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PubMed1991

An amplifiable and deletable chloramphenicol-resistance determinant of Streptomyces lividans 1326 encodes a putative transmembrane protein.

A genetically unstable chloramphenicol resistance gene from Streptomyces lividans 1326 was cloned and characterized. This gene and adjacent DNA regions can be lost spontaneously or amplify within variants. Biochemical studies proved that chloramphenicol is not modified by an acetyltransferase or any other enzyme and that ribosomes of the resistant strain are sensitive to chloramphenicol. Sequence data revealed that the resistance gene encodes a hydrophobic protein predicted to have 12 membrane-spanning alpha-helices and a hydropathic profile similar to the membrane of proteins required for the efflux of tetracycline. Variable proportions of the amino acids (about 16-24%) within the presumed chloramphenicol-resistant protein are identical to various aligned tetracycline-resistant proteins from Gram-negative and Gram-positive bacteria and to transporters for citrate in Klebsiella pneumonaie and for ferrichrome in Escherichia coli.

Amino Acid Sequence↗

PubMed1977

The in vivo effect of dimethyl sulphoxide (DMSO) on protein synthesis and the polyribosome profile in Paramecium.

When Paramecium tetraurelia in log phase growth is treated with 4% dimethyl sulphoxide (DMSO) for five minutes the amount of polyribosomes is reduced 3- to 4-fold while there is a corresponding increase in 80s ribosomal material. Reducing the concentration of DMSO to 1% allows immediate reversal of the condition. Paramecium polyribosomes subjected to 4% DMSO either in whole cell homogenates or during purification through sucrose density gradients appear unaffected while cycloheximide at concentrations up to 100 mug/ml did not prevent DMSO from exerting its effect in vivo. Analyses of 14C amino acid incorporation experiments indicated a strict correspondence between the effect of DMSO on polyribosomes and overall protein synthesis. The reduction of acid precipitable radioactivity in the polyribosomal region after DMSO treatment was associated with a corresponding increase in radioactivity in the 80s region. There was no comparable increase in the acid precipitable radioactivity in the soluble fraction. The overall results of the study suggest that DMSO acts on polyribosommes indirectly through some unknown primary reaction with cell constitutents, and that the mode of action is such as to cause the release of ribosomes from messenger RNA (mRNA) rather than to prevent initiation of the ribosome-mRNA complex. Our data suggest that the effect may be selective. Finally, it is of interest that high concentrations of DMSO (above 8%) appear to have the opposite effect of lower concentrations of DMSO, i.e., they appear to "freeze" the ribosomes to mRNA.

Animals↗

PubMed1998

Comparison of ARDRA and recA-RFLP analysis for genomic species identification of Acinetobacter spp.

The genus Acinetobacter is subdivided into genospecies on the basis of DNA relatedness of strains. Phenotypic tests are insufficient to identify the genospecies to which an isolate belongs. The effectiveness of two previously described PCR-based methods for genospeciating Acinetobacter spp. was compared using a group of 32 well-characterised strains representing six genospecies. Amplified ribosomal DNA restriction analysis (ARDRA) correctly identified all 32 strains. Using restriction fragment length polymorphism (RFLP) of recA PCR amplimers, only six of the 32 strains were correctly identified. Heterogeneity in the recA gene sequence was demonstrated within five of the genospecies. ARDRA proved to be a reliable method whereas analysis of recA RFLP profiles did not enable the genospecies of most of the isolates of Acinetobacter spp. to be determined.

Acinetobacter↗

PubMed1984

The apocytochrome b gene in maize mitochondria does not contain introns and is preceded by a potential ribosome binding site.

The apocytochrome b (COB) gene has been isolated from maize (Zea mays L.) mitochondrial DNA. Sequence analysis reveals that the coding region of the gene is 1164 bp long and, in contrast with the homologous gene from yeast, does not contain introns or TGA (Trp) codons. The predicted polypeptide encoded by the gene has a mol. wt. of 42 868 daltons, and shows 48% amino acid sequence homology with the corresponding yeast and mammalian polypeptides. Hydropathic profiles of the polypeptide indicate the presence of nine, membrane spanning hydrophobic domains suggesting that it is organised in the inner mitochondrial membrane in a similar fashion to that proposed for apocytochrome b in other organisms. The COB gene is preceded by a sequence 5' -AGTTGTCA-3' which may act as a ribosome binding site in the mRNA since: (i) it shows 67.5% complementarity with an octanucleotide at the 3' end of the maize mitochondrial 18S rRNA, located in a position homologous to that of the Escherichia coli Shine and Dalgarno sequence, and (ii) a similar sequence precedes several other plant mitochondrial genes at a distance of 15-20 nucleotides from the ATG initiation codon. RNA transcript analysis shows that the gene is transcribed in a complex manner with the presumed mature mRNA ( 2.25 kb) probably being derived by sequential processing from a larger primary transcript.

Journal Article↗

PubMed1995

Morphologic changes in superior vestibulo-ocular neurons and vestibular nerve following labyrinthectomy in the cat.

Morphologic changes in ipsilateral superior vestibulo-ocular neurons (SVON) and the vestibular nerve were measured in 4 cats 8 weeks after labyrinthectomy and 4 cats 1 year after labyrinthectomy. There is a 20% decrease in SVON size and 30% decrease in rough endoplasmic reticulum and ribosomes with no change in the volume fractions of Golgi apparatus or mitochondria. In the central nervous system degeneration of the vestibular nerve terminals ipsilateral to the labyrinthectomy was represented by a 25% loss of synaptic profiles (SP) on SVON at 8 weeks and 57% loss of SP at 1 year after labyrinthectomy. There was no significant loss of fiber number in the vestibular nerve at 8 weeks post lesion but a 35% loss of fibers primarily of the large size at 1 year post lesion.

Animals↗

PubMed1998

Species identification of Legionella via intergenic 16S-23S ribosomal spacer PCR analysis.

Species identification of Legionella in routine laboratory testing is hampered by the lack of highly discriminatory phenotypic tests. Amplification polymorphism of the intergenic 16S-23S spacer regions (ISR) has been previously developed for identification of species within the Legionellaceae [Hookey, J.V., Birtles, R.J. & Saunders, N.A. (1995). J Clin Microbiol 33, 2377-2381], but it did not provide enough resolution to distinguish all members of the bluish-white autofluorescent species and the red autofluorescent group of the Legionellaceae. By choosing new primers that target regions 4 (positions 1521-1541 of Escherichia coli 16S rRNA gene) and 6 (positions 114-132 of E.coli 23S rRNA gene) within the rDNA operon close to the 16S-23S intergenic spacer, 34 profiles were determined among the 79 type and reference strains representing 42 species that were tested. Analysis of the RFLP generated after Hinfl restriction digestion of the PCR products further improved the method, allowing complete discrimination among the species and subspecies of Legionella tested. Twenty-three well-identified strains from unrelated origins belonging to seven species gave amplification patterns identical to that of their type strain. The technique was also tested on 80 field isolates that could not be unequivocally assigned to groups by phenotypic methods. Seventy-two per cent (58/80) of these isolates had a profile identical to that of a type strain, while 27% (22/80) may correspond to new taxa since their ISR-PCR profiles did not match any of the known profiles.

Base Sequence↗

PubMed2005

Nocturnal changes in leaf growth of Populus deltoides are controlled by cytoplasmic growth.

Growing leaves do not expand at a constant rate but exhibit pronounced diel growth rhythms. However, the mechanisms giving rise to distinct diel growth dynamics in different species are still largely unknown. As a first step towards identifying genes controlling rate and timing of leaf growth, we analysed the transcriptomes of rapidly expanding and fully expanded leaves of Populus deltoides Bartr. ex. Marsh at points of high and low expansion at night. Tissues with well defined temporal growth rates were harvested using an online growth-monitoring system based on a digital image sequence processing method developed for quantitative mapping of dicot leaf growth. Unlike plants studied previously, leaf growth in P. deltoides was characterised by lack of a base-tip gradient across the lamina, and by maximal and minimal growth at dusk and dawn, respectively. Microarray analysis revealed that the nocturnal decline in growth coincided with a concerted down-regulation of ribosomal protein genes, indicating deceleration of cytoplasmic growth. In a subsequent time-course experiment, Northern blotting and real-time RT-PCR confirmed that the ribosomal protein gene RPL12 and a cell-cycle gene H2B were down-regulated after midnight following a decrease in cellular carbohydrate concentrations. Thus, we propose that the spatio-temporal growth pattern in leaves of P. deltoides primarily arises from cytoplasmic growth whose activity increases from afternoon to midnight and thereafter decreases in this species.

Carbohydrate Metabolism↗

PubMed2006

Ribosomal protein S27L is a direct p53 target that regulates apoptosis.

Ribosomal proteins were recently shown to regulate p53 activity by abrogating Mdm2-induced p53 degradation (L23, L11, L5) or by enhancing p53 translation (L26). Here, we report that a novel ribosomal protein, RPS27L (S27-like protein), is a direct p53 target. RPS27L, but not its family member RPS27, was identified as a p53 inducible gene in a genome-wide chip-profiling study. Further characterization revealed a p53-dependent induction of RPS27L in multiple cancer cell models. Indeed, a consensus p53-binding site was identified in the first intron of the RPS27L gene and a direct binding of p53 to this site was demonstrated both in vitro and in vivo. Characterization of a luciferase reporter driven by the RPS27L intron fragment revealed a p53-binding site-dependent transaction by wild-type p53, but not by several transactivating-deficient p53 mutants. This transactivation was enhanced by etoposide, a DNA damaging agent that activates p53 and was completely blocked by a dominant-negative p53 mutant. Functionally, overexpression of RPS27L within the physiological inducible levels promoted, whereas siRNA silencing of RPS27L inhibited, apoptosis induced by etoposide. This is the first report, to our knowledge, that p53 directly induces the expression of a ribosomal protein, RPS27L, which in turn promotes apoptosis.

Antineoplastic Agents, Phytogenic↗

PubMedDate not supplied

Expression profile of ovarian tumors: distinct signature of Sertoli-Leydig cell tumor.

Epithelial ovarian tumors are the most common subtype of ovarian cancer. In this study, we reveal distinct expression signatures of previously uncharacterized ovarian carcinoma subtypes, including endometrioid component of mixed ovarian tumor and Sertoli-Leydig tumor. Both subtypes were compared to the most common and well-characterized ovarian epithelial carcinoma of the serous type. These comparisons were performed by complementaryDNA (cDNA) microarrays allowing high-fidelity measurements of the expression levels of 39,360 human individual cDNA species representing both known and unknown human genes. Functional analysis of differentially expressed genes in Sertoli-Leydig tumor revealed an upregulation in sonic hedgehog pathway, deregulation of several metabolic pathways especially in amino acid metabolism and overexpression of genes associated with protein synthesis, including ribosomal genes.

Female↗

PubMed2005

Bacterial diversity in the rhizosphere of Proteaceae species.

The Cape Floral Kingdom is an area of unique plant biodiversity in South Africa with exceptional concentrations of rare and endemic species and experiencing drastic habitat loss. Here we present the first molecular study of the microbial diversity associated with the rhizosphere soil of endemic plants of the Proteaceae family (Leucospermum truncatulum and Leucadendron xanthoconus). Genomic DNA was extracted from L. truncatulum rhizosphere soil, L. xanthoconus rhizosphere and non-rhizosphere soil and used as a template for the polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA gene (rDNA). Construction and sequencing of 16S rDNA libraries revealed a high level of biodiversity and led to the identification of several novel bacterial phylotypes. The bacterial community profiles were compared by 16S rDNA denaturing gradient gel electrophoresis (DGGE). Cluster analysis and biodiversity indices revealed that the rhizosphere soil samples were more similar to each other than to non-rhizosphere soil and the rhizosphere soil contained a bacterial diversity that was richer and more equitable compared with non-rhizosphere soil. A Chloroflexus and an Azospirillum genospecies were restricted to the L. xanthoconus rhizosphere soil and Stenotrophomonas genospecies was identified in all rhizosphere soil samples but was not present in the non-rhizosphere soil. Taxon-specific nested PCR and DGGE-identified differences between the Proteaceae plant rhizosphere soil with a Frankia genospecies restricted the L. truncatulum rhizosphere. Archaea-specific rDNA PCR, DGGE and DNA sequencing revealed that Crenarcheote genospecies were excluded from the plant rhizosphere soil and only present in non-rhizosphere soil.

Bacteria↗

PubMed2000

In vitro coupled transcription translation: effects of modification in lysate preparation on protein composition and biosynthesis activity.

Cell-free extracts (lysates) from Escherichia coli were used for protein synthesis in vitro. Essential steps of the lysate preparation were modified and analyzed with respect to their impact on in vitro protein synthesis capacity, using the green fluorescent protein (GFP) as a target protein. Variably manufactured lysates of low, medium and higher protein synthesis activity, were examined by high resolution two-dimensional gel electrophoresis to determine whether the modifications result in substantial alterations in protein composition of the final lysate. The total number of proteins calculated from the gel maps did not vary for lysates with different activity and thus cannot serve as an evaluation parameter. Ribosomal proteins RP-S1, RP-L9, and RP-L10 were found in stoichiometric amounts for each of these lysates and in equal concentrations in comparison among the different lysates. Conversely, depending on the activity profiles, up to 7 different isoforms of the elongation factor EF-Ts were detected in the gel maps.

Bacterial Proteins↗

PubMed1987

Regulation of cardiac myosin synthesis: studies of RNA content in cultured heart cells.

Contraction regulates the myosin content and the rate of myosin synthesis in cultured neonatal rat heart cells. To further explore the mechanism for this regulation we examined various parameters of RNA content and RNA synthesis in contacting versus noncontracting myocytes. While contraction stimulated myosin heavy chain (MHC) synthesis by 72% compared to that of KCl-arrested cells, simultaneous analyses of polysome profiles were no different under the two culture conditions. Incorporation of [3H]uridine monophosphate into cellular RNA revealed no change in the rate of total RNA or ribosomal subunits synthesis. In vitro translation of cellular RNA yielded similar incorporation of [35S]methionine into trichloroacetic acid precipitable protein. Specific transcription of the MHC gene was examined by dot-blot analysis and was unaltered by contraction. Northern blot analysis of the MHC sequences detected by a cDNA probe revealed an mRNA sequence corresponding to a molecular weight of approximately 30 S. These data suggest that RNA synthesis and RNA content are unaltered by contraction in cultured heart cells and therefore the changes in myosin synthesis may be mediated at a post-transcriptional control level.

Animals↗

PubMed2006

Display technologies: application for the discovery of drug and gene delivery agents.

Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed.

Animals↗

PubMed2004

Ultrastructure of the rostral ventral respiratory group neurons in the ventrolateral medulla of the rat.

The neurons in the ventrolateral medulla that project to the spinal cord are called the rostral ventral respiratory group (rVRG) because they activate spinal respiratory motor neurons. We retrogradely labeled rVRG neurons with Fluoro-Gold (FG) injections into the fourth cervical spinal cord segment to determine their distribution. The rostral half of the rVRG was located in the area ventral to the semicompact formation of the nucleus ambiguus (AmS). A cluster of the neurons moved dorsally and intermingled with the palatopharyngeal motor neurons at the caudal end of the AmS. The caudal half of the rVRG was located in the area including the loose formation of the nucleus ambiguus caudal to the AmS. We also labeled the rVRG neurons retrogradely with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to determine their ultrastructural characteristics. The neurons of the rVRG were medium to large (38.1 x 22.1 microm), oval or ellipsoid in shape, and had a dark cytoplasm containing numerous free ribosomes, rough endoplasmic reticulum (rER), mitochondria, Golgi apparatuses, lipofuscin granules and a round nucleus with an invaginated nuclear membrane. The average number of axosomatic terminals in a profile was 33.2. The number of axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray's type I) was almost equal to those containing pleomorphic vesicles and making symmetric synaptic contacts (Gray's type II). The axodendritic terminals were large (1.55 microm), and about 60% of them were Gray's type I. The rVRG neurons have ultrastructural characteristics, which are different from the palatopharyngeal motor neurons or the prorpiobulbar neurons.

Animals↗

PubMed2005

Taxonomic note "Lactobacillus pastorianus" (Van Laer, 1892) a former synonym for Lactobacillus paracollinoides.

"Lactobacillus pastorianus" (Van Laer, 1892) is not a validly described species and is not included in the Approved List of Bacterial Names. The strain is available in multiple culture collections as Lactobacillus sp. DSM 20197, L. brevis ATCC 8291, "L. pastorianus" CECT 5926, L. brevis JCM 1113, and "L. pastorianus" LMG 11990. Nearly identical 16S rRNA sequences and protein encoding genes for 6-phosphogluconate dehydrogenase (99.9%) revealed this strain as L. paracollinoides. A 16S-23S rRNA intergenic spacer region-based PCR assay did not differentiate "L. pastorianus" DSM 20197 from L. paracollinoides DSM 15502(T). Highly similar RAPD profiles differentiated both strains below species level.

DNA Fingerprinting↗

PubMed2004

Revised definition of Actinobacillus sensu stricto isolated from animals. A review with special emphasis on diagnosis.

The taxonomy of the members of the genus Actinobacillus associated with animals has been reviewed with focus on classification and identification including molecular based characterization, typing and identification. Out of the 22 species or species like taxa reported as Actinobacillus, 19 are associated with animals. When classified on the basis of 16S rRNA sequence based phylogenetic analysis, DNA-DNA hybridizations and phenotypic analysis, Actinobacillus sensu stricto is restricted to include A. lignieresii, A. pleuropneumoniae, A. equuli subsp. equuli, A. equuli subsp. haemolyticus (taxon 11 of Bisgaard), A. hominis, A. suis, A. ureae, A. arthritidis (taxon 9 of Bisgaard), Actinobacillus genomospecies 1 and 2 and the taxa 8 and 26 of Bisgaard. The remaining 11 species of Actinobacillus are unrelated to A. sensu stricto and should consequently be grouped with other genera or be renamed as new genera depending on new data. Identification of members of Actinobacillus at species level is possible through phenotypic characterization combined with information on host of isolation. PCR tests are available for specific detection of A. pleuropneumoniae. Only A. pleuropneumoniae is presently considered as a primary pathogen. Based on different types of RTX genes it is possible to PCR type A. pleuropneumoniae to serotype level. PCR might also be used for the specific detection of A. equuli subsp. haemolyticus. Epidemiological investigations and surveillance have so far included serotyping, multilocus enzyme electrophoresis (MLEE), ribotyping and restriction fragment length profiling.

Actinobacillus↗

PubMed1978

In vivo effect of colchicine on hepatic protein synthesis and on the conversion of proalbumin to serum albumin.

Treatment of rats with 0.5-25 mumol/100 g body weight of colchicine for 1 h or more caused an inhibition of hepatic protein synthesis. This effect was not seen if animals were exposed to colchicine for less than 1 h. The delayed inhibition of protein synthesis affected both secretory and nonsecretory proteins. Treatment with colchicine (15 mumol/100 g) for 1 h or more caused the RNA content of membrane-bound polysomes to fall but did not change the polysomal profile of this fraction. By contrast, the total RNA content in the free polysome cell fraction was increased, and this was due to the presence of more ribosomal monomers and dimers. Electron microscope examination of the livers from rats treated for 3 h with colchicine showed an accumulation of secretory vesicles within the hepatocytes and a general distention of the endoplasmic reticulum. Administration of radioactive L-leucine to the rats led to an incorporation of radioactivity into two forms of intracellular albumin which were precipitable with antiserum to rat serum albumin but which were separable by diethylaminoethyl-cellulose chromatography. One form has arginine at the amino-terminal position and is proalbumin, and the other form, which more closely resembles serum albumin chromatographically, has glutamic acid at its amino terminus. Only proalbumin was found in rough and smooth endoplasmic reticulum fractions and in a Golgi cell fraction wich corresponds morphologically to mostly empty and partially filled secretory vesicles. However, in other Golgi cell fractions which were filled with secretory products, both radioactive proalbumin and serum albumin were found. This indicates that proalbumin is converted to serum albumin in these secretory vesicles just before exocytosis. Colchicine delayed the discharge of radioactive albumin from these filled secretory vesicles and caused an accumulation of both proalbumin and serum albumin within these cell fractions.

Animals↗

PubMed1995

Lentzea gen. nov., a new genus of the order Actinomycetales.

We describe a new genus of mesophilic actinomycetes, for which we propose the name Lentzea. The strains of this genus form abundant aerial hyphae that fragment into rod-shaped elements. Whole-cell hydrolysates contain the meso isomer of diaminopimelic acid and no characteristic sugar (wall chemotype III). The phospholipid pattern type is type PII (phosphatidylethanolamine is the characteristic phospholipid); the major menaquinone is MK-9. The fatty acid profile comprises saturated, unsaturated, and branched-chain fatty acids of the iso and anteiso types in addition to tuberculostearic acid (10Me-C18:0). A 16S ribosomal DNA sequence analysis revealed that the genus Lentzea is phylogenically related to the genera Actinosynnema, Saccharothrix, and Kutzneria. The type species of this genus is Lentzea albidocapillata sp. nov.; the type strain of this species is strain IMMIB D-958 (= DSM 44073).

Actinomycetales↗