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At least 343 records · Page 19Linked to original sources
PubMed1996

The infectious bronchitis virus nucleocapsid protein binds RNA sequences in the 3' terminus of the genome.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3' noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3' nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3' terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3' terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA.

Binding, Competitive↗
PubMed1992

The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence.

Among eukaryotic transcription trans-activators, the human immunodeficiency virus type 1 (HIV-1) Tat protein is exceptional in that its target site TAR is an RNA rather than a DNA sequence. Here, we confirm that fusion of Tat to the RNA-binding domain of the HIV-1 Rev protein permits the efficient activation of an HIV-1 long terminal repeat (LTR) promoter in which critical TAR sequences have been replaced by RNA sequences derived from the HIV-1 Rev response element (RRE). An RRE target sequence as small as 13 nucleotides is shown to form an effective in vivo target for Rev binding. More important, a fusion protein consisting of Rev attached to the VP16 transcription activation domain was also observed to efficiently activate the HIV-1 LTR from this nascent RNA target. These data demonstrate that trans-activation of transcription by acidic activation domains does not require a stable interaction with the promoter DNA and suggest that VP16, like Tat, can act on steps subsequent to the formation of the HIV-1 LTR preinitiation complex. The finding that the activation domains of VP16 and Tat are functionally interchangeable raises the possibility that these apparently disparate viral trans-activators may nevertheless act via similar mechanisms.

Base Sequence↗
PubMed1988

Human 28S ribosomal RNA sequence heterogeneity.

DNA sequencing of several cloned human 28S ribosomal RNA gene fragments has revealed sequence heterogeneity (1) but it was not clear whether these are inactive pseudogenes or are active genes that are transcribed and represented in ribosomes. S1 nuclease analysis allowed us to examine the population of ribosomal RNA molecules of a cell, and we found that 28S rRNA is a heterogeneous assortment of molecules in both mono- and polysomal preparations. Sequence variation, although largely concentrated in variable regions of the molecule, apparently also occurs in the conserved regions.

Base Sequence↗
PubMed2026

Comprehensive analysis of mRNA-microRNA-lncRNA expression profiles in post-traumatic elbow heterotopic ossification using RNA sequencing and experimental validation.

BACKGROUND: This study aimed to profile the molecular signatures of post-traumatic elbow heterotopic ossification (HO) to identify key regulators and potential therapeutic targets. METHODS: Total RNA from post-traumatic elbow HO tissues (n=4) and normal bone tissues (n=6) was subjected to high-throughput sequencing to identify differentially expressed mRNAs (DEGs), microRNAs (DEMs), and lncRNAs (DELs). Bioinformatics analyses included Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein-protein interaction network construction, and transcription factor (TF)-microRNA-mRNA network analysis. The expression trends of four most upregulated and four most downregulated DEGs were validated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: We identified 2,138 DEGs, 40 DEMs, and 905 DELs. DEGs were significantly enriched in biological process "bone mineralization," cellular component "plasma membrane," molecular function "integrin binding," and pathways including PI3K-Akt, NF-κB, JAK-STAT, and TNF signaling pathways. Hub genes with high connectivity included MMP9, IL6, MMP3, CTSK, and BGLAP. Integrated network analysis highlighted the transcription factor JUN and key microRNAs (hsa-miR-124-3p, hsa-miR-548c-3p, and hsa-miR-135b). The qRT-PCR results confirmed the expression trends of selected DEGs. CONCLUSIONS: This study, for the first time, profiled the differentially expressed mRNAs, microRNAs, and lncRNAs in post-traumatic elbow HO using high-throughput RNA sequencing. These findings provide valuable insights into the molecular mechanisms of HO following elbow trauma. The identified hub genes (MMP9, IL6, MMP3, CTSK, and BGLAP), key TF (JUN), and key microRNAs (hsa-miR-124-3p, hsa-miR-548c-3p, and hsa-miR-135b) may serve as potential therapeutic targets for preventing and treating post-traumatic elbow HO.

Humans↗
PubMed1982

Isolation of a mouse DNA fraction which encodes more informational than non informational RNA sequences.

A small fraction of single-stranded DNA (ssDNA), amounting to 1.5--2% of the total DNA, was isolated from native nuclear DNA of mouse Ascites tumour cells. In RNA-driven annealing reactions, 40--42% of the ssDNA, (labelled with [3H]-thymidine or 125I) could be hybridized to cytoplasmic RNA was compared with 4--4.5% for the non repetitious component of bulk DNA and 60--63% of the ssDNA could be hybridized to nuclear RNA as compared with 20--22% for bulk DNA. It was also found that most of the ssDNA sequences which are hybridizable to homologous RNA, consisted of non self-reassociating DNA regions (which can be reassociated with non repetitious bulk DNA). These findings complete earlier data obtained in other cell systems and demonstrate that ssDNA mainly originates from active DNA transcription sites which encode informational RNA sequences. It is proposed that ssDNA is formed via selective endogenous nuclease attacks presumably at an early stage of the DNA purification procedure.

Animals↗
PubMed2004

BayesFold: rational 2 degrees folds that combine thermodynamic, covariation, and chemical data for aligned RNA sequences.

BayesFold is a Web application that folds an alignment of closely related sequences and evaluates hypotheses about their shared structure. It uses Bayes's Theorem to combine information from several sources, including chemical mapping (if available), thermodynamic folding, and observed sequence variations. Its method provides a rational basis for integrating results, even when these methods conflict. On a gapped alignment of 86 tRNAPhe sequences each 77 bases long, BayesFold takes 31 sec to perform the calculations; the best structure contained 95% of the base pairs in the true structure, and the true structure was ranked second. Notably, similar results come from random samples of only 10 sequences from the alignment (running time 3 sec), suggesting that remarkably few sequences are required for good results. In contrast, folding single sequences with BayesFold produced structures 9.6 bp different, or with the Vienna package, 13.4 bp different, from the true structure. Similar results were obtained for other families of tRNAs. We especially recommend BayesFold for alignments of 3-50 closely related sequences, such as the sequence families frequently found in SELEX. In addition to providing a convenient way to explore the effects of each of the criteria on the plausibility of different structures, BayesFold also makes it easy to produce publication-quality secondary-structure graphics. The Web interface, available at http://bayes.colorado.edu/fold/, includes the flexibility to thread any of the sequences (or the consensus sequence) through any of the structures, including the one judged most probable.

Algorithms↗
PubMedDate not supplied

Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction.

The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.

Animals↗
PubMed1991

Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences.

The classification of species belonging to the genus Candida Berkhout is problematic. Therefore, we have determined the small ribosomal subunit RNA (srRNA) sequences of the type strains of three human pathogenic Candida species; Candida krusei, C. lusitaniae and C. tropicalis. The srRNA sequences were aligned with published eukaryotic srRNA sequences and evolutionary trees were inferred using a matrix optimization method. An evolutionary tree comprising all available eukaryotic srRNA sequences, including two other pathogenic Candida species, C. albicans and C. glabrata, showed that the yeasts diverge rather late in the course of eukaryote evolution, namely at the same depth as green plants, ciliates and some smaller taxa. The cluster of the higher fungi consists of 10 ascomycetes and ascomycete-like species with the first branches leading to Neurospora crassa, Pneumocystis carinii, Candida lusitaniae and C. krusei, in that order. Next there is a dichotomous divergence leading to a group consisting of Torulaspora delbrueckii, Saccharomyces cerevisiae, C. glabrata and Kluyveromyces lactis and a smaller group comprising C. tropicalis and C. albicans. The divergence pattern obtained on the basis of srRNA sequence data is also compared to various other chemotaxonomic data.

Base Sequence↗
PubMed2005

Pairwise local structural alignment of RNA sequences with sequence similarity less than 40%.

MOTIVATION: Searching for non-coding RNA (ncRNA) genes and structural RNA elements (eleRNA) are major challenges in gene finding today as these often are conserved in structure rather than in sequence. Even though the number of available methods is growing, it is still of interest to pairwise detect two genes with low sequence similarity, where the genes are part of a larger genomic region. RESULTS: Here we present such an approach for pairwise local alignment which is based on foldalign and the Sankoff algorithm for simultaneous structural alignment of multiple sequences. We include the ability to conduct mutual scans of two sequences of arbitrary length while searching for common local structural motifs of some maximum length. This drastically reduces the complexity of the algorithm. The scoring scheme includes structural parameters corresponding to those available for free energy as well as for substitution matrices similar to RIBOSUM. The new foldalign implementation is tested on a dataset where the ncRNAs and eleRNAs have sequence similarity <40% and where the ncRNAs and eleRNAs are energetically indistinguishable from the surrounding genomic sequence context. The method is tested in two ways: (1) its ability to find the common structure between the genes only and (2) its ability to locate ncRNAs and eleRNAs in a genomic context. In case (1), it makes sense to compare with methods like Dynalign, and the performances are very similar, but foldalign is substantially faster. The structure prediction performance for a family is typically around 0.7 using Matthews correlation coefficient. In case (2), the algorithm is successful at locating RNA families with an average sensitivity of 0.8 and a positive predictive value of 0.9 using a BLAST-like hit selection scheme. AVAILABILITY: The program is available online at http://foldalign.kvl.dk/

Algorithms↗
PubMed1975

Cell-free transcription of mammalian chromatin. Quantitative measurement of newly synthesized globin messenger RNA sequences.

Transcription of globin mRNA sequences from rabbit marrow chromatin was detected by hybridization with globin complementary DNA (cDNA). The presence of newly synthesized RNA in cDNA-globin mRNA hybrids isolated by Cs2SO4 density equilibrium centrifugation required the addition of both rabbit marrow chromatin and DNA-dependent RNA polymerase (Escherichia coli) to the transcription reaction. No globin mRNA sequences were detected in RNA transcribed from rabbit liver chromatin or from rabbit marrow DNA. Selective transcription of globin mRNA sequences was therefore tissue-specific and dependent on the presence of chromosomal proteins. Globin mRNA sequences synthesized by E. coli RNA polymerase were distinguished from those synthesized by chromatin-bound (endogenous) RNA polymerases by the use of alpha-amanitin. A typical reaction with rabbit marrow chromatin yielded 100 mug of purified RNA which contained approximately 5 ng (0.005%) of globin mRNA sequences synthesized by E. coli RNA polymerase, 1 ng (0.001%) of globin mRNA seqeences synthesized by endogenous RNA polymerases, and 4 ng (0.004%) of globin mRNA sequences derived from chromatin-associated (endogenous) RNA. Forty per cent of the globin mRNA sequences derived from endogenous RNA could be removed by poly(U)-Sepharose chromatography. The accurate measurement of globin mRNA sequences required improved conditions for the purification and hybridization of RNA transcribed from chromatin.

Animals↗
PubMed2004

Determination and augmentation of RNA sequence specificity of the Nova K-homology domains.

The Nova onconeural antigens are implicated in the pathogenesis of paraneoplastic opsoclonus-myoclonus-ataxia (POMA). The Nova antigens are neuron-specific RNA-binding proteins harboring three repeats of the K-homology (KH) motif; they have been implicated in the regulation of alternative splicing of a host of genes involved in inhibitory synaptic transmission. Although the third Nova KH domain (KH3) has been extensively characterized using biochemical and crystallographic techniques, the roles of the KH1 and KH2 domains remain unclear. Furthermore, the specificity determinants that distinguish the Nova KH domains from those of the closely related hnRNP E and hnRNP K proteins are undefined. We demonstrate through the use of RNA selection and biochemical analysis that the sequence specificity of the Nova KH1/2 domains is similar to that of Nova KH3. We also show that the mutagenesis of a Nova KH domain to render it similar to the KH domains of the heterogeneous nuclear ribonucleoprotein E (hnRNP E) and hnRNP K allow it to recognize longer RNA sequences. These data yield important insights into KH domain function and suggest a strategy by which to engineer KH domains with novel sequence preferences.

Antigens, Neoplasm↗
PubMed1993

Reconstructing evolution from eukaryotic small-ribosomal-subunit RNA sequences: calibration of the molecular clock.

The detailed descriptions now available for the secondary structure of small-ribosomal-subunit RNA, including areas of highly variable primary structure, facilitate the alignment of nucleotide sequences. However, for optimal exploitation of the information contained in the alignment, a method must be available that takes into account the local sequence variability in the computation of evolutionary distance. A quantitative definition for the variability of an alignment position is proposed in this study. It is a parameter in an equation which expresses the probability that the alignment position contains a different nucleotide in two sequences, as a function of the distance separating these sequences, i.e., the number of substitutions per nucleotide that occurred during their divergence. This parameter can be estimated from the distance matrix resulting from the conversion of pairwise sequence dissimilarities into pairwise distances. Alignment positions can then be subdivided into a number of sets of matching variability, and the average variability of each set can be derived. Next, the conversion of dissimilarity into distance can be recalculated for each set of alignment positions separately, using a modified version of the equation that corrects for multiple substitutions and changing for each set the parameter that reflects its average variability. The distances computed for each set are finally averaged, giving a more precise distance estimation. Trees constructed by the algorithm based on variability calibration have a topology markedly different from that of trees constructed from the same alignments in the absence of calibration. This is illustrated by means of trees constructed from small-ribosomal-subunit RNA sequences of Metazoa. A reconstruction of vertebrate evolution based on calibrated alignments matches the consensus view of paleontologists, contrary to trees based on uncalibrated alignments. In trees derived from sequences covering several metazoan phyla, artefacts in topology that are probably due to a high clock rate in certain lineages are avoided.

Algorithms↗
PubMed1991

Eukaryotic origins: string analysis of 5S ribosomal RNA sequences from some relevant organisms.

Using the PHYLOGEN tree-forming programs, we evaluate the published 5S rRNA sequences in certain of the files in the Berlin DataBank in an attempt to identify the connection between archaebacteria and the eukaryotic protists. These programs are based on methods of string analysis developed by Sankoff and others. Their discriminatory power is derived from their continuous realignment of sequences through repeated assessment of insertions and deletions as well as substitutions. The programs demonstrate that even these small molecules (ca. 120 bases) retain substantial records of evolutionary events that occurred over a billion years ago. The eukaryotes seem to have been derived from ancestors near the common origins of the halobacterial and Methanococcales groups. Identifying what might have been a primordial eukaryote is more difficult because several of the species considered as early derivatives from the common root are isolated species with large genetic differences from each other and from all other extant forms that have been sequenced. The ameboid, flagellated, and ciliated protists seem to have emerged nearly simultaneously from an ancient cluster, but the sarcodinid protozoa have preference as the group of most ancient origin. The euglenozoa and the ciliates are of later derivation. Our ability to tease plausible trees from such small molecules suggests that the mode of analysis rather than the size of the molecule is often a major limitation in the reconstruction of acceptable ancient phylogenics.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Phylogeny of the fish parasite Ichthyophthirius and its relatives Ophryoglena and Tetrahymena (Ciliophora, Hymenostomatia) inferred from 18S ribosomal RNA sequences.

Phylogenetic relationships within the subclass Hymenostomatia were inferred from the comparisons of three new SSrRNA gene sequences from Ichthyophthirius multifiliis (1,751 bp), Ophryoglena catenula (1,748 bp), and Tetrahymena corlissi (1,753 bp). Using maximum-parsimony and distance-matrix methods, Ichthyophthirius and Ophryoglena were consistently paired and formed a sister group to the tetrahymenines, consistent with their placement in the Ophryoglenina. Tetrahymenids formed a monophyletic group that was divided into main lineages: T. corlissi diverged from the base of the lineage that included T. thermophila.

Animals↗
PubMed1995

Phylogenetic position of Gluconobacter species as a coherent cluster separated from all Acetobacter species on the basis of 16S ribosomal RNA sequences.

The 16S rRNA sequences from the Gluconobacter species G. asaii, G. cerinus and G. frateurii were determined and compared with homologous sequences from published databases and sequences of G. oxydans and Acetobacter species previously described [Sievers, M., Ludwig, W. and Teuber, M. (1994) System. Appl. Microbiol. 17, 189-196]. The Gluconobacter species have unique 16S rRNA sequences and exhibit sequence similarity values of 97.4 to 99.1%, corresponding to 36 to 14 base differences. The phylogenetic tree inferring methods (distance matrix, maximum parsimony and maximum likelihood) show that the species of Gluconobacter form a coherent, closely related cluster. Based on the distance matrix method including Rhodopila globiformis as an outgroup reference organism, Gluconobacter is well separated from Acetobacter.

Acetobacter↗
PubMed1993

Molecular phylogeny of Drosophila based on ribosomal RNA sequences.

Nucleotide sequences of 72 species of Drosophilidae were determined for divergent D1 and D2 domains (representing 200 and 341 nucleotides respectively in D. melanogaster) of large ribosomal RNA, using the rRNA direct sequencing method. Molecular phylogenetic trees were reconstructed using both distance and parsimony methods and the robustness of the nodes was evaluated by the bootstrap procedure. The trees obtained by these methods revealed four main lineages or clades which do not correspond to the taxonomical hierarchy. In our results, the genus Chymomyza is associated with the subgenus Scaptodrosophila of the genus Drosophila and their cluster constitutes the most ancient clade. The two other clades are constituted of groups belonging to the subgenus Sophophora of the genus Drosophila: the so-called Neotropical clade including the willistoni and saltans groups and the obscura-melanogaster clade itself split into three lineages: (1) obscura group + ananassae subgroup, (2) montium subgroup, and (3) melanogaster + Oriental subgroups. The fourth clade, the Drosophila one, contains three lineages. D. polychaeta, D. iri, and D. fraburu are branched together and constitute the most ancient lineage; the second lineage includes the annulimana, bromeliae, dreyfusi, melanica, mesophragmatica, repleta, robusta, and virilis groups. The third lineage is composed of the immigrans and the cardini, funebris, guaramunu, guarani, histrio, pallidipennis, quinaria, and tripunctata groups. The genera Samoaia, Scaptomyza, and Zaprionus are branched within the Drosophila clade. Although these four clades appear regularly in almost all tree calculations, additional sequencing will be necessary to determine their precise relationships.

Animals↗
PubMed1986

Selective enrichment of specific DNA, cDNA and RNA sequences using biotinylated probes, avidin and copper-chelate agarose.

We have developed a general procedure for the rapid and efficient enrichment of specific DNA, RNA or cDNA sequences. Biotinylated DNA or RNA is used as a hybridization probe in solution, avidin is then added to label both the probe and hybrid molecules, and the hybridization mixture chromatographed over cupric-iminodiacetic acid agarose beads. Avidin-probe and avidin-hybrid molecules are selectively retained on the column; non-hybridized sequences are contained in the flow-through fraction. Sequences retained on the column are recovered in high yield by the addition of ethylenediamine tetracetic acid in the buffer. The method can be used in both subtractive enrichment and positive selection protocols. Here we report its application to the isolation of Neisseria gonorrhoeae specific genomic DNA clones and the purification of a cDNA subpopulation representing mRNA sequences that are over-expressed in murine Friend cells after dimethylsulfoxide induction.

Avidin↗