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PubMed1995

Assignment of Actinomyces pyogenes-like (CDC coryneform group E) bacteria to the genus Actinomyces as Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov.

In a previous study the authors reported the characterization of some facultatively anaerobic, Gram-positive, non-sporeforming rods which were found in mixed cultures from various infectious processes, including patients with otitis, empyema, perianal abscesses and decubitus ulcers. Phenotypically these organisms closely resembled Actinomyces pyogenes although their precise taxonomic position remained unknown. In the present investigation the authors have determined the 16S rRNA gene sequences of some representative strains of the Actinomyces pyogenes-like bacteria and report the results of a comparative sequence analysis. On the basis of the results of the present and earlier findings two new Actinomyces species, Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. are proposed. The type strains are DSM 9169T and DSM 9168T, respectively.

Actinomyces↗

PubMed1998

Phylogenetic diversity of Streptococcus suis strains of various serotypes as revealed by 16S rRNA gene sequence comparison.

The 16S rRNA gene sequences of reference strains of Streptococcus suis serotypes 1-34 and 1/2 were determined. A comparative sequence analysis showed that the degree of sequence similarity between S. suis reference strains ranged from 93.94 to 100%. A dendrogram was constructed from the similarity matrix. Thirty-two strains representing 32 serotypes fell into a major group divided into three clusters. The other strains, S. suis serotypes 32, 33 and 34, were more distant. Biochemical characterization of the six more distant strains, including S. suis serotypes 20, 22, 26, 32, 33 and 34, revealed a profile similar to that of other S. suis serotypes. Comparison of the 16S rRNA gene sequences of S. suis reference strains with sequences of other members of the genus Streptococcus indicated that, with the exception of S. suis serotypes 32, 33 and 34, reference strains did not cluster with any other species in the genus. In conclusion, 16S rRNA gene sequence analysis defined a major group of S. suis reference strains which were very closely related and a higher divergence for S. suis serotypes 32, 33 and 34. However, to date, there is no strong evidence to reclassify strains of these serotypes in another species.

Animals↗

PubMed1993

Nucleotide sequence analysis of the 5.8S rDNA and adjacent ITS2 region of Candida albicans and related species.

We have determined the nucleotide sequence for the DNA encoding the 5.8S RNAs and downstream internal transcribed spacer (ITS2) regions for Candida albicans and the taxonomically related species C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. Phylogenetic analysis of all known fungal 5.8S RNA sequences revealed a close relationship between C. tropicalis and C. parapsilosis, and to a lesser extent C. albicans within the yeast-like fungi. This group can itself be delineated from predominantly filamentous species. The more distal relationships between Candida (Torulopsis) glabrata and C. krusei support previous findings based on small (18S) ribosomal RNA sequence analysis, suggesting a greater degree of evolutionary divergence of these species from the C. albicans group. Among strains of C. albicans we observed conservation of the ITS2 region at the nucleotide level. Conservation was also observed for a more limited number of C. parapsilosis strains. Although the 3' region of the ITS spacer was species specific, sequence homology was observed in the 5' end within the albicans/parapsilosis/tropicalis group. Our findings suggest a rapid approach to species identification through the use of non-conserved regions flanked by highly conserved, functional domains.

Base Sequence↗

PubMed2004

The emergence in Japan of Sathuperi virus, a tropical Simbu serogroup virus of the genus Orthobunyavirus.

In 1999, two viruses were isolated from blood samples of sentinel cattle in the Western part of Japan. The physiochemical and morphological properties of these viruses indicated that they belonged to the family Bunyaviridae. Sequence analysis of the S segment indicates that the two viruses are closely related to Sathuperi virus (SATV). The N-terminal 168 amino acid of the G2 protein of the M segment was highly homologous with that of SATV (98.2%). Given these results, we conclude that the newly isolated viruses are closest to SATV, which was initially isolated in India and Nigeria over 30 years ago.

Amino Acid Sequence↗

PubMed1999

Morphine regulation of a novel uridine diphosphate glucuronosyl-transferase in guinea pig pups following in utero exposure.

The uridine diphosphate glucuronosyltransferases (UGTs) catalyze conjugation reactions between various substrates and glucuronic acid, UDPGA (uridine diphosphate glucuronic acid), within the endoplasmic reticulum. Conjugation with UDPGA (glucuronidation) is an important pathway in the elimination, detoxification, and activation of compounds including steroid hormones, xenobiotics, and quaternary ammonium substrates. The guinea pig, which has a placental structure and a glucuronidation profile for morphine that are similar to the human, serves as a good small animal model to study the ontogeny of UGTs and the effect of in utero exposure to morphine on UGTs. We examined type 2 UGTs expressed in the guinea pig using amplification and cloning of partial cDNAs from liver RNA. Sequence analysis revealed a novel UGT2 (subsequently named UGT2A3),(2) that has a 64% amino acid sequence similarity to a known UGT2.(3) Full-length cDNAs were isolated from a guinea pig liver cDNA library. Tissue distribution of UGT2A3 using Northern blot analysis showed expression of three distinct size UGT2A3 mRNAs with unique expression in liver and small intestine. UGT2A3 mRNA is expressed at high levels in liver and lower levels in kidney and small intestine. In utero exposure to chronic intermittent morphine resulted in the up regulation of mRNA in 7-day-old female pups' liver and kidney as determined by quantitative RT-PCR analysis. The conjugation profile for UGT2A3 using stable expression in CHO cells and thin-layer chromatography demonstrated active conjugation of phenolic substrates. Regulation of UGTs by in utero morphine exposure may play an important role in fetal development.

Amino Acid Sequence↗

PubMed2003

A new structural motif in the left terminal domain of large viroids identified by covariation analysis.

Sequence alignment of non-hammerhead viroids (Pospiviroidae) reveals that they can be divided into large and small viroids. Covariation analysis of these groups provides evidence for a rod-like secondary structure, but also for two proposed suboptimal structures, namely hairpin I and a bifurcation in the left terminus of large viroids. The strongest covariation within the branched T1 domain of all large Pospiviroidae indicates a new tertiary interaction, resulting in a double-pseudoknot within the (+) strand or an additional hairpin in the (-) strand. The structural element is expected to play a role in the viroid's life cycle.

Algorithms↗

PubMed1993

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

Base Sequence↗

PubMed2001

Taxonomy and important features of probiotic microorganisms in food and nutrition.

Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, eg, morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. Studies based on comparative 16S ribosomal RNA sequencing analysis, however, showed that some taxa generated on the basis of phenotypic features do not correspond with the suggested phylogenetic relations. Thus, some species are not readily distinguishable by phenotypic characteristics. This is especially true for the so-called Lactobacillus acidophilus group, the Lactobacillus casei and Lactobacillus paracasei group, and some bifidobacteria, strains of which have been introduced in many probiotic foods, eg, the novel yogurt-like commodities. Consequently, modern molecular techniques, including polymerase chain reaction-based and other genotyping methods, have become increasingly important for species identification or for the differentiation of probiotic strains. Probiotic strains are selected for potential application on the basis of particular physiologic and functional properties, some of which may be determined in vitro. The classification and identification of a probiotic strain may give a strong indication of its typical habitat and origin. The species, or even genus name, may also indicate the strain's safety and technical applicability for use in probiotic products. Molecular typing methods such as pulsed-field gel electrophoresis, repetitive polymerase chain reaction, and restriction fragment length polymorphism are extremely valuable for specific characterization and detection of such strains selected for application as probiotics.

Bifidobacterium↗

PubMed1994

Nuclear-encoded mitochondrial tRNAs of Trypanosoma brucei have a modified cytidine in the anticodon loop.

The mitochondrial genome of Trypanosoma brucei does not appear to encode any tRNA genes. Isolated organellar tRNAs hybridize to nuclear DNA, suggesting that they are synthesized in the nucleus and subsequently imported into the mitochondrion. Most imported tRNAs have cytosolic counterparts, showing identical mobility on two-dimensional polyacrylamide gels. We have compared three nuclear-encoded mitochondrial tRNAs (tRNA(Lys), tRNA(Leu), tRNA(Tyr)) with their cytosolic isoforms by direct enzymatic sequence analysis. Our findings indicate that the primary sequences of the mitochondrial and the corresponding cytosolic tRNAs are identical. However, we have identified a mitochondrion-specific nucleotide modification of each tRNA which is localized to a conserved cytidine residue at the penultimate position 5' of the anticodon. The modification present in mature mitochondrial tRNA(Tyr) was not found in a mutant tRNA(Tyr) defective in splicing in either cytosolic or mitochondrial fractions. The mutant tRNA(Tyr) has been expressed in transformed cells and its import into mitochondria has been demonstrated, suggesting that the modified cytidine residue is not required for import and therefore may be involved in adapting imported tRNAs to specific requirements of the mitochondrial translation machinery.

Animals↗

PubMed1998

Description of Arthrobacter creatinolyticus sp. nov., isolated from human urine.

Three strains of creatinine-hydrolysing bacteria isolated from human urine were characterized taxonomically. They were aerobic, non-spore-forming, Gram-positive rods with the peptidoglycan of the cell wall containing lysine. MK-8 and MK-9 were found to be the major types of menaquinone. The G + C content of the DNA was 66-67 mol%. The 16S rRNA sequence of one strain (GIFU 12498) was determined and aligned with other high-G + C-content Gram-positive rods from different genera. Following phylogenetic analysis, this strain was placed in the genus Arthrobacter. Arthrobacter protophormiae was the most closely related species in the phylogenetic tree, and this species also showed the highest sequence homology value (97%) with GIFU 12498. However, DNA-DNA hybridization indicated that GIFU 12498 did not belong to A. protophormiae (33.8 +/- 3.5% chromosomal similarity). The three urine strains belonged to one species because they shared more than 95% DNA-DNA similarity. It is proposed that these strains are placed in the genus Arthrobacter as a new species, Arthrobacter creatinolyticus sp. nov. The type strain of A. creatinolyticus is GIFU 12498, which has been deposited in the Japan Collection of Microorganisms (JCM) with the accession number JCM 10102.

Amino Acids↗

PubMed1997

Aerococcus urinae: intraspecies genetic and phenotypic relatedness.

A number of Aerococcus-like organisms were recently recognized as human pathogens. Five Aerococcus-like strains were proposed as members of the new species Aerococcus urinae (with type strain E2 [= NCTC 12142]) on the basis of the results of a 16S rRNA sequence analysis. The intraspecies phenotypic and genetic relatedness of 22 selected A. urinae strains was investigated, and a hitherto unrecognized esculin hydrolysis-positive biotype was identified. A total of 14 of the 15 more common esculin-negative strains exhibited very high DNA relatedness as determined by the hydroxyapatite method (the levels of relatedness were greater than 90% in 55 and 70 degrees C reactions, with 1.5% or less divergence in related sequences). The DNA relatedness among the six esculin-positive strains was more heterogeneous, and two DNA hybridization subgroups were formed. Our results are compatible with the hypothesis that both biotypes are members of the single species A. urinae, which contains two or more genetic subspecies. The putative subspecies have not been formally proposed since they cannot be definitively differentiated. The inclusion of A. urinae in the genus Aerococcus is supported by the results of 16S rRNA sequencing. The rRNA sequence data also is compatible with placing both biotypes in a single species.

Bacteriological Techniques↗

PubMed1994

Evolution of heliobacteria: implications for photosynthetic reaction center complexes.

The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein show rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to the core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.

Bacteria, Anaerobic↗

PubMed2002

Bacteria and pathogenesis of human salivary calculus.

The exact cause of salivary calculus formation is unknown and the aim of this study was to ascertain whether bacteria play a role. Sialoliths from nine patients with chronic obstructive sialadenitis of the submandibular gland were analysed. Bacterial gene fragments were amplified from DNA extracted from salivary calculi by means of polymerase chain reaction using a universal bacterial primer pair. Comparative 16S ribosomal RNA sequence analysis was used for identification. We detected and identified oral bacteria (predominantly Streptococcus species) in all samples. The present results suggest a potential role for bacteria in the etiopathogenesis of sialolithiasis.

Adult↗

PubMed1994

The ribosomal RNA gene region in Acanthamoeba castellanii mitochondrial DNA. A case of evolutionary transfer of introns between mitochondria and plastids?

Acanthamoeba castellanii, an amoeboid protozoan, occupies an intriguing position in phylogenetic trees based on nuclear rRNA sequences, branching together with or near (as an outgroup to) green algae and land plants. To gain insight into the organization, expression and evolutionary affiliations of the mtDNA of this non-photosynthetic protist, we determined the sequence of a 7778 base-pair region containing the single-copy large subunit (LSU) and small subunit (SSU) rRNA genes (rnl and rns, respectively) of the approximately 40 kilobase-pair A. castellanii mitochondrial genome. We also sequenced the 5'- and 3'-terminal portions of the corresponding LSU and SSU rRNAs. In A. castellanii mtDNA, rnl is flanked both upstream and downstream by a cluster of five tRNA genes, with rns and then cox1 (the cytochrome oxidase subunit 1 gene) following immediately further downstream. These genes are all in the same transcriptional orientation and are separated by only short non-coding spacers. Although rnl and rns are organized in a novel way in A. castellanii mtDNA, their SSU and LSU rRNA products are strikingly similar to their eubacterial homologs in primary sequence, secondary structure and post-transcriptional modification. In these characteristics, the A. castellanii mitochondrial rRNAs much more closely resemble their counterparts in land plants than do the corresponding mitochondrial rRNAs in the green alga, Chlamydomonas reinhardtii. Although no intervening sequences have so far been found in the mitochondrial rnl of angiosperms (flowering plants), A. castellanii mitochondrial rnl contains three group I introns, all located within highly conserved regions in the 3'-half of the gene and each possessing a free-standing open reading frame (ORF). The insertion site of one of these introns is identical to that of the single group I intron in the chloroplast rnl of C. reinhardtii, and sequence comparison reveals that these two introns (one mitochondrial, the other chloroplast) are structurally homologous both within the core region and within the ORFs they encode. These observations are indicative of intron movement between mitochondria and chloroplasts, either intracellularly in a photosynthetic, remote common ancestor of A. castellanii and C. reinhardtii or, more recently, as a result of an intercellular exchange of genetic information.

Acanthamoeba↗

PubMed1993

Molecular phylogeny of Drosophila based on ribosomal RNA sequences.

Nucleotide sequences of 72 species of Drosophilidae were determined for divergent D1 and D2 domains (representing 200 and 341 nucleotides respectively in D. melanogaster) of large ribosomal RNA, using the rRNA direct sequencing method. Molecular phylogenetic trees were reconstructed using both distance and parsimony methods and the robustness of the nodes was evaluated by the bootstrap procedure. The trees obtained by these methods revealed four main lineages or clades which do not correspond to the taxonomical hierarchy. In our results, the genus Chymomyza is associated with the subgenus Scaptodrosophila of the genus Drosophila and their cluster constitutes the most ancient clade. The two other clades are constituted of groups belonging to the subgenus Sophophora of the genus Drosophila: the so-called Neotropical clade including the willistoni and saltans groups and the obscura-melanogaster clade itself split into three lineages: (1) obscura group + ananassae subgroup, (2) montium subgroup, and (3) melanogaster + Oriental subgroups. The fourth clade, the Drosophila one, contains three lineages. D. polychaeta, D. iri, and D. fraburu are branched together and constitute the most ancient lineage; the second lineage includes the annulimana, bromeliae, dreyfusi, melanica, mesophragmatica, repleta, robusta, and virilis groups. The third lineage is composed of the immigrans and the cardini, funebris, guaramunu, guarani, histrio, pallidipennis, quinaria, and tripunctata groups. The genera Samoaia, Scaptomyza, and Zaprionus are branched within the Drosophila clade. Although these four clades appear regularly in almost all tree calculations, additional sequencing will be necessary to determine their precise relationships.

Animals↗

PubMed2004

A new virus related to Satsuma dwarf virus: the nucleotide sequence of the 3'-terminal regions of Hyuganatsu virus RNAs 1 and 2. Brief Report.

The sequences of the 3'-terminal 1306 and 2160 nucleotides of RNAs 1 and 2 of a virus serologically related to Satsuma dwarf virus (SDV) from Hyuganatsu ( Citrus tamurana Hort. ex Tan.) were determined, respectively. We found that the partial RNA-dependent RNA polymerase region in RNA1 and the coat proteins (CPs) region in RNA2 of the virus tentatively named Hyuganatsu virus (HV) have 78.3-84.0% and 76.9-80.7% amino acid sequence identities to those of known SDV-related viruses (SDV-RVs), i.e., SDV, Citrus mosaic virus, and Navel orange infectious mottling virus. Sequence analyses show that HV is classifiable as a new SDV-RV species.

Amino Acid Sequence↗

PubMed1997

Completion of molecular characterization of Toscana phlebovirus genome: nucleotide sequence, coding strategy of M genomic segment and its amino acid sequence comparison to other phleboviruses.

The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence determined. The M RNA segment is 4215 nucleotides in length, and it contains a single major open reading frame (ORF) in the viral-complementary sequence, between nucleotides 18 and 4034, which can encode for a polyprotein of 1339 amino acids (Mr 149 kDa). The viral segment is expressed via a unique mRNA containing 10-14 non-templated nucleotides at the 5' end and it is truncated at the 3' end by about 140 nucleotides in a purine-rich region. In M predicted amino acid sequences, several hydrophobic regions have been identified. They could function as a signal sequence or a transmembrane region for the different proteins. Comparison of the deduced amino acid sequence of M precursor product revealed 38, 36, and 25% identity and 58, 56, and 47% similarity with those of Rift Valley fever (RVF), Punta Toro (PT) and Unkuniemi (UUK) viruses, respectively. Residues conserved among the proteins are mainly located at the COOH-portion of the precursor, while the major divergence is in the NSm coding regions. Based on sequence comparison and similarity of hydropathic pattern of TOS M segment with other phleboviruses the N-termini of TOS GN and GC glycoproteins were placed at residues 297 and 936 of the precursor.

Amino Acid Sequence↗

PubMed1986

Archaebacterial phylogeny: perspectives on the urkingdoms.

Comparisons of complete 16S ribosomal RNA sequences have been used to confirm, refine and extend earlier concepts of archaebacterial phylogeny. The archaebacteria fall naturally into two major branches or divisions, I--the sulfur-dependent thermophilic archaebacteria, and II--the methanogenic archaebacteria and their relatives. Division I comprises a relatively closely related and phenotypically homogeneous collection of thermophilic sulfur-dependent species--encompassing the genera Sulfolobus, Thermoproteus, Pyrodictium and Desulfurococcus. The organisms of Division II, however, form a less compact grouping phylogenetically, and are also more diverse in phenotype. All three of the (major) methanogen groups are found in Division II, as are the extreme halophiles and two types of thermoacidophiles, Thermoplasma acidophilum and Thermococcus celer. This last species branches sufficiently deeply in the Division II line that it might be considered to represent a separate, third Division. However, both the extreme halophiles and Tp. acidophilum branch within the cluster of methanogens. The extreme halophiles are specifically related to the Methanomicrobiales, to the exclusion of both the Methanococcales and the Methanobacteriales. Tp. acidophilum is peripherally related to the halophile-Methanomicrobiales group. By 16S rRNA sequence measure the archaebacteria constitute a phylogenetically coherent grouping (clade), which excludes both the eubacteria and the eukaryotes--a conclusion that is supported by other sequence evidence as well. Alternative proposals for archaebacterial phylogeny, not based upon sequence evidence, are discussed and evaluated. In particular, proposals to rename (reclassify) various subgroups of the archaebacteria as new kingdoms are found wanting, for both their lack of proper experimental support and the taxonomic confusion they introduce.

Archaea↗