Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA sequencing analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources
PubMed2004

An improved beta-galactosidase reporter gene.

The coding sequence for the E. coli beta-galactosidase gene was codon-optimised for expression in mammalian cells. When expressed in mammalian cells the codon-optimised gene results in the expression of beta-galactosidase at levels 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. RNA analysis suggests the enhancement of beta-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. When used in a lentiviral construct the codon-optimised gene results in an approximately five-fold increase in apparent titre, as determined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, in comparison to an analogous construct containing the native E. coli gene. Southern blot analysis shows this is due to an increased efficiency of detection of transduced cells. In addition, codon-optimisation results in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells the codon-optimised beta-galactosidase gene has a number of advantages over the native E. coli gene sequence. A variant of the codon-optimised beta-galactosidase gene sequence that includes an effective nuclear localisation signal was also made.

Amino Acid Sequence↗
PubMed1991

De novo generation of cymbidium ringspot virus defective interfering RNA.

Nicotiana clevelandii plants were inoculated with cymbidium ringspot tombusvirus RNA synthesized in vitro, after which further passages were made by sap inoculation. During the third passage, low Mr RNA species appeared which had the characteristics of deletion mutants of genomic RNA. Sequence analysis of several of these defective interfering RNAs suggested a possible evolution of smaller from larger molecules. Computer-generated secondary structures of sequences surrounding recombination sites were extensive and stable and these sites occurred in interior or hairpin loops, thus providing a possible explanation for discontinuous RNA transcription and the formation of deletions in genomic RNA.

Base Sequence↗
PubMed1977

Initiator regions from the small size class of reovirus messenger RNA protected by rabbit reticulocyte ribosomes.

In order to investigate the general utility of the rabbit reticulocyte cell-free translating system for isolation of ribosome-protected initiator regions of eukaryotic mRNA, we have studied ribosome protection of 32P-labeled reovirus small mRNA species. Our studies show that rabbit reticulocyte ribosomes protect initiator regions in at least two of the four reovirus small mRNA molecules. RNA sequence analysis demonstrates that 40 S subunits protect additional sequences to the 5' side of the initiator AUG triplet.

Animals↗
PubMed2002

[Characterization of genome of A/Guangzhou/333/99(H9N2) virus].

BACKGROUND: To understand the characterization of genome of a strain of avian influenza A H9N2 virus repeatedly isolated from a child with influenza illness. Thereafter to reveal the origin of this H9N2 virus. METHODS: Viruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares. RESULTS: Genome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment derived from human influenza A virus. However, the genes encoding the HA,NA,NP and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA) proteins were derived from G1 lineage strain. CONCLUSIONS: A/Guangzhou/333/99 virus was a reassortant derived from reassortment betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most possibility is that it is derived from avian influenza A virus directly. The results do not only demonstrate that avian influenza A (H9N2) virus could infect men, but also firstly prove that the genetic reassortment could be occurred between different genetic lineages of avian influenza A (H9N2) viruses in the nature.

Animals↗
PubMed1990

The murine complement receptor gene family. IV. Alternative splicing of Cr2 gene transcripts predicts two distinct gene products that share homologous domains with both human CR2 and CR1.

The murine Cr2 gene encodes at least two related proteins. The first of these is predicted to include 1408 amino acids from a transcript including 4224 coding nucleotides. This protein is predicted to contain 21 60-amino acid repeats plus those residues encoding transmembrane and cytoplasmic regions for a total peptide m.w. of 155,307. The first six of these repeats are similar to human CR1 in sequence and organization. The second protein is encoded by an alternatively spliced Cr2 transcript that is lacking those sequences which encode the first six 60-amino acid repeats of the larger protein. This second, smaller protein, encoded by a transcript of 3096 coding nucleotides, is predicted to include 15 60-amino acid repeats, plus the transmembrane and cytoplasmic regions. This smaller protein contains 1032 amino acids for a peptide m.w. of 113,328. This second protein is extremely homologous in size and sequence to human CR2. Both proteins share the same signal sequence for membrane insertion. DNA sequence analysis, RNA protection studies and genomic phage mapping indicate the transcripts which encode these proteins are derived from the Cr2 gene via alternative splicing.

Amino Acid Sequence↗
PubMed1980

Identification of a cytidine-specific ribonuclease from chicken liver.

Rapid RNA sequencing technology was used to determine if the base specificities of an RNase recently purified from chicken liver would prove useful for RNA sequence analysis. Escherichia coli 5 S [5'-32P]rRNA or yeast 5.8 S [5'-32P]rRNA was digested with the enzyme and this digest, along with digests derived from RNases of known specificity (U2, T1, T2) were subjected to electrophoresis through denaturing polyacrylamide slab gels. Following autoradiography, the banding patterns arising from the activity of each enzyme were compared, and the base specificity of the unknown RNase was established. The chicken liver RNase was found to have a marked preference for phosphodiester bonds containing cytidylic acid residues, a property which should make the enzyme useful for distinguishing between pyrimidines in RNA sequencing.

Animals↗
PubMed2005

[Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

Cucumber Mosaic Virus Satellite↗
PubMed1982

Separation and sequence of the 3' termini of M double-stranded RNA from killer yeast.

Four subspecies of M double-stranded RNA from a killer strain of Saccharomyces cerevisiae were isolated. Each subspecies were susceptible to heat cleavage, presumably at an internal 190 base pair A,U-rich region, generating two discrete fragments corresponding to each side of the A, U-rich region. Enzymatic and chemical RNA sequence analysis defined the 3'-terminal 175 bases for the larger fragment (M-1) and 231 bases for the smaller fragment (M-2). All four subspecies of M have identical size and 3'-terminal sequences. Potential translation initiation codons are present on the corresponding 5' termini of both fragments, and a possible 18S ribosomal RNA binding site is also present on the 5' terminus of M-1. Stem and loop structures for the 5' and 3' termini of M-1 may function as recognition sites for replication, transcription, and translation.

Amino Acid Sequence↗
PubMed2003

RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis.

The mammalian translation initiation factor 4A (eIF4A) is a prototype member of the DEAD-box RNA helicase family that couples ATPase activity to RNA binding and unwinding. In the crystal form, eIF4A has a distended "dumbbell" structure consisting of two domains, which probably undergo a conformational change, on binding ATP, to form a compact, functional structure via the juxtaposition of the two domains. Moreover, additional conformational changes between two domains may be involved in the ATPase and helicase activity of eIF4A. The molecular basis of these conformational changes, however, is not understood. Here, we generated RNA aptamers with high affinity for eIF4A by in vitro RNA selection-amplification. On binding, the RNAs inhibit ATP hydrolysis. One class of RNAs contains members that exhibit dissociation constant of 27 nM for eIF4A and severely inhibit cap-dependent in vitro translation. The binding affinity was increased on Arg substitution in the conserved motif Ia of eIF4A, which probably improves a predicted arginine network to bind RNA substrates. Selected RNAs, however, failed to bind either domain of eIF4A that had been split at the linker site. These findings suggest that the selected RNAs interact cooperatively with both domains of eIF4A, either in the dumbbell or the compact form, and entrap it into a dead-end conformation, probably by blocking the conformational change of eIF4A. The selected RNAs, therefore, represent a new class of specific inhibitors that are suitable for the analysis of eukaryotic initiation, and which pose a potential therapeutic against malignancies that are caused by aberrant translational control.

Adenosine Triphosphate↗
PubMed1995

Saccharomyces species assignment by long range ribotyping.

Type strains of 10 genotypically distinct Saccharomyces species are differentiated by ribosomal DNA restriction fragment analysis (ribotyping). The full length of the chromosomal ribosomal repeat was amplified in two parts, the 18SrDNA including both ITS region (2600 bp) and the 25SrDNA (3300 bp). Restriction fragments generated by 9 enzymes from these two products yield characteristic patterns, by which unknown Saccharomyces isolates are assigned to the type strains. For convenient separation and detection only fragments longer than 200 bp were monitored. In contrast to molecular differentiation methods of highest resolution as RAPD-PCR or fingerprinting, the results from ribotyping are absolutely reproducible and thereby suitable for databases. The phylogeny computed from the discrete character matrix for presence/absence of fragments by the PHYLIP program package is in complete accordance to the phylogeny derived from ribosomal RNA sequence analysis. By this the field of application of the long range ribotyping can be regarded basically as equal to DNA sequence analysis of the same locus. Because distant relationships are recognized, misidentified genera were detected upon the species assignment. This cannot be done by methods of higher resolution like RAPD-PCR or fingerprinting.

Base Sequence↗
PubMed2000

Predicting U-turns in ribosomal RNA with comparative sequence analysis.

The U-turn is a well-known RNA motif characterized by a sharp reversal of the RNA backbone following a single-stranded uridine base. In experimentally determined U-turn motifs, the nucleotides 3' to the turn are frequently involved in tertiary interactions, rendering this motif particularly attractive in RNA modeling and functional studies. The U-turn signature is composed of an UNR sequence pattern flanked by a Y:Y, Y:A (Y=pyrimidine) or G:A base juxtaposition. We have identified 33 potential UNR-type U-turns and 25 related GNRA-type U-turns in a large set of aligned 16 S and 23 S rRNA sequences. U-turn candidates occur in hairpin loops (34 times) as well as in internal and multi-stem loops (24 times). These are classified into ten families based on loop type, sequence pattern (UNR or GNRA) and the nature of the closing base juxtaposition. In 13 cases, the bases on the 3' side of the turn, or on the immediate 5' side, are involved in tertiary covariations, making these sites strong candidates for tertiary interactions.

Animals↗
PubMed2005

CMfinder--a covariance model based RNA motif finding algorithm.

MOTIVATION: The recent discoveries of large numbers of non-coding RNAs and computational advances in genome-scale RNA search create a need for tools for automatic, high quality identification and characterization of conserved RNA motifs that can be readily used for database search. Previous tools fall short of this goal. RESULTS: CMfinder is a new tool to predict RNA motifs in unaligned sequences. It is an expectation maximization algorithm using covariance models for motif description, featuring novel integration of multiple techniques for effective search of motif space, and a Bayesian framework that blends mutual information-based and folding energy-based approaches to predict structure in a principled way. Extensive tests show that our method works well on datasets with either low or high sequence similarity, is robust to inclusion of lengthy extraneous flanking sequence and/or completely unrelated sequences, and is reasonably fast and scalable. In testing on 19 known ncRNA families, including some difficult cases with poor sequence conservation and large indels, our method demonstrates excellent average per-base-pair accuracy--79% compared with at most 60% for alternative methods. More importantly, the resulting probabilistic model can be directly used for homology search, allowing iterative refinement of structural models based on additional homologs. We have used this approach to obtain highly accurate covariance models of known RNA motifs based on small numbers of related sequences, which identified homologs in deeply-diverged species.

Algorithms↗
PubMed2003

Identification of genes expressed in a human scleral cDNA library.

PURPOSE: Clones established from a human scleral cDNA library were systematically sequenced. Public database sequence comparisons were performed to generate a profile of genes expressed in the human sclera and identify candidate genes for inherited diseases with scleral involvement. METHODS: A directionally cloned pCMV-PCR cDNA library was constructed from RNA isolated from scleras of human donor eyes with known plano refractive history. Plasmid DNA was extracted from randomly selected cDNA clones, and the insert sequences were determined by 5' end single-pass sequencing. Expressed sequence tags (ESTs) were generated and analyzed with the GenBank BLASTN program to identify sequence homologies to known genes. RESULTS: A total of 609 ESTs underwent BLAST analysis. Of these, 341 (56%) matched 228 known human genes and 4 non-human genes, 252 matched uncharacterized ESTs, and 16 showed no significant homology to human or non-human known sequences. The most redundant connective tissue-related genes were alphaA-crystalline, Xalpha-1 collagen, and beta-5 integrin. Other extracellular matrix gene matches were biglycan, syndecan, decorin, fibromodulin, proline arginine-rich end leucine-rich repeat protein, transgelin, TIMP-1, and fibulin 1. Human scleral expression of all but decorin and biglycan has not previously been reported. CONCLUSIONS: This effort provides the first partial list of genes expressed in human sclera. Identification of genes expressed in the sclera contributes to our understanding of scleral biology, and potentially provides positional candidate genes for scleral disorders such as high myopia.

Adult↗
PubMed1992

Structure of the L (polymerase) protein gene of sonchus yellow net virus.

The complete nucleotide sequence of the L protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined by dideoxynucleotide sequencing of cloned cDNAs derived from the negative-strand genomic RNA. The L protein gene is composed of 6401 nucleotides (nt) located between positions 7158 and 13558 relative to the 3' end of the genomic RNA. Sequence analysis suggests that the complementary mRNA contains a 44 nt untranslated 5' leader sequence preceding an open reading frame of 6348 nucleotides that is capable of encoding a polypeptide of 2116 amino acids with a deduced molecular weight of 241,569 Da. The L protein is positively charged, has a high proportion of the amino acids Leu and Ile, and contains putative polymerase and RNA binding domains. Extended alignment of the SYNV L protein amino acid sequence with those of other nonsegmented negative-strand RNA virus polymerases reveals conservation of sequences within 12 blocks that appear sequentially along the protein. A cluster dendrogram derived from the L protein alignments indicates that SYNV is more closely related to animal rhabdoviruses than to the paramyxoviruses and that the animal rhabdoviruses have diverged less from each other than from SYNV.

Amino Acid Sequence↗
PubMed1980

Nonviral oligonucleotides at the 5' terminus of cytoplasmic influenza viral mRNA deduced from cloned complete genomic sequences.

We obtained influenza viral DNA clones containing sequences derived from cytoplasmic viral mRNA and genomic viral RNA. Sequence analysis of terminal nucleotides of five independentlyisolated viral DNA segments showed that additional oligonucleotides were covalently linked to the 5' terminus of viral mRNA transcripts. The sequences of these additional nucleotides varied among DNA clones of the same gene and of different genes as well. These inserts also varied in length, ranging from 6 to 14 nucleotides. The heterogeneity of these sequences suggests that they were derived originally from cellular RNA molecules. These findings provide evidence that cellular RNA sequences are used to prime influenza viral mRNA transcription in infected cells. In addition, the sequences at both termini of vRNA were fully represented in clone pFV 88, indicating that the cloned DNA contained the complete viral gene coding for the hemagglutinin.

Base Sequence↗
PubMed1981

Yeast tRNA precursor mutated at a splice junction is correctly processed in vivo.

Yeast mutants with decreased expression of a tRNATyr gene were obtained by selection for functional inactivation of the tyrosine-inserting ochre suppressor SUP4 and subsequent screening for production of the tRNA gene product in vivo. One mutant with reduced suppressor activity was characterized by a decreased quantity of the suppressor-specific tRNA; a precursor to this tRNA, matured at both 5' and 3' termini but still containing a 14-nucleotide intervening sequence, was present in an amount greater than 7-fold that in the parent. By RNA sequence analysis of the accumulated precursor, we have identified the mutation as an A leads to G transition at the 5' splice junction. Similar analysis of the mature tRNA produced in this mutant demonstrated that the intervening sequence was accurately excised. We conclude that the specific sequence of nucleotides at this splice junction affects the efficiency but not the fidelity of processing.

Base Sequence↗
PubMed2006

4SALE--a tool for synchronous RNA sequence and secondary structure alignment and editing.

BACKGROUND: In sequence analysis the multiple alignment builds the fundament of all proceeding analyses. Errors in an alignment could strongly influence all succeeding analyses and therefore could lead to wrong predictions. Hand-crafted and hand-improved alignments are necessary and meanwhile good common practice. For RNA sequences often the primary sequence as well as a secondary structure consensus is well known, e.g., the cloverleaf structure of the t-RNA. Recently, some alignment editors are proposed that are able to include and model both kinds of information. However, with the advent of a large amount of reliable RNA sequences together with their solved secondary structures (available from e.g. the ITS2 Database), we are faced with the problem to handle sequences and their associated secondary structures synchronously. RESULTS: 4SALE fills this gap. The application allows a fast sequence and synchronous secondary structure alignment for large data sets and for the first time synchronous manual editing of aligned sequences and their secondary structures. This study describes an algorithm for the synchronous alignment of sequences and their associated secondary structures as well as the main features of 4SALE used for further analyses and editing. 4SALE builds an optimal and unique starting point for every RNA sequence and structure analysis. CONCLUSION: 4SALE, which provides an user-friendly and intuitive interface, is a comprehensive toolbox for RNA analysis based on sequence and secondary structure information. The program connects sequence and structure databases like the ITS2 Database to phylogeny programs as for example the CBCAnalyzer. 4SALE is written in JAVA and therefore platform independent. The software is freely available and distributed from the website at http://4sale.bioapps.biozentrum.uni-wuerzburg.de.

Algorithms↗
PubMed1999

Mapping sequences active in homologous RNA recombination in brome mosaic virus: prediction of recombination hot spots.

The mechanism of homologous recombination has been studied previously in brome mosaic virus (BMV), a tricomponent, positive-stranded RNA virus of plants, by using artificial sequences (reviewed by J. J. BujarskiP. D. Nagy (1996). Semin. Virol. 7, 363-372). Here we extend these studies over BMV-derived sequences to obtain clues on prediction of homologous recombination hot spots. First, mismatch mutations, which reduced the AU content, were introduced into the common 60-nt recombination hot-spot sequence, either in the RNA2 or in both RNA2RNA3 components. This decreased the frequency of targeted homologous RNA2/RNA3 recombinationchanged the distribution of junction sites. Second, several short BMV RNA1- or RNA2-derived sequences were introduced into the RNA3 component, homologous recombination activity of these sequences was compared with that observed for previously characterized artificial sequences. Third, sequences at homologous recombinant junctions were compared among a large number of targetednontargeted recombinants. All these studies revealed several factors important for homologous recombination including the length of sequence identity, the extent of sequence identity, the AU content of the common sequences, the relative position of the AU-rich segment vs a GC-rich segment,the presence of GC-rich sequences. Based on this novel model, we suggest that recombination hot spots can be predicted by means of RNA sequence analysis. In addition, we show that recombination can occur between positivenegative strands of BMV RNAs. This provides further clues toward the mechanism of recombination processes in BMV.

Base Sequence↗