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PubMed2004

Yeast Pho85 kinase is required for proper gene expression during the diauxic shift.

The budding yeast Saccharomyces cerevisiae changes its gene expression profile when environmental nutritional conditions are changed. Protein kinases including cyclic AMP-dependent kinase, Snf1 and Tor kinases play important roles in this process. Pho85 kinase, a member of the yeast cyclin-dependent kinase family, is involved in the regulation of phosphate metabolism and reserve carbohydrates, and thus is implicated to function as a nutrient-sensing kinase. Upon depletion of glucose in the medium, yeast cells undergo a diauxic shift, accompanied by a carbon metabolic pathway shift, stimulation of mitochondrial function and downregulation of ribosome biogenesis and protein synthesis. We analysed the effect of a pho85Delta mutation on the expression profiles of the genes in this process to investigate whether Pho85 kinase participates in the yeast diauxy. We found that, in the absence of PHO85, a majority of mitochondrial genes were not properly induced, that proteasome-related and chaperonin genes were more repressed, and that, when glucose was still present in the medium, a certain class of genes involved in ribosome biogenesis (ribosomal protein and rRNA processing genes) was repressed, whereas those involved in gluconeogenesis and the glyoxylate cycle were induced. We also found that PHO85 is required for proper expression of several metal sensor genes and their regulatory genes. These results suggest that Pho85 is required for proper onset of changes in expression profiles of genes responsible for the diauxic shift.

Cyclin-Dependent Kinases↗

PubMedDate not supplied

Monophyly of endosymbiont containing trypanosomatids: phylogeny versus taxonomy.

To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp. TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H. roitmani. The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA. The size of minicircles in both isolates is 4.2 kilobases. The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic. Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree. These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.

Animals↗

PubMed1963

NUCLEOTIDE COMPOSITION OF RIBONUCLEIC ACID FROM NEUROSPORA CRASSA.

Henney, H. (The University of Texas, Austin) and R. Storck. Nucleotide composition of ribonucleic acid from Neurospora crassa. J. Bacteriol. 85:822-826. 1963.-The nucleotide composition of total, ribosomal, and soluble ribonucleic acid (RNA) from the conidia and the hyphae of Neurospora crassa was determined. The corresponding RNA classes from the two morphological types had the same base ratio. Total, ribosomal, and soluble RNA from hyphae contained, respectively, 51.0, 50.2, and 61.6% guanylic acid (G) plus cytidylic acid (C), and from conidia, 50.6, 49.9, and 62.1%. The proportion of nucleotides in ribosomal RNA was in close agreement with that reported for ribosomal RNA from other organisms. Soluble RNA contained 2.8% pseudouridylic acid, 36.0% C, and 25.5% G, and differed from soluble RNA from other sources in its high C content. Centrifugation of purified RNA from conidia and hyphae, through a linear sucrose-density gradient, yielded identical sedimentation profiles.

Neurospora↗

PubMed2004

Denaturing gradient gel electrophoresis analysis of bacterial populations in 5-stage biological nutrient removal process with step feed system for wastewater treatment.

Changes in the bacterial populations of a 5-stage biological nutrient removal (BNR) process, with a step feed system for wastewater treatment, were monitored by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments. DGGE analysis indicated seasonal community changes were observed, however, community profiles of the total bacteria of each reactor showed only minor differences in the samples obtained from the same season. The number of major bands was higher in the summer samples, and decreased during the winter period, indicating that the microbial community structure became simpler at low temperatures. Since the nitrogen and phosphate removal efficiencies were highly maintained throughout the winter operation period, the bacteria which still remaining in the winter sample can be considered important, playing a key role in the present 5-stage BNR sludge. The prominent DGGE bands were excised, and sequenced to gain insight into the identities of the predominant bacterial populations present, and most were found to not be closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods for the quality control of wastewater treatment.

Bacteria↗

PubMed2004

Gene expression profiling of Escherichia coli expressing double Vitreoscilla haemoglobin.

In a recent investigation, expression of a double Vitreoscilla haemoglobin (two fused VHb molecules) in Escherichia coli grown in shake flasks resulted in higher final cell density and considerably higher levels of ribosomes and tRNA. In this study, we have investigated the E. coli transcriptome in cells expressing native VHb, double VHb and control cells lacking VHb by hybridising mRNA from the different constructs to high-density oligonucleotide arrays. Within the 95% confidence interval, 4 and 5% of all detected genes in native VHb cells were up- and down-regulated, respectively; in double VHb cells the corresponding numbers were 6 and 10%, respectively. Dividing the data into different functional groups revealed that genes involved in energy metabolism, central intermediary metabolism and cell processes were the most affected at the mRNA level. Particularly, the up-regulation of genes involved in translation and posttranslational modification observed in double VHb cells demonstrates a strong relationship between the regulation of ribosomal genes and the actual number of ribosomes.

Bacterial Proteins↗

PubMed1975

[Properties of ribosomes of normal and tumor cells (author's transl)].

On the basis of results showing that the extent and direction of protein biosynthesis essential for cell growth and cell differentiation can be regulated, aside from the transcription level, also on the level of ribosomes (translation), the main results of various properties of ribosomes from normal and tumor tissues are described. The main established difference between the two tissues concerns the organization and localization of the ribosomes within the cell. In tumors, polysomes and inactive dimers and monomeric particles are encountered in considerable amounts freely in the cytosol, i.e. not attached to the membranes. Furthermore, the polysomal profile of tumor cells compared with the normal tissue is characterized by a shift towards fractions with lower sedimentation coefficients. Monomeric ribosomes were not found to differ in physical and chemical properties. In smaller oligonucleotide fragments of ribosomal RNA, differences in the frequency of certain dinucleotides appear likely. Hybridization-competition experiments with DNA do suggest, however, that the ribosomal RNA from tumors is being synthesized at the same cistrons as ribosomal RNA from normal tissues, thus proving its overall sequence homology. Electrophoretic and immunologic methods have not so far revealed any clear differences in the properties of ribosomal proteins from normal and tumor tissues. In in-vitro systems, ribosomes from various tumors show approximately the same incorporation rates for amino acids as ribosomes from normal tissues. The enhanced protein synthesis of the cells, on the basis of data obtained from in-vitro experiments with cell free systems, is to be attributed mainly to the increased content of translation factors in the cytosol.

Animals↗

PubMed1990

Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes.

A protein-tyrosine-phosphatase (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), specific for phosphotyrosyl residues, was microinjected into Xenopus oocytes. This resulted in a 3- to 5-fold increase in PTPase activity over endogenous levels. The PTPase blocked the insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins, including a protein having a molecular mass in the same range as the beta subunit of the insulin or insulin-like growth factor I receptor. PTPase 1B also blocked the activation of an S6 peptide kinase--i.e., an enzyme recognizing a peptide having the sequence RRLSSLRA found in a segment of ribosomal protein S6 and known to be activated early in response to insulin. On the other hand, the insulin stimulation of an S6 kinase, detected by using 40S ribosomes as substrate, was unaffected even though PTPase 1B partially prevented the phosphorylation of ribosomal protein S6 in vivo. Mono Q chromatography of insulin-treated oocyte extracts revealed two main peaks of S6 kinase activity. Fractions from the first peak displayed S6 peptide kinase activity that was essentially abolished in profiles from PTPase 1B-injected oocytes. Material from the second peak, which was best revealed by using 40S ribosomes as substrate and had comparatively little S6 peptide kinase activity, was minimally affected by PTPase 1B. These observations suggest that at least two distinct "S6 kinases" are involved in ribosomal protein S6 phosphorylation in vivo and that the activation pathways for these enzymes differ in their sensitivity to PTPase 1B.

Animals↗

PubMed1975

Protein synthesis in cell-free systems from totally ischemic rat myocardium.

Ribosomes and cell sap were isolated at different times from excised rat hearts stored in a moist chamber at 37 degrees C and their capacity for protein synthesis was tested. The incorporation rate of labeled amino acids by ribosomes with cell sap from control hearts showed a slow decrease after the first hour of incubation. Polysome profiles as well as incorporation of [14C]phenylalanine in the presence of polyuridylic acid indicated an increase in the amount of monoribosomes during storage of the organ. The activity of cell sap prepared from such hearts after 1 hr and measured with ribosomes from control hearts was also decreased. The reduction in the capacity for protein synthesis noted separately with ribosomes and with cell sap from stored hearts is additive if both components are prepared from stored hearts.

Animals↗

PubMed1976

Surface topography of the Bacillus stearothermophilus ribosome.

The surface topography of the intact 70S ribosomes and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more 125I than did 50S subunits derived from 70S ribosomes, whereas, free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of 125I. Iodinated 70S ribosomes and subunits retained 62-78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosomes than in the free subunits. The locations of tyrosine residues in some homologous ribosomal proteins from B. stearothermophilus and E. coli are compared.

Electrophoresis, Polyacrylamide Gel↗

PubMed1978

Functional covalent complex between elongation factor Tu and an analog of lysyl-tRNA.

Complex formation between elongation factor Tu, GTP, and Nepsilon-bromoacetyl-Lys-tRNA results in the cross-linking of the protein and the modified Lys-tRNA. The efficiency of affinity labeling is greater than 50%. In the presence of unmodified Lys-tRNA, the amount of crosslinking is greatly decreased. There is no covalent reaction with elongation factor Tu in the absence of complex formation. Substantial purification of the crosslinked ternary complex can be achieved by gel filtration at low Mg2+ concentration and passage through nitrocellulose filters. The crosslinked complex exhibits message-dependent binding to ribosomes which is accompanied by the hydrolysis of the associated GTP, as shown by both filter assays and gel filtration profiles. The crosslinked complex therefore appears to function normally except for its inability to dissociate. These experiments demonstrate that the ternary complex is the true intermediate in the binding of aminoacyl-tRNA to the ribosomes.

Affinity Labels↗

PubMed1997

Characterization of translation initiation factor 5 (eIF5) from Saccharomyces cerevisiae. Functional homology with mammalian eIF5 and the effect of depletion of eIF5 on protein synthesis in vivo and in vitro.

Eukaryotic translation initiation factor 5 (eIF5) interacts in vitro with the 40 S initiation complex (40 S.AUG.Met-tRNAf.eIF2.GTP) to mediate the hydrolysis of ribosome-bound GTP. In Saccharomyces cerevisiae, eIF5 is encoded by a single copy essential gene, TIF5, that encodes a protein of 45,346 daltons. To understand the function of eIF5 in vivo, we constructed a conditional mutant yeast strain in which a functional but a rapidly degradable form of eIF5 fusion protein was synthesized from the repressible GAL promoter. Depletion of eIF5 from this mutant yeast strain resulted in inhibition of both cell growth and the rate of in vivo protein synthesis. Analysis of the polysome profiles of eIF5-depleted cells showed greatly diminished polysomes with simultaneous increase in free ribosomes. Furthermore, lysates of cells depleted of eIF5 were dependent on exogenously added yeast eIF5 for efficient translation of mRNAs in vitro. This is the first demonstration that the TIF5 gene encodes a protein involved in initiation of translation in eukaryotic cells. Additionally, we show that rat eIF5 can functionally substitute yeast eIF5 in translation of mRNAs in vitro as well as in complementing in vivo a genetic disruption in the chromosomal copy of TIF5.

Animals↗

PubMed1995

Characterization of the first European isolate of Neospora caninum (Dubey, Carpenter, Speer, Topper and Uggla).

Neospora caninum is an apicomplexan, protozoan parasite, which causes severe disease in dogs and cattle. It has previously been isolated only in the United States. A 5-week-old Boxer pup with a progressive hindlimb paresis was diagnosed as suffering from neosporosis on the basis of clinical signs and the presence of anti-Neospora antibodies in it, 2 litter-mates and its dam. Despite treatment with sulphonamides, the pup was euthanased 3 days later. The diagnosis of neosporosis was confirmed by immunohistochemical examination of muscle and CNS tissue sections from the pup. Parasites were isolated into Vero cell culture from the cerebrum, and confirmed as Neospora caninum by immunofluorescence with specific antibody, tachyzoite ultrastructure and 16S-like ribosomal RNA sequences. This isolate (designated NC-Liverpool) has been continuously passaged every 7-10 days. Its growth characteristics, ultrastructure and antigenic profile, as revealed by immunoblotting, have revealed no major differences from the American NC-1 isolate. Furthermore, no difference was seen when comparing the sequences of 16S-like ribosomal RNA and the ITS1 region of the two isolates.

Animals↗

PubMed2001

Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits.

Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission. With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function. We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs. The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)). Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family. The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites). The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit. Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal. In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.

Journal Article↗

PubMed1976

Hodgkin's disease and myelomonocytic leukemia: an ultrastructural and immunocytochemical study.

The ultrastructual and immunologic features of the initial Reed-Sternberg and Hodgkin cells are compared with the ultimate leukemic cell type in a child with Hodgkin's disease who subsequently developed acute myelomonocytic leukemia (AMML) following 29 months of chemotherapy. Hodgkin tumor cells contained cytoplasmic IgG and ultrastructurally resembled large immunoblasts, containing one or two round nuclei with large bizarre nucleoli, many polyribosomes, sparase endoplasmic reticulum, underdeveloped Golgi lamellae, and few cytoplasmic granules. The Hodgkin tumor cells displayed no evidence of phagocytosis. The leukemic monocytic cells did not contain cytoplasmic IgG and, ultrastrucally, exhibited and indented and irregular nuclear profile with less prominent nucleoli, numerous pleomorphic granules, a moderate number of free ribosomes, short segments of endoplasmic reticulum, and stacked Golgi lamellae. The cell surface was irregular and occasionally appeared involved in endocytic activity. These results indicate that the Hodgkin tumor cells originated from B lymphocytes rather than tissue macrophages, whereas the leukemic monocytes arose from the bone marrow-derived monocyte-macrophage series. The findings suggest further that AMML developing after Hodgkin's disease consitutes a second neoplasm rather than a leukemic transformation of Hodgkin tumor cells.

Bone Marrow↗

PubMed1980

Heterogeneity of the MtTw15 mammosomatotropic tumor. II. Characterization of parenchymal cells by superimposition immunocytochemistry and electron microscopy.

Large MtTw15 tumors, which secrete growth hormone (GH) and prolactin (PRL), are composed of ovoid, elongated, and angular cells which demonstrated interdigitating processes and junctional complexes. The majority of the cells were essentially agranular, but two types of granulated cells were identifiable. One class of granulated cells contained moderate to sparse populations of round dense-cored granules measuring up to 250 nm in diameter. Rod-shaped to filamentous mitochondria with an electron-dense matrix were characteristic of a second class of granulated cells with plemorphic granules of various sizes and electron densities. Images of exocytotic release of the round dense-cored granules were frequently seen, but were not observed with the pleomorphic granules, many of which were judged to be lysosomes. Superimposition immunocytochemistry revealed hormones only in the granulated cells with round to ovoid granules. Morphometry indicated that hormone specific subpopulations of tumor cells can be identified since PRL secretory granules were significantly smaller than GH secretory granules (149 +/- 6 nm for PRL versus 221 +/- 9 nm for GH, P less than 0.001). The vast majority of immunopositive cells contained only GH or PRL, but a few were observed containing both hormones. Ovoid to irregular-shaped nuclei, large lipid inclusions, numerous free ribosomes and polyribosomes, moderate development of the rough endoplasmic reticulum, and prominent Golig profiles were characteristics of all cell types. Irrespective of the presence or absence of cytoplasmic granular elements, particles resembling viruses were encountered in many tumor cells, and these frequently appeared to be budding into the cisternae of the endoplasmic reticulum.

Animals↗

PubMed1999

Autoregulation of the androgen receptor at the translational level: testosterone induces accumulation of androgen receptor mRNA in the rat ventral prostate polyribosomes.

Several studies have documented that androgens have the ability to autoregulate their own receptor levels; however, the mechanism of such autoregulation remains poorly understood. Along these lines, our laboratory has shown that testosterone increased androgen receptor (AR) protein levels and binding in the castrated rat ventral prostate within 1 h. Ongoing protein synthesis was required for the testosterone effect, as the protein synthesis inhibitor cycloheximide blocked this effect. Testosterone and/or actinomycin D, an mRNA synthesis inhibitor, did not affect the steady-state AR mRNA levels. Therefore, we suggest that the early events induced by testosterone are posttranscriptional and that protein synthesis is required for the maintenance of AR protein and AR mRNA levels. In addition, we hypothesize that the testosterone posttranscriptional effect is primarily through the sequestering of AR mRNA in the prostate polyribosomes. To test this hypothesis, total RNA was isolated from prostate polyribosomes of controls and testosterone-treated rats and AR mRNA levels were quantitated by competitive reverse transcription-polymerase chain reaction. Polyribosomes profiles on linear sucrose gradients showed no difference in the sedimentation characteristics of ribosomal particles from the vehicle-treated control or testosterone-treated animals. Furthermore, because both polyribosomal preparations can direct protein synthesis to the same extent in a cell-free system, testosterone does not increase the efficiency of translation. However, competitive reverse transcription-polymerase chain reaction revealed that testosterone increases AR mRNA associated with polyribosomes by threefold after 1 h of treatment compared with control. These data suggest a rapid testosterone-mediated posttranscriptional mechanism, in which testosterone regulates the stability of the AR mRNA by sequestering it in polyribosomes, and consequently increasing its translation.

Animals↗

PubMed2005

Gene expression changes in Arabidopsis thaliana seedling roots exposed to the munition hexahydro-1,3,5-trinitro-1,3,5-triazine.

Arabidopsis thaliana root transcriptome responses to the munition, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), were assessed using serial analysis of gene expression (SAGE). Sequencing of SAGE libraries from control and RDX-exposed root tissues revealed induction of genes known to respond to a variety of general stresses. Among the highly induced genes were several encoding molecular chaperones and transcription factors as well as vacuolar proteins and peroxidases. Strongly repressed transcripts included ones encoding ribosomal proteins, a cyclophilin, a katanin, and a peroxidase. Comparison of the transcriptional profile for the RDX response to a profile previously described for Arabidopsis roots exposed to trinitrotoluene (TNT) revealed significant differences in the inferred gene expression patterns. This suggests that Arabidopsis employs drastically different mechanisms for coping with these two compounds. With respect to the goal of engineering plants to better tolerate and degrade explosives at contaminated sites, these results suggest that enhancement of different genes and metabolic pathways may be required to deal effectively with each type of explosive. This has ramifications for phytoremediation efforts since many contaminated sites harbor both compounds.

Adaptation, Physiological↗

PubMed2004

Distinctive features of plant organs characterized by global analysis of gene expression in Arabidopsis.

The distinctive features of plant organs are primarily determined by organ-specific gene expression. We analyzed the expression specificity of 8809 genes in 7 organs of Arabidopsis using a cDNA macroarray system. Using relative expression (RE) values between organs, many known and unknown genes specifically expressed in each organ were identified. We also analyzed the organ specificity of various gene groups using the GRE (group relative expression) value, the average of the REs of all genes in a group. Consequently, we found that many gene groups even ribosomal protein genes, have strong organ-specific expression. Clustering of the expression profiles revealed that the 8809 genes were classified into 9 major categories. Although 3451 genes were clustered into the largest category, which showed constitutive gene expression, 266 and 1005 genes were found to be root- and silique-specific genes, respectively. By this clustering, particular gene groups which showed multi-organ-specific expression profiles, such as bud-flower-specific, stem-silique-specific or bud-flower-root-specific profiles, could be effectively identified. From these results, major features of plant organs could be characterized by their distinct profiles of global gene expression. These data of organ-specific gene expression are available at our web site: Arabidopsis thaliana Tissue-Specific Expression Database, ATTED (http://www.atted.bio.titech.ac.jp/).

Arabidopsis↗