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Sequence Databases (sequence + databases)
Selected AbstractsAnalysis of the distribution and diversity in recent Hawaiian volcanic deposits of a putative carbon monoxide dehydrogenase large subunit geneENVIRONMENTAL MICROBIOLOGY, Issue 9 2005Kari E. Dunfield Summary A putative carbon monoxide dehydrogenase large subunit gene (BMS putative coxL) was amplified from genomic DNA extracts of four recent (42,300 year old) Hawaiian volcanic deposits by polymerase chain reaction (PCR). Sequence databases derived from clone libraries constructed using PCR products were analysed phylogenetically and statistically. These analyses indicated that each of the deposits supported distinct BMS putative coxL gene assemblages. Statistical analyses also showed that the youngest deposit (42 years old) contained the least diverse sequences (P < 0.05), but that diversity did not vary significantly among three older deposits with ages from about 108,300 years. Although diversity indices did not vary among the older deposits, mismatch analyses suggested population structures increased in complexity with increasing deposit age. At each of the sites, most of the clone sequences appeared to originate from Proteobacteria not currently represented in culture or recognized as CO oxidizers. [source] Functional properties of the protein disulfide oxidoreductase from the archaeon Pyrococcus furiosusFEBS JOURNAL, Issue 16 2004A member of a novel protein family related to protein disulfide-isomerase Protein disulfide oxidoreductases are ubiquitous redox enzymes that catalyse dithiol,disulfide exchange reactions with a CXXC sequence motif at their active site. A disulfide oxidoreductase, a highly thermostable protein, was isolated from Pyrococcus furiosus (PfPDO), which is characterized by two redox sites (CXXC) and an unusual molecular mass. Its 3D structure at high resolution suggests that it may be related to the multidomain protein disulfide-isomerase (PDI), which is currently known only in eukaryotes. This work focuses on the functional characterization of PfPDO as well as its relation to the eukaryotic PDIs. Assays of oxidative, reductive, and isomerase activities of PfPDO were performed, which revealed that the archaeal protein not only has oxidative and reductive activity, but also isomerase activity. On the basis of structural data, two single mutants (C35S and C146S) and a double mutant (C35S/C146S) of PfPDO were constructed and analyzed to elucidate the specific roles of the two redox sites. The results indicate that the CPYC site in the C-terminal half of the protein is fundamental to reductive/oxidative activity, whereas isomerase activity requires both active sites. In comparison with PDI, the ATPase activity was tested for PfPDO, which was found to be cation-dependent with a basic pH optimum and an optimum temperature of 90 °C. These results and an investigation on genomic sequence databases indicate that PfPDO may be an ancestor of the eukaryotic PDI and belongs to a novel protein disulfide oxidoreductase family. [source] Genomic BLAST: custom-defined virtual databases for complete and unfinished genomesFEMS MICROBIOLOGY LETTERS, Issue 2 2002Leda Cummings Abstract BLAST (Basic Local Alignment Search Tool) searches against DNA and protein sequence databases have become an indispensable tool for biomedical research. The proliferation of the genome sequencing projects is steadily increasing the fraction of genome-derived sequences in the public databases and their importance as a public resource. We report here the availability of Genomic BLAST, a novel graphical tool for simplifying BLAST searches against complete and unfinished genome sequences. This tool allows the user to compare the query sequence against a virtual database of DNA and/or protein sequences from a selected group of organisms with finished or unfinished genomes. The organisms for such a database can be selected using either a graphic taxonomy-based tree or an alphabetical list of organism-specific sequences. The first option is designed to help explore the evolutionary relationships among organisms within a certain taxonomy group when performing BLAST searches. The use of an alphabetical list allows the user to perform a more elaborate set of selections, assembling any given number of organism-specific databases from unfinished or complete genomes. This tool, available at the NCBI web site http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi, currently provides access to over 170 bacterial and archaeal genomes and over 40 eukaryotic genomes. [source] Non-random reassortment in human influenza A virusesINFLUENZA AND OTHER RESPIRATORY VIRUSES, Issue 1 2008Raul Rabadan Background, The influenza A virus has two basic modes of evolution. Because of a high error rate in the process of replication by RNA polymerase, the viral genome drifts via accumulated mutations. The second mode of evolution is termed a shift, which results from the reassortment of the eight segments of this virus. When two different influenza viruses co-infect the same host cell, new virions can be released that contain segments from both parental strains. This type of shift has been the source of at least two of the influenza pandemics in the 20th century (H2N2 in 1957 and H3N2 in 1968). Objectives, The methods to measure these genetic shifts have not yet provided a quantitative answer to questions such as: what is the rate of genetic reassortment during a local epidemic? Are all possible reassortments equally likely or are there preferred patterns? Methods, To answer these questions and provide a quantitative way to measure genetic shifts, a new method for detecting reassortments from nucleotide sequence data was created that does not rely upon phylogenetic analysis. Two different sequence databases were used: human H3N2 viruses isolated in New York State between 1995 and 2006, and human H3N2 viruses isolated in New Zealand between 2000 and 2005. Results, Using this new method, we were able to reproduce all the reassortments found in earlier works, as well as detect, with very high confidence, many reassortments that were not detected by previous authors. We obtain a lower bound on the reassortment rate of 2,3 events per year, and find a clear preference for reassortments involving only one segment, most often hemagglutinin or neuraminidase. At a lower frequency several segments appear to reassort in vivo in defined groups as has been suggested previously in vitro. Conclusions, Our results strongly suggest that the patterns of reassortment in the viral population are not random. Deciphering these patterns can be a useful tool in attempting to understand and predict possible influenza pandemics. [source] Development of an ex vivo model for the study of microbial infection in human teethINTERNATIONAL ENDODONTIC JOURNAL, Issue 5 2007B. Patel Aims, (1) To infect human teeth artificially to mimic root canal and dentine infection, using the Constant Depth Film Fermenter (CDFF); (2) To verify the similarity of the infections to those found, in vivo, using culture and microscopy (SEM, LM and TEM). Methodology, Human teeth [n = 38 and n = 28, for phases I (preliminary) and II (definitive), respectively] were infected within the CDFF for a period of 28 days and at pre-selected time points were removed, externally decontaminated using validated protocols and subjected to either culture-dependent or microscopy protocols. The condition of the teeth was varied in phase I to establish the feasibility of the approach and identify optimal conditions. This informed the selection of optimal conditions for definitive test in phase II. For culture-dependent analysis in this phase, a dentine filing sample was obtained from the apical 5 mm of the root canal and cultured anaerobically to allow isolation of individual strains. Bacterial DNA was extracted from purified isolates, the 16S rRNA genes amplified by PCR and the amplicons sequenced for identity using sequence databases. Teeth assigned for microscopy were post-fixed in 3% gluteraldehyde after removal from the CDFF and then subjected to appropriate protocols prior to microscopic evaluation of the infection. Results, All three microscopy techniques and culture-dependent analysis confirmed infection of the human teeth using the CDFF, with root canal infections visually resembling closely those seen in vivo. Furthermore, partial 16S rRNA gene sequencing of DNA from cultured isolates confirmed a selective number of 7,9 genera/species in the apical portion of two teeth each at 7 and 28 days; these taxa are also commonly recovered from teeth with apical periodontitis, in vivo. There were no objective measures other than speciation and topographical evaluation to compare the artificial and real (in vivo) infections. Conclusions, The proposed ex vivo model has the potential for development into an investigative tool for studying the dynamics of bacterial ecology in infected root canals, both before and after treatment. Its advantage is the ability to control both the abiotic and biotic factors. There is a need for the development of objective measures to compare artificial and real bacterial biofilms. [source] Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB regionJOURNAL OF FISH DISEASES, Issue 7 2007S Izumi Abstract Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA. [source] Hemagglutinating activity and corresponding putative sequence identity from Curcuma aromatica rhizomeJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 6 2008Ponpimol Tiptara Abstract BACKGROUND:Curcuma aromatica is a medicinal plant belonging to the Zingiberaceae family with an incomplete genome sequence. It has been reported that extract from the rhizome of this plant contains haemagglutinating activity. In this study the profile of fractions containing hemagglutinating activity is described. RESULTS: Following extraction with saline buffer, the protein solution was fractionated by ammonium sulfate precipitation. Ion-exchange chromatography was completed on fast-flow SP-Sepharose, as well as gel filtration chromatography on Superdex 75. The active fractions were then separated by one-dimensional sodium dodecyl sulfate,polyacrylamide gel electrophoresis and labeled proteins were digested with trypsin. The digest bands were analyzed by reversed-phase liquid chromatography,tandem mass spectrometry. Inferred peptide sequences were used in Mascot searching and mass spectrometry-driven BLAST (MS-BLAST) homology searches allowed the recognition of related proteins in other species of Viridiplantae. Six putative proteins from nine bands showed similarity with lectin sequences. CONCLUSION: This study reports the identification of six lectins from the Curcuma aromatica rhizome achieved by mass spectrometry using MS-BLAST algorithms to search for homology between de novo determined peptide sequences and protein sequences available in sequence databases. Copyright © 2008 Society of Chemical Industry [source] The origin and evolution of human pathogensMOLECULAR MICROBIOLOGY, Issue 1 2005Eduardo A. Groisman Summary What are the genetic origins of human pathogens? An international group of scientists discussed this topic at a workshop that took place in late October 2004 in Baeza (Spain). Focusing primarily on bacterial pathogens, they examined the role that pathogenicity islands and bacteriophages play on determining the virulence properties that distinguish closely related members of a given species, such as host range and tissue specificity. They also discussed an instance in which closely related bacterial species differ in the production of a cell surface modification mediating resistance to an antibiotic as a result of the disparate regulation of homologous genes. In certain pathogens, genes normally carrying out housekeeping functions may adopt new functions, whereas in other organisms, genes that respond to stresses associated with non-host environments are silenced during infection to prevent the expression of products that interfere with the normal colonization process. The adaptive behaviour of certain pathogens relies on gene variation at certain loci that by virtue of containing polymeric repeats in regulatory or coding regions, can generate variants that may or may not express products that modify the cell surface of the organism. The meeting also addressed the properties of ORFan genes, which have no homologues in the sequence databases, as well as the creation of genes de novo by duplication and divergence. [source] Multiple bacteria encode metallothioneins and SmtA-like zinc fingersMOLECULAR MICROBIOLOGY, Issue 5 2002Claudia A. Blindauer Summary Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, 111Cd-NMR, and 111Cd-edited 1H-NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; 111Cd-NMR and 111Cd-edited 1H-NMR confirmed exclusive cysteine-coordination, and these cysteine residues reacted rapidly with 5,5,-dithiobis-(2-nitrobenzoic acid). 1H-NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA-like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA-like lone zinc fingers, devoid of a cluster, and designated GatA. We have identified 12 smtA -like genes in sequence databases including four of the gatA type. [source] 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversityNEW PHYTOLOGIST, Issue 2 2009M. Buée Summary ,,Soil fungi play a major role in ecological and biogeochemical processes in forests. Little is known, however, about the structure and richness of different fungal communities and the distribution of functional ecological groups (pathogens, saprobes and symbionts). ,,Here, we assessed the fungal diversity in six different forest soils using tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS-1). No less than 166 350 ITS reads were obtained from all samples. In each forest soil sample (4 g), approximately 30 000 reads were recovered, corresponding to around 1000 molecular operational taxonomic units. ,,Most operational taxonomic units (81%) belonged to the Dikarya subkingdom (Ascomycota and Basidiomycota). Richness, abundance and taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The ITS-1 sequences (73%) analysed corresponded to only 26 taxa. The most abundant operational taxonomic units showed the highest sequence similarity to Ceratobasidium sp., Cryptococcus podzolicus, Lactarius sp. and Scleroderma sp. ,,This study validates the effectiveness of high-throughput 454 sequencing technology for the survey of soil fungal diversity. The large proportion of unidentified sequences, however, calls for curated sequence databases. The use of pyrosequencing on soil samples will accelerate the study of the spatiotemporal dynamics of fungal communities in forest ecosystems. [source] Three monophyletic superfamilies account for the majority of the known glycosyltransferasesPROTEIN SCIENCE, Issue 7 2003Jing Liu Abstract Sixty-five families of glycosyltransferases (EC 2.4.x.y) have been recognized on the basis of high-sequence similarity to a founding member with experimentally demonstrated enzymatic activity. Although distant sequence relationships between some of these families have been reported, the natural history of glycosyltransferases is poorly understood. We used iterative searches of sequence databases, motif extraction, structural comparison, and analysis of completely sequenced genomes to track the origins of modern-type glycosyltransferases. We show that >75% of recognized glycosyltransferase families belong to one of only three monophyletic superfamilies of proteins, namely, (1) a recently described GPGTF/GT-B superfamily; (2) a nucleoside-diphosphosugar transferase (GT-A) superfamily, which is characterized by a DxD sequence signature and also includes nucleotidyltransferases; and (3) a GT-C superfamily of integral membrane glycosyltransferases with a modified DxD signature in the first extracellular loop. Several developmental regulators in Metazoans, including Fringe and Egghead homologs, belong to the second superfamily. Interestingly, Tout-velu/Exostosin family of developmental proteins found in all multicellular eukaryotes, contains separate domains belonging to the first and the second superfamilies, explaining multiple glycosyltransferase activities in one protein. [source] A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substratesTHE PLANT JOURNAL, Issue 1 2008Florina Vlad Summary Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants. [source] Transcriptome response of the Pacific oyster (Crassostrea gigas) to infection with Vibrio tubiashii using cDNA AFLP differential displayANIMAL GENETICS, Issue 5 2009N. Taris Summary We used qualitative complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) differential display analysis and real-time, quantitative PCR (RT-qPCR) to identify genes in the Pacific oyster Crassostrea gigas, whose transcription either changes in response to exposure to a pathogenic bacterium (Vibrio tubiashii) or varies between families known to differ in sensitivity to heat stress, before and at 12 and 36 h after bacterial exposure at a temperature of 25 °C. These conditions simulate those associated with summer mortality syndrome, a poorly understood cause of massive mortalities in cultured Pacific oysters in North America, Asia and Europe. Using 32 AFLP primer pairs, we identified 92 transcript-derived fragments that are qualitatively differentially expressed. We then cloned and sequenced 14 of these fragments, designed fragment-specific primers and quantified their transcription patterns using RT-qPCR. Most of the differences in transcription patterns between stress-tolerant and stress-sensitive families were evident before bacterial exposure, and genes that responded to bacterial exposure did so in parallel between stress-sensitive and stress-tolerant families. blast searches of sequence databases revealed that these fragments represent genes involved in immune response as well as genes related to metabolic processes. Our data support the hypothesis that family level differences in resistance to stress in Pacific oysters are largely attributable to constitutive differences in gene transcription or ,general vigour' that are detectable before and maintained after infection, rather than being due to induced responses at the transcriptome level. [source] | ||||||||||||||||