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At least 1,243 records · Page 69Linked to original sources
PubMed2003

16S ribosomal RNA sequence-based identification of veterinary clinical bacteria.

This study evaluated 16S rRNA gene sequence analysis methods as tools for identification of 22 phenotypically difficult to identify veterinary clinical bacterial isolates in a veterinary diagnostic laboratory. The study compared 16S rRNA gene sequencing and conventional phenotypic identification methods. Using 16S rRNA full-gene sequencing, 95% (21/22) of the isolates were identified to the genus level and 86% (19/22) to the species level. The conventional or commercially available manual identification phenotypic characterization methods presumptively identified 91% (20/22) of the isolates to the genus level and 1 isolate to the species level. However, only 55% (12/22) or 4.5% (1/22) of the phenotypic identifications were correct at the genus or species level when they were compared with the 16S rRNA full-gene sequencing. This study also compared 16S rRNA full-gene and partial-gene sequencing. The results demonstrated that the best 16S rRNA gene-sequencing approach is full-gene sequencing because it gives the most precise species identification. Sequencing of the variable regions 1, 2, and 3 of the 16S rRNA gene could be used for tentative identification because the ability of this sequencing to identify bacteria to the genus level is similar to that of the 16S rRNA full-gene sequencing. This method identified only 14% (3/22) isolates differently to the species level compared with the 16S rRNA full gene sequence. Sequencing of the variable regions 7, 8, and 9 is not recommended because it gives more ambiguous identifications. The cost of a 16S RNA full-gene-sequencing analysis was Can 160 dollars and Can 60 dollars for a partial 16S rRNA gene sequence, i.e., sequencing of variable regions 1, 2, and 3 or variable regions 7, 8 and 9.

Bacteria↗
PubMed2006

RNAMST: efficient and flexible approach for identifying RNA structural homologs.

RNA molecules fold into characteristic secondary structures for their diverse functional activities such as post-translational regulation of gene expression. Searching homologs of a pre-defined RNA structural motif, which may be a known functional element or a putative RNA structural motif, can provide useful information for deciphering RNA regulatory mechanisms. Since searching for the RNA structural homologs among the numerous RNA sequences is extremely time-consuming, this work develops a data preprocessing strategy to enhance the search efficiency and presents RNAMST, which is an efficient and flexible web server for rapidly identifying homologs of a pre-defined RNA structural motif among numerous RNA sequences. Intuitive user interface are provided on the web server to facilitate the predictive analysis. By comparing the proposed web server to other tools developed previously, RNAMST performs remarkably more efficiently and provides more effective and flexible functions. RNAMST is now available on the web at http://bioinfo.csie.ncu.edu.tw/~rnamst/.

Internet↗
PubMed1992

Structural requirements for the processing of Escherichia coli 5 S ribosomal RNA by RNase E in vitro.

Processing of 9 S precursor RNA in Escherichia coli requires the endoribonuclease RNase E, which makes two cleavages to liberate p5, the immature form of 5 S rRNA. The contributions of primary and secondary structure to RNase E-mediated cleavage of 9 S RNA were investigated. The structure of the 5' domain of 9 S RNA was probed by partial ribonuclease digestion and chemical modification. Our structural analysis of 9 S RNA supports a model in which the 5' spacer domain folds into tandem hairpins so that the first processing cleavage site 5' to the 5 S moiety resides in a stretch of single-stranded residues. Site-directed mutagenesis of a cloned 9 S RNA sequence was performed and synthetic transcripts derived from a variety of such mutant templates were assayed as substrates for RNase E-dependent endonuclease activity in fractionated extracts. Partial or complete deletion of the 5 S sequence did not eliminate site-specific processing of 9 S RNA. Mutations affecting the 5' domain revealed that secondary structure upstream from the first cleavage site is important in maintaining efficient processing. However, secondary structure downstream from either cleavage site is dispensable. Our results suggest that RNase E specifically recognizes and cleaves single-stranded RNA sequences only when presented in a proper conformational context. Adjacent secondary structures appear to play a direct and critical role in the enzyme's recognition of its substrate. Additionally, it may serve to anchor single-stranded regions to ensure the availability of the RNase E cleavage sites.

Base Sequence↗
PubMed1991

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.

Animals↗
PubMed1993

Identification of a tRNA-like molecule that copurifies with the 7SL RNA of Trypanosoma brucei.

During the purification of Trypanosoma brucei 7SL RNA, we detected a small RNA, 76 nucleotide-long (sRNA-76), that copurified with the 7SL RNP through several different separation steps. In this study, a partial RNA sequence of sRNA-76 was obtained and a complementary oligonucleotide to the RNA sequence was used to clone the corresponding gene. sRNA-76 is very similar to a tRNA molecule and is encoded by a single copy gene. The gene is located next to a tRNA(Val) which has 75.3% homology to T. brucei tRNA(Val) that exists in a different chromosomal locus. The highest homology of sRNA-76 is to mouse and rat tRNA(Asp) (69%), to mouse tRNA(Gly) (68.1%) and to yeast suppressor tRNA(Gly) (69.5%). However, sRNA-76 is neither a tRNA(Asp) nor a tRNA(Gly), since it has a Leu anticodon. In addition, sRNA-76 deviates from the canonical tRNA structure in 3 positions. A potential for base pairing between sRNA-76 and 7SL RNA was found in the 100 nt region of 7SL RNA, which is a highly conserved region in all 7SL RNAs.

Animals↗
PubMed2005

A comparison of RNA folding measures.

BACKGROUND: In the last few decades there has been a great deal of discussion concerning whether or not noncoding RNA sequences (ncRNAs) fold in a more well-defined manner than random sequences. In this paper, we investigate several existing measures for how well an RNA sequence folds, and compare the behaviour of these measures over a large range of Rfam ncRNA families. Such measures can be useful in, for example, identifying novel ncRNAs, and indicating the presence of alternate RNA foldings. RESULTS: Our analysis shows that ncRNAs, but not mRNAs, in general have lower minimal free energy (MFE) than random sequences with the same dinucleotide frequency. Moreover, even when the MFE is significant, many ncRNAs appear to not have a unique fold, but rather several alternative folds, at least when folded in silico. Furthermore, we find that the six investigated measures are correlated to varying degrees. CONCLUSION: Due to the correlations between the different measures we find that it is sufficient to use only two of them in RNA folding studies, one to test if the sequence in question has lower energy than a random sequence with the same dinucleotide frequency (the Z-score) and the other to see if the sequence has a unique fold (the average base-pair distance, D).

Models, Molecular↗
PubMed2005

Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1.

Sequence-specific RNA-protein interactions underlie regulation of many mRNAs. Here we analyze the RNA sequence specificity of Caenorhabditis elegans FBF-1, a founding member of the PUF protein family. Like other PUF proteins, FBF-1 binds to the 3' UTR of target mRNAs and decreases expression of those target genes. Here, we show that FBF-1 and its close relative, FBF-2, bind with similar affinity to multiple RNA sites. We use mutagenesis and in vivo selection experiments to identify nucleotides that are essential for FBF-1 binding. The binding elements comprise a "core" central region and flanking sequences. The core region is similar but distinct from the binding sites of other PUF proteins. We combine the identification of binding elements with informatics to predict new FBF-1 binding sites in a C. elegans 3' UTR database. These data identify a set of new candidate mRNA targets of FBF-1 and FBF-2.

Amino Acid Sequence↗
PubMed1991

Site-specific cleavage of natural mRNA sequences by newly designed hairpin catalytic RNAs.

The negative strand of tobacco ringspot virus satellite RNA is a self-cleaving RNA. Its catalytic domain and substrate domain have been identified, and the catalytic domain has been named hairpin catalytic RNA. Here we report the construction of a plasmid containing a modified hairpin catalytic RNA sequence that can be transcribed in vitro. Because this plasmid has two specific restriction enzyme recognition sites at both ends of the substrate binding site in the catalytic RNA sequence, it is possible to construct new plasmids by substituting different sequences in the substrate binding site. Using this plasmid, synthetic DNA, and in vitro transcription, we obtained three ribozymes designed to cleave Escherichia coli prolipoprotein signal peptidase (lsp) mRNA at specific sites. All three ribozymes cleaved the lsp mRNA sequence in vitro at the specific sites, and two of them cleaved it efficiently. Kinetic analyses showed that one had a higher kcat/Km value than that of the well-known hammerhead ribozyme. Problems associated with attaining the goal of expressing these ribozymes in vivo also are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1972

Strand orientation of simian virus 40 transcription in productively infected cells.

Strand orientation of transcription in BSC-1 cells infected by simian virus 40 (SV40) was investigated by annealing RNAs extracted from infected cells with asymmetric complementary RNA from SV40 DNA synthesized in vitro by RNA polymerase from Escherichia coli. The results suggest that the early viral RNA sequences (those made before replication of viral DNA) and the late RNA sequences (those synthesized concurrently with replication of viral DNA) are transcribed from opposite strands of the SV40 DNA. The RNA synthesized in vitro is of the same polarity as the early in vivo RNA and is complementary to the late in vivo RNA. Knowledge of strand selection in lytic infection is a step towards understanding the regulation of transcription of this viral DNA.

Animals↗
PubMed2003

Identification of novel alternative splice variants of APOBEC-1 complementation factor with different capacities to support apolipoprotein B mRNA editing.

Two novel mRNA transcripts have been identified that result from species- and tissue-specific, alternative polyadenylation and splicing of the pre-mRNA encoding the apolipoprotein B (apoB) editing catalytic subunit 1 (APOBEC-1) complementation factor (ACF) family of related proteins. The alternatively processed mRNAs encode 43- and 45-kDa proteins that are components of the previously identified p44 cluster of apoB RNA binding, editosomal proteins. Recombinant ACF45 displaced ACF64 and ACF43 in mooring sequence RNA binding but did not demonstrate strong binding to APOBEC-1. In contrast, ACF43 bound strongly to APOBEC-1 but demonstrated weak binding to mooring sequence RNA. Consequently ACF45/43 complemented APOBEC-1 in apoB mRNA editing with less efficiency than full-length ACF64. These data, together with the finding that all ACF variants were co-expressed in rat liver nuclei (the site of apoB mRNA editing), suggested that ACF variants might compete with one another for APOBEC-1 and apoB mRNA binding and thereby contribute to the regulation of apoB mRNA editing. In support for this hypothesis, the ratio of nuclear ACF65/64 to ACF45/43 decreased when hepatic editing was inhibited by fasting and increased when editing was re-stimulated by refeeding. These findings suggested a new model for the regulation of apoB mRNA editing in which the catalytic potential of editosomes is modulated at the level of their assembly by alterations in the relative abundance of multiple related RNA-binding auxiliary proteins and the expression level of APOBEC-1.

APOBEC-1 Deaminase↗
PubMed1975

Evidence for tertiary structural RNA-RNA interactions within the protein S4 binding site at the 5'-end of 16S ribosomal RNA of Escherichia coli.+.

Evidence is presented for tertiary structural interaction(s) (interactions(s) between two regions of an RNA molecule that are widely separated in the RNA sequence) within the 5'-one third of the 16S ribosomal RNA of Escherichia coli that constitutes the binding site of protein S4. The two main interacting RNA regions were separated by about 120 nucleotides (sections Q to M) of the 16S RNA sequence. A second, smaller gap, of 13 nucleotides, occurred within section C". The two main interacting regions contain about 150 nucleotides (sections H" to Q) and 160 nucleotides (sections M to C"). They are folded back on one another and, especially in the presence of protein S4, are strongly protected against ribonuclease digestion. The intermediate region (sections Q to M), however, is relatively accessible to ribonucleases in the S4-RNP. By partial removal of subfragments from the RNA complex it was possible to localise the two main interacting sites within sections H" - H and sections I" - C". Three main criteria for the specificity of the RNA-RNA interactions were invoked and satisfied. The possibility of other tertiary structural RNA-RNA interactions occurring in other regions of the 16S RNA is discussed. Finally, all the structural information on the S4-RNP is summarised and a tentative model is proposed.

Base Sequence↗
PubMed2006

Detection of non-coding RNAs on the basis of predicted secondary structure formation free energy change.

BACKGROUND: Non-coding RNAs (ncRNAs) have a multitude of roles in the cell, many of which remain to be discovered. However, it is difficult to detect novel ncRNAs in biochemical screens. To advance biological knowledge, computational methods that can accurately detect ncRNAs in sequenced genomes are therefore desirable. The increasing number of genomic sequences provides a rich dataset for computational comparative sequence analysis and detection of novel ncRNAs. RESULTS: Here, Dynalign, a program for predicting secondary structures common to two RNA sequences on the basis of minimizing folding free energy change, is utilized as a computational ncRNA detection tool. The Dynalign-computed optimal total free energy change, which scores the structural alignment and the free energy change of folding into a common structure for two RNA sequences, is shown to be an effective measure for distinguishing ncRNA from randomized sequences. To make the classification as a ncRNA, the total free energy change of an input sequence pair can either be compared with the total free energy changes of a set of control sequence pairs, or be used in combination with sequence length and nucleotide frequencies as input to a classification support vector machine. The latter method is much faster, but slightly less sensitive at a given specificity. Additionally, the classification support vector machine method is shown to be sensitive and specific on genomic ncRNA screens of two different Escherichia coli and Salmonella typhi genome alignments, in which many ncRNAs are known. The Dynalign computational experiments are also compared with two other ncRNA detection programs, RNAz and QRNA. CONCLUSION: The Dynalign-based support vector machine method is more sensitive for known ncRNAs in the test genomic screens than RNAz and QRNA. Additionally, both Dynalign-based methods are more sensitive than RNAz and QRNA at low sequence pair identities. Dynalign can be used as a comparable or more accurate tool than RNAz or QRNA in genomic screens, especially for low-identity regions. Dynalign provides a method for discovering ncRNAs in sequenced genomes that other methods may not identify. Significant improvements in Dynalign runtime have also been achieved.

Algorithms↗
PubMed1990

Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences.

Much of T and B lymphocyte receptor diversity derives from the addition of nontemplated N regions at the junctions of receptor gene elements, although fetal T cells expressing gamma/delta receptors lack N regions. I have sequenced immunoglobulin H chain variable regions of PCR-amplified DNA and cDNA from fetal and newborn mouse liver and spleen cells. These sequences showed an absence of N regions. Only 1/87 DNA sequences and 17/146 RNA sequences contained N regions, in striking contrast to adult Ig sequences. These data show that N region insertion is a developmentally regulated process in B cells as well as in T cells, and demonstrate that receptor diversity in neonatal B cells is limited by the absence of N regions as well as by biased usage of Vh genes.

Animals↗
PubMed1991

Nucleotide sequence and RNA hybridization analyses reveal an ambisense coding strategy for maize stripe virus RNA3.

The 2357-nt sequence of maize stripe virus (MStV) RNA3 was determined. Two nonoverlapping open reading frames (ORFs) of opposite polarities are contained in RNA3. A 591-nt ORF is located near the 5' end of MStV RNA3, while a second ORF of 948 nt is located near the 3' end in the viral complementary RNA (vcRNA). In vitro translation of transcripts derived from cDNA clones representing regions of each ORF was used to identify the respective proteins. A ca. 22,000 Mr protein was translated from the 591-nt ORF transcript. This protein comigrated in SDS-PAGE with a protein produced by in vitro translation of MStV RNA and is referred to as the NS3 protein. The protein from the 948-nt ORF transcript was specifically immunoprecipitated by antiserum to the MStV nucleocapsid protein (N protein). RNA hybridization analyses identified two subgenomic RNAs in total RNA extracts from MStV-infected Zea mays plants. These RNAs are ca. 650 and 1350 nt in size, are of opposite polarities, and correspond to regions of RNA3 containing the two ORFs. These data suggest that MStV RNA3 has an ambisense coding strategy similar to that found for the S RNA of the vertebrate-infecting phleboviruses, uukuviruses, and arenaviruses, as for tomato spotted wilt virus.

Amino Acid Sequence↗
PubMed1989

Secretory pancreatic stone protein messenger RNA. Nucleotide sequence and expression in chronic calcifying pancreatitis.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.

Adolescent↗
PubMed1993

Computer-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1.

For the biological and therapeutic application of antisense nucleic acids, there is a need to identify effective local target regions of given cellular target mRNAs or viral single-stranded nucleic acids. One critical parameter for the effectiveness of antisense nucleic acids could be the potential of intramolecular folding of a given sequence element of the target strand and the antisense strand, respectively. The folding potential of such subsequences was calculated by using an established secondary structure prediction algorithm. For the genomic RNA and the complementary RNA strand of the human immunodeficiency virus type 1 (HIV-1), an energy profile was calculated that monitors the local folding potential of each sequence position surrounded by a window of given length ranging from 50 to 400 nucleotides. The resulting energy profile was compared to the effectiveness of HIV-1-directed antisense RNAs. It was found that significant minima of the local folding potential (high delta G values) correlated with antisense RNA target regions involved in strong inhibition of HIV-1 replication that had been measured independently in two earlier studies by using different experimental approaches. Conversely, antisense RNAs directed against local subregions with a high folding potential (low delta G values) showed weak or no antiviral effect in human cells. The results indicate that analyses of the local folding potential of a given target RNA can support the selection of effective target sequences for antisense RNA.

Antiviral Agents↗
PubMed2003

The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana.

The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis: However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis: Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.

Amino Acid Sequence↗