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Sample records for acid sequence analyses

  1. Metazoan remaining genes for essential amino acid biosynthesis: sequence conservation and evolutionary analyses.

    PubMed

    Costa, Igor R; Thompson, Julie D; Ortega, José Miguel; Prosdocimi, Francisco

    2014-12-24

    Essential amino acids (EAA) consist of a group of nine amino acids that animals are unable to synthesize via de novo pathways. Recently, it has been found that most metazoans lack the same set of enzymes responsible for the de novo EAA biosynthesis. Here we investigate the sequence conservation and evolution of all the metazoan remaining genes for EAA pathways. Initially, the set of all 49 enzymes responsible for the EAA de novo biosynthesis in yeast was retrieved. These enzymes were used as BLAST queries to search for similar sequences in a database containing 10 complete metazoan genomes. Eight enzymes typically attributed to EAA pathways were found to be ubiquitous in metazoan genomes, suggesting a conserved functional role. In this study, we address the question of how these genes evolved after losing their pathway partners. To do this, we compared metazoan genes with their fungal and plant orthologs. Using phylogenetic analysis with maximum likelihood, we found that acetolactate synthase (ALS) and betaine-homocysteine S-methyltransferase (BHMT) diverged from the expected Tree of Life (ToL) relationships. High sequence conservation in the paraphyletic group Plant-Fungi was identified for these two genes using a newly developed Python algorithm. Selective pressure analysis of ALS and BHMT protein sequences showed higher non-synonymous mutation ratios in comparisons between metazoans/fungi and metazoans/plants, supporting the hypothesis that these two genes have undergone non-ToL evolution in animals.

  2. Homology analyses of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast

    SciTech Connect

    Chang, Soo-Ik ); Hammes, G.G. )

    1989-11-01

    Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.

  3. Identification and localization of amino acid substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses.

    PubMed Central

    Yuan, P M; Ryan, D E; Levin, W; Shively, J E

    1983-01-01

    Two isozymes of rat liver microsomal cytochrome P-450--P-450b and P-450e--were compared by micro sequence analyses of their NH2 termini and tryptic fragments. These two phenobarbital-inducible hemoproteins, which are immunochemically indistinguishable with antibody against cytochrome P-450b, have extensive sequence homology. Automated Edman degradation of the native proteins revealed identical amino acids for the first 35 residues. Sequence determinations of the tryptic peptides, which constitute approximately 75% of each protein molecule, have thus far shown 10 amino acid differences between the two isozymes. Results of our amino acid sequence analyses established that two of the cDNAs, pcP-450pb1 and pcP-450pb4, reported by Fujii-Kuriyama et al. [Fujii-Kuriyama, Y., Mizukami, Y., Kamajiri, K., Sogawa, K. & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. USA 79, 2793-2797] encode cytochrome P-450b whereas pcP-450pb2, a third cDNA whose nucleotide sequence differed slightly from that of the other two (six amino acid substitutions), encodes cytochrome P-450e. In addition to establishing the identity of these cloned cDNAs we provide direct evidence for seven additional amino acid differences between cytochromes P-450b and P-450e that occur beyond the region (Arg358) encoded by the cloned cDNA for cytochrome P-450e. Together, the amino acid sequences determined by micro sequence analysis and recombinant DNA techniques reveal 13 amino acid differences between these two isozymes. This report highlights the complementary nature of two different molecular approaches to elucidation of the amino acid sequences of isozymes with extensive structural homology. PMID:6572377

  4. Analyses of mitochondrial amino acid sequence datasets support the proposal that specimens of Hypodontus macropi from three species of macropodid hosts represent distinct species

    PubMed Central

    2013-01-01

    Background Hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies (macropodid marsupials). Based on previous multilocus enzyme electrophoresis (MEE) and nuclear ribosomal DNA sequence data sets, H. macropi has been proposed to be complex of species. To test this proposal using independent molecular data, we sequenced the whole mitochondrial (mt) genomes of individuals of H. macropi from three different species of hosts (Macropus robustus robustus, Thylogale billardierii and Macropus [Wallabia] bicolor) as well as that of Macropicola ocydromi (a related nematode), and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes. Results The mt genomes sequenced by next-generation (454) technology from H. macropi from the three host species varied from 13,634 bp to 13,699 bp in size. Pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of 5.8% to 18%. Phylogenetic analysis of the amino acid sequence data sets using Bayesian Inference (BI) showed that H. macropi from the three different host species formed distinct, well-supported clades. In addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of H. macropi in different macropodid hosts and geographical regions around Australia. Conclusions The present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that H. macropi from M. robustus robustus, M. bicolor and T. billardierii represent distinct species. PMID:24261823

  5. Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses

    NASA Technical Reports Server (NTRS)

    Zhao, H.; Yang, D.; Woese, C. R.; Bryant, M. P.

    1993-01-01

    After enrichment from Chinese rural anaerobic digestor sludge, anaerobic, sporing and nonsporing, saturated fatty acid-beta-oxidizing syntrophic bacteria were isolated as cocultures with H2- and formate-utilizing Methanospirillum hungatei or Desulfovibrio sp. strain G-11. The syntrophs degraded C4 to C8 saturated fatty acids, including isobutyrate and 2-methylbutyrate. They were adapted to grow on crotonate and were isolated as pure cultures. The crotonate-grown pure cultures alone did not grow on butyrate in either the presence or the absence of some common electron acceptors. However, when they were reconstituted with M. hungatei, growth on butyrate again occurred. In contrast, crotonate-grown Clostridium kluyveri and Clostridium sticklandii, as well as Clostridium sporogenes, failed to grow on butyrate when these organisms were cocultured with M. hungatei. The crotonate-grown pure subcultures of the syntrophs described above were subjected to 16S rRNA sequence analysis. Several previously documented fatty acid-beta-oxidizing syntrophs grown in pure cultures with crotonate were also subjected to comparative sequence analyses. The sequence analyses revealed that the new sporing and nonsporing isolates and other syntrophs that we sequenced, which had either gram-negative or gram-positive cell wall ultrastructure, all belonged to the phylogenetically gram-positive phylum. They were not closely related to any of the previously known subdivisions in the gram-positive phylum with which they were compared, but were closely related to each other, forming a new subdivision in the phylum. We recommend that this group be designated Syntrophomonadaceae fam. nov.; a description is given.

  6. Comparative sequence analyses of sixteen reptilian paramyxoviruses

    USGS Publications Warehouse

    Ahne, W.; Batts, W.N.; Kurath, G.; Winton, J.R.

    1999-01-01

    Viral genomic RNA of Fer-de-Lance virus (FDLV), a paramyxovirus highly pathogenic for reptiles, was reverse transcribed and cloned. Plasmids with significant sequence similarities to the hemagglutinin-neuraminidase (HN) and polymerase (L) genes of mammalian paramyxoviruses were identified by BLAST search. Partial sequences of the FDLV genes were used to design primers for amplification by nested polymerase chain reaction (PCR) and sequencing of 518-bp L gene and 352-bp HN gene fragments from a collection of 15 previously uncharacterized reptilian paramyxoviruses. Phylogenetic analyses of the partial L and HN sequences produced similar trees in which there were two distinct subgroups of isolates that were supported with maximum bootstrap values, and several intermediate isolates. Within each subgroup the nucleotide divergence values were less than 2.5%, while the divergence between the two subgroups was 20-22%. This indicated that the two subgroups represent distinct virus species containing multiple virus strains. The five intermediate isolates had nucleotide divergence values of 11-20% and may represent additional distinct species. In addition to establishing diversity among reptilian paramyxoviruses, the phylogenetic groupings showed some correlation with geographic location, and clearly demonstrated a low level of host species-specificity within these viruses. Copyright (C) 1999 Elsevier Science B.V.

  7. Composition for nucleic acid sequencing

    SciTech Connect

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2008-08-26

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  8. Analyses of Intestinal Microbiota: Culture versus Sequencing.

    PubMed

    Hiergeist, Andreas; Gläsner, Joachim; Reischl, Udo; Gessner, André

    2015-01-01

    Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed. PMID:26323632

  9. Amino acid analyses of Apollo 14 samples.

    NASA Technical Reports Server (NTRS)

    Gehrke, C. W.; Zumwalt, R. W.; Kuo, K.; Aue, W. A.; Stalling, D. L.; Kvenvolden, K. A.; Ponnamperuma, C.

    1972-01-01

    Detection limits were between 300 pg and 1 ng for different amino acids, in an analysis by gas-liquid chromatography of water extracts from Apollo 14 lunar fines in which amino acids were converted to their N-trifluoro-acetyl-n-butyl esters. Initial analyses of water and HCl extracts of sample 14240 and 14298 samples showed no amino acids above background levels.

  10. High speed nucleic acid sequencing

    SciTech Connect

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2011-05-17

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.

  11. Stem kernels for RNA sequence analyses.

    PubMed

    Sakakibara, Yasubumi; Popendorf, Kris; Ogawa, Nana; Asai, Kiyoshi; Sato, Kengo

    2007-10-01

    Several computational methods based on stochastic context-free grammars have been developed for modeling and analyzing functional RNA sequences. These grammatical methods have succeeded in modeling typical secondary structures of RNA, and are used for structural alignment of RNA sequences. However, such stochastic models cannot sufficiently discriminate member sequences of an RNA family from nonmembers and hence detect noncoding RNA regions from genome sequences. A novel kernel function, stem kernel, for the discrimination and detection of functional RNA sequences using support vector machines (SVMs) is proposed. The stem kernel is a natural extension of the string kernel, specifically the all-subsequences kernel, and is tailored to measure the similarity of two RNA sequences from the viewpoint of secondary structures. The stem kernel examines all possible common base pairs and stem structures of arbitrary lengths, including pseudoknots between two RNA sequences, and calculates the inner product of common stem structure counts. An efficient algorithm is developed to calculate the stem kernels based on dynamic programming. The stem kernels are then applied to discriminate members of an RNA family from nonmembers using SVMs. The study indicates that the discrimination ability of the stem kernel is strong compared with conventional methods. Furthermore, the potential application of the stem kernel is demonstrated by the detection of remotely homologous RNA families in terms of secondary structures. This is because the string kernel is proven to work for the remote homology detection of protein sequences. These experimental results have convinced us to apply the stem kernel in order to find novel RNA families from genome sequences. PMID:17933013

  12. A close relationship between Cercozoa and Foraminifera supported by phylogenetic analyses based on combined amino acid sequences of three cytoskeletal proteins (actin, alpha-tubulin, and beta-tubulin).

    PubMed

    Takishita, Kiyotaka; Inagaki, Yuji; Tsuchiya, Masashi; Sakaguchi, Miako; Maruyama, Tadashi

    2005-12-01

    Recently, there has been increasing molecular evidence of phylogenetic affinity between Cercozoa and Foraminifera in the eukaryotic lineage. We performed phylogenetic analyses based on the combined (concatenated) amino acid sequence data of actin, alpha-tubulin, and beta-tubulin from a wide variety of eukaryotes, including the foraminifers Planoglabratella opercularis and Reticulomyxa filosa, as well as cercomonad and chlorarachniophyte members of Cercozoa. A monophyletic lineage composed of two foraminiferan species branched with the centroheliozoan species Raphidiophrys contractilis was reconstructed in both Bayesian and maximum-likelihood (ML) analyses under 'linked' models, enforcing a single set of the parameters (the parameter for among-site rate variation and branch lengths) on the entire combined alignment. Considering the extremely divergent nature of Foraminifera and Raphidiophyrs tubulins, the union of these lineages recovered is most probably a long-branch attraction artifact due to ignoring gene-specific evolutionary processes. On the other hand, the foraminiferan lineage was within the radiation of Cercozoa in Bayesian analyses under 'unlinked' model conditions, accommodating differences in evolutionary processes across the three genes in the combined alignment. The Foraminifera+Cercozoa affinity recovered in the latter multi-gene analyses is most likely genuine, and thus our data presented here provide further support for the close relationship between these two protist lineages.

  13. Illumina sequencing-based analyses of bacterial communities during short-chain fatty-acid production from food waste and sewage sludge fermentation at different pH values.

    PubMed

    Cheng, Weixiao; Chen, Hong; Yan, ShuHai; Su, Jianqiang

    2014-09-01

    Short-chain fatty acids (SCFAs) can be produced by primary and waste activated sludge anaerobic fermentation. The yield and product spectrum distribution of SCFAs can be significantly affected by different initial pH values. However, most studies have focused on the physical and chemical aspects of SCFA production by waste activated sludge fermentation at different pH values. Information on the bacterial community structures during acidogenic fermentation is limited. In this study, comparisons of the bacterial communities during the co-substrate fermentation of food wastes and sewage sludge at different pH values were performed using the barcoded Illumina paired-end sequencing method. The results showed that different pH environments harbored a characteristic bacterial community, including sequences related to Lactobacillus, Prevotella, Mitsuokella, Treponema, Clostridium, and Ureibacillus. The most abundant bacterial operational taxonomic units in the different pH environments were those related to carbohydrate-degrading bacteria, which are associated with constituents of co-substrate fermentation. Further analyses showed that during organic matter fermentation, a core microbiota composed of Firmicutes, Proteobacteria, and Bacteroidetes existed. Comparison analyses revealed that the bacterial community during fermentation was significantly affected by the pH, and that the diverse product distribution was related to the shift in bacterial communities.

  14. Chip-based sequencing nucleic acids

    SciTech Connect

    Beer, Neil Reginald

    2014-08-26

    A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

  15. Distinguishing proteins from arbitrary amino acid sequences.

    PubMed

    Yau, Stephen S-T; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  16. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-06-06

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  17. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-05-30

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  18. Mouse BAC Ends Quality Assessment and Sequence Analyses

    PubMed Central

    Zhao, Shaying; Shatsman, Sofiya; Ayodeji, Bola; Geer, Keita; Tsegaye, Getahun; Krol, Margaret; Gebregeorgis, Elizabeth; Shvartsbeyn, Alla; Russell, Daniel; Overton, Larry; Jiang, Lingxia; Dimitrov, George; Tran, Kevin; Shetty, Jyoti; Malek, Joel A.; Feldblyum, Tamara; Nierman, William C.; Fraser, Claire M.

    2001-01-01

    A large-scale BAC end-sequencing project at The Institute for Genomic Research (TIGR) has generated one of the most extensive sets of sequence markers for the mouse genome to date. With a sequencing success rate of >80%, an average read length of 485 bp, and ABI3700 capillary sequencers, we have generated 449,234 nonredundant mouse BAC end sequences (mBESs) with 218 Mb total from 257,318 clones from libraries RPCI-23 and RPCI-24, representing 15× clone coverage, 7% sequence coverage, and a marker every 7 kb across the genome. A total of 191,916 BACs have sequences from both ends providing 12× genome coverage. The average Q20 length is 406 bp and 84% of the bases have phred quality scores ≥ 20. RPCI-24 mBESs have more Q20 bases and longer reads on average than RPCI-23 sequences. ABI3700 sequencers and the sample tracking system ensure that > 95% of mBESs are associated with the right clone identifiers. We have found that a significant fraction of mBESs contains L1 repeats and ∼48% of the clones have both ends with ≥ 100 bp contiguous unique Q20 bases. About 3% mBESs match ESTs and > 70% of matches were conserved between the mouse and the human or the rat. Approximately 0.1% mBESs contain STSs. About 0.2% mBESs match human finished sequences and > 70% of these sequences have EST hits. The analyses indicate that our high-quality mouse BAC end sequences will be a valuable resource to the community. PMID:11591651

  19. p53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture (pSILAC) and Next Generation Sequencing (NGS) Analyses*

    PubMed Central

    Hünten, Sabine; Kaller, Markus; Drepper, Friedel; Oeljeklaus, Silke; Bonfert, Thomas; Erhard, Florian; Dueck, Anne; Eichner, Norbert; Friedel, Caroline C.; Meister, Gunter; Zimmer, Ralf; Warscheid, Bettina; Hermeking, Heiko

    2015-01-01

    We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3′-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486–5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the

  20. p53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture (pSILAC) and Next Generation Sequencing (NGS) Analyses.

    PubMed

    Hünten, Sabine; Kaller, Markus; Drepper, Friedel; Oeljeklaus, Silke; Bonfert, Thomas; Erhard, Florian; Dueck, Anne; Eichner, Norbert; Friedel, Caroline C; Meister, Gunter; Zimmer, Ralf; Warscheid, Bettina; Hermeking, Heiko

    2015-10-01

    We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3'-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the

  1. Sequence and Structural Analyses for Functional Non-coding RNAs

    NASA Astrophysics Data System (ADS)

    Sakakibara, Yasubumi; Sato, Kengo

    Analysis and detection of functional RNAs are currently important topics in both molecular biology and bioinformatics research. Several computational methods based on stochastic context-free grammars (SCFGs) have been developed for modeling and analysing functional RNA sequences. These grammatical methods have succeeded in modeling typical secondary structures of RNAs and are used for structural alignments of RNA sequences. Such stochastic models, however, are not sufficient to discriminate member sequences of an RNA family from non-members, and hence to detect non-coding RNA regions from genome sequences. Recently, the support vector machine (SVM) and kernel function techniques have been actively studied and proposed as a solution to various problems in bioinformatics. SVMs are trained from positive and negative samples and have strong, accurate discrimination abilities, and hence are more appropriate for the discrimination tasks. A few kernel functions that extend the string kernel to measure the similarity of two RNA sequences from the viewpoint of secondary structures have been proposed. In this article, we give an overview of recent progress in SCFG-based methods for RNA sequence analysis and novel kernel functions tailored to measure the similarity of two RNA sequences and developed for use with support vector machines (SVM) in discriminating members of an RNA family from non-members.

  2. Sequencing and comparative analyses of the genomes of zoysiagrasses

    PubMed Central

    Tanaka, Hidenori; Hirakawa, Hideki; Kosugi, Shunichi; Nakayama, Shinobu; Ono, Akiko; Watanabe, Akiko; Hashiguchi, Masatsugu; Gondo, Takahiro; Ishigaki, Genki; Muguerza, Melody; Shimizu, Katsuya; Sawamura, Noriko; Inoue, Takayasu; Shigeki, Yuichi; Ohno, Naoki; Tabata, Satoshi; Akashi, Ryo; Sato, Shusei

    2016-01-01

    Zoysia is a warm-season turfgrass, which comprises 11 allotetraploid species (2n = 4x = 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession ‘Nagirizaki’ (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella ‘Wakaba’ and Z. pacifica ‘Zanpa’ were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica ‘Kyoto’, Z. japonica ‘Miyagi’ and Z. matrella ‘Chiba Fair Green’, were accumulated, and aligned against the reference genome of ‘Nagirizaki’ along with those from ‘Wakaba’ and ‘Zanpa’. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the ‘Zoysia Genome Database’ at http://zoysia.kazusa.or.jp. PMID:26975196

  3. Sequencing and comparative analyses of the genomes of zoysiagrasses.

    PubMed

    Tanaka, Hidenori; Hirakawa, Hideki; Kosugi, Shunichi; Nakayama, Shinobu; Ono, Akiko; Watanabe, Akiko; Hashiguchi, Masatsugu; Gondo, Takahiro; Ishigaki, Genki; Muguerza, Melody; Shimizu, Katsuya; Sawamura, Noriko; Inoue, Takayasu; Shigeki, Yuichi; Ohno, Naoki; Tabata, Satoshi; Akashi, Ryo; Sato, Shusei

    2016-04-01

    Zoysiais a warm-season turfgrass, which comprises 11 allotetraploid species (2n= 4x= 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession 'Nagirizaki' (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella 'Wakaba' and Z. pacifica 'Zanpa' were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica'Kyoto', Z. japonica'Miyagi' and Z. matrella'Chiba Fair Green', were accumulated, and aligned against the reference genome of 'Nagirizaki' along with those from 'Wakaba' and 'Zanpa'. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the 'Zoysia Genome Database' at http://zoysia.kazusa.or.jp.

  4. Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

    PubMed

    Fukushima, Nobuyuki; Ishii, Shoichi; Tsujiuchi, Toshifumi; Kagawa, Nao; Katoh, Kazutaka

    2015-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish. PMID:25732591

  5. Amino Acid Analyses of Acid Hydrolysates in Desert Varnish

    NASA Technical Reports Server (NTRS)

    Perry, Randall S.; Staley, James T.; Dworkin, Jason P.; Engel, Mike

    2001-01-01

    There has long been a debate as to whether rock varnish deposits are microbially mediated or are deposited by inorganic processes. Varnished rocks are found throughout the world primarily in arid and semi-arid regions. The varnish coats are typically up to 200 microns thick and are composed of clays and alternating layers enriched in manganese and iron oxides. The individual layers range in thickness from 1 micron to greater than 10 microns and may continue laterally for more than a 100 microns. Overlapping botryoidal structures are visible in thin section and scanning electron micrographs. The coatings also include small amounts of organic mater and detrital grains. Amino-acid hydrolysates offer a means of assessing the organic composition of rock varnish collected from the Sonoran Desert, near Phoenix, AZ. Chromatographic analyses of hydrolysates from powdered samples of rock varnish suggest that the interior of rock varnish is relatively enriched in amino acids and specifically in d-alanine and glutamic acid. Peptidoglycan (murein) is the main structural component of gram-positive bacterial cell walls. The d-enantiomer of alanine and glutamic acid are specific to peptidoglycan and are consequently an indicator for the presence of bacteria. D-alanine is also found in teichoic acid which is only found in gram-positive bacteria. Several researchers have cultured bacteria from the surface of rock varnish and most have been gram-positive, suggesting that gram-positive bacteria are intimately associated with varnish coatings and may play a role in the formation of varnish coatings.

  6. Bovine Parathyroid Hormone: Amino Acid Sequence

    PubMed Central

    Brewer, H. Bryan; Ronan, Rosemary

    1970-01-01

    Bovine parathyroid hormone has been isolated in homogeneous form, and its complete amino acid sequence determined. The bovine hormone is a single chain, 84 amino acids long. It contains amino-terminal alanine, and carboxyl-terminal glutamine. The bovine parathyroid hormone is approximately three times the length of the newly discovered hormone, thyrocalcitonin, whose action is reciprocal to parathyroid hormone. Images PMID:5275384

  7. Genetic analyses of GII.17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region.

    PubMed

    Matsushima, Y; Ishikawa, M; Shimizu, T; Komane, A; Kasuo, S; Shinohara, M; Nagasawa, K; Kimura, H; Ryo, A; Okabe, N; Haga, K; Doan, Y H; Katayama, K; Shimizu, H

    2015-01-01

    A novel GII.P17-GII.17 variant norovirus emerged as a major cause of norovirus outbreaks from December 2014 to March 2015 in Japan. Named Hu/GII/JP/2014/GII.P17-GII.17, this variant has a newly identified GII.P17 type RNA-dependent RNA polymerase, while the capsid sequence displays amino acid substitutions around histo-blood group antigen (HBGA) binding sites. Several variants caused by mutations in the capsid region have previously been observed in the GII.4 genotype. Monitoring the GII.17 variant's geographical spread and evolution is important.

  8. Whale song analyses using bioinformatics sequence analysis approaches

    NASA Astrophysics Data System (ADS)

    Chen, Yian A.; Almeida, Jonas S.; Chou, Lien-Siang

    2005-04-01

    Animal songs are frequently analyzed using discrete hierarchical units, such as units, themes and songs. Because animal songs and bio-sequences may be understood as analogous, bioinformatics analysis tools DNA/protein sequence alignment and alignment-free methods are proposed to quantify the theme similarities of the songs of false killer whales recorded off northeast Taiwan. The eighteen themes with discrete units that were identified in an earlier study [Y. A. Chen, masters thesis, University of Charleston, 2001] were compared quantitatively using several distance metrics. These metrics included the scores calculated using the Smith-Waterman algorithm with the repeated procedure; the standardized Euclidian distance and the angle metrics based on word frequencies. The theme classifications based on different metrics were summarized and compared in dendrograms using cluster analyses. The results agree with earlier classifications derived by human observation qualitatively. These methods further quantify the similarities among themes. These methods could be applied to the analyses of other animal songs on a larger scale. For instance, these techniques could be used to investigate song evolution and cultural transmission quantifying the dissimilarities of humpback whale songs across different seasons, years, populations, and geographic regions. [Work supported by SC Sea Grant, and Ilan County Government, Taiwan.

  9. Sequence and structural analyses of nuclear export signals in the NESdb database.

    PubMed

    Xu, Darui; Farmer, Alicia; Collett, Garen; Grishin, Nick V; Chook, Yuh Min

    2012-09-01

    We compiled >200 nuclear export signal (NES)-containing CRM1 cargoes in a database named NESdb. We analyzed the sequences and three-dimensional structures of natural, experimentally identified NESs and of false-positive NESs that were generated from the database in order to identify properties that might distinguish the two groups of sequences. Analyses of amino acid frequencies, sequence logos, and agreement with existing NES consensus sequences revealed strong preferences for the Φ1-X(3)-Φ2-X(2)-Φ3-X-Φ4 pattern and for negatively charged amino acids in the nonhydrophobic positions of experimentally identified NESs but not of false positives. Strong preferences against certain hydrophobic amino acids in the hydrophobic positions were also revealed. These findings led to a new and more precise NES consensus. More important, three-dimensional structures are now available for 68 NESs within 56 different cargo proteins. Analyses of these structures showed that experimentally identified NESs are more likely than the false positives to adopt α-helical conformations that transition to loops at their C-termini and more likely to be surface accessible within their protein domains or be present in disordered or unobserved parts of the structures. Such distinguishing features for real NESs might be useful in future NES prediction efforts. Finally, we also tested CRM1-binding of 40 NESs that were found in the 56 structures. We found that 16 of the NES peptides did not bind CRM1, hence illustrating how NESs are easily misidentified.

  10. Phenolic acid esterases, coding sequences and methods

    DOEpatents

    Blum, David L.; Kataeva, Irina; Li, Xin-Liang; Ljungdahl, Lars G.

    2002-01-01

    Described herein are four phenolic acid esterases, three of which correspond to domains of previously unknown function within bacterial xylanases, from XynY and XynZ of Clostridium thermocellum and from a xylanase of Ruminococcus. The fourth specifically exemplified xylanase is a protein encoded within the genome of Orpinomyces PC-2. The amino acids of these polypeptides and nucleotide sequences encoding them are provided. Recombinant host cells, expression vectors and methods for the recombinant production of phenolic acid esterases are also provided.

  11. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-07-21

    A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

  12. Method for identifying and quantifying nucleic acid sequence aberrations

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

  13. Optimization of short amino acid sequences classifier

    NASA Astrophysics Data System (ADS)

    Barcz, Aleksy; Szymański, Zbigniew

    This article describes processing methods used for short amino acid sequences classification. The data processed are 9-symbols string representations of amino acid sequences, divided into 49 data sets - each one containing samples labeled as reacting or not with given enzyme. The goal of the classification is to determine for a single enzyme, whether an amino acid sequence would react with it or not. Each data set is processed separately. Feature selection is performed to reduce the number of dimensions for each data set. The method used for feature selection consists of two phases. During the first phase, significant positions are selected using Classification and Regression Trees. Afterwards, symbols appearing at the selected positions are substituted with numeric values of amino acid properties taken from the AAindex database. In the second phase the new set of features is reduced using a correlation-based ranking formula and Gram-Schmidt orthogonalization. Finally, the preprocessed data is used for training LS-SVM classifiers. SPDE, an evolutionary algorithm, is used to obtain optimal hyperparameters for the LS-SVM classifier, such as error penalty parameter C and kernel-specific hyperparameters. A simple score penalty is used to adapt the SPDE algorithm to the task of selecting classifiers with best performance measures values.

  14. Methods for analyzing nucleic acid sequences

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2011-05-17

    The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.

  15. Development of an expert system for amino acid sequence identification.

    PubMed

    Hu, L; Saulinskas, E F; Johnson, P; Harrington, P B

    1996-08-01

    An expert system for amino acid sequence identification has been developed. The algorithm uses heuristic rules developed by human experts in protein sequencing. The system is applied to the chromatographic data of phenylthiohydantoin-amino acids acquired from an automated sequencer. The peak intensities in the current cycle are compared with those in the previous cycle, while the calibration and succeeding cycles are used as ancillary identification criteria when necessary. The retention time for each chromatographic peak in each cycle is corrected by the corresponding peak in the calibration cycle at the same run. The main improvement of our system compared with the onboard software used by the Applied Biosystems 477A Protein/Peptide Sequencer is that each peak in each cycle is assigned an identification name according to the corrected retention time to be used for the comparison with different cycles. The system was developed from analyses of ribonuclease A and evaluated by runs of four other protein samples that were not used in rule development. This paper demonstrates that rules developed by human experts can be automatically applied to sequence assignment. The expert system performed more accurately than the onboard software of the protein sequencer, in that the misidentification rates for the expert system were around 7%, whereas those for the onboard software were between 13 and 21%.

  16. Note on the chromatographic analyses of marine polyunsaturated fatty acids

    USGS Publications Warehouse

    Schultz, D.M.; Quinn, J.G.

    1977-01-01

    Gas-liquid chromatography was used to study the effects of saponification/methylation and thin-layer chromatographic isolation on the analyses of polyunsaturated fatty acids. Using selected procedures, the qualitative and quantitative distribution of these acids in marine organisms can be determined with a high degree of accuracy. ?? 1977 Springer-Verlag.

  17. Genomic sequencing and analyses of Lymantria xylina multiple nucleopolyhedrovirus

    PubMed Central

    2010-01-01

    Background Outbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylina larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed. Results The genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hrs) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV. Conclusion The gene parity plot analysis

  18. Sequence and structural analyses of nuclear export signals in the NESdb database

    PubMed Central

    Xu, Darui; Farmer, Alicia; Collett, Garen; Grishin, Nick V.; Chook, Yuh Min

    2012-01-01

    We compiled >200 nuclear export signal (NES)–containing CRM1 cargoes in a database named NESdb. We analyzed the sequences and three-dimensional structures of natural, experimentally identified NESs and of false-positive NESs that were generated from the database in order to identify properties that might distinguish the two groups of sequences. Analyses of amino acid frequencies, sequence logos, and agreement with existing NES consensus sequences revealed strong preferences for the Φ1-X3-Φ2-X2-Φ3-X-Φ4 pattern and for negatively charged amino acids in the nonhydrophobic positions of experimentally identified NESs but not of false positives. Strong preferences against certain hydrophobic amino acids in the hydrophobic positions were also revealed. These findings led to a new and more precise NES consensus. More important, three-dimensional structures are now available for 68 NESs within 56 different cargo proteins. Analyses of these structures showed that experimentally identified NESs are more likely than the false positives to adopt α-helical conformations that transition to loops at their C-termini and more likely to be surface accessible within their protein domains or be present in disordered or unobserved parts of the structures. Such distinguishing features for real NESs might be useful in future NES prediction efforts. Finally, we also tested CRM1-binding of 40 NESs that were found in the 56 structures. We found that 16 of the NES peptides did not bind CRM1, hence illustrating how NESs are easily misidentified. PMID:22833565

  19. 77 FR 65537 - Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-29

    ... Amino Acid Sequence Disclosures ACTION: Proposed collection; comment request. SUMMARY: The United States....'' SUPPLEMENTARY INFORMATION: I. Abstract Patent applications that contain nucleotide and/or amino acid...

  20. Detection of nucleic acid sequences by invader-directed cleavage

    DOEpatents

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  1. Analyses of Response-Stimulus Sequences in Descriptive Observations

    ERIC Educational Resources Information Center

    Samaha, Andrew L.; Vollmer, Timothy R.; Borrero, Carrie; Sloman, Kimberly; Pipkin, Claire St. Peter; Bourret, Jason

    2009-01-01

    Descriptive observations were conducted to record problem behavior displayed by participants and to record antecedents and consequences delivered by caregivers. Next, functional analyses were conducted to identify reinforcers for problem behavior. Then, using data from the descriptive observations, lag-sequential analyses were conducted to examine…

  2. [Pathological Diagnoses and Whole-genome Sequence Analyses of the Jaagsiekte Sheep Retrovirus in Xinjiang, China].

    PubMed

    Yang, Sufang; Liang, Tian; Zhao, Qingliang; Zhang, Dianqing; Si Junqiang; Zhang, Jing; Yang, Xia; Sheng, Jinliang

    2015-05-01

    To carry out pathologic diagnoses and whole-genome sequence analyses of the Jaagsiekte sheep retrovirus (JSRV) in Xinjiang, China, we first observed sheep suspected to have the JSRV. Then, the extracted virus suspension was observed by transmission electron microscopy (TEM). Total RNAs from lungs of JSRV-infected sheep were extracted and reverse-transcribed using a cDNA synthesis kit. Six pairs of primers were designed according to the exogenous reference virus strain (AF105220). Reverse transcription-polymerase chain reaction was carried out from JSRV-infected tissue, and the whole genome of the JSRV sequenced. Our results showed: flow of nasal fluid ("wheelbarrow test"); different sizes of adenoma lesions in the lungs; papillary hyperplasia of alveolar epithelial cells; alveolar cavity filled with macrophages; dissolute nuclei in central lesions. TEM revealed JSRV particles with a diameter of 88 nm to 125. 4 nm. The full-length of the viral genome sequence was 7456 bp. BLAST analyses showed nucleotide homology of 96% and 95% compared with that of the representative strain from the USA (AF105220) and UK (AF357971). Nucleotide homology was 89.8% and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus, Inner Mongolia strain (DQ838493) and USA strain (EF680300). The specific pathogenic amino-acid sequence "YXXM" was found in the TM district, similar to the exogenous JSRV: this gene has been reported to be oncogenic. This is the first report of the complete genomic sequence of the exogenous JSRV from Xinjiang, and could lay the foundation for study of the biological characteristics and pathogenic mechanisms of the pulmonary adenomatosis virus in sheep. PMID:26470525

  3. Nucleic acid renaturation and restriction endonuclease cleavage analyses show that the DNAs of a transforming and a nontransforming strain of Epstein-Barr virus share approximately 90% of their nucleotide sequences.

    PubMed Central

    Sugden, B; Summers, W C; Klein, G

    1976-01-01

    Viral DNA molecules were purified from a nontransforming and a transforming strain of Epstein-Barr virus. Each viral DNA was labeled in vitro and renatured in the presence of an excess of either one or the other unlabeled viral DNA. Both viral DNAs were also digested with the Eco R1 restriction endonuclease and subsequently labeled by using avian myeloblastosis virus DNA polymerase to repair either the EcoR1 nuclease-generated single-stranded ends of the DNAs or their single-stranded ends produced by a second digestion with exonuclease III after the first EcoR1 nuclease digestion. The results of these experiments support three general conclusions: (i) the DNAs of these two strains of Epstein-Barr virus share approximately 90% of their nucleotide sequences; (ii) both viral DNA populations are reasonably homogenous; and (iii) both DNAs contain repetitions or inverted repetitions of some of their nucleotide sequences. Images PMID:178907

  4. Hybridization and sequencing of nucleic acids using base pair mismatches

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    2001-01-01

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  5. Sequence and intramolecular distance scoring analyses of microbial rhodopsins

    PubMed Central

    Asano, Miki; Ide, Shunta; Kamata, Atsushi; Takahasi, Kiyohiro; Okada, Tetsuji

    2016-01-01

    Recent accumulation of sequence and structural data, in conjunction with systematical classification into a set of families, has significantly advanced our understanding of diverse and specific protein functions. Analysis and interpretation of protein family data requires comprehensive sequence and structural alignments. Here, we present a simple scheme for analyzing a set of experimental structures of a given protein or family of proteins, using microbial rhodopsins as an example. For a data set comprised of around a dozen highly similar structures to each other (overall pairwise root-mean-squared deviation < 2.3 Å), intramolecular distance scoring analysis yielded valuable information with respect to structural properties, such as differences in the relative variability of transmembrane helices. Furthermore, a comparison with recent results for G protein-coupled receptors demonstrates how the results of the present analysis can be interpreted and effectively utilized for structural characterization of diverse protein families in general. PMID:26998236

  6. Evidence for Balancing Selection from Nucleotide Sequence Analyses of Human G6PD

    PubMed Central

    Verrelli, Brian C.; McDonald, John H.; Argyropoulos, George; Destro-Bisol, Giovanni; Froment, Alain; Drousiotou, Anthi; Lefranc, Gerard; Helal, Ahmed N.; Loiselet, Jacques; Tishkoff, Sarah A.

    2002-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) mutations that result in reduced enzyme activity have been implicated in malarial resistance and constitute one of the best examples of selection in the human genome. In the present study, we characterize the nucleotide diversity across a 5.2-kb region of G6PD in a sample of 160 Africans and 56 non-Africans, to determine how selection has shaped patterns of DNA variation at this gene. Our global sample of enzymatically normal B alleles and A, A−, and Med alleles with reduced enzyme activities reveals many previously uncharacterized silent-site polymorphisms. In comparison with the absence of amino acid divergence between human and chimpanzee G6PD sequences, we find that the number of G6PD amino acid polymorphisms in human populations is significantly high. Unlike many other G6PD-activity alleles with reduced activity, we find that the age of the A variant, which is common in Africa, may not be consistent with the recent emergence of severe malaria and therefore may have originally had a historically different adaptive function. Overall, our observations strongly support previous genotype-phenotype association studies that proposed that balancing selection maintains G6PD deficiencies within human populations. The present study demonstrates that nucleotide sequence analyses can reveal signatures of both historical and recent selection in the genome and may elucidate the impact that infectious disease has had during human evolution. PMID:12378426

  7. Predicting intrinsic disorder from amino acid sequence.

    PubMed

    Obradovic, Zoran; Peng, Kang; Vucetic, Slobodan; Radivojac, Predrag; Brown, Celeste J; Dunker, A Keith

    2003-01-01

    Blind predictions of intrinsic order and disorder were made on 42 proteins subsequently revealed to contain 9,044 ordered residues, 284 disordered residues in 26 segments of length 30 residues or less, and 281 disordered residues in 2 disordered segments of length greater than 30 residues. The accuracies of the six predictors used in this experiment ranged from 77% to 91% for the ordered regions and from 56% to 78% for the disordered segments. The average of the order and disorder predictions ranged from 73% to 77%. The prediction of disorder in the shorter segments was poor, from 25% to 66% correct, while the prediction of disorder in the longer segments was better, from 75% to 95% correct. Four of the predictors were composed of ensembles of neural networks. This enabled them to deal more efficiently with the large asymmetry in the training data through diversified sampling from the significantly larger ordered set and achieve better accuracy on ordered and long disordered regions. The exclusive use of long disordered regions for predictor training likely contributed to the disparity of the predictions on long versus short disordered regions, while averaging the output values over 61-residue windows to eliminate short predictions of order or disorder probably contributed to the even greater disparity for three of the predictors. This experiment supports the predictability of intrinsic disorder from amino acid sequence. PMID:14579347

  8. Nucleotide Sequence Analyses and Predicted Coding of Bunyavirus Genome RNA Species

    PubMed Central

    Clerx-van Haaster, Corrie M.; Akashi, Hiroomi; Auperin, David D.; Bishop, David H. L.

    1982-01-01

    We performed 3′ RNA sequence analyses of [32P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3′-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3′-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3′-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3′-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the

  9. Phosphatidylinositol transfer proteins: sequence motifs in structural and evolutionary analyses

    PubMed Central

    Wyckoff, Gerald J.; Solidar, Ada; Yoden, Marilyn D.

    2016-01-01

    Phosphatidylinositol transfer proteins (PITP) are a family of monomeric proteins that bind and transfer phosphatidylinositol and phosphatidylcholine between membrane compartments. They are required for production of inositol and diacylglycerol second messengers, and are found in most metazoan organisms. While PITPs are known to carry out crucial cell-signaling roles in many organisms, the structure, function and evolution of the majority of family members remains unexplored; primarily because the ubiquity and diversity of the family thwarts traditional methods of global alignment. To surmount this obstacle, we instead took a novel approach, using MEME and a parsimony-based analysis to create a cladogram of conserved sequence motifs in 56 PITP family proteins from 26 species. In keeping with previous functional annotations, three clades were supported within our evolutionary analysis; two classes of soluble proteins and a class of membrane-associated proteins. By, focusing on conserved regions, the analysis allowed for in depth queries regarding possible functional roles of PITP proteins in both intra- and extra- cellular signaling. PMID:27429707

  10. Effects of fixed versus random condition sequencing during multielement functional analyses.

    PubMed

    Hammond, Jennifer L; Iwata, Brian A; Rooker, Griffin W; Fritz, Jennifer N; Bloom, Sarah E

    2013-01-01

    It has been suggested that a fixed condition sequence might facilitate differential responding during multielement functional analyses (FAs) by capitalizing on or limiting sequence effects (Iwata, Pace, et al., 1994); however, the effects of condition sequence have not been examined empirically. We conducted fixed- and random-sequence FAs for 7 individuals with developmental disabilities to determine the relative effects that sequence may have on assessment outcomes. Experimental conditions during the fixed sequence were conducted in the following order: ignore, attention, play, and demand; condition order during the random sequence was determined randomly. Results showed that sequence had no influence on the FA outcomes for 3 subjects, whereas differential responding emerged either faster (1 subject) or only (3 subjects) under the fixed sequence for the remaining subjects. These results suggest that the fixed sequence, a simple modification, should be used when conducting multielement FAs to accommodate the influence of establishing operations across assessment conditions.

  11. Comparison of the amino acid sequence of the major immunogen from three serotypes of foot and mouth disease virus.

    PubMed Central

    Makoff, A J; Paynter, C A; Rowlands, D J; Boothroyd, J C

    1982-01-01

    Cloned cDNA molecules from three serotypes of FMDV have been sequenced around the VP1-coding region. The predicted amino acid sequences for VP1 were compared with the published sequences and variable regions identified. The amino acid sequences were also analysed for hydrophilic regions. Two of the variable regions, numbered 129-160 and 193-204 overlapped hydrophilic regions, and were therefore identified as potentially immunogenic. These regions overlap regions shown by others to be immunogenic. PMID:6298715

  12. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2002-01-01

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  13. Methods and compositions for efficient nucleic acid sequencing

    DOEpatents

    Drmanac, Radoje

    2006-07-04

    Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

  14. Kit for detecting nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2001-01-01

    A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the

  15. Analysis and Annotation of Nucleic Acid Sequence

    SciTech Connect

    States, David J.

    2004-07-28

    The aims of this project were to develop improved methods for computational genome annotation and to apply these methods to improve the annotation of genomic sequence data with a specific focus on human genome sequencing. The project resulted in a substantial body of published work. Notable contributions of this project were the identification of basecalling and lane tracking as error processes in genome sequencing and contributions to improved methods for these steps in genome sequencing. This technology improved the accuracy and throughput of genome sequence analysis. Probabilistic methods for physical map construction were developed. Improved methods for sequence alignment, alternative splicing analysis, promoter identification and NF kappa B response gene prediction were also developed.

  16. Solid phase sequencing of double-stranded nucleic acids

    DOEpatents

    Fu, Dong-Jing; Cantor, Charles R.; Koster, Hubert; Smith, Cassandra L.

    2002-01-01

    This invention relates to methods for detecting and sequencing of target double-stranded nucleic acid sequences, to nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probe comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include nucleic acids in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated determination of molecular weights and identification of the target sequence.

  17. Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses.

    PubMed

    Ciarlet, M; Liprandi, F; Conner, M E; Estes, M K

    2000-01-01

    Previous sequence analyses of the rotavirus nonstructural NSP4 from human and some animal rotavirus strains revealed the presence of three distinct NSP4 alleles or genetic groups. To examine the species of origin relatedness and diversity of NSP4, the nucleotide and deduced amino acid sequences of the gene encoding the NSP4 from 15 animal rotavirus strains of porcine, equine, bovine, lapine and canine origin were determined and compared to human and other animal strains sequenced previously. Lapine and equine strains were shown to belong to the NSP4 genotype A. Murine NSP4 sequences formed a previously unrecognized fourth distinct NSP4 genotype (genotype D) that was more divergent compared to NSP4 genotype A, B, and C than the latter three are among each other. Within NSP4 genotypes, strains isolated from rabbits, horses, cows (genotype A) and pigs (genotype B) clustered according to species of origin, suggesting a conserved pattern of evolution within species. NSP4 sequence comparison among one wildtype and two tissue culture-adapted lapine strains, known to cause disease in neonatal rabbits, failed to identify amino acid changes within the variable region spanning amino acids 130 to 141, suggesting that disease in rabbits is the result of the lapine virus infection and replication, including production of the NSP4 enterotoxin. PMID:10752559

  18. Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses

    PubMed Central

    Lee, Joungmin; Jang, Yu-Sin; Han, Mee-Jung; Kim, Jin Young

    2016-01-01

    ABSTRACT Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. Due to limited knowledge on the genetic and metabolic characteristics of this strain, however, little progress has been made in metabolic engineering of this strain. Here we report the complete genome sequence of C. tyrobutyricum KCTC 5387 (ATCC 25755), which consists of a 3.07-Mbp chromosome and a 63-kbp plasmid. The results of genomic analyses suggested that C. tyrobutyricum produces butyrate from butyryl-coenzyme A (butyryl-CoA) through acetate reassimilation by CoA transferase, differently from Clostridium acetobutylicum, which uses the phosphotransbutyrylase-butyrate kinase pathway; this was validated by reverse transcription-PCR (RT-PCR) of related genes, protein expression levels, in vitro CoA transferase assay, and fed-batch fermentation. In addition, the changes in protein expression levels during the course of batch fermentations on glucose were examined by shotgun proteomics. Unlike C. acetobutylicum, the expression levels of proteins involved in glycolytic and fermentative pathways in C. tyrobutyricum did not decrease even at the stationary phase. Proteins related to energy conservation mechanisms, including Rnf complex, NfnAB, and pyruvate-phosphate dikinase that are absent in C. acetobutylicum, were identified. Such features explain why this organism can produce butyric acid to a much higher titer and better tolerate toxic metabolites. This study presenting the complete genome sequence, global protein expression profiles, and genome-based metabolic characteristics during the batch fermentation of C. tyrobutyricum will be valuable in designing strategies for metabolic engineering of this strain. PMID:27302759

  19. Molecular analyses of an acidic transthyretin Asn 90 variant.

    PubMed Central

    Saraiva, M J; Almeida, M R; Alves, I L; Moreira, P; Gawinowicz, M; Costa, P P; Rauh, S; Banhzoff, A; Altland, K

    1991-01-01

    A mutation in transthyretin (TTR Asn 90) has been identified in the Portuguese and German populations. This variant has a lower pI and was found by screening analyses in 2/4,000 German subjects and in 4/1,200 Portuguese by using either double one-dimensional (D1-D) electrophoresis with isoelectric focusing (IEF) or hybrid isoelectric focusing in immobilized pH gradient (HIEF) as the final separation step. The Portuguese population sample was from the area where TTR Met 30-associated familial amyloidotic polyneuropathy (FAP) prevails, and it was divided into (a) a group of 500 individuals belonging to FAP kindreds and (b) a group of 700 collected at random. HIEF showed two particular situations: (1) one case, from an FAP kindred, was simultaneously carrier of the Met 30 substitution and the acidic variant, and (2) one individual, from the randomly selected Portuguese sample, had only the acidic monomer. Comparative peptide mapping, by HPLC, of the acidic variant carriers and of normal TTR showed the presence of an abnormal tryptic peptide, not present in the normal TTR digests, with an asparagine-for-histidine substitution at position 90 explained by a single base change of adenine for cytosine in the histidine codon. This was confirmed at the DNA level by RFLP analyses of PCR-amplified material after digestion with SphI and BsmI. In all carriers of the Asn 90 substitution, no indicators were found for an association with traits characteristic for FAP. Images Figure 1 Figure 3 PMID:1850190

  20. Identification of food and beverage spoilage yeasts from DNA sequence analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection, identification, and classification of yeasts has undergone a major transformation in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of th...

  1. From Artificial Amino Acids to Sequence-Defined Targeted Oligoaminoamides.

    PubMed

    Morys, Stephan; Wagner, Ernst; Lächelt, Ulrich

    2016-01-01

    Artificial oligoamino acids with appropriate protecting groups can be used for the sequential assembly of oligoaminoamides on solid-phase. With the help of these oligoamino acids multifunctional nucleic acid (NA) carriers can be designed and produced in highly defined topologies. Here we describe the synthesis of the artificial oligoamino acid Fmoc-Stp(Boc3)-OH, the subsequent assembly into sequence-defined oligomers and the formulation of tumor-targeted plasmid DNA (pDNA) polyplexes. PMID:27436323

  2. Human retroviruses and AIDS 1996. A compilation and analysis of nucleic acid and amino acid sequences

    SciTech Connect

    Myers, G.; Foley, B.; Korber, B.; Mellors, J.W.; Jeang, K.T.; Wain-Hobson, S.

    1997-04-01

    This compendium and the accompanying floppy diskettes are the result of an effort to compile and rapidly publish all relevant molecular data concerning the human immunodeficiency viruses (HIV) and related retroviruses. The scope of the compendium and database is best summarized by the five parts that it comprises: (1) Nuclear Acid Alignments and Sequences; (2) Amino Acid Alignments; (3) Analysis; (4) Related Sequences; and (5) Database Communications. Information within all the parts is updated throughout the year on the Web site, http://hiv-web.lanl.gov. While this publication could take the form of a review or sequence monograph, it is not so conceived. Instead, the literature from which the database is derived has simply been summarized and some elementary computational analyses have been performed upon the data. Interpretation and commentary have been avoided insofar as possible so that the reader can form his or her own judgments concerning the complex information. In addition to the general descriptions of the parts of the compendium, the user should read the individual introductions for each part.

  3. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    SciTech Connect

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank

    2013-08-30

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  4. Segments of amino acid sequence similarity in beta-amylases.

    PubMed

    Friedberg, F; Rhodes, C

    1988-01-01

    In alpha-amylases from animals, plants and bacteria and in beta-amylases from plants and bacteria a number of segments exhibit amino acid sequence similarity specific to the alpha or to the beta type, respectively. In the case of the beta-amylases the similar sequence regions are extensive and they are disrupted only by short interspersed dissimilar regions. Close to the C terminus, however, no such sequence similarity exist. PMID:2464171

  5. Cloning, nucleotide sequence, and transcriptional analyses of the gene encoding a ferredoxin from Methanosarcina thermophila.

    PubMed Central

    Clements, A P; Ferry, J G

    1992-01-01

    A mixed 17-mer oligonucleotide deduced from the N terminus of a ferredoxin isolated from Methanosarcina thermophila was used to probe a lambda gt11 library prepared from M. thermophila genomic DNA; positive clones contained either a 5.7- or 2.1-kbp EcoRI insert. An open reading frame (fdxA) located within the 5.7-kbp insert had a deduced amino acid sequence that was identical to the first 26 N-terminal residues reported for the ferredoxin isolated from M. thermophila, with the exception of the initiator methionine. fdxA had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 2[4Fe-4S] ferredoxins. An open reading frame (ORF1) located within the 2.1-kbp EcoRI fragment also had the potential to encode a 2[4Fe-4S] bacterial-type ferredoxin (5,850 Da). fdxA and ORF1 were present as single copies in the genome, and each was transcribed on a monocistronic mRNA. While the fdxA- and ORF1-specific mRNAs were detected in cells grown on methanol and trimethylamine, only the fdxA-specific transcript was present in acetate-grown cells. The apparent transcriptional start sites of fdxA and ORF1, as determined by primer extension analyses, lay 21 to 28 bases downstream of sequences with high identity to the consensus methanogen promoter. Images PMID:1379583

  6. Comparative Sequence Analyses of La Crosse Virus Strain Isolated from Patient with Fatal Encephalitis, Tennessee, USA

    PubMed Central

    Fryxell, Rebecca Trout; Freyman, Kimberly; Ulloa, Armando; Velez, Jason O.; Paulsen, Dave; Lanciotti, Robert S.; Moncayo, Abelardo

    2015-01-01

    We characterized a La Crosse virus (LACV) isolate from the brain of a child who died of encephalitis-associated complications in eastern Tennessee, USA, during summer 2012. We compared the isolate with LACV sequences from mosquitoes collected near the child’s home just after his postmortem diagnosis. In addition, we conducted phylogenetic analyses of these and other sequences derived from LACV strains representing varied temporal, geographic, and ecologic origins. Consistent with historical findings, results of these analyses indicate that a limited range of LACV lineage I genotypes is associated with severe clinical outcomes. PMID:25898269

  7. Overview of BWR Severe Accident Sequence Analyses at Oak Ridge National Laboratory

    SciTech Connect

    Hodge, S.A.

    1983-01-01

    Since its inception in October 1980, the Severe Accident Sequence Analysis (SASA) program at Oak Ridge National Laboratory (ORNL) has completed four studies including Station Blackout, Scram Discharge Volume Break, Loss of Decay Heat Removal, and Loss of Injection accident sequences for the Browns Ferry Nuclear Plant. The accident analyses incorporated in a SASA study provide much greater detail than that practically achievable in a Probabilistic Risk Assessment (PRA). When applied to the candidate dominant accident sequences identified by a PRA, the detailed SASA results determine if factors neglected by the PRA would have a significant effect on the order of dominant sequences. Ongoing SASA work at ORNL involves the analysis of Anticipated Transients Without Scram (ATWS) sequences for Browns Ferry.

  8. The value of short amino acid sequence matches for prediction of protein allergenicity.

    PubMed

    Silvanovich, Andre; Nemeth, Margaret A; Song, Ping; Herman, Rod; Tagliani, Laura; Bannon, Gary A

    2006-03-01

    Typically, genetically engineered crops contain traits encoded by one or a few newly expressed proteins. The allergenicity assessment of newly expressed proteins is an important component in the safety evaluation of genetically engineered plants. One aspect of this assessment involves sequence searches that compare the amino acid sequence of the protein to all known allergens. Analyses are performed to determine the potential for immunologically based cross-reactivity where IgE directed against a known allergen could bind to the protein and elicit a clinical reaction in sensitized individuals. Bioinformatic searches are designed to detect global sequence similarity and short contiguous amino acid sequence identity. It has been suggested that potential allergen cross-reactivity may be predicted by identifying matches as short as six to eight contiguous amino acids between the protein of interest and a known allergen. A series of analyses were performed, and match probabilities were calculated for different size peptides to determine if there was a scientifically justified search window size that identified allergen sequence characteristics. Four probability modeling methods were tested: (1) a mock protein and a mock allergen database, (2) a mock protein and genuine allergen database, (3) a genuine allergen and genuine protein database, and (4) a genuine allergen and genuine protein database combined with a correction for repeating peptides. These analyses indicated that searches for short amino acid sequence matches of eight amino acids or fewer to identify proteins as potential cross-reactive allergens is a product of chance and adds little value to allergy assessments for newly expressed proteins.

  9. Amino acid sequences of proteins from Leptospira serovar pomona.

    PubMed

    Alves, S F; Lefebvre, R B; Probert, W

    2000-01-01

    This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

  10. The amino acid sequence of Staphylococcus aureus penicillinase.

    PubMed Central

    Ambler, R P

    1975-01-01

    The amino acid sequence of the penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Staphylococcus aureus strain PC1 was determined. The protein consists of a single polypeptide chain of 257 residues, and the sequence was determined by characterization of tryptic, chymotryptic, peptic and CNBr peptides, with some additional evidence from thermolysin and S. aureus proteinase peptides. A mistake in the preliminary report of the sequence is corrected; residues 113-116 are now thought to be -Lys-Lys-Val-Lys- rather than -Lys-Val-Lys-Lys-. Detailed evidence for the amino acid sequence has been deposited as Supplementary Publication SUP 50056 (91 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5. PMID:1218078

  11. Amino acid analyses of R and CK chondrites

    NASA Astrophysics Data System (ADS)

    Burton, Aaron S.; McLain, Hannah; Glavin, Daniel P.; Elsila, Jamie E.; Davidson, Jemma; Miller, Kelly E.; Andronikov, Alexander V.; Lauretta, Dante; Dworkin, Jason P.

    2015-03-01

    Exogenous delivery of amino acids and other organic molecules to planetary surfaces may have played an important role in the origins of life on Earth and other solar system bodies. Previous studies have revealed the presence of indigenous amino acids in a wide range of carbon-rich meteorites, with the abundances and structural distributions differing significantly depending on parent body mineralogy and alteration conditions. Here we report on the amino acid abundances of seven type 3-6 CK chondrites and two Rumuruti (R) chondrites. Amino acid measurements were made on hot water extracts from these meteorites by ultrahigh-performance liquid chromatography with fluorescence detection and time-of-flight mass spectrometry. Of the nine meteorites analyzed, four were depleted in amino acids, and one had experienced significant amino acid contamination by terrestrial biology. The remaining four, comprised of two R and two CK chondrites, contained low levels of amino acids that were predominantly the straight chain, amino-terminal (n-ω-amino) acids β-alanine, and γ-amino-n-butyric acid. This amino acid distribution is similar to what we reported previously for thermally altered ureilites and CV and CO chondrites, and these n-ω-amino acids appear to be indigenous to the meteorites and not the result of terrestrial contamination. The amino acids may have been formed by Fischer-Tropsch-type reactions, although this hypothesis needs further testing.

  12. Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses.

    PubMed

    Cheng, Tian-Fan; Jia, Xiao-Ming; Ma, Xiao-Hang; Lin, Hai-Ping; Zhao, Yu-Hua

    2004-01-01

    In this study, 18S rDNA and ITS-5.8S rDNA regions of four Shiraia bambusicola isolates collected from different species of bamboos were amplified by PCR with universal primer pairs NS1/NS8 and ITS5/ITS4, respectively, and sequenced. Phylogenetic analyses were conducted on three selected datasets of rDNA sequences. Maximum parsimony, distance and maximum likelihood criteria were used to infer trees. Morphological characteristics were also observed. The positioning of Shiraia in the order Pleosporales was well supported by bootstrap, which agreed with the placement by Amano (1980) according to their morphology. We did not find significant inter-hostal differences among these four isolates from different species of bamboos. From the results of analyses and comparison of their rDNA sequences, we conclude that Shiraia should be classified into Pleosporales as Amano (1980) proposed and suggest that it might be positioned in the family Phaeosphaeriaceae.

  13. Cloud-based bioinformatics workflow platform for large-scale next-generation sequencing analyses

    PubMed Central

    Liu, Bo; Madduri, Ravi K; Sotomayor, Borja; Chard, Kyle; Lacinski, Lukasz; Dave, Utpal J; Li, Jianqiang; Liu, Chunchen; Foster, Ian T

    2014-01-01

    Due to the upcoming data deluge of genome data, the need for storing and processing large-scale genome data, easy access to biomedical analyses tools, efficient data sharing and retrieval has presented significant challenges. The variability in data volume results in variable computing and storage requirements, therefore biomedical researchers are pursuing more reliable, dynamic and convenient methods for conducting sequencing analyses. This paper proposes a Cloud-based bioinformatics workflow platform for large-scale next-generation sequencing analyses, which enables reliable and highly scalable execution of sequencing analyses workflows in a fully automated manner. Our platform extends the existing Galaxy workflow system by adding data management capabilities for transferring large quantities of data efficiently and reliably (via Globus Transfer), domain-specific analyses tools preconfigured for immediate use by researchers (via user-specific tools integration), automatic deployment on Cloud for on-demand resource allocation and pay-as-you-go pricing (via Globus Provision), a Cloud provisioning tool for auto-scaling (via HTCondor scheduler), and the support for validating the correctness of workflows (via semantic verification tools). Two bioinformatics workflow use cases as well as performance evaluation are presented to validate the feasibility of the proposed approach. PMID:24462600

  14. Cloud-based bioinformatics workflow platform for large-scale next-generation sequencing analyses.

    PubMed

    Liu, Bo; Madduri, Ravi K; Sotomayor, Borja; Chard, Kyle; Lacinski, Lukasz; Dave, Utpal J; Li, Jianqiang; Liu, Chunchen; Foster, Ian T

    2014-06-01

    Due to the upcoming data deluge of genome data, the need for storing and processing large-scale genome data, easy access to biomedical analyses tools, efficient data sharing and retrieval has presented significant challenges. The variability in data volume results in variable computing and storage requirements, therefore biomedical researchers are pursuing more reliable, dynamic and convenient methods for conducting sequencing analyses. This paper proposes a Cloud-based bioinformatics workflow platform for large-scale next-generation sequencing analyses, which enables reliable and highly scalable execution of sequencing analyses workflows in a fully automated manner. Our platform extends the existing Galaxy workflow system by adding data management capabilities for transferring large quantities of data efficiently and reliably (via Globus Transfer), domain-specific analyses tools preconfigured for immediate use by researchers (via user-specific tools integration), automatic deployment on Cloud for on-demand resource allocation and pay-as-you-go pricing (via Globus Provision), a Cloud provisioning tool for auto-scaling (via HTCondor scheduler), and the support for validating the correctness of workflows (via semantic verification tools). Two bioinformatics workflow use cases as well as performance evaluation are presented to validate the feasibility of the proposed approach.

  15. The amino-acid sequence of kangaroo pancreatic ribonuclease.

    PubMed

    Gaastra, W; Welling, G W; Beintema, J J

    1978-05-01

    Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.

  16. Prebiotically plausible mechanisms increase compositional diversity of nucleic acid sequences

    PubMed Central

    Derr, Julien; Manapat, Michael L.; Rajamani, Sudha; Leu, Kevin; Xulvi-Brunet, Ramon; Joseph, Isaac; Nowak, Martin A.; Chen, Irene A.

    2012-01-01

    During the origin of life, the biological information of nucleic acid polymers must have increased to encode functional molecules (the RNA world). Ribozymes tend to be compositionally unbiased, as is the vast majority of possible sequence space. However, ribonucleotides vary greatly in synthetic yield, reactivity and degradation rate, and their non-enzymatic polymerization results in compositionally biased sequences. While natural selection could lead to complex sequences, molecules with some activity are required to begin this process. Was the emergence of compositionally diverse sequences a matter of chance, or could prebiotically plausible reactions counter chemical biases to increase the probability of finding a ribozyme? Our in silico simulations using a two-letter alphabet show that template-directed ligation and high concatenation rates counter compositional bias and shift the pool toward longer sequences, permitting greater exploration of sequence space and stable folding. We verified experimentally that unbiased DNA sequences are more efficient templates for ligation, thus increasing the compositional diversity of the pool. Our work suggests that prebiotically plausible chemical mechanisms of nucleic acid polymerization and ligation could predispose toward a diverse pool of longer, potentially structured molecules. Such mechanisms could have set the stage for the appearance of functional activity very early in the emergence of life. PMID:22319215

  17. The amino-acid sequence of kangaroo pancreatic ribonuclease.

    PubMed

    Gaastra, W; Welling, G W; Beintema, J J

    1978-05-01

    Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found. PMID:658039

  18. Comparative analyses of multi-species sequences from targeted genomic regions.

    PubMed

    Thomas, J W; Touchman, J W; Blakesley, R W; Bouffard, G G; Beckstrom-Sternberg, S M; Margulies, E H; Blanchette, M; Siepel, A C; Thomas, P J; McDowell, J C; Maskeri, B; Hansen, N F; Schwartz, M S; Weber, R J; Kent, W J; Karolchik, D; Bruen, T C; Bevan, R; Cutler, D J; Schwartz, S; Elnitski, L; Idol, J R; Prasad, A B; Lee-Lin, S-Q; Maduro, V V B; Summers, T J; Portnoy, M E; Dietrich, N L; Akhter, N; Ayele, K; Benjamin, B; Cariaga, K; Brinkley, C P; Brooks, S Y; Granite, S; Guan, X; Gupta, J; Haghighi, P; Ho, S-L; Huang, M C; Karlins, E; Laric, P L; Legaspi, R; Lim, M J; Maduro, Q L; Masiello, C A; Mastrian, S D; McCloskey, J C; Pearson, R; Stantripop, S; Tiongson, E E; Tran, J T; Tsurgeon, C; Vogt, J L; Walker, M A; Wetherby, K D; Wiggins, L S; Young, A C; Zhang, L-H; Osoegawa, K; Zhu, B; Zhao, B; Shu, C L; De Jong, P J; Lawrence, C E; Smit, A F; Chakravarti, A; Haussler, D; Green, P; Miller, W; Green, E D

    2003-08-14

    The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

  19. A weighted U-statistic for genetic association analyses of sequencing data.

    PubMed

    Wei, Changshuai; Li, Ming; He, Zihuai; Vsevolozhskaya, Olga; Schaid, Daniel J; Lu, Qing

    2014-12-01

    With advancements in next-generation sequencing technology, a massive amount of sequencing data is generated, which offers a great opportunity to comprehensively investigate the role of rare variants in the genetic etiology of complex diseases. Nevertheless, the high-dimensional sequencing data poses a great challenge for statistical analysis. The association analyses based on traditional statistical methods suffer substantial power loss because of the low frequency of genetic variants and the extremely high dimensionality of the data. We developed a Weighted U Sequencing test, referred to as WU-SEQ, for the high-dimensional association analysis of sequencing data. Based on a nonparametric U-statistic, WU-SEQ makes no assumption of the underlying disease model and phenotype distribution, and can be applied to a variety of phenotypes. Through simulation studies and an empirical study, we showed that WU-SEQ outperformed a commonly used sequence kernel association test (SKAT) method when the underlying assumptions were violated (e.g., the phenotype followed a heavy-tailed distribution). Even when the assumptions were satisfied, WU-SEQ still attained comparable performance to SKAT. Finally, we applied WU-SEQ to sequencing data from the Dallas Heart Study (DHS), and detected an association between ANGPTL 4 and very low density lipoprotein cholesterol.

  20. In silico comparative analysis of DNA and amino acid sequences for prion protein gene.

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences.

  1. Identification of a Herbal Powder by Deoxyribonucleic Acid Barcoding and Structural Analyses

    PubMed Central

    Sheth, Bhavisha P.; Thaker, Vrinda S.

    2015-01-01

    Background: Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. Objective: To identify a herbal powder obtained from a herbalist in the local vicinity of Rajkot, Gujarat, using deoxyribonucleic acid (DNA) barcoding and molecular tools. Materials and Methods: The DNA was extracted from a herbal powder and selected Cassia species, followed by the polymerase chain reaction (PCR) and sequencing of the rbcL barcode locus. Thereafter the sequences were subjected to National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis, followed by the protein three-dimension structure determination of the rbcL protein from the herbal powder and Cassia species namely Cassia fistula, Cassia tora and Cassia javanica (sequences obtained in the present study), Cassia Roxburghii, and Cassia abbreviata (sequences retrieved from Genbank). Further, the multiple and pairwise structural alignment were carried out in order to identify the herbal powder. Results: The nucleotide sequences obtained from the selected species of Cassia were submitted to Genbank (Accession No. JX141397, JX141405, JX141420). The NCBI BLAST analysis of the rbcL protein from the herbal powder showed an equal sequence similarity (with reference to different parameters like E value, maximum identity, total score, query coverage) to C. javanica and C. roxburghii. In order to solve the ambiguities of the BLAST result, a protein structural approach was implemented. The protein homology models obtained in the present study were submitted to the protein model database (PM0079748-PM0079753). The pairwise structural alignment of the herbal powder (as template) and C. javanica and C. roxburghii (as targets individually) revealed a close similarity of the herbal powder with C. javanica. Conclusion: A strategy as used here, incorporating the integrated use of DNA

  2. The genome of RNA tumor viruses contains polyadenylic acid sequences.

    PubMed

    Green, M; Cartas, M

    1972-04-01

    The 70S genome of two RNA tumor viruses, murine sarcoma virus and avian myeloblastosis virus, binds to Millipore filters in buffer with high salt concentration and to glass fiber filters containing poly(U). These observations suggest that 70S RNA contains adenylic acid-rich sequences. When digested by pancreatic RNase, 70S RNA of murine sarcoma virus yielded poly(A) sequences that contain 91% adenylic acid. These poly(A) sequences sedimented as a relatively homogenous peak in sucrose gradients with a sedimentation coefficient of 4-5 S, but had a mobility during polyacrylamide gel electrophoresis that corresponds to molecules that sediment at 6-7 S. If we estimate a molecular weight for each sequence of 30,000-60,000 (100-200 nucleotides) and a molecular weight for viral 70S RNA of 3-12 million, each viral genome could contain 1-8 poly(A) sequences. Possible functions of poly(A) in the infecting viral RNA may include a role in the initiation of viral DNA or RNA synthesis, in protein maturation, or in the assembly of the viral genome.

  3. Sequences Of Amino Acids For Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.

    1992-01-01

    Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

  4. Nucleic acid sequence design via efficient ensemble defect optimization.

    PubMed

    Zadeh, Joseph N; Wolfe, Brian R; Pierce, Niles A

    2011-02-01

    We describe an algorithm for designing the sequence of one or more interacting nucleic acid strands intended to adopt a target secondary structure at equilibrium. Sequence design is formulated as an optimization problem with the goal of reducing the ensemble defect below a user-specified stop condition. For a candidate sequence and a given target secondary structure, the ensemble defect is the average number of incorrectly paired nucleotides at equilibrium evaluated over the ensemble of unpseudoknotted secondary structures. To reduce the computational cost of accepting or rejecting mutations to a random initial sequence, candidate mutations are evaluated on the leaf nodes of a tree-decomposition of the target structure. During leaf optimization, defect-weighted mutation sampling is used to select each candidate mutation position with probability proportional to its contribution to the ensemble defect of the leaf. As subsequences are merged moving up the tree, emergent structural defects resulting from crosstalk between sibling sequences are eliminated via reoptimization within the defective subtree starting from new random subsequences. Using a Θ(N(3) ) dynamic program to evaluate the ensemble defect of a target structure with N nucleotides, this hierarchical approach implies an asymptotic optimality bound on design time: for sufficiently large N, the cost of sequence design is bounded below by 4/3 the cost of a single evaluation of the ensemble defect for the full sequence. Hence, the design algorithm has time complexity Ω(N(3) ). For target structures containing N ∈{100,200,400,800,1600,3200} nucleotides and duplex stems ranging from 1 to 30 base pairs, RNA sequence designs at 37°C typically succeed in satisfying a stop condition with ensemble defect less than N/100. Empirically, the sequence design algorithm exhibits asymptotic optimality and the exponent in the time complexity bound is sharp.

  5. Genome sequence analyses show that Neisseria oralis is the same species as 'Neisseria mucosa var. heidelbergensis'.

    PubMed

    Bennett, Julia S; Jolley, Keith A; Maiden, Martin C J

    2013-10-01

    Phylogenies generated from whole genome sequence (WGS) data provide definitive means of bacterial isolate characterization for typing and taxonomy. The species status of strains recently defined with conventional taxonomic approaches as representing Neisseria oralis was examined by the analysis of sequences derived from WGS data, specifically: (i) 53 Neisseria ribosomal protein subunit (rps) genes (ribosomal multi-locus sequence typing, rMLST); and (ii) 246 Neisseria core genes (core genome MLST, cgMLST). These data were compared with phylogenies derived from 16S and 23S rRNA gene sequences, demonstrating that the N. oralis strains were monophyletic with strains described previously as representing 'Neisseria mucosa var. heidelbergensis' and that this group was of equivalent taxonomic status to other well-described species of the genus Neisseria. Phylogenetic analyses also indicated that Neisseria sicca and Neisseria macacae should be considered the same species as Neisseria mucosa and that Neisseria flavescens should be considered the same species as Neisseria subflava. Analyses using rMLST showed that some strains currently defined as belonging to the genus Neisseria were more closely related to species belonging to other genera within the family; however, whole genome analysis of a more comprehensive selection of strains from within the family Neisseriaceae would be necessary to confirm this. We suggest that strains previously identified as representing 'N. mucosa var. heidelbergensis' and deposited in culture collections should be renamed N. oralis. Finally, one of the strains of N. oralis was able to ferment lactose, due to the presence of β-galactosidase and lactose permease genes, a characteristic previously thought to be unique to Neisseria lactamica, which therefore cannot be thought of as diagnostic for this species; however, the rMLST and cgMLST analyses confirm that N. oralis is most closely related to N. mucosa.

  6. On combining protein sequences and nucleic acid sequences in phylogenetic analysis: the homeobox protein case.

    PubMed

    Agosti, D; Jacobs, D; DeSalle, R

    1996-01-01

    Amino acid encoding genes contain character state information that may be useful for phylogenetic analysis on at least two levels. The nucleotide sequence and the translated amino acid sequences have both been employed separately as character states for cladistic studies of various taxa, including studies of the genealogy of genes in multigene families. In essence, amino acid sequences and nucleic acid sequences are two different ways of character coding the information in a gene. Silent positions in the nucleotide sequence (first or third positions in codons that can accrue change without changing the identity of the amino acid that the triplet codes for) may accrue change relatively rapidly and become saturated, losing the pattern of historical divergence. On the other hand, non-silent nucleotide alterations and their accompanying amino acid changes may evolve too slowly to reveal relationships among closely related taxa. In general, the dynamics of sequence change in silent and non-silent positions in protein coding genes result in homoplasy and lack of resolution, respectively. We suggest that the combination of nucleic acid and the translated amino acid coded character states into the same data matrix for phylogenetic analysis addresses some of the problems caused by the rapid change of silent nucleotide positions and overall slow rate of change of non-silent nucleotide positions and slowly changing amino acid positions. One major theoretical problem with this approach is the apparent non-independence of the two sources of characters. However, there are at least three possible outcomes when comparing protein coding nucleic acid sequences with their translated amino acids in a phylogenetic context on a codon by codon basis. First, the two character sets for a codon may be entirely congruent with respect to the information they convey about the relationships of a certain set of taxa. Second, one character set may display no information concerning a phylogenetic

  7. Nanopores and nucleic acids: prospects for ultrarapid sequencing

    NASA Technical Reports Server (NTRS)

    Deamer, D. W.; Akeson, M.

    2000-01-01

    DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.

  8. Phylogenetic Analyses of Novel Squamate Adenovirus Sequences in Wild-Caught Anolis Lizards

    PubMed Central

    Ascher, Jill M.; Geneva, Anthony J.; Ng, Julienne; Wyatt, Jeffrey D.; Glor, Richard E.

    2013-01-01

    Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity. PMID:23593364

  9. The amino acid sequence of Escherichia coli cyanase.

    PubMed

    Chin, C C; Anderson, P M; Wold, F

    1983-01-10

    The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

  10. Comparative sequence and genetic analyses of asparagus BACs reveal no microsynteny with onion or rice.

    PubMed

    Jakse, Jernej; Telgmann, Alexa; Jung, Christian; Khar, Anil; Melgar, Sergio; Cheung, Foo; Town, Christopher D; Havey, Michael J

    2006-12-01

    The Poales (includes the grasses) and Asparagales [includes onion (Allium cepa L.) and asparagus (Asparagus officinalis L.)] are the two most economically important monocot orders. The Poales are a member of the commelinoid monocots, a group of orders sister to the Asparagales. Comparative genomic analyses have revealed a high degree of synteny among the grasses; however, it is not known if this synteny extends to other major monocot groups such as the Asparagales. Although we previously reported no evidence for synteny at the recombinational level between onion and rice, microsynteny may exist across shorter genomic regions in the grasses and Asparagales. We sequenced nine asparagus BACs to reveal physically linked genic-like sequences and determined their most similar positions in the onion and rice genomes. Four of the asparagus BACs were selected using molecular markers tightly linked to the sex-determining M locus on chromosome 5 of asparagus. These BACs possessed only two putative coding regions and had long tracts of degenerated retroviral elements and transposons. Five asparagus BACs were selected after hybridization of three onion cDNAs that mapped to three different onion chromosomes. Genic-like sequences that were physically linked on the cDNA-selected BACs or genetically linked on the M-linked BACs showed significant similarities (e < -20) to expressed sequences on different rice chromosomes, revealing no evidence for microsynteny between asparagus and rice across these regions. Genic-like sequences that were linked in asparagus were used to identify highly similar (e < -20) expressed sequence tags (ESTs) of onion. These onion ESTs mapped to different onion chromosomes and no relationship was observed between physical or genetic linkages in asparagus and genetic linkages in onion. These results further indicate that synteny among grass genomes does not extend to a sister order in the monocots and that asparagus may not be an appropriate smaller genome

  11. Comparative sequence and genetic analyses of asparagus BACs reveal no microsynteny with onion or rice.

    PubMed

    Jakse, Jernej; Telgmann, Alexa; Jung, Christian; Khar, Anil; Melgar, Sergio; Cheung, Foo; Town, Christopher D; Havey, Michael J

    2006-12-01

    The Poales (includes the grasses) and Asparagales [includes onion (Allium cepa L.) and asparagus (Asparagus officinalis L.)] are the two most economically important monocot orders. The Poales are a member of the commelinoid monocots, a group of orders sister to the Asparagales. Comparative genomic analyses have revealed a high degree of synteny among the grasses; however, it is not known if this synteny extends to other major monocot groups such as the Asparagales. Although we previously reported no evidence for synteny at the recombinational level between onion and rice, microsynteny may exist across shorter genomic regions in the grasses and Asparagales. We sequenced nine asparagus BACs to reveal physically linked genic-like sequences and determined their most similar positions in the onion and rice genomes. Four of the asparagus BACs were selected using molecular markers tightly linked to the sex-determining M locus on chromosome 5 of asparagus. These BACs possessed only two putative coding regions and had long tracts of degenerated retroviral elements and transposons. Five asparagus BACs were selected after hybridization of three onion cDNAs that mapped to three different onion chromosomes. Genic-like sequences that were physically linked on the cDNA-selected BACs or genetically linked on the M-linked BACs showed significant similarities (e < -20) to expressed sequences on different rice chromosomes, revealing no evidence for microsynteny between asparagus and rice across these regions. Genic-like sequences that were linked in asparagus were used to identify highly similar (e < -20) expressed sequence tags (ESTs) of onion. These onion ESTs mapped to different onion chromosomes and no relationship was observed between physical or genetic linkages in asparagus and genetic linkages in onion. These results further indicate that synteny among grass genomes does not extend to a sister order in the monocots and that asparagus may not be an appropriate smaller genome

  12. Interpreting the biological relevance of bioinformatic analyses with T-DNA sequence for protein allergenicity.

    PubMed

    Harper, B; McClain, S; Ganko, E W

    2012-08-01

    Global regulatory agencies require bioinformatic sequence analysis as part of their safety evaluation for transgenic crops. Analysis typically focuses on encoded proteins and adjacent endogenous flanking sequences. Recently, regulatory expectations have expanded to include all reading frames of the inserted DNA. The intent is to provide biologically relevant results that can be used in the overall assessment of safety. This paper evaluates the relevance of assessing the allergenic potential of all DNA reading frames found in common food genes using methods considered for the analysis of T-DNA sequences used in transgenic crops. FASTA and BLASTX algorithms were used to compare genes from maize, rice, soybean, cucumber, melon, watermelon, and tomato using international regulatory guidance. Results show that BLASTX for maize yielded 7254 alignments that exceeded allergen similarity thresholds and 210,772 alignments that matched eight or more consecutive amino acids with an allergen; other crops produced similar results. This analysis suggests that each nontransgenic crop has a much greater potential for allergenic risk than what has been observed clinically. We demonstrate that a meaningful safety assessment is unlikely to be provided by using methods with inherently high frequencies of false positive alignments when broadly applied to all reading frames of DNA sequence.

  13. PeroxiBase: a powerful tool to collect and analyse peroxidase sequences from Viridiplantae.

    PubMed

    Oliva, Michele; Theiler, Grégory; Zamocky, Marcel; Koua, Dominique; Margis-Pinheiro, Marcia; Passardi, Filippo; Dunand, Christophe

    2009-01-01

    Peroxidases are enzymes that are implicated in several biological processes and are detected in all living organisms. The increasing number of sequencing projects and the poor quality of annotation justified the creation of an efficient tool that was suitable for collecting and annotating the huge quantity of data. Started in 2004 to collect only class III peroxidases, PeroxiBase has undergone important updates since then and, currently, the majority of peroxidase sequences from all kingdoms of life is stored in the database. In addition, the web site (http://peroxibase.isb-sib.ch) provides a series of bioinformatics tools and facilities suitable for analysing these stored sequences. In particular, the high number of isoforms in each organism makes phylogenetic studies extremely useful to elucidate the complex evolution of these enzymes, not only within the plant kingdom but also between the different kingdoms. This paper provides a general overview of PeroxiBase, focusing on its tools and the stored data. The main goal is to give researchers some guidelines to extract classified and annotated sequences from the data base in a quick and easy way in order to perform alignments and phylogenetic analysis. The description of the database is accompanied by the updates we have recently carried out in order to improve its completeness and make it more user-friendly.

  14. Quantum-Sequencing: Biophysics of quantum tunneling through nucleic acids

    NASA Astrophysics Data System (ADS)

    Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

    2014-03-01

    Tunneling microscopy and spectroscopy has extensively been used in physical surface sciences to study quantum tunneling to measure electronic local density of states of nanomaterials and to characterize adsorbed species. Quantum-Sequencing (Q-Seq) is a new method based on tunneling microscopy for electronic sequencing of single molecule of nucleic acids. A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free single-molecule sequencing method. Here, we present the unique ``electronic fingerprints'' for all nucleotides on DNA and RNA using Q-Seq along their intrinsic biophysical parameters. We have analyzed tunneling spectra for the nucleotides at different pH conditions and analyzed the HOMO, LUMO and energy gap for all of them. In addition we show a number of biophysical parameters to further characterize all nucleobases (electron and hole transition voltage and energy barriers). These results highlight the robustness of Q-Seq as a technique for next-generation sequencing.

  15. Candidate gene analyses by scanning or brute force fluorescent sequencing: a comparison of DOVAM-S with gel-based and capillary-based sequencing.

    PubMed

    Feng, Jinong; Yan, Jin; Li, Wenyan; Chen, Jiesheng; Sommer, Steve S

    2007-01-01

    For epidemiological and diagnostic applications, detection of virtually all mutations is desired. Herein, blinded analyses of DOVAM-S (Detection Of Virtually All Mutations-SSCP), a robotically enhanced multiplex SSCP method, demonstrate that all of 525 mutations (391 unique) are detected by the method. In addition, the costs of DOVAM-S, gel-based fluorescent sequencing and capillary-based fluorescent sequencing are compared. The relative cost effectiveness of gel-based and capillary-based sequence analysis depends on throughput and whether depreciation and service are considered. DOVAM-S reduces the cost of candidate gene analyses relative to brute force sequencing by about threefold. PMID:17949284

  16. Sequential multiple analyses of atmospheric nitrous acid and nitrogen oxides.

    PubMed

    Toda, Kei; Hato, Yuki; Mori, Kotaro; Ohira, Shin-Ichi; Namihira, Takao

    2007-03-15

    Sequential injection analysis (SIA) was applied to multi-gas monitoring for atmospheric analysis. HONO, NO(2) or NO was collected in an individual diffusion scrubber in which the channel array was filled with either HCl or triethanolamine solution. All analytes were collected in the form of nitrite ions in the scrubber, and were transferred via a 12-port selection valve into a 2.5-ml syringe. The reagent, 3-amino-1,5-naphthalenedisulfonic acid (C-acid) solution was subsequently introduced into the syringe, and inter-mixed with the nitrite sample, whereafter the mixed solution was transferred to a heated reactor and held for 3min at 100 degrees C. After that, the sample/reagent solution was returned to the syringe and alkalinized. Then, the final solution was analyzed using a homemade fluorescence detector. Atmospheric HONO, NO(2) and NO were successfully monitored 3 or 4times/h. The limits of detection were 0.22, 0.28 and 0.35ppbv for HONO, NO(2) and NO, respectively. It was demonstrated for the first time that SIA is a good tool for multi-gas atmospheric analysis. These nitrogen-oxygen compounds are interconvertible, and the simultaneous measurement of these gases is important. Especially, HONO is a source of OH radicals which contribute greatly to atmospheric pollution, and indeed atmospheric chemistry. This method allows the three gases to be measured using one system. The NO(2) and NO data obtained by SIA was compared with those obtained using chemiluminescence instrument. SIA has been successfully applied to atmospheric measurements. Interestingly, it was observed that HONO levels rose toward the end of periods of rain.

  17. Molecular Characterization of Five Potyviruses Infecting Korean Sweet Potatoes Based on Analyses of Complete Genome Sequences.

    PubMed

    Kwak, Hae-Ryun; Kim, Jaedeok; Kim, Mi-Kyeong; Seo, Jang-Kyun; Jung, Mi-Nam; Kim, Jeong-Soo; Lee, Sukchan; Choi, Hong-Soo

    2015-12-01

    Sweet potatoes (Ipomea batatas L.) are grown extensively, in tropical and temperate regions, and are important food crops worldwide. In Korea, potyviruses, including Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG), Sweet potato virus 2 (SPV2), and Sweet potato latent virus (SPLV), have been detected in sweet potato fields at a high (~95%) incidence. In the present work, complete genome sequences of 18 isolates, representing the five potyviruses mentioned above, were compared with previously reported genome sequences. The complete genomes consisted of 10,081 to 10,830 nucleotides, excluding the poly-A tails. Their genomic organizations were typical of the Potyvirus genus, including one target open reading frame coding for a putative polyprotein. Based on phylogenetic analyses and sequence comparisons, the Korean SPFMV isolates belonged to the strains RC and O with >98% nucleotide sequence identity. Korean SPVC isolates had 99% identity to the Japanese isolate SPVC-Bungo and 70% identity to the SPFMV isolates. The Korean SPVG isolates showed 99% identity to the three previously reported SPVG isolates. Korean SPV2 isolates had 97% identity to the SPV2 GWB-2 isolate from the USA. Korean SPLV isolates had a relatively low (88%) nucleotide sequence identity with the Taiwanese SPLV-TW isolates, and they were phylogenetically distantly related to SPFMV isolates. Recombination analysis revealed that possible recombination events occurred in the P1, HC-Pro and NIa-NIb regions of SPFMV and SPLV isolates and these regions were identified as hotspots for recombination in the sweet potato potyviruses.

  18. Molecular Characterization of Five Potyviruses Infecting Korean Sweet Potatoes Based on Analyses of Complete Genome Sequences.

    PubMed

    Kwak, Hae-Ryun; Kim, Jaedeok; Kim, Mi-Kyeong; Seo, Jang-Kyun; Jung, Mi-Nam; Kim, Jeong-Soo; Lee, Sukchan; Choi, Hong-Soo

    2015-12-01

    Sweet potatoes (Ipomea batatas L.) are grown extensively, in tropical and temperate regions, and are important food crops worldwide. In Korea, potyviruses, including Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG), Sweet potato virus 2 (SPV2), and Sweet potato latent virus (SPLV), have been detected in sweet potato fields at a high (~95%) incidence. In the present work, complete genome sequences of 18 isolates, representing the five potyviruses mentioned above, were compared with previously reported genome sequences. The complete genomes consisted of 10,081 to 10,830 nucleotides, excluding the poly-A tails. Their genomic organizations were typical of the Potyvirus genus, including one target open reading frame coding for a putative polyprotein. Based on phylogenetic analyses and sequence comparisons, the Korean SPFMV isolates belonged to the strains RC and O with >98% nucleotide sequence identity. Korean SPVC isolates had 99% identity to the Japanese isolate SPVC-Bungo and 70% identity to the SPFMV isolates. The Korean SPVG isolates showed 99% identity to the three previously reported SPVG isolates. Korean SPV2 isolates had 97% identity to the SPV2 GWB-2 isolate from the USA. Korean SPLV isolates had a relatively low (88%) nucleotide sequence identity with the Taiwanese SPLV-TW isolates, and they were phylogenetically distantly related to SPFMV isolates. Recombination analysis revealed that possible recombination events occurred in the P1, HC-Pro and NIa-NIb regions of SPFMV and SPLV isolates and these regions were identified as hotspots for recombination in the sweet potato potyviruses. PMID:26673876

  19. Molecular Characterization of Five Potyviruses Infecting Korean Sweet Potatoes Based on Analyses of Complete Genome Sequences

    PubMed Central

    Kwak, Hae-Ryun; Kim, Jaedeok; Kim, Mi-Kyeong; Seo, Jang-Kyun; Jung, Mi-Nam; Kim, Jeong-Soo; Lee, Sukchan; Choi, Hong-Soo

    2015-01-01

    Sweet potatoes (Ipomea batatas L.) are grown extensively, in tropical and temperate regions, and are important food crops worldwide. In Korea, potyviruses, including Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG), Sweet potato virus 2 (SPV2), and Sweet potato latent virus (SPLV), have been detected in sweet potato fields at a high (~95%) incidence. In the present work, complete genome sequences of 18 isolates, representing the five potyviruses mentioned above, were compared with previously reported genome sequences. The complete genomes consisted of 10,081 to 10,830 nucleotides, excluding the poly-A tails. Their genomic organizations were typical of the Potyvirus genus, including one target open reading frame coding for a putative polyprotein. Based on phylogenetic analyses and sequence comparisons, the Korean SPFMV isolates belonged to the strains RC and O with >98% nucleotide sequence identity. Korean SPVC isolates had 99% identity to the Japanese isolate SPVC-Bungo and 70% identity to the SPFMV isolates. The Korean SPVG isolates showed 99% identity to the three previously reported SPVG isolates. Korean SPV2 isolates had 97% identity to the SPV2 GWB-2 isolate from the USA. Korean SPLV isolates had a relatively low (88%) nucleotide sequence identity with the Taiwanese SPLV-TW isolates, and they were phylogenetically distantly related to SPFMV isolates. Recombination analysis revealed that possible recombination events occurred in the P1, HC-Pro and NIa-NIb regions of SPFMV and SPLV isolates and these regions were identified as hotspots for recombination in the sweet potato potyviruses. PMID:26673876

  20. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    SciTech Connect

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  1. Molecular cloning and amino acid sequence of human 5-lipoxygenase

    SciTech Connect

    Matsumoto, T.; Funk, C.D.; Radmark, O.; Hoeoeg, J.O.; Joernvall, H.; Samuelsson, B.

    1988-01-01

    5-Lipoxygenase (EC 1.13.11.34), a Ca/sup 2 +/- and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B/sub 4/. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the /sup 32/P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite DNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A/sub 4/ hydrolase or Ca/sup 2 +/-binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approx. = 2700 nucleotides in leukocytes, lung, and placenta.

  2. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed Central

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-01-01

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  3. Diversity and distribution of unicellular opisthokonts along the European coast analysed using high-throughput sequencing.

    PubMed

    Del Campo, Javier; Mallo, Diego; Massana, Ramon; de Vargas, Colomban; Richards, Thomas A; Ruiz-Trillo, Iñaki

    2015-09-01

    The opisthokonts are one of the major super groups of eukaryotes. It comprises two major clades: (i) the Metazoa and their unicellular relatives and (ii) the Fungi and their unicellular relatives. There is, however, little knowledge of the role of opisthokont microbes in many natural environments, especially among non-metazoan and non-fungal opisthokonts. Here, we begin to address this gap by analysing high-throughput 18S rDNA and 18S rRNA sequencing data from different European coastal sites, sampled at different size fractions and depths. In particular, we analyse the diversity and abundance of choanoflagellates, filastereans, ichthyosporeans, nucleariids, corallochytreans and their related lineages. Our results show the great diversity of choanoflagellates in coastal waters as well as a relevant representation of the ichthyosporeans and the uncultured marine opisthokonts (MAOP). Furthermore, we describe a new lineage of marine fonticulids (MAFO) that appears to be abundant in sediments. Taken together, our work points to a greater potential ecological role for unicellular opisthokonts than previously appreciated in marine environments, both in water column and sediments, and also provides evidence of novel opisthokont phylogenetic lineages. This study highlights the importance of high-throughput sequencing approaches to unravel the diversity and distribution of both known and novel eukaryotic lineages.

  4. Molecular phylogenetic and dating analyses using mitochondrial DNA sequences of eyelid geckos (Squamata: Eublepharidae).

    PubMed

    Jonniaux, Pierre; Kumazawa, Yoshinori

    2008-01-15

    Mitochondrial DNA sequences of approximately 2.3 kbp including the complete NADH dehydrogenase subunit 2 gene and its flanking genes, as well as parts of 12S and 16S rRNA genes were determined from major species of the eyelid gecko family Eublepharidae sensu [Kluge, A.G. 1987. Cladistic relationships in the Gekkonoidea (Squamata, Sauria). Misc. Publ. Mus. Zool. Univ. Michigan 173, 1-54.]. In contrast to previous morphological studies, phylogenetic analyses based on these sequences supported that Eublepharidae and Gekkonidae form a sister group with Pygopodidae, raising the possibility of homoplasious character change in some key features of geckos, such as reduction of movable eyelids and innovation of climbing toe pads. The phylogenetic analyses also provided a well-resolved tree for relationships between the eublepharid species. The Bayesian estimation of divergence times without assuming the molecular clock suggested the Jurassic divergence of Eublepharidae from Gekkonidae and radiations of most eublepharid genera around the Cretaceous. These dating results appeared to be robust against some conditional changes for time estimation, such as gene regions used, taxon representation, and data partitioning. Taken together with geological evidence, these results support the vicariant divergence of Eublepharidae and Gekkonidae by the breakup of Pangea into Laurasia and Gondwanaland, and recent dispersal of two African eublepharid genera from Eurasia to Africa after these landmasses were connected in the Early Miocene.

  5. Molecular phylogenetic and dating analyses using mitochondrial DNA sequences of eyelid geckos (Squamata: Eublepharidae).

    PubMed

    Jonniaux, Pierre; Kumazawa, Yoshinori

    2008-01-15

    Mitochondrial DNA sequences of approximately 2.3 kbp including the complete NADH dehydrogenase subunit 2 gene and its flanking genes, as well as parts of 12S and 16S rRNA genes were determined from major species of the eyelid gecko family Eublepharidae sensu [Kluge, A.G. 1987. Cladistic relationships in the Gekkonoidea (Squamata, Sauria). Misc. Publ. Mus. Zool. Univ. Michigan 173, 1-54.]. In contrast to previous morphological studies, phylogenetic analyses based on these sequences supported that Eublepharidae and Gekkonidae form a sister group with Pygopodidae, raising the possibility of homoplasious character change in some key features of geckos, such as reduction of movable eyelids and innovation of climbing toe pads. The phylogenetic analyses also provided a well-resolved tree for relationships between the eublepharid species. The Bayesian estimation of divergence times without assuming the molecular clock suggested the Jurassic divergence of Eublepharidae from Gekkonidae and radiations of most eublepharid genera around the Cretaceous. These dating results appeared to be robust against some conditional changes for time estimation, such as gene regions used, taxon representation, and data partitioning. Taken together with geological evidence, these results support the vicariant divergence of Eublepharidae and Gekkonidae by the breakup of Pangea into Laurasia and Gondwanaland, and recent dispersal of two African eublepharid genera from Eurasia to Africa after these landmasses were connected in the Early Miocene. PMID:18029117

  6. Identification of food and beverage spoilage yeasts from DNA sequence analyses.

    PubMed

    Kurtzman, Cletus P

    2015-11-20

    Detection, identification and classification of yeasts have undergone major changes in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of the nuclear large subunit rRNA gene and from ITS now permits many laboratories to identify species quickly and accurately, thus replacing the laborious and often inaccurate phenotypic tests previously used. Phylogenetic analysis of gene sequences has resulted in a major revision of yeast systematics resulting in redefinition of nearly all genera. This new understanding of species relationships has prompted a change of rules for naming and classifying yeasts and other fungi, and these new rules are presented in the recently implemented International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). The use of molecular methods for species identification and the impact of Code changes on classification will be discussed, especially in the context of food and beverage spoilage yeasts.

  7. New approaches for computer analysis of nucleic acid sequences.

    PubMed

    Karlin, S; Ghandour, G; Ost, F; Tavare, S; Korn, L J

    1983-09-01

    A new high-speed computer algorithm is outlined that ascertains within and between nucleic acid and protein sequences all direct repeats, dyad symmetries, and other structural relationships. Large repeats, repeats of high frequency, dyad symmetries of specified stem length and loop distance, and their distributions are determined. Significance of homologies is assessed by a hierarchy of permutation procedures. Applications are made to papovaviruses, the human papillomavirus HPV, lambda phage, the human and mouse mitochondrial genomes, and the human and mouse immunoglobulin kappa-chain genes. PMID:6577449

  8. DNA sequence analyses of blended herbal products including synthetic cannabinoids as designer drugs.

    PubMed

    Ogata, Jun; Uchiyama, Nahoko; Kikura-Hanajiri, Ruri; Goda, Yukihiro

    2013-04-10

    In recent years, various herbal products adulterated with synthetic cannabinoids have been distributed worldwide via the Internet. These herbal products are mostly sold as incense, and advertised as not for human consumption. Although their labels indicate that they contain mixtures of several potentially psychoactive plants, and numerous studies have reported that they contain a variety of synthetic cannabinoids, their exact botanical contents are not always clear. In this study, we investigated the origins of botanical materials in 62 Spice-like herbal products distributed on the illegal drug market in Japan, by DNA sequence analyses and BLAST searches. The nucleotide sequences of four regions were analyzed to identify the origins of each plant species in the herbal mixtures. The sequences of "Damiana" (Turnera diffusa) and Lamiaceae herbs (Mellissa, Mentha and Thymus) were frequently detected in a number of products. However, the sequences of other plant species indicated on the packaging labels were not detected. In a few products, DNA fragments of potent psychotropic plants were found, including marijuana (Cannabis sativa), "Diviner's Sage" (Salvia divinorum) and "Kratom" (Mitragyna speciosa). Their active constituents were also confirmed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), although these plant names were never indicated on the labels. Most plant species identified in the products were different from the plants indicated on the labels. The plant materials would be used mainly as diluents for the psychoactive synthetic compounds, because no reliable psychoactive effects have been reported for most of the identified plants, with the exception of the psychotropic plants named above. PMID:23092848

  9. DNA sequence analyses of blended herbal products including synthetic cannabinoids as designer drugs.

    PubMed

    Ogata, Jun; Uchiyama, Nahoko; Kikura-Hanajiri, Ruri; Goda, Yukihiro

    2013-04-10

    In recent years, various herbal products adulterated with synthetic cannabinoids have been distributed worldwide via the Internet. These herbal products are mostly sold as incense, and advertised as not for human consumption. Although their labels indicate that they contain mixtures of several potentially psychoactive plants, and numerous studies have reported that they contain a variety of synthetic cannabinoids, their exact botanical contents are not always clear. In this study, we investigated the origins of botanical materials in 62 Spice-like herbal products distributed on the illegal drug market in Japan, by DNA sequence analyses and BLAST searches. The nucleotide sequences of four regions were analyzed to identify the origins of each plant species in the herbal mixtures. The sequences of "Damiana" (Turnera diffusa) and Lamiaceae herbs (Mellissa, Mentha and Thymus) were frequently detected in a number of products. However, the sequences of other plant species indicated on the packaging labels were not detected. In a few products, DNA fragments of potent psychotropic plants were found, including marijuana (Cannabis sativa), "Diviner's Sage" (Salvia divinorum) and "Kratom" (Mitragyna speciosa). Their active constituents were also confirmed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), although these plant names were never indicated on the labels. Most plant species identified in the products were different from the plants indicated on the labels. The plant materials would be used mainly as diluents for the psychoactive synthetic compounds, because no reliable psychoactive effects have been reported for most of the identified plants, with the exception of the psychotropic plants named above.

  10. 5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

    NASA Technical Reports Server (NTRS)

    Van den Eynde, H.; De Baere, R.; Shah, H. N.; Gharbia, S. E.; Fox, G. E.; Michalik, J.; Van de Peer, Y.; De Wachter, R.

    1989-01-01

    The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

  11. Computer-aided analyses of transport protein sequences: gleaning evidence concerning function, structure, biogenesis, and evolution.

    PubMed Central

    Saier, M H

    1994-01-01

    Three-dimensional structures have been elucidated for very few integral membrane proteins. Computer methods can be used as guides for estimation of solute transport protein structure, function, biogenesis, and evolution. In this paper the application of currently available computer programs to over a dozen distinct families of transport proteins is reviewed. The reliability of sequence-based topological and localization analyses and the importance of sequence and residue conservation to structure and function are evaluated. Evidence concerning the nature and frequency of occurrence of domain shuffling, splicing, fusion, deletion, and duplication during evolution of specific transport protein families is also evaluated. Channel proteins are proposed to be functionally related to carriers. It is argued that energy coupling to transport was a late occurrence, superimposed on preexisting mechanisms of solute facilitation. It is shown that several transport protein families have evolved independently of each other, employing different routes, at different times in evolutionary history, to give topologically similar transmembrane protein complexes. The possible significance of this apparent topological convergence is discussed. PMID:8177172

  12. RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS

    PubMed Central

    Bowers, Holly A.; Tomas, Carmelo; Tengs, Torstein; Kempton, Jason W.; Lewitus, Alan J.; Oldach, David W.

    2010-01-01

    Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species. PMID:20411032

  13. Fatty acid analyses may provide insight into the progression of starvation among squamate reptiles.

    PubMed

    McCue, Marshall D

    2008-10-01

    Fasting-induced changes in fatty acid composition have been reported to occur within the body lipids of several types of animals; however, little is known about the changes in fatty acid profiles exhibited by reptiles subjected to prolonged fasting. This study characterizes the fatty acid profiles of six reptile species subjected to sublethal periods of fasting lasting 0, 56, 112, and 168 days. Analyses of fatty acid methyl esters (FAMEs) conducted on the total body lipids of rattlesnakes (Crotalus atrox), ratsnakes (Elaphe obsoleta), pythons (Python regius), boas (Boa constrictor), true vipers (Bitis gabonica), and monitor lizards (Varanus exanthematicus) revealed that all of the species exhibited similar characteristic changes in their fatty acid profiles during starvation stress. According to ANOVAs, the four most effective indicators of the onset of starvation were significant increases in the [1] fatty acid unsaturation index as well as ratios of [2] linoleic to palmitoleic acid, [3] oleic to palmitic, and [4] arachidonic to total fatty acid concentrations. The results of this study suggest that FAME analyses might be useful for identifying nutritional stress and/or starvation among squamate reptiles; however, forthcoming studies will be required to validate the generality of these responses. I also review the potential limitations of this approach, and suggest experiments that will be important for future applications of FAME analyses. Ultimately, it is hoped that FAME analyses can be used in conjunction with current practices as an additional tool to characterize the prevalence of starvation experienced by free-living reptiles.

  14. Human Retroviruses and AIDS. A compilation and analysis of nucleic acid and amino acid sequences: I--II; III--V

    SciTech Connect

    Myers, G.; Korber, B.; Wain-Hobson, S.; Smith, R.F.; Pavlakis, G.N.

    1993-12-31

    This compendium and the accompanying floppy diskettes are the result of an effort to compile and rapidly publish all relevant molecular data concerning the human immunodeficiency viruses (HIV) and related retroviruses. The scope of the compendium and database is best summarized by the five parts that it comprises: (I) HIV and SIV Nucleotide Sequences; (II) Amino Acid Sequences; (III) Analyses; (IV) Related Sequences; and (V) Database Communications. Information within all the parts is updated at least twice in each year, which accounts for the modes of binding and pagination in the compendium.

  15. The amino-acid sequence of the alpha-crystallin A chains of red kangaroo and Virginia opossum.

    PubMed

    De Jong, W W; Terwindt, E C

    1976-08-16

    The amino acid sequence of the A chain of the eye lens protein alpha-crystallin from the red kangaroo (Macropus rufus) was completely determined by manual Edman degradation of tryptic, thermolytic and cyanogen bromide peptides. The sequence of the alpha-crystallin A chain from the Virginia opossum (Didelphis marsupialis) was deduced from amino acid analyses and partial Edman degradation of peptides. The 173-residue A chains of kangaroo and opossum differ in six positions, whereas comparison with the bovine alpha-crystallin A chain reveals 17 and 22 substitutions, respectively. Most substitutions occur in the COOH-terminal part of the chain.

  16. Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing

    PubMed Central

    2013-01-01

    Background Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Results Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. Conclusions The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with

  17. Correlations Between Amino Acids at Different Sites in Local Sequences of Protein Fragments with Given Structural Patterns

    NASA Astrophysics Data System (ADS)

    Lu, Wen; Liu, Hai-yan

    2007-02-01

    Ample evidence suggests that the local structures of peptide fragments in native proteins are to some extent encoded by their local sequences. Detecting such local correlations is important but it is still an open question what would be the most appropriate method. This is partly because conventional sequence analyses treat amino acid preferences at each site of a protein sequence independently, while it is often the inter-site interactions that bring about local sequence-structure correlations. Here a new scheme is introduced to capture the correlation between amino acid preferences at different sites for different local structure types. A library of nine-residue fragments is constructed, and the fragments are divided into clusters based on their local structures. For each local structure cluster or type, chi-square tests are used to identify correlated preferences of amino acid combinations at pairs of sites. A score function is constructed including both the single site amino acid preferences and the dual-site amino acid combination preferences, which can be used to identify whether a sequence fragment would have a strong tendency to form a particular local structure in native proteins. The results show that, given a local structure pattern, dual-site amino acid combinations contain different information from single site amino acid preferences. Representative examples show that many of the statistically identified correlations agree with previously-proposed heuristic rules about local sequence-structure correlations, or are consistent with physical-chemical interactions required to stabilize particular local structures. Results also show that such dual-site correlations in the score function significantly improves the Z-score matching a sequence fragment to its native local structure relative to non-native local structures, and certain local structure types are highly predictable from the local sequence alone if inter-site correlations are considered.

  18. Predicting protein disorder by analyzing amino acid sequence

    PubMed Central

    Yang, Jack Y; Yang, Mary Qu

    2008-01-01

    Background Many protein regions and some entire proteins have no definite tertiary structure, presenting instead as dynamic, disorder ensembles under different physiochemical circumstances. These proteins and regions are known as Intrinsically Unstructured Proteins (IUP). IUP have been associated with a wide range of protein functions, along with roles in diseases characterized by protein misfolding and aggregation. Results Identifying IUP is important task in structural and functional genomics. We exact useful features from sequences and develop machine learning algorithms for the above task. We compare our IUP predictor with PONDRs (mainly neural-network-based predictors), disEMBL (also based on neural networks) and Globplot (based on disorder propensity). Conclusion We find that augmenting features derived from physiochemical properties of amino acids (such as hydrophobicity, complexity etc.) and using ensemble method proved beneficial. The IUP predictor is a viable alternative software tool for identifying IUP protein regions and proteins. PMID:18831799

  19. The Mitochondrial Genomes of Aquila fasciata and Buteo lagopus (Aves, Accipitriformes): Sequence, Structure and Phylogenetic Analyses

    PubMed Central

    Jiang, Lan; Chen, Juan; Wang, Ping; Ren, Qiongqiong; Yuan, Jian; Qian, Chaoju; Hua, Xinghong; Guo, Zhichun; Zhang, Lei; Yang, Jianke; Wang, Ying; Zhang, Qin; Ding, Hengwu; Bi, De; Zhang, Zongmeng; Wang, Qingqing; Chen, Dongsheng; Kan, Xianzhao

    2015-01-01

    The family Accipitridae is one of the largest groups of non-passerine birds, including 68 genera and 243 species globally distributed. In the present study, we determined the complete mitochondrial sequences of two species of accipitrid, namely Aquila fasciata and Buteo lagopus, and conducted a comparative mitogenome analysis across the family. The mitogenome length of A. fasciata and B. lagopus are 18,513 and 18,559 bp with an A + T content of 54.2% and 55.0%, respectively. For both the two accipitrid birds mtDNAs, obvious positive AT-skew and negative GC-skew biases were detected for all 12 PCGs encoded by the H strand, whereas the reverse was found in MT-ND6 encoded by the L strand. One extra nucleotide‘C’is present at the position 174 of MT-ND3 gene of A. fasciata, which is not observed at that of B. lagopus. Six conserved sequence boxes in the Domain II, named boxes F, E, D, C, CSBa, and CSBb, respectively, were recognized in the CRs of A. fasciata and B. lagopus. Rates and patterns of mitochondrial gene evolution within Accipitridae were also estimated. The highest dN/dS was detected for the MT-ATP8 gene (0.32493) among Accipitridae, while the lowest for the MT-CO1 gene (0.01415). Mitophylogenetic analysis supported the robust monophyly of Accipitriformes, and Cathartidae was basal to the balance of the order. Moreover, we performed phylogenetic analyses using two other data sets (two mitochondrial loci, and combined nuclear and mitochondrial loci). Our results indicate that the subfamily Aquilinae and all currently polytypic genera of this subfamily are monophyletic. These two novel mtDNA data will be useful in refining the phylogenetic relationships and evolutionary processes of Accipitriformes. PMID:26295156

  20. Phylogeny of tremellomycetous yeasts and related dimorphic and filamentous basidiomycetes reconstructed from multiple gene sequence analyses

    PubMed Central

    Liu, X.-Z.; Wang, Q.-M.; Theelen, B.; Groenewald, M.; Bai, F.-Y.; Boekhout, T.

    2015-01-01

    The Tremellomycetes (Basidiomycota) contains a large number of unicellular and dimorphic fungi with stable free-living unicellular states in their life cycles. These fungi have been conventionally classified as basidiomycetous yeasts based on physiological and biochemical characteristics. Many currently recognised genera of these yeasts are mainly defined based on phenotypical characters and are highly polyphyletic. Here we reconstructed the phylogeny of the majority of described anamorphic and teleomorphic tremellomycetous yeasts using Bayesian inference, maximum likelihood, and neighbour-joining analyses based on the sequences of seven genes, including three rRNA genes, namely the small subunit of the ribosomal DNA (rDNA), D1/D2 domains of the large subunit rDNA, and the internal transcribed spacer regions (ITS 1 and 2) of rDNA including 5.8S rDNA; and four protein-coding genes, namely the two subunits of the RNA polymerase II (RPB1 and RPB2), the translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB). With the consideration of morphological, physiological and chemotaxonomic characters and the congruence of phylogenies inferred from analyses using different algorithms based on different data sets consisting of the combined seven genes, the three rRNA genes, and the individual protein-coding genes, five major lineages corresponding to the orders Cystofilobasidiales, Filobasidiales, Holtermanniales, Tremellales, and Trichosporonales were resolved. A total of 45 strongly supported monophyletic clades with multiple species and 23 single species clades were recognised. This phylogenetic framework will be the basis for the proposal of an updated taxonomic system of tremellomycetous yeasts that will be compatible with the current taxonomic system of filamentous basidiomycetes accommodating the ‘one fungus, one name’ principle. PMID:26955196

  1. Heterogeneity of amino acid sequence in hippopotamus cytochrome c.

    PubMed

    Thompson, R B; Borden, D; Tarr, G E; Margoliash, E

    1978-12-25

    The amino acid sequences of chymotryptic and tryptic peptides of Hippopotamus amphibius cytochrome c were determined by a recent modification of the manual Edman sequential degradation procedure. They were ordered by comparison with the structure of the hog protein. The hippopotamus protein differs in three positions: serine, alanine, and glutamine replace alanine, glutamic acid, and lysine in positions 43, 92, and 100, respectively. Since the artiodactyl suborders diverged in the mid-Eocene some 50 million years ago, the fact that representatives of some of them show no differences in their cytochromes c (cow, sheep, and hog), while another exhibits as many as three such differences, verifies that even in relatively closely related lines of descent the rate at which cytochrome c changes in the course of evolution is not constant. Furthermore, 10.6% of the hippopotamus cytochrome c preparation was shown to contain isoleucine instead of valine at position 3, indicating that one of the four animals from which the protein was obtained was heterozygous in the cytochrome c gene. Such heterogeneity is a necessary condition of evolutionary variation and has not been previously observed in the cytochrome c of a wild mammalian population.

  2. Palindrome analyser - A new web-based server for predicting and evaluating inverted repeats in nucleotide sequences.

    PubMed

    Brázda, Václav; Kolomazník, Jan; Lýsek, Jiří; Hároníková, Lucia; Coufal, Jan; Št'astný, Jiří

    2016-09-30

    DNA cruciform structures play an important role in the regulation of natural processes including gene replication and expression, as well as nucleosome structure and recombination. They have also been implicated in the evolution and development of diseases such as cancer and neurodegenerative disorders. Cruciform structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling and protein binding. They have received broad attention because of their important roles in biology. Computational approaches to study inverted repeats have allowed detailed analysis of genomes. However, currently there are no easily accessible and user-friendly tools that can analyse inverted repeats, especially among long nucleotide sequences. We have developed a web-based server, Palindrome analyser, which is a user-friendly application for analysing inverted repeats in various DNA (or RNA) sequences including genome sequences and oligonucleotides. It allows users to search and retrieve desired gene/nucleotide sequence entries from the NCBI databases, and provides data on length, sequence, locations and energy required for cruciform formation. Palindrome analyser also features an interactive graphical data representation of the distribution of the inverted repeats, with options for sorting according to the length of inverted repeat, length of loop, and number of mismatches. Palindrome analyser can be accessed at http://bioinformatics.ibp.cz.

  3. Palindrome analyser - A new web-based server for predicting and evaluating inverted repeats in nucleotide sequences.

    PubMed

    Brázda, Václav; Kolomazník, Jan; Lýsek, Jiří; Hároníková, Lucia; Coufal, Jan; Št'astný, Jiří

    2016-09-30

    DNA cruciform structures play an important role in the regulation of natural processes including gene replication and expression, as well as nucleosome structure and recombination. They have also been implicated in the evolution and development of diseases such as cancer and neurodegenerative disorders. Cruciform structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling and protein binding. They have received broad attention because of their important roles in biology. Computational approaches to study inverted repeats have allowed detailed analysis of genomes. However, currently there are no easily accessible and user-friendly tools that can analyse inverted repeats, especially among long nucleotide sequences. We have developed a web-based server, Palindrome analyser, which is a user-friendly application for analysing inverted repeats in various DNA (or RNA) sequences including genome sequences and oligonucleotides. It allows users to search and retrieve desired gene/nucleotide sequence entries from the NCBI databases, and provides data on length, sequence, locations and energy required for cruciform formation. Palindrome analyser also features an interactive graphical data representation of the distribution of the inverted repeats, with options for sorting according to the length of inverted repeat, length of loop, and number of mismatches. Palindrome analyser can be accessed at http://bioinformatics.ibp.cz. PMID:27603574

  4. Sequence analyses of herpesviral enzymes suggest an ancient origin for human sexual behavior.

    PubMed Central

    Gentry, G A; Lowe, M; Alford, G; Nevins, R

    1988-01-01

    Comparison of the amino acid sequences of the deoxythymidine kinases of herpes simplex (HSV) and of marmoset herpes viruses (MHV) suggests a divergence time of 8 to 10 million years ago for HSV-1 and -2. Like MHV, HSV-1 and -2 cause local infections in their natural hosts, and direct contact between two individuals during the brief period of infectivity is needed for transmission. Because B virus, a nearer relative of HSV, depends on both oral and genital routes of transmission, we postulate that ancestral HSV (aHSV) was similar, and that for HSV-1 and -2 to diverge, genital and oral sites had to become microbiologically somewhat isolated from each other, while oral--oral and genital--genital contact had to be facilitated to maintain both aHSV strains. We propose that acquisition of continual sexual attractiveness by the ancestral human female and the adoption of close face-to-face mating, two hallmarks of human sexual behavior, provided the conditions for the divergence. PMID:3128793

  5. Sequence and Expression Analyses of Ethylene Response Factors Highly Expressed in Latex Cells from Hevea brasiliensis

    PubMed Central

    Piyatrakul, Piyanuch; Yang, Meng; Putranto, Riza-Arief; Pirrello, Julien; Dessailly, Florence; Hu, Songnian; Summo, Marilyne; Theeravatanasuk, Kannikar; Leclercq, Julie; Kuswanhadi; Montoro, Pascal

    2014-01-01

    The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors. PMID:24971876

  6. Genome sequence and origin analyses of the recombinant novel IBV virulent isolate SAIBK2.

    PubMed

    Wu, Xuan; Yang, Xin; Xu, Pengwei; Zhou, Long; Zhang, Zhikun; Wang, Hongning

    2016-08-01

    Recombination between infectious bronchitis viruses (IBVs), together with point mutations, insertions, and deletions, is thought to be responsible for the emergence of new IBV variants. SAIBK2 is a nephropathogenic strain isolated from layer flocks vaccinated with live attenuated H120 vaccine in Sichuan province, China in 2011. SAIBK2 causes severe kidney lesions and results in 50 % mortality in 30-day-old specific-pathogen-free chickens (with a dose of 10(5) EID50/0.1 mL SAIBK2 per chicken). The complete genome of SAIBK2 consists of 27669 nucleotides, excluding the poly-A tail at the 3' end. SAIBK2 has the highest identity to YX10 in terms of complete genome. Phylogenetic analysis of complete sequence showed that SAIBK2 belongs to the most dominant genotype in China. Comparison and recombination analyses with other IBV strains revealed that SAIBK2 may originate from recombination events among a YX10-, a YN-, and a Mass-like strain. Furthermore, whole gene 5 and parts of nsp 3, nsp 4, nsp 16, and N genes are involved in the recombination events, and the uptake of these regions from YN and Mass strains by SAIBK2 may increase its replication efficiency and be responsible for its increased virulence in specific-pathogen-free chickens. PMID:27108998

  7. Whole-genome analyses of Korean native and Holstein cattle breeds by massively parallel sequencing.

    PubMed

    Choi, Jung-Woo; Liao, Xiaoping; Stothard, Paul; Chung, Won-Hyong; Jeon, Heoyn-Jeong; Miller, Stephen P; Choi, So-Young; Lee, Jeong-Koo; Yang, Bokyoung; Lee, Kyung-Tai; Han, Kwang-Jin; Kim, Hyeong-Cheol; Jeong, Dongkee; Oh, Jae-Don; Kim, Namshin; Kim, Tae-Hun; Lee, Hak-Kyo; Lee, Sung-Jin

    2014-01-01

    A main goal of cattle genomics is to identify DNA differences that account for variations in economically important traits. In this study, we performed whole-genome analyses of three important cattle breeds in Korea--Hanwoo, Jeju Heugu, and Korean Holstein--using the Illumina HiSeq 2000 sequencing platform. We achieved 25.5-, 29.6-, and 29.5-fold coverage of the Hanwoo, Jeju Heugu, and Korean Holstein genomes, respectively, and identified a total of 10.4 million single nucleotide polymorphisms (SNPs), of which 54.12% were found to be novel. We also detected 1,063,267 insertions-deletions (InDels) across the genomes (78.92% novel). Annotations of the datasets identified a total of 31,503 nonsynonymous SNPs and 859 frameshift InDels that could affect phenotypic variations in traits of interest. Furthermore, genome-wide copy number variation regions (CNVRs) were detected by comparing the Hanwoo, Jeju Heugu, and previously published Chikso genomes against that of Korean Holstein. A total of 992, 284, and 1881 CNVRs, respectively, were detected throughout the genome. Moreover, 53, 65, 45, and 82 putative regions of homozygosity (ROH) were identified in Hanwoo, Jeju Heugu, Chikso, and Korean Holstein respectively. The results of this study provide a valuable foundation for further investigations to dissect the molecular mechanisms underlying variation in economically important traits in cattle and to develop genetic markers for use in cattle breeding.

  8. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses.

    PubMed

    Stelzer, Gil; Rosen, Naomi; Plaschkes, Inbar; Zimmerman, Shahar; Twik, Michal; Fishilevich, Simon; Stein, Tsippi Iny; Nudel, Ron; Lieder, Iris; Mazor, Yaron; Kaplan, Sergey; Dahary, Dvir; Warshawsky, David; Guan-Golan, Yaron; Kohn, Asher; Rappaport, Noa; Safran, Marilyn; Lancet, Doron

    2016-06-20

    GeneCards, the human gene compendium, enables researchers to effectively navigate and inter-relate the wide universe of human genes, diseases, variants, proteins, cells, and biological pathways. Our recently launched Version 4 has a revamped infrastructure facilitating faster data updates, better-targeted data queries, and friendlier user experience. It also provides a stronger foundation for the GeneCards suite of companion databases and analysis tools. Improved data unification includes gene-disease links via MalaCards and merged biological pathways via PathCards, as well as drug information and proteome expression. VarElect, another suite member, is a phenotype prioritizer for next-generation sequencing, leveraging the GeneCards and MalaCards knowledgebase. It automatically infers direct and indirect scored associations between hundreds or even thousands of variant-containing genes and disease phenotype terms. VarElect's capabilities, either independently or within TGex, our comprehensive variant analysis pipeline, help prepare for the challenge of clinical projects that involve thousands of exome/genome NGS analyses. © 2016 by John Wiley & Sons, Inc.

  9. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses.

    PubMed

    Stelzer, Gil; Rosen, Naomi; Plaschkes, Inbar; Zimmerman, Shahar; Twik, Michal; Fishilevich, Simon; Stein, Tsippi Iny; Nudel, Ron; Lieder, Iris; Mazor, Yaron; Kaplan, Sergey; Dahary, Dvir; Warshawsky, David; Guan-Golan, Yaron; Kohn, Asher; Rappaport, Noa; Safran, Marilyn; Lancet, Doron

    2016-01-01

    GeneCards, the human gene compendium, enables researchers to effectively navigate and inter-relate the wide universe of human genes, diseases, variants, proteins, cells, and biological pathways. Our recently launched Version 4 has a revamped infrastructure facilitating faster data updates, better-targeted data queries, and friendlier user experience. It also provides a stronger foundation for the GeneCards suite of companion databases and analysis tools. Improved data unification includes gene-disease links via MalaCards and merged biological pathways via PathCards, as well as drug information and proteome expression. VarElect, another suite member, is a phenotype prioritizer for next-generation sequencing, leveraging the GeneCards and MalaCards knowledgebase. It automatically infers direct and indirect scored associations between hundreds or even thousands of variant-containing genes and disease phenotype terms. VarElect's capabilities, either independently or within TGex, our comprehensive variant analysis pipeline, help prepare for the challenge of clinical projects that involve thousands of exome/genome NGS analyses. © 2016 by John Wiley & Sons, Inc. PMID:27322403

  10. Whole-Genome Analyses of Korean Native and Holstein Cattle Breeds by Massively Parallel Sequencing

    PubMed Central

    Stothard, Paul; Chung, Won-Hyong; Jeon, Heoyn-Jeong; Miller, Stephen P.; Choi, So-Young; Lee, Jeong-Koo; Yang, Bokyoung; Lee, Kyung-Tai; Han, Kwang-Jin; Kim, Hyeong-Cheol; Jeong, Dongkee; Oh, Jae-Don; Kim, Namshin; Kim, Tae-Hun; Lee, Hak-Kyo; Lee, Sung-Jin

    2014-01-01

    A main goal of cattle genomics is to identify DNA differences that account for variations in economically important traits. In this study, we performed whole-genome analyses of three important cattle breeds in Korea—Hanwoo, Jeju Heugu, and Korean Holstein—using the Illumina HiSeq 2000 sequencing platform. We achieved 25.5-, 29.6-, and 29.5-fold coverage of the Hanwoo, Jeju Heugu, and Korean Holstein genomes, respectively, and identified a total of 10.4 million single nucleotide polymorphisms (SNPs), of which 54.12% were found to be novel. We also detected 1,063,267 insertions–deletions (InDels) across the genomes (78.92% novel). Annotations of the datasets identified a total of 31,503 nonsynonymous SNPs and 859 frameshift InDels that could affect phenotypic variations in traits of interest. Furthermore, genome-wide copy number variation regions (CNVRs) were detected by comparing the Hanwoo, Jeju Heugu, and previously published Chikso genomes against that of Korean Holstein. A total of 992, 284, and 1881 CNVRs, respectively, were detected throughout the genome. Moreover, 53, 65, 45, and 82 putative regions of homozygosity (ROH) were identified in Hanwoo, Jeju Heugu, Chikso, and Korean Holstein respectively. The results of this study provide a valuable foundation for further investigations to dissect the molecular mechanisms underlying variation in economically important traits in cattle and to develop genetic markers for use in cattle breeding. PMID:24992012

  11. Sequence diversity and gene expression analyses of expansin-related proteins in the white-rot basidiomycete, Phanerochaete carnosa.

    PubMed

    Suzuki, Hitoshi; Vuong, Thu V; Gong, Yunchen; Chan, Kin; Ho, Chi-Yip; Master, Emma R; Kondo, Akihiko

    2014-11-01

    Expansin and expansin-related proteins loosen plant cell wall architectures and are widely distributed in several types of organisms, including plants, fungi and bacteria. Here we describe sequence diversity and unique gene expression profiles of multiple expansin-related proteins identified in the basidiomycete, Phanerochaete carnosa. The protein sequences were homologous to loosenin, an expansin-related protein reported in the basidiomycete, Bjerkandera adusta. We identified homologous sequences of each of those P. carnosa proteins in many basidiomycete species. Twelve P. carnosa loosenin-like proteins (LOOLs) were classified into two subgroups according to sequence homology. Conservation of polysaccharide-binding amino acid residues was stricter in subgroup A. Subgroup A sequences included a conserved 8-9 amino acid insertion in a polysaccharide-binding groove whereas subgroup B contained a 12-18 amino acid insertion next to the binding groove. The P. carnosa genome also encodes the expansin-related protein, DREX1, which adopts a loosenin-like structure but has lower sequence homology to other LOOLs. The gene expression analysis of those proteins showed distinct patterns that were not significantly related to subgroupings. The variation in the protein sequences and gene expression patterns, and wide distribution among the basidiomycota, suggest that the diverse cell wall loosening proteins contribute to effective plant cell wall association and utilization by basidiomycetes.

  12. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence.

    PubMed Central

    Law, S W; Grant, S M; Higuchi, K; Hospattankar, A; Lackner, K; Lee, N; Brewer, H B

    1986-01-01

    Human apolipoprotein B-100 (apoB-100), the ligand on low density lipoproteins that interacts with the low density lipoprotein receptor and initiates receptor-mediated endocytosis and low density lipoprotein catabolism, has been cloned, and the complete nucleic acid and derived amino acid sequences have been determined. ApoB-100 cDNAs were isolated from normal human liver cDNA libraries utilizing immunoscreening as well as filter hybridization with radiolabeled apoB-100 oligodeoxynucleotides. The apoB-100 mRNA is 14.1 kilobases long encoding a mature apoB-100 protein of 4536 amino acids with a calculated amino acid molecular weight of 512,723. ApoB-100 contains 20 potential glycosylation sites, and 12 of a total of 25 cysteine residues are located in the amino-terminal region of the apolipoprotein providing a potential globular structure of the amino terminus of the protein. ApoB-100 contains relatively few regions of amphipathic helices, but compared to other human apolipoproteins it is enriched in beta-structure. The delineation of the entire human apoB-100 sequence will now permit a detailed analysis of the conformation of the protein, the low density lipoprotein receptor binding domain(s), and the structural relationship between apoB-100 and apoB-48 and will provide the basis for the study of genetic defects in apoB-100 in patients with dyslipoproteinemias. PMID:3464946

  13. Searching for Extraterrestrial Amino Acids in a Contaminated Meteorite: Amino Acid Analyses of the Canakkale L6 Chondrite

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Elsila, J. E.; Glavin, D. P.; Dworkin, J. P.; Ornek, C. Y.; Esenoglu, H. H.; Unsalan, O.; Ozturk, B.

    2016-01-01

    Amino acids can serve as important markers of cosmochemistry, as their abundances and isomeric and isotopic compositions have been found to vary predictably with changes in parent body chemistry and alteration processes. Amino acids are also of astrobiological interest because they are essential for life on Earth. Analyses of a range of meteorites, including all groups of carbonaceous chondrites, along with H, R, and LL chondrites, ureilites, and a martian shergottite, have revealed that amino acids of plausible extraterrestrial origin can be formed in and persist after a wide range of parent body conditions. However, amino acid analyses of L6 chondrites to date have not provided evidence for indigenous amino acids. In the present study, we performed amino acid analysis on larger samples of a different L6 chondite, Canakkale, to determine whether or not trace levels of indigenous amino acids could be found. The Canakkale meteor was an observed fall in late July, 1964, near Canakkale, Turkey. The meteorite samples (1.36 and 1.09 g) analyzed in this study were allocated by C. Y. Ornek, along with a soil sample (1.5 g) collected near the Canakkale recovery site.

  14. Genomic Resources for Water Yam (Dioscorea alata L.): Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries

    PubMed Central

    Saski, Christopher A.; Bhattacharjee, Ranjana; Scheffler, Brian E.; Asiedu, Robert

    2015-01-01

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp.) is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST)-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS) profiles on two yam (Dioscorea alata L.) genotypes (TDa 95/00328 and TDa 95-310) was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using different approaches

  15. Genomic Resources for Water Yam (Dioscorea alata L.): Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries.

    PubMed

    Saski, Christopher A; Bhattacharjee, Ranjana; Scheffler, Brian E; Asiedu, Robert

    2015-01-01

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp.) is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST)-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS) profiles on two yam (Dioscorea alata L.) genotypes (TDa 95/00328 and TDa 95-310) was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using different approaches

  16. Genomic Resources for Water Yam (Dioscorea alata L.): Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries.

    PubMed

    Saski, Christopher A; Bhattacharjee, Ranjana; Scheffler, Brian E; Asiedu, Robert

    2015-01-01

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp.) is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST)-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS) profiles on two yam (Dioscorea alata L.) genotypes (TDa 95/00328 and TDa 95-310) was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using different approaches

  17. Associations between Homocysteine, Folic Acid, Vitamin B12 and Alzheimer's Disease: Insights from Meta-Analyses.

    PubMed

    Shen, Liang; Ji, Hong-Fang

    2015-01-01

    The associations between homocysteine (Hcy), folic acid, and vitamin B12 and Alzheimer's disease (AD) have gained much interest, while remaining controversial. We aim to perform meta-analyses to evaluate comprehensively: i) Hcy, folic acid, and vitamin B12 levels in AD patients in comparison with controls; and ii) the association between Hcy, folic acid, and vitamin B12 levels and risk of AD. A literature search was performed using Medline and Scopus databases. A total of 68 studies were identified and included in the meta-analyses. Stata 12.0 statistical software was used to perform the meta-analyses. First, AD patients may have higher level of Hcy, and lower levels of folate and vitamin B12 in plasma than controls. Further age-subgroup analysis showed no age effect for Hcy levels in plasma between AD patients and matched controls, while the differences in folate and vitamin B12 levels further enlarged with increased age. Second, data suggests that high Hcy and low folate levels may correlate with increased risk of AD occurrence. The comprehensive meta-analyses not only confirmed higher Hcy, lower folic acid, and vitamin B12 levels in AD patients than controls, but also implicated that high Hcy and low folic acid levels may be risk factors of AD. Further studies are encouraged to elucidate mechanisms linking these conditions.

  18. Specific catalysis of asparaginyl deamidation by carboxylic acids: kinetic, thermodynamic, and quantitative structure-property relationship analyses.

    PubMed

    Connolly, Brian D; Tran, Benjamin; Moore, Jamie M R; Sharma, Vikas K; Kosky, Andrew

    2014-04-01

    Asparaginyl (Asn) deamidation could lead to altered potency, safety, and/or pharmacokinetics of therapeutic protein drugs. In this study, we investigated the effects of several different carboxylic acids on Asn deamidation rates using an IgG1 monoclonal antibody (mAb1*) and a model hexapeptide (peptide1) with the sequence YGKNGG. Thermodynamic analyses of the kinetics data revealed that higher deamidation rates are associated with predominantly more negative ΔS and, to a lesser extent, more positive ΔH. The observed differences in deamidation rates were attributed to the unique ability of each type of carboxylic acid to stabilize the energetically unfavorable transition-state conformations required for imide formation. Quantitative structure property relationship (QSPR) analysis using kinetic data demonstrated that molecular descriptors encoding for the geometric spatial distribution of atomic properties on various carboxylic acids are effective determinants for the deamidation reaction. Specifically, the number of O-O and O-H atom pairs on carboxyl and hydroxyl groups with interatomic distances of 4-5 Å on a carboxylic acid buffer appears to determine the rate of deamidation. Collectively, the results from structural and thermodynamic analyses indicate that carboxylic acids presumably form multiple hydrogen bonds and charge-charge interactions with the relevant deamidation site and provide alignment between the reactive atoms on the side chain and backbone. We propose that carboxylic acids catalyze deamidation by stabilizing a specific, energetically unfavorable transition-state conformation of l-asparaginyl intermediate II that readily facilitates bond formation between the γ-carbonyl carbon and the deprotonated backbone nitrogen for cyclic imide formation.

  19. DNA sequence analyses reveal abundant diversity, endemism and evidence for Asian origin of the porcini mushrooms.

    PubMed

    Feng, Bang; Xu, Jianping; Wu, Gang; Hosen, Md Iqbal; Zeng, Nian-Kai; Li, Yan-Chun; Tolgor, Bau; Kost, Gerhard W; Yang, Zhu L

    2012-01-01

    The wild gourmet mushroom Boletus edulis and its close allies are of significant ecological and economic importance. They are found throughout the Northern Hemisphere, but despite their ubiquity there are still many unresolved issues with regard to the taxonomy, systematics and biogeography of this group of mushrooms. Most phylogenetic studies of Boletus so far have characterized samples from North America and Europe and little information is available on samples from other areas, including the ecologically and geographically diverse regions of China. Here we analyzed DNA sequence variation in three gene markers from samples of these mushrooms from across China and compared our findings with those from other representative regions. Our results revealed fifteen novel phylogenetic species (about one-third of the known species) and a newly identified lineage represented by Boletus sp. HKAS71346 from tropical Asia. The phylogenetic analyses support eastern Asia as the center of diversity for the porcini sensu stricto clade. Within this clade, B. edulis is the only known holarctic species. The majority of the other phylogenetic species are geographically restricted in their distributions. Furthermore, molecular dating and geological evidence suggest that this group of mushrooms originated during the Eocene in eastern Asia, followed by dispersal to and subsequent speciation in other parts of Asia, Europe, and the Americas from the middle Miocene through the early Pliocene. In contrast to the ancient dispersal of porcini in the strict sense in the Northern Hemisphere, the occurrence of B. reticulatus and B. edulis sensu lato in the Southern Hemisphere was probably due to recent human-mediated introductions.

  20. Transcriptome Sequencing in Response to Salicylic Acid in Salvia miltiorrhiza.

    PubMed

    Zhang, Xiaoru; Dong, Juane; Liu, Hailong; Wang, Jiao; Qi, Yuexin; Liang, Zongsuo

    2016-01-01

    Salvia miltiorrhiza is a traditional Chinese herbal medicine, whose quality and yield are often affected by diseases and environmental stresses during its growing season. Salicylic acid (SA) plays a significant role in plants responding to biotic and abiotic stresses, but the involved regulatory factors and their signaling mechanisms are largely unknown. In order to identify the genes involved in SA signaling, the RNA sequencing (RNA-seq) strategy was employed to evaluate the transcriptional profiles in S. miltiorrhiza cell cultures. A total of 50,778 unigenes were assembled, in which 5,316 unigenes were differentially expressed among 0-, 2-, and 8-h SA induction. The up-regulated genes were mainly involved in stimulus response and multi-organism process. A core set of candidate novel genes coding SA signaling component proteins was identified. Many transcription factors (e.g., WRKY, bHLH and GRAS) and genes involved in hormone signal transduction were differentially expressed in response to SA induction. Detailed analysis revealed that genes associated with defense signaling, such as antioxidant system genes, cytochrome P450s and ATP-binding cassette transporters, were significantly overexpressed, which can be used as genetic tools to investigate disease resistance. Our transcriptome analysis will help understand SA signaling and its mechanism of defense systems in S. miltiorrhiza. PMID:26808150

  1. Transcriptome Sequencing in Response to Salicylic Acid in Salvia miltiorrhiza

    PubMed Central

    Zhang, Xiaoru; Dong, Juane; Liu, Hailong; Wang, Jiao; Qi, Yuexin; Liang, Zongsuo

    2016-01-01

    Salvia miltiorrhiza is a traditional Chinese herbal medicine, whose quality and yield are often affected by diseases and environmental stresses during its growing season. Salicylic acid (SA) plays a significant role in plants responding to biotic and abiotic stresses, but the involved regulatory factors and their signaling mechanisms are largely unknown. In order to identify the genes involved in SA signaling, the RNA sequencing (RNA-seq) strategy was employed to evaluate the transcriptional profiles in S. miltiorrhiza cell cultures. A total of 50,778 unigenes were assembled, in which 5,316 unigenes were differentially expressed among 0-, 2-, and 8-h SA induction. The up-regulated genes were mainly involved in stimulus response and multi-organism process. A core set of candidate novel genes coding SA signaling component proteins was identified. Many transcription factors (e.g., WRKY, bHLH and GRAS) and genes involved in hormone signal transduction were differentially expressed in response to SA induction. Detailed analysis revealed that genes associated with defense signaling, such as antioxidant system genes, cytochrome P450s and ATP-binding cassette transporters, were significantly overexpressed, which can be used as genetic tools to investigate disease resistance. Our transcriptome analysis will help understand SA signaling and its mechanism of defense systems in S. miltiorrhiza. PMID:26808150

  2. Natural vs. random protein sequences: Discovering combinatorics properties on amino acid words.

    PubMed

    Santoni, Daniele; Felici, Giovanni; Vergni, Davide

    2016-02-21

    Casual mutations and natural selection have driven the evolution of protein amino acid sequences that we observe at present in nature. The question about which is the dominant force of proteins evolution is still lacking of an unambiguous answer. Casual mutations tend to randomize protein sequences while, in order to have the correct functionality, one expects that selection mechanisms impose rigid constraints on amino acid sequences. Moreover, one also has to consider that the space of all possible amino acid sequences is so astonishingly large that it could be reasonable to have a well tuned amino acid sequence indistinguishable from a random one. In order to study the possibility to discriminate between random and natural amino acid sequences, we introduce different measures of association between pairs of amino acids in a sequence, and apply them to a dataset of 1047 natural protein sequences and 10,470 random sequences, carefully generated in order to preserve the relative length and amino acid distribution of the natural proteins. We analyze the multidimensional measures with machine learning techniques and show that, to a reasonable extent, natural protein sequences can be differentiated from random ones.

  3. Natural vs. random protein sequences: Discovering combinatorics properties on amino acid words.

    PubMed

    Santoni, Daniele; Felici, Giovanni; Vergni, Davide

    2016-02-21

    Casual mutations and natural selection have driven the evolution of protein amino acid sequences that we observe at present in nature. The question about which is the dominant force of proteins evolution is still lacking of an unambiguous answer. Casual mutations tend to randomize protein sequences while, in order to have the correct functionality, one expects that selection mechanisms impose rigid constraints on amino acid sequences. Moreover, one also has to consider that the space of all possible amino acid sequences is so astonishingly large that it could be reasonable to have a well tuned amino acid sequence indistinguishable from a random one. In order to study the possibility to discriminate between random and natural amino acid sequences, we introduce different measures of association between pairs of amino acids in a sequence, and apply them to a dataset of 1047 natural protein sequences and 10,470 random sequences, carefully generated in order to preserve the relative length and amino acid distribution of the natural proteins. We analyze the multidimensional measures with machine learning techniques and show that, to a reasonable extent, natural protein sequences can be differentiated from random ones. PMID:26656109

  4. Cloning and sequencing of the Bet v 1-homologous allergen Fra a 1 in strawberry (Fragaria ananassa) shows the presence of an intron and little variability in amino acid sequence.

    PubMed

    Musidlowska-Persson, Anna; Alm, Rikard; Emanuelsson, Cecilia

    2007-02-01

    The Fra a 1 allergen in strawberry (Fragaria ananassa) is homologous to the major birch pollen allergen Bet v 1, which has numerous isoforms differing in terms of amino acid sequence and immunological impact. To map the extent of sequence differences in the Fra a 1 allergen, PCR cloning and sequencing was applied. Several genomic sequences of Fra a 1, with a length of either 584, 591 or 594 nucleotides, were obtained from three different strawberry varieties. All contained one intron, with the length of either 101 or 110 nucleotides. By sequencing 30 different clones, eight different DNA sequences were obtained, giving in total five potential Fra a 1 protein isoforms, with high sequence similarity (>97% sequence identity) and only seven positions of amino acid variability, which were largely confirmed by mass spectrometry of expressed proteins. We conclude that the sequence variability in the strawberry allergen Fra a 1 is small, within and between strawberry varieties, and that multiple spots, previously detected in 2DE, are presumably due to differences in post-translational modification rather than differences in amino acid sequence. The most abundant Fra a 1 isoform sequence, recombinantly expressed in Escherichia coli after removal of the intron, was recognized by IgE from strawberry allergic patients. It cross-reacted with antibodies to Bet v 1 and the homologous apple allergen Mal d 1 (61 and 78% sequence identity, respectively), and will be used in further analyses of variation in Fra a 1-expression.

  5. An on-line potentiometric sequential injection titration process analyser for the determination of acetic acid.

    PubMed

    van Staden, J F; Mashamba, Mulalo G; Stefan, Raluca I

    2002-09-01

    An on-line potentiometric sequential injection titration process analyser for the determination of acetic acid is proposed. A solution of 0.1 mol L(-1) sodium chloride is used as carrier. Titration is achieved by aspirating acetic acid samples between two strong base-zone volumes into a holding coil and by channelling the stack of well-defined zones with flow reversal through a reaction coil to a potentiometric sensor where the peak widths were measured. A linear relationship between peak width and logarithm of the acid concentration was obtained in the range 1-9 g/100 mL. Vinegar samples were analysed without any sample pre-treatment. The method has a relative standard deviation of 0.4% with a sample frequency of 28 samples per hour. The results revealed good agreement between the proposed sequential injection and an automated batch titration method.

  6. Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

    PubMed Central

    Fujii, Yuki; Tanaka, Shiho; Otsuki, Manami; Hoshino, Yasushi; Morimoto, Chinatsu; Kotani, Takuya; Harashima, Yuko; Endo, Haruka; Yoshizawa, Yasutaka; Sato, Ryoichi

    2012-01-01

    Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein. PMID:23145814

  7. Analyses of an Expressed Sequence Tag Library from Taenia solium, Cysticerca

    PubMed Central

    Lundström, Jonas; Salazar-Anton, Fernando; Sherwood, Ellen; Andersson, Björn; Lindh, Johan

    2010-01-01

    Background Neurocysticercosis is a disease caused by the oral ingestion of eggs from the human parasitic worm Taenia solium. Although drugs are available they are controversial because of the side effects and poor efficiency. An expressed sequence tag (EST) library is a method used to describe the gene expression profile and sequence of mRNA from a specific organism and stage. Such information can be used in order to find new targets for the development of drugs and to get a better understanding of the parasite biology. Methods and Findings Here an EST library consisting of 5760 sequences from the pig cysticerca stage has been constructed. In the library 1650 unique sequences were found and of these, 845 sequences (52%) were novel to T. solium and not identified within other EST libraries. Furthermore, 918 sequences (55%) were of unknown function. Amongst the 25 most frequently expressed sequences 6 had no relevant similarity to other sequences found in the Genbank NR DNA database. A prediction of putative signal peptides was also performed and 4 among the 25 were found to be predicted with a signal peptide. Proposed vaccine and diagnostic targets T24, Tsol18/HP6 and Tso31d could also be identified among the 25 most frequently expressed. Conclusions An EST library has been produced from pig cysticerca and analyzed. More than half of the different ESTs sequenced contained a sequence with no suggested function and 845 novel EST sequences have been identified. The library increases the knowledge about what genes are expressed and to what level. It can also be used to study different areas of research such as drug and diagnostic development together with parasite fitness via e.g. immune modulation. PMID:21200421

  8. FIGG: Simulating populations of whole genome sequences for heterogeneous data analyses

    PubMed Central

    2014-01-01

    Background High-throughput sequencing has become one of the primary tools for investigation of the molecular basis of disease. The increasing use of sequencing in investigations that aim to understand both individuals and populations is challenging our ability to develop analysis tools that scale with the data. This issue is of particular concern in studies that exhibit a wide degree of heterogeneity or deviation from the standard reference genome. The advent of population scale sequencing studies requires analysis tools that are developed and tested against matching quantities of heterogeneous data. Results We developed a large-scale whole genome simulation tool, FIGG, which generates large numbers of whole genomes with known sequence characteristics based on direct sampling of experimentally known or theorized variations. For normal variations we used publicly available data to determine the frequency of different mutation classes across the genome. FIGG then uses this information as a background to generate new sequences from a parent sequence with matching frequencies, but different actual mutations. The background can be normal variations, known disease variations, or a theoretical frequency distribution of variations. Conclusion In order to enable the creation of large numbers of genomes, FIGG generates simulated sequences from known genomic variation and iteratively mutates each genome separately. The result is multiple whole genome sequences with unique variations that can primarily be used to provide different reference genomes, model heterogeneous populations, and can offer a standard test environment for new analysis algorithms or bioinformatics tools. PMID:24885193

  9. Comparative metagenomic and metatranscriptomic analyses of microbial communities in acid mine drainage

    PubMed Central

    Chen, Lin-xing; Hu, Min; Huang, Li-nan; Hua, Zheng-shuang; Kuang, Jia-liang; Li, Sheng-jin; Shu, Wen-sheng

    2015-01-01

    The microbial communities in acid mine drainage have been extensively studied to reveal their roles in acid generation and adaption to this environment. Lacking, however, are integrated community- and organism-wide comparative gene transcriptional analyses that could reveal the response and adaptation mechanisms of these extraordinary microorganisms to different environmental conditions. In this study, comparative metagenomics and metatranscriptomics were performed on microbial assemblages collected from four geochemically distinct acid mine drainage (AMD) sites. Taxonomic analysis uncovered unexpectedly high microbial biodiversity of these extremely acidophilic communities, and the abundant taxa of Acidithiobacillus, Leptospirillum and Acidiphilium exhibited high transcriptional activities. Community-wide comparative analyses clearly showed that the AMD microorganisms adapted to the different environmental conditions via regulating the expression of genes involved in multiple in situ functional activities, including low-pH adaptation, carbon, nitrogen and phosphate assimilation, energy generation, environmental stress resistance, and other functions. Organism-wide comparative analyses of the active taxa revealed environment-dependent gene transcriptional profiles, especially the distinct strategies used by Acidithiobacillus ferrivorans and Leptospirillum ferrodiazotrophum in nutrients assimilation and energy generation for survival under different conditions. Overall, these findings demonstrate that the gene transcriptional profiles of AMD microorganisms are closely related to the site physiochemical characteristics, providing clues into the microbial response and adaptation mechanisms in the oligotrophic, extremely acidic environments. PMID:25535937

  10. Multiple Amino Acid Sequence Alignment Nitrogenase Component 1: Insights into Phylogenetics and Structure-Function Relationships

    PubMed Central

    Howard, James B.; Kechris, Katerina J.; Rees, Douglas C.; Glazer, Alexander N.

    2013-01-01

    Amino acid residues critical for a protein's structure-function are retained by natural selection and these residues are identified by the level of variance in co-aligned homologous protein sequences. The relevant residues in the nitrogen fixation Component 1 α- and β-subunits were identified by the alignment of 95 protein sequences. Proteins were included from species encompassing multiple microbial phyla and diverse ecological niches as well as the nitrogen fixation genotypes, anf, nif, and vnf, which encode proteins associated with cofactors differing at one metal site. After adjusting for differences in sequence length, insertions, and deletions, the remaining >85% of the sequence co-aligned the subunits from the three genotypes. Six Groups, designated Anf, Vnf , and Nif I-IV, were assigned based upon genetic origin, sequence adjustments, and conserved residues. Both subunits subdivided into the same groups. Invariant and single variant residues were identified and were defined as “core” for nitrogenase function. Three species in Group Nif-III, Candidatus Desulforudis audaxviator, Desulfotomaculum kuznetsovii, and Thermodesulfatator indicus, were found to have a seleno-cysteine that replaces one cysteinyl ligand of the 8Fe:7S, P-cluster. Subsets of invariant residues, limited to individual groups, were identified; these unique residues help identify the gene of origin (anf, nif, or vnf) yet should not be considered diagnostic of the metal content of associated cofactors. Fourteen of the 19 residues that compose the cofactor pocket are invariant or single variant; the other five residues are highly variable but do not correlate with the putative metal content of the cofactor. The variable residues are clustered on one side of the cofactor, away from other functional centers in the three dimensional structure. Many of the invariant and single variant residues were not previously recognized as potentially critical and their identification provides the bases

  11. Chances and pitfalls of leaf wax biomarker analyses applied to fluvial sediment sequences - the example of a Holocene fluvial sediment-paleosol sequence from the upper Alazani River, eastern Georgia

    NASA Astrophysics Data System (ADS)

    von Suchodoletz, Hans; Bliedtner, Marcel; Zielhofer, Christoph; Faust, Dominik; Zech, Roland

    2016-04-01

    During the last decades, fluvial sediment sequences in many regions have intensively been studied to reconstruct Late Quaternary palaeoenvironmental and palaeohydrological conditions. However, up to now analyses of leaf wax biomarkers that are increasingly used to reconstruct paleoenvironmental and -climate conditions e.g. from lake sediments or loess-paleosol sequences were not systematically applied to Late Quaternary fluvial sediments. Given the ubiquitous distribution of fluvial sediment sequences on the earth's surface such investigations could potentially strongly enhance the knowledge about former environmental conditions in many regions. For this conceptual study we exemplarily analysed leaf wax biomarker (long-chain n-alkanes, n-alkanoic acids) in a fluvial sediment palaeosol sequence from the upper Alazani River in eastern Georgia to discuss general possibilities and pitfalls: Generally, biomarker records from fluvial archives can be divided into i) a catchment signal recorded in the fluvial sediment layers and ii) a local in-situ signal recorded in the intercalated paleosols. This offers the great chance to reconstruct paleoenvironmental conditions in both the whole catchment and at the sampling site. However, potential pitfalls are, for example, that inherited catchment signals can bias the in-situ signal from paleosols, while intermediate sediment storage in the catchment prior to sediment deposition and postsedimentary processes may alter the original catchment signal in the fluvial sediment layers. Thus, when applying leaf wax biomarker analyses to fluvial sediment sequences one has to be careful: The interpretation of the biomarker record strongly depends on the specific geomorphological and sedimentological conditions of the investigated site and of the catchment area.

  12. Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    2000-01-01

    A method for detecting and isolating a target sequence in a sample of nucleic acids is provided using a bifunctional hybridization probe capable of hybridizing to the target sequence that includes a detectable marker and a first complexing agent capable of forming a binding pair with a second complexing agent. A kit is also provided for detecting a target sequence in a sample of nucleic acids using a bifunctional hybridization probe according to this method.

  13. Analyses of Arabidopsis ecotypes reveal metabolic diversity to convert D-amino acids.

    PubMed

    Gördes, Dirk; Koch, Grit; Thurow, Kerstin; Kolukisaoglu, Uner

    2013-01-01

    For a long time D-enantiomers of proteinogenic L-amino acids were assumed to be physiologically irrelevant for plants. But there is growing evidence that D-amino acids (D-AAs) also fulfil important physiological functions in these organisms. However, the knowledge about the metabolic fate of D-AAs in plants is still scarce and more information about it is needed. To close this gap we established an optimized protocol for the processing and analysis of D- and L-AAs from large numbers of Arabidopsis lines. This included the application of 18 different D-AAs to seedlings, the extraction of free amino acids from the samples and the determination of 16 L-AAs and their corresponding D-enantiomers. To validate our approach we searched for genetic accessions with aberrant amino acid metabolism. Therefore we applied D-AAs on 17 ecotypes of Arabidopsis thaliana and analysed their free amino acid contents. These analyses confirmed the suitability of the system for the analysis of large sets of plant samples with enhanced velocity and improved accuracy. Furthermore, the resulting data led to the definition of standard amino acid profiles in response to D-AAs of Arabidopsis seedlings. Within these analyses the ecotype Landsberg erecta was found with aberrant metabolic patterns like drastically reduced capabilities to convert different D-AAs to D-alanine and D-glutamate. The presented experimental setup and results of this study offer starting points to dissect the metabolic pathway of D-AAs in plants.

  14. Factorial Moments Analyses Show a Characteristic Length Scale in DNA Sequences

    NASA Astrophysics Data System (ADS)

    Mohanty, A. K.; Narayana Rao, A. V. S. S.

    2000-02-01

    A unique feature of most of the DNA sequences, found through the factorial moments analysis, is the existence of a characteristic length scale around which the density distribution is nearly Poissonian. Above this point, the DNA sequences, irrespective of their intron contents, show long range correlations with a significant deviation from the Gaussian statistics, while, below this point, the DNA statistics are essentially Gaussian. The famous DNA walk representation is also shown to be a special case of the present analysis.

  15. Molecular analyses of the genus Ilex (Aquifoliaceae) in southern South America, evidence from AFLP and ITS sequence data.

    PubMed

    Gottlieb, Alexandra M; Giberti, Gustavo C; Poggio, Lidia

    2005-02-01

    In order to clarify the relationships among southern South American (sSA) representatives of the genus Ilex, an amplified fragment length polymorphism (AFLP) analysis was accomplished. In addition, the phylogenetic relationships of the species were studied using ribosomal internal transcribed spacer (ITS) sequence data alone and in combination with AFLP data, taking into account the possible existence of paralogous sequences and the influence of alignment parameters. To explore stability of phylogenetic hypotheses, a sensitivity analysis was performed using 15 indel-substitution models. Within each species assayed, the AFLPs allowed the recognition of several diagnostic bands. Furthermore, the AFLP analysis revealed that individuals belonging to the same morpho-species formed coherent clades. In addition, some cases of geographical association were noted. Studies on ITS sequences revealed divergence between data obtained herein and sequence data downloaded from GenBank. The sensitivity analyses yielded different interspecific hypotheses of relationships. Notwithstanding, analyses of the ITS data alone and in combination with AFLPs, rendered clades stable to variation in the analytical parameters. Topologies obtained for the AFLPs, the ITS data alone and the combined analyses, demonstrated the existence of a group formed by I. argentina, I. brasiliensis, I. brevicuspis, I. integerrima, and I. theezans, and that I. dumosa and I. paraguariensis were distantly related to the former. Incongruence with traditional taxonomical treatments was found.

  16. Amino acid sequence of horseshoe crab, Tachypleus tridentatus, striated muscle troponin C.

    PubMed

    Kobayashi, T; Kagami, O; Takagi, T; Konishi, K

    1989-05-01

    The amino acid sequence of troponin C obtained from horseshoe crab, Tachypleus tridentatus, striated muscle was determined by sequence analysis and alignments of chemically and enzymatically cleaved peptides. Troponin C is composed of 153 amino acid residues with a blocked N-terminus and contains no tryptophan or cysteine residue. The site I, one of the four Ca2+-binding sites, is considered to have lost its ability to bind Ca2+ owing to the replacements of certain amino acid residues.

  17. Phylogenetic analysis of beta-papillomaviruses as inferred from nucleotide and amino acid sequence data.

    PubMed

    Gottschling, Marc; Köhler, Anja; Stockfleth, Eggert; Nindl, Ingo

    2007-01-01

    Human papillomaviruses (HPV) of the beta-group seem to be involved in the pathogenesis of non-melanoma skin cancer. Papillomaviruses are host specific and are considered closely co-evolving with their hosts. Evolutionary incongruence between early genes and late genes has been reported among oncogenic genital alpha-papillomaviruses and considerably challenge phylogenetic reconstructions. We investigated the relationships of 29 beta-HPV (25 types plus four putative new types, subtypes, or variants) as inferred from codon aligned and amino acid sequence data of the genes E1, E2, E6, E7, L1, and L2 using likelihood, distance, and parsimony approaches. An analysis of a L1 fragment included additional nucleotide and amino acid sequences from seven non-human beta-papillomaviruses. Early genes and late genes evolution did not conflict significantly in beta-papillomaviruses based on partition homogeneity tests (p > or = 0.001). As inferred from the complete genome analyses, beta-papillomaviruses were monophyletic and segregated into four highly supported monophyletic assemblages corresponding to the species 1, 2, 3, and fused 4/5. They basically split into the species 1 and the remainder of beta-papillomaviruses, whose species 3, 4, and 5 constituted the sistergroup of species 2. beta-Papillomaviruses have been isolated from humans, apes, and monkeys, and phylogenetic analyses of the L1 fragment showed non-human papillomaviruses highly polyphyletic nesting within the HPV species. Thus, host and virus phylogenies were not congruent in beta-papillomaviruses, and multiple invasions across species borders may contribute (additionally to host-linked evolution) to their diversification.

  18. The Complete Chloroplast Genome Sequences of Five Epimedium Species: Lights into Phylogenetic and Taxonomic Analyses

    PubMed Central

    Zhang, Yanjun; Du, Liuwen; Liu, Ao; Chen, Jianjun; Wu, Li; Hu, Weiming; Zhang, Wei; Kim, Kyunghee; Lee, Sang-Choon; Yang, Tae-Jin; Wang, Ying

    2016-01-01

    Epimedium L. is a phylogenetically and economically important genus in the family Berberidaceae. We here sequenced the complete chloroplast (cp) genomes of four Epimedium species using Illumina sequencing technology via a combination of de novo and reference-guided assembly, which was also the first comprehensive cp genome analysis on Epimedium combining the cp genome sequence of E. koreanum previously reported. The five Epimedium cp genomes exhibited typical quadripartite and circular structure that was rather conserved in genomic structure and the synteny of gene order. However, these cp genomes presented obvious variations at the boundaries of the four regions because of the expansion and contraction of the inverted repeat (IR) region and the single-copy (SC) boundary regions. The trnQ-UUG duplication occurred in the five Epimedium cp genomes, which was not found in the other basal eudicotyledons. The rapidly evolving cp genome regions were detected among the five cp genomes, as well as the difference of simple sequence repeats (SSR) and repeat sequence were identified. Phylogenetic relationships among the five Epimedium species based on their cp genomes showed accordance with the updated system of the genus on the whole, but reminded that the evolutionary relationships and the divisions of the genus need further investigation applying more evidences. The availability of these cp genomes provided valuable genetic information for accurately identifying species, taxonomy and phylogenetic resolution and evolution of Epimedium, and assist in exploration and utilization of Epimedium plants. PMID:27014326

  19. Comprehensive Functional Analyses of Expressed Sequence Tags in Common Wheat (Triticum aestivum)

    PubMed Central

    Manickavelu, Alagu; Kawaura, Kanako; Oishi, Kazuko; Shin-I, Tadasu; Kohara, Yuji; Yahiaoui, Nabila; Keller, Beat; Abe, Reina; Suzuki, Ayako; Nagayama, Taishi; Yano, Kentaro; Ogihara, Yasunari

    2012-01-01

    About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37 138 contigs and 215 199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics. PMID:22334568

  20. Isotopic and molecular analyses of hydrocarbons and monocarboxylic acids of the Murchison meteorite

    NASA Technical Reports Server (NTRS)

    Krishnamurthy, R. V.; Epstein, S.; Cronin, John R.; Pizzarello, Sandra; Yuen, George U.

    1992-01-01

    The monocarboxylic acids and hydrocarbons of the Murchison meteorite (CM2) were isolated for isotropic analysis. The nonvolatile hydrocarbons were analyzed as crude methanol and benzene-methanol extracts and also after separation by silica gel chromatography into predominantly aliphatic, aromatic, and polar hydrocarbon fractions. The volatile hydrocarbons were obtained after progressive decomposition of the meteorite matrix by freeze-thaw, hot water, and acid treatment. Molecular analyses of the aromatic hydrocarbons showed them to comprise a complex suite of compounds in which pyrene, fluoranthene, phenanthrene, and acenaphthene were the most abundant components, a result similar to earlier analyses. The polar hydrocarbons also comprise a very complex mixture in which aromatic ketones, nitrogen, and sulfur heterocycles were identified. The monocarboxylic acids, aliphatic, aromatic, and polar hydrocarbons, and the indigenous volatile hydrocarbons were found to be D-rich. The deuterium enrichment observed in these compounds is suggestive. In two separate analyses, the delta-D values of the nonvolatile hydrocarbons were observed to increase in the following order: aliphatic-aromatic-polar. This finding is consistent with an early solar system or parent body conversion of aromatic to aliphatic compounds as well as the suggestion of pyrolytic formation of aromatic from aliphatic compounds.

  1. Analyses of DNA Base Sequences for Eukaryotes in Terms of Power Spectrum Method

    NASA Astrophysics Data System (ADS)

    Isohata, Yasuhiko; Hayashi, Masaki

    2005-02-01

    By adopting a power spectrum method we have analyzed long-range correlations in the gene base sequences, exons and introns for five or six eukaryote species. As a measure of the long-range correlations, we have used an exponent α in 1/fα, which is an approximation of a power spectrum in a low-frequency region. We have analyzed frequency distributions of α and the dependence of its average values <α> on the sequence length for the five or six species, paying particular attention to the species dependence. We have shown that long-range correlations have been formed mainly due to the intron's elongation as well as by the sequence structures of introns acquired over the course of evolution.

  2. Whole genome sequence analyses of Xylella fastidiosa PD strains from different geographical regions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome sequences were determined for two Pierce’s disease (PD) causing Xylella fastidiosa (Xf) strains, one from Florida and one from Taiwan. The Florida strain was ATCC 35879, the type of strain used as a standard reference for related taxonomy research. By contrast, the Taiwan strain used was only...

  3. spads 1.0: a toolbox to perform spatial analyses on DNA sequence data sets.

    PubMed

    Dellicour, Simon; Mardulyn, Patrick

    2014-05-01

    SPADS 1.0 (for 'Spatial and Population Analysis of DNA Sequences') is a population genetic toolbox for characterizing genetic variability within and among populations from DNA sequences. In view of the drastic increase in genetic information available through sequencing methods, spads was specifically designed to deal with multilocus data sets of DNA sequences. It computes several summary statistics from populations or groups of populations, performs input file conversions for other population genetic programs and implements locus-by-locus and multilocus versions of two clustering algorithms to study the genetic structure of populations. The toolbox also includes two MATLAB and r functions, GDISPAL and GDIVPAL, to display differentiation and diversity patterns across landscapes. These functions aim to generate interpolating surfaces based on multilocus distance and diversity indices. In the case of multiple loci, such surfaces can represent a useful alternative to multiple pie charts maps traditionally used in phylogeography to represent the spatial distribution of genetic diversity. These coloured surfaces can also be used to compare different data sets or different diversity and/or distance measures estimated on the same data set.

  4. Trichomonas vaginalis acidic phospholipase A2: isolation and partial amino acid sequence.

    PubMed

    Escobedo-Guajardo, Brenda L; González-Salazar, Francisco; Palacios-Corona, Rebeca; Torres de la Cruz, Víctor M; Morales-Vallarta, Mario; Mata-Cárdenas, Benito D; Garza-González, Jesús N; Rivera-Silva, Gerardo; Vargas-Villarreal, Javier

    2013-12-01

    Sexually transmitted diseases are a major cause of acute disease worldwide, and trichomoniasis is the most common and curable disease, generating more than 170 million cases annually worldwide. Trichomonas vaginalis is the causal agent of trichomoniasis and has the ability to destroy in vitro cell monolayers of the vaginal mucosa, where the phospholipases A2 (PLA2) have been reported as potential virulence factors. These enzymes have been partially characterized from the subcellular fraction S30 of pathogenic T. vaginalis strains. The main objective of this study was to purify a phospholipase A2 from T. vaginalis, make a partial characterization, obtain a partial amino acid sequence, and determine its enzymatic participation as hemolytic factor causing lysis of erythrocytes. Trichomonas S30, RF30 and UFF30 sub-fractions from GT-15 strain have the capacity to hydrolyze [2-(14)C-PA]-PC at pH 6.0. Proteins from the UFF30 sub-fraction were separated by affinity chromatography into two eluted fractions with detectable PLA A2 activity. The EDTA-eluted fraction was analyzed by HPLC using on-line HPLC-tandem mass spectrometry and two protein peaks were observed at 8.2 and 13 kDa. Peptide sequences were identified from the proteins present in the eluted EDTA UFF30 fraction; bioinformatic analysis using Protein Link Global Server charged with T. vaginalis protein database suggests that eluted peptides correspond a putative ubiquitin protein in the 8.2 kDa fraction and a phospholipase preserved in the 13 kDa fraction. The EDTA-eluted fraction hydrolyzed [2-(14)C-PA]-PC lyses erythrocytes from Sprague-Dawley in a time and dose-dependent manner. The acidic hemolytic activity decreased by 84% with the addition of 100 μM of Rosenthal's inhibitor. PMID:24338313

  5. Trichomonas vaginalis acidic phospholipase A2: isolation and partial amino acid sequence.

    PubMed

    Escobedo-Guajardo, Brenda L; González-Salazar, Francisco; Palacios-Corona, Rebeca; Torres de la Cruz, Víctor M; Morales-Vallarta, Mario; Mata-Cárdenas, Benito D; Garza-González, Jesús N; Rivera-Silva, Gerardo; Vargas-Villarreal, Javier

    2013-12-01

    Sexually transmitted diseases are a major cause of acute disease worldwide, and trichomoniasis is the most common and curable disease, generating more than 170 million cases annually worldwide. Trichomonas vaginalis is the causal agent of trichomoniasis and has the ability to destroy in vitro cell monolayers of the vaginal mucosa, where the phospholipases A2 (PLA2) have been reported as potential virulence factors. These enzymes have been partially characterized from the subcellular fraction S30 of pathogenic T. vaginalis strains. The main objective of this study was to purify a phospholipase A2 from T. vaginalis, make a partial characterization, obtain a partial amino acid sequence, and determine its enzymatic participation as hemolytic factor causing lysis of erythrocytes. Trichomonas S30, RF30 and UFF30 sub-fractions from GT-15 strain have the capacity to hydrolyze [2-(14)C-PA]-PC at pH 6.0. Proteins from the UFF30 sub-fraction were separated by affinity chromatography into two eluted fractions with detectable PLA A2 activity. The EDTA-eluted fraction was analyzed by HPLC using on-line HPLC-tandem mass spectrometry and two protein peaks were observed at 8.2 and 13 kDa. Peptide sequences were identified from the proteins present in the eluted EDTA UFF30 fraction; bioinformatic analysis using Protein Link Global Server charged with T. vaginalis protein database suggests that eluted peptides correspond a putative ubiquitin protein in the 8.2 kDa fraction and a phospholipase preserved in the 13 kDa fraction. The EDTA-eluted fraction hydrolyzed [2-(14)C-PA]-PC lyses erythrocytes from Sprague-Dawley in a time and dose-dependent manner. The acidic hemolytic activity decreased by 84% with the addition of 100 μM of Rosenthal's inhibitor.

  6. The complete chloroplast DNA sequence of Eleutherococcus senticosus (Araliaceae); comparative evolutionary analyses with other three asterids.

    PubMed

    Yi, Dong-Keun; Lee, Hae-Lim; Sun, Byung-Yun; Chung, Mi Yoon; Kim, Ki-Joong

    2012-05-01

    This study reports the complete chloroplast (cp) DNA sequence of Eleutherococcus senticosus (GenBank: JN 637765), an endangered endemic species. The genome is 156,768 bp in length, and contains a pair of inverted repeat (IR) regions of 25,930 bp each, a large single copy (LSC) region of 86,755 bp and a small single copy (SSC) region of 18,153 bp. The structural organization, gene and intron contents, gene order, AT content, codon usage, and transcription units of the E. senticosus chloroplast genome are similar to that of typical land plant cp DNA. We aligned and analyzed the sequences of 86 coding genes, 19 introns and 113 intergenic spacers (IGS) in three different taxonomic hierarchies; Eleutherococcus vs. Panax, Eleutherococcus vs. Daucus, and Eleutherococcus vs. Nicotiana. The distribution of indels, the number of polymorphic sites and nucleotide diversity indicate that positional constraint is more important than functional constraint for the evolution of cp genome sequences in Asterids. For example, the intron sequences in the LSC region exhibited base substitution rates 5-11-times higher than that of the IR regions, while the intron sequences in the SSC region evolved 7-14-times faster than those in the IR region. Furthermore, the Ka/Ks ratio of the gene coding sequences supports a stronger evolutionary constraint in the IR region than in the LSC or SSC regions. Therefore, our data suggest that selective sweeps by base collection mechanisms more frequently eliminate polymorphisms in the IR region than in other regions. Chloroplast genome regions that have high levels of base substitutions also show higher incidences of indels. Thirty-five simple sequence repeat (SSR) loci were identified in the Eleutherococcus chloroplast genome. Of these, 27 are homopolymers, while six are di-polymers and two are tri-polymers. In addition to the SSR loci, we also identified 18 medium size repeat units ranging from 22 to 79 bp, 11 of which are distributed in the IGS or

  7. The Complete Chloroplast DNA Sequence of Eleutherococcus senticosus (Araliaceae); Comparative Evolutionary Analyses with Other Three Asterids

    PubMed Central

    Yi, Dong-Keun; Lee, Hae-Lim; Sun, Byung-Yun; Chung, Mi Yoon; Kim, Ki-Joong

    2012-01-01

    This study reports the complete chloroplast (cp) DNA sequence of Eleutherococcus senticosus (GenBank: JN 637765), an endangered endemic species. The genome is 156,768 bp in length, and contains a pair of inverted repeat (IR) regions of 25,930 bp each, a large single copy (LSC) region of 86,755 bp and a small single copy (SSC) region of 18,153 bp. The structural organization, gene and intron contents, gene order, AT content, codon usage, and transcription units of the E. senticosus chloroplast genome are similar to that of typical land plant cp DNA. We aligned and analyzed the sequences of 86 coding genes, 19 introns and 113 intergenic spacers (IGS) in three different taxonomic hierarchies; Eleutherococcus vs. Panax, Eleutherococcus vs. Daucus, and Eleutherococcus vs. Nicotiana. The distribution of indels, the number of polymorphic sites and nucleotide diversity indicate that positional constraint is more important than functional constraint for the evolution of cp genome sequences in Asterids. For example, the intron sequences in the LSC region exhibited base substitution rates 5-11-times higher than that of the IR regions, while the intron sequences in the SSC region evolved 7-14-times faster than those in the IR region. Furthermore, the Ka/Ks ratio of the gene coding sequences supports a stronger evolutionary constraint in the IR region than in the LSC or SSC regions. Therefore, our data suggest that selective sweeps by base collection mechanisms more frequently eliminate polymorphisms in the IR region than in other regions. Chloroplast genome regions that have high levels of base substitutions also show higher incidences of indels. Thirty-five simple sequence repeat (SSR) loci were identified in the Eleutherococcus chloroplast genome. Of these, 27 are homopolymers, while six are di-polymers and two are tri-polymers. In addition to the SSR loci, we also identified 18 medium size repeat units ranging from 22 to 79 bp, 11 of which are distributed in the IGS or

  8. tax and rex Sequences of bovine leukaemia virus from globally diverse isolates: rex amino acid sequence more variable than tax.

    PubMed

    McGirr, K M; Buehring, G C

    2005-02-01

    Bovine leukaemia virus (BLV) is an important agricultural problem with high costs to the dairy industry. Here, we examine the variation of the tax and rex genes of BLV. The tax and rex genes share 420 bases and have overlapping reading frames. The tax gene encodes a protein that functions as a transactivator of the BLV promoter, is required for viral replication, acts on cellular promoters, and is responsible for oncogenesis. The rex facilitates the export of viral mRNAs from the nucleus and regulates transcription. We have sequenced five new isolates of the tax/rex gene. We examined the five new and three previously published tax/rex DNA and predicted amino acid sequences of BLV isolates from cattle in representative regions worldwide. The highest variation among nucleic acid sequences for tax and rex was 7% and 5%, respectively; among predicted amino acid sequences for Tax and Rex, 9% and 11%, respectively. Significantly more nucleotide changes resulted in predicted amino acid changes in the rex gene than in the tax gene (P < or = 0.0006). This variability is higher than previously reported for any region of the viral genome. This research may also have implications for the development of Tax-based vaccines. PMID:15702995

  9. A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences.

    PubMed Central

    Blais, B W; Turner, G; Sooknanan, R; Malek, L T

    1997-01-01

    A nucleic acid sequence-based amplification system primarily targeting mRNA from the Listeria monocytogenes hlyA gene was developed. This system enabled the detection of low numbers (< 10 CFU/g) of L. monocytogenes cells inoculated into a variety of dairy and egg products after 48 h of enrichment in modified listeria enrichment broth. PMID:8979357

  10. Molecular Characterization and Variation of the Broad bean wilt virus 2 Isolates Based on Analyses of Complete Genome Sequences.

    PubMed

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Lee, Ye-Ji; Seo, Jang-Kyun; Kim, Jeong-Soo; Kim, Kook-Hyung; Cha, Byeongjin; Choi, Hong-Soo

    2013-12-01

    The full-genome sequences of fourteen isolates of Broad bean wilt virus 2 (BBWV2), collected from broad bean, pea, spinach, bell pepper and paprika plants in Korea during the years 2006-2012, were determined and analyzed comparatively along with fifteen previously reported BBWV2 genome sequences. Sequence analyses showed that RNA-1 and RNA-2 sequences of BBWV2 Korean isolates consisted of 5950-5956 and 3568-3604 nucleotides, respectively. Full-length genome sequence-based phylogenetic analyses revealed that the BBWV2 Korean isolates could be divided into three major groups comprising GS-I (isolates BB2 and RP7) along with isolate IP, GS-II (isolates BB5, P2, P3 and RP3) along with isolate B935, and GS-III including 16 BBWV2 Korean isolates. Interestingly, GS-III appears to be newly emerged and predominant in Korea. Recombination analyses identified two recombination events in the analyzed BBWV2 population: one in the RNA-1 of isolate K and another one in the RNA-2 of isolate XJ14-3. However, no recombination events were detected in the other 21 Korean isolates. On the other hand, out of 29 BBWV2 isolates, 16 isolates were found to be reassortants, of which each RNA segment (i.e. RNA1 and RNA2) was originated from different parental isolates. Our findings suggested that reassortment rather than recombination is a major evolutionary force in the genetic diversification of BBWV population in Korea. PMID:25288968

  11. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1998-01-01

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration.

  12. Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1998-03-24

    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. 14 figs.

  13. Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens.

    PubMed

    Chen, Jie; Moinard, Magalie; Xu, Jianping; Wang, Shouxian; Foulongne-Oriol, Marie; Zhao, Ruilin; Hyde, Kevin D; Callac, Philippe

    2016-01-01

    The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated. PMID:27228131

  14. Phylogeny of the Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analyses.

    PubMed

    Siering, P L; Ghiorse, W C

    1996-01-01

    Phase-contrast light microscopy revealed that only one of eight cultivated strains belonging to the Sphaerotilus-Leptothrix group of sheathed bacteria actually produced a sheath in standard growth media. Two Sphaerotilus natans strains produced branched cells, but other morphological characteristics that were used to identify these bacteria were consistent with previously published descriptions. Genomic fingerprints, which were obtained by performing PCR amplification with primers corresponding to enterobacterial repetitive intergenic consensus sequences, were useful for distinguishing between the genera Sphaerotilus and Leptothrix, as well as among individual strains. The complete 16S ribosomal DNA (rDNA) sequences of two strains of "Leptothrix discophora" (strains SP-6 and SS-1) were determined. In addition, partial sequences (approximately 300 nucleotides) of one strain of Leptothrix cholodnii (strain LMG 7171), an unidentified Leptothrix strain (strain NC-1), and four strains of Sphaerotilus natans (strains ATCC 13338T [T = type strain], ATCC 15291, ATCC 29329, and ATCC 29330) were determined. We found that two of the S. natans strains (ATCC 15291 and ATCC 13338T), which differed in morphology and in their genomic fingerprints, had identical sequences in the 300-nucleotide region sequenced. Both parsimony and distance matrix methods were used to infer the evolutionary relationships of the eight strains in a comparison of the 16S rDNA sequences of these organisms with 16S rDNA sequences obtained from ribosomal sequence databases. All of the strains clustered in the Rubrivivax subdivision of the beta subclass of the Proteobacteria, which confirmed previously published conclusions concerning selected individual strains. Additional analyses revealed that all of the S. natans strains clustered in one closely related group, while the Leptothrix strains clustered in two separate lineages that were approximately equidistant from the S. natans cluster. This finding

  15. Phylogeny of the Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analyses.

    PubMed

    Siering, P L; Ghiorse, W C

    1996-01-01

    Phase-contrast light microscopy revealed that only one of eight cultivated strains belonging to the Sphaerotilus-Leptothrix group of sheathed bacteria actually produced a sheath in standard growth media. Two Sphaerotilus natans strains produced branched cells, but other morphological characteristics that were used to identify these bacteria were consistent with previously published descriptions. Genomic fingerprints, which were obtained by performing PCR amplification with primers corresponding to enterobacterial repetitive intergenic consensus sequences, were useful for distinguishing between the genera Sphaerotilus and Leptothrix, as well as among individual strains. The complete 16S ribosomal DNA (rDNA) sequences of two strains of "Leptothrix discophora" (strains SP-6 and SS-1) were determined. In addition, partial sequences (approximately 300 nucleotides) of one strain of Leptothrix cholodnii (strain LMG 7171), an unidentified Leptothrix strain (strain NC-1), and four strains of Sphaerotilus natans (strains ATCC 13338T [T = type strain], ATCC 15291, ATCC 29329, and ATCC 29330) were determined. We found that two of the S. natans strains (ATCC 15291 and ATCC 13338T), which differed in morphology and in their genomic fingerprints, had identical sequences in the 300-nucleotide region sequenced. Both parsimony and distance matrix methods were used to infer the evolutionary relationships of the eight strains in a comparison of the 16S rDNA sequences of these organisms with 16S rDNA sequences obtained from ribosomal sequence databases. All of the strains clustered in the Rubrivivax subdivision of the beta subclass of the Proteobacteria, which confirmed previously published conclusions concerning selected individual strains. Additional analyses revealed that all of the S. natans strains clustered in one closely related group, while the Leptothrix strains clustered in two separate lineages that were approximately equidistant from the S. natans cluster. This finding

  16. Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens

    PubMed Central

    Chen, Jie; Moinard, Magalie; Xu, Jianping; Wang, Shouxian; Foulongne-Oriol, Marie; Zhao, Ruilin; Hyde, Kevin D.; Callac, Philippe

    2016-01-01

    The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated. PMID:27228131

  17. The amino acid sequence of elephant (Elephas maximus) myoglobin and the phylogeny of Proboscidea.

    PubMed

    Dene, H; Goodman, M; Romero-Herrera, A E

    1980-02-13

    The complete amino acid sequence of skeletal myoglobin from the Asian elephant (Elephas maximus) is reported. The functional significance of variations seen when this sequence is compared with that of sperm whale myoglobin is explored in the light of the crystallographic model available for the latter molecule. The phylogenetic implications of the elephant myoglobin amino acid sequence are evaluated by using the maximum parsimony technique. A similar analysis is also presented which incorporates all of the proteins sequenced from the elephant. These results are discussed with respect to current views on proboscidean phylogeny.

  18. Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus.

    PubMed

    Reddy, Puli Chandramouli; Sinha, Ishani; Kelkar, Ashwin; Habib, Farhat; Pradhan, Saurabh J; Sukumar, Raman; Galande, Sanjeev

    2015-12-01

    The Asian elephant Elephas maximus and the African elephant Loxodonta africana that diverged 5-7 million years ago exhibit differences in their physiology, behaviour and morphology. A comparative genomics approach would be useful and necessary for evolutionary and functional genetic studies of elephants. We performed sequencing of E. maximus and map to L. africana at ~15X coverage. Through comparative sequence analyses, we have identified Asian elephant specific homozygous, non-synonymous single nucleotide variants (SNVs) that map to 1514 protein coding genes, many of which are involved in olfaction. We also present the first report of a high-coverage transcriptome sequence in E. maximus from peripheral blood lymphocytes. We have identified 103 novel protein coding transcripts and 66-long non-coding (lnc)RNAs. We also report the presence of 181 protein domains unique to elephants when compared to other Afrotheria species. Each of these findings can be further investigated to gain a better understanding of functional differences unique to elephant species, as well as those unique to elephantids in comparison with other mammals. This work therefore provides a valuable resource to explore the immense research potential of comparative analyses of transcriptome and genome sequences in the Asian elephant.

  19. Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus.

    PubMed

    Reddy, Puli Chandramouli; Sinha, Ishani; Kelkar, Ashwin; Habib, Farhat; Pradhan, Saurabh J; Sukumar, Raman; Galande, Sanjeev

    2015-12-01

    The Asian elephant Elephas maximus and the African elephant Loxodonta africana that diverged 5-7 million years ago exhibit differences in their physiology, behaviour and morphology. A comparative genomics approach would be useful and necessary for evolutionary and functional genetic studies of elephants. We performed sequencing of E. maximus and map to L. africana at ~15X coverage. Through comparative sequence analyses, we have identified Asian elephant specific homozygous, non-synonymous single nucleotide variants (SNVs) that map to 1514 protein coding genes, many of which are involved in olfaction. We also present the first report of a high-coverage transcriptome sequence in E. maximus from peripheral blood lymphocytes. We have identified 103 novel protein coding transcripts and 66-long non-coding (lnc)RNAs. We also report the presence of 181 protein domains unique to elephants when compared to other Afrotheria species. Each of these findings can be further investigated to gain a better understanding of functional differences unique to elephant species, as well as those unique to elephantids in comparison with other mammals. This work therefore provides a valuable resource to explore the immense research potential of comparative analyses of transcriptome and genome sequences in the Asian elephant. PMID:26648035

  20. [Whole-sequence Analyses for 12 HBV C/D Recombinants from a Population in Tibet (China)].

    PubMed

    Liu, Tiezhu; Shen, Liping; Yin, Wenjiao; Wang, Feng; Wang, Fuzhen; Zhang, Guomin; Zheng, Hui; Dunzhu, Duoji; Bi, Shengli; Cui, Fuqiang

    2016-03-01

    We wished to undertake molecular genetic typing and evaluate recombinants of the hepatitis-B virus (HBV) in Tibet (China). Multistage random sampling was used to collect HBsAg-positive samples. Nested polymerase chain reactions were used to amplify the whole sequence of the HBV. DNAstar, MEGA6 and SimPlot were used to assemble sequences, create phylogenetic trees, and undertake recombination analyses. Twelve whole sequences of the HBV of a Tibetan population were collected using these methods. Results showed that all 12 strains were C/D recombinants. Nine of the recombinations were at nt750, and the other three at nt1526. Therefore, the 12 strains could be divided into two types of recombinants: C/Da and C/Db. Analyses of the sequence of the whole genome revealed that the 12 strains belonged to genotype C, and that the nucleotide distance was > 4% between the 12 strains and sub-genotypes C1 to C15 in Genbank. The most likely sub-genotype was C1. Individuals with C/Da were from central and northern Tibet (e.g., Lasa, Linzhi, Ali) and those with C/Db recombinants were from Shannan in southern Tibet. These data suggest that the two types of recombinants had a good distribution in Tibet. Also, they can provide important information for studies on HBV recombination, gene features, virus evolution, as well as the control and prevention of HBV infection in Tibet.

  1. Naked but not Hairless: the pitfalls of analyses of molecular adaptation based on few genome sequence comparisons.

    PubMed

    Delsuc, Frédéric; Tilak, Marie-Ka

    2015-02-20

    The naked mole-rat (Heterocephalus glaber) is the only rodent species that naturally lacks fur. Genome sequencing of this atypical rodent species recently shed light on a number of its morphological and physiological adaptations. More specifically, its hairless phenotype has been traced back to a single amino acid change (C397W) in the hair growth associated (HR) protein (or Hairless). By considering the available species diversity, we show that this specific position is in fact variable across mammals, including in the horse that was misleadingly reported to have the ancestral Cysteine. Moreover, by sequencing the corresponding HR exon in additional rodent species, we demonstrate that the C397W substitution is actually not a peculiarity of the naked mole-rat. Instead, this specific amino acid substitution is present in all hystricognath rodents investigated, which are all fully furred, including the naked mole-rat closest relative, the Damaraland mole-rat (Fukomys damarensis). Overall, we found no statistical correlation between amino acid changes at position 397 of the HR protein and reduced pilosity across the mammalian phylogeny. This demonstrates that this single amino acid change does not explain the naked mole-rat hairless phenotype. Our case study calls for caution before making strong claims regarding the molecular basis of phenotypic adaptation based on the screening of specific amino acid substitutions using only few model species in genome sequence comparisons. It also exposes the more general problem of the dilution of essential information in the supplementary material of genome papers thereby increasing the probability that misleading results will escape the scrutiny of editors, reviewers, and ultimately readers.

  2. Root of the Eukaryota tree as inferred from combined maximum likelihood analyses of multiple molecular sequence data.

    PubMed

    Arisue, Nobuko; Hasegawa, Masami; Hashimoto, Tetsuo

    2005-03-01

    Extensive studies aiming to establish the structure and root of the Eukaryota tree by phylogenetic analyses of molecular sequences have thus far not resulted in a generally accepted tree. To re-examine the eukaryotic phylogeny using alternative genes, and to obtain a more robust inference for the root of the tree as well as the relationship among major eukaryotic groups, we sequenced the genes encoding isoleucyl-tRNA and valyl-tRNA synthetases, cytosolic-type heat shock protein 90, and the largest subunit of RNA polymerase II from several protists. Combined maximum likelihood analyses of 22 protein-coding genes including the above four genes clearly demonstrated that Diplomonadida and Parabasala shared a common ancestor in the rooted tree of Eukaryota, but only when the fast-evolving sites were excluded from the original data sets. The combined analyses, together with recent findings on the distribution of a fused dihydrofolate reductase-thymidylate synthetase gene, narrowed the possible position of the root of the Eukaryota tree on the branch leading to Opisthokonta or to the common ancestor of Diplomonadida/Parabasala. However, the analyses did not agree with the position of the root located on the common ancestor of Opisthokonta and Amoebozoa, which was argued by Stechmann and Cavalier-Smith [Curr. Biol. 13:R665-666, 2003] based on the presence or absence of a three-gene fusion of the pyrimidine biosynthetic pathway: carbamoyl-phosphate synthetase II, dihydroorotase, and aspartate carbamoyltransferase. The presence of the three-gene fusion recently found in the Cyanidioschyzon merolae (Rhodophyta) genome sequence data supported our analyses against the Stechmann and Cavalier-Smith-rooting in 2003.

  3. Rtools: a web server for various secondary structural analyses on single RNA sequences.

    PubMed

    Hamada, Michiaki; Ono, Yukiteru; Kiryu, Hisanori; Sato, Kengo; Kato, Yuki; Fukunaga, Tsukasa; Mori, Ryota; Asai, Kiyoshi

    2016-07-01

    The secondary structures, as well as the nucleotide sequences, are the important features of RNA molecules to characterize their functions. According to the thermodynamic model, however, the probability of any secondary structure is very small. As a consequence, any tool to predict the secondary structures of RNAs has limited accuracy. On the other hand, there are a few tools to compensate the imperfect predictions by calculating and visualizing the secondary structural information from RNA sequences. It is desirable to obtain the rich information from those tools through a friendly interface. We implemented a web server of the tools to predict secondary structures and to calculate various structural features based on the energy models of secondary structures. By just giving an RNA sequence to the web server, the user can get the different types of solutions of the secondary structures, the marginal probabilities such as base-paring probabilities, loop probabilities and accessibilities of the local bases, the energy changes by arbitrary base mutations as well as the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp, which integrates software tools, CentroidFold, CentroidHomfold, IPKnot, CapR, Raccess, Rchange and RintD. PMID:27131356

  4. Rtools: a web server for various secondary structural analyses on single RNA sequences

    PubMed Central

    Hamada, Michiaki; Ono, Yukiteru; Kiryu, Hisanori; Sato, Kengo; Kato, Yuki; Fukunaga, Tsukasa; Mori, Ryota; Asai, Kiyoshi

    2016-01-01

    The secondary structures, as well as the nucleotide sequences, are the important features of RNA molecules to characterize their functions. According to the thermodynamic model, however, the probability of any secondary structure is very small. As a consequence, any tool to predict the secondary structures of RNAs has limited accuracy. On the other hand, there are a few tools to compensate the imperfect predictions by calculating and visualizing the secondary structural information from RNA sequences. It is desirable to obtain the rich information from those tools through a friendly interface. We implemented a web server of the tools to predict secondary structures and to calculate various structural features based on the energy models of secondary structures. By just giving an RNA sequence to the web server, the user can get the different types of solutions of the secondary structures, the marginal probabilities such as base-paring probabilities, loop probabilities and accessibilities of the local bases, the energy changes by arbitrary base mutations as well as the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp, which integrates software tools, CentroidFold, CentroidHomfold, IPKnot, CapR, Raccess, Rchange and RintD. PMID:27131356

  5. Complete nuclear ribosomal DNA sequence amplification and molecular analyses of Bangia (Bangiales, Rhodophyta) from China

    NASA Astrophysics Data System (ADS)

    Xu, Jiajie; Jiang, Bo; Chai, Sanming; He, Yuan; Zhu, Jianyi; Shen, Zonggen; Shen, Songdong

    2016-09-01

    Filamentous Bangia, which are distributed extensively throughout the world, have simple and similar morphological characteristics. Scientists can classify these organisms using molecular markers in combination with morphology. We successfully sequenced the complete nuclear ribosomal DNA, approximately 13 kb in length, from a marine Bangia population. We further analyzed the small subunit ribosomal DNA gene (nrSSU) and the internal transcribed spacer (ITS) sequence regions along with nine other marine, and two freshwater Bangia samples from China. Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences (00.003; 0-0.006, respectively) from each other, but high divergences (0.123-0.126; 0.198, respectively) from freshwater samples. An exception is the marine sample collected from Weihai, which shows high divergence from both other marine samples (0.063-0.065; 0.129, respectively) and the freshwater samples (0.097; 0.120, respectively). A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades, with the two marine sample clades containing Bangia spp. from North America, Europe, Asia, and Australia; and one freshwater clade, containing Bangia atropurpurea from North America and China.

  6. Facile Analysis and Sequencing of Linear and Branched Peptide Boronic Acids by MALDI Mass Spectrometry

    PubMed Central

    Crumpton, Jason; Zhang, Wenyu; Santos, Webster

    2011-01-01

    Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. PMID:21449540

  7. Evolution of an Enzyme from a Noncatalytic Nucleic Acid Sequence.

    PubMed

    Gysbers, Rachel; Tram, Kha; Gu, Jimmy; Li, Yingfu

    2015-01-01

    The mechanism by which enzymes arose from both abiotic and biological worlds remains an unsolved natural mystery. We postulate that an enzyme can emerge from any sequence of any functional polymer under permissive evolutionary conditions. To support this premise, we have arbitrarily chosen a 50-nucleotide DNA fragment encoding for the Bos taurus (cattle) albumin mRNA and subjected it to test-tube evolution to derive a catalytic DNA (DNAzyme) with RNA-cleavage activity. After only a few weeks, a DNAzyme with significant catalytic activity has surfaced. Sequence comparison reveals that seven nucleotides are responsible for the conversion of the noncatalytic sequence into the enzyme. Deep sequencing analysis of DNA pools along the evolution trajectory has identified individual mutations as the progressive drivers of the molecular evolution. Our findings demonstrate that an enzyme can indeed arise from a sequence of a functional polymer via permissive molecular evolution, a mechanism that may have been exploited by nature for the creation of the enormous repertoire of enzymes in the biological world today. PMID:26091540

  8. Development and application of new nucleic acid-based technologies for microbial community analyses in foods.

    PubMed

    Rudi, Knut; Nogva, Hege K; Moen, Birgitte; Nissen, Hilde; Bredholt, Sylvia; Møretrø, Trond; Naterstad, Kristine; Holck, Askild

    2002-09-15

    Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.

  9. Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

    PubMed Central

    Grant, P. T.; Reid, K. B. M.

    1968-01-01

    1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain. PMID:4866431

  10. Computer Simulation of the Determination of Amino Acid Sequences in Polypeptides

    ERIC Educational Resources Information Center

    Daubert, Stephen D.; Sontum, Stephen F.

    1977-01-01

    Describes a computer program that generates a random string of amino acids and guides the student in determining the correct sequence of a given protein by using experimental analytic data for that protein. (MLH)

  11. Analyses of MYMIV-induced transcriptome in Vigna mungo as revealed by next generation sequencing.

    PubMed

    Ganguli, Sayak; Dey, Avishek; Banik, Rahul; Kundu, Anirban; Pal, Amita

    2016-03-01

    Mungbean Yellow Mosaic Virus (MYMIV) is the viral pathogen that causes yellow mosaic disease to a number of legumes including Vigna mungo. VM84 is a recombinant inbred line resistant to MYMIV, developed in our laboratory through introgression of resistance trait from V. mungo line VM-1. Here we present the quality control passed transcriptome data of mock inoculated (control) and MYMIV-infected VM84, those have already been submitted in Sequence Read Archive (SRX1032950, SRX1082731) of NCBI. QC reports of FASTQ files generated by 'SeqQC V2.2' bioinformatics tool.

  12. Analyses of MYMIV-induced transcriptome in Vigna mungo as revealed by next generation sequencing

    PubMed Central

    Ganguli, Sayak; Dey, Avishek; Banik, Rahul; Kundu, Anirban; Pal, Amita

    2016-01-01

    Mungbean Yellow Mosaic Virus (MYMIV) is the viral pathogen that causes yellow mosaic disease to a number of legumes including Vigna mungo. VM84 is a recombinant inbred line resistant to MYMIV, developed in our laboratory through introgression of resistance trait from V. mungo line VM-1. Here we present the quality control passed transcriptome data of mock inoculated (control) and MYMIV-infected VM84, those have already been submitted in Sequence Read Archive (SRX1032950, SRX1082731) of NCBI. QC reports of FASTQ files generated by ‘SeqQC V2.2’ bioinformatics tool. PMID:26981413

  13. Complete Genome Sequence and Immunoproteomic Analyses of the Bacterial Fish Pathogen Streptococcus parauberis▿†

    PubMed Central

    Nho, Seong Won; Hikima, Jun-ichi; Cha, In Seok; Park, Seong Bin; Jang, Ho Bin; del Castillo, Carmelo S.; Kondo, Hidehiro; Hirono, Ikuo; Aoki, Takashi; Jung, Tae Sung

    2011-01-01

    Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae. PMID:21531805

  14. Quod erat demonstrandum? The mystery of experimental validation of apparently erroneous computational analyses of protein sequences

    PubMed Central

    Iyer, Lakshminarayan M; Aravind, L; Bork, Peer; Hofmann, Kay; Mushegian, Arcady R; Zhulin, Igor B; Koonin, Eugene V

    2001-01-01

    Background Computational predictions are critical for directing the experimental study of protein functions. Therefore it is paradoxical when an apparently erroneous computational prediction seems to be supported by experiment. Results We analyzed six cases where application of novel or conventional computational methods for protein sequence and structure analysis led to non-trivial predictions that were subsequently supported by direct experiments. We show that, on all six occasions, the original prediction was unjustified, and in at least three cases, an alternative, well-supported computational prediction, incompatible with the original one, could be derived. The most unusual cases involved the identification of an archaeal cysteinyl-tRNA synthetase, a dihydropteroate synthase and a thymidylate synthase, for which experimental verifications of apparently erroneous computational predictions were reported. Using sequence-profile analysis, multiple alignment and secondary-structure prediction, we have identified the unique archaeal 'cysteinyl-tRNA synthetase' as a homolog of extracellular polygalactosaminidases, and the 'dihydropteroate synthase' as a member of the β-lactamase-like superfamily of metal-dependent hydrolases. Conclusions In each of the analyzed cases, the original computational predictions could be refuted and, in some instances, alternative strongly supported predictions were obtained. The nature of the experimental evidence that appears to support these predictions remains an open question. Some of these experiments might signify discovery of extremely unusual forms of the respective enzymes, whereas the results of others could be due to artifacts. PMID:11790254

  15. The amino acid sequence of monal pheasant lysozyme and its activity.

    PubMed

    Araki, T; Matsumoto, T; Torikata, T

    1998-10-01

    The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).

  16. The amino acid sequence of monal pheasant lysozyme and its activity.

    PubMed

    Araki, T; Matsumoto, T; Torikata, T

    1998-10-01

    The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly). PMID:9836434

  17. cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins.

    PubMed

    Iwanami, Kentaro; Mita, Hajime; Yamamoto, Yasuhiko; Fujise, Yoshihiro; Yamada, Tadasu; Suzuki, Tomohiko

    2006-10-01

    We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level. PMID:16962803

  18. Studies on monotreme proteins. VII. Amino acid sequence of myoglobin from the platypus, Ornithoryhynchus anatinus.

    PubMed

    Fisher, W K; Thompson, E O

    1976-03-01

    Myoglobin isolated from skeletal muscle of the platypus contains 153 amino acid residues. The complete amino acid sequence has been determined following cleavage with cyanogen bromide and further digestion of the four fragments with trypsin, chymotrypsin, pepsin and thermolysin. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed 25 differences from human myoglobin and 24 from kangaroo myoglobin. Amino acid sequences in myoglobins are more conserved than sequences in the alpha- and beta-globin chains, and platypus myoglobin shows a similar number of variations in sequence to kangaroo myoglobin when compared with myoglobin of other species. The date of divergence of the platypus from other mammals was estimated at 102 +/- 31 million years, based on the number of amino acid differences between species and allowing for mutations during the evolutionary period. This estimate differs widely from the estimate given by similar treatment of the alpha- and beta-chain sequences and a constant rate of mutation of globin chains is not supported. PMID:962722

  19. Isotopic analyses of nitrogenous compounds from the Murchison meteorite: ammonia, amines, amino acids, and polar hydrocarbons

    NASA Technical Reports Server (NTRS)

    Pizzarello, S.; Feng, X.; Epstein, S.; Cronin, J. R.

    1994-01-01

    The combined volatile bases (ammonia, aliphatic amines, and possibly other bases), ammonia, amino acids, and polar hydrocarbons were prepared from the Murchison meteorite for isotopic analyses. The volatile bases were obtained by cryogenic transfer after acid-hydrolysis of a hot-water extract and analyzed by combined gas chromatography-mass spectrometry of pentafluoropropionyl derivatives. The aliphatic amines present in this preparation comprise a mixture that includes both primary and secondary isomers through C5 at a total concentration of > or = 100 nmoles g-1. As commonly observed for meteoritic organic compounds, almost all isomers through C5 are present, and the concentrations within homologous series decrease with increasing chain length. Ammonia was chromatographically separated from the other volatile bases and found at a concentration of 1.1-1.3 micromoles g-1 meteorite. The ammonia analyzed includes contributions from ammonium salts and the hydrolysis of extractable organic compounds, e.g., carboxamides. Stable isotope analyses showed the volatile bases to be substantially enriched in the heavier isotopes, relative to comparable terrestrial compounds delta D < or = +1221%; delta 13C = +22%; delta 15N = +93%). Ammonia, per se, was found to have a somewhat lower delta 15N value (+69%) than the total volatile bases; consequently, a higher delta 15N (>93%) can be inferred for the other bases, which include the amines. Solvent-extractable polar hydrocarbons obtained separately were found to be enriched in 15N (delta 15N = +104%). Total amino acids, prepared from a hydrolyzed hot-water extract by cation exchange chromatography, gave a delta 15N of +94%, a value in good agreement with that obtained previously. Nitrogen isotopic data are also given for amino acid fractions separated chromatographically. The delta 15N values of the Murchison soluble organic compounds analyzed to date fall within a rather narrow range (delta 15N = +94 +/- 8%), an observation

  20. Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

    DOE PAGES

    Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.; Ryken, Scott A.; Yost, Will; Jha, Ramesh K.; Patterson, Johnathan; Monnat, Raymond J.; Barlow, Steven B.; Starkenburg, Shawn R.; et al

    2015-09-23

    Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. Themore » nuclear genome of C. tobin is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. In conclusion, the Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.« less

  1. Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

    PubMed Central

    Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.; Ryken, Scott A.; Yost, Will; Jha, Ramesh K.; Patterson, Johnathan; Monnat, Raymond J.; Barlow, Steven B.; Starkenburg, Shawn R.; Cattolico, Rose Ann

    2015-01-01

    Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes. PMID:26397803

  2. Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

    SciTech Connect

    Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.; Ryken, Scott A.; Yost, Will; Jha, Ramesh K.; Patterson, Johnathan; Monnat, Raymond J.; Barlow, Steven B.; Starkenburg, Shawn R.; Cattolico, Rose Ann; Richardson, Paul M.

    2015-09-23

    Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. In conclusion, the Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.

  3. Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae).

    PubMed

    Hovde, Blake T; Deodato, Chloe R; Hunsperger, Heather M; Ryken, Scott A; Yost, Will; Jha, Ramesh K; Patterson, Johnathan; Monnat, Raymond J; Barlow, Steven B; Starkenburg, Shawn R; Cattolico, Rose Ann

    2015-01-01

    Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼ 40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two "red" RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.

  4. Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae).

    PubMed

    Hovde, Blake T; Deodato, Chloe R; Hunsperger, Heather M; Ryken, Scott A; Yost, Will; Jha, Ramesh K; Patterson, Johnathan; Monnat, Raymond J; Barlow, Steven B; Starkenburg, Shawn R; Cattolico, Rose Ann

    2015-01-01

    Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼ 40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two "red" RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes. PMID:26397803

  5. Genome-wide analyses of Epstein-Barr virus reveal conserved RNA structures and a novel stable intronic sequence RNA

    PubMed Central

    2013-01-01

    Background Epstein-Barr virus (EBV) is a human herpesvirus implicated in cancer and autoimmune disorders. Little is known concerning the roles of RNA structure in this important human pathogen. This study provides the first comprehensive genome-wide survey of RNA and RNA structure in EBV. Results Novel EBV RNAs and RNA structures were identified by computational modeling and RNA-Seq analyses of EBV. Scans of the genomic sequences of four EBV strains (EBV-1, EBV-2, GD1, and GD2) and of the closely related Macacine herpesvirus 4 using the RNAz program discovered 265 regions with high probability of forming conserved RNA structures. Secondary structure models are proposed for these regions based on a combination of free energy minimization and comparative sequence analysis. The analysis of RNA-Seq data uncovered the first observation of a stable intronic sequence RNA (sisRNA) in EBV. The abundance of this sisRNA rivals that of the well-known and highly expressed EBV-encoded non-coding RNAs (EBERs). Conclusion This work identifies regions of the EBV genome likely to generate functional RNAs and RNA structures, provides structural models for these regions, and discusses potential functions suggested by the modeled structures. Enhanced understanding of the EBV transcriptome will guide future experimental analyses of the discovered RNAs and RNA structures. PMID:23937650

  6. Multiple Genome Sequences of Important Beer-Spoiling Lactic Acid Bacteria

    PubMed Central

    Geissler, Andreas J.; Vogel, Rudi F.

    2016-01-01

    Seven strains of important beer-spoiling lactic acid bacteria were sequenced using single-molecule real-time sequencing. Complete genomes were obtained for strains of Lactobacillus paracollinoides, Lactobacillus lindneri, and Pediococcus claussenii. The analysis of these genomes emphasizes the role of plasmids as the genomic foundation of beer-spoiling ability. PMID:27795248

  7. Does more sequence data improve estimates of galliform phylogeny? Analyses of a rapid radiation using a complete data matrix

    PubMed Central

    Braun, Edward L.

    2014-01-01

    The resolution of rapid evolutionary radiations or “bushes” in the tree of life has been one of the most difficult and interesting problems in phylogenetics. The avian order Galliformes appears to have undergone several rapid radiations that have limited the resolution of prior studies and obscured the position of taxa important both agriculturally and as model systems (chicken, turkey, Japanese quail). Here we present analyses of a multi-locus data matrix comprising over 15,000 sites, primarily from nuclear introns but also including three mitochondrial regions, from 46 galliform taxa with all gene regions sampled for all taxa. The increased sampling of unlinked nuclear genes provided strong bootstrap support for all but a small number of relationships. Coalescent-based methods to combine individual gene trees and analyses of datasets that are independent of published data indicated that this well-supported topology is likely to reflect the galliform species tree. The inclusion or exclusion of mitochondrial data had a limited impact upon analyses upon analyses using either concatenated data or multispecies coalescent methods. Some of the key phylogenetic findings include support for a second major clade within the core phasianids that includes the chicken and Japanese quail and clarification of the phylogenetic relationships of turkey. Jackknifed datasets suggested that there is an advantage to sampling many independent regions across the genome rather than obtaining long sequences for a small number of loci, possibly reflecting the differences among gene trees that differ due to incomplete lineage sorting. Despite the novel insights we obtained using this increased sampling of gene regions, some nodes remain unresolved, likely due to periods of rapid diversification. Resolving these remaining groups will likely require sequencing a very large number of gene regions, but our analyses now appear to support a robust backbone for this order. PMID:24795852

  8. Computational Analyses of Simple Sequence Repeats on Human Tissue Specific Genes Promoters

    NASA Astrophysics Data System (ADS)

    FeiFei, Zhao; XiuJun, Gong; XinMi, Liu; LiFeng, Dong

    Promoter region of gene closely related with tissue specific expression and SSRs (simple sequence repeats) have been shown to have a variety of effects on an organism. This paper used a heuristic method to find SSRs and compared the most frequently SSRs on promoter region of both human tissues specific genes and human housekeeping genes. We used kidney and testis tissue as examples to show the final results. Especially, we found that (AGG)n is kidney specific SSR and (GCG)n is testis specific SSR. We also analyzed the SSRs frequency density distribution on different promoter regions of both tissue specific genes and housekeeping genes, and we found the density of housekeeping genes on core-promoter region is much higher than on other promoter regions.

  9. Novel evolutionary lineages in Labeobarbus (Cypriniformes; Cyprinidae) based on phylogenetic analyses of mtDNA sequences.

    PubMed

    Beshera, Kebede A; Harris, Phillip M; Mayden, Richard L

    2016-01-01

    Phylogenetic relationships within Labeobarbus, the large-sized hexaploid cyprinids, were examined using cytochrome b gene sequences from a broad range of geographic localities and multiple taxa. Maximum likelihood and Bayesian methods revealed novel lineages from previously unsampled drainages in central (Congo River), eastern (Genale River) and southeastern (Revue and Mussapa Grande rivers) Africa. Relationships of some species of Varicorhinus in Africa (excluding 'V.' maroccanus) render Labeobarbus as paraphyletic. 'Varicorhinus' beso, 'V.' jubae, 'V.' mariae, 'V.' nelspruitensis, and 'V.' steindachneri are transferred to Labeobarbus. Bayesian estimation of time to most recent common ancestor suggested that Labeobarbus originated in the Late Miocene while lineage diversification began during the Late Miocene-Early Pliocene and continued to the late Pleistocene. The relationships presented herein provide phylogenetic resolution within Labeobarbus and advances our knowledge of genetic diversity within the lineage as well as provides some interesting insight into the hydrographic and geologic history of Africa. PMID:27394501

  10. Acinetobacter seifertii Isolated from China: Genomic Sequence and Molecular Epidemiology Analyses.

    PubMed

    Yang, Yunxing; Wang, Jianfeng; Fu, Ying; Ruan, Zhi; Yu, Yunsong

    2016-03-01

    Clinical infections caused by Acinetobacter spp. have increasing public health concerns because of their global occurrence and ability to acquire multidrug resistance. Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex encompasses A. calcoaceticus, A. baumannii, A. pittii (formerly genomic species 3), and A nosocomial (formerly genomic species 13TU), which are predominantly responsible for clinical pathogenesis in the Acinetobacter genus. In our previous study, a putative novel species isolated from 385 non-A. baumannii spp. strains based on the rpoB gene phylogenetic tree was reported. Here, the putative novel species was identified as A. seifertii based on the whole-genome phylogenetic tree. A. seifertii was recognized as a novel member of the ACB complex and close to A. baumannii and A. nosocomials. Furthermore, we studied the characteristics of 10 A. seifertii isolates, which were distributed widely in 6 provinces in China and mainly caused infections in the elderly or children. To define the taxonomic status and characteristics, the biochemical reactions, antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and whole-genome sequence analysis were performed. The phenotypic characteristics failed to distinguish A. serfertii from other species in the ACB complex. Most of the A. seifertii isolates were susceptible to antibiotics commonly used for nosocomial Acinetobacter spp. infections, but one isolate (strain A362) was resistant to ampicillin/sulbactam, ceftazidime and amikacin. The different patterns of MLST and PFGE suggested that the 10 isolates were not identical and lacked clonal relatedness. Our study reported for the first time the molecular epidemiological and genomic features of widely disseminated A. seifertii in China. These observations could enrich the knowledge of infections caused by non-A. baumannii and may provide a scientific basis for future clinical treatment. PMID

  11. Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

    PubMed Central

    1990-01-01

    The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation. PMID:2202734

  12. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  13. Draft Genome Sequences of Two Novel Acidimicrobiaceae Members from an Acid Mine Drainage Biofilm Metagenome

    PubMed Central

    Pinto, Ameet J.; Sharp, Jonathan O.; Yoder, Michael J.

    2016-01-01

    Bacteria belonging to the family Acidimicrobiaceae are frequently encountered in heavy metal-contaminated acidic environments. However, their phylogenetic and metabolic diversity is poorly resolved. We present draft genome sequences of two novel and phylogenetically distinct Acidimicrobiaceae members assembled from an acid mine drainage biofilm metagenome. PMID:26769942

  14. Complete Genome Sequence of Streptomyces clavuligerus F613-1, an Industrial Producer of Clavulanic Acid.

    PubMed

    Cao, Guangxiang; Zhong, Chuanqing; Zong, Gongli; Fu, Jiafang; Liu, Zhong; Zhang, Guimin; Qin, Ronghuo

    2016-01-01

    Streptomyces clavuligerus strain F613-1 is an industrial strain with high-yield clavulanic acid production. In this study, the complete genome sequence of S. clavuligerus strain F613-1 was determined, including one linear chromosome and one linear plasmid, carrying numerous sets of genes involving in the biosynthesis of clavulanic acid.

  15. Complete Genome Sequence of Streptomyces clavuligerus F613-1, an Industrial Producer of Clavulanic Acid.

    PubMed

    Cao, Guangxiang; Zhong, Chuanqing; Zong, Gongli; Fu, Jiafang; Liu, Zhong; Zhang, Guimin; Qin, Ronghuo

    2016-01-01

    Streptomyces clavuligerus strain F613-1 is an industrial strain with high-yield clavulanic acid production. In this study, the complete genome sequence of S. clavuligerus strain F613-1 was determined, including one linear chromosome and one linear plasmid, carrying numerous sets of genes involving in the biosynthesis of clavulanic acid. PMID:27660792

  16. Complete Genome Sequence of Streptomyces clavuligerus F613-1, an Industrial Producer of Clavulanic Acid

    PubMed Central

    Zhong, Chuanqing; Zong, Gongli; Fu, Jiafang; Liu, Zhong; Zhang, Guimin; Qin, Ronghuo

    2016-01-01

    Streptomyces clavuligerus strain F613-1 is an industrial strain with high-yield clavulanic acid production. In this study, the complete genome sequence of S. clavuligerus strain F613-1 was determined, including one linear chromosome and one linear plasmid, carrying numerous sets of genes involving in the biosynthesis of clavulanic acid. PMID:27660792

  17. Discrimination of prey species of juvenile swordfish Xiphias gladius (Linnaeus, 1758) using signature fatty acid analyses

    NASA Astrophysics Data System (ADS)

    Young, Jock W.; Guest, Michaela A.; Lansdell, Matt; Phleger, Charles F.; Nichols, Peter D.

    2010-07-01

    Signature lipid and fatty acid analysis were used to discriminate the diet of swordfish ( Xiphias gladius, orbital fork length: 60-203 cm) from waters off eastern Australia. The fatty acid (FA) composition of a range of known prey (squid, myctophids, and other fishes) of swordfish, taken from stomach samples and from net tows, was compared with that of the white muscle tissue (WMT) of swordfish from the same region. Swordfish muscle was lipid rich (average 24-42% dry weight), as was the skeleton (28-41%). The robustness of the approach was also tested by comparison against a key squid prey species that was collected and stored using different protocols: (i) fresh frozen, (ii) fresh frozen, then thawed, and (iii) stomach content collection. The FA profiles were generally similar, with the ratio of docosahexaenoic acid (DHA) and palmitic acid (16:0) in particular showing no significant difference. Major fatty acids in swordfish WMT were 18:1ω9c, 16:0, 22:6ω3, and 18:0. Multidimensional scaling showed that the swordfish WMT grouped closely with small fish prey including myctophids, and not with squid. Squid contained markedly higher 22:6ω3 than swordfish. Individual prey species of the myctophidae could also be separated by the same technique. These results were supported by traditional stomach content analyses (SCA) that showed fish were the dominant prey for small swordfish sampled from southern waters whereas squid were the main prey in more northern waters, matching the FA patterns we found for the two regions. We propose that where general diet patterns are established, signature FA analysis has good potential to compliment or in some cases, replace temporal and spatial monitoring of trophic pathways for swordfish and other marine species.

  18. Reprint of "Sequence and phylogenetic analyses of novel totivirus-like double-stranded RNAs from field-collected powdery mildew fungi".

    PubMed

    Kondo, Hideki; Hisano, Sakae; Chiba, Sotaro; Maruyama, Kazuyuki; Andika, Ida Bagus; Toyoda, Kazuhiro; Fujimori, Fumihiro; Suzuki, Nobuhiro

    2016-07-01

    The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a -1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of

  19. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

    PubMed Central

    Wang, Lijie; Zhang, Tong; Watson, David G.; Silva, Ana Marta; Coombs, Graham H.

    2015-01-01

    Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts. PMID:26368322

  20. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism.

    PubMed

    Westrop, Gareth D; Williams, Roderick A M; Wang, Lijie; Zhang, Tong; Watson, David G; Silva, Ana Marta; Coombs, Graham H

    2015-01-01

    Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

  1. Parvalbumins from coelacanth muscle. III. Amino acid sequence of the major component.

    PubMed

    Jauregui-Adell, J; Pechere, J F

    1978-09-26

    The primary structure of the major parvalbumin (pI = 4.52) from coelacanth muscle (Latimeria chalumnae) has been determined. Sequence analysis of the tryptic peptides, in some cases obtained with beta-trypsin, accounts for the total amino acid content of the protein. Chymotryptic peptides provide appropriate sequence overlaps, to complete the localization of the tryptic peptides. Examination of the amino acid sequence of this protein shows the typical structure of a beta-parvalbumin. Its position in the dendrogram of related calcium-binding proteins corresponds to that usually accepted for crossopterygians.

  2. Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify ABCB6 as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria

    PubMed Central

    Wang, Na; Wang, Chuan; Chen, Xuechao; Sheng, Donglai; Fu, Xi’an; See, Kelvin; Foo, Jia Nee; Low, Huiqi; Liany, Herty; Irwan, Ishak Darryl; Liu, Jian; Yang, Baoqi; Chen, Mingfei; Yu, Yongxiang; Yu, Gongqi; Niu, Guiye; You, Jiabao; Zhou, Yan; Ma, Shanshan; Wang, Ting; Yan, Xiaoxiao; Goh, Boon Kee; Common, John E. A.; Lane, Birgitte E.; Sun, Yonghu; Zhou, Guizhi; Lu, Xianmei; Wang, Zhenhua; Tian, Hongqing; Cao, Yuanhua; Chen, Shumin; Liu, Qiji; Liu, Jianjun; Zhang, Furen

    2014-01-01

    Background As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH. Methodology We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation. Results Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified ABCB6 as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of ABCB6 in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of ABCB6 in melanocytes and pigmentation. Given the involvement of ABCB6 mutations in coloboma, we performed ophthalmological examination of the DUH carriers of ABCB6 mutations and found ocular abnormalities in them. Conclusion Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma. PMID:24498303

  3. [Sequencing Analyses of the Hypervariable Region within the VP2 Gene of a Strain of the Aleutian Mink Disease Virus].

    PubMed

    Zhang, Lei; Hu, Bo; Bai, Xue; Zhang, Hailing; Zhao, Jianjun; Wang, Zhenjun; Ma, Fanshu; Yan, Xijun; Wu, Wei; Xu, Shujuan

    2015-05-01

    To analyze the molecular mechanisms of cross-host transmission of the Aleutian mink disease vi rus (ADV), the hypervariable region fragment of the VP2 gene of the ADV in Jilin Province (China) was amplified. Sequencing analyses showed diversity at residue 174 by comparison with other VP2 genes in GenBank. The phylogenetic tree indicated that the ADV-JL strain had a close relationship with the highly pathogenic strain from Denmark: ADV-K. Results implied that residue 174 may be associated with ADV infectivity. PMID:26470526

  4. Sequencing and computational analysis of complete genome sequences of Citrus yellow mosaic badna virus from acid lime and pummelo.

    PubMed

    Borah, Basanta K; Johnson, A M Anthony; Sai Gopal, D V R; Dasgupta, Indranil

    2009-08-01

    Citrus yellow mosaic badna virus (CMBV), a member of the Family Caulimoviridae, Genus Badnavirus, is the causative agent of Citrus mosaic disease in India. Although the virus has been detected in several citrus species, only two full-length genomes, one each from Sweet orange and Rangpur lime, are available in publicly accessible databases. In order to obtain a better understanding of the genetic variability of the virus in other citrus mosaic-affected citrus species, we performed the cloning and sequence analysis of complete genomes of CMBV from two additional citrus species, Acid lime and Pummelo. We show that CMBV genomes from the two hosts share high homology with previously reported CMBV sequences and hence conclude that the new isolates represent variants of the virus present in these species. Based on in silico sequence analysis, we predict the possible function of the protein encoded by one of the five ORFs.

  5. Amino acid sequence of anionic peroxidase from the windmill palm tree Trachycarpus fortunei.

    PubMed

    Baker, Margaret R; Zhao, Hongwei; Sakharov, Ivan Yu; Li, Qing X

    2014-12-10

    Palm peroxidases are extremely stable and have uncommon substrate specificity. This study was designed to fill in the knowledge gap about the structures of a peroxidase from the windmill palm tree Trachycarpus fortunei. The complete amino acid sequence and partial glycosylation were determined by MALDI-top-down sequencing of native windmill palm tree peroxidase (WPTP), MALDI-TOF/TOF MS/MS of WPTP tryptic peptides, and cDNA sequencing. The propeptide of WPTP contained N- and C-terminal signal sequences which contained 21 and 17 amino acid residues, respectively. Mature WPTP was 306 amino acids in length, and its carbohydrate content ranged from 21% to 29%. Comparison to closely related royal palm tree peroxidase revealed structural features that may explain differences in their substrate specificity. The results can be used to guide engineering of WPTP and its novel applications.

  6. Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

    PubMed Central

    Velours, J; Esparza, M; Hoppe, J; Sebald, W; Guerin, B

    1984-01-01

    The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans. Images Fig. 2. PMID:6323165

  7. TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations.

    PubMed

    Abascal, Federico; Zardoya, Rafael; Telford, Maximilian J

    2010-07-01

    We present TranslatorX, a web server designed to align protein-coding nucleotide sequences based on their corresponding amino acid translations. Many comparisons between biological sequences (nucleic acids and proteins) involve the construction of multiple alignments. Alignments represent a statement regarding the homology between individual nucleotides or amino acids within homologous genes. As protein-coding DNA sequences evolve as triplets of nucleotides (codons) and it is known that sequence similarity degrades more rapidly at the DNA than at the amino acid level, alignments are generally more accurate when based on amino acids than on their corresponding nucleotides. TranslatorX novelties include: (i) use of all documented genetic codes and the possibility of assigning different genetic codes for each sequence; (ii) a battery of different multiple alignment programs; (iii) translation of ambiguous codons when possible; (iv) an innovative criterion to clean nucleotide alignments with GBlocks based on protein information; and (v) a rich output, including Jalview-powered graphical visualization of the alignments, codon-based alignments coloured according to the corresponding amino acids, measures of compositional bias and first, second and third codon position specific alignments. The TranslatorX server is freely available at http://translatorx.co.uk.

  8. Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses.

    PubMed

    Ludyga, Natalie; Grünwald, Barbara; Azimzadeh, Omid; Englert, Sonja; Höfler, Heinz; Tapio, Soile; Aubele, Michaela

    2012-02-01

    Tissues used for clinical diagnostics are mostly formalin-fixed and paraffin-embedded (FFPE) which provides many advantages. However, the quality of the obtained nucleic acids (NA) is reduced and this turns out to be a challenge for further molecular analyses. Although the spectrum of analyses of NA extracted from FFPE tissue has increased, the standard operating procedures for NA isolation from old tissue blocks still need to be improved. Here, we compared the efficiency of different NA extraction methods, using FFPE tissues of variable age and origin, with respect to downstream analyses. Our study showed that the phenol-chloroform isoamyl alcohol (PCI) and the commercial Qiagen protocol yielded samples with highest purity. The PCI protocol delivered the longest amplicons even from samples from the 1970s. We developed a short (1 h) tissue lysis procedure that turned out to be highly time- and cost-effective when DNA quality was tested using single and multiplex PCR. Compared to a 1-day lysis-protocol, the amplicons were only 100 bp shorter. In addition, single-copy genes used in daily routine were successfully amplified from long-term stored FFPE samples following 1-h tissue-lysis. The RNA integrity numbers (RIN) determined on RNA isolated from FFPE tissues indicated degraded RNA; however, all RINs were above the generally agreed threshold of 1.4. We showed that, depending on the purpose of the analysis, NA retrieved from FFPE tissues older than 40 years may be successfully used for molecular analysis.

  9. Evolution and biogeography of Centaurea section Acrocentron inferred from nuclear and plastid DNA sequence analyses

    PubMed Central

    Font, Mònica; Garcia-Jacas, Núria; Vilatersana, Roser; Roquet, Cristina; Susanna, Alfonso

    2009-01-01

    Background and Aims Section Acrocentron of the genus Centaurea is one of the largest sections of Centaurea with approx. 100 species. The geographic distribution, centred in the Mediterranean, makes it an excellent example for studies of the biogeographic history of this biodiversity-rich region. Methods Plastid (trnH-psbA) and nuclear (ITS and ETS) DNA sequence analysis was used for phylogenetic reconstruction. Ancestral biogeographic patterns were inferred by dispersal-vicariance analysis (DIVA). Key Results The resulting phylogeny has implications for the sectional classification of Acrocentron and confirms merging sect. Chamaecyanus into Acrocentron as a subsection. Previous suggestions of an eastern Mediterranean origin of the group are confirmed. The main centres of diversification established in previous studies are now strongly supported. Expansion of the group in two different radiations that followed patently diverse paths is inferred. Conclusions Radiation followed two waves, widely separated in time scale. The oldest one, from Turkey to Greece and the northern Balkans and then to North Africa and Iberia, should be dated at the end of the Miocene in the Messinian period. It reached the Iberian Peninsula from the south, following a route that is landmarked by several relictic taxa in Sicily and North Africa. A later radiation during the Holocene interglacial periods followed, involving species from the north of the Balkan Peninsula, along a Eurasian pathway running from Central Iberia to the steppes of Kazakhstan. A generalized pattern of reticulation is also evident from the results, indicating past contacts between presently separated species. Molecular data also confirmed the extent of hybridization within Acrocentron and were successful in reconstructing the paleogeography of the section. PMID:19228702

  10. Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase. A new family of fungal peroxidases.

    PubMed

    Baunsgaard, L; Dalbøge, H; Houen, G; Rasmussen, E M; Welinder, K G

    1993-04-01

    Sequence analysis and cDNA cloning of Coprinus peroxidase (CIP) were undertaken to expand the understanding of the relationships of structure, function and molecular genetics of the secretory heme peroxidases from fungi and plants. Amino acid sequencing of Coprinus macrorhizus peroxidase, and cDNA sequencing of Coprinus cinereus peroxidase showed that the mature proteins are identical in amino acid sequence, 343 residues in size and preceded by a 20-residue signal peptide. Their likely identity to peroxidase from Arthromyces ramosus is discussed. CIP has an 8-residue, glycine-rich N-terminal extension blocked with a pyroglutamate residue which is absent in other fungal peroxidases. The presence of pyroglutamate, formed by cyclization of glutamine, and the finding of a minor fraction of a variant form lacking the N-terminal residue, indicate that signal peptidase cleavage is followed by further enzymic processing. CIP is 40-45% identical in amino-acid sequence to 11 lignin peroxidases from four fungal species, and 42-43% identical to the two known Mn-peroxidases. Like these white-rot fungal peroxidases, CIP has an additional segment of approximately 40 residues at the C-terminus which is absent in plant peroxidases. Although CIP is much more similar to horseradish peroxidase (HRP C) in substrate specificity, specific activity and pH optimum than to white-rot fungal peroxidases, the sequences of CIP and HRP C showed only 18% identity. Hence, CIP qualifies as the first member of a new family of fungal peroxidases. The nine invariant residues present in all plant, fungal and bacterial heme peroxidases are also found in CIP. The present data support the hypothesis that only one chromosomal CIP gene exists. In contrast, a large number of secretory plant and fungal peroxidases are expressed from several peroxidase gene clusters. Analyses of three batches of CIP protein and of 49 CIP clones revealed the existence of only two highly similar alleles indicating less

  11. Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin.

    PubMed

    Theerasilp, S; Hitotsuya, H; Nakajo, S; Nakaya, K; Nakamura, Y; Kurihara, Y

    1989-04-25

    The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 263, 11536-11539) was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor. PMID:2708331

  12. Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.

    PubMed Central

    Takio, K; Towatari, T; Katunuma, N; Teller, D C; Titani, K

    1983-01-01

    The amino acid sequences of rat liver lysosomal thiol endopeptidases, cathepsins B and H, are presented and compared with that of the plant thiol protease papain. The 252-residue sequence of cathepsin B and the 220-residue sequence of cathepsin H were determined largely by automated Edman degradation of their intact polypeptide chains and of the two chains of each enzyme generated by limited proteolysis. Subfragments of the chains were produced by enzymatic digestion and by chemical cleavage of methionyl and tryptophanyl bonds. Comparison of the amino acid sequences of cathepsins B and H with each other and with that of papain demonstrates a striking homology among their primary structures. Sequence identity is extremely high in regions which, according to the three-dimensional structure of papain, constitute the catalytic site. The results not only reveal the first structural features of mammalian thiol endopeptidases but also provide insight into the evolutionary relationships among plant and mammalian thiol proteases. PMID:6574504

  13. Complete cDNA and derived amino acid sequence of human factor V.

    PubMed Central

    Jenny, R J; Pittman, D D; Toole, J J; Kriz, R W; Aldape, R A; Hewick, R M; Kaufman, R J; Mann, K G

    1987-01-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. Images PMID:3110773

  14. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  15. NCI-60 whole exome sequencing and pharmacological CellMiner analyses.

    PubMed

    Reinhold, William C; Varma, Sudhir; Sousa, Fabricio; Sunshine, Margot; Abaan, Ogan D; Davis, Sean R; Reinhold, Spencer W; Kohn, Kurt W; Morris, Joel; Meltzer, Paul S; Doroshow, James H; Pommier, Yves

    2014-01-01

    Exome sequencing provides unprecedented insights into cancer biology and pharmacological response. Here we assess these two parameters for the NCI-60, which is among the richest genomic and pharmacological publicly available cancer cell line databases. Homozygous genetic variants that putatively affect protein function were identified in 1,199 genes (approximately 6% of all genes). Variants that are either enriched or depleted compared to non-cancerous genomes, and thus may be influential in cancer progression and differential drug response were identified for 2,546 genes. Potential gene knockouts are made available. Assessment of cell line response to 19,940 compounds, including 110 FDA-approved drugs, reveals ≈80-fold range in resistance versus sensitivity response across cell lines. 103,422 gene variants were significantly correlated with at least one compound (at p<0.0002). These include genes of known pharmacological importance such as IGF1R, BRAF, RAD52, MTOR, STAT2 and TSC2 as well as a large number of candidate genes such as NOM1, TLL2, and XDH. We introduce two new web-based CellMiner applications that enable exploration of variant-to-compound relationships for a broad range of researchers, especially those without bioinformatics support. The first tool, "Genetic variant versus drug visualization", provides a visualization of significant correlations between drug activity-gene variant combinations. Examples are given for the known vemurafenib-BRAF, and novel ifosfamide-RAD52 pairings. The second, "Genetic variant summation" allows an assessment of cumulative genetic variations for up to 150 combined genes together; and is designed to identify the variant burden for molecular pathways or functional grouping of genes. An example of its use is provided for the EGFR-ERBB2 pathway gene variant data and the identification of correlated EGFR, ERBB2, MTOR, BRAF, MEK and ERK inhibitors. The new tools are implemented as an updated web-based CellMiner version, for

  16. NCI-60 Whole Exome Sequencing and Pharmacological CellMiner Analyses

    PubMed Central

    Reinhold, William C.; Varma, Sudhir; Sousa, Fabricio; Sunshine, Margot; Abaan, Ogan D.; Davis, Sean R.; Reinhold, Spencer W.; Kohn, Kurt W.; Morris, Joel; Meltzer, Paul S.; Doroshow, James H.; Pommier, Yves

    2014-01-01

    Exome sequencing provides unprecedented insights into cancer biology and pharmacological response. Here we assess these two parameters for the NCI-60, which is among the richest genomic and pharmacological publicly available cancer cell line databases. Homozygous genetic variants that putatively affect protein function were identified in 1,199 genes (approximately 6% of all genes). Variants that are either enriched or depleted compared to non-cancerous genomes, and thus may be influential in cancer progression and differential drug response were identified for 2,546 genes. Potential gene knockouts are made available. Assessment of cell line response to 19,940 compounds, including 110 FDA-approved drugs, reveals ≈80-fold range in resistance versus sensitivity response across cell lines. 103,422 gene variants were significantly correlated with at least one compound (at p<0.0002). These include genes of known pharmacological importance such as IGF1R, BRAF, RAD52, MTOR, STAT2 and TSC2 as well as a large number of candidate genes such as NOM1, TLL2, and XDH. We introduce two new web-based CellMiner applications that enable exploration of variant-to-compound relationships for a broad range of researchers, especially those without bioinformatics support. The first tool, “Genetic variant versus drug visualization”, provides a visualization of significant correlations between drug activity-gene variant combinations. Examples are given for the known vemurafenib-BRAF, and novel ifosfamide-RAD52 pairings. The second, “Genetic variant summation” allows an assessment of cumulative genetic variations for up to 150 combined genes together; and is designed to identify the variant burden for molecular pathways or functional grouping of genes. An example of its use is provided for the EGFR-ERBB2 pathway gene variant data and the identification of correlated EGFR, ERBB2, MTOR, BRAF, MEK and ERK inhibitors. The new tools are implemented as an updated web-based Cell

  17. Characterization of the mechanism of prolonged adaptation to osmotic stress of Jeotgalibacillus malaysiensis via genome and transcriptome sequencing analyses

    PubMed Central

    Yaakop, Amira Suriaty; Chan, Kok-Gan; Ee, Robson; Lim, Yan Lue; Lee, Siew-Kim; Manan, Fazilah Abd; Goh, Kian Mau

    2016-01-01

    Jeotgalibacillus malaysiensis, a moderate halophilic bacterium isolated from a pelagic area, can endure higher concentrations of sodium chloride (NaCl) than other Jeotgalibacillus type strains. In this study, we therefore chose to sequence and assemble the entire J. malaysiensis genome. This is the first report to provide a detailed analysis of the genomic features of J. malaysiensis, and to perform genetic comparisons between this microorganism and other halophiles. J. malaysiensis encodes a native megaplasmid (pJeoMA), which is greater than 600 kilobases in size, that is absent from other sequenced species of Jeotgalibacillus. Subsequently, RNA-Seq-based transcriptome analysis was utilised to examine adaptations of J. malaysiensis to osmotic stress. Specifically, the eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) and KEGG (Kyoto Encyclopaedia of Genes and Genomes) databases were used to elucidate the overall effects of osmotic stress on the organism. Generally, saline stress significantly affected carbohydrate, energy, and amino acid metabolism, as well as fatty acid biosynthesis. Our findings also indicate that J. malaysiensis adopted a combination of approaches, including the uptake or synthesis of osmoprotectants, for surviving salt stress. Among these, proline synthesis appeared to be the preferred method for withstanding prolonged osmotic stress in J. malaysiensis. PMID:27641516

  18. Characterization of the mechanism of prolonged adaptation to osmotic stress of Jeotgalibacillus malaysiensis via genome and transcriptome sequencing analyses.

    PubMed

    Yaakop, Amira Suriaty; Chan, Kok-Gan; Ee, Robson; Lim, Yan Lue; Lee, Siew-Kim; Manan, Fazilah Abd; Goh, Kian Mau

    2016-01-01

    Jeotgalibacillus malaysiensis, a moderate halophilic bacterium isolated from a pelagic area, can endure higher concentrations of sodium chloride (NaCl) than other Jeotgalibacillus type strains. In this study, we therefore chose to sequence and assemble the entire J. malaysiensis genome. This is the first report to provide a detailed analysis of the genomic features of J. malaysiensis, and to perform genetic comparisons between this microorganism and other halophiles. J. malaysiensis encodes a native megaplasmid (pJeoMA), which is greater than 600 kilobases in size, that is absent from other sequenced species of Jeotgalibacillus. Subsequently, RNA-Seq-based transcriptome analysis was utilised to examine adaptations of J. malaysiensis to osmotic stress. Specifically, the eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) and KEGG (Kyoto Encyclopaedia of Genes and Genomes) databases were used to elucidate the overall effects of osmotic stress on the organism. Generally, saline stress significantly affected carbohydrate, energy, and amino acid metabolism, as well as fatty acid biosynthesis. Our findings also indicate that J. malaysiensis adopted a combination of approaches, including the uptake or synthesis of osmoprotectants, for surviving salt stress. Among these, proline synthesis appeared to be the preferred method for withstanding prolonged osmotic stress in J. malaysiensis. PMID:27641516

  19. Fad7 gene identification and fatty acids phenotypic variation in an olive collection by EcoTILLING and sequencing approaches.

    PubMed

    Sabetta, Wilma; Blanco, Antonio; Zelasco, Samanta; Lombardo, Luca; Perri, Enzo; Mangini, Giacomo; Montemurro, Cinzia

    2013-08-01

    The ω-3 fatty acid desaturases (FADs) are enzymes responsible for catalyzing the conversion of linoleic acid to α-linolenic acid localized in the plastid or in the endoplasmic reticulum. In this research we report the genotypic and phenotypic variation of Italian Olea europaea L. germoplasm for the fatty acid composition. The phenotypic oil characterization was followed by the molecular analysis of the plastidial-type ω-3 FAD gene (fad7) (EC 1.14.19), whose full-length sequence has been here identified in cultivar Leccino. The gene consisted of 2635 bp with 8 exons and 5'- and 3'-UTRs of 336 and 282 bp respectively, and showed a high level of heterozygousity (1/110 bp). The natural allelic variation was investigated both by a LiCOR EcoTILLING assay and the PCR product direct sequencing. Only three haplotypes were identified among the 96 analysed cultivars, highlighting the strong degree of conservation of this gene. PMID:23685785

  20. An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification.

    PubMed

    Smith, Matthew C; Steimle, George; Ivanov, Stan; Holly, Mark; Fries, David P

    2007-08-29

    A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board/Micro Electro Mechanical Systems (PCB/MEMS) reaction detection/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled in planar geometry, rolled into a tubular format and packaged with custom control electronics to form the hand-held reactor. Positive or negative results for each reaction are displayed to the user using an LED interface. Reaction data is stored in FLASH memory for retrieval via an in-built USB connection. Operating on one disposable 3 V lithium battery >12, 60 min reactions can be performed. Maximum dimensions of the system are 150 mm (h) x 48 mm (d) x 40 mm (w), the total instrument weight (with battery) is 140 g. The system produces comparable results to laboratory instrumentation when performing a real-time nucleic acid sequence-based amplification (NASBA) reaction, and also displayed comparable precision, accuracy and resolution to laboratory-based real-time nucleic acid amplification instrumentation. A good linear response (R2 = 0.948) to fluorescein gradients ranging from 0.5 to 10 microM was also obtained from the instrument indicating that it may be utilized for other fluorometric assays. This instrument enables an inexpensive, compact approach to in-field genetic screening, providing results comparable to laboratory equipment with rapid user feedback as to the status of the reaction. PMID:17719904

  1. Amino acid sequence heterogeneity of the chromosomal encoded Borrelia burgdorferi sensu lato major antigen P100.

    PubMed

    Fellinger, W; Farencena, A; Redl, B; Sambri, V; Cevenini, R; Stöffler, G

    1995-04-01

    The entire nucleotide sequence of the chromosomal encoded major antigen p100 of the European Borrelia garinii isolate B29 was determined and the deduced amino acid sequence was compared to the homologous antigen p83 of the North American Borrelia burgdorferi sensu stricto strain B31 and the p100 of the European Borrelia afzelii (group VS461) strain PKo. p100 of strain B29 shows 87% amino acid sequence identity to strain B31 and 79.2% to strain PKo, p100 of strain B31 and PKo shows 62.5% identity to each other. In addition, partial nucleotide sequences of the most heterogeneous region of the p100 gene of two other Borrelia garinii isolates (PBi and VS286) have been determined and the deduced amino acid sequences were compared with all p100 of Borrelia garinii published so far. We found an amino acid sequence identity between 88.6 and 100% within the same genospecies. The N-terminal part of the p100 proteins is highly conserved whereas a striking heterogeneous region within the C-terminal part of the proteins was observed.

  2. Organic Analysis in the Miller Range 090657 CR2 Chondrite: Part 2 Amino Acid Analyses

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Cao, T.; Nakamura-Messenger, K.; Berger, E. L.; Messenger, S.; Clemett, S. J.; Aponte, J. C.; Elsila, J. E.

    2016-01-01

    Primitive carbonaceous chondrites contain a wide variety of organic material, ranging from soluble discrete molecules to insoluble, unstructured kerogen-like components, as well as structured nano-globules of macromolecular carbon. The relationship between the soluble organic molecules, macromolecular organic material, and host minerals are poorly understood. Due to the differences in extractability of soluble and insoluble organic materials, the analysis methods for each differ and are often performed independently. The combination of soluble and insoluble analyses, when performed concurrently, can provide a wider understanding of spatial distribution, and elemental, structural and isotopic composition of organic material in primitive meteorites. Using macroscale extraction and analysis techniques in combination with in situ microscale observation, we have been studying both insoluble and soluble organic material in the primitive CR2 chondrite Miller Range (MIL) 090657. In accompanying abstracts (Cao et al. and Messenger et al.) we discuss insoluble organic material in the samples. By performing the consortium studies, we aim to improve our understanding of the relationship between the meteorite minerals and the soluble and insoluble organic phases and to delineate which species formed within the meteorite and those that formed in nebular or presolar environments. In this abstract, we present the results of amino acid analyses of MIL 090657 by ultra performance liquid chromatography with fluorescence detection and quadrupole-time of flight mass spectrometry. Amino acids are of interest because they are essential to life on Earth, and because they are present in sufficient structural, enantiomeric and isotopic diversity to allow insights into early solar system chemical processes. Furthermore, these are among the most isotopically anomalous species, yet at least some fraction are thought to have formed by aqueously-mediated processes during parent body alteration.

  3. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

    1997-01-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided.

  4. Detection and isolation of nucleic acid sequences using competitive hybridization probes

    DOEpatents

    Lucas, J.N.; Straume, T.; Bogen, K.T.

    1997-04-01

    A method for detecting a target nucleic acid sequence in a sample is provided using hybridization probes which competitively hybridize to a target nucleic acid. According to the method, a target nucleic acid sequence is hybridized to first and second hybridization probes which are complementary to overlapping portions of the target nucleic acid sequence, the first hybridization probe including a first complexing agent capable of forming a binding pair with a second complexing agent and the second hybridization probe including a detectable marker. The first complexing agent attached to the first hybridization probe is contacted with a second complexing agent, the second complexing agent being attached to a solid support such that when the first and second complexing agents are attached, target nucleic acid sequences hybridized to the first hybridization probe become immobilized on to the solid support. The immobilized target nucleic acids are then separated and detected by detecting the detectable marker attached to the second hybridization probe. A kit for performing the method is also provided. 7 figs.

  5. Analyses of transcriptome sequences reveal multiple ancient large-scale duplication events in the ancestor of Sphagnopsida (Bryophyta).

    PubMed

    Devos, Nicolas; Szövényi, Péter; Weston, David J; Rothfels, Carl J; Johnson, Matthew G; Shaw, A Jonathan

    2016-07-01

    The goal of this research was to investigate whether there has been a whole-genome duplication (WGD) in the ancestry of Sphagnum (peatmoss) or the class Sphagnopsida, and to determine if the timing of any such duplication(s) and patterns of paralog retention could help explain the rapid radiation and current ecological dominance of peatmosses. RNA sequencing (RNA-seq) data were generated for nine taxa in Sphagnopsida (Bryophyta). Analyses of frequency plots for synonymous substitutions per synonymous site (Ks ) between paralogous gene pairs and reconciliation of 578 gene trees were conducted to assess evidence of large-scale or genome-wide duplication events in each transcriptome. Both Ks frequency plots and gene tree-based analyses indicate multiple duplication events in the history of the Sphagnopsida. The most recent WGD event predates divergence of Sphagnum from the two other genera of Sphagnopsida. Duplicate retention is highly variable across species, which might be best explained by local adaptation. Our analyses indicate that the last WGD could have been an important factor underlying the diversification of peatmosses and facilitated their rise to ecological dominance in peatlands. The timing of the duplication events and their significance in the evolutionary history of peat mosses are discussed. PMID:26900928

  6. Analyses of transcriptome sequences reveal multiple ancient large-scale duplication events in the ancestor of Sphagnopsida (Bryophyta).

    PubMed

    Devos, Nicolas; Szövényi, Péter; Weston, David J; Rothfels, Carl J; Johnson, Matthew G; Shaw, A Jonathan

    2016-07-01

    The goal of this research was to investigate whether there has been a whole-genome duplication (WGD) in the ancestry of Sphagnum (peatmoss) or the class Sphagnopsida, and to determine if the timing of any such duplication(s) and patterns of paralog retention could help explain the rapid radiation and current ecological dominance of peatmosses. RNA sequencing (RNA-seq) data were generated for nine taxa in Sphagnopsida (Bryophyta). Analyses of frequency plots for synonymous substitutions per synonymous site (Ks ) between paralogous gene pairs and reconciliation of 578 gene trees were conducted to assess evidence of large-scale or genome-wide duplication events in each transcriptome. Both Ks frequency plots and gene tree-based analyses indicate multiple duplication events in the history of the Sphagnopsida. The most recent WGD event predates divergence of Sphagnum from the two other genera of Sphagnopsida. Duplicate retention is highly variable across species, which might be best explained by local adaptation. Our analyses indicate that the last WGD could have been an important factor underlying the diversification of peatmosses and facilitated their rise to ecological dominance in peatlands. The timing of the duplication events and their significance in the evolutionary history of peat mosses are discussed.

  7. HPLC and ELISA analyses of larval bile acids from Pacific and western brook lampreys

    USGS Publications Warehouse

    Yun, S.-S.; Scott, A.P.; Bayer, J.M.; Seelye, J.G.; Close, D.A.; Li, W.

    2003-01-01

    Comparative studies were performed on two native lamprey species, Pacific lamprey (Lampetra tridentata) and western brook lamprey (Lampetra richardsoni) from the Pacific coast along with sea lamprey (Petromyzon marinus) from the Great Lakes, to investigate their bile acid production and release. HPLC and ELISA analyses of the gall bladders and liver extract revealed that the major bile acid compound from Pacific and western brook larval lampreys was petromyzonol sulfate (PZS), previously identified as a migratory pheromone in larval sea lamprey. An ELISA for PZS has been developed in a working range of 20pg-10ng per well. The tissue concentrations of PZS in gall bladder were 127.40, 145.86, and 276.96??g/g body mass in sea lamprey, Pacific lamprey, and western brook lamprey, respectively. Releasing rates for PZS in the three species were measured using ELISA to find that western brook and sea lamprey released PZS 20 times higher than Pacific lamprey did. Further studies are required to determine whether PZS is a chemical cue in Pacific and western brook lampreys. ?? 2003 Elsevier Inc. All rights reserved.

  8. Trophic position of deep-sea fish—Assessment through fatty acid and stable isotope analyses

    NASA Astrophysics Data System (ADS)

    Stowasser, G.; McAllen, R.; Pierce, G. J.; Collins, M. A.; Moffat, C. F.; Priede, I. G.; Pond, D. W.

    2009-05-01

    To investigate the trophic ecology of two of the dominant families of deep-sea fish (Macrouridae and Moridae) fatty acid and stable isotope analyses were applied to liver and muscle samples of five abundant species from the NE Atlantic. In conjunction with stomach content data these methods made it possible to identify differences in feeding strategies between the five study species as well as variation in feeding in relation to increasing depth and body size. Biomarkers identified strong similarities between Coryphaenoides armatus and Antimora rostrata though differences were found associating C. armatus more with the benthic food web whereas A. rostrata showed stronger links to the pelagic food web. While Lepidion eques was classified as a species linking benthic and benthopelagic food webs, both fatty acid and stable isotope data suggested that Coryphaenoides guentheri fed on an exclusively benthic diet . Coryphaenoides rupestris on the other hand were largely dependent on a copepod-based food web. Ontogenetic changes in feeding were found for both A. rostrata and C. armatus with the indication of a switch from active predation to scavenging occurring with increasing body size. Biomarkers also reflected the seasonal influx from the photic zone though changes were species-specific and probably reflected the variation in prey availability and abundance in response to these inputs. Our findings have thus demonstrated that the combined use of these biomarkers can elucidate trophic specialisations in situations where conventional methods alone previously provided insufficient data.

  9. Stable isotope and signature fatty acid analyses suggest reef manta rays feed on demersal zooplankton.

    PubMed

    Couturier, Lydie I E; Rohner, Christoph A; Richardson, Anthony J; Marshall, Andrea D; Jaine, Fabrice R A; Bennett, Michael B; Townsend, Kathy A; Weeks, Scarla J; Nichols, Peter D

    2013-01-01

    Assessing the trophic role and interaction of an animal is key to understanding its general ecology and dynamics. Conventional techniques used to elucidate diet, such as stomach content analysis, are not suitable for large threatened marine species. Non-lethal sampling combined with biochemical methods provides a practical alternative for investigating the feeding ecology of these species. Stable isotope and signature fatty acid analyses of muscle tissue were used for the first time to examine assimilated diet of the reef manta ray Manta alfredi, and were compared with different zooplankton functional groups (i.e. near-surface zooplankton collected during manta ray feeding events and non-feeding periods, epipelagic zooplankton, demersal zooplankton and several different zooplankton taxa). Stable isotope δ(15)N values confirmed that the reef manta ray is a secondary consumer. This species had relatively high levels of docosahexaenoic acid (DHA) indicating a flagellate-based food source in the diet, which likely reflects feeding on DHA-rich near-surface and epipelagic zooplankton. However, high levels of ω6 polyunsaturated fatty acids and slightly enriched δ(13)C values in reef manta ray tissue suggest that they do not feed solely on pelagic zooplankton, but rather obtain part of their diet from another origin. The closest match was with demersal zooplankton, suggesting it is an important component of the reef manta ray diet. The ability to feed on demersal zooplankton is likely linked to the horizontal and vertical movement patterns of this giant planktivore. These new insights into the habitat use and feeding ecology of the reef manta ray will assist in the effective evaluation of its conservation needs.

  10. Characterization of sources for southern African aerosols through fatty acid and trajectory analyses

    NASA Astrophysics Data System (ADS)

    Billmark, Kaycie A.; Swap, Robert J.; Macko, Stephen A.

    2003-07-01

    Biogeochemical cycles in southern Africa are affected by emissions from extensive biomass burning. Emitted trace gases and aerosols frequently accumulate and recirculate in the well-defined synoptic pattern that persists for long time periods over southern Africa. The role of organic aerosols during atmospheric transport and the influence of neighboring air masses on biogeochemical dynamics in this nutrient-limited region are insufficiently studied. The Southern African Regional Science Initiative (SAFARI 2000) was conducted in part to investigate the impacts of this large-scale transport and deposition of increasingly anthropogenic emissions on southern African biogeochemical cycling. This study explores the understanding of regional atmospheric transport through the identification of chemical biomarkers to describe aerosols collected during the SAFARI 2000 dry season research campaign. Total suspended particulate aerosol samples were collected diurnally for a period of two weeks in Mongu, Zambia. Mongu is bordered by the Zambezi River on the west and the Miombo woodland savanna in all other directions. It also lies on the northern extent of the Kalahari Desert. This region is characterized by high biomass burning emissions of river floodplain grasses and woodland savanna during the dry season. Fatty acids were extracted from the collected aerosols and analyzed using gas chromatography. The resultant fatty acid compositions were examined for temporal patterns and trends. Furthermore, these results were compared to both synoptic meteorological patterns over the region, as well as to modeled air parcel trajectories, to gain insight into changes in aerosol composition resulting from changes in atmospheric transports from regions of different vegetation. The results of these analyses confirm that abundances of fatty acids are dependent on local and synoptic meteorology and can thus be used as an additional geochemical tracer to better describe aerosol sources and

  11. Stable Isotope and Signature Fatty Acid Analyses Suggest Reef Manta Rays Feed on Demersal Zooplankton

    PubMed Central

    Couturier, Lydie I. E.; Rohner, Christoph A.; Richardson, Anthony J.; Marshall, Andrea D.; Jaine, Fabrice R. A.; Bennett, Michael B.; Townsend, Kathy A.; Weeks, Scarla J.; Nichols, Peter D.

    2013-01-01

    Assessing the trophic role and interaction of an animal is key to understanding its general ecology and dynamics. Conventional techniques used to elucidate diet, such as stomach content analysis, are not suitable for large threatened marine species. Non-lethal sampling combined with biochemical methods provides a practical alternative for investigating the feeding ecology of these species. Stable isotope and signature fatty acid analyses of muscle tissue were used for the first time to examine assimilated diet of the reef manta ray Manta alfredi, and were compared with different zooplankton functional groups (i.e. near-surface zooplankton collected during manta ray feeding events and non-feeding periods, epipelagic zooplankton, demersal zooplankton and several different zooplankton taxa). Stable isotope δ15N values confirmed that the reef manta ray is a secondary consumer. This species had relatively high levels of docosahexaenoic acid (DHA) indicating a flagellate-based food source in the diet, which likely reflects feeding on DHA-rich near-surface and epipelagic zooplankton. However, high levels of ω6 polyunsaturated fatty acids and slightly enriched δ13C values in reef manta ray tissue suggest that they do not feed solely on pelagic zooplankton, but rather obtain part of their diet from another origin. The closest match was with demersal zooplankton, suggesting it is an important component of the reef manta ray diet. The ability to feed on demersal zooplankton is likely linked to the horizontal and vertical movement patterns of this giant planktivore. These new insights into the habitat use and feeding ecology of the reef manta ray will assist in the effective evaluation of its conservation needs. PMID:24167562

  12. Ligation with nucleic acid sequence-based amplification.

    PubMed

    Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M; Artenstein, Andrew W; Tripathi, Anubhav

    2012-01-01

    This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays. PMID:22449695

  13. The amino acid sequence of mitogenic lectin-B from the roots of pokeweed (Phytolacca americana).

    PubMed

    Yamaguchi, K; Yurino, N; Kino, M; Ishiguro, M; Funatsu, G

    1997-04-01

    The complete amino acid sequence of pokeweed lectin-B (PL-B) has been analyzed by first sequencing seven lysylendopeptidase peptides derived from the reduced and S-pyridylethylated PL-B and then connecting them by analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides linked to Asn96 and Asn139, and has a molecular mass of 34,493 Da. PL-B is composed of seven repetitive chitin-binding domains having 48-79% sequence homology with each other. Twelve amino acid residues including eight cysteine residues in these domains are absolutely conserved in all other chitin-binding domains of plant lectins and class I chitinases. Also, it was strongly suggested that the extremely high hemagglutinating and mitogenic activities of PL-B may be ascribed to its seven-domain structure.

  14. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids.

    PubMed

    Ashkenazy, Haim; Erez, Elana; Martz, Eric; Pupko, Tal; Ben-Tal, Nir

    2010-07-01

    It is informative to detect highly conserved positions in proteins and nucleic acid sequence/structure since they are often indicative of structural and/or functional importance. ConSurf (http://consurf.tau.ac.il) and ConSeq (http://conseq.tau.ac.il) are two well-established web servers for calculating the evolutionary conservation of amino acid positions in proteins using an empirical Bayesian inference, starting from protein structure and sequence, respectively. Here, we present the new version of the ConSurf web server that combines the two independent servers, providing an easier and more intuitive step-by-step interface, while offering the user more flexibility during the process. In addition, the new version of ConSurf calculates the evolutionary rates for nucleic acid sequences. The new version is freely available at: http://consurf.tau.ac.il/.

  15. Amino acid repeats cause extraordinary coding sequence variation in the social amoeba Dictyostelium discoideum.

    PubMed

    Scala, Clea; Tian, Xiangjun; Mehdiabadi, Natasha J; Smith, Margaret H; Saxer, Gerda; Stephens, Katie; Buzombo, Prince; Strassmann, Joan E; Queller, David C

    2012-01-01

    Protein sequences are normally the most conserved elements of genomes owing to purifying selection to maintain their functions. We document an extraordinary amount of within-species protein sequence variation in the model eukaryote Dictyostelium discoideum stemming from triplet DNA repeats coding for long strings of single amino acids. D. discoideum has a very large number of such strings, many of which are polyglutamine repeats, the same sequence that causes various human neurological disorders in humans, like Huntington's disease. We show here that D. discoideum coding repeat loci are highly variable among individuals, making D. discoideum a candidate for the most variable proteome. The coding repeat loci are not significantly less variable than similar non-coding triplet repeats. This pattern is consistent with these amino-acid repeats being largely non-functional sequences evolving primarily by mutation and drift. PMID:23029418

  16. Conservation of Shannon's redundancy for proteins. [information theory applied to amino acid sequences

    NASA Technical Reports Server (NTRS)

    Gatlin, L. L.

    1974-01-01

    Concepts of information theory are applied to examine various proteins in terms of their redundancy in natural originators such as animals and plants. The Monte Carlo method is used to derive information parameters for random protein sequences. Real protein sequence parameters are compared with the standard parameters of protein sequences having a specific length. The tendency of a chain to contain some amino acids more frequently than others and the tendency of a chain to contain certain amino acid pairs more frequently than other pairs are used as randomness measures of individual protein sequences. Non-periodic proteins are generally found to have random Shannon redundancies except in cases of constraints due to short chain length and genetic codes. Redundant characteristics of highly periodic proteins are discussed. A degree of periodicity parameter is derived.

  17. Shark myoglobins. II. Isolation, characterization and amino acid sequence of myoglobin from Galeorhinus japonicus.

    PubMed

    Suzuki, T; Suzuki, T; Yata, T

    1985-01-01

    Native oxymyoglobin (MbO2) was isolated from red muscle of G. japonicus by chromatographic separation from metmyoglobin (metMb) on DEAE-cellulose and the amino acid sequence of the major chain was determined with the aid of sequence homology with that of G. australis. It was shown to differ in amino acid sequence from that of G. australis by 10 replacements, to be acetylated at the amino terminus and to contain glutamine at the distal (E7) residue. It was also shown to have a spectrum very similar to that of mammalian MbO2. However, the pH-dependence for the autoxidation of MbO2 was seen to be quite different from that of sperm whale (Physeter catodon) MbO2. Although the sequence homology between sperm whale and G. japonicus myoglobins is about 40%, their hydropathy profiles were very similar, indicating that they have a similar geometry in their globin folding.

  18. Conversion of amino-acid sequence in proteins to classical music: search for auditory patterns

    PubMed Central

    2007-01-01

    We have converted genome-encoded protein sequences into musical notes to reveal auditory patterns without compromising musicality. We derived a reduced range of 13 base notes by pairing similar amino acids and distinguishing them using variations of three-note chords and codon distribution to dictate rhythm. The conversion will help make genomic coding sequences more approachable for the general public, young children, and vision-impaired scientists. PMID:17477882

  19. Visible sensing of nucleic acid sequences using a genetically encodable unmodified mRNA probe.

    PubMed

    Narita, Atsushi; Ogawa, Kazumasa; Sando, Shinsuke; Aoyama, Yasuhiro

    2006-01-01

    We previously reported a molecular beacon-mRNA (MB-mRNA) strategy for nucleic acid detection/sensing in a cell-free translation system using unmodified RNA as a probe. Here in this presentation, we report that a combination with RNase H activity, which induces an additional process of irreversible cleavage of MB-domain, achieves an improved sequence selectivity (one nucleotide selectivity) and an enhanced sensitivity. This improved system finally enabled visible sensing of target nucleic acid sequence at a single nucleotide resolution under isothermal conditions.

  20. Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia

    PubMed Central

    Engström, Å.; Xanthopoulos, K. G.; Boman, H. G.; Bennich, H.

    1985-01-01

    The amino acid and cDNA sequences of lysozyme from the giant silk moth Hyalophora cecropia have been determined. This enzyme is one of several immune proteins produced by the diapausing pupae after injection of bacteria. Cecropia lysozyme is composed of 120 amino acids, has a mol. wt. of 13.8 kd and shows great similarity with vertebrate lysozymes of the chicken type. The amino acid residues responsible for the catalytic activity and for the binding of substrate are essentially conserved. Three allelic variants of the Cecropia enzyme are identified. A comparison of the chicken and the Cecropia lysozymes shows that there is a 40% identity at both the amino acid and the nucleotide level. Some evolutionary aspects of the sequence data are discussed. PMID:16453632

  1. Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses.

    PubMed

    Yan, Liying; Huang, Lei; Xu, Liya; Huang, Jin; Ma, Fei; Zhu, Xiaohui; Tang, Yaqiong; Liu, Mingshan; Lian, Ying; Liu, Ping; Li, Rong; Lu, Sijia; Tang, Fuchou; Qiao, Jie; Xie, X Sunney

    2015-12-29

    In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), and preimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosome abnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the false-positive and false-negative single-nucleotide variations (SNVs), which are not acceptable in IVF and can be circumvented by linkage analyses, such as short tandem repeats or karyomapping. It is noteworthy that existing methods of detecting SNV/copy number variation (CNV) and linkage analysis often require separate procedures for the same embryo. Here we report an NGS-based PGD/PGS procedure that can simultaneously detect a single-gene disorder and aneuploidy and is capable of linkage analysis in a cost-effective way. This method, called "mutated allele revealed by sequencing with aneuploidy and linkage analyses" (MARSALA), involves multiple annealing and looping-based amplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated with the monogenic diseases are detected by PCR amplification of the MALBAC product. The false-positive and -negative SNVs are avoided by an NGS-based linkage analysis. Two healthy babies, free of the monogenic diseases of their parents, were born after such embryo selection. The monogenic diseases originated from a single base mutation on the autosome and the X-chromosome of the disease-carrying father and mother, respectively. PMID:26712022

  2. Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses.

    PubMed

    Yan, Liying; Huang, Lei; Xu, Liya; Huang, Jin; Ma, Fei; Zhu, Xiaohui; Tang, Yaqiong; Liu, Mingshan; Lian, Ying; Liu, Ping; Li, Rong; Lu, Sijia; Tang, Fuchou; Qiao, Jie; Xie, X Sunney

    2015-12-29

    In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), and preimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosome abnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the false-positive and false-negative single-nucleotide variations (SNVs), which are not acceptable in IVF and can be circumvented by linkage analyses, such as short tandem repeats or karyomapping. It is noteworthy that existing methods of detecting SNV/copy number variation (CNV) and linkage analysis often require separate procedures for the same embryo. Here we report an NGS-based PGD/PGS procedure that can simultaneously detect a single-gene disorder and aneuploidy and is capable of linkage analysis in a cost-effective way. This method, called "mutated allele revealed by sequencing with aneuploidy and linkage analyses" (MARSALA), involves multiple annealing and looping-based amplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated with the monogenic diseases are detected by PCR amplification of the MALBAC product. The false-positive and -negative SNVs are avoided by an NGS-based linkage analysis. Two healthy babies, free of the monogenic diseases of their parents, were born after such embryo selection. The monogenic diseases originated from a single base mutation on the autosome and the X-chromosome of the disease-carrying father and mother, respectively.

  3. Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66.

    PubMed

    Liu, Bin; Ertesvåg, Helga; Aasen, Inga Marie; Vadstein, Olav; Brautaset, Trygve; Heggeset, Tonje Marita Bjerkan

    2016-06-01

    Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids. PMID:27222814

  4. Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66.

    PubMed

    Liu, Bin; Ertesvåg, Helga; Aasen, Inga Marie; Vadstein, Olav; Brautaset, Trygve; Heggeset, Tonje Marita Bjerkan

    2016-06-01

    Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids.

  5. Deep sequencing and in silico analyses identify MYB-regulated gene networks and signaling pathways in pancreatic cancer

    PubMed Central

    Azim, Shafquat; Zubair, Haseeb; Srivastava, Sanjeev K.; Bhardwaj, Arun; Zubair, Asif; Ahmad, Aamir; Singh, Seema; Khushman, Moh’d.; Singh, Ajay P.

    2016-01-01

    We have recently demonstrated that the transcription factor MYB can modulate several cancer-associated phenotypes in pancreatic cancer. In order to understand the molecular basis of these MYB-associated changes, we conducted deep-sequencing of transcriptome of MYB-overexpressing and -silenced pancreatic cancer cells, followed by in silico pathway analysis. We identified significant modulation of 774 genes upon MYB-silencing (p < 0.05) that were assigned to 25 gene networks by in silico analysis. Further analyses placed genes in our RNA sequencing-generated dataset to several canonical signalling pathways, such as cell-cycle control, DNA-damage and -repair responses, p53 and HIF1α. Importantly, we observed downregulation of the pancreatic adenocarcinoma signaling pathway in MYB-silenced pancreatic cancer cells exhibiting suppression of EGFR and NF-κB. Decreased expression of EGFR and RELA was validated by both qPCR and immunoblotting and they were both shown to be under direct transcriptional control of MYB. These observations were further confirmed in a converse approach wherein MYB was overexpressed ectopically in a MYB-null pancreatic cancer cell line. Our findings thus suggest that MYB potentially regulates growth and genomic stability of pancreatic cancer cells via targeting complex gene networks and signaling pathways. Further in-depth functional studies are warranted to fully understand MYB signaling in pancreatic cancer. PMID:27354262

  6. Deep sequencing and in silico analyses identify MYB-regulated gene networks and signaling pathways in pancreatic cancer.

    PubMed

    Azim, Shafquat; Zubair, Haseeb; Srivastava, Sanjeev K; Bhardwaj, Arun; Zubair, Asif; Ahmad, Aamir; Singh, Seema; Khushman, Moh'd; Singh, Ajay P

    2016-06-29

    We have recently demonstrated that the transcription factor MYB can modulate several cancer-associated phenotypes in pancreatic cancer. In order to understand the molecular basis of these MYB-associated changes, we conducted deep-sequencing of transcriptome of MYB-overexpressing and -silenced pancreatic cancer cells, followed by in silico pathway analysis. We identified significant modulation of 774 genes upon MYB-silencing (p < 0.05) that were assigned to 25 gene networks by in silico analysis. Further analyses placed genes in our RNA sequencing-generated dataset to several canonical signalling pathways, such as cell-cycle control, DNA-damage and -repair responses, p53 and HIF1α. Importantly, we observed downregulation of the pancreatic adenocarcinoma signaling pathway in MYB-silenced pancreatic cancer cells exhibiting suppression of EGFR and NF-κB. Decreased expression of EGFR and RELA was validated by both qPCR and immunoblotting and they were both shown to be under direct transcriptional control of MYB. These observations were further confirmed in a converse approach wherein MYB was overexpressed ectopically in a MYB-null pancreatic cancer cell line. Our findings thus suggest that MYB potentially regulates growth and genomic stability of pancreatic cancer cells via targeting complex gene networks and signaling pathways. Further in-depth functional studies are warranted to fully understand MYB signaling in pancreatic cancer.

  7. Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

    PubMed

    Soyer, Jessica L; Möller, Mareike; Schotanus, Klaas; Connolly, Lanelle R; Galazka, Jonathan M; Freitag, Michael; Stukenbrock, Eva H

    2015-06-01

    The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.

  8. AcalPred: a sequence-based tool for discriminating between acidic and alkaline enzymes.

    PubMed

    Lin, Hao; Chen, Wei; Ding, Hui

    2013-01-01

    The structure and activity of enzymes are influenced by pH value of their surroundings. Although many enzymes work well in the pH range from 6 to 8, some specific enzymes have good efficiencies only in acidic (pH<5) or alkaline (pH>9) solution. Studies have demonstrated that the activities of enzymes correlate with their primary sequences. It is crucial to judge enzyme adaptation to acidic or alkaline environment from its amino acid sequence in molecular mechanism clarification and the design of high efficient enzymes. In this study, we developed a sequence-based method to discriminate acidic enzymes from alkaline enzymes. The analysis of variance was used to choose the optimized discriminating features derived from g-gap dipeptide compositions. And support vector machine was utilized to establish the prediction model. In the rigorous jackknife cross-validation, the overall accuracy of 96.7% was achieved. The method can correctly predict 96.3% acidic and 97.1% alkaline enzymes. Through the comparison between the proposed method and previous methods, it is demonstrated that the proposed method is more accurate. On the basis of this proposed method, we have built an online web-server called AcalPred which can be freely accessed from the website (http://lin.uestc.edu.cn/server/AcalPred). We believe that the AcalPred will become a powerful tool to study enzyme adaptation to acidic or alkaline environment.

  9. AcalPred: A Sequence-Based Tool for Discriminating between Acidic and Alkaline Enzymes

    PubMed Central

    Lin, Hao; Chen, Wei; Ding, Hui

    2013-01-01

    The structure and activity of enzymes are influenced by pH value of their surroundings. Although many enzymes work well in the pH range from 6 to 8, some specific enzymes have good efficiencies only in acidic (pH<5) or alkaline (pH>9) solution. Studies have demonstrated that the activities of enzymes correlate with their primary sequences. It is crucial to judge enzyme adaptation to acidic or alkaline environment from its amino acid sequence in molecular mechanism clarification and the design of high efficient enzymes. In this study, we developed a sequence-based method to discriminate acidic enzymes from alkaline enzymes. The analysis of variance was used to choose the optimized discriminating features derived from g-gap dipeptide compositions. And support vector machine was utilized to establish the prediction model. In the rigorous jackknife cross-validation, the overall accuracy of 96.7% was achieved. The method can correctly predict 96.3% acidic and 97.1% alkaline enzymes. Through the comparison between the proposed method and previous methods, it is demonstrated that the proposed method is more accurate. On the basis of this proposed method, we have built an online web-server called AcalPred which can be freely accessed from the website (http://lin.uestc.edu.cn/server/AcalPred). We believe that the AcalPred will become a powerful tool to study enzyme adaptation to acidic or alkaline environment. PMID:24130738

  10. Antibody-specific model of amino acid substitution for immunological inferences from alignments of antibody sequences.

    PubMed

    Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria

    2015-03-01

    Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, higher affinity and selectivity to the pathogenic target. As somatic hypermutation involves fast mutation of antibody sequences, this process can be described using a Markov substitution model of molecular evolution. Here, using large sets of antibody sequences from mice and humans, we infer an empirical amino acid substitution model AB, which is specific to antibody sequences. Compared with existing general amino acid models, we show that the AB model provides significantly better description for the somatic evolution of mice and human antibody sequences, as demonstrated on large next generation sequencing (NGS) antibody data. General amino acid models are reflective of conservation at the protein level due to functional constraints, with most frequent amino acids exchanges taking place between residues with the same or similar physicochemical properties. In contrast, within the variable part of antibody sequences we observed an elevated frequency of exchanges between amino acids with distinct physicochemical properties. This is indicative of a sui generis mutational mechanism, specific to antibody somatic hypermutation. We illustrate this property of antibody sequences by a comparative analysis of the network modularity implied by the AB model and general amino acid substitution models. We recommend using the new model for computational studies of antibody sequence maturation, including inference of alignments and phylogenetic trees describing antibody somatic hypermutation in

  11. Genetic diversity and population structure of black Dahe pig based on DNA sequences analyses of mitochondrial and nuclear genes.

    PubMed

    Tang, Lizhou; Yu, Long; Wang, Junjie; Liu, Chao; Shi, Xiaodong; Ding, Wei; Zhu, Lei; Guo, Songchang

    2016-01-01

    To investigate the genetic diversity and population structure of black Dahe pigs, we collected 175 samples from 5 local populations and sequenced them using a combination of two selected molecular markers for mitochondrial cytochrome b and Major Histocompatibility Complex (MHC) DRB. Overall, the results of AMOVA and phylogenetic tree and gene flow analyses detected high levels of gene flow among the five populations, particularly individual pigs from Dahe town (Pop1) or Yingshang town (Pop2) to other populations (Pop3, Pop4, and Pop5). The genetic diversity analyses showed that the diversity indices of the five populations did not vary significantly, but they were much lower than those of other Chinese pig species. These results suggest that distinct gene flow, unstable population pattern, and lower genetic diversity have been influenced mainly by human introductions for economic ends. These findings provide genetic information that could be used for the preservation and further genetic improvement of the black Dahe pig, as well as an important reference for the evaluation, conservation, and utilization of the genetic resources of this breed.

  12. Folic acid alone or multivitamin containing folic acid intake during pregnancy and the risk of gestational hypertension and preeclampsia through meta-analyses

    PubMed Central

    Shim, Sang-Min; Yun, Yeo-Ul

    2016-01-01

    Objective The objective of this study was to assess the effect of folic acid and multivitamin use during pregnancy on the risk of developing of hypertensive disorder of pregnancy. Methods Two reviewers independently determined all prospective cohort study, retrospective cohort study, large population based cohort study, retrospective secondary analysis, and double blinded, placebo-controlled, randomized clinical trial published using PubMed Medline database, KERIS (Korea Education and Research Information Service), Scopus, and the Cochrane Central Register of controlled trials comparing before conception throughout pregnancy intake oral multivitamin containing folic acid or folic acid alone. Meta-analyses were estimated with odds ratios and 95% confidence intervals (CIs) using random effect analysis according to heterogeneity of studies. Results Data from six effect sizes from six studies involving 201,661 patients were enrolled. These meta-analyses showed multivitamin containing folic acid or folic acid alone was not significantly effective in reducing gestational hypertension or preeclampsia incidence (odds ratio, 0.91; 95% CI, 0.81 to 1.03) than the placebo. And the difference of effective sizes of preeclampsia and gestational hypertension according to two dependent variables, multivitamin and folic acid were not significant, respectively (point estimate, 0.66; 95% CI, 0.46 to 0.96). Conclusion These meta-analyses demonstrate multivitamin containing folic acid or folic acid alone was not significantly effective in reducing gestational hypertension or preeclampsia incidence. PMID:27004201

  13. Quantitative detection of Aspergillus spp. by real-time nucleic acid sequence-based amplification.

    PubMed

    Zhao, Yanan; Perlin, David S

    2013-01-01

    Rapid and quantitative detection of Aspergillus from clinical samples may facilitate an early diagnosis of invasive pulmonary aspergillosis (IPA). As nucleic acid-based detection is a viable option, we demonstrate that Aspergillus burdens can be rapidly and accurately detected by a novel real-time nucleic acid assay other than qPCR by using the combination of nucleic acid sequence-based amplification (NASBA) and the molecular beacon (MB) technology. Here, we detail a real-time NASBA assay to determine quantitative Aspergillus burdens in lungs and bronchoalveolar lavage (BAL) fluids of rats with experimental IPA.

  14. Draft Genome Sequence of the Butyric Acid Producer Clostridium tyrobutyricum Strain CIP I-776 (IFP923)

    PubMed Central

    Clément, Benjamin; Lopes Ferreira, Nicolas

    2016-01-01

    Here, we report the draft genome sequence of Clostridium tyrobutyricum CIP I-776 (IFP923), an efficient producer of butyric acid. The genome consists of a single chromosome of 3.19 Mb and provides useful data concerning the metabolic capacities of the strain. PMID:26941139

  15. Amino acid sequence of the encephalitogenic basic protein from human myelin

    PubMed Central

    Carnegie, P. R.

    1971-01-01

    Myelin from the central nervous system contains an unusual basic protein, which can induce experimental autoimmune encephalomyelitis. The basic protein from human brain was digested with trypsin and other enzymes and the sequence of the 170 amino acids was determined. The localization of the encephalitogenic determinants was described. Possible roles for the protein in the structure and function of myelin are discussed. PMID:4108501

  16. Sequence-specific formation of d-amino acids in a monoclonal antibody during light exposure.

    PubMed

    Mozziconacci, Olivier; Schöneich, Christian

    2014-11-01

    The photoirradiation of a monoclonal antibody 1 (mAb1) at λ = 254 nm and λmax = 305 nm resulted in the sequence-specific generation of d-Val, d-Tyr, and potentially d-Ala and d-Arg, in the heavy chain sequence [95-101] YCARVVY. d-Amino acid formation is most likely the product of reversible intermediary carbon-centered radical formation at the (α)C-positions of the respective amino acids ((α)C(•) radicals) through the action of Cys thiyl radicals (CysS(•)). The latter can be generated photochemically either through direct homolysis of cystine or through photoinduced electron transfer from Trp and/or Tyr residues. The potential of mAb1 sequences to undergo epimerization was first evaluated through covalent H/D exchange during photoirradiation in D2O, and proteolytic peptides exhibiting deuterium incorporation were monitored by HPLC-MS/MS analysis. Subsequently, mAb1 was photoirradiated in H2O, and peptides, for which deuterium incorporation in D2O had been documented, were purified by HPLC and subjected to hydrolysis and amino acid analysis. Importantly, not all peptide sequences which incorporated deuterium during photoirradiation in D2O also exhibited photoinduced d-amino acid formation. For example, the heavy chain sequence [12-18] VQPGGSL showed significant deuterium incorporation during photoirradiation in D2O, but no photoinduced formation of d-amino acids was detected. Instead this sequence contained ca. 22% d-Val in both a photoirradiated and a control sample. This observation could indicate that d-Val may have been generated either during production and/or storage or during sample preparation. While sample preparation did not lead to the formation of d-Val or other d-amino acids in the control sample for the heavy chain sequence [95-101] YCARVVY, we may have to consider that during hydrolysis N-terminal residues (such as in VQPGGSL) may be more prone to epimerization. We conclude that the photoinduced, radical-dependent formation of d-amino acids

  17. The complete amino acid sequence of chitinase-B from the leaves of pokeweed (Phytolacca americana).

    PubMed

    Tanigawa, M; Yamagami, T; Funatsu, G

    1995-05-01

    The complete amino acid sequence of pokeweed leaf chitinase-B (PLC-B) has been determined by first sequencing all 19 tryptic peptides derived from the reduced and S-carboxymethylated (RCm-) PLC-B and then connecting them by analyzing the chymotryptic peptides from three fragments produced by cyanogen bromide cleavage of RCm-PLC-B. PLC-B consists of 274 amino acid residues and has a molecular mass of 29,473 Da. Six cysteine residues are linked by disulfide bonds between Cys20 and Cys67, Cys50 and Cys57, and Cys159 and Cys188. From 58-68% sequence homology of PLC-B with five class III chitinases, it was concluded that PLC-B is a basic class III chitinase.

  18. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  19. The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

    PubMed

    Goncerzewicz, Anna; Misiewicz, Anna

    2015-01-01

    Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S

  20. Pyruvate decarboxylase from Pisum sativum. Properties, nucleotide and amino acid sequences.

    PubMed

    Mücke, U; Wohlfarth, T; Fiedler, U; Bäumlein, H; Rücknagel, K P; König, S

    1996-04-15

    To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography, it was necessary to elucidate the protein sequence. Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequences were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'- and 5'-rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzyme. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR.

  1. Allelic polymorphism in arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease.

    PubMed

    Welling, G W; Mulder, H; Beintema, J J

    1976-04-01

    Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position. PMID:962846

  2. Comparative sequence analyses of rhodopsin and RPE65 reveal patterns of selective constraint across hereditary retinal disease mutations.

    PubMed

    Hauser, Frances E; Schott, Ryan K; Castiglione, Gianni M; Van Nynatten, Alexander; Kosyakov, Alexander; Tang, Portia L; Gow, Daniel A; Chang, Belinda S W

    2016-01-01

    Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (d N/d S) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur. PMID:26750628

  3. Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications

    PubMed Central

    Post, Deborah M. B.; Ketterer, Margaret R.; Coffin, Jeremy E.; Reinders, Lorri M.; Munson, Robert S.; Bair, Thomas; Murphy, Timothy F.; Foster, Eric D.; Gibson, Bradford W.

    2016-01-01

    Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease. PMID:26729761

  4. Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses

    PubMed Central

    Yan, Liying; Huang, Lei; Xu, Liya; Huang, Jin; Ma, Fei; Zhu, Xiaohui; Tang, Yaqiong; Liu, Mingshan; Lian, Ying; Liu, Ping; Li, Rong; Lu, Sijia; Tang, Fuchou; Qiao, Jie; Xie, X. Sunney

    2015-01-01

    In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), and preimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosome abnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the false-positive and false-negative single-nucleotide variations (SNVs), which are not acceptable in IVF and can be circumvented by linkage analyses, such as short tandem repeats or karyomapping. It is noteworthy that existing methods of detecting SNV/copy number variation (CNV) and linkage analysis often require separate procedures for the same embryo. Here we report an NGS-based PGD/PGS procedure that can simultaneously detect a single-gene disorder and aneuploidy and is capable of linkage analysis in a cost-effective way. This method, called “mutated allele revealed by sequencing with aneuploidy and linkage analyses” (MARSALA), involves multiple annealing and looping-based amplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated with the monogenic diseases are detected by PCR amplification of the MALBAC product. The false-positive and -negative SNVs are avoided by an NGS-based linkage analysis. Two healthy babies, free of the monogenic diseases of their parents, were born after such embryo selection. The monogenic diseases originated from a single base mutation on the autosome and the X-chromosome of the disease-carrying father and mother, respectively. PMID:26712022

  5. Minding the gap: Frequency of indels in mtDNA control region sequence data and influence on population genetic analyses

    USGS Publications Warehouse

    Pearce, J.M.

    2006-01-01

    Insertions and deletions (indels) result in sequences of various lengths when homologous gene regions are compared among individuals or species. Although indels are typically phylogenetically informative, occurrence and incorporation of these characters as gaps in intraspecific population genetic data sets are rarely discussed. Moreover, the impact of gaps on estimates of fixation indices, such as FST, has not been reviewed. Here, I summarize the occurrence and population genetic signal of indels among 60 published studies that involved alignments of multiple sequences from the mitochondrial DNA (mtDNA) control region of vertebrate taxa. Among 30 studies observing indels, an average of 12% of both variable and parsimony-informative sites were composed of these sites. There was no consistent trend between levels of population differentiation and the number of gap characters in a data block. Across all studies, the average influence on estimates of ??ST was small, explaining only an additional 1.8% of among population variance (range 0.0-8.0%). Studies most likely to observe an increase in ??ST with the inclusion of gap characters were those with < 20 variable sites, but a near equal number of studies with few variable sites did not show an increase. In contrast to studies at interspecific levels, the influence of indels for intraspecific population genetic analyses of control region DNA appears small, dependent upon total number of variable sites in the data block, and related to species-specific characteristics and the spatial distribution of mtDNA lineages that contain indels. ?? 2006 Blackwell Publishing Ltd.

  6. Nucleotide sequence of Crithidia fasciculata cytosol 5S ribosomal ribonucleic acid.

    PubMed

    MacKay, R M; Gray, M W; Doolittle, W F

    1980-11-11

    The complete nucleotide sequence of the cytosol 5S ribosomal ribonucleic acid of the trypanosomatid protozoan Crithidia fasciculata has been determined by a combination of T1-oligonucleotide catalog and gel sequencing techniques. The sequence is: GAGUACGACCAUACUUGAGUGAAAACACCAUAUCCCGUCCGAUUUGUGAAGUUAAGCACC CACAGGCUUAGUUAGUACUGAGGUCAGUGAUGACUCGGGAACCCUGAGUGCCGUACUCCCOH. This 5S ribosomal RNA is unique in having GAUU in place of the GAAC or GAUC found in all other prokaryotic and eukaryotic 5S RNAs, and thought to be involved in interactions with tRNAs. Comparisons to other eukaryotic cytosol 5S ribosomal RNA sequences indicate that the four major eukaryotic kingdoms (animals, plants, fungi, and protists) are about equally remote from each other, and that the latter kingdom may be the most internally diverse.

  7. Pattern recognition in nucleic acid sequences. II. An efficient method for finding locally stable secondary structures.

    PubMed Central

    Kanehisa, M I; Goad, W B

    1982-01-01

    We present a method for calculating all possible single hairpin loop secondary structures in a nucleic acid sequence by the order of N2 operations where N is the total number of bases. Each structure may contain any number of bulges and internal loops. Most natural sequences are found to be indistinguishable from random sequences in the potential of forming secondary structures, which is defined by the frequency of possible secondary structures calculated by the method. There is a strong correlation between the higher G+C content and the higher structure forming potential. Interestingly, the removal of intervening sequences in mRNAs is almost always accompanied by an increase in the G+C content, which may suggest an involvement of structural stabilization in the mRNA maturation. PMID:6174936

  8. Amino acid analyses of type 3 chondrites Colony, Ornans, Chainpur, and Bishunpur

    NASA Astrophysics Data System (ADS)

    Chan, H.-S.; Martins, Zita; Sephton, Mark A.

    2012-09-01

    The CO3s Colony and Ornans and LL3s Chainpur and Bishunpur were analyzed for the first time for amino acids using gas chromatography-mass spectrometry (GC-MS). Type 3 chondrites have relatively unaltered metamorphic and petrological histories. Chainpur was the most amino acid rich of the four type 3 chondrites with a total amino acid abundance of 3330 parts per billion (ppb). The other type 3 chondrites had total amino acid abundances that ranged from 660 to 1110 ppb. A D/L ratio of <0.7 for all proteic amino acids suggests at least some amino acid terrestrial contamination. However, a small fraction of indigenous extraterrestrial amino acids cannot be excluded because of the presence of the nonprotein amino acid α-aminoisobutyric acid (α-AIB), and unusually high relative abundances (to glycine) of β-alanine and γ-ABA. The comparisons between the free and total amino acid contents of the samples also indicate a low free/total amino acid ratio (ranging from about 1:4 in CO chondrites to about 1:50 in Chainpur), which indicate that amino acids are present mainly in the bound form and were made detectable after acid hydrolysis.

  9. Evolutionary History and Phylodynamics of Influenza A and B Neuraminidase (NA) Genes Inferred from Large-Scale Sequence Analyses

    PubMed Central

    Xu, Jianpeng; Davis, C. Todd; Christman, Mary C.; Rivailler, Pierre; Zhong, Haizhen; Donis, Ruben O.; Lu, Guoqing

    2012-01-01

    Background Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking. Methodology/Principal findings Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The dN/dS ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding. Conclusions/Significance The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance. PMID:22808012

  10. Stability Test and Quantitative and Qualitative Analyses of the Amino Acids in Pharmacopuncture Extracted from Scolopendra subspinipes mutilans

    PubMed Central

    Cho, GyeYoon; Han, KyuChul; Yoon, JinYoung

    2015-01-01

    Objectives: Scolopendra subspinipes mutilans (S. subspinipes mutilans) is known as a traditional medicine and includes various amino acids, peptides and proteins. The amino acids in the pharmacopuncture extracted from S. subspinipes mutilans by using derivatization methods were analyzed quantitatively and qualitatively by using high performance liquid chromatography (HPLC) over a 12 month period to confirm its stability. Methods: Amino acids of pharmacopuncture extracted from S. subspinipes mutilans were derived by using O-phthaldialdehyde (OPA) & 9-fluorenyl methoxy carbonyl chloride (FMOC) reagent and were analyzed using HPLC. The amino acids were detected by using a diode array detector (DAD) and a fluorescence detector (FLD) to compare a mixed amino acid standard (STD) to the pharmacopuncture from centipedes. The stability tests on the pharmacopuncture from centipedes were done using HPLC for three conditions: a room temperature test chamber, an acceleration test chamber, and a cold test chamber. Results: The pharmacopuncture from centipedes was prepared by using the method of the Korean Pharmacopuncture Institute (KPI) and through quantitative analyses was shown to contain 9 amino acids of the 16 amino acids in the mixed amino acid STD. The amounts of the amino acids in the pharmacopuncture from centipedes were 34.37 ppm of aspartate, 123.72 ppm of arginine, 170.63 ppm of alanine, 59.55 ppm of leucine and 57 ppm of lysine. The relative standard deviation (RSD %) results for the pharmacopuncture from centipedes had a maximum value of 14.95% and minimum value of 1.795% on the room temperature test chamber, the acceleration test chamber and the cold test chamber stability tests. Conclusion: Stability tests on and quantitative and qualitative analyses of the amino acids in the pharmacopuncture extracted from centipedes by using derivatization methods were performed by using HPLC. Through research, we hope to determine the relationship between time and the

  11. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

    PubMed

    Anahtar, Melis N; Bowman, Brittany A; Kwon, Douglas S

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  12. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

    PubMed Central

    Anahtar, Melis N.; Bowman, Brittany A.; Kwon, Douglas S.

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  13. Design of nucleic acid sequences for DNA computing based on a thermodynamic approach.

    PubMed

    Tanaka, Fumiaki; Kameda, Atsushi; Yamamoto, Masahito; Ohuchi, Azuma

    2005-01-01

    We have developed an algorithm for designing multiple sequences of nucleic acids that have a uniform melting temperature between the sequence and its complement and that do not hybridize non-specifically with each other based on the minimum free energy (DeltaG (min)). Sequences that satisfy these constraints can be utilized in computations, various engineering applications such as microarrays, and nano-fabrications. Our algorithm is a random generate-and-test algorithm: it generates a candidate sequence randomly and tests whether the sequence satisfies the constraints. The novelty of our algorithm is that the filtering method uses a greedy search to calculate DeltaG (min). This effectively excludes inappropriate sequences before DeltaG (min) is calculated, thereby reducing computation time drastically when compared with an algorithm without the filtering. Experimental results in silico showed the superiority of the greedy search over the traditional approach based on the hamming distance. In addition, experimental results in vitro demonstrated that the experimental free energy (DeltaG (exp)) of 126 sequences correlated well with DeltaG (min) (|R| = 0.90) than with the hamming distance (|R| = 0.80). These results validate the rationality of a thermodynamic approach. We implemented our algorithm in a graphic user interface-based program written in Java.

  14. Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast

    PubMed Central

    Misra, Ashish; Conway, Matthew F.; Johnnie, Joseph; Qureshi, Tabish M.; Lige, Bao; Derrick, Anne M.; Agbo, Eddy C.; Sriram, Ganesh

    2013-01-01

    Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo 13C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast. PMID:23898325

  15. Metabolic analyses elucidate non-trivial gene targets for amplifying dihydroartemisinic acid production in yeast.

    PubMed

    Misra, Ashish; Conway, Matthew F; Johnnie, Joseph; Qureshi, Tabish M; Lige, Bao; Derrick, Anne M; Agbo, Eddy C; Sriram, Ganesh

    2013-01-01

    Synthetic biology enables metabolic engineering of industrial microbes to synthesize value-added molecules. In this, a major challenge is the efficient redirection of carbon to the desired metabolic pathways. Pinpointing strategies toward this goal requires an in-depth investigation of the metabolic landscape of the organism, particularly primary metabolism, to identify precursor and cofactor availability for the target compound. The potent antimalarial therapeutic artemisinin and its precursors are promising candidate molecules for production in microbial hosts. Recent advances have demonstrated the production of artemisinin precursors in engineered yeast strains as an alternative to extraction from plants. We report the application of in silico and in vivo metabolic pathway analyses to identify metabolic engineering targets to improve the yield of the direct artemisinin precursor dihydroartemisinic acid (DHA) in yeast. First, in silico extreme pathway (ExPa) analysis identified NADPH-malic enzyme and the oxidative pentose phosphate pathway (PPP) as mechanisms to meet NADPH demand for DHA synthesis. Next, we compared key DHA-synthesizing ExPas to the metabolic flux distributions obtained from in vivo (13)C metabolic flux analysis of a DHA-synthesizing strain. This comparison revealed that knocking out ethanol synthesis and overexpressing glucose-6-phosphate dehydrogenase in the oxidative PPP (gene YNL241C) or the NADPH-malic enzyme ME2 (YKL029C) are vital steps toward overproducing DHA. Finally, we employed in silico flux balance analysis and minimization of metabolic adjustment on a yeast genome-scale model to identify gene knockouts for improving DHA yields. The best strategy involved knockout of an oxaloacetate transporter (YKL120W) and an aspartate aminotransferase (YKL106W), and was predicted to improve DHA yields by 70-fold. Collectively, our work elucidates multiple non-trivial metabolic engineering strategies for improving DHA yield in yeast.

  16. Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    ScienceCinema

    Patel, Kamlesh D [Ken; SNL,

    2016-07-12

    Kamlesh (Ken) Patel from Sandia National Laboratories (Livermore, California) presents "Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology " at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  17. Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    SciTech Connect

    Patel, Kamlesh D; SNL,

    2012-06-01

    Kamlesh (Ken) Patel from Sandia National Laboratories (Livermore, California) presents "Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology " at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  18. Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses.

    PubMed

    Todorovic Balint, Milena; Jelicic, Jelena; Mihaljevic, Biljana; Kostic, Jelena; Stanic, Bojana; Balint, Bela; Pejanovic, Nadja; Lucic, Bojana; Tosic, Natasa; Marjanovic, Irena; Stojiljkovic, Maja; Karan-Djurasevic, Teodora; Perisic, Ognjen; Rakocevic, Goran; Popovic, Milos; Raicevic, Sava; Bila, Jelena; Antic, Darko; Andjelic, Bosko; Pavlovic, Sonja

    2016-01-01

    The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach. PMID:27164089

  19. Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses

    PubMed Central

    Todorovic Balint, Milena; Jelicic, Jelena; Mihaljevic, Biljana; Kostic, Jelena; Stanic, Bojana; Balint, Bela; Pejanovic, Nadja; Lucic, Bojana; Tosic, Natasa; Marjanovic, Irena; Stojiljkovic, Maja; Karan-Djurasevic, Teodora; Perisic, Ognjen; Rakocevic, Goran; Popovic, Milos; Raicevic, Sava; Bila, Jelena; Antic, Darko; Andjelic, Bosko; Pavlovic, Sonja

    2016-01-01

    The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach. PMID:27164089

  20. Molecular systematics of the Amphisphaeriaceae based on cladistic analyses of partial LSU rDNA gene sequences.

    PubMed

    Jeewon, Rajesh; Liew, Edward C Y; Hyde, Kevin D

    2003-12-01

    The Amphisphaeriaceae is an important family of ascomycetes within the Xylariales. There has been, however, disagreement regarding the taxonomic placement of many genera within this family and whether it should be confined to ascomycetes producing Pestalotiopsis-like anamorphs. In this study, phylogenetic relationships among members of the Amphisphaeriaceae are investigated using partial sequences of the 28S rDNA. Molecular data provided further evidence to support the association of several coelomycetous genera with the ascomycetous Amphisphaeriaceae. Phylogenetic analyses also show that all ascomycetous genera possessing Pestalotiopsis-like anamorphs are monophyletic and confirm the anamorphic-teleomorphic connections of some. There is, however, insufficient evidence to support the restriction of Amphisphaeriaceae to genera, which produce Pestalotiopsis-like anamorphs, because the phylogenetic placement of Amphisphaeria umbrina is not fully resolved and its affinities with other members received low bootstrap support. The results also indicate that Iodosphaeria and Arecophila should be excluded from the Amphisphaeriaceae. The placement of Lanceispora in the Amphisphaeriaceae is doubtful. A broad concept of the family Amphisphaeriaceae is advocated until further data are available.

  1. Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase.

    PubMed

    Kuby, S A; Palmieri, R H; Frischat, A; Fischer, A H; Wu, L H; Maland, L; Manship, M

    1984-05-22

    The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Deduced amino acid sequence of human pulmonary surfactant proteolipid: SPL(pVal)

    SciTech Connect

    Whitsett, J.A.; Glasser, S.W.; Korfhagen, T.R.; Weaver, T.E.; Clark, J.; Pilot-Matias, T.; Meuth, J.; Fox, J.L.

    1987-05-01

    Hydrophobic, proteolipid-like protein of Mr 6500 was isolated from ether/ethanol extracts of human, canine and bovine pulmonary surfactant. Amino acid composition of the protein demonstrated a remarkable abundance of hydrophobic residues, particularly valine and leucine. The N-terminal amino acid sequence of the human protein was determined: N-Leu-Ile-Pro-Cys-Cys-Pro-Val-Asn-Leu-Lys-Arg-Leu-Leu-Ile-Val4... An oligonucleotide probe was used to screen an adult human lung cDNA library and resulted in detection of cDNA clones with predicted amino acid sequence with close identity to the N-terminal amino acid sequence of the human peptide. SPL(pVal) was found within the reading frame of a larger peptide. SPL(pVal) results from proteolytic processing of a larger preprotein. Northern blot analysis detected in a single 1.0 kilobase SPL(pVal) RNA which was less abundant in fetal than in adult lung. Mixtures of purified canine and bovine SPL(pVal) and synthetic phospholipids display properties of rapid adsorption and surface tension lowering activity characteristic of surfactant. Human SPL(pVal) is a pulmonary surfactant proteolipid which may therefore be useful in combination with phospholipids and/or other surfactant proteins for the treatment of surfactant deficiency such as hyaline membrane disease in newborn infants.

  3. Complete amino acid sequence of a human monocyte chemoattractant, a putative mediator of cellular immune reactions.

    PubMed Central

    Robinson, E A; Yoshimura, T; Leonard, E J; Tanaka, S; Griffin, P R; Shabanowitz, J; Hunt, D F; Appella, E

    1989-01-01

    In a study of the structural basis for leukocyte specificity of chemoattractants, we determined the complete amino acid sequence of human glioma-derived monocyte chemotactic factor (GDCF-2), a peptide that attracts human monocytes but not neutrophils. The choice of a tumor cell product for analysis was dictated by its relative abundance and an amino acid composition indistinguishable from that of lymphocyte-derived chemotactic factor (LDCF), the agonist thought to account for monocyte accumulation in cellular immune reactions. By a combination of Edman degradation and mass spectrometry, it was established that GDCF-2 comprises 76 amino acid residues, commencing at the N terminus with pyroglutamic acid. The peptide contains four half-cystines, at positions 11, 12, 36, and 52, which create a pair of loops, clustered at the disulfide bridges. The relative positions of the half-cystines are almost identical to those of monocyte-derived neutrophil chemotactic factor (MDNCF), a peptide of similar mass but with only 24% sequence identity to GDCF. Thus, GDCF and MDNCF have a similar gross secondary structure because of the loops formed by the clustered disulfides, and their different leukocyte specificities are most likely determined by the large differences in primary sequence. PMID:2648385

  4. Amino acid sequences of lower vertebrate parvalbumins and their evolution: parvalbumins of boa, turtle, and salamander.

    PubMed

    Maeda, N; Zhu, D X; Fitch, W M

    1984-11-01

    One major parvalbumin each was isolated from the skeletal muscle of two reptiles, a boa snake, Boa constrictor, and a map turtle, Graptemys geographica, while two parvalbumins were isolated from an amphibian, the salamander Amphiuma means. The amino acid sequences of all four parvalbumins were determined from the sequences of their tryptic peptides, which were ordered partially by homology to other parvalbumins. Phylogenetic study of these and 16 other parvalbumin sequences revealed that the turtle parvalbumin belongs to beta lineage, while the salamander sequences belong, one each, to the alpha and beta lineages defined by Goodman and Pechère (1977). Boa parvalbumin, however, while belonging to the beta lineage, clusters within the fish in all reasonably parsimonious trees. The most parsimonious trees show many parallel or back mutations in the evolution of many parvalbumin residues, although the residues responsible for Ca2+ binding are very well conserved. These most parsimonious trees show an actinopterygian rather than a crossoptyrigian origin of the tetrapods in both the alpha and beta groups. One of two electric eel parvalbumins is evolving more than 10 times faster than its paralogous partner, suggesting it may be on its way to becoming a pseudogene. It is concluded that varying rates of amino acid replacement, much homoplasy, considerable gene duplication, plus complicated lineages make the set of parvalbumin sequences unsuitable for systematic study of the origin of the tetrapods and other higher-taxa divergence, although it may be suitable within a genus or family.

  5. Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.

    PubMed

    Kinumi, Tomoya; Goto, Mari; Eyama, Sakae; Kato, Megumi; Kasama, Takeshi; Takatsu, Akiko

    2012-07-01

    A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of β-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

  6. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  7. Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase

    SciTech Connect

    Henthorn, P.S.; Raducha, M.; Edwards, Y.H.; Weiss, M.J.; Slaughter, C.; Lafferty, M.A.; Harris, H.

    1987-03-01

    A cDNA clone for human adult intestinal alkaline phosphatase (ALP) (orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1) was isolated from a lambdagt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.

  8. Genome Sequence of Azospirillum brasilense CBG497 and Comparative Analyses of Azospirillum Core and Accessory Genomes provide Insight into Niche Adaptation

    PubMed Central

    Wisniewski-Dyé, Florence; Lozano, Luis; Acosta-Cruz, Erika; Borland, Stéphanie; Drogue, Benoît; Prigent-Combaret, Claire; Rouy, Zoé; Barbe, Valérie; Mendoza Herrera, Alberto; González, Victor; Mavingui, Patrick

    2012-01-01

    Bacteria of the genus Azospirillum colonize roots of important cereals and grasses, and promote plant growth by several mechanisms, notably phytohormone synthesis. The genomes of several Azospirillum strains belonging to different species, isolated from various host plants and locations, were recently sequenced and published. In this study, an additional genome of an A. brasilense strain, isolated from maize grown on an alkaline soil in the northeast of Mexico, strain CBG497, was obtained. Comparative genomic analyses were performed on this new genome and three other genomes (A. brasilense Sp245, A. lipoferum 4B and Azospirillum sp. B510). The Azospirillum core genome was established and consists of 2,328 proteins, representing between 30% to 38% of the total encoded proteins within a genome. It is mainly chromosomally-encoded and contains 74% of genes of ancestral origin shared with some aquatic relatives. The non-ancestral part of the core genome is enriched in genes involved in signal transduction, in transport and in metabolism of carbohydrates and amino-acids, and in surface properties features linked to adaptation in fluctuating environments, such as soil and rhizosphere. Many genes involved in colonization of plant roots, plant-growth promotion (such as those involved in phytohormone biosynthesis), and properties involved in rhizosphere adaptation (such as catabolism of phenolic compounds, uptake of iron) are restricted to a particular strain and/or species, strongly suggesting niche-specific adaptation. PMID:24705077

  9. Leaf waxes, compound-specific D/H and 14C analyses in the Loess Paleosol Sequence Möhlin, Switzerland

    NASA Astrophysics Data System (ADS)

    Wüthrich, Lorenz; Bliedtner, Marcel; Kathrin Schäfer, Imke; Zech, Jana; Gaar, Dorian; Preusser, Frank; Zech, Roland

    2016-04-01

    Leaf waxes, such as long-chain n-alkanes and n-alkanoic acids, and their D/H isotopic composition, are increasingly used for paleoenvironmental and -climate reconstructions. Recent technological innovations now also allow to perform radiocarbon analyses on leaf waxes. For this study, we analyzed leaf waxes and their δD and 14C composition in the 7 m Loess Paleosol Sequence Möhlin, Switzerland. The chain length patterns in the upper part of the profile indicate n-alkane contribution from deciduous trees, while the underlying loess is dominated by inputs from grasses and herbs. Our δD record does not show depleted, glacial values compared to the Holocene, as we had expected in analogy to the Greenland ice core records. Values are most enriched at 1 m depth, i.e. well below the topsoil. Further research is needed to disentangle source effects and evapotranspirative enrichment, before the δD record can be interpreted robustly. Our radiocarbon ages for the leaf waxes are in very good agreement with independent age control based on luminescence ages, corroborating that massive loess accumulation occurred already at 35 ka. Only the uppermost 3 m were deposited during the last glacial maximum.

  10. Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

    PubMed

    Novikov, Alexey; Shah, Nita R; AlBitar-Nehme, Sami; Basheer, Soorej M; Trento, Ilaria; Tirsoaga, Alina; Moksa, Michelle; Hirst, Martin; Perry, Malcolm B; Hamidi, Asmaa El; Fernandez, Rachel C; Caroff, Martine

    2014-08-01

    Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

  11. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F.W.

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient. 2 figs.

  12. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F. William

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

  13. Reaction sequences in simulated neutralized current acid waste slurry during processing with formic acid

    SciTech Connect

    Smith, H.D.; Wiemers, K.D.; Langowski, M.H.; Powell, M.R.; Larson, D.E.

    1993-11-01

    The Hanford Waste Vitrification Plant (HWVP) is being designed for the Department of Energy to immobilize high-level and transuranic wastes as glass for permanent disposal. Pacific Northwest Laboratory is supporting the HWVP design activities by conducting laboratory-scale studies using a HWVP simulated waste slurry. Conditions which affect the slurry processing chemistry were evaluated in terms of offgas composition and peak generation rate and changes in slurry composition. A standard offgas profile defined in terms of three reaction phases, decomposition of H{sub 2}CO{sub 3}, destruction of NO{sub 2}{sup {minus}}, and production of H{sub 2} and NH{sub 3} was used as a baseline against which changes were evaluated. The test variables include nitrite concentration, acid neutralization capacity, temperature, and formic acid addition rate. Results to date indicate that pH is an important parameter influencing the N{sub 2}O/NO{sub x} generation ratio; nitrite can both inhibit and activate rhodium as a catalyst for formic acid decomposition to CO{sub 2} and H{sub 2}; and a separate reduced metal phase forms in the reducing environment. These data are being compiled to provide a basis for predicting the HWVP feed processing chemistry as a function of feed composition and operation variables, recommending criteria for chemical adjustments, and providing guidelines with respect to important control parameters to consider during routine and upset plant operation.

  14. The complete amino acid sequence of lectin-C from the roots of pokeweed (Phytolacca americana).

    PubMed

    Yamaguchi, K; Mori, A; Funatsu, G

    1995-07-01

    The complete amino acid sequence of pokeweed lectin-C (PL-C) consisting of 126 residues has been determined. PL-C is an acidic simple protein with molecular mass of 13,747 Da and consists of three cysteine-rich domains with 51-63% homology. PL-C shows homology to chitin-binding proteins such as wheat germ agglutinin, and all eight cysteine residues in the three domains of PL-C are completely conserved in all other chitin-binding domains.

  15. Amino-acid sequence of a cooperative, dimeric myoglobin from the gastropod mollusc, Buccinum undatum L.

    PubMed

    Wen, D; Laursen, R A

    1994-10-19

    The complete amino-acid sequence of a dimeric myoglobin from the radular mussel of the gastropod mollusc, Buccinum undatum L. has been determined. The globin, which shows cooperative binding of oxygen, contains 146 amino acids, is N-terminal aminoacetylated, and has histidine residues at position 65 and 97, corresponding to the heme-binding histidines seen in mammalian myoglobins. It shows about 75% and 50% homology, respectively, with the dimeric molluscan myoglobins from Busycon canaliculatum and Cerithidea rhizophorarum, the former of which also shows weak cooperatively, but much less similarity to other species of myoglobin and hemoglobin.

  16. The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp. lactis IL1403

    PubMed Central

    Bolotin, Alexander; Wincker, Patrick; Mauger, Stéphane; Jaillon, Olivier; Malarme, Karine; Weissenbach, Jean; Ehrlich, S. Dusko; Sorokin, Alexei

    2001-01-01

    Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group. [The sequence data described in this paper has been submitted to the GenBank data library under accession no. AE005176.] PMID:11337471

  17. Amino acid sequence of human cholinesterase. Annual report, 30 September 1984-30 September 1985

    SciTech Connect

    Lockridge, O.

    1985-10-01

    The active-site serine residue is located 198 amino acids from the N-terminal. The active-site peptide was isolated from three different genetic types of human serum cholinesterase: from usual, atypical, and atypical-silent genotypes. It was found that the amino acid sequence of the active-site peptide was identical in all three genotypes. Comparison of the complete sequences of cholinesterase from human serum and acetylcholinesterase from the electric organ of Torpedo californica shows an identity of 53%. Cholinesterase is of interest to the Department of Defense because cholinesterase protects against organophosphate poisons of the type used in chemical warfare. The structural results presented here will serve as the basis for cloning the gene for cholinesterase. The potential uses of large amounts of cholinesterase would be for cleaning up spills of organophosphates and possibly for detoxifying exposed personnel.

  18. Amino acid sequence differences in pancreatic ribonucleases from water buffalo breeds from Indonesia and Italy.

    PubMed

    Sidik, A; Martena, B; Beintema, J J

    1979-12-01

    The amino acid sequences of the pancreatic ribonucleases from river-breed water buffaloes from Italy and swamp-breed water buffaloes from Indonesia differ at three positions. One of the differences involves a replacement of asparagine-34, with covalently attached carbohydrate on all molecules, in the river-breed enzyme by serine in the swamp-breed enzyme. The ribonuclease content of the pancreas differs considerably between breeds and is lower in river buffaloes. A ribonuclease preparation from two swamp buffaloes contained a minor glycosylated component. Preliminary evidence was obtained that the amino acid sequence of this component has factors in common with the main component of the swamp-breed ribonuclease and with the river-breed enzyme.

  19. Stereochemical Sequence Ion Selectivity: Proline versus Pipecolic-acid-containing Protonated Peptides

    NASA Astrophysics Data System (ADS)

    Abutokaikah, Maha T.; Guan, Shanshan; Bythell, Benjamin J.

    2016-10-01

    Substitution of proline by pipecolic acid, the six-membered ring congener of proline, results in vastly different tandem mass spectra. The well-known proline effect is eliminated and amide bond cleavage C-terminal to pipecolic acid dominates instead. Why do these two ostensibly similar residues produce dramatically differing spectra? Recent evidence indicates that the proton affinities of these residues are similar, so are unlikely to explain the result [Raulfs et al., J. Am. Soc. Mass Spectrom. 25, 1705-1715 (2014)]. An additional hypothesis based on increased flexibility was also advocated. Here, we provide a computational investigation of the "pipecolic acid effect," to test this and other hypotheses to determine if theory can shed additional light on this fascinating result. Our calculations provide evidence for both the increased flexibility of pipecolic-acid-containing peptides, and structural changes in the transition structures necessary to produce the sequence ions. The most striking computational finding is inversion of the stereochemistry of the transition structures leading to "proline effect"-type amide bond fragmentation between the proline/pipecolic acid-congeners: R (proline) to S (pipecolic acid). Additionally, our calculations predict substantial stabilization of the amide bond cleavage barriers for the pipecolic acid congeners by reduction in deleterious steric interactions and provide evidence for the importance of experimental energy regime in rationalizing the spectra.

  20. On human disease-causing amino acid variants: statistical study of sequence and structural patterns

    PubMed Central

    Alexov, Emil

    2015-01-01

    Statistical analysis was carried out on large set of naturally occurring human amino acid variations and it was demonstrated that there is a preference for some amino acid substitutions to be associated with diseases. At an amino acid sequence level, it was shown that the disease-causing variants frequently involve drastic changes of amino acid physico-chemical properties of proteins such as charge, hydrophobicity and geometry. Structural analysis of variants involved in diseases and being frequently observed in human population showed similar trends: disease-causing variants tend to cause more changes of hydrogen bond network and salt bridges as compared with harmless amino acid mutations. Analysis of thermodynamics data reported in literature, both experimental and computational, indicated that disease-causing variants tend to destabilize proteins and their interactions, which prompted us to investigate the effects of amino acid mutations on large databases of experimentally measured energy changes in unrelated proteins. Although the experimental datasets were linked neither to diseases nor exclusory to human proteins, the observed trends were the same: amino acid mutations tend to destabilize proteins and their interactions. Having in mind that structural and thermodynamics properties are interrelated, it is pointed out that any large change of any of them is anticipated to cause a disease. PMID:25689729

  1. Characterization of rapeseed (Brassica napus) oils by bulk C, O, H, and fatty acid C stable isotope analyses.

    PubMed

    Richter, Eva Katharina; Spangenberg, Jorge E; Kreuzer, Michael; Leiber, Florian

    2010-07-14

    Rapeseed ( Brassica napus ) oils differing in cultivar, sites of growth, and harvest year were characterized by fatty acid concentrations and carbon, hydrogen, and oxygen stable isotope analyses of bulk oils (delta(13)C(bulk), delta(2)H(bulk), delta(18)O(bulk) values) and individual fatty acids (delta(13)C(FA)). The delta(13)C(bulk), delta(2)H(bulk), and delta(18)O(bulk) values were determined by continuous flow combustion and high-temperature conversion elemental analyzer-isotope ratio mass spectrometry (EA/IRMS, TC-EA/IRMS). The delta(13)C(FA) values were determined using gas chromatography--combustion-isotope ratio mass spectrometry (GC/C/IRMS). For comparison, other C(3) vegetable oils rich in linolenic acid (flax and false flax oils) and rich in linoleic acid (poppy, sunflower, and safflower oils) were submitted to the same chemical and isotopic analyses. The bulk and molecular delta(13)C values were typical for C(3) plants. The delta(13)C value of palmitic acid (delta(13)C(16:0)) and n-3 alpha-linolenic acid (delta(13)C(18:3n-3)) differed (p < 0.001) between rape, flax, and poppy oils. Also within species, significant differences of delta(13)C(FA) were observed (p < 0.01). The hydrogen and oxygen isotope compositions of rape oil differed between cultivars (p < 0.05). Major differences in the individual delta(13)C(FA) values were found. A plant-specific carbon isotope fractionation occurs during the biosynthesis of the fatty acids and particularly during desaturation of C(18) acids in rape and flax. Bulk oil and specific fatty acid stable isotope analysis might be useful in tracing dietary lipids differing in their origin.

  2. Self-sequencing of amino acids and origins of polyfunctional protocells

    NASA Technical Reports Server (NTRS)

    Fox, S. W.

    1984-01-01

    The role of proteins in the origin of living things is discussed. It has been experimentally established that amino acids can sequence themselves under simulated geological conditions with highly nonrandom products which accordingly contain diverse information. Multiple copies of each type of macromolecule are formed, resulting in greater power for any protoenzymic molecule than would accrue from a single copy of each type. Thermal proteins are readily incorporated into laboratory protocells. The experimental evidence for original polyfunctional protocells is discussed.

  3. Comparisons of the Distribution of Nucleotides and Common Sequences in Deoxyribonucleic Acid from Selected Bacteriophages

    PubMed Central

    Skalka, A.; Hanson, P.

    1972-01-01

    Results from comparisons of deoxyribonucleic acid (DNA) from several classes of bacteriophages suggest that most phage chromosomes contain either a homogeneous distribution of nucleotides or are made up of a few, rather large segments of different quanine plus cytosine (G + C) contents which are internally homogeneous. Among those temperate phages tested, most contained segmented DNA. Comparisons of sequence similarities among segments from lambdoid phage DNA species revealed the following order in relatedness to λ: 82 (and 434) > 21 > 424 > φ80. Most common sequences are found in the highest G + C segments, which in λ contain head and tail genes. Hybridization tests with λ and 186 or P2 DNA species verified that the lambdoids and 186 and P2 belong to two distinct groups. There are fewer homologous sequences between the DNA species of coliphages λ and P2 or 186 than there are between the DNA species of coliphage λ and salmonella phage P22. PMID:4553679

  4. Structure of the fully modified left-handed cyclohexene nucleic acid sequence GTGTACAC.

    PubMed

    Robeyns, Koen; Herdewijn, Piet; Van Meervelt, Luc

    2008-02-13

    CeNA oligonucleotides consist of a phosphorylated backbone where the deoxyribose sugars are replaced by cyclohexene moieties. The X-ray structure determination and analysis of a fully modified octamer sequence GTGTACAC, which is the first crystal structure of a carbocyclic-based nucleic acid, is presented. This particular sequence was built with left-handed building blocks and crystallizes as a left-handed double helix. The helix can be characterized as belonging to the (mirrored) A-type family. Crystallographic data were processed up to 1.53 A, and the octamer sequence crystallizes in the space group R32. The sugar puckering is found to adopt the 3H2 half-chair conformation which mimics the C3'-endo conformation of the ribose sugar. The double helices stack on top of each other to form continuous helices, and static disorder is observed due to this end-to-end stacking.

  5. Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

    PubMed

    Papayannopoulos, I A; Biemann, K

    1992-02-01

    The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

  6. Fatty Acid Profile and Unigene-Derived Simple Sequence Repeat Markers in Tung Tree (Vernicia fordii)

    PubMed Central

    Zhang, Lin; Jia, Baoguang; Tan, Xiaofeng; Thammina, Chandra S.; Long, Hongxu; Liu, Min; Wen, Shanna; Song, Xianliang; Cao, Heping

    2014-01-01

    Tung tree (Vernicia fordii) provides the sole source of tung oil widely used in industry. Lack of fatty acid composition and molecular markers hinders biochemical, genetic and breeding research. The objectives of this study were to determine fatty acid profiles and develop unigene-derived simple sequence repeat (SSR) markers in tung tree. Fatty acid profiles of 41 accessions showed that the ratio of α-eleostearic acid was increasing continuously with a parallel trend to the amount of tung oil accumulation while the ratios of other fatty acids were decreasing in different stages of the seeds and that α-eleostearic acid (18∶3) consisted of 77% of the total fatty acids in tung oil. Transcriptome sequencing identified 81,805 unigenes from tung cDNA library constructed using seed mRNA and discovered 6,366 SSRs in 5,404 unigenes. The di- and tri-nucleotide microsatellites accounted for 92% of the SSRs with AG/CT and AAG/CTT being the most abundant SSR motifs. Fifteen polymorphic genic-SSR markers were developed from 98 unigene loci tested in 41 cultivated tung accessions by agarose gel and capillary electrophoresis. Genbank database search identified 10 of them putatively coding for functional proteins. Quantitative PCR demonstrated that all 15 polymorphic SSR-associated unigenes were expressed in tung seeds and some of them were highly correlated with oil composition in the seeds. Dendrogram revealed that most of the 41 accessions were clustered according to the geographic region. These new polymorphic genic-SSR markers will facilitate future studies on genetic diversity, molecular fingerprinting, comparative genomics and genetic mapping in tung tree. The lipid profiles in the seeds of 41 tung accessions will be valuable for biochemical and breeding studies. PMID:25167054

  7. The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses.

    PubMed

    Yergeau, Etienne; Hogues, Hervé; Whyte, Lyle G; Greer, Charles W

    2010-09-01

    The fate of the carbon stocked in permafrost following global warming and permafrost thaw is of major concern in view of the potential for increased CH(4) and CO(2) emissions from these soils. Complex carbon compound degradation and greenhouse gas emissions are due to soil microbial communities, but no comprehensive study has yet addressed their composition and functional potential in permafrost. Here, a 2-m deep permafrost sample and its overlying active layer soil were subjected to metagenomic sequencing, quantitative PCR (qPCR) and microarray analyses. The active layer soil and the 2-m permafrost microbial community structures were very similar, with Actinobacteria being the dominant phylum. The two samples also possessed a highly similar spectrum of functional genes, especially when compared with other already published metagenomes. Key genes related to methane generation, methane oxidation and organic matter degradation were highly diverse for both samples in the metagenomic libraries and some (for example, pmoA) showed relatively high abundance in qPCR assays. Genes related to nitrogen fixation and ammonia oxidation, which could have important roles following climatic change in these nitrogen-limited environments, showed low diversity but high abundance. The 2-m permafrost showed lower abundance and diversity for all the assessed genes and taxa. Experimental biases were also evaluated using qPCR and showed that the whole-community genome amplification technique used caused representational biases in the metagenomic libraries by increasing the abundance of Bacteroidetes and decreasing the abundance of Actinobacteria. This study describes for the first time the detailed functional potential of permafrost-affected soils.

  8. Cloning and nucleotide sequence analyses of 11 genome segments of two American and one British equine rotavirus strains.

    PubMed

    Ma, Yongping; Wen, Xiaobo; Hoshino, Yasutaka; Yuan, L

    2015-03-23

    Group A equine rotavirus (ERV) is the main cause of diarrhea in foals and causes severe economic loss due to morbidity and mortality on stud farming worldwide. Molecular evolution of equine rotaviruses remains understudies. In this study, whole-genomic analysis of 2 group A ERV, FI-14 (G3P[12]), H-2 (G3P[12]) isolated from American, and FI23 (G14P[12]) from British was carried out and genotype constellations were determined as G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 for FI-14; G14-P[12]-I2-R2-C2-M3-A10-N2-T3-E2-H7 for FI23; and G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 for H-2, respectively. With the exception of the VP7 and VP6 gene, 2 G3P[12] strains (FI-14 and H-2) and one G14P[12] strain (FI23) were highly related genetically. Of note, the VP6 genotype of H-2 strain was previously reported to be I2, however, sequence and phylogenetic analyses demonstrated that it was I6. Therefore, it showed that G3P[12] ERV strains and G14P[12] ERV strains bore a distinct VP6 genotype: I6 for G3P[12] strains and I2 for G14P[12] strains. Moreover, it demonstrated that T-cell epitope 299P-300P/Q residues (PP/Q) of VP6 may be considered as I2 ERV typical molecular marker, which facilitates the analysis of the molecular evolution of equine rotaviruses. PMID:25631250

  9. Some properties and amino acid sequence of plastocyanin from a green alga, Ulva arasakii.

    PubMed

    Yoshizaki, F; Fukazawa, T; Mishina, Y; Sugimura, Y

    1989-08-01

    Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775 nm, and shoulders at 259, 265, 269, and 282.5 nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356 V at pH 7.0 with a ferri- and ferrocyanide system. The molecular weight was estimated to be 10,200 and 11,000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10,236 including one copper atom. The complete sequence is as follows: AQIVKLGGDDGALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETV VRKLSTPGVY G VYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closet to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants. PMID:2509442

  10. Characterization of the microbial acid mine drainage microbial community using culturing and direct sequencing techniques.

    PubMed

    Auld, Ryan R; Myre, Maxine; Mykytczuk, Nadia C S; Leduc, Leo G; Merritt, Thomas J S

    2013-05-01

    We characterized the bacterial community from an AMD tailings pond using both classical culturing and modern direct sequencing techniques and compared the two methods. Acid mine drainage (AMD) is produced by the environmental and microbial oxidation of minerals dissolved from mining waste. Surprisingly, we know little about the microbial communities associated with AMD, despite the fundamental ecological roles of these organisms and large-scale economic impact of these waste sites. AMD microbial communities have classically been characterized by laboratory culturing-based techniques and more recently by direct sequencing of marker gene sequences, primarily the 16S rRNA gene. In our comparison of the techniques, we find that their results are complementary, overall indicating very similar community structure with similar dominant species, but with each method identifying some species that were missed by the other. We were able to culture the majority of species that our direct sequencing results indicated were present, primarily species within the Acidithiobacillus and Acidiphilium genera, although estimates of relative species abundance were only obtained from direct sequencing. Interestingly, our culture-based methods recovered four species that had been overlooked from our sequencing results because of the rarity of the marker gene sequences, likely members of the rare biosphere. Further, direct sequencing indicated that a single genus, completely missed in our culture-based study, Legionella, was a dominant member of the microbial community. Our results suggest that while either method does a reasonable job of identifying the dominant members of the AMD microbial community, together the methods combine to give a more complete picture of the true diversity of this environment. PMID:23485423

  11. Complete amino acid sequence of chitinase-A from leaves of pokeweed (Phytolacca americana).

    PubMed

    Yamagami, T; Tanigawa, M; Ishiguro, M; Funatsu, G

    1998-04-01

    The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.

  12. The amino acid sequence of the aspartate aminotransferase from baker's yeast (Saccharomyces cerevisiae).

    PubMed Central

    Cronin, V B; Maras, B; Barra, D; Doonan, S

    1991-01-01

    1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5. PMID:1859361

  13. [MOLECULAR EVOLUTION OF ION CHANNELS: AMINO ACID SEQUENCES AND 3D STRUCTURES].

    PubMed

    Korkosh, V S; Zhorov, B S; Tikhonov, D B

    2016-01-01

    An integral part of modern evolutionary biology is comparative analysis of structure and function of macromolecules such as proteins. The first and critical step to understand evolution of homologous proteins is their amino acid sequence alignment. However, standard algorithms fop not provide unambiguous sequence alignments for proteins of poor homology. More reliable results can be obtained by comparing experimental 3D structures obtained at atomic resolution, for instance, with the aid of X-ray structural analysis. If such structures are lacking, homology modeling is used, which may take into account indirect experimental data on functional roles of individual amino-acid residues. An important problem is that the sequence alignment, which reflects genetic modifications, does not necessarily correspond to the functional homology. The latter depends on three-dimensional structures which are critical for natural selection. Since alignment techniques relying only on the analysis of primary structures carry no information on the functional properties of proteins, including 3D structures into consideration is very important. Here we consider several examples involving ion channels and demonstrate that alignment of their three-dimensional structures can significantly improve sequence alignments obtained by traditional methods.

  14. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

    PubMed Central

    Rhee, Mun Su; Moritz, Brélan E.; Xie, Gary; Glavina del Rio, T.; Dalin, E.; Tice, H.; Bruce, D.; Goodwin, L.; Chertkov, O.; Brettin, T.; Han, C.; Detter, C.; Pitluck, S.; Land, Miriam L.; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O.; Shanmugam, K. T.

    2011-01-01

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed. PMID:22675583

  15. BeadCons: detection of nucleic acid sequences by flow cytometry.

    PubMed

    Horejsh, Douglas; Martini, Federico; Capobianchi, Maria Rosaria

    2005-11-01

    Molecular beacons are single-stranded nucleic acid structures with a terminal fluorophore and a distal, terminal quencher. These molecules are typically used in real-time PCR assays, but have also been conjugated with solid matrices. This unit describes protocols related to molecular beacon-conjugated beads (BeadCons), whose specific hybridization with complementary target sequences can be resolved by cytometry. Assay sensitivity is achieved through the concentration of fluorescence signal on discrete particles. By using molecular beacons with different fluorophores and microspheres of different sizes, it is possible to construct a fluid array system with each bead corresponding to a specific target nucleic acid. Methods are presented for the design, construction, and use of BeadCons for the specific, multiplexed detection of unlabeled nucleic acids in solution. The use of bead-based detection methods will likely lead to the design of new multiplex molecular diagnostic tools.

  16. Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

    PubMed Central

    Qin, Peter Z; Haworth, Ian S; Cai, Qi; Kusnetzow, Ana K; Grant, Gian Paola G; Price, Eric A; Sowa, Glenna Z; Popova, Anna; Herreros, Bruno; He, Honghang

    2008-01-01

    This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron–electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes. PMID:17947978

  17. [Partial sequence homology of FtsZ in phylogenetics analysis of lactic acid bacteria].

    PubMed

    Zhang, Bin; Dong, Xiu-zhu

    2005-10-01

    FtsZ is a structurally conserved protein, which is universal among the prokaryotes. It plays a key role in prokaryote cell division. A partial fragment of the ftsZ gene about 800bp in length was amplified and sequenced and a partial FtsZ protein phylogenetic tree for the lactic acid bacteria was constructed. By comparing the FtsZ phylogenetic tree with the 16S rDNA tree, it was shown that the two trees were similar in topology. Both trees revealed that Pediococcus spp. were closely related with L. casei group of Lactobacillus spp. , but less related with other lactic acid cocci such as Enterococcus and Streptococcus. The results also showed that the discriminative power of FtsZ was higher than that of 16S rDNA for either inter-species or inter-genus and could be a very useful tool in species identification of lactic acid bacteria. PMID:16342751

  18. The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes.

    PubMed

    Araki, T; Kuramoto, M; Torikata, T

    1990-09-01

    The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group.

  19. The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes.

    PubMed

    Araki, T; Kuramoto, M; Torikata, T

    1990-09-01

    The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group. PMID:1368578

  20. Fatty acid composition analyses of the DCMU resistant mutants of Nannochloropsis oculata (eustigmatophyceae)

    NASA Astrophysics Data System (ADS)

    Jimin, Zhang; Shuang, Liu; Xue, Sun; Guanpin, Yang; Xuecheng, Zhang; Zhenhui, Gao

    2003-04-01

    Ultraviolet mutagenesis was applied to Nannochloropsis oculata and three mutants resistant to 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU) were isolated. The cellular chlorophyll a and total lipid content of the wild are higher in the medium supplemented with DCMU than in the control without DCMU. Without DCMU, the growth rates and chlorophyll a contents of the mutants are similar to those of the wild. Significant changes of fatty acid content and composition have occurred in DCMU-resistant mutants growing in the medium supplemented with DCMU. The total lipid, palmitic acid (16:0), palmitoleic acid (16:1ω9) and oleic (18:1ω9) contents decrease significantly, while the vaccenic acid (18:1ω11) increases significantly and the EPA content of dried powder increases slightly in the mutants. The study may provide a basis to improve EPA content in Nannochloropsis oculata in the future.

  1. THERMAL AND SPECTROSCOPIC ANALYSES OF CAUSTIC LIDE SOLVENT EXTRACTION SOLVENT CONTACTED WITH 16 MOLAR AND 8 MOLAR NITRIC ACID

    SciTech Connect

    Fondeur, F; David Hobbs, D; Samuel Fink, S

    2007-07-12

    Thermal and spectroscopic analyses were performed on multiple layers formed from contacting Caustic Side Solvent Extraction (CSSX) solvent with 1 M or 3 M nitric acid. A slow chemical reaction occurs (i.e., over several weeks) between the solvent and 1 M or 3 M nitric acid as evidenced by color changes and the detection of nitro groups in the infrared spectrum of the aged samples. Thermal analysis revealed that decomposition of the resulting mixture does not meet the definition of explosive or deflagrating material.

  2. THERMAL AND SPECTROSCOPIC ANALYSES OF CAUSTIC SIDE SOLVENT EXTRACTION SOLVENT CONTACTED WITH 1 MOLARAND 3 MOLAR NITRIC ACID

    SciTech Connect

    Fondeur, F; David Hobbs, D; Samuel Fink, S

    2007-07-23

    Thermal and spectroscopic analyses were performed on multiple layers formed from contacting Caustic Side Solvent Extraction (CSSX) solvent with 1 M or 3 M nitric acid. A slow chemical reaction occurs (i.e., over several weeks) between the solvent and 1 M or 3 M nitric acid as evidenced by color changes and the detection of nitro groups in the infrared spectrum of the aged samples. Thermal analysis revealed that decomposition of the resulting mixture does not meet the definition of explosive or deflagrating material.

  3. Conservation Weighting Functions Enable Covariance Analyses to Detect Functionally Important Amino Acids

    PubMed Central

    Colwell, Lucy J.; Brenner, Michael P.; Murray, Andrew W.

    2014-01-01

    The explosive growth in the number of protein sequences gives rise to the possibility of using the natural variation in sequences of homologous proteins to find residues that control different protein phenotypes. Because in many cases different phenotypes are each controlled by a group of residues, the mutations that separate one version of a phenotype from another will be correlated. Here we incorporate biological knowledge about protein phenotypes and their variability in the sequence alignment of interest into algorithms that detect correlated mutations, improving their ability to detect the residues that control those phenotypes. We demonstrate the power of this approach using simulations and recent experimental data. Applying these principles to the protein families encoded by Dscam and Protocadherin allows us to make testable predictions about the residues that dictate the specificity of molecular interactions. PMID:25379728

  4. A case study on discovery of novel Citrus leprosis virus cytoplasmic type 2 utilizing small RNA libraries by next generation sequencing and bioinformatic analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The identification of novel plant viruses is a tricky matter. Most plant virus diagnostics are based on immunological or nucleic acid based assays, where prior characterization of the virus (either antibodies or genetic sequence) is required for reagent production. There are no universal nucleic a...

  5. N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

    PubMed

    Maruyama, Masugi; Sugiki, Masahiko; Anai, Keita; Yoshida, Etsuo

    2002-08-01

    We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells. PMID:12165326

  6. Protein sequence analysis by incorporating modified chaos game and physicochemical properties into Chou's general pseudo amino acid composition.

    PubMed

    Xu, Chunrui; Sun, Dandan; Liu, Shenghui; Zhang, Yusen

    2016-10-01

    In this contribution we introduced a novel graphical method to compare protein sequences. By mapping a protein sequence into 3D space based on codons and physicochemical properties of 20 amino acids, we are able to get a unique P-vector from the 3D curve. This approach is consistent with wobble theory of amino acids. We compute the distance between sequences by their P-vectors to measure similarities/dissimilarities among protein sequences. Finally, we use our method to analyze four datasets and get better results compared with previous approaches. PMID:27375218

  7. Comparative genomic sequence and expression analyses of Medicago truncatula and alfalfa subspecies falcata COLD-ACCLIMATION-SPECIFIC genes.

    PubMed

    Pennycooke, Joyce C; Cheng, Hongmei; Stockinger, Eric J

    2008-03-01

    In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key step in cold acclimation. CBFs in turn activate a battery of downstream genes known as the CBF regulon, which collectively act to increase tolerance to low temperatures. Fundamental questions are: What determines the size and scope of the CBF regulon, and is this is a major determinant of the low-temperature tolerance capacity of individual plant species? Here we have begun to address these questions through comparative analyses of Medicago truncatula and Medicago sativa subsp. falcata. M. truncatula survived to -4 degrees C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived -14 degrees C. Both species possessed low-temperature-induced CBFs but differed in the expression of the COLD-ACCLIMATION-SPECIFIC (CAS) genes, which are candidate CBF targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31 homolog was not. M. falcata also possessed additional CAS30 homologs in comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple pairs of closely spaced C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motifs, the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one CRT/DRE partner of the two proximal partner pairs. CAS genes also shared a promoter structure comprising modules proximal and distal to the coding sequence. CAS15, highly cold-responsive in both species, harbored numerous CRT/DRE motifs, but only in the distal module. However, fusion of the MtCAS15 promoter, including the distal module, to a reporter gene did not result in low-temperature responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and MfCAS30 promoter fusions were low-temperature responsive, although the MfCAS31 fusion was less robust than the MfCAS30 fusion. From these studies we conclude that CAS genes harbor CRT/DRE motifs, their

  8. Data on the evolutionary history of the V(D)J recombination-activating protein 1 - RAG1 coupled with sequence and variant analyses.

    PubMed

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J; Muppavarapu, Sekhar; Tandon, Ravi

    2016-09-01

    RAG1 protein is one of the key component of RAG complex regulating the V(D)J recombination. There are only few studies for RAG1 concerning evolutionary history, detailed sequence and mutational hotspots. Herein, we present out datasets used for the recent comprehensive study of RAG1 based on sequence, phylogenetic and genetic variant analyses (Kumar et al., 2015) [1]. Protein sequence alignment helped in characterizing the conserved domains and regions of RAG1. It also aided in unraveling ancestral RAG1 in the sea urchin. Human genetic variant analyses revealed 751 mutational hotspots, located both in the coding and the non-coding regions. For further analysis and discussion, see (Kumar et al., 2015) [1]. PMID:27284568

  9. Spectroscopic analyses and studies on respective interaction of cyanuric acid and uric acid with bovine serum albumin and melamine

    NASA Astrophysics Data System (ADS)

    Chen, Dandan; Wu, Qiong; Wang, Jun; Wang, Qi; Qiao, Heng

    2015-01-01

    In this work, the fluorescence quenching was used to study the interaction of cyanuric acid (CYA) and uric acid (UA) with bovine serum albumin (BSA) at two different temperatures (283 K and 310 K). The bimolecular quenching constant (Kq), apparent quenching constant (Ksv), effective binding constant (KA) and corresponding dissociation constant (KD), binding site number (n) and binding distance (r) were calculated by adopting Stern-Volmer, Lineweaver-Burk, Double logarithm and overlap integral equations. The results show that CYA and UA are both able to obviously bind to BSA, but the binding strength order is BSA + CYA < BSA + UA. And then, the interactions of CYA and UA with melamine (MEL) under the same conditions were also studied by using similar methods. The results indicates that both CYA and UA can bind together closely with melamine (MEL). It is wished that these research results would facilitate the understanding the formation of kidney stones and gout in the body after ingesting excess MEL.

  10. Purification to homogeneity and amino acid sequence analysis of two anionic species of human interleukin 1

    PubMed Central

    1986-01-01

    Two anionic species of human IL-1 have been purified to homogeneity. These molecules were characterized as having pI of 5.4 and 5.2 and molecular weights identical to IL-1/6.8 (17,500). The specific activities of IL-1/5.4 and IL-1/5.2, as measured in the mouse thymocyte co-mitogenic assay, were identical to that of IL-1/6.8, namely 1.2 X 10(7) U/mg, with half-maximal stimulation observed at 2 X 10(-11) M. IL- 1/5.4 and IL-1/5.2 were found to be antigenically distinct from IL- 1/6.8 in an ELISA. IL-1/5.4 was structurally distinct from IL-1/6.8 based on reverse-phase HPLC or CNBr peptides. Intact IL-1/5.2 and three intact CNBr peptides of IL-1/5.4 were sequenced, with the identification of 74 amino acid residues. These sequences were found to correspond exactly with the amino acid sequence deduced from the IL-1- alpha cDNA reported by March et al. PMID:3487613

  11. Protein meta-functional signatures from combining sequence, structure, evolution, and amino acid property information.

    PubMed

    Wang, Kai; Horst, Jeremy A; Cheng, Gong; Nickle, David C; Samudrala, Ram

    2008-09-26

    Protein function is mediated by different amino acid residues, both their positions and types, in a protein sequence. Some amino acids are responsible for the stability or overall shape of the protein, playing an indirect role in protein function. Others play a functionally important role as part of active or binding sites of the protein. For a given protein sequence, the residues and their degree of functional importance can be thought of as a signature representing the function of the protein. We have developed a combination of knowledge- and biophysics-based function prediction approaches to elucidate the relationships between the structural and the functional roles of individual residues and positions. Such a meta-functional signature (MFS), which is a collection of continuous values representing the functional significance of each residue in a protein, may be used to study proteins of known function in greater detail and to aid in experimental characterization of proteins of unknown function. We demonstrate the superior performance of MFS in predicting protein functional sites and also present four real-world examples to apply MFS in a wide range of settings to elucidate protein sequence-structure-function relationships. Our results indicate that the MFS approach, which can combine multiple sources of information and also give biological interpretation to each component, greatly facilitates the understanding and characterization of protein function.

  12. Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid.

    PubMed

    Mohn, W W

    1995-06-01

    Eleven isolates capable of growth on the resin acid dehydroabietic acid (DhA) were obtained from a sequencing batch reactor designed to treat a high-strength process stream from a paper mill. The isolates belonged to two groups, represented by strains DhA-33 and DhA-35, which were characterized. In the bioreactor, bacteria like DhA-35 were more abundant than those like DhA-33. The population in the bioreactor of organisms capable of growth on DhA was estimated to be 1.1 x 10(6) propagules per ml, based on a most-probable-number determination. Analysis of small-subunit rRNA partial sequences indicated that DhA-33 was most closely related to Sphingomonas yanoikuyae (Sab = 0.875) and that DhA-35 was most closely related to Zoogloea ramigera (Sab = 0.849). Both isolates additionally grew on other abietanes, i.e., abietic and palustric acids, but not on the pimaranes, pimaric and isopimaric acids. For DhA-33 and DhA-35 with DhA as the sole organic substrate, doubling times were 2.7 and 2.2 h, respectively, and growth yields were 0.30 and 0.25 g of protein per g of DhA, respectively. Glucose as a cosubstrate stimulated growth of DhA-33 on DhA and stimulated DhA degradation by the culture. Pyruvate as a cosubstrate did not stimulate growth of DhA-35 on DhA and reduced the specific rate of DhA degradation of the culture. DhA induced DhA and abietic acid degradation activities in both strains, and these activities were heat labile. Cell suspensions of both strains consumed DhA at a rate of 6 mumol mg of protein-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Development of a SCAR (sequence-characterised amplified region) marker for acid resistance-related gene in Lactobacillus plantarum.

    PubMed

    Liu, Shu-Wen; Li, Kai; Yang, Shi-Ling; Tian, Shu-Fen; He, Ling

    2015-03-01

    A sequence characterised amplified region marker was developed to determine an acid resistance-related gene in Lactobacillus plantarum. A random amplified polymorphic DNA marker named S116-680 was reported to be closely related to the acid resistance of the strains. The DNA band corresponding to this marker was cloned and sequenced with the induction of specific designed PCR primers. The results of PCR test helped to amplify a clear specific band of 680 bp in the tested acid-resistant strains. S116-680 marker would be useful to explore the acid-resistant mechanism of L. plantarum and to screen desirable malolactic fermentation strains.

  14. Robust gene expression and mutation analyses of RNA-sequencing of formalin-fixed diagnostic tumor samples.

    PubMed

    Graw, Stefan; Meier, Richard; Minn, Kay; Bloomer, Clark; Godwin, Andrew K; Fridley, Brooke; Vlad, Anda; Beyerlein, Peter; Chien, Jeremy

    2015-01-01

    Current genomic studies are limited by the availability of fresh tissue samples. Here, we show that Illumina RNA sequencing of formalin-fixed diagnostic tumor samples produces gene expression that is strongly correlated with matched frozen tumor samples (r > 0.89). In addition, sequence variations identified from FFPE RNA show 99.67% concordance with that from exome sequencing of matched frozen tumor samples. Because FFPE is a routine diagnostic sample preparation, the feasibility results reported here will facilitate the setup of large-scale research and clinical studies in medical genomics that are currently limited by the availability of fresh frozen samples. PMID:26202458

  15. Nucleic and amino acid sequences relating to a novel transketolase, and methods for the expression thereof

    DOEpatents

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Lange, Bernd Markus; McCaskill, David G.

    2001-01-01

    cDNAs encoding 1-deoxyxylulose-5-phosphate synthase from peppermint (Mentha piperita) have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) are provided which code for the expression of 1-deoxyxylulose-5-phosphate synthase from plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, replicable recombinant cloning vehicles are provided which code for plant 1-deoxyxylulose-5-phosphate synthases, or for a base sequence sufficiently complementary to at least a portion of 1-deoxyxylulose-5-phosphate synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a plant 1-deoxyxylulose-5-phosphate synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant 1-deoxyxylulose-5-phosphate synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant 1-deoxyxylulose-5-phosphate synthase may be used to obtain expression or enhanced expression of 1-deoxyxylulose-5-phosphate synthase in plants in order to enhance the production of 1-deoxyxylulose-5-phosphate, or its derivatives such as isopentenyl diphosphate (BP), or may be otherwise employed for the regulation or expression of 1-deoxyxylulose-5-phosphate synthase, or the production of its products.

  16. Community Genomic and Proteomic Analyses of Chemoautotrophic Iron-Oxidizing "Leptospirillum rubarum" (Group II) and "Leptospirillum ferrodiazotrophum" (Group III) Bacteria in Acid Mine Drainage Biofilms

    SciTech Connect

    Goltsman, Daniela; Denef, Vincent; Singer, Steven; Verberkmoes, Nathan C; Lefsrud, Mark G; Mueller, Ryan; Dick, Gregory J.; Sun, Christine; Wheeler, Korin; Zelma, Adam; Baker, Brett J.; Hauser, Loren John; Land, Miriam L; Shah, Manesh B; Thelen, Michael P.; Hettich, Robert {Bob} L; Banfield, Jillian F.

    2009-01-01

    We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.

  17. Genome Sequence Analysis of the Naphthenic Acid Degrading and Metal Resistant Bacterium Cupriavidus gilardii CR3

    PubMed Central

    Xiao, Jingfa; Hao, Lirui; Crowley, David E.; Zhang, Zhewen; Yu, Jun; Huang, Ning; Huo, Mingxin; Wu, Jiayan

    2015-01-01

    Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds, although the degradation pathways and substrate versatilities remain largely unknown. Here we studied the bacterium Cupriavidus gilardii strain CR3, which was isolated from a natural asphalt deposit, and which was shown to utilize naphthenic acids as a sole carbon source. Genome sequencing of C. gilardii CR3 was carried out to elucidate possible mechanisms for the naphthenic acid biodegradation. The genome of C. gilardii CR3 was composed of two circular chromosomes chr1 and chr2 of respectively 3,539,530 bp and 2,039,213 bp in size. The genome for strain CR3 encoded 4,502 putative protein-coding genes, 59 tRNA genes, and many other non-coding genes. Many genes were associated with xenobiotic biodegradation and metal resistance functions. Pathway prediction for degradation of cyclohexanecarboxylic acid, a representative naphthenic acid, suggested that naphthenic acid undergoes initial ring-cleavage, after which the ring fission products can be degraded via several plausible degradation pathways including a mechanism similar to that used for fatty acid oxidation. The final metabolic products of these pathways are unstable or volatile compounds that were not toxic to CR3. Strain CR3 was also shown to have tolerance to at least 10 heavy metals, which was mainly achieved by self-detoxification through ion efflux, metal-complexation and metal-reduction, and a powerful DNA self-repair mechanism. Our genomic analysis suggests that CR3 is well adapted to survive the harsh environment in natural asphalts containing naphthenic acids and high concentrations of heavy metals. PMID:26301592

  18. Genome Sequence Analysis of the Naphthenic Acid Degrading and Metal Resistant Bacterium Cupriavidus gilardii CR3.

    PubMed

    Wang, Xiaoyu; Chen, Meili; Xiao, Jingfa; Hao, Lirui; Crowley, David E; Zhang, Zhewen; Yu, Jun; Huang, Ning; Huo, Mingxin; Wu, Jiayan

    2015-01-01

    Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds, although the degradation pathways and substrate versatilities remain largely unknown. Here we studied the bacterium Cupriavidus gilardii strain CR3, which was isolated from a natural asphalt deposit, and which was shown to utilize naphthenic acids as a sole carbon source. Genome sequencing of C. gilardii CR3 was carried out to elucidate possible mechanisms for the naphthenic acid biodegradation. The genome of C. gilardii CR3 was composed of two circular chromosomes chr1 and chr2 of respectively 3,539,530 bp and 2,039,213 bp in size. The genome for strain CR3 encoded 4,502 putative protein-coding genes, 59 tRNA genes, and many other non-coding genes. Many genes were associated with xenobiotic biodegradation and metal resistance functions. Pathway prediction for degradation of cyclohexanecarboxylic acid, a representative naphthenic acid, suggested that naphthenic acid undergoes initial ring-cleavage, after which the ring fission products can be degraded via several plausible degradation pathways including a mechanism similar to that used for fatty acid oxidation. The final metabolic products of these pathways are unstable or volatile compounds that were not toxic to CR3. Strain CR3 was also shown to have tolerance to at least 10 heavy metals, which was mainly achieved by self-detoxification through ion efflux, metal-complexation and metal-reduction, and a powerful DNA self-repair mechanism. Our genomic analysis suggests that CR3 is well adapted to survive the harsh environment in natural asphalts containing naphthenic acids and high concentrations of heavy metals. PMID:26301592

  19. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, Heinz-Ulrich G.; Gray, Joe W.

    1995-01-01

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers ard probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity.

  20. Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

    DOEpatents

    Weier, H.U.G.; Gray, J.W.

    1995-06-27

    A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers and probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity. 18 figs.

  1. Unconventional amino acid sequence of the sun anemone (Stoichactis helianthus) polypeptide neurotoxin

    SciTech Connect

    Kem, W.; Dunn, B.; Parten, B.; Pennington, M.; Price, D.

    1986-05-01

    A 5000 dalton polypeptide neurotoxin (Sh-NI) purified by G50 Sephadex, P-cellulose, and SP-Sephadex chromatography was homogeneous by isoelectric focusing. Sh-NI was highly toxic to crayfish (LD/sub 50/ 0.6 ..mu..g/kg) but without effect upon mice at 15,000 ..mu..g/kg (i.p. injection). The reduced, /sup 3/H-carboxymethylated toxin and its fragments were subjected to automatic Edman degradation and the resulting PTH-amino acids were identified by HPLC, back hydrolysis, and scintillation counting. Peptides resulting from proteolytic (clostripain, staphylococcal protease) and chemical (tryptophan) cleavage were sequenced. The sequence is: AACKCDDEGPDIRTAPLTGTVDLGSCNAGWEKCASYYTIIADCCRKKK. This sequence differs considerably from the homologous Anemonia and Anthopleura toxins; many of the identical residues (6 half-cystines, G9, P10, R13, G19, G29, W30) are probably critical for folding rather than receptor recognition. However, the Sh-NI sequence closely resembles Radioanthus macrodactylus neurotoxin III and r. paumotensis II. The authors propose that Sh-NI and related Radioanthus toxins act upon a different site on the sodium channel.

  2. Sequence-defined bioactive macrocycles via an acid-catalysed cascade reaction

    NASA Astrophysics Data System (ADS)

    Porel, Mintu; Thornlow, Dana N.; Phan, Ngoc N.; Alabi, Christopher A.

    2016-06-01

    Synthetic macrocycles derived from sequence-defined oligomers are a unique structural class whose ring size, sequence and structure can be tuned via precise organization of the primary sequence. Similar to peptides and other peptidomimetics, these well-defined synthetic macromolecules become pharmacologically relevant when bioactive side chains are incorporated into their primary sequence. In this article, we report the synthesis of oligothioetheramide (oligoTEA) macrocycles via a one-pot acid-catalysed cascade reaction. The versatility of the cyclization chemistry and modularity of the assembly process was demonstrated via the synthesis of >20 diverse oligoTEA macrocycles. Structural characterization via NMR spectroscopy revealed the presence of conformational isomers, which enabled the determination of local chain dynamics within the macromolecular structure. Finally, we demonstrate the biological activity of oligoTEA macrocycles designed to mimic facially amphiphilic antimicrobial peptides. The preliminary results indicate that macrocyclic oligoTEAs with just two-to-three cationic charge centres can elicit potent antibacterial activity against Gram-positive and Gram-negative bacteria.

  3. A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning.

    PubMed

    Shao, F; Hu, Z; Xiong, Y M; Huang, Q Z; WangCG; Zhu, R H; Wang, D C

    1999-03-19

    An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.

  4. Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

    PubMed

    Arai, Naoki; Nishimura, Emi; Kikuchi, Yo; Ohyama, Takashi

    2015-09-11

    Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation.

  5. Lysosomal acid lipase deficiency in rats: Lipid analyses and lipase activities in liver and spleen

    SciTech Connect

    Kuriyama, M.; Yoshida, H.; Suzuki, M.; Fujiyama, J.; Igata, A. )

    1990-09-01

    We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for (14C)triolein, (14C)cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans.

  6. Quantitative analyses of tartaric acid based on terahertz time domain spectroscopy

    NASA Astrophysics Data System (ADS)

    Cao, Binghua; Fan, Mengbao

    2010-10-01

    Terahertz wave is the electromagnetic spectrum situated between microwave and infrared wave. Quantitative analysis based on terahertz spectroscopy is very important for the application of terahertz techniques. But how to realize it is still under study. L-tartaric acid is widely used as acidulant in beverage, and other food, such as soft drinks, wine, candy, bread and some colloidal sweetmeats. In this paper, terahertz time-domain spectroscopy is applied to quantify the tartaric acid. Two methods are employed to process the terahertz spectra of different samples with different content of tartaric acid. The first one is linear regression combining correlation analysis. The second is partial least square (PLS), in which the absorption spectra in the 0.8-1.4THz region are used to quantify the tartaric acid. To compare the performance of these two principles, the relative error of the two methods is analyzed. For this experiment, the first method does better than the second one. But the first method is suitable for the quantitative analysis of materials which has obvious terahertz absorption peaks, while for material which has no obvious terahertz absorption peaks, the second one is more appropriate.

  7. Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

    PubMed

    Arai, Naoki; Nishimura, Emi; Kikuchi, Yo; Ohyama, Takashi

    2015-09-11

    Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation. PMID:26235877

  8. Sequence and Transcriptional Analyses of the Fish Retroviruses Walleye Epidermal Hyperplasia Virus Types 1 and 2: Evidence for a Gene Duplication

    PubMed Central

    LaPierre, Lorie A.; Holzschu, Donald L.; Bowser, Paul R.; Casey, James W.

    1999-01-01

    Walleye epidermal hyperplasia virus types 1 and 2 (WEHV1 and WEHV2, respectively) are associated with a hyperproliferative skin lesion on walleyes that appears and regresses seasonally. We have determined the complete nucleotide sequences and transcriptional profiles of these viruses. WEHV1 and WEHV2 are large, complex retroviruses of 12,999 and 13,125 kb in length, respectively, that are closely related to one another and to walleye dermal sarcoma virus (WDSV). These walleye retroviruses contain three open reading frames, orfA, orfB, and orfC, in addition to gag, pol, and env. orfA and orfB are adjacent to one another and located downstream of env. The OrfA proteins were previously identified as cyclin D homologs that may contribute to the induction of cell proliferation leading to epidermal hyperplasia and dermal sarcoma. The sequence analysis of WEHV1 and WEHV2 revealed that the OrfB proteins are distantly related to the OrfA proteins, suggesting that orfB arose by gene duplication. Presuming that the precursor of orfA and orfB was derived from a cellular cyclin, these genes are the first accessory genes of complex retroviruses that can be traced to a cellular origin. WEHV1, WEHV2, and WDSV are the only retroviruses that have an open reading frame, orfC, of considerable size (ca. 130 amino acids) in the leader region preceding gag. While we were unable to predict a function for the OrfC proteins, they are more conserved than OrfA and OrfB, suggesting that they may be biologically important to the viruses. The transcriptional profiles of WEHV1 and WEHV2 were also similar to that of WDSV; Northern blot analyses detected only low levels of the orfA transcripts in developing lesions, whereas abundant levels of genomic, env, orfA, and orfB transcripts were detected in regressing lesions. The splice donors and acceptors of individual transcripts were identified by reverse transcriptase PCR. The similarities of WEHV1, WEHV2, and WDSV suggest that these viruses use

  9. Amino acid sequence and variant forms of favin, a lectin from Vicia faba.

    PubMed

    Hopp, T P; Hemperly, J J; Cunningham, B A

    1982-04-25

    We have determined the complete amino acid sequence (182 residues) of the beta chain of favin, the glucose-binding lectin from fava beans (Vicia faba), and have established that the carbohydrate moiety is attached to Asn 168. Together with the sequence of the alpha chain previously reported (Hemperly, J. J., Hopp, T. P., Becker, J. W., and Cunningham, B. A. (1979) J. Biol. Chem. 254, 6803-6810), these data complete the analysis of the primary structure of the lectin. We have also examined minor polypeptides that appear in all preparations of favin. Two lower molecular weight species (Mr = 9,500-11,600) appear to be fragments of the beta chain resulting from cleavage following Asn 76, whereas six high molecular weight forms (Mr = 25,000 or greater) appear to include aggregates of the beta chain and possibly some alternative products of chain processing. PMID:7068646

  10. Pyrosequencing on templates generated by asymmetric nucleic acid sequence-based amplification (asymmetric-NASBA).

    PubMed

    Jia, Huning; Chen, Zhiyao; Wu, Haiping; Ye, Hui; Yan, Zhengyu; Zhou, Guohua

    2011-12-21

    Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.

  11. Evolution of alpha-lactalbumins. The complete amino acid sequence of the alpha-lactalbumin from a marsupial (Macropus rufogriseus) and corrections to regions of sequence in bovine and goat alpha-lactalbumins.

    PubMed

    Shewale, J G; Sinha, S K; Brew, K

    1984-04-25

    alpha-Lactalbumin was purified from a whey protein fraction of the milk of the red-necked wallaby (Macropus rufogriseus). The complete amino acid sequence was determined from the results of automatic sequenator analyses of the intact protein, the three cyanogen bromide fragments, and of peptides generated from the larger, COOH-terminal CNBr fragment by digestion with trypsin or staphylococcal protease. This is the first sequence to be determined of an alpha-lactalbumin from a marsupial and differs from known eutherian alpha-lactalbumins in size and locations of deletions in alignments with the homologous type c lysozymes, as well as in having amino acid substitutions at 8 sites that are invariant in known eutherian proteins. Some corrections are also reported for two regions of sequence in both bovine and goat alpha-lactalbumins. The new and previously published information on alpha-lactalbumin sequences is analyzed in relation to the evolutionary history of the alpha-lactalbumin line as well as the relationship of structure to function in these proteins. PMID:6715332

  12. Morphological tranformation of calcite crystal growth by prismatic "acidic" polypeptide sequences.

    SciTech Connect

    Kim, I; Giocondi, J L; Orme, C A; Collino, J; Evans, J S

    2007-02-13

    Many of the interesting mechanical and materials properties of the mollusk shell are thought to stem from the prismatic calcite crystal assemblies within this composite structure. It is now evident that proteins play a major role in the formation of these assemblies. Recently, a superfamily of 7 conserved prismatic layer-specific mollusk shell proteins, Asprich, were sequenced, and the 42 AA C-terminal sequence region of this protein superfamily was found to introduce surface voids or porosities on calcite crystals in vitro. Using AFM imaging techniques, we further investigate the effect that this 42 AA domain (Fragment-2) and its constituent subdomains, DEAD-17 and Acidic-2, have on the morphology and growth kinetics of calcite dislocation hillocks. We find that Fragment-2 adsorbs on terrace surfaces and pins acute steps, accelerates then decelerates the growth of obtuse steps, forms clusters and voids on terrace surfaces, and transforms calcite hillock morphology from a rhombohedral form to a rounded one. These results mirror yet are distinct from some of the earlier findings obtained for nacreous polypeptides. The subdomains Acidic-2 and DEAD-17 were found to accelerate then decelerate obtuse steps and induce oval rather than rounded hillock morphologies. Unlike DEAD-17, Acidic-2 does form clusters on terrace surfaces and exhibits stronger obtuse velocity inhibition effects than either DEAD-17 or Fragment-2. Interestingly, a 1:1 mixture of both subdomains induces an irregular polygonal morphology to hillocks, and exhibits the highest degree of acute step pinning and obtuse step velocity inhibition. This suggests that there is some interplay between subdomains within an intra (Fragment-2) or intermolecular (1:1 mixture) context, and sequence interplay phenomena may be employed by biomineralization proteins to exert net effects on crystal growth and morphology.

  13. Negative-ion Electrospray Tandem Mass Spectrometry and Microarray Analyses of Developmentally-regulated Antigens Based on Type 1 and Type 2 Backbone Sequences

    PubMed Central

    Gao, Chao; Zhang, Yibing; Liu, Yan; Feizi, Ten; Chai, Wengang

    2016-01-01

    Type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) sequences are constituents of the backbones of a large family of glycans of glycoproteins and glycolipids whose branching and peripheral substitutions are developmentally-regulated. It is highly desirable to have micro-sequencing methods that can be used to precisely identify and monitor these oligosaccharide sequences with high sensitivity. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation has been used for characterization of branching points, peripheral substitutions and partial assignment of linkages in reducing oligosaccharides. We now extend this method to characterizing entire sequences of linear type 1 and type 2 chain-based glycans, focusing on the type 1 and -2 units in the internal regions including the linkages connecting type 1 and type 2 disaccharide units. We apply the principles to sequence analysis of closely related isomeric oligosaccharides and demonstrate by microarray analyses distinct binding activities of antibodies and a lectin toward various combinations of type 1 and 2 units joined by 1,3- and 1,6-linkages. These sequence-specific carbohydrate-binding proteins are in turn valuable tools for detecting and distinguishing the type 1 and type 2-based developmentally-regulated glycan sequences. PMID:26530895

  14. The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY.

    PubMed

    Cutfield, S M; Carne, A; Cutfield, J F

    1987-04-01

    Two pancreatic peptides, somatostatin-28 and peptide YY, have been isolated from the Brockmann bodies of the teleost fish Cottus scorpius (daddy sculpin). Following purification by reverse-phase HPLC, each peptide was sequenced completely through to the carboxyl-terminus by gas-phase Edman degradation. Somatostatin-28 was the major form of somatostatin detected and is similar to the gene II product from anglerfish. Peptide YY (36 amino acids) more closely resembles porcine neuropeptide YY and intestinal peptide YY than it does the pancreatic polypeptides. PMID:2883025

  15. Sequence selective recognition of double-stranded RNA using triple helix-forming peptide nucleic acids.

    PubMed

    Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks

    2014-01-01

    Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

  16. Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

    PubMed Central

    Demidov, V; Frank-Kamenetskii, M D; Egholm, M; Buchardt, O; Nielsen, P E

    1993-01-01

    A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease. Images PMID:8502550

  17. Fast computational methods for predicting protein structure from primary amino acid sequence

    DOEpatents

    Agarwal, Pratul Kumar

    2011-07-19

    The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

  18. WinGene/WinPep: user-friendly software for the analysis of amino acid sequences.

    PubMed

    Hennig, L

    1999-06-01

    WinGene1.0/WinPep1.2 is a pair of Microsoft Windows programs designed to read nucleotide or amino acid sequence data. These versatile programs have the following capabilities: (i) searches for open reading frames and their translation, (ii) assisting the design of primers for PCR and (iii) calculation of molecular weight, isoelectric point and molar absorbtion coefficients of polypeptides. Furthermore, hydropathic plots and helical wheel displays are easily produced. The programs run with an intuitive Windows interface, contain a comprehensive help file and enable data exchange with other applications by means of the Copy&Paste command. The software is free for academic and noncommercial users.

  19. Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

    PubMed

    Kato, Hiroaki; Shiwa, Yuh; Oshima, Kenshiro; Machii, Miki; Araya-Kojima, Tomoko; Zendo, Takeshi; Shimizu-Kadota, Mariko; Hattori, Masahira; Sonomoto, Kenji; Yoshikawa, Hirofumi

    2012-04-01

    We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.

  20. Purification and amino acid sequence of aminopeptidase P from pig kidney.

    PubMed

    Vergas Romero, C; Neudorfer, I; Mann, K; Schäfer, W

    1995-04-01

    Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane-bound glycoprotein with n-butanol. A coupled enzymic assay, using Gly-Pro-Pro-NH-Nap as substrate and dipeptidyl-peptidase IV as auxilliary enzyme, was used to monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six N-glycosylation sites, all contained in the N-terminal half of the protein, were characterized. PMID:7744038

  1. Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen.

    PubMed

    Kaysheva, A L; Ivanov, Yu D; Frantsuzov, P A; Krohin, N V; Pavlova, T I; Uchaikin, V F; Konev, V А; Kovalev, O B; Ziborov, V S; Archakov, A I

    2016-03-01

    A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

  2. Complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera, and comparative analyses with other grass genomes

    PubMed Central

    Saski, Christopher; Lee, Seung-Bum; Fjellheim, Siri; Guda, Chittibabu; Jansen, Robert K.; Luo, Hong; Tomkins, Jeffrey; Rognli, Odd Arne; Clarke, Jihong Liu

    2009-01-01

    Comparisons of complete chloroplast genome sequences of Hordeum vulgare, Sorghum bicolor and Agrostis stolonifera to six published grass chloroplast genomes reveal that gene content and order are similar but two microstructural changes have occurred. First, the expansion of the IR at the SSC/IRa boundary that duplicates a portion of the 5′ end of ndhH is restricted to the three genera of the subfamily Pooideae (Agrostis, Hordeum and Triticum). Second, a 6 bp deletion in ndhK is shared by Agrostis, Hordeum, Oryza and Triticum, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis identified 19–37 direct and inverted repeats 30 bp or longer with a sequence identity of at least 90%. Seventeen of the 26 shared repeats are found in all the grass chloroplast genomes examined and are located in the same genes or intergenic spacer (IGS) regions. Examination of simple sequence repeats (SSRs) identified 16–21 potential polymorphic SSRs. Five IGS regions have 100% sequence identity among Zea mays, Saccharum officinarum and Sorghum bicolor, whereas no spacer regions were identical among Oryza sativa, Triticum aestivum, H. vulgare and A. stolonifera despite their close phylogenetic relationship. Alignment of EST sequences and DNA coding sequences identified six C–U conversions in both Sorghum bicolor and H. vulgare but only one in A. stolonifera. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae. PMID:17534593

  3. Draft Genome Sequence of Bacillus subtilis subsp. natto Strain CGMCC 2108, a High Producer of Poly-γ-Glutamic Acid

    PubMed Central

    Tan, Siyuan; Su, Anping; Zhang, Chen; Ren, Yuanyuan

    2016-01-01

    Here, we report the 4.1-Mb draft genome sequence of Bacillus subtilis subsp. natto strain CGMCC 2108, a high producer of poly-γ-glutamic acid (γ-PGA). This sequence will provide further help for the biosynthesis of γ-PGA and will greatly facilitate research efforts in metabolic engineering of B. subtilis subsp. natto strain CGMCC 2108. PMID:27231363

  4. WAViS server for handling, visualization and presentation of multiple alignments of nucleotide or amino acids sequences.

    PubMed

    Zika, Radek; Paces, Jan; Pavlícek, Adam; Paces, Václav

    2004-07-01

    Web Alignment Visualization Server contains a set of web-tools designed for quick generation of publication-quality color figures of multiple alignments of nucleotide or amino acids sequences. It can be used for identification of conserved regions and gaps within many sequences using only common web browsers. The server is accessible at http://wavis.img.cas.cz.

  5. ANTICALIgN: visualizing, editing and analyzing combined nucleotide and amino acid sequence alignments for combinatorial protein engineering.

    PubMed

    Jarasch, Alexander; Kopp, Melanie; Eggenstein, Evelyn; Richter, Antonia; Gebauer, Michaela; Skerra, Arne

    2016-07-01

    ANTIC ALIGN: is an interactive software developed to simultaneously visualize, analyze and modify alignments of DNA and/or protein sequences that arise during combinatorial protein engineering, design and selection. ANTIC ALIGN: combines powerful functions known from currently available sequence analysis tools with unique features for protein engineering, in particular the possibility to display and manipulate nucleotide sequences and their translated amino acid sequences at the same time. ANTIC ALIGN: offers both template-based multiple sequence alignment (MSA), using the unmutated protein as reference, and conventional global alignment, to compare sequences that share an evolutionary relationship. The application of similarity-based clustering algorithms facilitates the identification of duplicates or of conserved sequence features among a set of selected clones. Imported nucleotide sequences from DNA sequence analysis are automatically translated into the corresponding amino acid sequences and displayed, offering numerous options for selecting reading frames, highlighting of sequence features and graphical layout of the MSA. The MSA complexity can be reduced by hiding the conserved nucleotide and/or amino acid residues, thus putting emphasis on the relevant mutated positions. ANTIC ALIGN: is also able to handle suppressed stop codons or even to incorporate non-natural amino acids into a coding sequence. We demonstrate crucial functions of ANTIC ALIGN: in an example of Anticalins selected from a lipocalin random library against the fibronectin extradomain B (ED-B), an established marker of tumor vasculature. Apart from engineered protein scaffolds, ANTIC ALIGN: provides a powerful tool in the area of antibody engineering and for directed enzyme evolution.

  6. 3-d structure-based amino acid sequence alignment of esterases, lipases and related proteins

    SciTech Connect

    Gentry, M.K.; Doctor, B.P.; Cygler, M.; Schrag, J.D.; Sussman, J.L.

    1993-05-13

    Acetylcholinesterase and butyrylcholinesterase, enzymes with potential as pretreatment drugs for organophosphate toxicity, are members of a larger family of homologous proteins that includes carboxylesterases, cholesterol esterases, lipases, and several nonhydrolytic proteins. A computer-generated alignment of 18 of the proteins, the acetylcholinesases, butyrylcholinesterases, carboxylesterases, some esterases, and the nonenzymatic proteins has been previously presented. More recently, the three-dimensional structures of two enzymes enzymes in this group, acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum, have been determined. Based on the x-ray structures and the superposition of these two enzymes, it was possible to obtain an improved amino acid sequence alignment of 32 members of this family of proteins. Examination of this alignment reveals that 24 amino acids are invariant in all of the hydrolytic proteins, and an additional 49 are well conserved. Conserved amino acids include those of the active site, the disulfide bridges, the salt bridges, in the core of the proteins, and at the edges of secondary structural elements. Comparison of the three-dimensional structures makes it possible to find a well-defined structural basis for the conservation of many of these amino acids.

  7. A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance

    PubMed Central

    Ahrenfeldt, Johanne; Cisneros, Jose Luis Bellod; Jurtz, Vanessa; Larsen, Mette Voldby; Hasman, Henrik; Aarestrup, Frank Møller; Lund, Ole

    2016-01-01

    Recent advances in whole genome sequencing have made the technology available for routine use in microbiological laboratories. However, a major obstacle for using this technology is the availability of simple and automatic bioinformatics tools. Based on previously published and already available web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the individual services. The reported results enable a rapid overview of the major results, and comparing that to the previously found results showed that the platform is reliable and able to correctly predict the species and find most of the expected genes automatically. In conclusion, a combined bioinformatics platform was developed and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 and it is the intention that it will continue to be expanded with new features as these become available. PMID:27327771

  8. Draft Genome Sequences of Gluconobacter cerinus CECT 9110 and Gluconobacter japonicus CECT 8443, Acetic Acid Bacteria Isolated from Grape Must

    PubMed Central

    Sainz, Florencia

    2016-01-01

    We report here the draft genome sequences of Gluconobacter cerinus strain CECT9110 and Gluconobacter japonicus CECT8443, acetic acid bacteria isolated from grape must. Gluconobacter species are well known for their ability to oxidize sugar alcohols into the corresponding acids. Our objective was to select strains to oxidize effectively d-glucose. PMID:27365351

  9. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed Central

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-01-01

    A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses. Images PMID:3025846

  10. Partial amino acid sequences around sulfhydryl groups of soybean beta-amylase.

    PubMed

    Nomura, K; Mikami, B; Morita, Y

    1987-08-01

    Sulfhydryl (SH) groups of soybean beta-amylase were modified with 5-(iodoaceto-amidoethyl)aminonaphthalene-1-sulfonate (IAEDANS) and the SH-containing peptides exhibiting fluorescence were purified after chymotryptic digestion of the modified enzyme. The sequence analysis of the peptides derived from the modification of all SH groups in the denatured enzyme revealed the existence of six SH groups, in contrast to five reported previously. One of them was found to have extremely low reactivity toward SH-reagents without reduction. In the native state, IAEDANS reacted with 2 mol of SH groups per mol of the enzyme (SH1 and SH2) accompanied with inactivation of the enzyme owing to the modification of SH2 located near the active site of this enzyme. The selective modification of SH2 with IAEDANS was attained after the blocking of SH1 with 5,5'-dithiobis-(2-nitrobenzoic acid). The amino acid sequences of the peptides containing SH1 and SH2 were determined to be Cys-Ala-Asn-Pro-Gln and His-Gln-Cys-Gly-Gly-Asn-Val-Gly-Asp-Ile-Val-Asn-Ile-Pro-Ile-Pro-Gln-Trp, respectively.

  11. Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA).

    PubMed

    Starkey, William G; Millar, Rose Mary; Jenkins, Mary E; Ireland, Jacqueline H; Muir, K Fiona; Richards, Randolph H

    2004-05-01

    Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.

  12. From amino acid sequence to bioactivity: The biomedical potential of antitumor peptides.

    PubMed

    Blanco-Míguez, Aitor; Gutiérrez-Jácome, Alberto; Pérez-Pérez, Martín; Pérez-Rodríguez, Gael; Catalán-García, Sandra; Fdez-Riverola, Florentino; Lourenço, Anália; Sánchez, Borja

    2016-06-01

    Chemoprevention is the use of natural and/or synthetic substances to block, reverse, or retard the process of carcinogenesis. In this field, the use of antitumor peptides is of interest as, (i) these molecules are small in size, (ii) they show good cell diffusion and permeability, (iii) they affect one or more specific molecular pathways involved in carcinogenesis, and (iv) they are not usually genotoxic. We have checked the Web of Science Database (23/11/2015) in order to collect papers reporting on bioactive peptide (1691 registers), which was further filtered searching terms such as "antiproliferative," "antitumoral," or "apoptosis" among others. Works reporting the amino acid sequence of an antiproliferative peptide were kept (60 registers), and this was complemented with the peptides included in CancerPPD, an extensive resource for antiproliferative peptides and proteins. Peptides were grouped according to one of the following mechanism of action: inhibition of cell migration, inhibition of tumor angiogenesis, antioxidative mechanisms, inhibition of gene transcription/cell proliferation, induction of apoptosis, disorganization of tubulin structure, cytotoxicity, or unknown mechanisms. The main mechanisms of action of those antiproliferative peptides with known amino acid sequences are presented and finally, their potential clinical usefulness and future challenges on their application is discussed.

  13. Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II.

    PubMed

    Ansari, A A; Shenbagamurthi, P; Marsh, D G

    1989-07-01

    The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p II was determined by automated Edman degradation of the protein and selected fragments. Cleavage of the protein by enzymatic and chemical techniques established an unambiguous sequence for the protein. Lol p II contains 97 amino acid residues, with a calculated molecular weight of 10,882. The protein lacks cysteine and glutamine and shows no evidence of glycosylation. Theoretical predictions by Fraga's (Fraga, S. (1982) Can. J. Chem. 60, 2606-2610) and Hopp and Woods' (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828) methods indicate the presence of four hydrophilic regions, which may contribute to sequential or parts of conformational B-cell epitopes. Analysis of amphipathic regions by Berzofsky's method indicates the presence of a highly amphipathic region, which may contain, or contribute to, an Ia/T-cell epitope. This latter segment of Lol p II was found to be highly homologous with an antibody-binding segment of the major rye allergen Lol p I and may explain why immune responsiveness to both the allergens is associated with HLA-DR3.

  14. Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

    PubMed

    Rodriguez, J F; Kahn, J S; Esteban, M

    1986-12-01

    A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses.

  15. Using Synthetic Nanopores for Single-Molecule Analyses: Detecting SNPs, Trapping DNA Molecules, and the Prospects for Sequencing DNA

    ERIC Educational Resources Information Center

    Dimitrov, Valentin V.

    2009-01-01

    This work focuses on studying properties of DNA molecules and DNA-protein interactions using synthetic nanopores, and it examines the prospects of sequencing DNA using synthetic nanopores. We have developed a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. There exists…

  16. Learning Hypotheses and an Associated Tool to Design and to Analyse Teaching-Learning Sequences. Special Issue

    ERIC Educational Resources Information Center

    Buty, Christian; Tiberghien, Andree; Le Marechal, Jean-Francois

    2004-01-01

    This contribution presents a tool elaborated from a theoretical framework linking epistemological, learning and didactical hypotheses. This framework lead to design teaching sequences from a socio-constructivist perspective, and is based on the role of models in physics or chemistry, and on the role of students' initial knowledge in learning…

  17. Isolation and amino acid sequences of squirrel monkey (Saimiri sciurea) insulin and glucagon.

    PubMed Central

    Yu, J H; Eng, J; Yalow, R S

    1990-01-01

    It was reported two decades ago that insulin was not detectable in the glucose-stimulated state in Saimiri sciurea, the New World squirrel monkey, by a radioimmunoassay system developed with guinea pig anti-pork insulin antibody and labeled pork insulin. With the same system, reasonable levels were observed in rhesus monkeys and chimpanzees. This suggested that New World monkeys, like the New World hystricomorph rodents such as the guinea pig and the coypu, might have insulins whose sequences differ markedly from those of Old World mammals. In this report we describe the purification and amino acid sequences of squirrel monkey insulin and glucagon. We demonstrate that the substitutions at B29, B27, A2, A4, and A17 of squirrel monkey insulin are identical with those previously found in another New World primate, the owl monkey (Aotus trivirgatus). The immunologic cross-reactivity of this insulin in our immunoassay system is only a few percent of that of human insulin. Squirrel monkey glucagon is identical with the usual glucagon found in Old World mammals, which predicts that the glucagons of other New World monkeys would not differ from the usual Old World mammalian glucagon. It appears that the peptides of the New World monkeys have diverged less from those of the Old World mammals than have those of the New World hystricomorph rodents. The striking improvements in peptide purification and sequencing have the potential for adding new information concerning the evolutionary divergence of species. PMID:2263627

  18. Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

    PubMed

    Jones, B N; Wang, C C; Dwulet, F E; Lehman, L D; Meuth, J L; Bogardt, R A; Gurd, F R

    1979-04-25

    The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea. PMID:454657

  19. Isolation and amino acid sequences of squirrel monkey (Saimiri sciurea) insulin and glucagon

    SciTech Connect

    Yu, Jinghua ); Eng, J.; Yalow, R.S. City Univ. of New York, NY )

    1990-12-01

    It was reported two decades ago that insulin was not detectable in the glucose-stimulated state in Saimiri sciurea, the New World squirrel monkey, by a radioimmunoassay system developed with guinea pig anti-pork insulin antibody and labeled park insulin. With the same system, reasonable levels were observed in rhesus monkeys and chimpanzees. This suggested that New World monkeys, like the New World hystricomorph rodents such as the guinea pig and the coypu, might have insulins whose sequences differ markedly from those of Old World mammals. In this report the authors describe the purification and amino acid sequences of squirrel monkey insulin and glucagon. They demonstrate that the substitutions at B29, B27, A2, A4, and A17 of squirrel monkey insulin are identical with those previously found in another New World primate, the owl monkey (Aotus trivirgatus). The immunologic cross-reactivity of this insulin in their immunoassay system is only a few percent of that of human insulin. It appears that the peptides of the New World monkeys have diverged less from those of the Old World mammals than have those of the New World hystricomorph rodents. The striking improvements in peptide purification and sequencing have the potential for adding new information concerning the evolutionary divergence of species.

  20. Purification, amino acid sequence and characterisation of kangaroo IGF-I.

    PubMed

    Yandell, C A; Francis, G L; Wheldrake, J F; Upton, Z

    1998-01-01

    Insulin-like growth factor-I (IGF-I) and IGF-II have been purified to homogeneity from kangaroo (Macropus fuliginosus) serum, thus this represents the first report of the purification, sequencing and characterisation of marsupial IGFs. N-Terminal protein sequencing reveals that there are six amino acid differences between kangaroo and human IGF-I. Kangaroo IGF-II has been partially sequenced and no differences were found between human and kangaroo IGF-II in the 53 residues identified. Thus the IGFs appear to be remarkably structurally conserved during mammalian radiation. In addition, in vitro characterisation of kangaroo IGF-I demonstrated that the functional properties of human, kangaroo and chicken IGF-I are very similar. In an assay measuring the ability of the proteins to stimulate protein synthesis in rat L6 myoblasts, all IGF-I proteins were found to be equally potent. The ability of all three proteins to compete for binding with radiolabelled human IGF-I to type-1 IGF receptors in L6 myoblasts and in Sminthopsis crassicaudata transformed lung fibroblasts, a marsupial cell line, was comparable. Furthermore, kangaroo and human IGF-I react equally in a human IGF-I RIA using a human reference standard, radiolabelled human IGF-I and a polyclonal antibody raised against recombinant human IGF-I. This study indicates that not only is the primary structure of eutherian and metatherian IGF-I conserved, but also the proteins appear to be functionally similar.

  1. The quest for the best: The impact of different EPI sequences on the sensitivity of random effect fMRI group analyses.

    PubMed

    Kirilina, Evgeniya; Lutti, Antoine; Poser, Benedikt A; Blankenburg, Felix; Weiskopf, Nikolaus

    2016-02-01

    We compared the sensitivity of standard single-shot 2D echo planar imaging (EPI) to three advanced EPI sequences, i.e., 2D multi-echo EPI, 3D high resolution EPI and 3D dual-echo fast EPI in fixed effect and random effects group level fMRI analyses at 3T. The study focused on how well the variance reduction in fixed effect analyses achieved by advanced EPI sequences translates into increased sensitivity in the random effects group level analysis. The sensitivity was estimated in a functional MRI experiment of an emotional learning and a reward based learning tasks in a group of 24 volunteers. Each experiment was acquired with the four different sequences. The task-related response amplitude, contrast level and respective t-value were proxies for the functional sensitivity across the brain. All three advanced EPI methods increased the sensitivity in the fixed effects analyses, but standard single-shot 2D EPI provided a comparable performance in random effects group analysis when whole brain coverage and moderate resolution are required. In this experiment inter-subject variability determined the sensitivity of the random effects analysis for most brain regions, making the impact of EPI pulse sequence improvements less relevant or even negligible for random effects analyses. An exception concerns the optimization of EPI reducing susceptibility-related signal loss that translates into an enhanced sensitivity e.g. in the orbitofrontal cortex for multi-echo EPI. Thus, future optimization strategies may best aim at reducing inter-subject variability for higher sensitivity in standard fMRI group studies at moderate spatial resolution. PMID:26515905

  2. Ribosomal PCR and DNA sequencing for detection and identification of bacteria: experience from 6 years of routine analyses of patient samples.

    PubMed

    Jensen, Kristine Helander; Dargis, Rimtas; Christensen, Jens Jørgen; Kemp, Michael

    2014-03-01

    The use of broad range PCR and DNA sequencing of bacterial 16S ribosomal RNA genes for routine diagnostics of bacterial infections was evaluated. Here, the results from more than 2600 analyses during a 6-year period (2003-2009) are presented. Almost half of the samples were from joints and bones, and the second most frequent origin of samples was from the central nervous system. Overall, 26% of all samples were positive for bacterial DNA and bacterial identification was obtained in 80% of the PCR-positive samples by subsequent DNA sequencing. Ambiguous species identification was noticed among non-haemolytic streptococci, especially within the mitis group. The data show that ribosomal PCR with subsequent DNA sequencing of the PCR product is a most valuable supplement to culture for identifying bacterial agents of both acute and prolonged infections. However, some bacteria, including non-haemolytic streptococci, may not be precisely identified.

  3. The evolution of proteins from random amino acid sequences: II. Evidence from the statistical distributions of the lengths of modern protein sequences.

    PubMed

    White, S H

    1994-04-01

    This paper continues an examination of the hypothesis that modern proteins evolved from random heteropeptide sequences. In support of the hypothesis, White and Jacobs (1993, J Mol Evol 36:79-95) have shown that any sequence chosen randomly from a large collection of nonhomologous proteins has a 90% or better chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. The goal of the present study was to investigate the possibility that the random-origin hypothesis could explain the lengths of modern protein sequences without invoking specific mechanisms such as gene duplication or exon splicing. The sets of sequences examined were taken from the 1989 PIR database and consisted of 1,792 "super-family" proteins selected to have little sequence identity, 623 E. coli sequences, and 398 human sequences. The length distributions of the proteins could be described with high significance by either of two closely related probability density functions: The gamma distribution with parameter 2 or the distribution for the sum of two exponential random independent variables. A simple theory for the distributions was developed which assumes that (1) protoprotein sequences had exponentially distributed random independent lengths, (2) the length dependence of protein stability determined which of these protoproteins could fold into compact primitive proteins and thereby attain the potential for biochemical activity, (3) the useful protein sequences were preserved by the primitive genome, and (4) the resulting distribution of sequence lengths is reflected by modern proteins. The theory successfully predicts the two observed distributions which can be distinguished by the functional form of the dependence of protein stability on length. The theory leads to three interesting conclusions. First, it predicts that a tetra-nucleotide was the signal for primitive translation termination. This prediction is

  4. Molar ratio iron: zinc and folic acid in Brazilian biscuits and snacks and test for classification using principal component analyses.

    PubMed

    Godoy, Adriana Teixeira; Rebelatto, Ana Paula; Borin-Nogueira, Alessandra; Lima-Pallone, Juliana Azevedo

    2014-06-01

    The aim of the present work was to evaluate molar ratio iron: zinc and the levels of folic acid in biscuit and snacks commercialized in Brazil, prepared with folic acid and iron fortified flours. These nutrients are important for human nutrition; however, iron can have a negative effect on zinc absorption. Molar ratio iron:zinc can indicate if there will be any problems for absorption of these nutrients. The folic acid content varied from 58 to 433 μg/100 g and iron and zinc levels varied from 2.9 to 9.4 mg/100 g and from 0.2 to 1.3 mg/100 g, respectively, for 75 analyzed samples. The average iron contents observed in the products and molar ratio iron:zinc (in average 8:1 for biscuits and 12.8:1 for snacks) could result in problems with the zinc absorption. Moreover, principal compo- nent analyses (PCA) indicated low uniformity in the distribution of minerals and vitamin in the majority of the samples, mainly among brands. The results indicated that for the majority of the samples tested folic acid and iron content was higher than expected for flours and could be useful to governmental authorities in their evaluation program of flour fortification.

  5. Global trophic position comparison of two dominant mesopelagic fish families (Myctophidae, Stomiidae) using amino acid nitrogen isotopic analyses

    USGS Publications Warehouse

    Choy, C. Anela; Davison, Peter C.; Drazen, Jeffrey C.; Flynn, Adrian; Gier, Elizabeth J.; Hoffman, Joel C.; McClain-Counts, Jennifer P.; Miller, Todd W.; Popp, Brian N.; Ross, Steve W.; Sutton, Tracey T.

    2012-01-01

    The δ15N values of organisms are commonly used across diverse ecosystems to estimate trophic position and infer trophic connectivity. We undertook a novel cross-basin comparison of trophic position in two ecologically well-characterized and different groups of dominant mid-water fish consumers using amino acid nitrogen isotope compositions. We found that trophic positions estimated from the δ15N values of individual amino acids are nearly uniform within both families of these fishes across five global regions despite great variability in bulk tissue δ15N values. Regional differences in the δ15N values of phenylalanine confirmed that bulk tissue δ15N values reflect region-specific water mass biogeochemistry controlling δ15N values at the base of the food web. Trophic positions calculated from amino acid isotopic analyses (AA-TP) for lanternfishes (family Myctophidae) (AA-TP ~2.9) largely align with expectations from stomach content studies (TP ~3.2), while AA-TPs for dragonfishes (family Stomiidae) (AA-TP ~3.2) were lower than TPs derived from stomach content studies (TP~4.1). We demonstrate that amino acid nitrogen isotope analysis can overcome shortcomings of bulk tissue isotope analysis across biogeochemically distinct systems to provide globally comparative information regarding marine food web structure.

  6. Global Trophic Position Comparison of Two Dominant Mesopelagic Fish Families (Myctophidae, Stomiidae) Using Amino Acid Nitrogen Isotopic Analyses

    PubMed Central

    Choy, C. Anela; Davison, Peter C.; Drazen, Jeffrey C.; Flynn, Adrian; Gier, Elizabeth J.; Hoffman, Joel C.; McClain-Counts, Jennifer P.; Miller, Todd W.; Popp, Brian N.; Ross, Steve W.; Sutton, Tracey T.

    2012-01-01

    The δ15N values of organisms are commonly used across diverse ecosystems to estimate trophic position and infer trophic connectivity. We undertook a novel cross-basin comparison of trophic position in two ecologically well-characterized and different groups of dominant mid-water fish consumers using amino acid nitrogen isotope compositions. We found that trophic positions estimated from the δ15N values of individual amino acids are nearly uniform within both families of these fishes across five global regions despite great variability in bulk tissue δ15N values. Regional differences in the δ15N values of phenylalanine confirmed that bulk tissue δ15N values reflect region-specific water mass biogeochemistry controlling δ15N values at the base of the food web. Trophic positions calculated from amino acid isotopic analyses (AA-TP) for lanternfishes (family Myctophidae) (AA-TP ∼2.9) largely align with expectations from stomach content studies (TP ∼3.2), while AA-TPs for dragonfishes (family Stomiidae) (AA-TP ∼3.2) were lower than TPs derived from stomach content studies (TP∼4.1). We demonstrate that amino acid nitrogen isotope analysis can overcome shortcomings of bulk tissue isotope analysis across biogeochemically distinct systems to provide globally comparative information regarding marine food web structure. PMID:23209656

  7. Sequence, secondary structure, and phylogenetic analyses of the ribosomal internal transcribed spacer 2 (ITS2) in members of the North American Signifera Group of Orthopodomyia (Diptera: Culicidae).

    PubMed

    Byrd, Brian D; Harrison, Bruce A; Zavortink, Thomas J; Wesson, Dawn M

    2012-11-01

    Mosquitoes of the genus Orthopodomyia (Diptera: Culicidae) are little known and of uncertain epidemiological importance. In the United States, there are three Orthopodomyia species (i.e., Or. signifera (Coquillett), Or. alba Baker, and Or. kummi Edwards); they are all members of the Signifera Group based on the current morphological taxonomy. In the course of identifying recently collected specimens, a problem was found with the current key morphological characters for separating the fourth instar larvae of Or. signifera and Or. kummi. Internal transcribed spacer two sequences of the rDNA were obtained to resolve the identities. The Orthopodomyia internal transcribed spacer two ranged in size from 193 (Or. kummi) to 244 bp (Or. signifera) (mean = 218 bp) and were slightly Adenine/Thymine enriched (44.7% Guanine/Cytosine on average). Putative secondary structures reveal structural homologies (four domains) consistent between species that also feature conserved sequences specific to mosquitoes (e.g., a conserved motif on the 3' aspect of the longest helix: GARTACATCC). Sequence analyses suggest that in certain areas of southwestern North America, hybridization may occur between Or. kummi and Or. signifera. Furthermore, our analyses confirm that Or. californica (a junior synonym of Or. signifera) is indeed Or. signifera. To our knowledge, this is the first sequence-based phylogenetic and molecular analysis of the Orthopodomyia.

  8. Molecular cloning of the. alpha. -subunit of human prolyl 4-hydroxylase: The complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts

    SciTech Connect

    Helaakoski, T.; Vuori, K.; Myllylae, R.; Kivirikko, K.I.; Pihlajaniemi, T. )

    1989-06-01

    Prolyl 4-hydroxylase an {alpha}{sub 2}{beta}{sub 2} tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. The authors report here on the isolation of cDNA clones encoding the {alpha}-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3,000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the {beta}-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Gly-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The {alpha}-subunit does not have this C-terminal sequence, and thus one function of the {beta}-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Southern blot analyses of human genomic DNA with a cDNA probe for the {alpha}-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

  9. Sequence Design for a Test Tube of Interacting Nucleic Acid Strands.

    PubMed

    Wolfe, Brian R; Pierce, Niles A

    2015-10-16

    We describe an algorithm for designing the equilibrium base-pairing properties of a test tube of interacting nucleic acid strands. A target test tube is specified as a set of desired "on-target" complexes, each with a target secondary structure and target concentration, and a set of undesired "off-target" complexes, each with vanishing target concentration. Sequence design is performed by optimizing the test tube ensemble defect, corresponding to the concentration of incorrectly paired nucleotides at equilibrium evaluated over the ensemble of the test tube. To reduce the computational cost of accepting or rejecting mutations to a random initial sequence, the structural ensemble of each on-target complex is hierarchically decomposed into a tree of conditional subensembles, yielding a forest of decomposition trees. Candidate sequences are evaluated efficiently at the leaf level of the decomposition forest by estimating the test tube ensemble defect from conditional physical properties calculated over the leaf subensembles. As optimized subsequences are merged toward the root level of the forest, any emergent defects are eliminated via ensemble redecomposition and sequence reoptimization. After successfully merging subsequences to the root level, the exact test tube ensemble defect is calculated for the first time, explicitly checking for the effect of the previously neglected off-target complexes. Any off-target complexes that form at appreciable concentration are hierarchically decomposed, added to the decomposition forest, and actively destabilized during subsequent forest reoptimization. For target test tubes representative of design challenges in the molecular programming and synthetic biology communities, our test tube design algorithm typically succeeds in achieving a normalized test tube ensemble defect ≤1% at a design cost within an order of magnitude of the cost of test tube analysis.

  10. Sequence-Specific Electrical Purification of Nucleic Acids with Nanoporous Gold Electrodes.

    PubMed

    Daggumati, Pallavi; Appelt, Sandra; Matharu, Zimple; Marco, Maria L; Seker, Erkin

    2016-06-22

    Nucleic-acid-based biosensors have enabled rapid and sensitive detection of pathogenic targets; however, these devices often require purified nucleic acids for analysis since the constituents of complex biological fluids adversely affect sensor performance. This purification step is typically performed outside the device, thereby increasing sample-to-answer time and introducing contaminants. We report a novel approach using a multifunctional matrix, nanoporous gold (np-Au), which enables both detection of specific target sequences in a complex biological sample and their subsequent purification. The np-Au electrodes modified with 26-mer DNA probes (via thiol-gold chemistry) enabled sensitive detection and capture of complementary DNA targets in the presence of complex media (fetal bovine serum) and other interfering DNA fragments in the range of 50-1500 base pairs. Upon capture, the noncomplementary DNA fragments and serum constituents of varying sizes were washed away. Finally, the surface-bound DNA-DNA hybrids were released by electrochemically cleaving the thiol-gold linkage, and the hybrids were iontophoretically eluted from the nanoporous matrix. The optical and electrophoretic characterization of the analytes before and after the detection-purification process revealed that low target DNA concentrations (80 pg/μL) can be successfully detected in complex biological fluids and subsequently released to yield pure hybrids free of polydisperse digested DNA fragments and serum biomolecules. Taken together, this multifunctional platform is expected to enable seamless integration of detection and purification of nucleic acid biomarkers of pathogens and diseases in miniaturized diagnostic devices.

  11. Amino acid sequence analysis and characterization of a ribonuclease from starfish Asterias amurensis.

    PubMed

    Motoyoshi, Naomi; Kobayashi, Hiroko; Itagaki, Tadashi; Inokuchi, Norio

    2016-09-01

    The aim of this study was to phylogenetically characterize the location of the RNase T2 enzyme in the starfish (Asterias amurensis). We isolated an RNase T2 ribonuclease (RNase Aa) from the ovaries of starfish and determined its amino acid sequence by protein chemistry and cloning cDNA encoding RNase Aa. The isolated protein had 231 amino acid residues, a predicted molecular mass of 25,906 Da, and an optimal pH of 5.0. RNase Aa preferentially released guanylic acid from the RNA. The catalytic sites of the RNase T2 family are conserved in RNase Aa; furthermore, the distribution of the cysteine residues in RNase Aa is similar to that in other animal and plant T2 RNases. RNase Aa is cleaved at two points: 21 residues from the N-terminus and 29 residues from the C-terminus; however, both fragments may remain attached to the protein via disulfide bridges, leading to the maintenance of its conformation, as suggested by circular dichroism spectrum analysis. The phylogenetic analysis revealed that starfish RNase Aa is evolutionarily an intermediate between protozoan and oyster RNases. PMID:26920046

  12. Sequence analyses of two neuropeptides of the AKH/RPCH-family from the lubber grasshopper, Romalea microptera.

    PubMed

    Gäde, G; Hilbich, C; Beyreuther, K; Rinehart, K L

    1988-01-01

    Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high-performance liquid chromatography from the corpus cardiacum of the lubber grasshopper, Romalea microptera. The sequences of both peptides, designated Ro I and Ro II, were determined by gas-phase sequencing employing Edman degradation after the N-terminal pyroglutamate residue was enzymatically deblocked, as well as by fast atom bombardment mass spectrometry. Ro I was found to be a decapeptide with the primary structure: pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2, whereas Ro II is an octapeptide with the structure: pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2. Ro II is identical with AKH-G isolated from the cricket Gryllus bimaculatus. Synthetic materials having the assigned structures were found to be chromatographically, mass spectrometrically, and biologically indistinguishable from the natural peptides, confirming the sequences and establishing the Romalea peptides as members of the AKH/RPCH-family of peptides. PMID:3226948

  13. Authentication of Cordyceps sinensis by DNA Analyses: Comparison of ITS Sequence Analysis and RAPD-Derived Molecular Markers.

    PubMed

    Lam, Kelly Y C; Chan, Gallant K L; Xin, Gui-Zhong; Xu, Hong; Ku, Chuen-Fai; Chen, Jian-Ping; Yao, Ping; Lin, Huang-Quan; Dong, Tina T X; Tsim, Karl W K

    2015-01-01

    Cordyceps sinensis is an endoparasitic fungus widely used as a tonic and medicinal food in the practice of traditional Chinese medicine (TCM). In historical usage, Cordyceps specifically is referring to the species of C. sinensis. However, a number of closely related species are named themselves as Cordyceps, and they are sold commonly as C. sinensis. The substitutes and adulterants of C. sinensis are often introduced either intentionally or accidentally in the herbal market, which seriously affects the therapeutic effects or even leads to life-threatening poisoning. Here, we aim to identify Cordyceps by DNA sequencing technology. Two different DNA-based approaches were compared. The internal transcribed spacer (ITS) sequences and the random amplified polymorphic DNA (RAPD)-sequence characterized amplified region (SCAR) were developed here to authenticate different species of Cordyceps. Both approaches generally enabled discrimination of C. sinensis from others. The application of the two methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, the SCAR markers derived from the RAPD results provide a new method for quick authentication of Cordyceps. PMID:26694332

  14. Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids

    PubMed Central

    Jansen, Robert K; Kaittanis, Charalambos; Saski, Christopher; Lee, Seung-Bum; Tomkins, Jeffrey; Alverson, Andrew J; Daniell, Henry

    2006-01-01

    Background The Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids. Results The Vitis vinifera chloroplast genome is 160,928 bp in length, including a pair of inverted repeats of 26,358 bp that are separated by small and large single copy regions of 19,065 bp and 89,147 bp, respectively. The gene content and order of Vitis is identical to many other unrearranged angiosperm chloroplast genomes, including tobacco. Phylogenetic analyses using maximum parsimony and maximum likelihood were performed on DNA sequences of 61 protein-coding genes for two datasets with 28 or 29 taxa, including eight or nine taxa from four of the seven currently recognized major clades of rosids. Parsimony and likelihood phylogenies of both data sets provide strong support for the placement of Vitaceae as sister to the remaining rosids. However, the position of the Myrtales and support for the monophyly of the eurosid I clade differs between the two data sets and the two methods of analysis. In parsimony analyses, the inclusion of Gossypium is necessary to obtain trees that support the monophyly of the eurosid I clade. However, maximum

  15. The genetic diversity of genus Bacillus and the related genera revealed by 16s rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey

    PubMed Central

    Cihan, Arzu Coleri; Tekin, Nilgun; Ozcan, Birgul; Cokmus, Cumhur

    2012-01-01

    Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4–100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies. PMID:24031834

  16. Retention and loss of amino acid biosynthetic pathways based on analysis of whole-genome sequences.

    PubMed

    Payne, Samuel H; Loomis, William F

    2006-02-01

    Plants and fungi can synthesize each of the 20 amino acids by using biosynthetic pathways inherited from their bacterial ancestors. However, the ability to synthesize nine amino acids (Phe, Trp, Ile, Leu, Val, Lys, His, Thr, and Met) was lost in a wide variety of eukaryotes that evolved the ability to feed on other organisms. Since the biosynthetic pathways and their respective enzymes are well characterized, orthologs can be recognized in whole genomes to understand when in evolution pathways were lost. The pattern of pathway loss and retention was analyzed in the complete genomes of three early-diverging protist parasites, the amoeba Dictyostelium, and six animals. The nine pathways were lost independently in animals, Dictyostelium, Leishmania, Plasmodium, and Cryptosporidium. Seven additional pathways appear to have been lost in one or another parasite, demonstrating that they are dispensable in a nutrition-rich environment. Our predictions of pathways retained and pathways lost based on computational analyses of whole genomes are validated by minimal-medium studies with mammals, fish, worms, and Dictyostelium. The apparent selective advantages of retaining biosynthetic capabilities for amino acids available in the diet are considered.

  17. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning

    SciTech Connect

    Takahashi, N.; Takahashi, Y.; Blumberg, B.S.; Putnam, F.W.

    1987-07-01

    The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO/sub 4//PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.

  18. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning.

    PubMed

    Takahashi, N; Takahashi, Y; Blumberg, B S; Putnam, F W

    1987-07-01

    The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO4/PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.

  19. Amino acid sequences of neuropeptides in the sinus gland of the land crab Cardisoma carnifex: a novel neuropeptide proteolysis site.

    PubMed

    Newcomb, R W

    1987-08-01

    The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.

  20. GENOME-WIDE ASSOCIATION ANALYSES BASED ON WHOLE-GENOME SEQUENCING IN SARDINIA PROVIDE INSIGHTS INTO REGULATION OF HEMOGLOBIN LEVELS

    PubMed Central

    Danjou, Fabrice; Zoledziewska, Magdalena; Sidore, Carlo; Steri, Maristella; Busonero, Fabio; Maschio, Andrea; Mulas, Antonella; Perseu, Lucia; Barella, Susanna; Porcu, Eleonora; Pistis, Giorgio; Pitzalis, Maristella; Pala, Mauro; Menzel, Stephan; Metrustry, Sarah; Spector, Timothy D.; Leoni, Lidia; Angius, Andrea; Uda, Manuela; Moi, Paolo; Thein, Swee Lay; Galanello, Renzo; Abecasis, Gonçalo R.; Schlessinger, David; Sanna, Serena; Cucca, Francesco

    2015-01-01

    We report GWAS results for the levels of A1, A2 and fetal hemoglobins, analyzed for the first time concurrently. Integrating high-density array genotyping and whole-genome sequencing in a large general population cohort from Sardinia, we detected 23 associations at 10 loci. Five are due to variants at previously undetected loci: MPHOSPH9, PLTP-PCIF1, FOG1, NFIX, and CCND3. Among those at known loci, 10 are new lead variants and 4 are novel independent signals. Half of all variants also showed pleiotropic associations with different hemoglobins, which further corroborated some of the detected associations and revealed features of coordinated hemoglobin species production. PMID:26366553

  1. Enzyme-free translation of DNA into sequence-defined synthetic polymers structurally unrelated to nucleic acids

    NASA Astrophysics Data System (ADS)

    Niu, Jia; Hili, Ryan; Liu, David R.

    2013-04-01

    The translation of DNA sequences into corresponding biopolymers enables the production, function and evolution of the macromolecules of life. In contrast, methods to generate sequence-defined synthetic polymers with similar levels of control have remained elusive. Here, we report the development of a DNA-templated translation system that enables the enzyme-free translation of DNA templates into sequence-defined synthetic polymers that have no necessary structural relationship with nucleic acids. We demonstrate the efficiency, sequence-specificity and generality of this translation system by oligomerizing building blocks including polyethylene glycol, α-(D)-peptides, and β-peptides in a DNA-programmed manner. Sequence-defined synthetic polymers with molecular weights of 26 kDa containing 16 consecutively coupled building blocks and 90 densely functionalized β-amino acid residues were translated from DNA templates using this strategy. We integrated the DNA-templated translation system developed here into a complete cycle of translation, coding sequence replication, template regeneration and re-translation suitable for the iterated in vitro selection of functional sequence-defined synthetic polymers unrelated in structure to nucleic acids.

  2. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    PubMed Central

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  3. Kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences.

    PubMed

    Vilasi, Silvia; Dosi, Roberta; Iannuzzi, Clara; Malmo, Clorinda; Parente, Augusto; Irace, Gaetano; Sirangelo, Ivana

    2006-03-01

    In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.

  4. GAWK, a novel human pituitary polypeptide: isolation, immunocytochemical localization and complete amino acid sequence.

    PubMed

    Benjannet, S; Leduc, R; Lazure, C; Seidah, N G; Marcinkiewicz, M; Chrétien, M

    1985-01-16

    During the course of reverse-phase high pressure liquid chromatography (RP-HPLC) purification of a postulated big ACTH (1) from human pituitary gland extracts, a highly purified peptide bearing no resemblance to any known polypeptide was isolated. The complete sequence of this 74 amino acid polypeptide, called GAWK, has been determined. Search on a computer data bank on the possible homology to any known protein or fragment, using a mutation data matrix, failed to reveal any homology greater than 30%. An antibody produced against a synthetic fragment allowed us to detect several immunoreactive forms. The antisera also enabled us to localize the polypeptide, by immunocytochemistry, in the anterior lobe of the pituitary gland.

  5. Evolutionary connections of biological kingdoms based on protein and nucleic acid sequence evidence

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.

    1983-01-01

    Prokaryotic and eukaryotic evolutionary trees are developed from protein and nucleic-acid sequences by the methods of numerical taxonomy. Trees are presented for bacterial ferredoxins, 5S ribosomal RNA, c-type cytochromes , cytochromes c2 and c', and 5.8S ribosomal RNA; the implications for early evolution are discussed; and a composite tree showing the branching of the anaerobes, aerobes, archaebacteria, and eukaryotes is shown. Single lines are found for all oxygen-evolving photosynthetic forms and for the salt-loving and high-temperature forms of archaebacteria. It is argued that the eukaryote mitochondria, chloroplasts, and cytoplasmic host material are descended from free-living prokaryotes that formed symbiotic associations, with more than one symbiotic event involved in the evolution of each organelle.

  6. Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

  7. Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii.

    PubMed

    Schmitter, J M; Jacquot, J P; de Lamotte-Guéry, F; Beauvallet, C; Dutka, S; Gadal, P; Decottignies, P

    1988-03-01

    The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.

  8. Real-time nucleic acid sequence-based amplification in nanoliter volumes.

    PubMed

    Gulliksen, Anja; Solli, Lars; Karlsen, Frank; Rogne, Henrik; Hovig, Eivind; Nordstrøm, Trine; Sirevåg, Reidun

    2004-01-01

    Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.

  9. Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus.

    PubMed

    Abd el-Galil, Khaled H; el-Sokkary, M A; Kheira, S M; Salazar, Andre M; Yates, Marylynn V; Chen, Wilfred; Mulchandani, Ashok

    2005-11-01

    A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

  10. Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification.

    PubMed

    Starkey, William G; Smail, David A; Bleie, Hogne; Muir, K Fiona; Ireland, Jacqueline H; Richards, Randolph H

    2006-10-17

    We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100x more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.

  11. Sequence-defined shuttles for targeted nucleic acid and protein delivery.

    PubMed

    Röder, Ruth; Wagner, Ernst

    2014-01-01

    Molecular medicine opens into a space of novel specific therapeutic agents: intracellularly active drugs such as peptides, proteins or nucleic acids, which are not able to cross cell membranes and enter the intracellular space on their own. Through the development of cell-targeted shuttles for specific delivery, this restriction in delivery has the potential to be converted into an advantage. On the one hand, due to the multiple extra- and intracellular barriers, such carrier systems need to be multifunctional. On the other hand, they must be precise and reproducibly manufactured due to pharmaceutical reasons. Here we review the design of precise sequence-defined delivery carriers, including solid-phase synthesized peptides and nonpeptidic oligomers, or nucleotide-based carriers such as aptamers and origami nanoboxes.

  12. Parameters of proteome evolution from histograms of amino-acid sequence identities of paralogous proteins

    PubMed Central

    Axelsen, Jacob Bock; Yan, Koon-Kiu; Maslov, Sergei

    2007-01-01

    Background The evolution of the full repertoire of proteins encoded in a given genome is mostly driven by gene duplications, deletions, and sequence modifications of existing proteins. Indirect information about relative rates and other intrinsic parameters of these three basic processes is contained in the proteome-wide distribution of sequence identities of pairs of paralogous proteins. Results We introduce a simple mathematical framework based on a stochastic birth-and-death model that allows one to extract some of this information and apply it to the set of all pairs of paralogous proteins in H. pylori, E. coli, S. cerevisiae, C. elegans, D. melanogaster, and H. sapiens. It was found that the histogram of sequence identities p generated by an all-to-all alignment of all protein sequences encoded in a genome is well fitted with a power-law form ~ p-γ with the value of the exponent γ around 4 for the majority of organisms used in this study. This implies that the intra-protein variability of substitution rates is best described by the Gamma-distribution with the exponent α ≈ 0.33. Different features of the shape of such histograms allow us to quantify the ratio between the genome-wide average deletion/duplication rates and the amino-acid substitution rate. Conclusion We separately measure the short-term ("raw") duplication and deletion rates rdup∗, rdel∗ which include gene copies that will be removed soon after the duplication event and their dramatically reduced long-term counterparts rdup, rdel. High deletion rate among recently duplicated proteins is consistent with a scenario in which they didn't have enough time to significantly change their functional roles and thus are to a large degree disposable. Systematic trends of each of the four duplication/deletion rates with the total number of genes in the genome were analyzed. All but the deletion rate of recent duplicates rdel∗ were shown to systematically increase with Ngenes. Abnormally flat shapes

  13. Molecular phylogeography of the brown bear (Ursus arctos) in Northeastern Asia based on analyses of complete mitochondrial DNA sequences.

    PubMed

    Hirata, Daisuke; Mano, Tsutomu; Abramov, Alexei V; Baryshnikov, Gennady F; Kosintsev, Pavel A; Vorobiev, Alexandr A; Raichev, Evgeny G; Tsunoda, Hiroshi; Kaneko, Yayoi; Murata, Koichi; Fukui, Daisuke; Masuda, Ryuichi

    2013-07-01

    To further elucidate the migration history of the brown bears (Ursus arctos) on Hokkaido Island, Japan, we analyzed the complete mitochondrial DNA (mtDNA) sequences of 35 brown bears from Hokkaido, the southern Kuril Islands (Etorofu and Kunashiri), Sakhalin Island, and the Eurasian Continent (continental Russia, Bulgaria, and Tibet), and those of four polar bears. Based on these sequences, we reconstructed the maternal phylogeny of the brown bear and estimated divergence times to investigate the timing of brown bear migrations, especially in northeastern Eurasia. Our gene tree showed the mtDNA haplotypes of all 73 brown and polar bears to be divided into eight divergent lineages. The brown bear on Hokkaido was divided into three lineages (central, eastern, and southern). The Sakhalin brown bear grouped with eastern European and western Alaskan brown bears. Etorofu and Kunashiri brown bears were closely related to eastern Hokkaido brown bears and could have diverged from the eastern Hokkaido lineage after formation of the channel between Hokkaido and the southern Kuril Islands. Tibetan brown bears diverged early in the eastern lineage. Southern Hokkaido brown bears were closely related to North American brown bears.

  14. Molecular evolution and diversity of Conus peptide toxins, as revealed by gene structure and intron sequence analyses.

    PubMed

    Wu, Yun; Wang, Lei; Zhou, Maojun; You, Yuwen; Zhu, Xiaoyan; Qiang, Yuanyuan; Qin, Mengying; Luo, Shaonan; Ren, Zhenghua; Xu, Anlong

    2013-01-01

    Cone snails, which are predatory marine gastropods, produce a cocktail of venoms used for predation, defense and competition. The major venom component, conotoxin, has received significant attention because it is useful in neuroscience research, drug development and molecular diversity studies. In this study, we report the genomic characterization of nine conotoxin gene superfamilies from 18 Conus species and investigate the relationships among conotoxin gene structure, molecular evolution and diversity. The I1, I2, M, O2, O3, P, S, and T superfamily precursors all contain three exons and two introns, while A superfamily members contain two exons and one intron. The introns are conserved within a certain gene superfamily, and also conserved across different Conus species, but divergent among different superfamilies. The intronic sequences contain many simple repeat sequences and regulatory elements that may influence conotoxin gene expression. Furthermore, due to the unique gene structure of conotoxins, the base substitution rates and the number of positively selected sites vary greatly among exons. Many more point mutations and trinucleotide indels were observed in the mature peptide exon than in the other exons. In addition, the first example of alternative splicing in conotoxin genes was found. These results suggest that the diversity of conotoxin genes has been shaped by point mutations and indels, as well as rare gene recombination or alternative splicing events, and that the unique gene structures could have made a contribution to the evolution of conotoxin genes.

  15. Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: purification, properties, and amino acid sequences.

    PubMed

    Haldar, U C; Saha, S K; Beavis, R C; Sinha, N K

    1996-02-01

    Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is at pH 4.55 for LA-1 and at pH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 A. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0 x 10(9) M-1 sec-1 for LA-1 and 0.8 x 10(9) M-1 sec-1 for LA-2 and that of K2HPO4 quenching is 1.6 x 10(11) M-1 sec-1 for LA-1 and 1.2 x 10(11) M-1 sec-1 for LA-2. Analysis of the circular dichroic spectra yields 40% alpha-helix and 60% beta-turn for La-1 and 45% alpha-helix and 55% beta-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzyme-inhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors. PMID:8924202

  16. Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization

    PubMed Central

    Wang, Jingjing; Morabito, Kenneth; Tang, Jay X.; Tripathi, Anubhav

    2013-01-01

    The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein. PMID:24404041

  17. Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification.

    PubMed

    Fykse, Else M; Skogan, Gunnar; Davies, William; Olsen, Jaran Strand; Blatny, Janet M

    2007-03-01

    A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

  18. The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes.

    PubMed

    Fushitani, K; Higashiyama, K; Moriyama, E N; Imai, K; Hosokawa, K

    1996-09-01

    To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.

  19. The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes.

    PubMed

    Fushitani, K; Higashiyama, K; Moriyama, E N; Imai, K; Hosokawa, K

    1996-09-01

    To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds. PMID:8752011

  20. A single molecular beacon probe is sufficient for the analysis of multiple nucleic acid sequences.

    PubMed

    Gerasimova, Yulia V; Hayson, Aaron; Ballantyne, Jack; Kolpashchikov, Dmitry M

    2010-08-16

    Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping.

  1. Complete mitochondrial genome sequences of three bats species and whole genome mitochondrial analyses reveal patterns of codon bias and lend support to a basal split in Chiroptera.

    PubMed

    Meganathan, P R; Pagan, Heidi J T; McCulloch, Eve S; Stevens, Richard D; Ray, David A

    2012-01-15

    Order Chiroptera is a unique group of mammals whose members have attained self-powered flight as their main mode of locomotion. Much speculation persists regarding bat evolution; however, lack of sufficient molecular data hampers evolutionary and conservation studies. Of ~1200 species, complete mitochondrial genome sequences are available for only eleven. Additional sequences should be generated if we are to resolve many questions concerning these fascinating mammals. Herein, we describe the complete mitochondrial genomes of three bats: Corynorhinus rafinesquii, Lasiurus borealis and Artibeus lituratus. We also compare the currently available mitochondrial genomes and analyze codon usage in Chiroptera. C. rafinesquii, L. borealis and A. lituratus mitochondrial genomes are 16438 bp, 17048 bp and 16709 bp, respectively. Genome organization and gene arrangements are similar to other bats. Phylogenetic analyses using complete mitochondrial genome sequences support previously established phylogenetic relationships and suggest utility in future studies focusing on the evolutionary aspects of these species. Comprehensive analyses of available bat mitochondrial genomes reveal distinct nucleotide patterns and synonymous codon preferences corresponding to different chiropteran families. These patterns suggest that mutational and selection forces are acting to different extents within Chiroptera and shape their mitochondrial genomes.

  2. Taxonomic relationships among Turkish water frogs as revealed by phylogenetic analyses using mtDNA gene sequences.

    PubMed

    Bülbül, Ufuk; Matsui, Masafumi; Kutrup, Bilal; Eto, Koshiro

    2011-12-01

    We assessed taxonomic relationships among Turkish water frogs through estimation of phylogenetic relationships among 62 adult specimens from 44 distinct populations inhabiting seven main geographical regions of Turkey using 2897 bp sequences of the mitochondrial Cytb, 12S rRNA and 16S rRNA genes with equally-weighted parsimony, likelihood, and Bayesian methods of inference. Monophyletic clade (Clade A) of the northwesternmost (Thrace) samples is identified as Pelophylax ridibundus. The other clade (Clade B) consisted of two monophyletic subclades. One of these contains specimens from southernmost populations that are regarded as an unnamed species. The other subclade consists of two lineages, of which one corresponds to P. caralitanus and another to P. bedriagae. Taxonomic relationships of these two species are discussed and recognition of P. caralitanus as a subspecies of P. bedriagae is proposed.

  3. Canine amino acid transport system Xc(-): cDNA sequence, distribution and cystine transport activity in lens epithelial cells.

    PubMed

    Maruo, Takuya; Kanemaki, Nobuyuki; Onda, Ken; Sato, Reiichiro; Ichihara, Nobuteru; Ochiai, Hideharu

    2014-04-01

    The cystine transport activity of a lens epithelial cell line originated from a canine mature cataract was investigated. The distinct cystine transport activity was observed, which was inhibited to 28% by extracellular 1 mM glutamate. The cDNA sequences of canine cysteine/glutamate exchanger (xCT) and 4F2hc were determined. The predicted amino acid sequences were 527 and 533 amino acid polypeptides, respectively. The amino acid sequences of canine xCT and 4F2hc showed high similarities (>80%) to those of humans. The expression of xCT in lens epithelial cell line was confirmed by western blot analysis. RT-PCR analysis revealed high level expression only in the brain, and it was below the detectable level in other tissues.

  4. Sequence and expression analyses of Cytophaga-like hydrolases in a Western arctic metagenomic library and the Sargasso Sea.

    PubMed

    Cottrell, Matthew T; Yu, Liying; Kirchman, David L

    2005-12-01

    Sequence analysis of environmental DNA promises to provide new insights into the ecology and biogeochemistry of uncultured marine microbes. In this study we used the Sargasso Sea Whole Genome Sequence (WGS) data set to search for hydrolases used by Cytophaga-like bacteria to degrade biopolymers such as polysaccharides and proteins. Analysis of the Sargasso WGS data for contigs bearing both the 16S rRNA genes of Cytophaga-like bacteria and hydrolase genes revealed a cellulase gene (celM) most similar to the gene found in Cytophaga hutchinsonii. A BLAST search of the entire Sargasso Sea WGS data set indicated that celM was the most abundant cellulase-like gene in the Sargasso Sea. However, the similarity between CelM-like cellulases and peptidases belonging to metalloprotease family M42 led us to question whether CelM is involved in the degradation of polysaccharides or proteins. PCR primers were designed for the celM genes in the Sargasso Sea WGS data set and used to identify celM in a fosmid library constructed with prokaryotic DNA from the western Arctic Ocean. Expression analysis of the Cytophaga-like Arctic CelM, which is 63% identical and 77% similar to CelM in C. hutchinsonii, indicated that there was peptidase activity, whereas cellulase activity was not detected. Our analysis suggests that the celM gene plays a role in the degradation of protein by Cytophaga-like bacteria. The abundance of peptidase genes in the Cytophaga-like fosmid clone provides further evidence for the importance of Cytophaga-like bacteria in the degradation of protein in high-molecular-weight dissolved organic matter.

  5. Lactic acid production from potato peel waste by anaerobic sequencing batch fermentation using undefined mixed culture.

    PubMed

    Liang, Shaobo; McDonald, Armando G; Coats, Erik R

    2015-11-01

    Lactic acid (LA) is a necessary industrial feedstock for producing the bioplastic, polylactic acid (PLA), which is currently produced by pure culture fermentation of food carbohydrates. This work presents an alternative to produce LA from potato peel waste (PPW) by anaerobic fermentation in a sequencing batch reactor (SBR) inoculated with undefined mixed culture from a municipal wastewater treatment plant. A statistical design of experiments approach was employed using set of 0.8L SBRs using gelatinized PPW at a solids content range from 30 to 50 g L(-1), solids retention time of 2-4 days for yield and productivity optimization. The maximum LA production yield of 0.25 g g(-1) PPW and highest productivity of 125 mg g(-1) d(-1) were achieved. A scale-up SBR trial using neat gelatinized PPW (at 80 g L(-1) solids content) at the 3 L scale was employed and the highest LA yield of 0.14 g g(-1) PPW and a productivity of 138 mg g(-1) d(-1) were achieved with a 1 d SRT.

  6. Amino acid sequence surrounding the chondroitin sulfate attachment site of thrombomodulin regulates chondroitin polymerization.

    PubMed

    Izumikawa, Tomomi; Kitagawa, Hiroshi

    2015-05-01

    Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization.

  7. Lactic acid production from potato peel waste by anaerobic sequencing batch fermentation using undefined mixed culture.

    PubMed

    Liang, Shaobo; McDonald, Armando G; Coats, Erik R

    2015-11-01

    Lactic acid (LA) is a necessary industrial feedstock for producing the bioplastic, polylactic acid (PLA), which is currently produced by pure culture fermentation of food carbohydrates. This work presents an alternative to produce LA from potato peel waste (PPW) by anaerobic fermentation in a sequencing batch reactor (SBR) inoculated with undefined mixed culture from a municipal wastewater treatment plant. A statistical design of experiments approach was employed using set of 0.8L SBRs using gelatinized PPW at a solids content range from 30 to 50 g L(-1), solids retention time of 2-4 days for yield and productivity optimization. The maximum LA production yield of 0.25 g g(-1) PPW and highest productivity of 125 mg g(-1) d(-1) were achieved. A scale-up SBR trial using neat gelatinized PPW (at 80 g L(-1) solids content) at the 3 L scale was employed and the highest LA yield of 0.14 g g(-1) PPW and a productivity of 138 mg g(-1) d(-1) were achieved with a 1 d SRT. PMID:25708409

  8. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

    PubMed

    Hegeman, C E; Grabau, E A

    2001-08-01

    Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.

  9. Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov

    NASA Technical Reports Server (NTRS)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.; Deinhard, G.; Poralla, K.

    1992-01-01

    Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

  10. Genetic variability among Syphacia obvelata isolates from laboratory mice in four different geographical locations of China revealed by sequence analyses of five mitochondrial genes.

    PubMed

    Wang, Chun-Ren; Lou, Yan; Zhang, Yan; Wang, Wen-Tao; Zheng, Xu; Xu, Wen-Wen; Zhang, Ying; Tian, Si-Qin; Na, Lu; Chang, Qiao-Cheng

    2015-04-01

    Syphacia obvelata is a rodent nematode with high prevalence in laboratory mice. In the present study, we examined the genetic variability of S. obvelata from naturally infected laboratory mice in four different provinces, China. Five mitochondrial (mt) DNA regions, namely cytochrome c oxidase subunit 1 (pcox1), cytochrome b (pcytb), large subunit ribosomal RNA (prrnL) and NADH dehydrogenase subunits 1 and 5 (pnad1 and pnad5), were amplified separately from individual nematodes by PCR, and then sequenced directly. The size of the sequences of pcox1, pcytb, prrnL, pnad1 and pnad5 was 628 bp, 555 bp, 548 bp, 548 bp and 561 bp, respectively. While the intra-specific sequence variations within S. obvelata were 0-1.0% for pcox1, 0-1.6% for pcytb, 0-2.8% for prrnL, 0-2.0% for pnad1 and 0-1.8% for pnad5, the inter-specific sequence differences among members of the Oxyuridae were significantly higher, being 14.0-17.5% for pcox1, 27.5-32.9% for pcytb, 35.8-37.2% for prrnL, 22.2-26.8% for pnad1 and 22.3-25.2% for pnad5, respectively. Phylogenetic analyses based on combined sequences of four mt protein-coding genes, using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods, indicated that all of the S. obvelata samples grouped together with high statistical support, but samples from the same geographical origin did not always cluster together. These findings demonstrated the existence of low-level intra-specific variation in five mtDNA sequences among S. obvelata isolates from laboratory mice, but no obvious geographical distinction among S. obvelata isolates from laboratory mice in different geographic regions in China. These results provide basic information for further studies of systematics and population genetics of S. obvelata.

  11. The complete mitochondrial genome sequence of the network pipefish (Corythoichthys flavofasciatus) and the analyses of phylogenetic relationships within the Syngnathidae species.

    PubMed

    Zhang, Huixian; Zhang, Yanhong; Qin, Geng; Lin, Qiang

    2015-02-01

    This study determined the mitochondrial genome sequence of the network pipefish (Corythoichthys flavofasciatus) (Gasterosteiformes: Syngnathidae). The mitogenome was a circular molecule consisting of 16,961 nucleotides, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and a control region. The nucleotide composition of the genome was biased toward A+T content at 59.3%. All tRNA genes had typical cloverleaf secondary structure except for tRNA(Ser (AGY)), in which the dihydrouridine arm was missing. The C. flavofasciatus control region of 1130 bp contained several features common to other teleost, including conserved sequence blocks. We also performed comparative analysis of the network pipefish mitogenome to the available mitogenome sequences of other Syngnathidae species, and phylogenetic relationship of the Syngnathidae species was constructed based on the data sets including all the concatenated nucleotide sequences of the mitogenomes except the third codon positions. Partitioned Bayesian inference and maximum likelihood analyses showed that all seahorse species formed a monophyletic group of Hippocampus with 100% PPs and BPs, but the pipefish species did not form a monophyletic group. Corythoichthys flavofasciatus was placed as a sister relationship to the Hippocampus clade by strong node-supporting values. The availability of mitogenome of the network pipefish will shed light on the molecular systematics, biogeography and genetic differentiation in this species.

  12. Sequence analyses of the distal-less homeobox gene family in East African cichlid fishes reveal signatures of positive selection

    PubMed Central

    2013-01-01

    Background Gen(om)e duplication events are hypothesized as key mechanisms underlying the origin of phenotypic diversity and evolutionary innovation. The diverse and species-rich lineage of teleost fishes is a renowned example of this scenario, because of the fish-specific genome duplication. Gene families, generated by this and other gene duplication events, have been previously found to play a role in the evolution and development of innovations in cichlid fishes - a prime model system to study the genetic basis of rapid speciation, adaptation and evolutionary innovation. The distal-less homeobox genes are particularly interesting candidate genes for evolutionary novelties, such as the pharyngeal jaw apparatus and the anal fin egg-spots. Here we study the dlx repertoire in 23 East African cichlid fishes to determine the rate of evolution and the signatures of selection pressure. Results Four intact dlx clusters were retrieved from cichlid draft genomes. Phylogenetic analyses of these eight dlx loci in ten teleost species, followed by an in-depth analysis of 23 East African cichlid species, show that there is disparity in the rates of evolution of the dlx paralogs. Dlx3a and dlx4b are the fastest evolving dlx genes, while dlx1a and dlx6a evolved more slowly. Subsequent analyses of the nonsynonymous-synonymous substitution rate ratios indicate that dlx3b, dlx4a and dlx5a evolved under purifying selection, while signs of positive selection were found for dlx1a, dlx2a, dlx3a and dlx4b. Conclusions Our results indicate that the dlx repertoire of teleost fishes and cichlid fishes in particular, is shaped by differential selection pressures and rates of evolution after gene duplication. Although the divergence of the dlx paralogs are putative signs of new or altered functions, comparisons with available expression patterns indicate that the three dlx loci under strong purifying selection, dlx3b, dlx4a and dlx5a, are transcribed at high levels in the cichlids

  13. Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

    PubMed

    Reiz, Bela; Li, Liang

    2010-09-01

    Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.

  14. Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

    NASA Astrophysics Data System (ADS)

    McMillen, Chelsea L.; Wright, Patience M.; Cassady, Carolyn J.

    2016-05-01

    Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

  15. Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21.

    PubMed

    Barrientos, Álvaro; Merino, Estefanía; Casabon, Israël; Rodríguez, Joaquín; Crowe, Adam M; Holert, Johannes; Philipp, Bodo; Eltis, Lindsay D; Olivera, Elías R; Luengo, José M

    2015-01-01

    Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

  16. Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

    PubMed

    Whetsell, M; Mosley, R L; Whetsell, L; Schaefer, F V; Miller, K S; Klein, J R

    1991-12-01

    The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.

  17. Compact variant-rich customized sequence database and a fast and sensitive database search for efficient proteogenomic analyses.

    PubMed

    Park, Heejin; Bae, Junwoo; Kim, Hyunwoo; Kim, Sangok; Kim, Hokeun; Mun, Dong-Gi; Joh, Yoonsung; Lee, Wonyeop; Chae, Sehyun; Lee, Sanghyuk; Kim, Hark Kyun; Hwang, Daehee; Lee, Sang-Won; Paek, Eunok

    2014-12-01

    In proteogenomic analysis, construction of a compact, customized database from mRNA-seq data and a sensitive search of both reference and customized databases are essential to accurately determine protein abundances and structural variations at the protein level. However, these tasks have not been systematically explored, but rather performed in an ad-hoc fashion. Here, we present an effective method for constructing a compact database containing comprehensive sequences of sample-specific variants--single nucleotide variants, insertions/deletions, and stop-codon mutations derived from Exome-seq and RNA-seq data. It, however, occupies less space by storing variant peptides, not variant proteins. We also present an efficient search method for both customized and reference databases. The separate searches of the two databases increase the search time, and a unified search is less sensitive to identify variant peptides due to the smaller size of the customized database, compared to the reference database, in the target-decoy setting. Our method searches the unified database once, but performs target-decoy validations separately. Experimental results show that our approach is as fast as the unified search and as sensitive as the separate searches. Our customized database includes mutation information in the headers of variant peptides, thereby facilitating the inspection of peptide-spectrum matches.

  18. Transcriptome Sequencing Analyses between the Cytoplasmic Male Sterile Line and Its Maintainer Line in Welsh Onion (Allium fistulosum L.)

    PubMed Central

    Liu, Qianchun; Lan, Yanping; Wen, Changlong; Zhao, Hong; Wang, Jian; Wang, Yongqin

    2016-01-01

    Cytoplasmic male sterility (CMS) is important for exploiting heterosis in crop plants and also serves as a model for investigating nuclear–cytoplasmic interaction. The molecular mechanism of cytoplasmic male sterility and fertility restoration was investigated in several important economic crops but remains poorly understood in the Welsh onion. Therefore, we compared the differences between the CMS line 64-2 and its maintainer line 64-1 using transcriptome sequencing with the aim of determining critical genes and pathways associated with male sterility. This study combined two years of RNA-seq data; there were 1504 unigenes (in May 2013) and 2928 unigenes (in May 2014) that were differentially expressed between the CMS and cytoplasmic male maintainer Welsh onion varieties. Known CMS-related genes were found in the set of differentially expressed genes and checked by qPCR. These genes included F-type ATPase, NADH dehydrogenase, cytochrome c oxidase, etc. Overall, this study demonstrated that the CMS regulatory genes and pathways may be associated with the mitochondria and nucleus in the Welsh onion. We believe that this transcriptome dataset will accelerate the research on CMS gene clones and other functional genomics research on A. fistulosum L. PMID:27376286

  19. Ancient DNA analyses reveal high mitochondrial DNA sequence diversity and parallel morphological evolution of late pleistocene cave bears.

    PubMed

    Hofreiter, Michael; Capelli, Cristian; Krings, Matthias; Waits, Lisette; Conard, Nicholas; Münzel, Susanne; Rabeder, Gernot; Nagel, Doris; Paunovic, Maja; Jambrĕsić, Gordana; Meyer, Sonja; Weiss, Gunter; Pääbo, Svante

    2002-08-01

    Cave bears (Ursus spelaeus) existed in Europe and western Asia until the end of the last glaciation some 10,000 years ago. To investigate the genetic diversity, population history, and relationship among different cave bear populations, we have determined mitochondrial DNA sequences from 12 cave bears that range in age from about 26,500 to at least 49,000 years and originate from nine caves. The samples include one individual from the type specimen population, as well as two small-sized high-Alpine bears. The results show that about 49,000 years ago, the mtDNA diversity among cave bears was about 1.8-fold lower than the current species-wide diversity of brown bears (Ursus arctos). However, the current brown bear mtDNA gene pool consists of three clades, and cave bear mtDNA diversity is similar to the diversity observed within each of these clades. The results also show that geographically separated populations of the high-Alpine cave bear form were polyphyletic with respect to their mtDNA. This suggests that small size may have been an ancestral trait in cave bears and that large size evolved at least twice independently.

  20. Phylogenetic Analysis of Bolivian Bat Trypanosomes of the Subgenus Schizotrypanum Based on Cytochrome b Sequence and Minicircle Analyses

    PubMed Central

    García, Lineth; Ortiz, Sylvia; Osorio, Gonzalo; Torrico, Mary Cruz; Torrico, Faustino; Solari, Aldo

    2012-01-01

    The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed. PMID:22590570

  1. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    PubMed

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution.

  2. Transcriptome Sequencing Analyses between the Cytoplasmic Male Sterile Line and Its Maintainer Line in Welsh Onion (Allium fistulosum L.).

    PubMed

    Liu, Qianchun; Lan, Yanping; Wen, Changlong; Zhao, Hong; Wang, Jian; Wang, Yongqin

    2016-01-01

    Cytoplasmic male sterility (CMS) is important for exploiting heterosis in crop plants and also serves as a model for investigating nuclear-cytoplasmic interaction. The molecular mechanism of cytoplasmic male sterility and fertility restoration was investigated in several important economic crops but remains poorly understood in the Welsh onion. Therefore, we compared the differences between the CMS line 64-2 and its maintainer line 64-1 using transcriptome sequencing with the aim of determining critical genes and pathways associated with male sterility. This study combined two years of RNA-seq data; there were 1504 unigenes (in May 2013) and 2928 unigenes (in May 2014) that were differentially expressed between the CMS and cytoplasmic male maintainer Welsh onion varieties. Known CMS-related genes were found in the set of differentially expressed genes and checked by qPCR. These genes included F-type ATPase, NADH dehydrogenase, cytochrome c oxidase, etc. Overall, this study demonstrated that the CMS regulatory genes and pathways may be associated with the mitochondria and nucleus in the Welsh onion. We believe that this transcriptome dataset will accelerate the research on CMS gene clones and other functional genomics research on A. fistulosum L. PMID:27376286

  3. Polyvinyl-alcohol-based magnetic beads for rapid and efficient separation of specific or unspecific nucleic acid sequences

    NASA Astrophysics Data System (ADS)

    Oster, Jürgen; Parker, Jeffrey; à Brassard, Lothar

    2001-01-01

    The versatile application of polyvinyl-alcohol-based magnetic M-PVA beads is demonstrated in the separation of genomic DNA, sequence specific nucleic acid purification, and binding of bacteria for subsequent DNA extraction and detection. It is shown that nucleic acids can be obtained in high yield and purity using M-PVA beads, making sample preparation efficient, fast and highly adaptable for automation processes.

  4. Unifying bacteria from decaying wood with various ubiquitous Gibbsiella species as G. acetica sp. nov. based on nucleotide sequence similarities and their acetic acid secretion.

    PubMed

    Geider, Klaus; Gernold, Marina; Jock, Susanne; Wensing, Annette; Völksch, Beate; Gross, Jürgen; Spiteller, Dieter

    2015-12-01

    Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37 °C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov.

  5. Full Vector Analyses of the Short-Term Behavior Recorded in Long Volcanic Sequences in Hawaii, USA.

    NASA Astrophysics Data System (ADS)

    Herrero-Bervera, E.

    2007-05-01

    The Hawaiian volcanoes, in principle, offer the opportunity of observing the geomagnetic field behavior from present back to 5.72 Ma (from the Big Island of Hawaii to the island of Kauai). Thus, new paleomagnetic measurements coupled with radioisotopic dating are revolutionizing our understanding of the geodynamo by providing terrestrial lava records of the short-term behavior of the paleomagnetic field. As part of our investigations of some of these Hawaiian volcanoes, we have sampled long volcanic sequences of the Waianae, Koolau (island of O'ahu) and Mauna Loa (Big Island of Hawaii) volcanoes. These volcanic edifices have collapsed in the past leaving highly dissected lava sequences ideal for paleomagnetic sampling. We have sampled and studied in detail the directional characteristics and their respective absolute paleointensities of three successive reversals, namely, Gilbert-Gauss, Lower and Upper Mammoth polarity transitions recorded on Waianae lavas, Cryptochron C2r.2r-1 (ca. 2.514 +/- 0.030 Ma) as well as the Kaena Subchron recorded in the Koolau Volcano and the Laschamp and Pringle Falls excursions recorded on lavas from the Mauna Loa volcano (Big Island of Hawaii). The records of the three successive Gilbert-Gauss, Lower and Upper Mammoth reversals confirm that large oscillations of directions precede or follow the reversals, which reminds waveforms typical of paleosecular variation with their amplitude being considerably amplified by the decrease of the dipole. Determinations of absolute paleointensity were attempted on more than 750 samples. Special care was taken at selecting data obtained from segments covering more than 50% of the remanent magnetization of the samples. This procedure limited the success rate to 13% for the Waianae lavas, 70% for the Cryptochron flows and 25% for the Kaena samples but provided consistent and reliable paleointensities. In addition to other time intervals, the results document the field variations surrounding the five

  6. Statistical analyses of soil properties on a quaternary terrace sequence in the upper sava river valley, Slovenia, Yugoslavia

    USGS Publications Warehouse

    Vidic, N.; Pavich, M.; Lobnik, F.

    1991-01-01

    Alpine glaciations, climatic changes and tectonic movements have created a Quaternary sequence of gravely carbonate sediments in the upper Sava River Valley, Slovenia, Yugoslavia. The names for terraces, assigned in this model, Gu??nz, Mindel, Riss and Wu??rm in order of decreasing age, are used as morphostratigraphic terms. Soil chronosequence on the terraces was examined to evaluate which soil properties are time dependent and can be used to help constrain the ages of glaciofluvial sedimentation. Soil thickness, thickness of Bt horizons, amount and continuity of clay coatings and amount of Fe and Me concretions increase with soil age. The main source of variability consists of solutions of carbonate, leaching of basic cations and acidification of soils, which are time dependent and increase with the age of soils. The second source of variability is the content of organic matter, which is less time dependent, but varies more within soil profiles. Textural changes are significant, presented by solution of carbonate pebbles and sand, and formation is silt loam matrix, which with age becomes finer, with clay loam or clayey texture. The oldest, Gu??nz, terrace shows slight deviation from general progressive trends of changes of soil properties with time. The hypothesis of single versus multiple depositional periods of deposition was tested with one-way analysis of variance (ANOVA) on a staggered, nested hierarchical sampling design on a terrace of largest extent and greatest gravel volume, the Wu??rm terrace. The variability of soil properties is generally higher within subareas than between areas of the terrace, except for the soil thickness. Observed differences in soil thickness between the areas of the terrace could be due to multiple periods of gravel deposition, or to the initial differences of texture of the deposits. ?? 1991.

  7. Pancreatic ribonucleases of mammals with ruminant-like digestion. Amino-acid sequences of hippopotamus and sloth ribonucleases.

    PubMed

    Havinga, J; Beintema, J J

    1980-09-01

    High levels of pancreatic ribonucleases are found in ruminants, species that have a ruminant-like digestion and several species with coecal digestion. Pancreatic ribonucleases from several independently evolved species with ruminant-like digestion were investigated to test a hypothesis that glycosylation of ribonucleases may have some function in species with coecal digestion and that glycosylation of the enzyme may not be advantageous for ruminants. Ribonucleases from the hippopotamus, two-toed sloth and three-toed sloth were isolated by extraction with sulfuric acid and affinity chromatography. Complete amino acid sequences were determined for the ribonucleases from the hippopotamus and two-toed sloth and a partial sequence for the enzyme from the three-toed sloth. The amino acids 75-78 of hippopotamus ribonuclease were positioned by homology with other artiodactyl ribonucleases. In hippopotamus ribonuclease a heterogeneity was found at position 37, half of the molecules containing glutamine acid the other half lysine. Hippopotamus ribonuclease differs less from pig and bovine ribonuclease than these differ from each other, because more ancestral characteristics have been retained. Although hippopotamus ribonuclease contains all four Asn-X-Ser/Thr sequences previously found to be glycosylation sites in one or more pancreatic ribonucleases, only the sequence Ans-Met-Thr (34-36) is glycosylated in the variant with glutamine at position 37, while the variant with lysine at this position is carbohydrate-free. Both sloth ribonucleases are completely glycosylated at the sequence Ans-Met-Thr (34-36) with a simple type of carbohydrate chain. The amino acid sequence of two-toed sloth ribonuclease shows some interesting coupled replacements.

  8. Effects of simple acid leaching of crushed and powdered geological materials on high-precision Pb isotope analyses

    NASA Astrophysics Data System (ADS)

    Todd, Erin; Stracke, Andreas; Scherer, Erik E.

    2015-07-01

    We present new results of simple acid leaching experiments on the Pb isotope composition of USGS standard reference material powders and on ocean island basalt whole rock splits and powders. Rock samples were leached with cold 6 N HCl in an ultrasonic bath, then on a hot plate, and washed with ultrapure H2O before sample digestion in HF-HNO3 and chromatographic purification of Pb. Lead isotope analyses were measured by Tl-doped MC-ICPMS. Intrasession and intersession analytical reproducibilities of repeated analyses of both synthetic Pb solutions and Pb from single digests of chemically processed natural samples were generally better than 100 ppm (2 SD). The comparison of leached and unleached samples shows that leaching consistently removes variable amounts of contaminants that differ in Pb isotopic composition for different starting materials. For repeated digests of a single sample, analyses of leached samples reproduce better than those of unleached ones, confirming that leaching effectively removes most of the heterogeneously distributed extraneous Pb. Nevertheless, the external reproducibility of leached samples is still up to an order of magnitude worse than that of Pb solution standards (˜100 ppm). More complex leaching methods employed by earlier studies yield Pb isotope ratios within error of those produced by our method and at similar levels of reproducibility, demonstrating that our simple leaching method is as effective as more complex leaching techniques. Therefore, any Pb isotope heterogeneity among multiple leached digests of samples in excess of the external reproducibility is attributed to inherent isotopic heterogeneity of the sample. The external precision of ˜100 ppm (2 SD) achieved for Pb isotope ratio determination by Tl-doped MC-ICPMS is thus sufficient for most rocks. The full advantage of the most precise Pb isotope analytical methods is only realized in cases where the natural isotopic heterogeneity among samples in a studied suite is

  9. Method for the detection of specific nucleic acid sequences by polymerase nucleotide incorporation

    DOEpatents

    Castro, Alonso

    2004-06-01

    A method for rapid and efficient detection of a target DNA or RNA sequence is provided. A primer having a 3'-hydroxyl group at one end and having a sequence of nucleotides sufficiently homologous with an identifying sequence of nucleotides in the target DNA is selected. The primer is hybridized to the identifying sequence of nucleotides on the DNA or RNA sequence and a reporter molecule is synthesized on the target sequence by progressively binding complementary nucleotides to the primer, where the complementary nucleotides include nucleotides labeled with a fluorophore. Fluorescence emitted by fluorophores on single reporter molecules is detected to identify the target DNA or RNA sequence.

  10. Detection of Dengue Viral RNA Using a Nucleic Acid Sequence-Based Amplification Assay

    PubMed Central

    Wu, Shuenn-Jue L.; Lee, Eun Mi; Putvatana, Ravithat; Shurtliff, Roxanne N.; Porter, Kevin R.; Suharyono, Wuryadi; Watts, Douglas M.; King, Chwan-Chuen; Murphy, Gerald S.; Hayes, Curtis G.; Romano, Joseph W.

    2001-01-01

    Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The “gold standard” used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections. PMID:11473994

  11. Depositional sequences of offshore Canterbury, New Zealand, and preliminary results of stable isotope analyses of the samples from IODP Expedition 317

    NASA Astrophysics Data System (ADS)

    Hoyanagi, K.; Koto, S.; Kawagata, S.; Fulthorpe, C.; Blum, P.; Shipboard Scientific Party, E.

    2010-12-01

    INTRODUCTION Integrated Ocean Drilling Program Expedition 317 was devoted to understanding the relative importance of global sea level (eustasy) versus local tectonic and sedimentary processes in controlling continental-margin sedimentary cycles. In order to achieve these objectives, upper Miocene to Recent sedimentary sequences were cored in a transect of three sites on the continental shelf (landward to basinward, Sites U1353, U1354, U1351). Highest recovery was achieved in cores of upper Pliocene (3.5 Ma) to Recent sediments. We also drilled one site (Site U1352) on the continental slope, reaching a depth of 1927.5 m below sea floor and obtaining Eocene samples. CORRELATION OF SEISMIC SEQUENCE BOUNDARIES AND DISCONTINUITIES IN THE CORES Nineteen regional seismic sequence boundaries (U1-U19, in ascending order) were idendified in the middle Miocene to recent shelf-slope sediment prism of the offshore Canterbury Basin (Lu and Fulthope, 2004). Discontinuities identified in cores may correlate to U19-U8 at Site U1353, and to U19-U10 at Sites U1354 and U1351. We estimate the ages of the discontinuities, based on shipboard analyses, to correspond to both Marine Isotope Stages (Lisiecki and Raymo, 2005) and global sequence boundaries (Haq et al., 1987). STABLE ISOTOPE MEASUREMENTS OF THE ORGANIC MATTER AND FORAMINIFERA TESTS We are analyzing carbon isotopic ratios of organic matter in the sediments and oxygen isotopic ratios of foraminifer tests. Carbon isotopic ratio indicates whether the origin of the organic matter is terrestrial or marine. Samples for stable isotope analysis of organic carbon are treated with HCl to dissolve calcium carbonate. Analyses are carried out at the Faculty of Science, Shinshu University, using an elemental analyzer (FlashEA1122, ThermoQuest Ltd.) and a mass spectrometer (Delta V, ThermoQuest Ltd.). We are picking foraminifera tests from core samples from slope Site U1352 and measure oxygen isotope ratios of the calcium carbonate to

  12. Identification of tropomyosins as major allergens in antarctic krill and mantis shrimp and their amino acid sequence characteristics.

    PubMed

    Motoyama, Kanna; Suma, Yota; Ishizaki, Shoichiro; Nagashima, Yuji; Lu, Ying; Ushio, Hideki; Shiomi, Kazuo

    2008-01-01

    Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the tropomyosins from both species with decapod tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod tropomyosins, especially with fast-type tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13-42, the amino acid sequence of which is highly conserved for decapod tropomyosins, and hence, it shares somewhat lower sequence identity (82.4-89.8% identity) with decapod tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin. PMID:18521668

  13. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus CFL1, a Lactic Acid Bacterium Isolated from French Handcrafted Fermented Milk

    PubMed Central

    Meneghel, Julie; Irlinger, Françoise; Loux, Valentin; Vidal, Marie; Passot, Stéphanie; Béal, Catherine; Layec, Séverine

    2016-01-01

    Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a lactic acid bacterium widely used for the production of yogurt and cheeses. Here, we report the genome sequence of L. bulgaricus CFL1 to improve our knowledge on its stress-induced damages following production and end-use processes. PMID:26941141

  14. Update of PROFEAT: a web server for computing structural and physicochemical features of proteins and peptides from amino acid sequence.

    PubMed

    Rao, H B; Zhu, F; Yang, G B; Li, Z R; Chen, Y Z

    2011-07-01

    Sequence-derived structural and physicochemical features have been extensively used for analyzing and predicting structural, functional, expression and interaction profiles of proteins and peptides. PROFEAT has been developed as a web server for computing commonly used features of proteins and peptides from amino acid sequence. To facilitate more extensive studies of protein and peptides, numerous improvements and updates have been made to PROFEAT. We added new functions for computing descriptors of protein-protein and protein-small molecule interactions, segment descriptors for local properties of protein sequences, topological descriptors for peptide sequences and small molecule structures. We also added new feature groups for proteins and peptides (pseudo-amino acid composition, amphiphilic pseudo-amino acid composition, total amino acid properties and atomic-level topological descriptors) as well as for small molecules (atomic-level topological descriptors). Overall, PROFEAT computes 11 feature groups of descriptors for proteins and peptides, and a feature group of more than 400 descriptors for small molecules plus the derived features for protein-protein and protein-small molecule interactions. Our computational algorithms have been extensively tested and used in a number of published works for predicting proteins of specific structural or functional classes, protein-protein interactions, peptides of specific functions and quantitative structure activity relationships of small molecules. PROFEAT is accessible free of charge at http://bidd.cz3.nus.edu.sg/cgi-bin/prof/protein/profnew.cgi.

  15. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing

    PubMed Central

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G.

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA. PMID:27587826

  16. Draft Genome Sequence of Burkholderia stabilis LA20W, a Trehalose Producer That Uses Levulinic Acid as a Substrate

    PubMed Central

    Sato, Yuya; Koike, Hideaki; Kondo, Susumu; Hori, Tomoyuki; Kanno, Manabu; Kimura, Nobutada; Morita, Tomotake; Kirimura, Kohtaro

    2016-01-01

    Burkholderia stabilis LA20W produces trehalose using levulinic acid (LA) as a substrate. Here, we report the 7.97-Mb draft genome sequence of B. stabilis LA20W, which will be useful in investigations of the enzymes involved in LA metabolism and the mechanism of LA-induced trehalose production. PMID:27491978

  17. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus CFL1, a Lactic Acid Bacterium Isolated from French Handcrafted Fermented Milk.

    PubMed

    Meneghel, Julie; Dugat-Bony, Eric; Irlinger, Françoise; Loux, Valentin; Vidal, Marie; Passot, Stéphanie; Béal, Catherine; Layec, Séverine; Fonseca, Fernanda

    2016-01-01

    Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a lactic acid bacterium widely used for the production of yogurt and cheeses. Here, we report the genome sequence of L. bulgaricus CFL1 to improve our knowledge on its stress-induced damages following production and end-use processes. PMID:26941141

  18. Draft Genome Sequence of Acetobacter tropicalis Type Strain NBRC16470, a Producer of Optically Pure d-Glyceric Acid

    PubMed Central

    Koike, Hideaki; Sato, Shun; Morita, Tomotake; Fukuoka, Tokuma

    2014-01-01

    Here we report the 3.7-Mb draft genome sequence of Acetobacter tropicalis NBRC16470T, which can produce optically pure d-glyceric acid (d-GA; 99% enantiomeric excess) from raw glycerol feedstock derived from biodiesel fuel production processes. PMID:25523780

  19. Complete genome sequence of Lactobacillus plantarum ZS2058, a probiotic strain with high conjugated linoleic acid production ability.

    PubMed

    Yang, Bo; Chen, Haiqin; Tian, Fengwei; Zhao, Jianxin; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Wei

    2015-11-20

    Lactobacillus plantarum ZS2058 was isolated from sauerkraut and identified to synthesize the beneficial metabolite conjugated linoleic acid. The genome contains a 319,7363-bp chromosome and three plasmids. The sequence will facilitate identification and characterization of the genetic determinants for its putative biological benefits.

  20. Draft Genome Sequence of Cutaneotrichosporon curvatus DSM 101032 (Formerly Cryptococcus curvatus), an Oleaginous Yeast Producing Polyunsaturated Fatty Acids

    PubMed Central

    Hofmeyer, Thomas; Hackenschmidt, Silke; Nadler, Florian; Thürmer, Andrea; Daniel, Rolf

    2016-01-01

    Cutaneotrichosporon curvatus DSM 101032 is an oleaginous yeast that can be isolated from various habitats and is capable of producing substantial amounts of polyunsaturated fatty acids. Here, we present the first draft genome sequence of any C. curvatus species. PMID:27174275

  1. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus CFL1, a Lactic Acid Bacterium Isolated from French Handcrafted Fermented Milk.

    PubMed

    Meneghel, Julie; Dugat-Bony, Eric; Irlinger, Françoise; Loux, Valentin; Vidal, Marie; Passot, Stéphanie; Béal, Catherine; Layec, Séverine; Fonseca, Fernanda

    2016-03-03

    Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a lactic acid bacterium widely used for the production of yogurt and cheeses. Here, we report the genome sequence of L. bulgaricus CFL1 to improve our knowledge on its stress-induced damages following production and end-use processes.

  2. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing.

    PubMed

    Trösemeier, Jan-Hendrik; Musso, Didier; Blümel, Johannes; Thézé, Julien; Pybus, Oliver G; Baylis, Sally A

    2016-01-01

    We report here the sequence of a candidate reference strain of Zika virus (ZIKV) developed on behalf of the World Health Organization (WHO). The ZIKV reference strain is intended for use in nucleic acid amplification (NAT)-based assays for the detection and quantification of ZIKV RNA. PMID:27587826

  3. Ultra high-throughput nucleic acid sequencing as a tool for virus discovery in the turkey gut.