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Results for “Ribosome Profiling”

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PubMed1975

Potentiation of hemoglobin messenger ribonucleic acid. A step in protein synthesis initiation involving interaction of messenger with 18 S ribosomal ribonucleic acid.

The polyadenylic acid-containing messenger ribonucleic acid from rabbit reticulocyte polyribosomes, isolated by a rapid and very gentle procedure (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084), sediments in a sucrose gradient in three sharp peaks, at 9 S, 17 to 18 S, and 28 S. The alpha and beta globin messenger activity follows the absorbance profile in the sucrose gradients and has its major peak at 17 to 18 S. The larger messengers are more active than 9 S messenger by approximately 2-fold per mass unit of ribonucleic acid or by at least 8-fold per molecule. The major 17 to 18 S form of globin messenger was examined further and was shown to be a 1:1 complex of 9 S messenger and 18 S ribosomal ribonucleic acid. The effect of 18 S ribosomal ribonucleic acid on translation of purified 9 S globin messenger was analyzed in a messenger-dependent protein-synthesizing system (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084). In the absence of exogenous ribosomal ribonucleic acid, 9 S messenger is inefficiently translated; a large excess of messenger is required to saturate the system; and globin is synthesized mainly on di- and monoribosomes. Exogenous liver or reticulocyte 18 S ribosomal ribonucleic acid potentiates 9 S messenger translation and renders it at least 10 times more efficient. The potentiation reaction can also be accomplished by increasing the concentration of ribosomes in the assay system. However, transfer or messenger ribonucleic acids cannot carry out this reaction. It is proposed that 9 S globin messenger ribonucleic acid is an inactive molecule which is normally potentiated by specific reversible base pairing with an accessible region of ribosomal ribonucleic acid contained in a 40 S ribosomal subunit. The potentiated messenger interacts with initiation factors and with other ribosomal subunits to synthesize protein. Potentiation is the first specific function in protein synthesis demonstrated for the ribosomal ribonucleic acid portion of ribosomes.

Adenine Nucleotides↗

PubMed2005

Comparison of proteome and antigenic proteome between two Neospora caninum isolates.

This study was conducted to explore the relationship between two isolates of Neospora caninum (N. caninum) (KBA-2 and VMDL-1) using proteomics. To achieve the goal, proteins of N. caninum tachyzoite lysates of KBA-2 and VMDL-1 were separated by two-dimensional gel electrophoresis (2-DE), stained with silver-nitrate and analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to compare protein profiles. In addition, proteins separated by 2-DE were transferred to membranes, probed with bovine anti-N. caninum KBA-2 immunoglobulin G, and reactive proteins were visualized and compared between the two isolates. Most spots on 2-DE profiles and antigenic spots on 2-DE immunoblot profiles were located at similar locations in terms of isoelectric point and molecular weight. Proteins common to both isolates included the following: heat shock protein 70, subtilisin-like serine protease, nucleoside triphosphatase, heat shock protein 60, pyruvate kinase, tubulin alpha, tubulin beta, enolase, putative protein disulfide isomerase, actin, fructase-1,6-bisphosphatase, putative ribosomal protein S2, microneme protein Nc-P38, lactate dihydrogenase, fructose-1,6-bisphosphatase aldolase, serine threonine phosphatase 2C, 14-3-3 protein homologue, N. caninum dense granule-1 and NcGRA2. As a consequence, even though N. caninum KBA-2 and VMDL-1 isolates were isolated from geographically distinct locations there were significant homology in the proteome and antigenic proteome profiles. In addition, proteomic approach was verified as a useful tool for understanding of host immune response against different isolates of protozoa.

Animals↗

PubMed1997

Classification of Austrian rhizobia and the Mexican isolate FL27 obtained from Phaseolus vulgaris L. as Rhizobium gallicum.

The phylogenetic positions of four rhizobial strains obtained from nodules of common bean plants (Phaseolus vulgaris L.) grown in an Austrian soil and of the Mexican bean isolate FL27 are described. Analysis of the 16S rRNA genes revealed sequences almost identical to that of the Rhizobium gallicum type strain, R602sp, with a maximum of two nucleotide substitutions. Comparison of the 16S rRNA gene sequences with those from other bacteria indicated highest similarity to Rhizobium sp. strain OK-50, Rhizobium leguminosarum IAM 12609, and Rhizobium etli. DNA homology determined by DNA-DNA hybridization was high among the Austrian isolates and R602spT (45 to 90%) and ranged from 21 to 65% with FL27, but hybridization analysis revealed very low homology to the recognized common bean-nodulating species, R. leguminosarum bv. phaseoli, R. etli, and Rhizobium tropici. Ribosomal gene organization was studied by Southern hybridization with the 16S rRNA gene and temperature gradient gel electrophoresis, indicating identical organizations and the presence of three identical 16S rRNA copies in the genome of this species. The six strains investigated showed different plasmid profiles based on their geographical origins. We propose that the Austrian isolates and the Mexican strain FL27 are members of the species R. gallicum.

Austria↗

PubMed2004

An electrophoretic tool for the genetic characterisation and delineation of lungworms.

In the present study, PCR-based single-strand conformation polymorphism (SSCP) analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was applied to the genetic characterisation of Dictyocaulus from red deer from New Zealand and to its differentiation from species of lungworm from cattle and other hosts. Based on SSCP profiles, Dictyocaulus individuals from red deer from different geographical localities in New Zealand could be readily distinguished from those representing other lungworms examined, irrespective of low-level sequence variability in the ITS-2 (0.4-2.6%) detectable among individuals. The ITS-2 of Dictyocaulus from red deer differed in sequence by approximately 7-35% from congeners from other cervid hosts, demonstrating that this parasite is genetically distinct from other species of Dictyocaulus for which ITS-2 sequence data are presently available. The results emphasize the need for a large-scale molecular systematic study of Dictyocaulus specimens from various species of cervid and other ruminant hosts and the usefulness of mutation scanning for taxonomic, epidemiological and population genetic investigations.

Animals↗

PubMed2000

Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA-based approaches and quinone profiling.

Depth-related changes in whole-community structure were evaluated in a coastal marine sediment using a molecular fingerprinting method, terminal restriction fragment length polymorphism (T-RFLP) analysis, and a chemotaxonomic technique (quinone profiling). Dendrograms derived from both T-RFLP analysis and quinone profiling indicated a significant variation in microbial community structure between the 0-2 cm layer and deeper layers. This corresponded to the dramatic change in the redox potential, acid-volatile sulphide-sulphur and bacterial numbers observed at 0-2 cm and 2-4 cm depths. A significant change in the number of terminal restriction fragments (T-RFs) was also detected at this transition depth. However, the change in major T-RFs with depth was not seen in electropherograms. The population changes were primarily variations in minor ribotypes. Most quinone homologues were detected at all depths, although the quinone composition changed with depth. Therefore, quinone profiling also suggested that the depth-related variation was primarily attributable to minor bacterial groups rather than change in the major population structure. 16S rDNA clone library analysis revealed that clones belonging to the genera Vibrio and Serratia predominated as major bacterial groups at all depths. Our data suggested that the sediment community might result from sedimentation effects of sinking particles. Overall, our results demonstrated that the combined methods of T-RFLP analysis and quinone profiling were effective for assessing depth-related microbial populations.

DNA, Ribosomal↗

PubMed1979

Preparation polyacrylamide-agarose gel electrophoresis of proteins and RNAs by the use of new device.

Quantitatively reproducible results were obtained by using a new device for preparation gel electrophoresis combined with polyacrylamide-agarose composite gel. When an adequate gel-buffer system was selected according to the procedure described in this paper, proteins and RNA's were well separated and recovered. The new device for preparative gel electrophoresis and the method for preparation of polyacrylamide-agarose composite gel are presented together with the elution profiles of the recovered substances.

Blood Proteins↗

PubMed2005

Multi-metric and multi-substructure biclustering analysis for gene expression data.

A good number of biclustering algorithms have been proposed for grouping gene expression data. Many of them have adopted matrix norms to define the similarity score of a bicluster. We shall show that almost all matrix metrics can be converted into vector norms while preserving the rank equivalence. Vector norms provide a much more efficient vehicle for biclustering analysis and computation. The advantages are two folds: ease of analysis and saving of computation. Most existing biclustering algorithms have also implicitly assumed the use of univariate (i.e., single metric) evaluation for identifying biclusters. Such an approach however overlooks the fundamental principle that genes (even though they may belong to the same gene group) (1) may be subdivided into different substructures; and (2) they may be co-expressed via a diversity of coherence models (a gene may participate in multiple pathways that may or may not be co-active under all conditions). The former leads to the adoption of a multi-substurcture analysis, while the latter to the multivariate analysis. This paper will show that the proposed multivariate and multi-subscluster analysis is very effective in identifying and classifying biologically relevant groups in genes and conditions. For example, it has successfully yielded highly discriminant and accurate classification based on known ribosomal gene groups.

Algorithms↗

PubMed2004

Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma.

Using messenger RNA (mRNA) differential display, we identified a single complementary DNA (cDNA) fragment (HG23T1) that was over-expressed in a hepatocellular carcinoma (HCC) specimen. We cloned the full-length HG23T1 gene by the rapid amplification of cDNA end (RACE) polymerase chain reaction (PCR) method. It perfectly matched the gene encoding human ribosomal protein L36a (RPL36A also referred to as RPL44). RPL36A mRNA was preferentially over-expressed in 34 of 40 HCC cases (85%, P <.001) and in all of 8 HCC cell lines. Ectopically over-expressed L36a ribosomal protein localized in the nucleoli of cells, and this localization seemed to be controlled by the N-terminal or the internal tetrapeptide consensus with its adjacent N-terminal domain. Over-expression of L36a led to enhanced colony formation and cell proliferation, which may have resulted from rapid cell cycling, and an antisense cDNA effectively reversed these alterations. In conclusion, RPL36A plays a role in tumor cell proliferation and may be a potential target for anticancer therapy of HCC.

Animals↗

PubMed2006

Genes associated with telomerase activity levels in esophageal carcinoma cell lines.

Telomerase activity levels have been shown to correlate with tumor progression in several malignancies. However, the genetic regulation of telomerase activity levels is not fully understood. The aim of the present study has been to identify a gene expression profile, predicting correlation with the telomerase-activity test. Ten human esophageal carcinoma cell lines were investigated using the telomerase activity assay (TRAPeze) Telomerase Detection Kit), followed by further characterization using the GeneChip Human Genome U133A 2.0 Array (Affymetrics Inc., USA), including 14 500 human genes. Telomerase activity levels were detected in all cell lines with a broad range of activity levels. Using a high correlation coefficient, r > 0.90, the following genes were found to be positively correlated with telomerase activity levels: N-myristoyltransferase 2; ribosomal protein L3; retinoblastoma-like 2 (pRb2/p130); and cyclin G2. Only one gene was negatively correlated with telomerase activity levels, zinc finger protein 207. In conclusion, the present microarray data provide primary validation data indicating possible candidates for prognostic and prediction factors in esophageal cancer in relation to telomerase activity.

Acyltransferases↗

PubMed2005

Gene expression profiling of the PPAR-alpha agonist ciprofibrate in the cynomolgus monkey liver.

Fibrates, such as ciprofibrate, fenofibrate, and clofibrate, are peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists that have been in clinical use for many decades for treatment of dyslipidemia. When mice and rats are given PPARalpha agonists, these drugs cause hepatic peroxisome proliferation, hypertrophy, hyperplasia, and eventually hepatocarcinogenesis. Importantly, primates are relatively refractory to these effects; however, the mechanisms for the species differences are not clearly understood. Cynomolgus monkeys were exposed to ciprofibrate at various dose levels for either 4 or 15 days, and the liver transcriptional profiles were examined using Affymetrix human GeneChips. Strong upregulation of many genes relating to fatty acid metabolism and mitochondrial oxidative phosphorylation was observed; this reflects the known pharmacology and activity of the fibrates. In addition, (1) many genes related to ribosome and proteasome biosynthesis were upregulated, (2) a large number of genes downregulated were in the complement and coagulation cascades, (3) a number of key regulatory genes, including members of the JUN, MYC, and NFkappaB families were downregulated, which appears to be in contrast to the rodent, where JUN and MYC are reported to upregulated after PPARalpha agonist treatment, (4) no transcriptional signal for DNA damage or oxidative stress was observed, and (5) transcriptional signals consistent with an anti-proliferative and a pro-apoptotic effect were seen. We also compared the primate data to literature reports of hepatic transcriptional profiling in PPARalpha-treated rodents, which showed that the magnitude of induction in beta-oxidation pathways was substantially greater in the rodent than the primate.

Animals↗

PubMed2005

Gene expression profiles among immature and adult reproductive castes of the termite Reticulitermes flavipes.

Array-based genomic studies were conducted with the goal of identifying immature (i.e. nymph) and adult reproductive caste-biased gene expression in the termite Reticulitermes flavipes. Using cDNA macro-arrays, we identified thirty-four nymph-biased genes falling into eight ontogenic categories. Based on gene expression profiles among diverse castes and developmental stages (determined by quantitative PCR), several important trends emerged. These findings highlight the importance of several developmental and survival-based factors among immature and adult termite reproductives, including: vitellogenesis, nutrient storage, juvenile hormone sequestration, ribosomal translational and filtering mechanisms, fatty acid biosynthesis, apoptosis inhibition, and both endogenous and symbiont cellulase-assisted nutrition. These findings are highly significant as they are the first to elucidate the molecular biology underlying termite reproductive caste differentiation and reproductive caste-specific biology. Other gene expression results are in agreement with previous findings that suggest roles for vitellogenin-like haemolymph proteins in soldier caste differentiation.

AMP Deaminase↗

PubMed1997

Immunization with recombinant Trypanosoma cruzi ribosomal P2beta protein induces changes in the electrocardiogram of immunized mice.

Molecular expression cloning techniques revealed that patients with severe chronic Chagas heart disease showed a strong humoral response against the cloned C-terminal portion of the Trypanosoma cruzi ribosomal P2beta protein, previously named JL5. The main linear epitope of this polypeptide was mapped to the 13 C-terminal amino acid sequence EEEDDDMGFGLFD (named R13), which is almost identical to the mammalian ribosomal P consensus sequence EESDDDMGFGLFD (named H13). Enzyme-linked immunosorbent assay measurements demonstrated that sera from patients with chronic Chagas heart disease presented a very specific anti-P humoral response with high anti-R13, but low H13 antibody levels. We attempted to develop an animal model that would reproduce, at least partially, two features of the human infection: (1) the serological pattern of the anti-P response, and (2) specific cardiac symptoms. To this effect, mice were immunized with T. cruzi P2beta recombinant protein. Immunization reproduced the typical anti-P antibody profile defined for chronic infections, but did not induce cardiac inflammatory lesions. However, it altered significantly the electrocardiograms of immunized mice. It is suggested that this assay represents a functional test for assessing the biological activity of antibodies against T. cruzi ribosomal P protein on cardiac muscle.

Animals↗

PubMedDate not supplied

Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene.

Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

Animals↗

PubMed2006

Development and validation of endogenous reference genes for expression profiling of medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR.

The quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) technique has been increasingly used in endocrine disrupting chemicals (EDCs) research. Usually, an appropriate endogenous control gene is critical for Q-RT-PCR to normalize the errors and sample-to-sample variations that occur in the course of tissue collection, RNA isolation, and RT-PCR. In this study, we cloned ribosomal protein L7 (RPL-7) from medaka (Oryzias latipes), and then used Q-RT-PCR to study its transcription characteristics and those of glyceraldehyde-3-phosphate dehydrogenase, beta-actin, mitochondrial 16S ribosomal RNA (16S rRNA), and 18S rRNA. Of the five genes, RPL-7 and 18S rRNA were expressed with the less variance among the same tissue samples, in different tissues, and stages of development and were unaffected by EDCs exposure. The expression levels of RPL-7 among different tissues were between 9.76 x 10(6) +/- 9.49 x 10(5) and 1.39 x 10(7) +/- 1.69 x 10(6) copies/microg RNA but those of 18S rRNA were as high as 4.48 x 10(11) +/- 5.95 x 10(10) to 5.90 x 10(11) +/- 1.21 x 10(10) copies/microg RNA, which is above the usual detection scope of Q-RT-PCR if no complementary DNA reaction dilution is performed. As a result, RPL-7 is the single suitable endogenous control gene for expression profiling in future studies, especially in studies on the EDCs issue using medaka.

Amino Acid Sequence↗

PubMed2005

The dynamics of single-substrate continuous cultures: the role of ribosomes.

When a chemostat is perturbed from its steady state, it displays complex dynamics. For instance, if the identity of the growth-limiting substrate is switched abruptly, the substrate concentration and cell density undergo a pronounced excursion from the steady state that can last several days. These dynamics occur because certain physiological variables respond slowly. In the literature, several physiological variables have been postulated as potential sources of the slow response. These include transport enzymes, biosynthetic enzymes, and ribosomes. We have been addressing this problem by systematically exploring the role of these variables. In previous work Shoemaker et al. (J. Theor. Biol., 222 (2003) 307-322), we studied the role of transport enzymes, and we showed that transients starting from low transport enzyme levels could be quantitatively captured by a model taking due account of transport enzyme synthesis. However, there is some experimental data indicating that slow responses occur even if the initial enzyme levels are high. Here, we analyse this data to show that in these cases, the sluggish response is most probably due to slow adjustment of the ribosome levels. To test this hypothesis, we extend our previous model by accounting for the evolution of both the transport enzyme and the ribosomes. Based on a kinetic analysis of the data in the literature, we assume that the specific protein synthesis rate is proportional to the ribosome level, and the specific ribosome synthesis rate is autocatalytic. Simulations of the model show remarkable agreement with experimentally observed steady states and the transients. Specifically, the model predictions are in good agreement with (1) the steady-state profiles of the cell density, substrate concentration, RNA, proteins, and transport enzymes, (2) the instantaneous specific substrate uptake, growth, and respiration rates in response to a continuous-to-batch shift, and (3) the transient profiles of the cell density, substrate concentration, and RNA in response to feed switches and dilution rate shifts. Time-scale analysis of the model reveals that every transient response is a combination of two fundamental (and simpler) dynamics, namely, substrate-sufficient batch dynamics and cell-sufficient fed-batch dynamics. We obtain further insight into the transient response by analysing the equations describing these fundamental dynamics. The analysis reveals that in feed switches or dilution rate shift-ups, the transport enzyme reaches a maximum before RNA achieves its maximum, and in dilution rate shift-downs the cell density reaches a maximum before RNA achieves a minimum.

Bacterial Physiological Phenomena↗

PubMed1992

Translational initiation factors IF-1 and eIF-2 alpha share an RNA-binding motif with prokaryotic ribosomal protein S1 and polynucleotide phosphorylase.

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2 alpha chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2 alpha may bind to RNA.

Amino Acid Sequence↗

PubMed1973

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo.

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2-3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[(14)C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.

Animals↗

PubMed2001

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Adult↗