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At least 703 records · Page 39Linked to original sources
PubMed2001

5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure.

Oligonucleotide conjugates bearing two pyrene residues attached to 5'-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (lambda(max) 382 nm) and a broad emission peak with lambda(max )476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5'-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5'-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Enzymatic replacement of RNA sequences in vitro; use of FPLC for rapid and efficient preparation of tRNA variants.

A chromatographic procedure using columns of Superose-12 (gel filtration) and PBA-30 (phenylboronate agarose) in tandem has been developed. This procedure is very useful for rapidly separating and efficiently recovering each of the components in the reaction mixture of joining reaction with RNA ligase, and thereby is also beneficial to rapid preparation of tRNA variants in a high yield.

Chromatography, Gel↗
PubMed1990

Effect of the preS1 RNA sequence on the efficiency of the hepatitis B virus preS2 and S protein translation.

The gene coding for hepatitis B virus surface antigen consists of preS1, preS2, and S regions. Two species of mRNAs of this gene are transcribed. The larger species covers all three regions and is translated solely into preS1 protein, whereas the smaller one covers the preS2 and S regions and is translated into preS2 and S proteins. This study examines the influence of the 5' upstream sequence lying in the preS1 region on the synthesis of preS2 and S proteins. For this purpose, several expression plasmids were constructed by inserting various portions of the preS1 region between the retroviral LTR promoter and the preS2/S coding region, and preS2/S protein production was examined in the transfected CHL cells. All the transcripts were initiated in the LTR. A sequence located in the region between 102 and 38 nucleotides upstream from the preS2 initiation codon was found to reduce the production of preS2/S proteins probably at the level of translation. Expression of the heterologous chloramphenicol acetyltransferase gene was similarly inhibited when it was placed downstream of the preS1-102/-38 sequence.

Animals↗
PubMed2000

Comparison of six nucleic acid extraction methods for detection of viral DNA or RNA sequences in four different non-serum specimen types.

BACKGROUND: Extraction of viral nucleic acids from serum samples is widely used in diagnostic pathology tests. However, the heterogeneous nature of non-serum samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. OBJECTIVES: Six different methods were compared for optimal extraction of viral DNA or RNA from four types of non-serum specimens. STUDY DESIGN: The DNA viruses used were herpes simplex virus and cytomegalovirus. The RNA viruses were poliovirus, rotavirus and small round structured virus. The specimens used were from respiratory, genital, faecal and peripheral blood mononuclear cell samples. The extracted nucleic acids were amplified by PCR and detected in an enzyme immunoassay using digoxygenin-labelled amplicons. RESULTS AND CONCLUSIONS: For extraction of viral DNA, the phenol-chloroform method yielded the highest amount of DNA as judged by endpoint titration. The three methods compared for extraction of viral RNA used guanidine isothiocyanate and the QiaRNA kit was shown to yield the highest amount of viral RNA.

Cytomegalovirus↗
PubMed1981

Translational activity and functional stability of human fibroblast beta 1 and beta 2 interferon mRNAs lacking 3'-terminal RNA sequences.

Polyadenylylated mRNA was purified from poly(I).poly(C)- and cycloheximide-superinduced human fibroblast (FS-4) cultures. The mRNA was subjected to electrophoresis through an agarose/CH3HgOH gel, and human fibroblast beta 1 and beta 2 interferon mRNAs were isolated. Each mRNA preparation was phosphorolyzed at 0 degrees C for 20 min by using a molar excess of polynucleotide phosphorylase to produce RNAs lacking poly(A) and then incubated at 37 degrees C for varying lengths of time to allow the phosphorylase to further digest the deadenylylated RNA from the 3' end in a processive and synchronous manner. Removal of the poly(A) (less than or equal to 100 residues) and approximately 100 adjacent residues from human fibroblast beta 1 interferon mRNA (native length, 900 residues, including a 3'-noncoding region of 203 residues) did not alter the translational activity or the functional stability of this mRNA in Xenopus oocytes, whereas deletion of the poly(A) and approximately 200 adjacent residues decreased its translational efficiency. On the other hand, removal of the poly(A) (approximately 200 residues) and approximately 200 adjacent residues from human fibroblast beta 2 interferon mRNA (native length, 1300 residues) did not alter the translational activity or the functional stability of this molecule in oocytes. Thus, neither the poly(A) nor large segments of the 3'-noncoding region (which includes the hexanucleotide A-A-U-A-A-A sequence, at least in the case of beta 1 mRNA) are required for the maintenance of the functional stability of human beta 1 and beta 2 interferon mRNAs in Xenopus oocytes.

Animals↗
PubMed1997

Positive and negative strand of hepatitis C virus RNA sequences in peripheral blood mononuclear cells in patients with chronic hepatitis C: no correlation with viral genotypes 1b, 2a, and 2b.

Hepatitis C virus (HCV) has many genotypes which are closely associated with the severity of chronic hepatitis and the response to antiviral therapy. Although HCV is essentially hepatotropic, several lines of evidence suggest that this virus can infect peripheral blood mononuclear cells (PBMC) in most patients with chronic HCV infection. However, the methods used previously to detect negative-strand HCV RNA have been questioned, and the PBMC tropism of different HCV genotypes remains unknown. A stringent method was used to investigate the prevalence of positive- and negative-strand HCV RNA in the PBMC of 106 patients with chronic hepatitis C and to analyze the influence of HCV genotype on the tropism of PBMC. HCV type 1b was the predominant strain in the patients. Positive-strand RNA in PBMC was detected in 83 (78%) and 40% had negative-strand RNA. The demographic and clinical features were comparable among different patients grouped by the replication status of HCV in the plasma and PBMC samples. In addition, there was no significant difference of PBMC tropism between type 1b and non-1b HCV. In summary, HCV does indeed infect actively the PBMC of chronic hepatitis C patients and such infection is not correlated to the pathogenesis of liver cell damage. Moreover, the genotype is not associated specifically with PBMC tropism of HCV.

Adult↗
PubMed2006

Unweaving the meanings of messenger RNA sequences.

In addition to protein-coding information, mRNAs harbor regulatory sequences necessary for appropriate processing of their precursors. Goren et al. (2006) and Wang et al. (2006) explore the diversity of these signals and the rules by which they function.

Alternative Splicing↗
PubMed2005

A chimeric GB virus B with 5' nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins.

Only humans and chimpanzees are fully permissive for replication of hepatitis C virus (HCV), an important cause of liver cirrhosis and cancer worldwide. The absence of suitable animal models limits opportunities for in vivo evaluation of candidate hepatitis C therapeutics and slows progress in the field. Here, we describe a chimeric virus derived from GB virus B (GBV-B), an unclassified hepatotropic member of the family Flaviviridae that is closely related to HCV and infects tamarins (Saguinus sp.), in which a functionally important HCV regulatory sequence replaced an analogous sequence in the 5' nontranslated region (5'NTR) of the GBV-B genome. The transplanted sequence comprised domain III of the internal ribosome entry site (IRES), which directly binds the 40S ribosome subunit and is a target for candidate therapeutics. The chimeric 5'NTR retained ribosome binding activity and was competent in directing protein translation both in cell-free translation reactions and in transfected primary tamarin hepatocyte cultures. Virus rescued from the chimeric RNA replicated in the liver of tamarins, causing biochemical and histopathological changes typical of viral hepatitis. However, adaptive mutations were required elsewhere in the genome for efficient replication. Virus was not rescued from other, translationally competent, chimeric RNAs in which domain II of the IRES was exchanged. Thus, the 5'NTR appears to contain virus-specific replication signals that interact with other sites within the viral genome or with viral proteins. In conclusion, such novel chimeric flaviviruses offer opportunities for new insights into HCV replication mechanisms, while potentially facilitating the evaluation of candidate therapeutics in vivo.

5' Untranslated Regions↗
PubMed1993

Molecular approaches to diagnosis of Chagas' disease: use of defined antigens and a target ribosomal RNA sequence.

We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two "polar" strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system.

Animals↗
PubMed2006

Ribosomal RNA sequences of Clostridium piliforme isolated from rodent and rabbit: re-examining the phylogeny of the Tyzzer's disease agent and development of a diagnostic polymerase chain reaction assay.

We used polymerase chain reaction (PCR) technology to amplify the 16S rRNA gene, the intergenic spacer, and most of the 23S rRNA gene from 6 isolates (2 mice, 1 hamster, 1 rat, and 2 rabbit isolates) of the Tyzzer's disease agent (Clostridium piliforme) and C. colinum. Sequence similarity searches of GenBank identified 45 closely related bacteria, which we used for phylogenetic analysis by parsimony and maximum-likelihood methods using Escherichia coli to root the resulting phylogram. Microorganisms identified as C. piliforme form 3 clusters within a single clade; the nearest related distinguishable species is C. colinum. Other bacterial clades closely related to C. piliforme are clostridia previously identified by molecular methods in the bovine, porcine, and human gastrointestinal tracts. DNA sequence alignment highlighting sequence differences were used to design a rodent and rabbit C. piliforme-specific PCR assay, which targets a 639-basepair region at the 3' end of the 16S rRNA gene and the 5' end of the intergenic spacer. We used this PCR assay to examine 4 rat fecal samples from C. piliformeseropositive rats and reexamine 2 rabbit fecal samples previously identified as containing DNA sequences consistent with C. piliforme infection by 16S PCR assay. Our new assay did not detect the presence of C. piliforme DNA sequences in either the rat or rabbit fecal DNA samples, consistent with the absence of clinical disease in the colonies evaluated.

Animals↗