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At least 631 records · Page 35Linked to original sources
PubMed1998

Heterologous protection of mice from a lethal human H1N1 influenza A virus infection by H3N8 equine defective interfering virus: comparison of defective RNA sequences isolated from the DI inoculum and mouse lung.

We have examined the RNAs involved in the heterologous protection of adult mice from otherwise lethal intranasal infection with mouse-adapted human A/WSN (H1N1) by defective interfering (DI) equine A/equine/Newmarket/7339/79 (H3N8: EQV) influenza virus, as well as the RNAs involved in the protection of WSN- or EQV-infected mice by their homologous DI viruses. The aim of this study was to describe the types of defective RNAs present in protected mice in order to guide the design of potentially protective DI RNAs. The interfering and mouse-protecting activity of DI virus was destroyed by prolonged UV irradiation (iDI virus) demonstrating that protection correlated with an active DI genome, and not viral antigen. Protected mice were all infected but suffered a lower degree of morbidity than those given iDI virus. The DI EQV inoculum contained defective segment 1-8 RNAs while DI WSN inoculum contained only defective segment 1-6 RNAs. However lungs of mice given EQV + DI EQV contained only defective segments 1-4 or 1-6 RNAs (mouse-to-mouse variation), while control mice given EQV or EQV + iDI EQV contained few very defective RNAs. Thus prevention of death was the result of quantitative and/or qualitative differences in defective RNAs administered to the mice. Only defective segments 1-3 RNAs were isolated from the lungs of mice given WSN + DI WSN, confirming the earlier report of Noble and Dimmock (1995). A detailed analysis showed that most defective RNAs isolated from the lungs of mice protected from a lethal WSN infection by DI EQV were EQV in origin. Thus, as no infectious EQV was present, these defective RNAs from the DI EQV inoculum must have been heterologously replicated in mouse lung by WSN. All defective segment 3-6 RNAs isolated were of EQV origin, indicating that they were replicated by WSN in preference to its own. Defective segments 1 and 2 were a mixture of EQV and WSN RNAs. Of 17 defective EQV segment 1-3 sequences from mouse lung, all but three differed in their primary central deletion from 20 defective RNAs isolated from the inoculum. No bias in the break points was evident. A number of minor deletions of 2 or more nts were also present in defective EQV and WSN RNAs in segments 1 and 2, but none in segment 3. Their 5', but not 3', breakpoints were heterogeneous, suggesting that defective RNAs were generated during positive strand synthesis. Two cloned EQV-defective segment 3 RNAs were chimeras containing a 30 nt insert from segment 1. Most defective RNAs possessed at least 178 nts from the 5' end of vRNA. The amount of 5' sequence present in those RNAs correlated with the segment of origin, suggesting that this was the minimum required for propagation of viral RNA in mouse lung and hence possibly for protection also.

Animals↗
PubMed1987

Nonuniformity of nucleotide substitution rates in molecular evolution: computer simulation and analysis of 5S ribosomal RNA sequences.

The effects of temporal (among different branches of a phylogeny) and spatial (among different nucleotide sites within a gene) nonuniformities of nucleotide substitution rates on the construction of phylogenetic trees from nucleotide sequences are addressed. Spatial nonuniformity may be estimated by using Shannon's (1948) entropy formula to measure the Relative Nucleotide Variability (RNV) at each nucleotide site in an aligned set of sequences; this is demonstrated by a comparative analysis of 5S rRNAs. New methods of constructing phylogenetic trees are proposed that augment the Unweighted Pair-Group Using Arithmetic Averages (UPGMA) algorithm by estimating and compensating for both spatial and temporal nonuniformity in substitution rates. These methods are evaluated by computer simulations of 5S rRNA evolution that include both kinds of nonuniformities. It was found that the proposed Reference Ratio Method improved both the ability to reconstruct the correct topology of a tree and also the estimation of branch lengths as compared to UPGMA. A previous method (Farris et al. 1970; Klotz et al. 1979; Li 1981) was found to be less successful in reconstructing topologies when there is high probability of multiple mutations at some sites. Phylogenetic analyses of 5S rRNA sequences support the endosymbiotic origins of both chloroplasts and mitochondria, even though the latter exhibit an accelerated rate of nucleotide substitution. Phylogenetic trees also reveal an adaptive radiation within the eubacteria and another within the eukaryotes for the origins of most major phyla within each group during the Precambrian era.

Animals↗
PubMed2006

Unbiased in vitro selection reveals the unique character of the self-cleaving antigenomic HDV RNA sequence.

In order to revisit the architecture of the catalytic center of the antigenomic hepatitis delta virus (HDV) ribozyme we developed an unbiased in vitro selection procedure that efficiently selected novel variants from a relatively small set of sequences. Using this procedure we examined all possible variants from a pool of HDV ribozymes that had been randomized at 25 positions (4(25)). The isolated set of sequences shows more variability than do the natural variants. Nucleotide variations were found at all randomized positions, even at positions when the general belief was that the specific base was absolutely required for catalytic activity. Covariation analysis supports the presence of several base pairs, although it failed to propose any new tertiary contacts. HDV ribozyme appears to possess a greater number of constraints, in terms of sequences capable of supporting the catalysed cleavage, than do other catalytic RNAs. This supports the idea that the appearance of this catalytic RNA structure has a low probability (i.e. is a rare event), which may explain why to date it has been found in nature only in the HDV. These contrasts with the hammerhead self-cleaving motif that is proposed to have multiple origins, and that is widespread among different organisms. Thus, just because a self-cleaving RNA motif is small does not imply that it occurs easily.

Catalytic Domain↗
PubMed1992

Amplification and analysis of specific DNA and RNA sequences of bovine leukemia virus from infected cows by polymerase chain reaction.

Bovine leukemia virus (BLV) is the etiologic agent of leukemia in cattle and is believed to cause decreases in milk productivity, fertility, and life span in infected cows. BLV is a type C retrovirus in the Oncovirinae subfamily. It is most closely related to human T-cell lymphoma/leukemia virus type I (HTLV-I) and type II (HTLV-II). Since the polymerase chain reaction (PCR) provides rapid and efficient amplification of DNA sequences, primers were designed to amplify regions of the polymerase (pol) and pX genes specific for BLV targets. These sets of primers consistently amplified as few as 10 copies of BLV DNA contained in a plasmid in the background of 1 microgram of either human or bovine chromosomal DNA. In addition, no amplification products were detected from cell lines infected with HTLV-I, HTLV-II, or human immunodeficiency virus type 1 or 2 by the BLV PCR systems. Samples of peripheral blood mononuclear cells from 18 cows, previously determined to be serologically positive or negative, were correctly identified in a blind study as containing proviral DNA by use of the BLV primers and probes. Cloning and sequencing of amplified products revealed finite sequence variations among a previously cloned BLV isolate, the wild-type virus, and the published genome. Reverse transcriptase-directed PCR with the primers for both BLV pol and BLV pX was performed on plasma from a BLV-infected cow and detected in vivo BLV RNA expression. In summary, we have developed a specific and sensitive assay using PCR for the detection and identification of BLV infections; this assay can now be applied to clinical and basic research questions in veterinary medicine.

Animals↗
PubMed1992

Detection of hepatitis C virus (HCV) RNA sequences in liver tissue by in situ hybridization.

In situ hybridization was used to identify the cell types infected by hepatitis C virus (HCV) in the liver. Using an antisense HCV-RNA probe from the 5' non-coding region, HCV-RNAs molecules were detected in liver sections of 4/11 patients with chronic hepatitis C. These 4 positive subjects were also infected by HIV. HCV-RNA-positive strands were detected in scattered hepatocytes as well as in cells identified as mononuclear cells within the inflammatory infiltrates. HCV-RNA negative strands, likely replicative intermediates, were also detected in these cells. This study therefore indicates that replication of HCV may occur in both hepatocytes and mononuclear liver cells.

Antigens, Viral↗
PubMed1982

Ribosomal RNA sequence conservation and gene number in the larval brine shrimp.

The haploid genome size of Artemia is determined to be about 0.9 X 10(12), as evidenced both by Feulgen microspectrophotometry of individual diploid class nuclei, which are but one of five polyploid classes present within the larvae, and by analysis of the reassociation kinetics of the isolated single copy DNA component. Polysomes isolated from 24-h incubation stage larvae contain an average of 10 ribosomes per messenger RNA molecule. Their rRNAs are found to have sedimentation coefficients of 18 S and 26 S, corresponding to molecular weights of 0.70 X 10(6) and 1.40 X 10(6), respectively, as determined by polyacrylamide electrophoresis and also by sucrose density centrifugation. Denaturation in glyoxal followed by agarose gel electrophoresis shows that unlike deuterostome rRNAs, Artemia 26 S rRNA contains a cryptic nick about midway in the molecule, which is not found in the 18 S molecule. Isolated rRNAs were labelled in vitro with 125I and hybridized with filter-immobilized DNA to saturation, which occurred at 0.051% for Xenopus, and at 0.074% for Artemia. From these results, it is calculated that in the haploid Artemia genome there are about 320 copies of the (18 S + 26 S) ribosomal RNA genes. Reciprocal heterologous hybridizations between these two species show that they share about 30% homology between their rDNA coding sequences.

Animals↗
PubMed1987

A family of lambda phage cDNA cloning vectors, lambda SWAJ, allowing the amplification of RNA sequences.

This paper describes the construction and characterization of a family of lambda phage cDNA cloning vectors that allows high-efficiency directional cDNA cloning and selective amplification of either sense or antisense cRNA sequences. These vectors contain several unique restriction sites (EcoRI, XbaI, and SacI) positioned between two specific phage promoters, SP6 and T7. This system facilitates the in vitro preparation of single-stranded (ss) RNA molecules that should be useful in subtractive hybridization and in situ hybridization procedures. Using subtractive hybridization and this vector system, it should be possible to identify sequences present in one cDNA library and not another. In addition, it should be possible to construct subtracted cDNA libraries in these vectors and to generate high specific activity, ss, antisense cRNA probes directly from DNA prepared from the whole subtracted library or from individual clones.

Bacteriophage lambda↗
PubMed2001

HGV RNA sequences in liver and PBMC.

HGV has been identified in patients with chronic hepatitis of unknown aetiology and the possibility that HGV may be the cause has been raised. We have analysed liver biopsies and PBMC (peripheral blood mononuclear cells) from 80 patients with chronic hepatitis and HGV-RNA in serum by PCR. In ten patients HGV-RNA was detected in liver, in five patients it was detected in PBMC and seven were positive in both specimens by PCR. Whether this agent resides and replicates in hepatocytes remains controversial and needs more studies.

Base Sequence↗