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PubMed1984

Epidemiology of rotavirus gastroenteritis in infants from the area of Bochum as revealed by electrophoresis of genome RNA.

Rotaviruses were detected by enzyme-linked immunosorbent assay (ELISA) in stool samples from 504 infants admitted with gastroenteritis to the children's hospital of the University Bochum in the period from 1979 to 1984. In most infants rotaviruses persisted in the stools for 6-8 days. Rotavirus RNA was extracted from stool samples from 203 infants. By acrylamide gel electrophoresis, bands characteristic of rotavirus double-stranded RNA were detected in 172 (85%) of these 203 samples in which the presence of rotavirus had been demonstrated by ELISA. Of the RNA-positive samples 87% had a "long" RNA pattern. Six different electropherotypes co-circulated and one electropherotype predominated for more than 4 years. In stool samples of one infant 12 genome segments were found, suggesting the possibility of simultaneous infection by more than one electropherotype or the occurrence of modification in the length of RNA segments during infection. In another infant the sequential infection by two different electropherotypes was observed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

The Escherichia coli ribosomal RNA leader nut region interacts specifically with mature 16S RNA.

All ribosomal RNAs are preceded by leader sequences not present in the final ribosome particles. The highly conserved leader sequences of bacterial rRNAs are known to be important for the folding and assembly of functional ribosomes. Very likely transient binding of the leader to mature parts of the 16S RNA occurs during transcription. To better understand the mechanistic details of these functions we have performed a secondary structural analysis of E. coli ribosomal RNA leader transcripts by chemical modification and enzymatic hydrolysis studies. The data were combined with results from thermodynamic stability calculations to yield a generalized structural model. The same secondary structure of the leader core, comprising the nut-like sequences up to the mature 5' end of the 16S RNA, was deduced, irrespective if transcripts started at promoter P1 or 120 nucleotides downstream at P2. Employing gelshift and cross-linking studies we were able to demonstrate that a part of the leader core, namely the nut-like sequence elements bind directly to specific regions within the mature 16S RNA. The sites of RNA-RNA cross-linking could be localized by sequencing. They map in the 16S RNA 5' domain at nucleotide positions G27 to G42, C48, G68, G117 and G126. The results may explain the recently observed scaffolding function of the leader RNA during ribosome biogenesis.

Base Sequence↗
PubMed1995

A secondary-structure model for the self-cleaving region of Neurospora VS RNA.

Neurospora VS RNA performs an RNA-mediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis delta virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region of VS RNA, supported by site-directed mutagenesis and chemical modification structure probing data. The secondary structure of VS RNA is distinct from those of the other naturally occurring RNA self-cleaving domains. In addition to a unique secondary structure, several Mg-dependent interactions occur during the folding of VS RNA into its active tertiary conformation.

Cloning, Molecular↗
PubMed1997

Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster.

Early embryonic germ cells in C. elegans and D. melanogaster fail to express many messenger RNAs expressed in somatic cells. In contrast, we find that ribosomal RNAs are expressed in both cell types. We show that this deficiency in mRNA production correlates with the absence of a specific phosphoepitope on the carboxy-terminal domain of RNA polymerase II. In both C. elegans and Drosophila embryos, this phosphoepitope appears in somatic nuclei coincident with the onset of embryonic transcription, but remains absent from germ cells until these cells associate with the gut primordium during gastrulation. In contrast, a second distinct RNA polymerase II phosphoepitope is present continuously in both somatic and germ cells. The germ-line-specific factor PIE-1 is required to block mRNA production in the germ lineage of early C. elegans embryos (Seydoux, G., Mello, C. C., Pettitt, J., Wood, W. B., Priess, J. R. and Fire, A. (1996) Nature 382, 713-716). We show here that PIE-1 is also required for the germ-line-specific pattern of RNA polymerase II phosphorylation. These observations link inhibition of mRNA production in embryonic germ cells to a specific modification in the phosphorylation pattern of RNA polymerase II and suggest that repression of RNA polymerase II activity may be part of an evolutionarily conserved mechanism that distinguishes germ line from soma during early embryogenesis. In addition, these studies also suggest that different phosphorylated isoforms of RNA polymerase II perform distinct functions.

Animals↗
PubMed1993

Secondary structure of RNase MRP RNA as predicted by phylogenetic comparison.

RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to cleave mitochondrial primer RNA sequences from a variety of sources. The bulk of RNase MRP activity is found in the nucleus where its function remains unknown. Two different approaches have resulted in predictions of distinct secondary structures for RNase MRP RNA. In order to analyze more definitively the higher-order structure of RNase MRP RNA, we have conducted a phylogenetic comparison of the available RNase MRP RNA sequences from human, mouse, rat, cow, toad, and yeast. The resulting secondary structure shares features in common with previously described structures for prokaryotic and eukaryotic RNase P RNAs (1) and RNase MRP RNAs (2, 3). In addition, the phylogenetic structure is consistent with available chemical modification data on RNase MRP RNA and with the detailed analysis of the To antigen binding domain located near the 5' end of the RNase MRP RNA. The structure is not limited to RNase MRP RNAs, but can be expanded to cover both eukaryotic RNase P RNAs and RNase P/MRP RNAs from plants.

Animals↗
PubMedDate not supplied

Predicted secondary structure of the foraminiferal SSU 3' major domain reveals a molecular synapomorphy for granuloreticulosean protists.

The small subunit ribosomal RNA genes of foraminiferal protists are the largest and most divergent of any eukaryote. We demonstrate that this foraminiferal sequence alteration represents a substantial modification to the small subunit ribosomal RNA structure, including a large (up to 350 nt) novel helix in a very well-conserved portion of the head domain. This modification dates from the beginning of the foraminiferal radiation and, within modern orders, is partially conserved at the sequence level, suggesting that it is a functional part of the ribosome. The pattern of conservation makes it particularly useful for determining lower-taxon relationships in morphologically ambiguous allogromiid foraminifera.

Animals↗
PubMed2003

The solution structure of a DNA*RNA duplex containing 5-propynyl U and C; comparison with 5-Me modifications.

The addition of the propynyl group at the 5 position of pyrimidine nucleotides is highly stabilising. We have determined the thermodynamic stability of the DNA.RNA hybrid r(GAAGAGAAGC)*d(GC(p)U(p)U(p)C(p)U(p) C(p)U(p)U(p)C) where p is the propynyl group at the 5 position and compared it with that of the unmodified duplex and the effects of methyl substitutions. The incorporation of the propyne group at the 5 position gives rise to a very large stabilisation of the hybrid duplex compared with the analogous 5-Me modification. The duplexes have been characterised by gel electrophoresis and NMR spectroscopy, which indicate that methyl substitutions have a smaller influence on local and global conformation than the propynyl groups. The increased NMR spectral dispersion of the propyne-modified duplex allowed a larger number of experimental restraints to be measured. Restrained molecular dynamics in a fully solvated system showed that the propyne modification leads to substantial conformational rearrangements stabilising a more A-like structure. The propynyl groups occupy a large part of the major groove and make favourable van der Waals interactions with their nearest neighbours and the atoms of the rings. This enhanced overlap may account at least in part for the increased thermodynamic stability. Furthermore, the simulations show a spine of hydration in the major groove as well as in the minor groove involving the RNA hydroxyl groups.

DNA↗
PubMed2004

Antisense-mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1-based vector results in severely attenuated mutants incapable of developing resistance.

We have constructed a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector expressing a 937-base antisense sequence against the HIV-1 envelope gene. Transduction of CD4(+) T lymphocytes with this vector results in expression of the therapeutic antisense sequence and subsequent inhibition of productive HIV-1 replication. In this report, we examined the effect of antisense-mediated suppression on the potential development of virus escape mutants using a permissive T-cell line cultured under conditions that over serial passages specifically allowed for generation and amplification of mutants selected for by antisense pressure. In the resulting virus clones, we found a significant increase in the number of deletions at the envelope target region (91% compared to 27.5% in wild-type HIV). Deletions were most often greater than 1 kb in length. These data demonstrate for the first time that during antisense-mediated suppression of HIV, mutants develop as a direct result of selective pressure on the HIV genomic RNA. Interestingly, in clones where deletions were not observed, there was a high rate of A-G transitions in mutants at the antisense target region but not outside this region, which is consistent with those mutations that are predicted as a result of antisense-mediated modification of double-stranded RNA by the enzyme double-stranded RNA-specific adenosine deaminase. These clones were not found to be escape mutants, as their replicative ability was severely attenuated, and they did not replicate in the presence of vector.

Base Sequence↗
PubMed2004

In vitro evolution of RNA aptamers recognizing carcinogenic aromatic amines.

The modification of cellular DNA by environmental substances is thought to be a crucial event in chemical induced carcinogenesis. Among the environmental carcinogens, aromatic amines are known for the fact that they can induce several types of cancers through the formation of so-called DNA adducts. We took advantage of the potential of the SELEX method to select for highly specific RNA ligands that recognize specific genotoxic aromatic amines. The aromatic amine 4,4'-methylenedianiline (MDA) was used as a target. Following in vitro selection, we obtained specific MDA-binding RNA molecules based on an affinity chromatography assay. These results open the possibility of using the SELEX technique to generate RNA molecules as diagnostic tools for the detection of DNA damaging compounds and ultimately DNA adducts.

Amines↗
PubMed1999

Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression.

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.

Base Sequence↗
PubMed1975

Properties of feline leukemia virus. III. Analysis of the RNA.

The kinetics of virus labeling was used to study the maturation of viral RNA in the Rickard strain of feline leukemia virus. Viral RNA labeled over differing intervals was characterized by gel electrophoresis and velocity sedimentation in sucrose gradients made up in aqueous buffer and 99% dimethyl sulfoxide. Labeled virus was found within 30 min after adding radioactive uridine to the cells and production of labeled virus reached a maximum at 4 to 5 h after pulse labeling. Native RNA from feline leukemia virus resolved into three size classes when analyzed by electrophoresis on 2.0% polyacrylamide-0.5% agarose gels: a 6.2 x 10(6) to 7.1 x 10(6) mol wt (50 to 60S) class, an 8.7 x 10(4) mol wt (approximately 8S) class, and a 2.5 x 10(4) mol wt (4 to 5S) class. From two experiments during which RNA degradation appeared minimal, these made up to 57 to 76%, 2 to 5%, and 6 to 12%, respectively, of the total RNA. The 8S RNA in feline leukemia virus has not previously been reported. The 50 to 60S RNA from virus harvested after 4 h of labeling electrophoretically migrated faster and sedimented more slowly in sucrose gradients than did the same RNA species harvested after 20 h of labeling. This argues for an intravirion modification of the high-molecular-weight RNA. The large subunits of denatured viral RNA from both 4- and 20-h labeled-viral RNA electrophoretically migrated with an estimated molecular weight of 3.2 x 10(6) but sedimented with 28S ribosomal RNA (1.8 X 10(6) mol wt) when analyzed by velocity sedimentation through 99% dimethyl sulfoxide.

Cell Line↗
PubMed2002

Delivery of unmodified bioactive ribozymes by an RNA-stabilizing polyethylenimine (LMW-PEI) efficiently down-regulates gene expression.

The sequence-specific cleavage of RNA molecules through ribozyme targeting is particularly attractive since it allows the effective abrogation of protein expression. So far, however, use of enzymatically active RNA molecules (ribozymes) has, without chemical modification, been severely hampered by ribozyme instability and poor cellular uptake. In this paper, we present a method for protection and cellular delivery of ribozymes by complexation with a low molecular weight polyethylenimine (LMW-PEI). We show that LMW-PEI almost completely stabilizes ribozymes or any RNA against degradation in vitro. Upon their highly efficient cellular uptake, non-toxic LMW-PEI-complexed ribozymes display intracellular bioactivity already at low concentrations as demonstrated by down-regulation of two different genes in different cell lines. In vivo, LMW-PEI-complexed ribozymes were stabilized after intraperitoneal (i.p.) injections, showed prolonged circulation time and intact ribozymes were detected in the subcutaneous (s.c.) tumor mass 60 min after the injection. In addition, i.p. injections of LMW-PEI-complexed ribozymes targeted against the growth factor pleiotrophin (PTN) resulted in marked reduction of s.c. human melanoma tumor growth and of intratumoral PTN levels in a mouse xenograft model. Thus, this paper describes a novel method for exogenous delivery of any bioactive RNA ribozyme in vitro and in vivo without chemical modification.

Animals↗
PubMed2006

siRNA and isRNA: two edges of one sword.

RNA interference mediated by small interfering RNAs (siRNA) has emerged as a powerful tool to target specific knockdown of gene expression in cell culture. siRNA is now the gold standard technique to study gene function, and expectations for the development of new target-specific drugs are high. In addition to the gene-silencing activity of siRNA, a number of recent studies have pointed to immunological effects of siRNAs, including the induction of proinflammatory cytokines and type I interferon. There is good evidence that gene silencing and immunostimulation are two independent functional characteristics of RNA oligonucleotides. Immunorecognition of RNA depends on certain molecular features such as length, double- versus single-strand configuration, sequence motifs, and nucleoside modifications such as triphosphate residues. RNA-sensing immunoreceptors include three members of the Toll-like receptor (TLR) family (TLR3, TLR7, TLR8) and cytosolic RNA-binding proteins like PKR and the helicases RIG-I and Mda5. Detection of RNA molecules occurs during viral infection and triggers antiviral innate defense mechanisms including the induction of type I interferons (IFN-alpha, IFN-beta) and downregulation of gene expression. Type I interferon induction by synthetic siRNAs requires TLR7 and is sequence dependent, similar to the detection of CpG motifs in DNA by TLR9. Identification of the exact molecular mechanisms of immunorecognition of RNA will allow the development of methods to avoid immunostimulation of siRNA and the design of potent immunostimulatory RNA (isRNA) oligonucleotides, depending on the aim. Furthermore, the combination of both gene-silencing and immunostimulation in one RNA molecule may lead to novel drugs that use both functional activities of RNA as two edges of one sword for effective treatment of viral infection and cancer.

Animals↗
PubMed1999

Sampling lymphoid tissue cells by ultrasound-guided fine needle aspiration of lymph nodes in HIV-infected patients. Swiss HIV Cohort Study.

OBJECTIVE: To establish the feasibility of using ultrasound-guided lymph node needle aspiration as a means to obtain lymphoid tissue cells for the determination of a series of immunologic and virologic measures in HIV-infected patients. DESIGN: First, a comparison of the characteristics of cell populations obtained by simultaneous needle aspiration and standard excisional biopsy in six patients. Second, use of lymph node needle aspiration to assess longitudinally T-cell subset changes in patients initiating highly effective antiretroviral treatment. METHODS: T-cell subsets (CD4 and CD8) and percentage Ki67+ cycling T cells were measured in lymph node cell populations harvested by ultrasound-guided aspiration or standard biopsy by flow cytometry. Cellular RNA content was assessed by a modification of the Roche Amplicor HIV-1 Monitor test. RESULTS: CD4 and CD8 T-cell percentage and HIV RNA cell content of lymph node cell suspensions obtained from the simultaneous performance of ultrasound-guided needle aspiration and excisional biopsy in the same patients were correlated (n = 6). Among the 87 aspiration sessions reported here, mononuclear cell suspensions were obtained in 100% of the sessions, in numbers ranging between 4x10(4) to 6.7x10(6) cells (median: 7x10(5)). This limited number of cells did not allow to perform all type of analyses in all patients. By prioritizing the cells for the determination of T-cell subsets and proliferation rate, this approach was instrumental for demonstrating the normalization of the T-cell subset ratio and the kinetic of normalization of proliferating rates of CD4 and CD8 T cells, as well as the decrease in HIV-1 viral load in the lymph node following HAART initiation. CONCLUSION: Ultrasound-guided aspiration appears to be a non-invasive and ad libitum, safe and repeatable procedure for the longitudinal monitoring of changes in lymph nodes.

Anti-HIV Agents↗
PubMed1975

Chemical modification of ribosomes with dimethyl sulfate: a probe to the structural organization of ribosomal proteins and RNA.

Ribosomal proteins from [14-C]dimethyl sulfate treated with 30S and 50S subunits of Escherichia coli ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and the degree of methylation of each protein was determined. Comparison of the results from this relatively non-specific chemical modification procedure with results from the milerd lysine-specific reductive alkylation procedure reported previously (Moore, G. & Crichton, R.R. (1974) Biochem. J. 143, 607-612) has permitted a topographical classification of ribosomal proteins in terms of 'degree of exposure' in the 30S and 50S subunits. The reaction of dimethyl sulfate with ribosomal RNA, both in intact subunits and after isolation from the subunits, has indicated that approximately half of the RNA in 30S and 50S subunits is exposed on the surface of the ribonucleoprotein complexes, and that no large sections (extended sequences) of 16S RNA are concealed in the 30S subunit. It is proposed that modification of ribosomes with dimethyl sulfate is a potentially useful technique for probing exposed and hidden regions and also exposed single-stranded regions of RNA in ribosomes.

Bacterial Proteins↗
PubMed2002

Synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides.

A versatile synthetic route has been developed for the synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2'-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2'-O-DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2'-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2'-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2'-O-(2-hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2'-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2'-O-[2-[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3'-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2'-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t(1/2) > 24 h.

Animals↗
PubMed2003

Effects of modification of the transcription initiation site context on citrus tristeza virus subgenomic RNA synthesis.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3'-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5' termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5' or 3' of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.

Base Sequence↗
PubMed1991

An improved method for isolating RNA from porcine adipose tissue.

In the present study, we describe a method that we developed to isolate total RNA from porcine adipose tissue. This method entails homogenizing porcine adipose tissue in 10 ml of 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% Sarcosyl, 0.1 M beta-mercaptoethanol, pH 7.0, and then performing two CHCl3 extractions to remove lipid before following the procedure described by P. Chomczynski and N. Sacchi (1987, Anal. Biochem. 162, 156-159). This modification improved the yield of RNA approximately threefold (yield was 88 +/- 7 micrograms total RNA/g of tissue) without affecting RNA quality.

Adipose Tissue↗