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At least 937 records · Page 52Linked to original sources
PubMed2005

Epigenomic mapping in Arabidopsis using tiling microarrays.

In addition to genetic information, chromosomes transmit epigenetic information from cell to cell during division, and sometimes from generation to generation. While genetic information is encoded directly in the DNA sequence, epigenetic information is not, although it is usually associated with specific chromosomal regions. Epigenetic modifications in plants include cytosine methylation as well as modification of histones and other chromosomal proteins. Small interfering RNA play major roles in targeting these modifications to specific regions. Genomic tiling microarrays are powerful tools for analysing epigenetic information, and we review their application in building epigenomic maps in the model plant, Arabidopsis.

Arabidopsis↗
PubMed1992

Structural requirements for the processing of Escherichia coli 5 S ribosomal RNA by RNase E in vitro.

Processing of 9 S precursor RNA in Escherichia coli requires the endoribonuclease RNase E, which makes two cleavages to liberate p5, the immature form of 5 S rRNA. The contributions of primary and secondary structure to RNase E-mediated cleavage of 9 S RNA were investigated. The structure of the 5' domain of 9 S RNA was probed by partial ribonuclease digestion and chemical modification. Our structural analysis of 9 S RNA supports a model in which the 5' spacer domain folds into tandem hairpins so that the first processing cleavage site 5' to the 5 S moiety resides in a stretch of single-stranded residues. Site-directed mutagenesis of a cloned 9 S RNA sequence was performed and synthetic transcripts derived from a variety of such mutant templates were assayed as substrates for RNase E-dependent endonuclease activity in fractionated extracts. Partial or complete deletion of the 5 S sequence did not eliminate site-specific processing of 9 S RNA. Mutations affecting the 5' domain revealed that secondary structure upstream from the first cleavage site is important in maintaining efficient processing. However, secondary structure downstream from either cleavage site is dispensable. Our results suggest that RNase E specifically recognizes and cleaves single-stranded RNA sequences only when presented in a proper conformational context. Adjacent secondary structures appear to play a direct and critical role in the enzyme's recognition of its substrate. Additionally, it may serve to anchor single-stranded regions to ensure the availability of the RNase E cleavage sites.

Base Sequence↗
PubMed2003

Widespread inosine-containing mRNA in lymphocytes regulated by ADAR1 in response to inflammation.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of pre-mRNA catalysed by an RNA-specific adenosine deaminase (ADAR). A-to-I RNA editing has been previously reported in the pre-mRNAs of brain glutamate and serotonin receptors and in lung tissue during inflammation. Here we report that systemic inflammation markedly induces inosine-containing mRNA to approximately 5% of adenosine in total mRNA. Induction was the result of up-regulation of A-to-I RNA editing as both dsRNA editing activity and ADAR1 expression were increased in the spleen, thymus and peripheral lymphocytes from endotoxin-treated mice. Up-regulation of ADAR1 was confirmed in vitro in T lymphocytes and macrophages stimulated with a variety of inflammatory mediators including tumour necrosis factor-alpha and interferon-gamma. A late induction of RNA editing was detected in concanavalin A-activated splenocytes stimulated with interleukin-2 in vitro. Taken together, these data suggest that a large number of inosine-containing mRNAs are produced during acute inflammation via up-regulation of ADAR1-mediated RNA editing. These events may affect the inflammatory and immune response through modulation of protein production.

Acute Disease↗
PubMed2001

Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome.

During the translocation step of the elongation cycle, two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. The movement is catalyzed by the binding of elongation factor G (EF-G) and driven by GTP hydrolysis. Here we study structural changes of the ribosome related to EF-G binding and translocation by monitoring the accessibility of ribosomal RNA (rRNA) for chemical modification by dimethyl sulfate or cleavage by hydroxyl radicals generated by Fe(II)-EDTA. In the state of the ribosome that is formed upon binding of EF-G but before the movement of the tRNAs takes place, residues 1054,1196, and 1201 in helix 34 in 16S rRNA are strongly protected. The protections depend on EF-G binding, but do not require GTP hydrolysis, and are lost upon translocation. Mutants of EF-G, which are active in ribosome binding and GTP hydrolysis but impaired in translocation, do not bring about the protections. According to cryo-electron microscopy (Stark et al., Cell, 2000, 100:301-309), there is no contact of EF-G with the protected residues of helix 34 in the pretranslocation state, suggesting that the observed protections are due to an induced conformational change. Thus, the present results indicate that EF-G binding to the pretranslocation ribosome induces a structural change of the head of the 30S subunit that is essential for subsequent tRNA-mRNA movement in translocation.

Guanosine Triphosphate↗
PubMed1987

Inhibition of eukaryotic translation by analogues of messenger RNA 5'-cap: chemical and biological consequences of 5'-phosphate modifications of 7-methylguanosine 5'-monophosphate.

New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of -O with -H, -CH3, or -NH2. Additional analogues were synthesized with 8-methyl- or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 1H and 31P NMR for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by -H and -CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by -NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the alpha-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation.

Animals↗
PubMed2004

Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication.

Hepatitis delta virus (HDV) contains a circular RNA which encodes a single protein, hepatitis delta antigen (HDAg). HDAg exists in two forms, a small form (S-HDAg) and a large form (L-HDAg). S-HDAg can transactivate HDV RNA replication. Recent studies have shown that posttranslational modifications, such as phosphorylation and acetylation, of S-HDAg can modulate HDV RNA replication. Here we show that S-HDAg can be methylated by protein arginine methyltransferase (PRMT1) in vitro and in vivo. The major methylation site is at arginine-13 (R13), which is in the RGGR motif of an RNA-binding domain. The methylation of S-HDAg is essential for HDV RNA replication, especially for replication of the antigenomic RNA strand to form the genomic RNA strand. An R13A mutation in S-HDAg inhibited HDV RNA replication. The presence of a methylation inhibitor, S-adenosyl-homocysteine, also inhibited HDV RNA replication. We further found that the methylation of S-HDAg affected its subcellular localization. Methylation-defective HDAg lost the ability to form a speckled structure in the nucleus and also permeated into the cytoplasm. These results thus revealed a novel posttranslational modification of HDAg and indicated its importance for HDV RNA replication. This and other results further showed that, unlike replication of the HDV genomic RNA strand, replication of the antigenomic RNA strand requires multiple types of posttranslational modification, including the phosphorylation and methylation of HDAg.

Amino Acid Sequence↗
PubMed2005

The PML-nuclear inclusion of human supraoptic neurons: a new compartment with SUMO-1- and ubiquitin-proteasome-associated domains.

It is well known that the cell nucleus is organized in structural and functional compartments involved in transcription, RNA processing and protein modifications such as conjugation with SUMO-1 and proteolysis. Promyelocytic leukaemia (PML) bodies are dynamic nuclear structures that concentrate PML protein, SUMO-1 and several sumoylated and non-sumoylated protein regulators of nuclear functions. PML bodies and their associated CBP has been involved in neuronal survival. By light and electron microscopy immunocytochemistry and in situ hybridization we reported the presence, in non-pathological conditions, of a large PML-nuclear inclusion (PML-NI) in human supraoptic neurons. This inclusion appears as a single nuclear structure composed of a capsule enriched in PML, SUMO-1 and CBP proteins and a central lattice of filaments immunoreactive for class III beta-tubulin, ubiquitinated proteins and proteasomes. Furthermore, the PML-NI concentrates the SUMO-conjugating enzyme E2 (UBC9). The PML-NI may be considered a nuclear factory involved in sumoylation and proteolysis via ubiquitin-proteasome system, two nuclear pathways engaged in the control of the nucleoplasmic concentration of active transcriptional regulators. Interestingly, the structural and molecular organization of the PML-NI is related to the Marinesco bodies, age-associated ubiquitinated intranuclear inclusions, and to the intranuclear rodlets enriched in class III beta-tubulin, which are nuclear structures markedly decreased in Alzheimer's disease.

Adolescent↗
PubMed2001

Extraction of total RNA from adipocytes.

RNA isolation from adipocytes presents with several technical problems and yields unacceptable results when following standard protocols. Here, we will describe additional steps and modifications necessary for the use of different RNA isolation protocols in terms of RNA yield, RNA quality and preparation time. Using five times the recommended quantity of lysis buffer, incubating the lysate at 37 degrees C, repeatedly passing the lysate through a cannula, and centrifugation to remove the lipid layer are essential additional steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12-30 microg total RNA from 2 x 10(6) cells. Preparation times were similar for all but the CsCl gradient method. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA. CsCl gradient ultracentrifugation is suggested for situations where DNase I digestion is impractical.

Adipocytes↗
PubMed1999

Differential expressions of CD44 variants in tumors affecting the central nervous system.

BACKGROUND: The polymorphic cell adhesion molecule CD44 exists as a family of proteins generated by extensive alternative splicing of the CD44 pre-messenger RNA and marked posttranslational modification. The differential expression of CD44 isoforms in a variety of human cancers has been proposed to influence tumorigenesis and metastasis. In this study, CD44 gene expression was analyzed in primary and metastatic tumors and in cell lines derived from tumors that affect the central nervous system (CNS), including tumors metastatic to the spine. MATERIALS AND METHODS: Fifty-four samples were subjected to semiquantitative reverse-transcriptase polymerase chain reaction with CD44-specific primers and hybridized individually with probes specific for the CD44 variant (CD44v) exons v3 to v10. RESULTS: Compared with CD44v-positive breast cancer cell lines and CD44v-negative normal brain tissue, CD44v expression was weak in primary brain tumors and cell lines derived from normal brain and tumor tissue. However, high levels of isoforms encoding multiple-variant exons were shown in all metastatic brain tumors. In contrast, tumors metastatic to the spine were virtually negative for CD44v expression. Several rare CD44 isoforms composed of single-variant exons v3, v4, v6, or v9 were identified in primary brain tumors and may reflect their invasive potential or culturability in vitro. CONCLUSION: These data suggest differential expression of CD44v may substantially influence the end-organ site of metastasis for tumor cells destined for the CNS.

Antigens, Neoplasm↗
PubMed1980

Modification of cytidines in a Q beta replicase template: analysis of conformation and localization of lethal nucleotide substitutions.

The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in vitro by Q beta replicase, was investigated with sodium bisulfite, a reagent that selectively converts single-stranded cytidines to uridines. The reactivity of 45 of the 76 cytidines in MDV-1( + ) RNA was determined by nucleotide sequence analysis. Only 14 of these 45 cytidines were converted to uridine. Treatment of the RNA with methoxyamine, another single-strand-specific cytidine modification reagent, gave results in good agreement with the bisulfite data. The limited reactivity of MDV-1 ( + ) RNA with these reagents indicates that it is a highly structured molecule. A secondary structure consistent with the chemical modification data is proposed. Modification of MDV-1 ( + ) RNA by bisulfite renders it inactive as a template for RNA replication. This inactivation and the modification of the cytidines at the 3' end of the molecule occur at very similar rates. By using a short complementary RNA "mask" to protect just these cytidines, we demonstrated that the loss of activity resulted from their modification. This implies that one or more of the cytidines in the 3'-terminal sequence is required for template activity and that changes within this sequence can have lethal consequences. The effects of modification elsewhere in the sequence are discussed.

Base Sequence↗
PubMed1976

The study of DNA-RNA-polymerase complexes by kinetic formaldehyde method.

A modification of the kinetic formaldehyde method has been proposed providing a possibility for locally denatured regions (defects) formed in DNA preincubated with RNA polymerase (in the absence of nucleoside triphosphates) to be detected. This modification consists in a previous fixation of DNA-enzyme complex with small concentrations of formaldehyde, which do not induce formation of defects in DNA alone. The method has been calibrated under the conditions favourable to RNA synthesis. Studies of the effect of the fixation conditions on the number of defects in DNA interacting with RNA polymerase have shown that the number of defects is constant with formaldehyde fixation concentration between 0.05% and 0.3-0.5% and with fixation time between 2 min and 100 min. The dependence of the number of defects in DNA on RNA polymerase concentration at low ionic strength (0.05 M KCl) is presented by a curve with a plateau. From the initial linear part of the curve it has been found that the enzyme bound to DNA as a monomer. At the excess of the enzyme the mean number of nucleotide pairs between defects is 400-500. Increase of ionic strength results in decrease of the number of defects in DNA. The number of defects depends on temperature of preincubation of the complex. There were no defects in DNA at temperatures below 20 degrees C. At temperatures above 30 degrees C the number of defects reaches saturation. A sharp transition occurs in the range of temperatures between 20 degrees C and 30 degrees C. Analysis of the experimental and literature data, concerning the interaction of formaldehyde and amino acid methylol derivatives with DNA bases, leads to the conclusion that the mechanism of the formation of defects in helical DNA most likely consists in its unwinding or sharp weakening upon binding of RNA polymerase, prior to addition of formaldehyde.

Binding Sites↗
PubMed2006

Analysis of IRF5 gene functional polymorphisms in rheumatoid arthritis.

OBJECTIVE: Recent findings suggest that interferon regulatory factor 5 (IRF-5) may play a crucial role in several cellular processes, including the transcription of genes for inflammatory cytokines. Two genetic variants of the IRF5 gene (rs2004640 in exon 1 and rs2280714 in the 3'-untranslated region) have been shown to exert functional modifications affecting IRF5 messenger RNA splicing and expression, and have been associated with genetic predisposition to systemic lupus erythematosus (SLE). The aim of this study was to analyze the possible contribution of the IRF5 gene to the predisposition to rheumatoid arthritis (RA). METHODS: Three case-control cohorts from Spain (724 RA patients and 542 healthy controls), Sweden (281 RA patients 474 healthy controls), and Argentina (284 RA patients and 286 healthy controls) were independently analyzed. Genotyping for IRF5 rs2004640 and rs2280714 was performed using a TaqMan 5' allele-discrimination assay. RESULTS: In the 3 cohorts studied, no statistically significant differences in allele or genotype frequencies of the rs2004640 and rs2280714 IRF5 polymorphisms were observed between RA patients and controls. Accordingly, haplotype analysis revealed that none of the IRF5 haplotypes was associated with genetic predisposition to RA. CONCLUSION: Our results suggest that the IRF5 functional polymorphisms analyzed do not seem to be implicated in genetic susceptibility to RA.

Argentina↗
PubMed2002

In vitro high-throughput screening assay for modulators of transcription.

We developed a 96-well microtiter-plate high-throughput screening (HTS) assay for the detection of modulators of transcription. This HTS assay consists of three steps: (1) the in vitro transcription reaction; (2) modification and hybridization of RNA products; and (3) washing and quantification. During the first step, a DNA template containing the promoter of interest upstream of a cassette lacking guanosine residues in one of its strands (G-less cassette) is incubated with nuclear extract and the necessary cofactors/activators and substrates. During the second step, the in vitro synthesized transcripts are digested with RNase T1 and hybridized to two DNA oligonucleotides. One oligonucleotide is biotinylated for trapping of the RNA products to a streptavidin-coated plate, and the other is europium-labeled for detection by time-resolved fluorescence. We show that this assay is highly reproducible and robust, yielding results comparable to those obtained by standard methodologies employing radioactive nucleotide incorporation and gel electrophoresis while offering a very significant advantage in terms of throughput (>2000 assay points per operator per day). We demonstrate the usefulness of the assay for the discovery of small molecule inhibitors of transcription, and applications of this approach for the high-throughput discovery of transcriptional modulators are discussed.

Amanitins↗
PubMed1993

Interferon rapidly induces modifications of chromatin sensitivity to DNase I in Daudi lymphoma cells.

The DNase I sensitivity of total chromatin has been analyzed in nuclei isolated from control and interferon-treated Daudi cells. The electrophoretic analysis of DNA has evidenced a different pattern of DNA fragment size produced by DNase I in nuclei isolated from control cells compared to interferon-treated samples. This feature is supported by a different recovery of acid soluble chromatin and is accompanied by modifications of in vitro RNA synthesis along with initiation and elongation of RNA chains. No changes have been evidenced in nuclei isolated from Daudi-resistant cells under the same experimental conditions. These data might be interpreted as a transient modulation, induced by interferon, of chromatin structure in terms of chromatin condensation which, in turn, activates the RNA synthesis after the transduction into the nucleus of the interferon-generated signals.

Burkitt Lymphoma↗
PubMed1987

Antibodies to a fusion protein identify a cDNA clone encoding msp130, a primary mesenchyme-specific cell surface protein of the sea urchin embryo.

In this report we identify a 130-kDa protein encoded by a sea urchin primary mesenchyme-specific cDNA clone, 18C6. The cDNA clone has been partially sequenced, and an open reading frame has been identified. A portion of this open reading frame has been expressed as a beta-galactosidase fusion protein in Escherichia coli, and antibodies to the fusion protein have been generated. These antibodies recognize a 130-kDa protein localized at the surface of primary mesenchyme cells and designated msp130. This is demonstrated to be the same 130-kDa protein recognized by the primary mesenchyme-specific monoclonal antibody B2C2, which recognizes a post-translational modification of the protein. RNA gel blots show that the transcript encoding msp130 is undetectable in egg RNA or 16-cell RNA but can be first detected in premesenchyme blastula embryos. The transcript accumulates significantly after primary mesenchyme cell ingression. Analysis of the expression of msp130 by indirect immunofluorescence staining of embryos and by immunoblots using fusion protein antibodies shows that the msp130 protein is first detectable soon after primary mesenchyme cell ingression.

Amino Acid Sequence↗
PubMed2006

Selenoprotein synthesis: UGA does not end the story.

It is well established that the beneficial effects of the trace element selenium are mediated by its major biological product, the amino acid selenocysteine, present in the active site of selenoproteins. These fulfill different functions, as varied as oxidation-reduction of metabolites in bacteria, reduction of reactive oxygen species, control of the redox status of the cell or thyroid hormone maturation. This review will focus on the singularities of the selenocysteine biosynthesis pathway and its unique incorporation mechanism into eukaryal selenoproteins. Selenocysteine biosynthesis from serine is achieved on tRNA(Sec) and requires four proteins. As this amino acid is encoded by an in-frame UGA codon, otherwise signaling termination of translation, ribosomes must be told not to stop at this position in the mRNA. Several molecular partners acting in cis or in trans have been identified, but their knowledge has not enabled yet to firmly establish the molecular events underlying this mechanism. Data suggest that other, so far uncharacterized factors might exist. In this survey, we attempted to compile all the data available in the literature and to describe the latest developments in the field.

Models, Molecular↗
PubMed2005

Mitochondrial signal peptidases of yeast: the rhomboid peptidase Pcp1 and its substrate cytochrome C peroxidase.

The rhomboid peptidase Pcp1 of yeast is the first mitochondrial enzyme of this new class of serine peptidases. Pcp1 is an integral part of the inner membrane and was identified by its signal peptidase activity responsible for processing of the intermediate of cytochrome c peroxidase (iCcp1) to the mature enzyme. Here we describe studies on the expression of the PCP1 gene. Proteolytic processing of Pcp1 itself was found. The precursor and the intermediate of Ccp1 were localized to the inner membrane. The results confirm our previous report on a two-step processing pathway of cytochrome c peroxidase and the identification of the signal peptidases involved.

Cytochrome-c Peroxidase↗
PubMed2006

Translational fidelity mutations in 18S rRNA affect the catalytic activity of ribosomes and the oxidative balance of yeast cells.

The function of mutations rdn1A, rdn1T, and rdn2 in 18S rRNA of Saccharomyces cerevisiae is investigated. The mutations correspond to substitutions C1054A, C1054U in helix 34, and G517A in helix 18 of 16S rRNA in Escherichia coli, respectively, in which the first and third mutations caused nonsense suppression, while C1054U caused no suppression. In yeast, rdn1A caused phenotypic suppression at nonsense codons, whereas rdn1T and rdn2 caused antisuppression. We provide in vitro evidence that, in addition, rdn1A decreases translational accuracy at sense codons as well, by a factor of 8, accompanied by extreme sensitivity to paromomycin, compatible with its error-prone character. Mutations rdn1T andrdn2 exhibit hyperaccuracy and paromomycin resistance. Thus, mutations in conserved rRNA regions may affect the same functions in the various species but in opposite directions. Mutation rdn1A, but not rdn1T or rdn2, affected also the catalytic activity of the ribosome, a 60S subunit activity. The rate of peptide bond formation was reduced to half its normal value, indicating a communication between the two subunits. Moreover, error-prone mutation rdn1A was less susceptible to oxidative modifications than wild type, indicated by decreased lipid peroxidation and nonprotein/protein disulfides, as well as by increased protein thiols. In contrast, hyperaccurate mutations rdn1T and rdn2 displayed increased oxidative stress. Our results suggest that the cells may consume more energy to achieve hyperaccuracy leading to increased oxidative modifications.

Base Sequence↗