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PubMed1982

Isolation and characterization of a single-stranded specific endoribonuclease from Ehrlich cell nucleoli.

An endoribonuclease which cleaves only single-stranded RNA has been purified from nucleoli of Ehrlich ascites tumor cells. The molecular weight of the ribonuclease is 50,000 to 52,000 as estimated from sedimentation in glycerol density gradients and by gel filtration on Sephadex G-100. The endoribonuclease requires Mg2+ or Mn2+ (0.2 mM) for optimum activity. Monovalent cations including K+, Na+, and NH+4 are inhibitory. The ribonuclease gave an apparent Km for single-stranded RNA of 30 microM. Using ribohomopolymers, we found that the enzyme could digest single-stranded, poly(C), poly(U), and poly(A) equally well, but would not degrade duplex poly(C) . poly(I) or poly(A) . poly(U). The lack of base specificity was further demonstrated using RNA sequence analysis of partial digest products of yeast 5.8 S RNA. The ribonuclease activity is sensitive to EDTA and N-ethylmaleimide, but is not inhibited by human placental RNase inhibitor. The enzyme makes endonucleolytic cleavages which generate 5'-phosphate-terminated oligonucleotides.

Animals↗
PubMed1995

Complete genomic sequence of the fish rhabdovirus infectious haematopoietic necrosis virus.

The complete nucleotide sequence of the genome of the fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) has been determined after cDNA cloning of the viral genomic RNA. Sequence analysis showed the presence of six open reading frames encoding the nucleoprotein N, the matrix proteins M1 and M2, the glycoprotein G, a so-called non-structural protein NV, and the RNA polymerase L. The genome organization is 3'N-M1-M2-G-NV-L 5'. The extreme 5' and 3' ends of the genome were sequenced after RNA ligation or RACE. Prokaryotic expression products of the open reading frames predicted to encode the matrix proteins M1 and M2, the glycoprotein G and the NV protein reacted with rabbit anti-IHNV serum thereby confirming their identity. This is the first complete nucleotide sequence of a fish rhabdovirus. Knowledge of the complete sequence is an essential prerequisite for future manipulation of the genome and also serves to provide gene- and protein specific reagents for use in further examination of the replication of the fish rhabdoviruses.

Amino Acid Sequence↗
PubMed1976

In vitro site-directed mutagenesis: generation and properties of an infectious extracistronic mutant of bacteriophage Qbeta.

An infectious extracistronic mutant of phage Qbeta has been prepared by site-directed mutagenesis. Qbeta RNA minus strands containing the mutagenic base analog N4-hydroxy-CMP instead of UMP at position 39 from the 5' end were synthesized in vitro and used as template for Qbeta replicase to synthesize one generation of plus strands. E. coli spheroplasts were infected with the newly synthesized plus strands and phage recovered from single plaques. RNA sequence analysis revealed that four out of the eighteen phage clones analyzed contained RNA with an A leads to G transition at position 40 from the 3' end (which corresponds to position 39 of the minus strand). Thus, the viability of phage Qbeta does not depend on a unique nucleotide sequence in the 3'-extracistronic RNA segment. Upon in vivo propagation of mutant 40, spontaneous true revertants arose with high frequency and overgrew the parental clone within about 10 passages, indicating a selective disadvantage of the extracistronic mutant. Replication of mixtures of wild type and mutant RNA in vitro resulted in a decrease of the proportion of mutated RNA in the progeny plus strands. The fact that Qbeta RNA containing an A leads to G transition in nucleotide--40 of Qbeta RNA is less efficiently replicated in vitro may explain the selective disadvantage of the mutant phage in vivo. The preparation of an infectious mutated RNA by site-directed mutagenesis shows that the method is suitable to produce specific nucleotide exchanges without impairing the biological competence of the RNA.

Bacteriophages↗
PubMed2006

Ribosomal RNA as molecular barcodes: a simple correlation analysis without sequence alignment.

MOTIVATION: We explored the feasibility of using unaligned rRNA gene sequences as DNA barcodes, based on correlation analysis of composition vectors (CVs) derived from nucleotide strings. We tested this method with seven rRNA (including 12, 16, 18, 26 and 28S) datasets from a wide variety of organisms (from archaea to tetrapods) at taxonomic levels ranging from class to species. RESULT: Our results indicate that grouping of taxa based on CV analysis is always in good agreement with the phylogenetic trees generated by traditional approaches, although in some cases the relationships among the higher systemic groups may differ. The effectiveness of our analysis might be related to the length and divergence among sequences in a dataset. Nevertheless, the correct grouping of sequences and accurate assignment of unknown taxa make our analysis a reliable and convenient approach in analyzing unaligned sequence datasets of various rRNAs for barcoding purposes. AVAILABILITY: The newly designed software (CVTree 1.0) is publicly available at the Composition Vector Tree (CVTree) web server http://cvtree.cbi.pku.edu.cn.

Algorithms↗
PubMed1997

Characterisation of genomic RNA of Coxsackievirus B3 in murine myocarditis: reliability of direct sequencing of reverse transcription-nested polymerase chain reaction products.

SWR mice develop viral myocarditis histologically similar to the human disease following inoculation with a cardiovirulent Coxsackievirus B3 (CVB3), reactivated from a sequenced cDNA clone of Nancy strain. A sequence of 215 nucleotides, or 628 nucleotides in representative cases, of the 5'non-translated region (5'NTR) of CVB3 genome was amplified from myocardial samples of the infected mice by reverse transcription-nested polymerase chain reaction (RT-NPCR). In order to verify the viral nucleotide sequence and detect the mutation frequency of the viral RNA, the nucleotide sequence of NPCR products were determined by direct sequencing in both orientations. The amplified products from mouse heart on day 1-13 post-inoculation were sequenced and, in each case, the consensus sequence was identical to the published sequence of CVB3 (Nancy strain). To evaluate further the reproducibility of these techniques, three tissue samples from the same infected mouse heart were processed independently. Sequences of their RT-NPCR products were identical to each other as well as to the published sequence. When two attenuated CVB3 mutants were amplified and sequenced, single mutations were detected. To evaluate the overall fidelity of these two combined techniques, genomic RNA of a different CVB3 Nancy strain stock, Coxsackievirus A9 or poliovirus sabin 1 was amplified and the NPCR products sequenced. Each product showed 100% homology with its published sequence. These results demonstrate that the coupled technique of the enterovirus RT-NPCR with direct sequencing of NPCR products generates accurate consensus sequence data and this technique proved to be useful in verification of enteroviral amplicons and in detection of nucleotide mutations. In addition, a low mutation frequency was found in the 5'NTR of CVB3 detected in myocardial samples of immunocompetent mice up to 13 days.

Animals↗
PubMedDate not supplied

Acetogenic coccoid spore-forming bacteria isolated from the rumen.

Thirteen strains of a new acetogenic bacterium were isolated from the rumen contents of lambs, llamas and bisons. This paper is the first report of Gram-positive coccoid spore-forming bacteria occurring in chains and able to use H2 + CO2 as energy source and produce acetate from this gas mixture. One of them, chosen as the reference strain for its efficiency in utilizing H2/CO2 likely via the acetyl-CoA pathway, was characterized in detail. The G + C ratio of the DNA of the organism was 46.5 mol%. The temperature and pH optimum were 37 degrees-40 degrees C and 6.3-6.8, respectively. Numerous organic substrates including some o-methylate aromatic compounds were used heterotrophically. The full 16S rRNA gene sequence was determined. The phylogeny, physiology, morphology and numerous features described here are sufficiently different from those of any bacteria described today to justify the definition of a new species. The name "New acetogenic bacterium" is temporarily proposed, awaiting a future taxonomic revision of the genus Clostridium.

Acetic Acid↗
PubMed2003

Finding the common structure shared by two homologous RNAs.

MOTIVATION: CARNAC is a new method for pairwise folding of RNA sequences. The program takes into account local similarity, stem energy, and covariations to produce the common folding. It can handle all RNA types, and has also been adapted to align a new homologous sequence along a reference structured sequence. RESULTS: Using different data sets, we show that CARNAC provides a good partial prediction for a wide range of sequences (16S ssu rRNA, RNase P RNA, viruses) with only two sequences. In presence of a whole family of sequences, we also show that CARNAC can be used to detect whether the sequences actually share the same structure. AVAILABILITY: CARNAC is available at the URLhttp://www.lifl.fr/~perrique/rna/.

Algorithms↗
PubMed2004

A graph theoretical approach for predicting common RNA secondary structure motifs including pseudoknots in unaligned sequences.

MOTIVATION: RNA structure motifs contained in mRNAs have been found to play important roles in regulating gene expression. However, identification of novel RNA regulatory motifs using computational methods has not been widely explored. Effective tools for predicting novel RNA regulatory motifs based on genomic sequences are needed. RESULTS: We present a new method for predicting common RNA secondary structure motifs in a set of functionally or evolutionarily related RNA sequences. This method is based on comparison of stems (palindromic helices) between sequences and is implemented by applying graph-theoretical approaches. It first finds all possible stable stems in each sequence and compares stems pairwise between sequences by some defined features to find stems conserved across any two sequences. Then by applying a maximum clique finding algorithm, it finds all significant stems conserved across at least k sequences. Finally, it assembles in topological order all possible compatible conserved stems shared by at least k sequences and reports a number of the best assembled stem sets as the best candidate common structure motifs. This method does not require prior structural alignment of the sequences and is able to detect pseudoknot structures. We have tested this approach on some RNA sequences with known secondary structures, in which it is capable of detecting the real structures completely or partially correctly and outperforms other existing programs for similar purposes. AVAILABILITY: The algorithm has been implemented in C++ in a program called comRNA, which is available at http://ural.wustl.edu/softwares.html

Algorithms↗
PubMed2005

MARNA: multiple alignment and consensus structure prediction of RNAs based on sequence structure comparisons.

MOTIVATION: Due to the importance of considering secondary structures in aligning functional RNAs, several pairwise sequence-structure alignment methods have been developed. They use extended alignment scores that evaluate secondary structure information in addition to sequence information. However, two problems for the multiple alignment step remain. First, how to combine pairwise sequence-structure alignments into a multiple alignment and second, how to generate secondary structure information for sequences whose explicit structural information is missing. RESULTS: We describe a novel approach for multiple alignment of RNAs (MARNA) taking into consideration both the primary and the secondary structures. It is based on pairwise sequence-structure comparisons of RNAs. From these sequence-structure alignments, libraries of weighted alignment edges are generated. The weights reflect the sequential and structural conservation. For sequences whose secondary structures are missing, the libraries are generated by sampling low energy conformations. The libraries are then processed by the T-Coffee system, which is a consistency based multiple alignment method. Furthermore, we are able to extract a consensus-sequence and -structure from a multiple alignment. We have successfully tested MARNA on several datasets taken from the Rfam database.

Algorithms↗
PubMed2006

Phylogenetic and biologic evidence that Babesia divergens is not endemic in the United States.

The causative agent of human babesiosis in a Kentucky case, which was first identified as Babesia divergens, is identical to a parasite of eastern cottontail rabbits on Nantucket Island, Massachusetts based on piroplasm size, morphology, and ribosomal RNA sequence analysis. Studies showing differential infectivity for cattle, host erythrocyte specificity in vitro, parasite size and morphology in vitro, and ribosomal RNA sequences clearly demonstrate that the parasite from the rabbit (conspecific with the human Kentucky agent) is not the same organism as B. divergens.

Animals↗
PubMed2006

A GCUA tetranucleotide loop found in the poliovirus oriL by in vivo SELEX (un)expectedly forms a YNMG-like structure: Extending the YNMG family with GYYA.

The cloverleaf structure in the 5'-untranslated region of enterovirus RNA that regulates viral RNA replication contains an evolutionarily conserved YNMG tetraloop closed by a Y-G base pair. This loop is believed to interact specifically with the viral protease 3C. To further characterize the specificity of this interaction, the tetraloop and two flanking base pairs of the poliovirus RNA were randomized, and viable viral clones were obtained using in vivo SELEX. Among many different mutants with the canonical YNMG sequences to be described elsewhere, a large-plaque-forming clone contained a deviating uGCUAg sequence. The NMR structure of a small hairpin capped with uGCUAg that we present here shows that the GCUA tetraloop adopts a novel fold, which is highly similar to that of the YNMG tetraloop with common stacking properties and hydrogen-bond interactions including an unusual syn conformation of the adenosine. Thermodynamic studies show moderate stabilities of hairpins with canonical YNMG and the novel GCUA loops, which, together with the similarity of spatial structures, illustrates that the tetraloop structure itself is crucial for the RNA-protein interaction required for the viral replication. A re-evaluation of the ribosomal secondary structure database reveals a hairpin containing a GCUA loop, which covaries with YNMG and is involved in a tertiary interaction, and in the 50S ribosomal subunit from Haloarcula marismortui the structurally comparable apex of stem-loop 35a is a recognition site for protein L2. These observations show a more general occurrence and importance of the so-far unrecognized GYYA hairpin loops.

Base Pairing↗
PubMed1995

A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe).

An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E. coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned. The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da. Amino acid sequence comparison with the three other psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the catalysis of a common reaction. The gene was cloned behind a (His)6 leader for affinity purification. Upon overexpression, most of the enzyme remained soluble in the cell cytoplasm and could be purified to homogeneity on a Ni(2+)-containing resin. The enzyme reacted with both full-length 23S RNA or a fragment from residues 1-847, forming 1 mol psi/mol RNA at position 746, a normal site for psi. The enzyme has no dependence on Mg2+. The same yield was obtained in 1 mM EDTA as in 10 mM Mg2+, and the rate was faster in EDTA than in Mg2+. Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were not modified in either EDTA or Mg2+. tRNA(Phe) transcripts, however, were modified with a yield of 1 mol psi/mol transcript at a rate in EDTA like that of 23S RNA. Sequencing showed all of the psi to be at position 32, a normal site for psi in this tRNA. Both 23S rRNA psi 746 and tRNA psi 32 occur in single-stranded segments of the same sequence, psi UGAAAA, closed by a stem. Therefore, this synthase may require for recognition only a short stretch of primary sequence 3' to the site of pseudouridylation. This is the first example of a dual-specificity modifying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA. The essentiality of these psi residues can now be assessed by disruption of the synthase gene.

Amino Acid Sequence↗
PubMed1997

Sequence analysis of the Puumala hantavirus Sotkamo strain L segment.

Sequence of the Puumala virus (PUU) Sotkamo strain L segment is provided, completing the total genome of this prototype PUU virus strain. The L segment is 6530 nucleotides long and it can encode 2156 amino-acids-long L protein, RNA-dependent RNA polymerase. The strain Sotkamo, originally isolated in Finland, showed for the L genome segment nucleotide (84.6%) and amino acid (97.3%) homology to a previously sequenced PUU Russian isolate, strain Bashkiria/CG1820 (B1820) and the L genome segment appeared to be at least as conserved as the S segment. Phylogenetic analysis based on the S, M and L segment sequences proposes that the three viral genes have a similar evolutionary history with no evidence for genome segment reassortment. Precise sequencing of the L segment termini demonstrated that the Puumala strains differ from the conserved sequences of the other hantaviruses at two positions.

Amino Acid Sequence↗
PubMed1999

Complete nucleotide sequence of bean pod mottle virus RNA1: sequence comparisons and evolutionary relationships to other comoviruses.

The complete nucleotide sequence of bean pod mottle comovirus (BPMV) RNA 1 was determined. It is 5983 nucleotides long, excluding the poly(A) tail, and encodes a polyprotein of 1850 amino acid (aa) residues. Multiple alignments of the deduced aa sequence of BPMV polyprotein with those of cowpea mosaic virus (CPMV), red clover mottle virus (RCMV) and cowpea severe mosaic virus (CPSMV) indicated that BPMV RNA1 encodes the predicted set of five mature proteins: the equivalent of CPMV 32K protease cofactor, 58K putative helicase, VPg, 24K protease and 87K putative RNA-dependent RNA polymerase. Of the four proposed cleavage sites in BPMV RNA1 polyprotein, the one at the 32K/58K site (Q/A) is distinct for BPMV polyprotein and those at the 58K/VPg and VPg/24K junctions (Q/S and Q/M, respectively) are identical in all four comovirus polyproteins. Sequence comparison and phylogenetic analysis revealed that BPMV RNA1 is more closely related to CPSMV than to CPMV or to RCMV.

Amino Acid Sequence↗
PubMed1993

Two distinct mutations in a single dystrophin gene: identification of an altered splice-site as the primary Becker muscular dystrophy mutation.

A single base change in the 5' splice-site of intron 19 has been identified as the cause of the Becker muscular dystrophy in a family which had previously been deduced to carry both a major deletion and another, at that stage unidentified, mutation in the same dystrophin gene [Laing et al., 1992]. RNA from a muscle biopsy of one of the Becker muscular dystrophy patients in the family was analysed using the reverse transcriptase-polymerase chain reaction (RT-PCR) to study the mature gene transcript. Exon 19 was deleted from the dystrophin mRNA but present at the genomic level. The loss of exon 19 in the mature mRNA was found to be associated with an A to C mutation in the 5' splice site of intron 19. Deletion of exon 19 should alter the reading frame of the mRNA and be associated with a severe form of muscular dystrophy; however, low levels of normal-size dystrophin message and dystrophin were present in this patient. The distance between the splice-site mutation and the secondary deletion in the dystrophin gene is such that it would seem unlikely that the initial base change could act as a premutation for the deletion. Specific primers to detect the splice-site mutation have been designed and used to genotype all relatives.

Alleles↗
PubMed1995

Human enteric caliciviruses have a unique genome structure and are distinct from the Norwalk-like viruses.

Classic human enteric caliciviruses (HuCVs) have a distinctive morphology and are primarily associated with pediatric acute gastroenteritis. Although morphologically distinct from the small round structured viruses (SRSVs), the classic HuCVs are thought to be closely related and were anticipated to have a similar genome organisation. We report the first genome sequence and molecular characterisation of a classic human enteric calicivirus associated with a case of acute vomiting and diarrhoea in an infant. The RNA genome (7266 nt) is smaller than the genome of SRSVs from the two genetic groups and has a unique arrangement of open reading frames. Further analysis of the 3' terminal 3 kb from a second unrelated isolate confirmed this genomic organisation. Analysis of capsid and RNA polymerase sequences together with the unique genomic organisation of classic HuCV suggest these viruses are more closely related to the animal caliciviruses than the enteric SRSV group of viruses.

Amino Acid Sequence↗