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PubMed2001

Cymbidium ringspot tombusvirus coat protein coding sequence acts as an avirulent RNA.

Avirulent genes either directly or indirectly produce elicitors that are recognized by specific receptors of plant resistance genes, leading to the induction of host defense responses such as hypersensitive reaction (HR). HR is characterized by the development of a necrotic lesion at the site of infection which results in confinement of the invader to this area. Artificial chimeras and mutants of cymbidium ringspot (CymRSV) and the pepper isolate of tomato bushy stunt (TBSV-P) tombusviruses were used to determine viral factors involved in the HR resistance phenotype of Datura stramonium upon infection with CymRSV. A series of constructs carrying deletions and frameshifts of the CymRSV coat protein (CP) undoubtedly clarified that an 860-nucleotide (nt)-long RNA sequence in the CymRSV CP coding region (between nt 2666 and 3526) is the elicitor of a very rapid HR-like response of D. stramonium which limits the virus spread. This finding provides the first evidence that an untranslatable RNA can trigger an HR-like resistance response in virus-infected plants. The effectiveness of the resistance response might indicate that other nonhost resistance could also be due to RNA-mediated HR. It is an appealing explanation that RNA-mediated HR has evolved as an alternative defense strategy against RNA viruses.

Amino Acid Sequence↗
PubMed2005

Consensus shapes: an alternative to the Sankoff algorithm for RNA consensus structure prediction.

MOTIVATION: The well-known Sankoff algorithm for simultaneous RNA sequence alignment and folding is currently considered an ideal, but computationally over-expensive method. Available tools implement this algorithm under various pragmatic restrictions. They are still expensive to use, and it is difficult to judge if the moderate quality of results is because of the underlying model or to its imperfect implementation. RESULTS: We propose to redefine the consensus structure prediction problem in a way that does not imply a multiple sequence alignment step. For a family of RNA sequences, our method explicitly and independently enumerates the near-optimal abstract shape space, and predicts as the consensus an abstract shape common to all sequences. For each sequence, it delivers the thermodynamically best structure which has this common shape. Since the shape space is much smaller than the structure space, and identification of common shapes can be done in linear time (in the number of shapes considered), the method is essentially linear in the number of sequences. Our evaluation shows that the new method compares favorably with available alternatives. AVAILABILITY: The new method has been implemented in the program RNAcast and is available on the Bielefeld Bioinformatics Server. CONTACT: jreeder@TechFak.Uni-Bielefeld.DE, robert@TechFak.Uni-Bielefeld.DE SUPPLEMENTARY INFORMATION: Available at http://bibiserv.techfak.uni-bielefeld.de/rnacast/supplementary.html

Algorithms↗
PubMed1994

Phylogeny of the attine ant fungi based on analysis of small subunit ribosomal RNA gene sequences.

Complete 16S-like ribosomal RNA coding regions were obtained from the fungal symbiont of five genera of attine (leaf-cutting) ants and two free-living fungi. Phylogenetic analyses with distance matrix, maximum likelihood, and parsimony methods revealed that the attine fungal symbionts are homobasidiomycetes in the order Agaricales. Comparison of the topology of the attine fungal symbiont phylogenetic tree with a tree based on attine ant morphology revealed a congruent branching pattern of the more derived attine ants and their fungal symbionts. The parallel branching pattern suggests a long-term coevolution of derived leaf-cutting attine ants and their fungal symbionts.

Agaricales↗
PubMed2001

Detection of germline mutations in the BRCA1 gene by RNA-based sequencing.

BRCA1 mutation detection is expensive and has sensitivity limitations, which might at least partially be overcome by RNA-based sequencing. There are claims that RNA tests are unreliable due to differential splicing, exon skipping, or nonsense-mediated mRNA decay that results in either the absence or low expression of mRNA harboring mutations. The major aim of this study was to determine if the application of specific high temperature annealing primers can assure high sensitivity of detection of BRCA1 sequence alterations by cDNA sequencing. The study group comprised 21 Polish cancer families with aggregations of breast and/or ovarian cancer. We detected mutations in 10 out of 21 unrelated patients. These were: nucleotide substitutions (c.309T>C; c.300T>G); nucleotide insertions (c.5382insC) three cases; nucleotide deletions (c.4154delA) one case, (c. 185delAG) one case, (c.3819delGTAAA) two cases; and the deletion of the entire sequence of exon 22, one case. In addition, we identified three transcript variants resulting from alternative splice sites affecting the last six nucleotides of exon 1a (GTAAAG), and the first three nucleotides (CAG) of exon 8 and exon 14. In all cases these were cDNA heterozygous changes. Two of these splice site changes have not been previously described. Sequencing of genomic DNA "exon by exon" did not result in the detection of any additional abnormalities. The sensitivity of our analyses was sufficient to reliably detect mutations without the necessity of tissue culturing to obtain enough template cDNA for analysis.

Alternative Splicing↗
PubMed1997

Poliovirus-encoded 2C polypeptide specifically binds to the 3'-terminal sequences of viral negative-strand RNA.

The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5' untranslated region of the positive strand and its complementary 3'-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3'-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3'-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3'-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.

Base Sequence↗
PubMed1991

Nucleotide sequence of RNA 2 of a Czechoslovakian isolate of red clover necrotic mosaic virus.

The complete nucleotide sequence (1448 nucleotides) of RNA 2 of a Czechoslovakian isolate TpM-34 of red clover necrotic mosaic virus (RCNMV-TpM-34) has been determined. The sequence contained one major open reading frame (ORF) with the potential to encode a protein of 326 amino acids (Mr 35755), designated P2. The nucleotide sequence of RNA 2 of RCNMV-TpM-34 and the previously published sequence of RNA 2 of an Australian isolate of the virus (RCNMV-Aus) were 83% identical and there was 80% amino acid sequence identity between the P2 proteins of these isolates. However the N-terminal two-thirds of the P2 proteins shared a higher degree of similarity than the C-terminal regions which were predicted to have a more flexible structure. An ORF in the 3' portion of RNA 2 of RCNMV-Aus, which could encode a protein of Mr 5000, was not present in RNA 2 of RCNMV-TpM-34. RNAs 1 and 2 of RCNMV-TpM-34 and RCNMV-Aus are bilaterally compatible.

Amino Acid Sequence↗
PubMed1979

Fv-2 locus controls expression of Friend spleen focus-forming virus-specific sequences in normal and infected mice.

We have recently demonstrated that normal hemopoietic cells express RNA sequences that are homologous to sequences specific for the Friend erythroleukemia virus genome [Bernstein, A., Gamble, C., Penrose, D. & Mak, T. W. (1979) Proc. Natl. Acad. Sci. USA 76, 4455-4459]. In this communication, we report that the Fv-2 locus, the major genetic determinant controlling host susceptibility to erythroleukemia induction by Friend leukemia virus, also controls the expression of endogenous sequences related to the replication-defective component of Friend leukemia virus, Friend spleen focus-forming virus (SFFV), in normal uninfected mice. Two independent congeneic pairs of mice [C57BL/6 (B6) and B6.S; B6 and B6.C(H-7(b))], differing only in a small region of the mouse genome including the Fv-2 locus, were used for this purpose. In both cases, molecular hybridization analysis indicated that the presence of SFFV-related RNA sequences in normal mice was associated with the Fv-2(s) allele: bone marrow or spleen cellular RNA from Fv-2(rr) B6 mice contained no detectable SFFV-related sequences, whereas their congeneic Fv-2(ss) pairs contained relatively high levels of these RNA sequences. The absence of these RNA sequences in Fv-2(rr) mice was not due to deletion of these sequences from the DNA of Fv-2(rr) mice. Repopulation of lethally irradiated Fv-2(rr) mice with syngeneic Fv-2(rr) bone marrow cells did not lead to any increase in the levels of these SFFV-related RNA sequences, suggesting that the expression of these sequences is still reduced or inhibited in actively cycling Fv-2(rr) hemopoietic cells. Infection with Friend leukemia virus resulted in the appearance of high levels of RNA homologous to SFFV-specific sequences in the leukemic spleens of B6.S (Fv-2(ss)) mice, whereas these cellular RNA sequences could not be detected in the spleens of Friend virus-infected B6 (Fv-2(rr)) mice. The demonstration that the same gene locus controls both the expression of exogenous SFFV-specific sequences and erythroleukemia induction by Friend leukemia virus suggests that these sequences may be necessary for erythroleukemic transformation. In addition, the finding that the Fv-2 gene locus controls the expression of endogenous SFFV-related sequences suggests that these sequences may also be involved in normal hemopoiesis.

Animals↗
PubMed1993

The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences.

Partial sequences of the 16S ribosomal RNA genes of eleven autotrophic ammonia-oxidizing bacteria were determined by PCR amplification from small amounts of heat-lysed biomass followed by direct sequencing of PCR products. The sequences were aligned with those of representative Proteobacteria and phylogenetic trees inferred using both parsimony and distance matrix methods. This confirmed that the autotrophic ammonia-oxidizers comprise two major lines of descent within the Proteobacteria. Nitrosomonas spp., Nitrosococcus mobilis, and strains of Nitrosovibrio, Nitrosospira and Nitrosolobus were located in the beta-subdivision. The recovery of Nitrosococcus oceanus strains as a deep branch in the gamma-subdivision supported the RNA catalogue data which had indicated that the genus Nitrosococcus is polyphyletic. The autotrophic ammonia-oxidizing bacteria of the beta-Proteobacteria formed a coherent group which is interpreted as representing a single family. Within this clade, the genera Nitrosovibrio, Nitrosospira and Nitrosolobus exhibited very high levels of homology in their 16S ribosomal RNA gene sequences and can be accommodated within a single genus. Separation of these genera is currently based entirely on gross morphological differences and these can now be considered more appropriate for the identification of species within this group. It is therefore proposed that Nitrosolobus, Nitrosovibrio and Nitrosospira strains be reclassified in a single genus for which the name Nitrosospira has priority.

Ammonia↗
PubMed2005

Sequence to Structure (S2S): display, manipulate and interconnect RNA data from sequence to structure.

SUMMARY: Efficient RNA sequence manipulations (such as multiple alignments) need to be constrained by rules of RNA structure folding. The structural knowledge has increased dramatically in the last years with the accumulation of several large RNA structures similar to those of the bacterial ribosome subunits. However, no tool in the RNA community provides an easy way to link and integrate progress made at the sequence level using the available three-dimensional information. Sequence to Structure (S2S) proposes a framework in which an user can easily display, manipulate and interconnect heterogeneous RNA data, such as multiple sequence alignments, secondary and tertiary structures. S2S has been implemented using the Java language and has been developed and tested under UNIX systems, such as Linux and MacOSX. AVAILABILITY: S2S is available at http://bioinformatics.org/S2S/.

Algorithms↗
PubMed1998

Nucleotide sequencing of S-RNA segment and sequence analysis of the nucleocapsid protein gene of the newly isolated Akabane virus PT-17 strain.

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.

Amino Acid Sequence↗
PubMed1998

Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55.

Purine-rich exonic splicing enhancers (ESEs) have been identified in many alternatively spliced exons. Alternative splicing of several ESE-containing exons has been shown to depend on subsets of the SR protein family of pre-mRNA splicing factors. In this report, we show that purified SR protein family member SRp55 by itself binds a 30-nt ESE-containing exon, the alternatively spliced exon 5 of avian cardiac troponin T. We show that purified SRp55 binds specifically to this RNA sequence with an apparent Kd of 60 nM as assayed by gel mobility retardation experiments. Mutations in the exon 5 sequence that increase or decrease exon 5 inclusion in vivo and in vitro have correspondingly different affinities for SRp55 in our assays. The exon 5 sequence contains two purine-rich motifs, common to many ESEs, and both are required for SRp55 binding. Hill plot analysis of binding titration reactions indicates that there is a cooperative binding of at least two SRp55 proteins to the exon sequence. Chemical modification interference studies using kethoxal show that SRp55 binding to exon 5 requires the N1 and/or the N2 of almost every G residue in the exon. Dimethylsulfate modification interference studies indicate that none of the N1 positions of A residues in the exon are important for binding. We postulate that SRp55 may recognize both primary sequence and RNA secondary structural elements within pre-mRNA.

Adenosine↗
PubMed1982

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.

Adenoviruses, Human↗
PubMed1975

The 3' terminal oligonucleotide of E. coli 16S ribosomal RNA: the sequence in both wild-type and RNase iii- cells is complementary to the polypurine tracts common to mRNA initiator regions.

Application of Sanger techniques to the analysis of the 3' terminal oligonucleotide from E. coli 32-P-labelled 16 S rRNA yields the sequence AUCACCUCCUUAOH. This sequence is identical in RNA isolated from two wild-type strains (MRE600 and E. coli B, SY106) and from a mutant strain (AB301/105) defective in RNase III. Data presented here explains the previous derivation of an incorrect sequence (AUCCUCACUUCAOH) by others. The functional significance of complementarity between the 3' terminus of 16S rRNA and poly-purine tracts commonly found in mRNA initiator regions is discussed.

Base Sequence↗
PubMed1989

Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5 beta from pre-mRNA for cytochrome c oxidase subunit I.

The nuclear PET54 gene of Saccharomyces cerevisiae was cloned and a pet54::LEU2 gene disruption strain was constructed. Analysis of the phenotype of this strain revealed a defect in expression of two mitochondrial genes: COX1, which encodes cytochrome c oxidase subunit I, and COX3, which encodes cytochrome c oxidase subunit III. The defect in COX1 gene expression in the pet54 mutant was shown to be the result of inefficient excision of COX1 intron aI5 beta. Two lines of evidence indicate that inefficient excision of intron aI5 beta is the sole defect in COX1 gene expression. First, a pet54::LEU2 cytoductant bearing the 'short' mitochondrial genome that lacks both COX1 introns aI5 alpha and aI5 beta is defective only in COX3 gene expression and not in COX1 mRNA splicing or mRNA translation. Second, Northern analysis of COX1 transcipts from the pet54 mutant showed that a 3.8 kb COX1 transcript containing unexcised intron aI5 beta and lacking intron aI5 alpha is accumulated while the amount of 2.2 kb mature COX1 mRNA is diminished. In an effort to relate the role of the PET54 gene product in splicing of COX1 pre-mRNA to the previously characterized role for PET54 in translation of mitochondrial COX3 mRNA, the sequence of the PET54-responsive portion of the COX3 5' untranslated leader region was compared to the COX1 intron aI5 beta sequence. Two blocks of RNA sequence present in COX3 have similar counterparts within intron aI5 beta of COX1. The possibility that the PET54 protein binds to one or the other of these blocks of RNA sequence and the potential consequences of this interaction are discussed.

Base Sequence↗
PubMed1986

Nucleotide sequence of RNA segment 7 of influenza B/Singapore/222/79: maintenance of a second large open reading frame.

The nucleotide sequence of RNA segment 7 of influenza B/Singapore/222/79 has been determined. The deduced amino acid sequence of the M1 protein indicates that it contains 248 amino acids of which 243 are identical in the B/Lee/40 M1 protein. A second overlapping open reading frame with a maximum coding region of 195 amino acids has also been maintained in the 39 years separating the two isolates. The deduced amino acid sequence of the second coding region displays 86% homology with the comparable B/Lee/40 sequence (27 changes). The conservation of the second open reading frame suggests that this region is important in the replicative cycle of influenza B viruses.

Base Sequence↗
PubMed1986

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.

Amino Acid Sequence↗
PubMed1997

Mutational analysis reveals that an array of GCAAG/CTTGC motifs between sprit promoter sequences for RNA polymerase III is essential for neural BC1 RNA transcription.

BC1 RNA is expressed from an identifier (ID) sequence by RNA polymerase III (Pol III) and occurs in neural cells as a ribonucleoprotein particle (BC1 RNP). On the BC1 RNA gene, between the Pol III promoter A and B boxes, there is a region which contains short inverted repeats, including three GCAAG/CTTGC motifs. We found that a nuclear protein binds specifically to this region and, using an in vitro transcription system, demonstrated that point mutations within these motifs markedly inhibit BC1 RNA transcription. These results suggest that the GCAAG/CTTGC motif region and its binding protein may play a role in the transcription of BC1 RNA. Moreover, we demonstrated that transcription is repressed by a concomitant molar excess of BC1 RNA and that the BC1 RNA transcribed by this system forms an RNP with nuclear protein(s), suggesting some interaction of BC1 RNA with transcription factor(s).

Animals↗