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PubMed2003

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences.

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.

Animals↗
PubMedDate not supplied

Phylogeny of trichomonads inferred from small-subunit rRNA sequences.

Small subunit (16S-like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to the early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S-like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free-living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free-living ancestral trichomonad.

Animals↗
PubMed1984

Deletions, duplications and rearrangements in mycoplasma ribosomal RNA gene sequences.

Mycoplasma genomes lack several entire ribosomal RNA gene sets, and the remaining RNA genes lack some nucleotides. Analysis of mycoplasma 5S RNAs utilizing generalized models for prokaryotic 5S RNA secondary structure allows us to specify the molecular locations and identify many of the nucleotides missing from mycoplasma 5S RNAs and to detect the presence of unusual structural features. The most extensive alterations occur in the 5S RNAs from M. mycoides subsp. capri and M. capricolum, which have a similar pattern. Each of these 5S RNAs, as well as the Spiroplasma BC3 5S RNA, has a concentration of deletions in helix V, and they also share several unusual structures. We also noted the occurrence in mycoplasma 5S RNAs of numerous repeated sequences. The genetic process of unequal crossing-over is suggested as the explanation for the repeated sequences observed in mycoplasma RNAs.

Base Sequence↗
PubMed1995

Characterization of multiple circular RNAs derived from a plant viroid-like RNA by sequence deletions and duplications.

Northern blot hybridizations with a probe complementary to a 275-nt circular RNA isolated from carnation plants revealed that this RNA co-exists in vivo with minor amounts of other small circular RNAs. Sequencing of cDNA clones of nine RNA species demonstrated deletions and duplications of the predominant 275-nt RNA. Minor sequence heterogeneities were observed at the crossover sites. Deletions mapped to three arms of the cruciform structure of lowest free energy obtained previously for the parental 275-nt RNA, whereas repeats encompassed mostly regions of the arm where deletions were not found. Some of the deleted and duplicated regions corresponded to sequences forming part of the two hammerhead structures involved in the in vitro self-cleavage of the plus and minus strands of the 275-nt RNA. A copy choice model is proposed for the emergence of deletions and duplications, where the RNA polymerase with the nascent strand dissociates from the template at regions rich in secondary and possibly tertiary structures, and reinitiates synthesis at different upstream and downstream positions.

Base Sequence↗
PubMed1979

Sequence studies of poliovirus RNA. IV. Nucleotide sequence complexities of poliovirus type 1, type 2 and two type 1 defective interfering particles RNAs, and fingerprint of the poliovirus type 3 genome.

The 32P-labelled genomes of poliovirus type 1, 2 and 3 have been digested with RNase T1 and the products separated by two-dimensional gel electrophoresis. All three fingerprints differ in the separation pattern of the large oligonucleotides. The molar yields of the large RNase T1-resistant oligonucleotides of type 1 and type 2 RNA of poliovirus RNA are close to one. By comparing the yields of these oligonucleotides to the amount of RNA from which they originated, the chain length of type 1 poliovirus RNA was found to be 7851 +/- 567 nucleotides (mol. wt. 2.66 +/- 0.19 x 10(6) and that of poliovirus type 2, 8181 +/- 578 nucleotides (mol. wt. 2.77 +/- 0.19 x 10(6). The chain length of two defective interfering particle (DI) RNAs of poliovirus type 1 were determined to be 7042 +/- 999 nucleotides for DI(1) and 6639 +/- 674 nucleotides for DI(2).

Base Composition↗
PubMed1997

Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.

Localization of mRNA has emerged as a fundamental mechanism for generating polarity during development. In vertebrates, one example of this phenomenon is Vg1 RNA, which is localized to the vegetal cortex of Xenopus oocytes. Vegetal localization of Vg1 RNA is directed by a 340-nt sequence element contained within its 3' untranslated region. To investigate how such cis-acting elements function in the localization process, we have undertaken a detailed analysis of the precise sequence requirements for vegetal localization within the 340-nt localization element. We present evidence for considerable redundancy within the localization element and demonstrate that critical sequences lie at the ends of the element. Importantly, we show that a subelement from the 5' end of the Vg1 localization element is, when duplicated, sufficient to direct vegetal localization. We suggest that the Vg1 localization element is composed of smaller, redundant sequence motifs and identify one such 6-nt motif as essential for localization. These results allow insight into what constitutes an RNA localization signal and how RNA sequence elements may act in the localization process.

Animals↗
PubMed1999

Purification and characterization of an RNA dodecamer sequence binding protein from mitochondria of Saccharomyces cerevisiae.

Saccharomyces cerevisiae mitochondrial mRNAs terminate at their 3' ends with a conserved dodecamer sequence, 5'-AAUAA(U/C)AUUCUU-3'. We have identified a nuclear-encoded protein (DBP) which specifically binds to the dodecamer sequence and have purified it to apparent homogeneity by RNA affinity chromatography. DBP consists of a single polypeptide of 55 kDa and binds to its RNA substrate with a 1:1 stoichiometry. Scatchard analysis determines that K(d) is 0.93 nM for the canonical dodecamer sequence (5'-AAUAAUAUUCUU-3') and 0.46 nM for the only naturally occurring variant (5'-AAUAACAUUCUU-3') unique to oli1 gene. Based on the studies using mutant oligonucleotides, DBP appears to recognize primarily the nucleotide sequence of an RNA rather than its potential secondary structure.

Base Sequence↗
PubMed1989

Cloning and sequencing of RNA-1 cDNA from cucumber mosaic virus strain O.

The complete nucleotide (nt) sequence (3369 nt) of RNA 1 of cucumber mosaic virus strain O (CMV-O) was determined. One open reading frame (ORF; 993 aa) could be deduced from the nt sequence. The homologies of the ORF between CMV-O and CMV-Q or CMV-Fny were calculated to be 85% or 97%, respectively. For CMV-O and CMV-Q, the first one-third of the ORF showed a higher degree of homology (89%), as compared with the other portions (82-85%); the first 224 aa showed more than 93% homology. A comparative study of the three viruses revealed that CMV-O is more homologous to CMV-Fny (subgroup I) [corrected]) than to CMV-Q (subgroup II) [corrected].

Amino Acid Sequence↗
PubMed2001

Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species with ambiguous biochemical profile from a renal transplant recipient.

Traditional ways of identification of bacteria by phenotypic characteristics cannot be used for non-cultivable organisms and organisms with unusual biochemical profiles. In this study, an Enterobacteriaceae was isolated in pure growth from the mid-stream urine of a 67-year old renal transplant recipient with urinary tract infection. Conventional biochemical tests did not reveal a pattern resembling any known member of the Enterobacteriaceae family. The Vitek system (GNI+) showed that it was 18% Leclercia adecarboxylata and 55% Klebsiella ozaenae; whereas the API system (20E) showed that it was 99.8% Rahnella aquatilis. 16S ribosomal RNA gene sequencing showed that there was 7 base differences between the isolate and Enterobacter cloacae, 18 base differences between the isolate and Enterobacter asburiae, 17 base differences between the isolate and Enterobacter cancerogenus, 35 base differences between the isolate and K. ozaenae, 27 base differences between the isolate and L. adecarboxylata, and 72 base differences between the isolate and R. aquatilis, indicating that the isolate most closely resembled a strain of E. cloacae. Identification of the organism in this study is important, as the choice of antibiotics would be radically different. In this case, cephalosporins should be avoided regardless of in-vitro susceptibility as cephalosporins are well-known to select for AmpC derepressed mutants in Enterobacter, and previous administration of third-generation cephalosporins is more likely to be associated with multidrug resistant Enterobacter isolates than is administration of antibiotics that do not include a third-generation cephalosporin.

Aged↗
PubMed1997

Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 1. Characterization and analysis of the reaction.

The kinetics of the ATP-dependent RNA-DNA helicase activity of Escherichia colitranscription termination factor rho have been analyzed. Helicase substrates were assembled using 255 nt and 391 nt RNA sequences from the trp t' RNA transcript of E. coli. These RNA sequences each carry a rho "loading site" at a position near the 5'-end, and a rho-dependent terminator sequence at the 3'-end to which complementary approximately 20 nt DNA oligonucleotides have been annealed. A rapid ( approximately 30 s) pre-steady-state burst of helicase activity (DNA oligomer release), followed by a slow linear phase, is observed in reactions carried out at low salt concentrations (50 mM KCl). Using poly(rC) or poly(dC) as traps for the rho that is released after one round of activity, we have shown that the first (burst) phase of the reaction represents the processive translocation of prebound rho hexamers from the rho loading site to the 3'-end of the RNA molecule. The slow phase of the reaction is complex and represents a combination of many different processes, including the slow release of RNA from rho, the reannealing of complementary DNA oligonucleotides to the RNA substrate, and the recycling of rho hexamers onto additional RNA molecules. Reactions carried out at higher salt concentrations (150 mM KCl) consist of only one phase, since under these conditions rho dissociates more rapidly from the RNA, with an amplitude corresponding to several DNA oligomers removed per rho hexamer. Thus, rho can recycle and function as a catalytic helicase under reaction conditions resembling those found in the cell.

Adenosine Triphosphate↗
PubMed1974

An association between globin messenger RNA and 60S RNA derived from Friend leukemia virus.

The association between certain cellular RNAs and purified RNA tumor viruses prompted us to examine the possibility that specific host messenger RNAs might also be incorporated into RNA tumor viruses. Using a mouse cell line infected with Friend leukemia virus, T-3-Cl-2, which can be induced to accumulate mouse-globin messenger RNA, we show that mouse-globin messenger RNA sequences are present in viral particles purified from the culture medium of globin-producing cells. These globin messenger RNA sequences are absent from viral particles derived from T-3-Cl-2 cells that are not producing globin messenger RNA. Virus-associated globin messenger RNA sequences sediment in association with the 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin messenger RNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin messenger RNA does not become associated with the 60S viral RNA complex; and that globin messenger RNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin messenger RNA.

Animals↗
PubMed1994

Editing of Trypanosoma brucei maxicircle CR5 mRNA generates variable carboxy terminal predicted protein sequences.

RNA editing post-transcriptionally modifies several mRNAs from the maxicircle of kinetoplastid parasites by addition and removal of uridine residues. We report here that maxicircle CR5 transcripts of Trypanosoma brucei are edited in two domains separated by an eight nucleotide sequence that remains unedited. The large 5' domain is edited to a consensus sequence while the smaller 3' domain is edited to multiple final sequences. In all, 205-217 Us are inserted and 13-16 encoded uridines are deleted from the CR5 mRNA, producing a mature transcript 75-80% larger than the unedited transcript. The edited RNAs predict small, highly hydrophobic proteins. The carboxy terminal 15-30% of these predicted proteins have multiple different amino acid sequences as a result of the variable edited 3' mRNA sequence, but these fall into two families of sequence. Limited amino acid sequence and hydrophobicity profile similarities suggest that the protein encoded by edited CR5 mRNA may be a subunit of NADH dehydrogenase.

Amino Acid Sequence↗
PubMed2003

Prediction and statistics of pseudoknots in RNA structures using exactly clustered stochastic simulations.

Ab initio RNA secondary structure predictions have long dismissed helices interior to loops, so-called pseudoknots, despite their structural importance. Here we report that many pseudoknots can be predicted through long-time-scale RNA-folding simulations, which follow the stochastic closing and opening of individual RNA helices. The numerical efficacy of these stochastic simulations relies on an O(n2) clustering algorithm that computes time averages over a continuously updated set of n reference structures. Applying this exact stochastic clustering approach, we typically obtain a 5- to 100-fold simulation speed-up for RNA sequences up to 400 bases, while the effective acceleration can be as high as 105-fold for short, multistable molecules (<or=150 bases). We performed extensive folding statistics on random and natural RNA sequences and found that pseudoknots are distributed unevenly among RNA structures and account for up to 30% of base pairs in G+C-rich RNA sequences (online RNA-folding kinetics server including pseudoknots: http://kinefold.u-strasbg.fr).

Algorithms↗
PubMed1988

A sequence-specific conformational epitope on U1 RNA is recognized by a unique autoantibody.

An autoantibody from a patient with lupus-overlap syndrome was found to bind a specific region of U1 RNA. By using RNA sequence analysis, immunoprecipitation, and competition experiments with in vitro synthesized fragments of U1 RNA, a region of 40 nucleotides representing a stem-loop secondary structure was found to be an immunoreactive domain. This antibody recognized a conformational epitope because neither the RNA stem nor the RNA loop alone was immunoprecipitable. Antisense U1 RNA, U1 DNA, and other small RNAs were not reactive with the antibody. While the origins of nucleic acid-binding antibodies are unknown, this RNA-specific autoantibody probably originated by direct presentation to the immune system or as an anti-idiotype against a more common U1 small nuclear ribonucleoprotein-specific autoantibody. Thus, these findings have implications for the mechanisms of autoimmune recognition and provide an immunological approach to probing RNA structure and protein-RNA interactions.

Autoantibodies↗
PubMed2001

Complete nucleotide sequence of RNA-2 of Olive latent ringspot virus.

The complete nucleotide sequence of Olive latent ringspot virus (OLRSV) RNA-2 was determined. This RNA is 3969 nucleotides in length and contains a single open reading frame of 3448 nt, that encodes a polypeptide of 1146 amino acids, with a calculated Mr of 126,044. OLRSV RNA-2 has a structural organization typical of nepoviruses, with the coat protein (CP) cistron located in the C-terminal and the putative movement protein (MP) in the N-terminal regions of the polyprotein. Computer-assisted comparison of coat proteins of OLRSV and other nepoviruses disclosed relationships that tally with subgrouping based on physicochemical properties.

Amino Acid Sequence↗
PubMed1992

Association of hY4 pseudogenes with Alu repeats and abundance of hY RNA-like sequences in the human genome.

Three loci having homology with the small human cytoplasmic RNA, hY4, were isolated from human genomic DNA libraries and sequenced. Each sequence contains dispersed mismatches as compared with hY4 RNA, is followed by an A-rich or A + T-rich sequence, and is bordered by direct repeats. Each of these loci, therefore, appears to constitute a small RNA class-III pseudogene. Surprisingly, two of the three loci are associated with Alu repeats. In the hY4.B7 locus, the hY4 sequence has integrated into the tail of an Alu element and in the hY4.F2 locus, an Alu sequence has inserted into the hY4 tail, confirming that A-rich tracts are preferential targets for retroposition. In addition, Southern blots with probes for each of the four hY RNAs indicate that hY RNA-like sequences are abundant in the human genome.

Base Sequence↗
PubMed1979

Rapid print-readout technique for sequencing of RNA's containing modified nucleotides.

A rapid, simple, and highly sensitive method for sequence analysis of RNA was developed, which consists of the following steps: (i) controlled hydrolysis of the RNA by brief heating in water; (ii) (32P)-labeling of 5'-hydroxyl groups of the fragments produced in (i); (iii) resolution of labeled fragments by size on polyacrylamide gels giving the familiar "ladder"; (iv) contact transfer ("print") of the ladder from the gel to a PEI-cellulose thin layer; (v) in situ treatment of the ladder with RNase T2 resulting in the release of 5'-(32P)-labeled nucleoside-3',5' diphosphates; (vi) contact transfer and thin-layer separation of (32P)-labeled nucleotides on PEI-cellulose in ammonium sulfate and ammonium formate solvents; (vii) autoradiography. The chromatographic behavior of the 4 major and 18 modified nucleotides was determined. The positions of major and modified nucleotides in the sequence can be read directly from the separation patterns displayed on X-ray film. As this is the only sequencing method presently available that allows one to display and identify directly the positions in the RNA chain of major and modified nucleotides, no additional procedures are required to analyze the latter.

Base Sequence↗
PubMed1996

The complete nucleotide sequence of RNA-2 of a fungally-transmitted UK isolate of barley mild mosaic bymovirus and identification of amino acid combinations possibly involved in fungus transmission.

The complete nucleotide sequence of RNA-2 of a fungally-transmitted UK isolate of barley mild mosaic bymovirus (BaMMV isolate UK-F) was determined and compared with other published sequences, particularly UK-M, an isolate derived from the same source but which has been mechanically passaged for several years, has a deletion of about 1 kb and cannot be fungally transmitted. From an alignment of the BaMMV RNA-2 encoded protein with that for barley yellow mosaic bymovirus (BaYMV), several regions of consistent homology were identified and extensive searches made for similarities with the proteins of other fungally-transmitted viruses, especially amongst the furovirus capsid readthrough proteins which seem especially prone to deletion and which have already been implicated in fungus transmission. The amino acid combinations ER (glutamic acid-arginine) or QR (glutamine-arginine) were found consistently in all of the viruses. They occurred in positions predicted to be on the outside of the protein, and therefore available for interaction with the fungus vector, and were also within the regions prone to spontaneous deletion. In view of the lack of other structural or sequence homologies, it is suggested that these motifs are strong candidates for involvement in fungus transmission.

Amino Acid Sequence↗