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At least 199 records · Page 11Linked to original sources
PubMed1994

Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.

Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia- and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor's photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.

Ammonia↗
PubMed2006

Globicatella bacteraemia identified by 16S ribosomal RNA gene sequencing.

BACKGROUND: Globicatella are streptococcus-like organisms that have been rarely isolated from clinical specimens. Their epidemiology and clinical significance remain largely unknown. AIMS: To describe two cases of Globicatella bacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing. METHODS: Two unidentified streptococcus-like bacteria isolated from blood cultures of patients were subject to 16S rRNA gene sequencing. RESULTS: Two cases of Globicatella bacteraemia were identified by 16S rRNA gene sequencing. In the first case, a gram positive coccus was isolated from the blood culture of an 80 year old woman with diabetes mellitus and nosocomial sepsis, who died the day after developing the bacteraemia. The bacterium was unidentified by conventional phenotypic tests, the Vitek (gram positive identification) and the ATB expression (ID32 Strep) systems. In the second case, a similar bacterium was isolated from the blood culture of a 92 year old woman with polymicrobial acute pyelonephritis complicated by septic shock, who subsequently recovered after antibiotic treatment. 16S rRNA gene sequencing of the two isolates showed 0.5% nucleotide difference from that of G. sulfidifaciens and 0.7% nucleotide difference from that of G. sanguinis, indicating that they were Globicatella species. CONCLUSIONS: Because Globicatella is rarely encountered in clinical microbiology laboratories, it may have been overlooked or misidentified in these cases. 16S rRNA gene sequencing is a useful tool to better characterise the epidemiology and clinical significance of Globicatella.

Aged, 80 and over↗
PubMed2005

Approximate matching of structured motifs in DNA sequences.

Several methods have been developed for identifying more or less complex RNA structures in a genome. All these methods are based on the search for conserved primary and secondary sub-structures. In this paper, we present a simple formal representation of a helix, which is a combination of sequence and folding constraints, as a constrained regular expression. This representation allows us to develop a well-founded algorithm that searches for all approximate matches of a helix in a genome. The algorithm is based on an alignment graph constructed from several copies of a pushdown automaton, arranged one on top of another. This is a first attempt to take advantage of the possibilities of pushdown automata in the context of approximate matching. The worst time complexity is O(krpn), where k is the error threshold, n the size of the genome, p the size of the secondary expression, and r its number of union symbols. We then extend the algorithm to search for pseudo-knots and secondary structures containing an arbitrary number of helices.

Algorithms↗
PubMed2006

Automatic generation of gene finders for eukaryotic species.

BACKGROUND: The number of sequenced eukaryotic genomes is rapidly increasing. This means that over time it will be hard to keep supplying customised gene finders for each genome. This calls for procedures to automatically generate species-specific gene finders and to re-train them as the quantity and quality of reliable gene annotation grows. RESULTS: We present a procedure, Agene, that automatically generates a species-specific gene predictor from a set of reliable mRNA sequences and a genome. We apply a Hidden Markov model (HMM) that implements explicit length distribution modelling for all gene structure blocks using acyclic discrete phase type distributions. The state structure of the each HMM is generated dynamically from an array of sub-models to include only gene features represented in the training set. CONCLUSION: Acyclic discrete phase type distributions are well suited to model sequence length distributions. The performance of each individual gene predictor on each individual genome is comparable to the best of the manually optimised species-specific gene finders. It is shown that species-specific gene finders are superior to gene finders trained on other species.

Algorithms↗
PubMed2006

U1-like snRNAs lacking complementarity to canonical 5' splice sites.

We have detected a surprising heterogeneity among human spliceosomal U1 small nuclear RNA (snRNA). Most interestingly, we have identified three U1 snRNA variants that lack complementarity to the canonical 5' splice site (5'SS) GU dinucleotide. Furthermore, we have observed heterogeneity among the identified variant U1 snRNA genes caused by single nucleotide polymorphism (SNP). The identified snRNAs were ubiquitously expressed in a variety of human tissues representing different stages of development and displayed features of functional spliceosomal snRNAs, i.e., trimethylated cap structures, association with Sm proteins and presence in nuclear RNA-protein complexes. The unanticipated heterogeneity among spliceosomal snRNAs could contribute to the complexity of vertebrates by expanding the coding capacity of their genomes.

Base Pairing↗
PubMed2003

A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya.

Putative pseudouridine synthase genes are members of a class consisting of four subgroups that possess characteristic amino acid sequence motifs. These genes have been found in all organisms sequenced to date. In Escherichia coli, 10 such genes have been identified, and the 10 synthase gene products have been shown to function in making all of the pseudouridines found in tRNA and ribosomal RNA except for tRNA(Glu) pseudouridine13. In this work, a protein able to make this pseudouridine was purified by standard biochemical procedures. Amino-terminal sequencing of the isolated protein identified the synthase as YgbO. Deletion of the ygbO gene caused the loss of tRNA(Glu) pseudouridine13 and plasmid-borne restoration of the structural gene restored pseudouridine13. Reaction of the overexpressed gene product, renamed TruD, with a tRNA(Glu) transcript made in vitro also yielded only pseudouridine13. A search of the database detected 58 homologs of TruD spanning all three phylogenetic domains, including ancient organisms. Thus, we have identified a new wide-spread class of pseudouridine synthase with no sequence homology to the previously known four subgroups. The only completely conserved sequence motif in all 59 organisms that contained aspartate was GXKD, in motif II. This aspartate was essential for in vitro activity.

Amino Acid Motifs↗
PubMed2005

Complete nucleotide sequence of Radish mosaic virus RNA polymerase gene and phylogenetic relationships in the genus Comovirus.

The 3'-terminal part of RNA1 genome segment of Radish mosaic virus (RaMV) including complete RNA polymerase gene was sequenced. The 207 amino acids long polymerase is matured from a polyprotein precursor by cleavage at putative Q/H site by viral protease. The alignment of available amino acid sequences of RNA polymerase genes of comoviruses revealed a closest (55%) identity of RaMV to Red clover mottle virus (RCMV).

Amino Acid Sequence↗
PubMed1994

Disseminated neoplastic cells in Mytilus trossulus: verification of host species origin by (16S-like) rRNA sequence comparison.

Disseminated neoplasia is a leukemia-like disease that occurs in many species of bivalve molluscs worldwide, including the bay mussel (Mytilus trossulus). The etiology of the disease is undetermined, but an early report proposed that the anomalous bivalve cells were actually an invasive parasite rather than cancerous cells of host origin. Comparison of partial sequences of small subunit rRNA from normal and putative cancer cells was performed to resolve this issue. These studies showed a close phylogenetic relationship of the different forms of cancer cells to each other (similarity coefficient, 0.982), to the normal hemocytes (similarity coefficient, 0.990, 0.992), and to the oyster, Crassostrea virginica (similarity coefficient, 0.895-0.927). A large phylogenetic distance separates all 3 mussel hemocyte types from several representative protists (similarity coefficient, 0.702-0.761). These results indicate that the disseminated neoplastic cells in mussels are indeed proliferative host cells and not unicellular parasites.

Animals↗
PubMed2005

Precise characterization of rDNA genes by intraspecies and inter-loci comparison of rDNA sequences and biochemical analysis of ribosomal RNA molecules in Agrobacterium tumefaciens.

Annotation of rRNA genes has been incomplete in Agrobacterium species although a number of Agrobacterial rDNA fragments have been sequenced. In this study, precise characterization of rRNA operons (rrn) was carried out in two biovar 1 strains, C58 and MAFF301001. Complete DNA sequencing of four rrns in MAFF301001 indicated that each operon codes for 16S, 23S and 5S rRNA as well as three tRNAs, trn(Ile), trn(Ala) and trn(Met). The genes and 16S-23S ITS of a given locus were exactly identical with those in the other three loci, except for a T-base loss in the 23S rRNA gene of rrnA and in the 5S rRNA gene of rrnB. Comparison with the four C58 rDNAs available in the DNA database indicated extensive sequence and size variations in the 23S rRNA gene, suggesting the presence of an intervening sequence (IVS). Biochemical RNA analysis, including Northern hybridization and 5' end mapping, in MAFF301001 revealed 2886-base and 2571-base precursors, two 1.3-kb major fragments, a 150-base fragment and removal of an IVS for 23S rRNA. We confirmed similar biochemical characteristics in the C58 strain. The features of rDNA detected here enable correction of previously reported information about Agrobacterial rRNAs and rRNA genes and should be useful for phylogenetic considerations.

Agrobacterium tumefaciens↗
PubMed1991

Specific transcription of an Acanthamoeba castellanii 5S RNA gene in homologous nuclear extracts.

An RNA polymerase III in vitro transcription system has been developed from the protist Acanthamoeba castellanii. The system is dependent on a cloned 5S RNA gene and utilizes a nuclear extract which contains all the necessary protein components. The system is assembled from completely homologous components. Primer extension and RNA sequencing analysis confirm that the in vitro 5S RNA transcript is identical to the 5S RNA isolated from cells. The transcription complex forms unusually rapidly on the 5S RNA gene and is stable to challenge by excess competitor templates. Several 5' deletion mutants were constructed and indicate that the region upstream of -33 is dispensable. Deletion to +16 show the region between -33 and +16 to be required for transcription, a region outside the canonical internal control region.

Acanthamoeba↗
PubMed1995

Replication of cymbidium ringspot virus satellite RNA mutants.

Cymbidium ringspot tombusvirus (CyRSV) supports the replication of an RNA molecule known to be a satellite (sat-RNA). Sequence analysis of wild-type sat-RNA showed that the 3' terminus is heterogeneous and terminates with one, two, or three C residues. Transcripts from mutant clones which lacked up to eight nucleotides at the 3' end were biologically active and yielded progeny RNA that had the 3' end restored as in wild-type RNA. Deletions or substitutions at the 5' end produced nonviable molecules. Studies with mutants containing deletions in internal regions showed that replication was affected essentially by the location of deletions; viable mutants were much less encapsidated than the wild type, showing that size also may be important in the encapsidation and survival of CyRSV sat-RNA.

Base Sequence↗
PubMed1982

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.

Animals↗
PubMed2005

Complete nucleotide sequences and genome characterization of double-stranded RNA 1 and RNA 2 in the Raphanus sativus-root cv. Yidianhong [corrected].

Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yidianhong [corrected] with yellowing at the leaf edge in China. Purified viral particles of 28-30 nm in diameter contained dsRNA segments with the same number and mobility as these extracted directly from radish leaves. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained a single open reading frame (ORF) on their plus-stranded RNA, putatively encoding a RNA dependent RNA polymerase and a capsid protein similar to that encoded by members of the family Partitiviridae. In addition, both RasR 1 and RasR 2 were highly conserved at the 5' untranslated regions (UTR) and had an adenosine-uracil rich stretch at the 3' UTR, with an identical terminal motif (5'-AAAAUAAAACC-3'). Taken together, these results suggest that the two major dsRNA segments constitute the genome of a partitivirus infecting radish.

3' Untranslated Regions↗
PubMed2004

Sequence analysis of L RNA of Lassa virus.

The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses.

Amino Acid Sequence↗
PubMed2025

Extraction, Purification, and Next-Generation Sequencing (NGS) Analysis of DNA and RNA from Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue.

Formalin fixed paraffin embedded (FFPE) tissues have long been used for immunohistological analyses. FFPE tissues can be stored at room temperature for several years enabling analyses to be performed later. Ease of storage and transport makes these tissues an attractive source of biological material. However, formalin fixation results in chemical modifications of proteins and nucleic acids that poses a major challenge to any type of analysis. Recovery of nucleic acids for quantitative assays is rendered difficult due to degradation resulting from fixation and long-term storage, producing low usable yields. Extensive efforts in the last 20 years have led to significant improvements in use of FFPE tissues for DNA and RNA analyses and resulted in development of sensitive assays for a wide range of applications, including next-generation sequencing. In this chapter, we describe the optimization of methods for sequential extraction of DNA and RNA from FFPE tissue and subsequent preparation of DNA-seq and RNA-seq libraries for use with the Illumina platform using commercially available reagents/kits.

Paraffin Embedding↗
PubMed1989

Characterization of immunoglobulin mRNA expression in Burkitt lymphoma cell lines.

Immunoglobulin heavy- and light-chain mRNA of 11 Burkitt lymphoma (BL) cell lines (9 African and 2 American) were analyzed for various structural characteristics. In agreement with previous results at the protein level, all the BL cell lines express heavy-chain mRNA transcripts of the mu class. Surprisingly, a high mu s/mu m mRNA ratio was found in 2 IgM-producing BL cell lines (Raji and CCL85), that do not secrete immunoglobulin. Variable region gene use was also assessed in the cell lines: while 4 out of 7 endemic BL cell lines use VH genes that belong to the VH3 gene family, no clear bias in the expression of particular VH or VL gene families among this sampling of BL lines was found. Northern blot analysis of immunoglobulin transcripts in endemic BL cell lines did show that 2 such lines (AG876 and HTB62) expressed truncated heavy-chain transcripts; RNA sequence analysis of the VH region demonstrated different abnormal 5'-localized RNA splicing events for the 2 shortened mu transcripts. The light-chain mRNA in these 2 cell lines also showed structural abnormalities and, in the case of HTB62, 3 different kappa light-chain transcripts are produced (of elongated, native and truncated sizes). In vitro translation of mRNA from HTB62 showed mu and kappa chain proteins corresponding with the relative size for each message.

Amino Acid Sequence↗
PubMed1989

Sequence analysis reveals extensive polymorphism and evidence of deletions within the E2 glycoprotein gene of several strains of murine hepatitis virus.

Direct RNA sequence analysis of the E2 gene of wild-type MHV-4 and of neutralization resistant, neuroattenuated variants has identified a polymorphic region with respect to deletions. These variants had large deletions of 142 to 159 amino acids mapping to a localized region in the amino-terminal domain of the peplomer glycoprotein. The nucleotide sequence of the E2 gene for wild-type strain MHV-4 was found to be very similar to that of MHV-JHM but had an insertion of 423 nucleotides resulting in the addition of a stretch of 141 unique amino acids in the amino-terminal domain of E2. We propose that deletions reflect a major source of heterogeneity in the E2 protein of MHV.

Amino Acid Sequence↗
PubMedDate not supplied

Rapid ribosequencing--an effective diagnostic tool for detecting microbial infection.

BACKGROUND: Rapid and reliable identification of microorganisms is a prerequisite for the diagnosis and subsequent treatment of infectious diseases. The identification of pathogenic bacteria is traditionally based on their isolation from clinical samples and propagation on culture medium in the routine laboratory. However, despite clinical signs of infection, culture of the pathogenic agent often fails. This may be due to a low number of microorganisms, prior antibiotic treatment, nonculturable microorganisms or specific culture requirements for presently unknown pathogens. Amplification and sequencing of the entire prokaryotic 16S-rRNA is time consuming, labor intensive and expensive. MATERIALS AND METHODS: We describe here a procedure for the identification of a wide range of known and unknown clinically relevant microorganisms by sequencing a small, but highly informative region of the prokaryotic 16S-rRNA gene. This rapid ribosequencing method was evaluated with various reference strains and with clinical samples including eye anterior chamber fluid, cerebrospinal fluid (CSF) and blood cultures. RESULTS: All sequences obtained from the reference strains corresponded to the sequences in databases. We correlated severe eye infection with the isolation of Pseudomonas putida, neurological disorder with Tropheryma whippelii and disseminated visceral abscesses in a child with Blastobacter denitrificans. CONCLUSION: We consider the rapid ribosequencing method as a promising new tool for the analysis of infectious agents in primarily sterile body fluids where conventional culturing of microorganisms fails.

Bacteria↗