detected and quantified, 4% are NGF-regulated by 6-fold or more. Here, we used database information to identify transcripts in our SAGE libraries that encode ribosomal proteins and have compared the effect of NGF on their relative levels of expression. RESULTS: Among the transcripts detected in our SAGE analysis, 74 were identified as encoding ribosomal proteins. Ribosomal protein transcripts were among the most abundantly expressed and, for naive and NGF-treated PC12 cells, represented 5.2% and 3.5%, respectively, of total transcripts analyzed. Surprisingly, nearly half of ribosomal protein transcripts underwent statistically significant NGF-promoted alterations in relative abundance, with changes of up to 5-fold. Of the changes, approximately 2/3 represented decreases. A time course revealed that the relative abundance of transcripts encoding RPL9 increases within 1 hr of NGF treatment and is maximally elevated by 8 hr. CONCLUSIONS: These data establish that NGF selectively changes expression of ribosomal protein transcripts. These findings raise potential roles for regulation of ribosomal protein transcripts in NGF-promoted withdrawal from the cell cycle and neuronal differentiation and indicate that regulation of individual ribosomal protein transcripts is cell- and stimulus-specific.

Animals↗

PubMed2001

Heat treatment adaptations in Clostridium perfringens vegetative cells.

Vegetative cells of Clostridium perfringens enterotoxigenic strains NCTC 8679, NCTC 8238. and H6 were grown at 37 degrees C followed by a 60-min exposure to 28 degrees C or 46 degrees C. D10-values, as a measure of thermal resistance at 60 degrees C, were significantly lower for 28 degrees C exposures as compared with cultures given 37 and 46 degrees C exposures. Following refrigeration at 4 degrees C for 24 h, D10-values for the 37 and 46 degrees C samples could not be differentiated from 28 degrees C samples. Western immunoblot analyses of lysates from heat-adapted cells also detected the increased expression of proteins reacting with antiserum directed against the molecular chaperonins from Escherichia coli; GroEL, DnaJ, and the small acid soluble protein from Bacillus subtilis, SspC. Differential scanning calorimetry (DSC) identified thermal transitions corresponding to ribosomal protein denaturations at 72.1 +/- 0.5 degrees C. Any cellular heat adaptations in the DSC profiles were lost following refrigeration for several days to simulate minimally processed food storage conditions. Further analyses of high-speed pellets from crude cell extract fractions using two-dimensional gel electrophoresis detected the differential gene expression of at least four major proteins in heat-adapted vegetative cells of C. perfringens. N-terminal amino acid analyses identified two of the proteins as glyceraldehyde 3-phosphate dehydrogenase and rubrerythrin. Both appear to have roles in this anaerobe under stressful conditions.

Adaptation, Physiological↗

PubMed2004

Characterization of humus microbial communities in adjacent forest types that differ in nitrogen availability.

To address the link between soil microbial community composition and soil processes, we investigated the microbial communities in forest floors of two forest types that differ substantially in nitrogen availability. Cedar-hemlock (CH) and hemlock-amabilis fir (HA) forests are both common on northern Vancouver Island, B.C., occurring adjacently across the landscape. CH forest floors have low nitrogen availability and HA high nitrogen availability. Total microbial biomass was assessed using chloroform fumigation-extraction and community composition was assessed using several cultivation-independent approaches: denaturing gradient gel electrophoresis (DGGE) of the bacterial communities, ribosomal intergenic spacer analysis (RISA) of the bacterial and fungal communities, and phospholipid fatty acid (PLFA) profiles of the whole microbial community. We did not detect differences in the bacterial communities of each forest type using DGGE and RISA, but differences in the fungal communities were detected using RISA. PLFA analysis detected subtle differences in overall composition of the microbial community between the forest types, as well as in particular groups of organisms. Fungal PLFAs were more abundant in the nitrogen-poor CH forests. Bacteria were proportionally more abundant in HA forests than CH in the lower humus layer, and Gram-positive bacteria were proportionally more abundant in HA forests irrespective of layer. Bacterial and fungal communities were distinct in the F, upper humus, and lower humus layers of the forest floor and total biomass decreased in deeper layers. These results indicate that there are distinct patterns in forest floor microbial community composition at the landscape scale, which may be important for understanding nutrient availability to forest vegetation.

Bacteria↗

PubMed1997

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.

Bacterial Typing Techniques↗

PubMed2005

Comparison of 16S rRNA and 16S rDNA T-RFLP approaches to study bacterial communities in soil microcosms treated with chromate as perturbing agent.

Transcripts of ribosomal RNA have been used for assessing the structure and dynamics of active bacterial populations; however, it remains unclear whether the information provided by community profiling derived from RNA is different from that derived from DNA, particularly when a selective pressure is applied on the bacterial community. In the present work, terminal-restriction fragment length polymorphism (T-RFLP) community profiles based on DNA and RNA extracted from soil microcosms treated with a toxic concentration of chromate were compared. Microcosms of a nonpolluted agricultural soil and of a heavy-metal-rich soil (serpentine) were treated with chromate and DNA and RNA were extracted. T-RFLP analysis was performed on amplified and retro-amplified 16SrRNA gene sequences, and band profiles obtained from samples of DNA and of RNA were compared. Some of the T-RFLP bands, identified as peculiar peaks in the profiles, were cloned and sequenced for taxonomic interpretation. Results indicated that: (1) community profiles derived from RNA and DNA were partly overlapping; (2) there was a strong correlation between the dynamics shown by RNA- and DNA-based T-RFLP profiles; (3) chromate addition exerted a clear effect on both agricultural and serpentine soil bacterial communities, either at the DNA and at the RNA level; however, the profiles derived from RNA showed sharper differences between treated and control samples than that of DNA-based profiles.

Agriculture↗

PubMed1987

The primary structure of the ribosomal A-protein (L12) from the halophilic eubacterium Haloanaerobium praevalens.

The ribosomal A-protein, equivalent to the ribosomal protein L12 from Escherichia coli, has been sequenced from the anaerobic halophilic eubacterium Haloanaerobium praevalens (DSM 2228). The protein contains 122 amino acids, has a composition of Asp6, Asn2, Thr2, Ser6, Glu22, Pro2, Gly13, Ala19, Val12, Met4, Ile5, Leu11, Phe3, Lys14, Arg1 and has a molecular weight of 12,691. The hydrophilicity profile was determined for this protein. A phylogenetic or cluster tree was calculated from computer analysis of the sequence data on eubacterial ribosomal A-proteins. H. praevalens clusters with a group that includes Bacillus subtilis, Micrococcus lysodeikticus, Bacillus stearothermophilus and Clostridium pasteurianum.

Amino Acid Sequence↗

PubMed1998

Differentiation of oral Actinomyces species by 16S ribosomal DNA polymerase chain reaction-restriction fragment length polymorphism.

16S rDNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to generate restriction profiles of the reference strains, including the American Type Culture Collection type strains, of oral Actinomyces spp., i.e., A. israelii, A. gerencseriae, A. naeslundii genospecies 1 and 2, A. odontolyticus, A. meyeri and A. georgiae, and 23 Actinomyces strains isolated from human dental plaque. The 16S rRNA gene sequences from isolated genomic DNA samples were amplified by PCR. The PCR products were purified and characterized by single digestion with four restriction endonucleases, i.e., MnlI, HaeIII, CfoI, or HpaII. Among them, MnlI was found to discriminate the respective reference strains. The clinical isolates were assigned to one of the species, i.e., A. gerencseriae, A. naeslundii genospecies 1 and 2 and A. odontolyticus, on the basis of their restriction profiles by single digestion with MnlI. Thus, 16S rDNA PCR-RFLP, using MnlI, is a rapid and reliable method for the differentiation of oral Actinomyces spp.

Actinomyces↗

PubMed2005

Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling.

BACKGROUND: The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling. RESULTS: Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus, Streptococcus, Weissella, Staphylococcus, and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products. CONCLUSION: Lactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies.

Animals↗

PubMed1966

The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid.

1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture.

Animals↗

PubMed2007

A genome-wide transcriptional analysis of producer and non-producer NS0 myeloma cell lines.

'Genome-wide' or 'global' gene expression profiling provides a powerful approach to the characterization of a cell's transcriptional state. Such technology has been used in animal cell culture to create genome-wide snapshots of transcriptional activity in response to environmental factors or cellular triggers under bioprocessing conditions. Furthermore, it allows us to have a fundamental understanding of genetic mechanisms involved in recombinant protein production. One such mechanism adversely affecting the growth of recombinant bacteria is the increased metabolic burden resulting from the maintenance of plasmid copy number and heterologous protein expression. There have also been some reports on the effect of metabolic burden in mammalian cell systems. In the present study, we have used a mouse array representing 6400 genes to assess the expression profile of a WT (wild-type) mouse plasmacytoma cell line, NS0 WT, and a GS (glutamine synthetase)-NS0 6A1-100 cell line expressing chimaeric monoclonal antibody. The producer cells did not exhibit a slower growth as the result of any metabolic burden, but showed differences in metabolic activity. Gene expression profiling revealed that the producer cell line was selected for a higher expression of chromosomal genes, genes for zinc-finger proteins as well as cell-cycle-related events. On the other hand, protein synthesis is greater and ribosomal genes were more expressed in the WT cells. A possible shift from expressing antigen presenting proteins to recombinant protein could also be seen. Hence, gene expression profiling suggests that the effect of the metabolic burden in slowing growth can be mostly negated in producer cell lines by careful clonal selection, where stable transfected cells are selected for both high productivity as well as high growth rates.

Animals↗

PubMed1978

Rat adrenocortical carcinoma 494: an integrated structural, stereological, and biochemical analysis.

Snell adrenocortical tumor 494 was implanted into male Sprague-Dawley rats and recovered 7, 14, 21, 28 or 35 days following initial detection by palpation (7-10 days following transplantation). Electron microscopic, stereological and biochemical analyses of the tumor were compared to adrenals of normal animals to serve as a baseline for further studies of the effects of chemotherapeutic agents on tumor cells. Tumor cells possessed oval or elongated mitochondrial profiles with tubular cristae, one or two very large (greater than 5 micrometer) lipid droplets, abundant ribosomes and coated vesicles, and sparse rough and smooth endoplasmic reticulum. Stereologic evaluation revealed that tumor lipid volume was 41% and mitochondrial volume 29% that of the normal adrenal controls. Tumor nuclei were 2.5 times larger than adrenocortical nuclei while cellular volumes were similar. On a net weight basis, tumor cholesterol was 55%, cholesterol ester 2.2%, and lipid phosphate 25% of respective mean values for normal adrenal glands. The tumor cholesterol: cholesterol ester ratio progressively decreased with time but remained 18-fold greater than the normal adrenal. Plasma corticosterone levels in tumor-bearing rats were elevated 3-fold by 14 days and initial detection. The adrenals of the tumor-bearing host exhibited marked involution, the extent of which was directly related to tumor size.

Adrenal Cortex Neoplasms↗

PubMed2005

Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis.

Cell-free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell-free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli-based CFPS reactions was 1.6 +/- 0.1 microM, with 72 +/- 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 +/- 0.2 amino acids per second per ribosome and an initiation rate of 8.2 x 10(-9) +/- 0.3 x 10(-9) M/s, which correlates to, on average, one initiation every 60 +/- 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell-free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell-free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation.

Cell-Free System↗

PubMed2006

New species of rumen treponemes.

Three strains of rumen treponemes were isolated and partially characterized. The strains differed significantly one from another in morphology, fermentation characteristics and plasmid profiles. Their genetic variability was assayed using DNA-based molecular approaches. Easily differentiated ARDRA (amplified ribosomal DNA restriction analysis) patterns indicated that the strains represent different bacterial species.

Animals↗

PubMed1999

Novel predicted RNA-binding domains associated with the translation machinery.

Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60-65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions.

Amino Acid Sequence↗

PubMed1990

Immunocytochemical visualization of muscarinic cholinoceptors in the human cerebral cortex.

The monoclonal antibody M 35 was used to study the cellular and subcellular distribution of muscarinic acetylcholine receptors in the human cerebral cortex. M35, raised against purified muscarinic receptor protein, exerts muscarinic agonist-like effects at cholinergic synapses. Applied to human cortical tissue immediately fixed upon neurosurgical removal, light microscopically M 35 revealed immunoreactive perikarya predominantly of the pyramidal cell type in layers II/III and V, their labeled apical dendrites extending into the superficial layers. Furthermore, immunoprecipitate decorated distinct regions of the perikaryal cytoplasm, numerous dendritic profiles and synapses of both symmetric and asymmetric appearance. In the perikarya immunoprecipitate was associated with ribosomes, the endoplasmic reticulum and the Golgi apparatus. In dendrites the microtubular system, in synaptic complexes the postsynaptic membranes were decorated. The results suggest that novel informations about the cell type specific and subcellular distribution of human muscarinic cholinoceptors can be obtained by M 35 immunocytochemistry.

Adult↗

PubMed2004

Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application.

The Eph family of receptors, with 14 members in humans, makes up the largest group of receptor tyrosine kinases. These Eph receptors, along with their ligands, the 8 members of the ephrin family of ligands are involved in diverse developmental functions, including hindbrain development in vertebrates, tissue patterning, and angiogenesis. These Eph receptors and ephrin ligands have also been identified as important regulators in the development and progression of cancer. We have presented here a systematic and comprehensive investigation of the Eph/ephrin expression profiles of MCF-10A, MCF-7, and MDA-MB-231 cells representing normal breast, non-invasive breast tumor, and invasive tumor, respectively, based on their characteristic phenotypes in Matrigel matrix. The data have allowed us to correlate the gene expression profile with the cell phenotype that has potential application in tumor diagnostics. We demonstrate here that upregulation of EphA2, A7, A10, and ephrinA2 and B3 is likely involved in tumorigenesis and/or invasiveness, while downregulation of EphA1, A3, A4, A8, B3, B4, B6, and ephrinA1 and B1 may be particularly important in invasiveness. Based on these results we discuss the role of EphA2 and ephrinA1 combination in malignancy. The data have provided clues as to the importance of these molecules in the progression of breast cancer and specifically identified EphB6, a kinase-deficient receptor, which is downregulated in the most aggressive cell line, as reported for several other cancer types including neuroblastoma and melanoma suggesting its potential as a prognostic indicator in breast cancer as well.

Biomarkers, Tumor↗

PubMed2005

Real-time quantitative PCR assay for analysis of platelet glycoprotein IIIa gene expression.

A quantitative detection assay for analysis of platelet glycoprotein GPIIIa gene expression is presented. The assay uses two fluorescently labeled TaqMan MGB probes to detect the polymorphic site in GPIIIa nucleotide sequence, leading to antigens HPA-1a and HPA-1b. In order to avoid the influence of DNA contamination on RNA quantification, a forward primer was constructed to span an exon-exon junction. The assay is therefore applicable to expression studies also in samples containing only a small amount of contaminating DNA. To standardize the amount of sample cDNA added to the reaction, amplification of endogenous control 18SrRNA was included in a separate well. The amplification validation experiment showed a high real-time PCR efficiency for HPA-1a, HPA-1b and 18SrRNA. Relative quantification was therefore performed using the comparative C(T) method. The assay was optimized on a reversely transcribed total RNA from platelets, and the specificity rate was determined by sequencing. The amount of cDNA at which amplification was still clearly detectable was 5 ng. This newly developed real-time quantitative PCR assay is a sensitive, reproducible and reliable method. It is suitable for studying different stages of megakaryopoiesis, monitoring molecular alteration in defective platelets and determining differences in the GPIIIa expression level between normal and pathological megakaryocytic differentiation pathways.

Antigens, Human Platelet↗