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Sample records for acid sequence variability

  1. 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

  2. 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.

  3. Complete amino acid sequence of the variable domains of two human IgM anti-gamma globulins (Lay/Pom) with shared idiotypic specificities.

    PubMed

    Capra, J D; Klapper, D G

    1976-01-01

    On the basis of extensive shared idiotypic specificities, two human IgM anti-gamma-globulins (Lay/Pom) were selected for complete amino acid sequence analysis of their variable domains. Previous studies on the variable regions of the heavy chains of these proteins had shown but eight amino acid differences, only one of which was within a complementarity-determining hypervariable region. The complete amino acid sequence of the variable regions of the light chains of these two proteins is the subject of this report. Protein Lay is a typical VchiI protein with only five 'framework' differences when compared with protein Roy. Protein Pom is best classified as a VchiII, but in the 'framework' there are 16 differences between it and protein Ti. Although there are extensive differences in the first hypervariable region, the second and third light-chain hypervariable regions have an identical sequence. The finding of two identical light-chain and two identical heavy-chain hypervariable regions in these two proteins, which were selected on the basis of their combining specificities and their idiotypic cross-reactions, strongly implicates hypervariable regions in the constitution of the idiotypic determinants and the antibody combining site. Additionally, the finding of identical hypervariable regions in light chains of different V-region subgroups fulfills a prediction of the gene-interaction concept of antibody variability. PMID:824717

  4. Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm

    PubMed Central

    Öhrmalm, Christina; Jobs, Magnus; Eriksson, Ronnie; Golbob, Sultan; Elfaitouri, Amal; Benachenhou, Farid; Strømme, Maria; Blomberg, Jonas

    2010-01-01

    One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes. PMID:20864443

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Sequence Variability of a Human Pseudogene

    PubMed Central

    Martínez-Arias, Rosa; Calafell, Francesc; Mateu, Eva; Comas, David; Andrés, Aida; Bertranpetit, Jaume

    2001-01-01

    We have obtained haplotypes from the autosomal glucocerebrosidase pseudogene (psGBA) for 100 human chromosomes from worldwide populations, as well as for four chimpanzee and four gorilla chromosomes. In humans, in a 5420-nucleotide stretch analyzed, variation comprises 17 substitutions, a 3-bp deletion, and a length polymorphism at a polyadenine tract. The substitution rate on the pseudogene (1.23 ± 0.22 × 10−9 per nucleotide and year) is within the range of previous estimates considering phylogenetic estimations. Recombination within the pseudogene was recognized, although the low variability of this locus prevented an accurate measure of recombination rates. At least 13% of the psGBA sequence could be attributed to gene conversion from the contiguous GBA gene, whereas the reciprocal event has been shown to lead to Gaucher disease. Human psGBA sequences showed a recent coalescence time (∼200,000 yr ago), and the most ancestral haplotype was found only in Africans; both observations are compatible with the replacement hypothesis of human origins. In a deeper timeframe, phylogenetic analysis showed that the duplication event that created psGBA could be dated at ∼27 million years ago, in agreement with previous estimates. PMID:11381033

  11. 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.

  12. 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.

  13. Sequence Variability in Staphylococcal Enterotoxin Genes seb, sec, and sed

    PubMed Central

    Johler, Sophia; Sihto, Henna-Maria; Macori, Guerrino; Stephan, Roger

    2016-01-01

    Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences. PMID:27258311

  14. Sequence Variability in Staphylococcal Enterotoxin Genes seb, sec, and sed.

    PubMed

    Johler, Sophia; Sihto, Henna-Maria; Macori, Guerrino; Stephan, Roger

    2016-01-01

    Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences.

  15. Method to amplify variable sequences without imposing primer sequences

    DOEpatents

    Bradbury, Andrew M.; Zeytun, Ahmet

    2006-11-14

    The present invention provides methods of amplifying target sequences without including regions flanking the target sequence in the amplified product or imposing amplification primer sequences on the amplified product. Also provided are methods of preparing a library from such amplified target sequences.

  16. 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

  17. Variable depth recursion algorithm for leaf sequencing

    SciTech Connect

    Siochi, R. Alfredo C.

    2007-02-15

    The processes of extraction and sweep are basic segmentation steps that are used in leaf sequencing algorithms. A modified version of a commercial leaf sequencer changed the way that the extracts are selected and expanded the search space, but the modification maintained the basic search paradigm of evaluating multiple solutions, each one consisting of up to 12 extracts and a sweep sequence. While it generated the best solutions compared to other published algorithms, it used more computation time. A new, faster algorithm selects one extract at a time but calls itself as an evaluation function a user-specified number of times, after which it uses the bidirectional sweeping window algorithm as the final evaluation function. To achieve a performance comparable to that of the modified commercial leaf sequencer, 2-3 calls were needed, and in all test cases, there were only slight improvements beyond two calls. For the 13 clinical test maps, computation speeds improved by a factor between 12 and 43, depending on the constraints, namely the ability to interdigitate and the avoidance of the tongue-and-groove under dose. The new algorithm was compared to the original and modified versions of the commercial leaf sequencer. It was also compared to other published algorithms for 1400, random, 15x15, test maps with 3-16 intensity levels. In every single case the new algorithm provided the best solution.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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...

  4. 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

  5. Variable speed wind turbine generator with zero-sequence filter

    DOEpatents

    Muljadi, Eduard

    1998-01-01

    A variable speed wind turbine generator system to convert mechanical power into electrical power or energy and to recover the electrical power or energy in the form of three phase alternating current and return the power or energy to a utility or other load with single phase sinusoidal waveform at sixty (60) hertz and unity power factor includes an excitation controller for generating three phase commanded current, a generator, and a zero sequence filter. Each commanded current signal includes two components: a positive sequence variable frequency current signal to provide the balanced three phase excitation currents required in the stator windings of the generator to generate the rotating magnetic field needed to recover an optimum level of real power from the generator; and a zero frequency sixty (60) hertz current signal to allow the real power generated by the generator to be supplied to the utility. The positive sequence current signals are balanced three phase signals and are prevented from entering the utility by the zero sequence filter. The zero sequence current signals have zero phase displacement from each other and are prevented from entering the generator by the star connected stator windings. The zero sequence filter allows the zero sequence current signals to pass through to deliver power to the utility.

  6. Variable Speed Wind Turbine Generator with Zero-sequence Filter

    DOEpatents

    Muljadi, Eduard

    1998-08-25

    A variable speed wind turbine generator system to convert mechanical power into electrical power or energy and to recover the electrical power or energy in the form of three phase alternating current and return the power or energy to a utility or other load with single phase sinusoidal waveform at sixty (60) hertz and unity power factor includes an excitation controller for generating three phase commanded current, a generator, and a zero sequence filter. Each commanded current signal includes two components: a positive sequence variable frequency current signal to provide the balanced three phase excitation currents required in the stator windings of the generator to generate the rotating magnetic field needed to recover an optimum level of real power from the generator; and a zero frequency sixty (60) hertz current signal to allow the real power generated by the generator to be supplied to the utility. The positive sequence current signals are balanced three phase signals and are prevented from entering the utility by the zero sequence filter. The zero sequence current signals have zero phase displacement from each other and are prevented from entering the generator by the star connected stator windings. The zero sequence filter allows the zero sequence current signals to pass through to deliver power to the utility.

  7. Variable speed wind turbine generator with zero-sequence filter

    DOEpatents

    Muljadi, E.

    1998-08-25

    A variable speed wind turbine generator system to convert mechanical power into electrical power or energy and to recover the electrical power or energy in the form of three phase alternating current and return the power or energy to a utility or other load with single phase sinusoidal waveform at sixty (60) hertz and unity power factor includes an excitation controller for generating three phase commanded current, a generator, and a zero sequence filter. Each commanded current signal includes two components: a positive sequence variable frequency current signal to provide the balanced three phase excitation currents required in the stator windings of the generator to generate the rotating magnetic field needed to recover an optimum level of real power from the generator; and a zero frequency sixty (60) hertz current signal to allow the real power generated by the generator to be supplied to the utility. The positive sequence current signals are balanced three phase signals and are prevented from entering the utility by the zero sequence filter. The zero sequence current signals have zero phase displacement from each other and are prevented from entering the generator by the star connected stator windings. The zero sequence filter allows the zero sequence current signals to pass through to deliver power to the utility. 14 figs.

  8. 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.

  9. 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.

  10. SAW: a graphical user interface for the analysis of immunoglobulin variable domain sequences.

    PubMed

    Elgavish, R A; Schroeder, H W

    1993-12-01

    The Sequence Analysis Workshop (SAW) is an interactive program for sequence analysis of immunoglobulin variable domains. Sequences for SAW can be obtained from GenBank or from a standard text file. SAW can compare a variable domain to as many as 100 different sequences, calculate the extent of homology, sort the sequences by their degree of similarity, translate the nucleotide codons into amino acids and then display the results in either a graphical or text format. These comparisons allow the investigator to determine the likely germ-line progenitors of a variable domain and to visualize how it differs from other antibody genes by functional region. SAW supports replacement and silent site substitution analysis by either codon or region, thus providing rapid insight into the forces that have shaped mutations. The sequence comparisons can be printed out as an aid for paper analysis or for preparation of figures for publication. SAW is written in Microsoft C for use with the Microsoft Windows graphics environment. The use of color and graphics, the generation of subsidiary windows that contain the results of specific analyses and the mouse-driven control of the program make SAW an easy-to-use tool for immunoglobulin sequence comparison. PMID:8292340

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Gene Sequence Variability of the Three Surface Proteins of Human Respiratory Syncytial Virus (HRSV) in Texas

    PubMed Central

    Tapia, Lorena I.; Shaw, Chad A.; Aideyan, Letisha O.; Jewell, Alan M.; Dawson, Brian C.; Haq, Taha R.; Piedra, Pedro A.

    2014-01-01

    Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004–2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community. PMID:24625544

  18. Detection of sequence variability of the collagen type IIalpha 1 3' variable number of tandem repeat.

    PubMed

    van Meurs, J B; Arp, P P; Fang, Y; Slagboom, P E; Meulenbelt, I; van Leeuwen, J P; Pols, H A; Uitterlinden, A G

    2000-11-01

    The variable number of tandem repeat (VNTR) 3' of the collagen type II (COL2A1) gene has been shown to be highly variable with a complex molecular structure. In a previous pilot experiment we observed discordance between methods to genotype this informative marker. To further investigate the extent and molecular nature of this discordance, we genotyped a random sample of 207 Caucasian individuals with two genotyping methods and sequenced new alleles. We compared single-strand (SS) analysis, which is based on detection of size differences between the different alleles, and heteroduplex analysis (HA), which is sensitive to both size and sequence differences. Overall, 26% of discordance between the two methods was detected. Approximately two thirds of this discordance was caused by subdivision of SS-alleles 13R1 and 14R2 into HA-alleles 4A + 4B and 3B + 3C, respectively. Sequence analysis of the COL2A1 VNTR alleles 4B and 3C showed that these alleles differed in sequence, but not in size, from already described SS-alleles, which explains why they escape detection by SS. The 4B allele is a frequent allele in the population (14%) and is, therefore, important to distinguish in association studies. We conclude that HA is a reliable method when the described optimized electrophoretic conditions are used. HA is a sensitive genotyping method to document allelic diversity at this locus, which can distinguish more alleles compared to the SS method.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome.

    PubMed

    Aggarwala, Varun; Voight, Benjamin F

    2016-04-01

    The rate of single-nucleotide polymorphism varies substantially across the human genome and fundamentally influences evolution and incidence of genetic disease. Previous studies have only considered the immediately flanking nucleotides around a polymorphic site--the site's trinucleotide sequence context--to study polymorphism levels across the genome. Moreover, the impact of larger sequence contexts has not been fully clarified, even though context substantially influences rates of polymorphism. Using a new statistical framework and data from the 1000 Genomes Project, we demonstrate that a heptanucleotide context explains >81% of variability in substitution probabilities, highlighting new mutation-promoting motifs at ApT dinucleotide, CAAT and TACG sequences. Our approach also identifies previously undocumented variability in C-to-T substitutions at CpG sites, which is not immediately explained by differential methylation intensity. Using our model, we present informative substitution intolerance scores for genes and a new intolerance score for amino acids, and we demonstrate clinical use of the model in neuropsychiatric diseases. PMID:26878723

  4. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome

    PubMed Central

    Aggarwala, Varun; Voight, Benjamin F.

    2016-01-01

    The rate of single nucleotide polymorphism varies substantially across the human genome and fundamentally influences evolution and incidence of genetic disease. Previous studies have only considered the immediate flanking nucleotides around a polymorphic site –the site’s trinucleotide sequence context– to study polymorph levels across the genome. Moreover, the impact of larger sequence contexts has not been fully clarified, even though context substantially influences rates of polymorphism. Using a new statistical framework and data from the 1000 Genomes Project, we demonstrate that a heptanucleotide context explains >81% of variability in substitution probabilities, revealing new mutation-promoting motifs at ApT dinucleotide, CAAT, and TACG sequences. Our approach also identifies previously undocumented variability in C-to-T substitutions at CpG sites, which is not immediately explained by differential methylation intensity. Using our model, we present informative substitution intolerance scores for genes and a new intolerance score for amino acids, and we demonstrate clinical use of the model in neuropsychiatric diseases. PMID:26878723

  5. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome.

    PubMed

    Aggarwala, Varun; Voight, Benjamin F

    2016-04-01

    The rate of single-nucleotide polymorphism varies substantially across the human genome and fundamentally influences evolution and incidence of genetic disease. Previous studies have only considered the immediately flanking nucleotides around a polymorphic site--the site's trinucleotide sequence context--to study polymorphism levels across the genome. Moreover, the impact of larger sequence contexts has not been fully clarified, even though context substantially influences rates of polymorphism. Using a new statistical framework and data from the 1000 Genomes Project, we demonstrate that a heptanucleotide context explains >81% of variability in substitution probabilities, highlighting new mutation-promoting motifs at ApT dinucleotide, CAAT and TACG sequences. Our approach also identifies previously undocumented variability in C-to-T substitutions at CpG sites, which is not immediately explained by differential methylation intensity. Using our model, we present informative substitution intolerance scores for genes and a new intolerance score for amino acids, and we demonstrate clinical use of the model in neuropsychiatric diseases.

  6. 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.

  7. 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

  8. Genetic Diagnosis Using Whole Exome Sequencing in Common Variable Immunodeficiency

    PubMed Central

    Maffucci, Patrick; Filion, Charles A.; Boisson, Bertrand; Itan, Yuval; Shang, Lei; Casanova, Jean-Laurent; Cunningham-Rundles, Charlotte

    2016-01-01

    Whole exome sequencing (WES) has proven an effective tool for the discovery of genetic defects in patients with primary immunodeficiencies (PIDs). However, success in dissecting the genetic etiology of common variable immunodeficiency (CVID) has been limited. We outline a practical framework for using WES to identify causative genetic defects in these subjects. WES was performed on 50 subjects diagnosed with CVID who had at least one of the following criteria: early onset, autoimmune/inflammatory manifestations, low B lymphocytes, and/or familial history of hypogammaglobulinemia. Following alignment and variant calling, exomes were screened for mutations in 269 PID-causing genes. Variants were filtered based on the mode of inheritance and reported frequency in the general population. Each variant was assessed by study of familial segregation and computational predictions of deleteriousness. Out of 433 variations in PID-associated genes, we identified 17 probable disease-causing mutations in 15 patients (30%). These variations were rare or private and included monoallelic mutations in NFKB1, STAT3, CTLA4, PIK3CD, and IKZF1, and biallelic mutations in LRBA and STXBP2. Forty-two other damaging variants were found but were not considered likely disease-causing based on the mode of inheritance and/or patient phenotype. WES combined with analysis of PID-associated genes is a cost-effective approach to identify disease-causing mutations in CVID patients with severe phenotypes and was successful in 30% of our cohort. As targeted therapeutics are becoming the mainstay of treatment for non-infectious manifestations in CVID, this approach will improve management of patients with more severe phenotypes. PMID:27379089

  9. Genetic Diagnosis Using Whole Exome Sequencing in Common Variable Immunodeficiency.

    PubMed

    Maffucci, Patrick; Filion, Charles A; Boisson, Bertrand; Itan, Yuval; Shang, Lei; Casanova, Jean-Laurent; Cunningham-Rundles, Charlotte

    2016-01-01

    Whole exome sequencing (WES) has proven an effective tool for the discovery of genetic defects in patients with primary immunodeficiencies (PIDs). However, success in dissecting the genetic etiology of common variable immunodeficiency (CVID) has been limited. We outline a practical framework for using WES to identify causative genetic defects in these subjects. WES was performed on 50 subjects diagnosed with CVID who had at least one of the following criteria: early onset, autoimmune/inflammatory manifestations, low B lymphocytes, and/or familial history of hypogammaglobulinemia. Following alignment and variant calling, exomes were screened for mutations in 269 PID-causing genes. Variants were filtered based on the mode of inheritance and reported frequency in the general population. Each variant was assessed by study of familial segregation and computational predictions of deleteriousness. Out of 433 variations in PID-associated genes, we identified 17 probable disease-causing mutations in 15 patients (30%). These variations were rare or private and included monoallelic mutations in NFKB1, STAT3, CTLA4, PIK3CD, and IKZF1, and biallelic mutations in LRBA and STXBP2. Forty-two other damaging variants were found but were not considered likely disease-causing based on the mode of inheritance and/or patient phenotype. WES combined with analysis of PID-associated genes is a cost-effective approach to identify disease-causing mutations in CVID patients with severe phenotypes and was successful in 30% of our cohort. As targeted therapeutics are becoming the mainstay of treatment for non-infectious manifestations in CVID, this approach will improve management of patients with more severe phenotypes. PMID:27379089

  10. Variable major proteins of Borrelia hermsii. Epitope mapping and partial sequence analysis of CNBr peptides

    PubMed Central

    1985-01-01

    The variable major proteins (VMP) of serotypes 7 and 21 of the relapsing fever agent Borrelia hermsii were isolated by detergent extraction and high performance liquid chromatography. Cyanogen bromide (CNBr) digestion of the isolated VMP yielded two peptides of apparent molecular weights 20,000 (20 K) and 16 K from VMP7, and three peptides of 14.5, 14, and 7 K mol wt from VMP21. Serotype-specific monoclonal antibodies bound in Western blots to one of each of the two or three CNBr fragments from the homologous VMP. A single monoclonal antibody bound to the whole cells, the isolated VMP, and a CNBr fragment of both serotype 7 and serotype 21. (This crossreactive antibody did not, however, bind to any of four other serotypes examined.) Regional conservation of structure between VMP7 and VMP21 was also shown by amino acid sequence analysis of the N-termini of the five CNBr fragments. One pair of aligned fragments from VMP7 and VMP21 had 80% amino acid homology in sequence; a second pair had 40% homology. The partial amino acid homologies between two VMP suggest that these proteins are products of members of a polygene family. PMID:2409197

  11. 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.

  12. 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

  13. 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

  14. 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%.

  15. Exploring pre-main-sequence variables of the ONC: the new variables

    NASA Astrophysics Data System (ADS)

    Parihar, Padmakar; Messina, Sergio; Distefano, Elisa; Shantikumar, N. S.; Medhi, Biman J.

    2009-12-01

    Since 2004, we have been engaged in a long-term observing programme to monitor young stellar objects (YSOs) in the Orion Nebula Cluster (ONC). We have collected about 2000 frames in V, R and I broad-band filters on more than 200 nights distributed over five consecutive observing seasons. The high-quality and time-extended photometric data give us an opportunity to address various phenomena associated with young stars. The prime motivations of this project are (i) to explore various manifestations of stellar magnetic activity in very young low-mass stars, (ii) to search for new pre-main-sequence eclipsing binaries and (iii) to look for any EXor and FUor-like transient activities associated with YSOs. Since this is the first paper on this programme, we give a detailed description of the science drivers, the observation and the data reduction strategies as well. In addition to these, we also present a large number of new periodic variables detected from our first 5 yr of time-series photometric data. Our study reveals that about 72 per cent of classical T Tauri stars (CTTS) in our field of view are periodic, whereas only 32 per cent of weak-lined T Tauri stars (WTTS) are periodic. This indicates that inhomogeneity patterns on the surface of CTTS of the ONC stars are much more stable than on WTTS. From our multiyear monitoring campaign, we found that the photometric surveys based on single season are incapable of identifying all periodic variables. And any study on evolution of angular momentum based on single-season surveys must be carried out with caution.

  16. 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.

  17. Evolution of tRNA-like sequences and genome variability.

    PubMed

    Frenkel, Felix E; Chaley, Maria B; Korotkov, Eugene V; Skryabin, Konstantin G

    2004-06-23

    Transfer RNA (tRNA)-like sequences were searched for in the nine basic taxonomic divisions of GenBank-121 (viruses, phages, bacteria, plants, invertebrates, vertebrates, rodents, mammals, and primates) by an original program package implementing a dynamic profile alignment approach for the genetic texts' analysis, in using 22 profiles of tRNAs of different isotypes. In total, 175,901 previously unknown tRNA-like sequences were revealed. The locations of the tRNA-likes were considered over the regions whose functional meaning is described by standard Feature Keys in GenBank. Many regions containing the tRNA-like sequences were recognized as known repeats. A mode of distribution of the tRNA-like sequences in a genome was proposed as expansion in a content of the various transposable elements. An analysis of the integrity of RNA polymerase III inner promoters in the tRNA-like sequences over the GenBank divisions has shown a high possibility of generating new copies of short interspersed nuclear element (SINE) repeats in all divisions, excepting primates. The numerous tRNA-likes found in the regions of RNA polymerase II promoters have suggested an adaptation of RNA polymerase III promoter to a binding of RNA polymerase II. PMID:15194190

  18. Genetic variability of Taenia saginata inferred from mitochondrial DNA sequences.

    PubMed

    Rostami, Sima; Salavati, Reza; Beech, Robin N; Babaei, Zahra; Sharbatkhori, Mitra; Harandi, Majid Fasihi

    2015-04-01

    Taenia saginata is an important tapeworm, infecting humans in many parts of the world. The present study was undertaken to identify inter- and intraspecific variation of T. saginata isolated from cattle in different parts of Iran using two mitochondrial CO1 and 12S rRNA genes. Up to 105 bovine specimens of T. saginata were collected from 20 slaughterhouses in three provinces of Iran. DNA were extracted from the metacestode Cysticercus bovis. After PCR amplification, sequencing of CO1 and 12S rRNA genes were carried out and two phylogenetic analyses of the sequence data were generated by Bayesian inference on CO1 and 12S rRNA sequences. Sequence analyses of CO1 and 12S rRNA genes showed 11 and 29 representative profiles respectively. The level of pairwise nucleotide variation between individual haplotypes of CO1 gene was 0.3-2.4% while the overall nucleotide variation among all 11 haplotypes was 4.6%. For 12S rRNA sequence data, level of pairwise nucleotide variation was 0.2-2.5% and the overall nucleotide variation was determined as 5.8% among 29 haplotypes of 12S rRNA gene. Considerable genetic diversity was found in both mitochondrial genes particularly in 12S rRNA gene. PMID:25687521

  19. SEMIREGULAR VARIABLES WITH PERIODS LYING BETWEEN THE PERIOD-LUMINOSITY SEQUENCES C', C, AND D

    SciTech Connect

    Soszynski, I.; Wood, P. R. E-mail: wood@mso.anu.edu.au

    2013-02-15

    We analyze the distribution of semiregular variables and Mira stars in the period-luminosity plane. Our sample consists of 6169 oxygen-rich long-period variables in the Large Magellanic Cloud included in the OGLE-III Catalog of Variable Stars. There are many stars with periods that lie between the well-known sequences C and C'. Most of these stars are multi-periodic and the period ratios suggest that these stars oscillate in the same mode as the sequence C stars. Models suggest that this mode is the fundamental radial pulsation mode. The stars with primary periods between sequences C and C' preferentially lie on an additional sequence (named F), and a large fraction of these stars also have long secondary periods (LSPs) that lie between sequences C and D. There are also a small number of stars with primary periods lying between sequences C and D. The origin of this long-period variability is unknown, as is the cause of sequence D variability. In addition, the origin of sequence F is unknown but we speculate that sequence F variability may be excited by the same phenomenon that causes the LSPs.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. Topic Sequence and Emphasis Variability of Selected Organic Chemistry Textbooks

    ERIC Educational Resources Information Center

    Houseknecht, Justin B.

    2010-01-01

    Textbook choice has a significant effect upon course success. Among the factors that influence this decision, two of the most important are material organization and emphasis. This paper examines the sequencing of 19 organic chemistry topics, 21 concepts and skills, and 7 biological topics within nine of the currently available organic textbooks.…

  5. Sequence variability of Rhizobiales orthologs and relationship with physico-chemical characteristics of proteins

    PubMed Central

    2011-01-01

    Background Chromosomal orthologs can reveal the shared ancestral gene set and their evolutionary trends. Additionally, physico-chemical properties of encoded proteins could provide information about functional adaptation and ecological niche requirements. Results We analyzed 7080 genes (five groups of 1416 orthologs each) from Rhizobiales species (S. meliloti, R. etli, and M. loti, plant symbionts; A. tumefaciens, a plant pathogen; and B. melitensis, an animal pathogen). We evaluated their phylogenetic relationships and observed three main topologies. The first, with closer association of R. etli to A. tumefaciens; the second with R. etli closer to S. meliloti; and the third with A. tumefaciens and S. meliloti as the closest pair. This was not unusual, given the close relatedness of these three species. We calculated the synonymous (dS) and nonsynonymous (dN) substitution rates of these orthologs, and found that informational and metabolic functions showed relatively low dN rates; in contrast, genes from hypothetical functions and cellular processes showed high dN rates. An alternative measure of sequence variability, percentage of changes by species, was used to evaluate the most specific proportion of amino acid residues from alignments. When dN was compared with that measure a high correlation was obtained, revealing that much of evolutive information was extracted with the percentage of changes by species at the amino acid level. By analyzing the sequence variability of orthologs with a set of five properties (polarity, electrostatic charge, formation of secondary structures, molecular volume, and amino acid composition), we found that physico-chemical characteristics of proteins correlated with specific functional roles, and association of species did not follow their typical phylogeny, probably reflecting more adaptation to their life styles and niche preferences. In addition, orthologs with low dN rates had residues with more positive values of polarity

  6. 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.

  7. Sequence variability and geographic distribution of Lassa virus, Sierra Leone.

    PubMed

    Leski, Tomasz A; Stockelman, Michael G; Moses, Lina M; Park, Matthew; Stenger, David A; Ansumana, Rashid; Bausch, Daniel G; Lin, Baochuan

    2015-04-01

    Lassa virus (LASV) is endemic to parts of West Africa and causes highly fatal hemorrhagic fever. The multimammate rat (Mastomys natalensis) is the only known reservoir of LASV. Most human infections result from zoonotic transmission. The very diverse LASV genome has 4 major lineages associated with different geographic locations. We used reverse transcription PCR and resequencing microarrays to detect LASV in 41 of 214 samples from rodents captured at 8 locations in Sierra Leone. Phylogenetic analysis of partial sequences of nucleoprotein (NP), glycoprotein precursor (GPC), and polymerase (L) genes showed 5 separate clades within lineage IV of LASV in this country. The sequence diversity was higher than previously observed; mean diversity was 7.01% for nucleoprotein gene at the nucleotide level. These results may have major implications for designing diagnostic tests and therapeutic agents for LASV infections in Sierra Leone.

  8. Sequence Variability and Geographic Distribution of Lassa Virus, Sierra Leone

    PubMed Central

    Stockelman, Michael G.; Moses, Lina M.; Park, Matthew; Stenger, David A.; Ansumana, Rashid; Bausch, Daniel G.; Lin, Baochuan

    2015-01-01

    Lassa virus (LASV) is endemic to parts of West Africa and causes highly fatal hemorrhagic fever. The multimammate rat (Mastomys natalensis) is the only known reservoir of LASV. Most human infections result from zoonotic transmission. The very diverse LASV genome has 4 major lineages associated with different geographic locations. We used reverse transcription PCR and resequencing microarrays to detect LASV in 41 of 214 samples from rodents captured at 8 locations in Sierra Leone. Phylogenetic analysis of partial sequences of nucleoprotein (NP), glycoprotein precursor (GPC), and polymerase (L) genes showed 5 separate clades within lineage IV of LASV in this country. The sequence diversity was higher than previously observed; mean diversity was 7.01% for nucleoprotein gene at the nucleotide level. These results may have major implications for designing diagnostic tests and therapeutic agents for LASV infections in Sierra Leone. PMID:25811712

  9. 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

  10. 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.

  11. Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence

    PubMed Central

    Shchyolkina, Anna K.; Kaluzhny, Dmitry N.; Borisova, Olga F.; Arndt-Jovin, Donna J.; Jovin, Thomas M.; Zhurkin, Victor B.

    2016-01-01

    Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na+ than for Li+. Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance. PMID:26308235

  12. Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence.

    PubMed

    Shchyolkina, Anna K; Kaluzhny, Dmitry N; Borisova, Olga F; Arndt-Jovin, Donna J; Jovin, Thomas M; Zhurkin, Victor B

    2016-06-01

    Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na(+) than for Li(+). Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance. PMID:26308235

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. The Effects of Delayed Reinforcement on Variability and Repetition of Response Sequences

    ERIC Educational Resources Information Center

    Odum, Amy L.; Ward, Ryan D.; Burke, K. Anne; Barnes, Christopher A.

    2006-01-01

    Four experiments examined the effects of delays to reinforcement on key peck sequences of pigeons maintained under multiple schedules of contingencies that produced variable or repetitive behavior. In Experiments 1, 2, and 4, in the repeat component only the sequence right-right-left-left earned food, and in the vary component four-response…

  19. 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

  20. Manifold Learning for Multivariate Variable-Length Sequences With an Application to Similarity Search.

    PubMed

    Ho, Shen-Shyang; Dai, Peng; Rudzicz, Frank

    2016-06-01

    Multivariate variable-length sequence data are becoming ubiquitous with the technological advancement in mobile devices and sensor networks. Such data are difficult to compare, visualize, and analyze due to the nonmetric nature of data sequence similarity measures. In this paper, we propose a general manifold learning framework for arbitrary-length multivariate data sequences driven by similarity/distance (parameter) learning in both the original data sequence space and the learned manifold. Our proposed algorithm transforms the data sequences in a nonmetric data sequence space into feature vectors in a manifold that preserves the data sequence space structure. In particular, the feature vectors in the manifold representing similar data sequences remain close to one another and far from the feature points corresponding to dissimilar data sequences. To achieve this objective, we assume a semisupervised setting where we have knowledge about whether some of data sequences are similar or dissimilar, called the instance-level constraints. Using this information, one learns the similarity measure for the data sequence space and the distance measures for the manifold. Moreover, we describe an approach to handle the similarity search problem given user-defined instance level constraints in the learned manifold using a consensus voting scheme. Experimental results on both synthetic data and real tropical cyclone sequence data are presented to demonstrate the feasibility of our manifold learning framework and the robustness of performing similarity search in the learned manifold.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Effects of variable sequences of food availability on interval time-place learning by pigeons.

    PubMed

    García-Gallardo, Daniel; Carpio, Claudio

    2016-09-01

    The effects of within session variability of the sequences of food availability in a 16 period Time Place Learning (TPL) task on the performance of pigeons were assessed. Two groups of birds were exposed to two conditions. For group 1 (N=3), the first condition consisted of a TPL task in which food could be obtained according to a Random Interval (RI) 25s schedule of reinforcement in one of four feeders, the correct feeder changed every 3min. The same sequence was repeated four times within every training session (Fixed Sequence). The second condition was exactly the same as the first one with the exception that the sequence in which the correct feeder changed was randomized, yielding a total of four randomized sequences of food availability each session (Variable Sequence). An Open Hopper Test (OHT) was conducted at the end of each condition. Birds in group 2 (N=3) experienced the same conditions but in the reverse order. Results showed high percent correct responses for both group of birds under both conditions. However, birds were able to time the availability period's duration only under the Fixed Sequence condition, as shown by anticipation, anticipation of depletion and persistence of visiting patterns on the OHT. The implications of these results to Gallistels (1990) tripartite time-place-event memory code model are discussed, pointing out that these results are in line with previous findings about the important role that spatial parameters of a TPL task can play, for accurate timing was precluded when a variable sequence was employed. PMID:27425658

  6. A survey for rapid variability among early main sequence A-stars

    NASA Astrophysics Data System (ADS)

    Schutt, Randy L.

    This thesis is a survey of non-peculiar early (A0 to A5) main sequence A-stars for rapid (4 to 30 minutes), low amplitude (less than 10 millimagnitudes) variability. Peculiar stars (roAp stars) are presently the only objects known to exhibit this behavior on or near the main sequence. There are also reasons for suspecting variability in normal stars; survey objects are in close proximity, in an Hertzsprung-Russell (HR) diagram, to the cepheid instability strip where many pulsational variables are found (i.e. the delta Scuti and roAp stars), and there is evidence of pulsational variability (at slightly longer periods) in the non-peculiar delta Scuti stars. The survey is also an independent test of the main sequence mass-loss theory proposed by Wilson et al. 1986. Finally, surveys of this type may produce objects of asteroseismological interest. The purpose of the survey is to detect variability, not to resolve all frequencies that may be involved. All observations were gathered with the University of Wisconsin Two-Star Photometer. This instrument coupled with computerized high-speed data collection used the small (16 to 24 in.) telescopes at Pine Bluff Observatory and Table Mountain Observatory. Several period-searching methods were used to analyze time series of differential photometric data. The survey produced a few stars suspected of variability, however, there is no evidence for large scale rapid variability among the non-peculiar main sequence A-stars. The survey also produced several low-amplitude delta Scuti stars, which are in or blueward of the recognized instability strip. These stars verify predictions that delta Scuti stars exist at lower amplitudes, and may also indicate they are present at earlier spectral types.

  7. Sequence variability is correlated with weak immunogenicity in Streptococcus pyogenes M protein

    PubMed Central

    Lannergård, Jonas; Kristensen, Bodil M; Gustafsson, Mattias C U; Persson, Jenny J; Norrby-Teglund, Anna; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

    2015-01-01

    The M protein of Streptococcus pyogenes, a major bacterial virulence factor, has an amino-terminal hypervariable region (HVR) that is a target for type-specific protective antibodies. Intriguingly, the HVR elicits a weak antibody response, indicating that it escapes host immunity by two mechanisms, sequence variability and weak immunogenicity. However, the properties influencing the immunogenicity of regions in an M protein remain poorly understood. Here, we studied the antibody response to different regions of the classical M1 and M5 proteins, in which not only the HVR but also the adjacent fibrinogen-binding B repeat region exhibits extensive sequence divergence. Analysis of antisera from S. pyogenes-infected patients, infected mice, and immunized mice showed that both the HVR and the B repeat region elicited weak antibody responses, while the conserved carboxy-terminal part was immunodominant. Thus, we identified a correlation between sequence variability and weak immunogenicity for M protein regions. A potential explanation for the weak immunogenicity was provided by the demonstration that protease digestion selectively eliminated the HVR-B part from whole M protein-expressing bacteria. These data support a coherent model, in which the entire variable HVR-B part evades antibody attack, not only by sequence variability but also by weak immunogenicity resulting from protease attack. PMID:26175306

  8. Variable-Interval Sequenced-Action Camera (VINSAC). Dissemination Document No. 1.

    ERIC Educational Resources Information Center

    Ward, Ted

    The 16 millimeter (mm) Variable-Interval Sequenced-Action Camera (VINSAC) is designed for inexpensive photographic recording of effective teacher instruction and use of instructional materials for teacher education and research purposes. The camera photographs single frames at preselected time intervals (.5 second to 20 seconds) which are…

  9. Long Recording Sequences: How to Track the Intra-Individual Variability of Acoustic Signals

    PubMed Central

    Lengagne, Thierry; Gomez, Doris; Josserand, Rémy; Voituron, Yann

    2015-01-01

    Recently developed acoustic technologies - like automatic recording units - allow the recording of long sequences in natural environments. These devices are used for biodiversity survey but they could also help researchers to estimate global signal variability at various (individual, population, species) scales. While sexually-selected signals are expected to show a low intra-individual variability at relatively short time scale, this variability has never been estimated so far. Yet, measuring signal variability in controlled conditions should prove useful to understand sexual selection processes and should help design acoustic sampling schedules and to analyse long call recordings. We here use the overall call production of 36 male treefrogs (Hyla arborea) during one night to evaluate within-individual variability in call dominant frequency and to test the efficiency of different sampling methods at capturing such variability. Our results confirm that using low number of calls underestimates call dominant frequency variation of about 35% in the tree frog and suggest that the assessment of this variability is better by using 2 or 3 short and well-distributed records than by using samples made of consecutive calls. Hence, 3 well-distributed 2-minutes records (beginning, middle and end of the calling period) are sufficient to capture on average all the nightly variability, whereas a sample of 10 000 consecutive calls captures only 86% of it. From a biological point of view, the call dominant frequency variability observed in H. arborea (116Hz on average but up to 470 Hz of variability during the course of the night for one male) challenge about its reliability in mate quality assessment. Automatic acoustic recording units will provide long call sequences in the near future and it will be then possible to confirm such results on large samples recorded in more complex field conditions. PMID:25970183

  10. Impact of Pre-Analytical Variables on Cancer Targeted Gene Sequencing Efficiency.

    PubMed

    Araujo, Luiz H; Timmers, Cynthia; Shilo, Konstantin; Zhao, Weiqiang; Zhang, Jianying; Yu, Lianbo; Natarajan, Thanemozhi G; Miller, Clinton J; Yilmaz, Ayse Selen; Liu, Tom; Amann, Joseph; Lapa E Silva, José Roberto; Ferreira, Carlos Gil; Carbone, David P

    2015-01-01

    Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized. PMID:26605948

  11. 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.

  12. High sequence variability, diverse subcellular localizations, and ecological implications of alkaline phosphatase in dinoflagellates and other eukaryotic phytoplankton.

    PubMed

    Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

    2012-01-01

    Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the "red-type" eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort.

  13. High Sequence Variability, Diverse Subcellular Localizations, and Ecological Implications of Alkaline Phosphatase in Dinoflagellates and Other Eukaryotic Phytoplankton

    PubMed Central

    Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

    2012-01-01

    Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the “red-type” eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

    PubMed Central

    Sweeney, R; Chen, L; Yao, M C

    1994-01-01

    Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably. Images PMID:8196658

  20. 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.

  1. Amino acid and structural variability of Yersinia pestis LcrV protein

    SciTech Connect

    Anisimov, A P; Dentovskaya, S V; Panfertsev, E A; Svetoch, T E; Kopylov, P K; Segelke, B W; Zemla, A; Telepnev, M V; Motin, V L

    2009-11-09

    The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium which is generally conserved between the epidemic strains of Yersinia pestis. They investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from ten Y. pestis strains belonging to different phylogenetic groups (subspecies) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324-326. These major variations, together with other minor substitutions in amino acid sequences, allowed them to classify the LcrV alleles into five sequence types (A-E). They observed that the strains of different Y. pestis subspecies can have the same typ of LcrV, and different types of LcrV can exist within the same natural plague focus. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in oligomerization of LcrV. The importance of the latter property in emergence of epidemic strains of Y. pestis during evolution of this pathogen will need to be further investigated.

  2. 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

  3. 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.

  4. An analysis of rotor blade twist variables associated with different Euler sequences and pretwist treatments

    NASA Technical Reports Server (NTRS)

    Alkire, K.

    1984-01-01

    A nonlinear analysis which is necessary to adequately model elastic helicopter rotor blades experiencing moderately large deformations was examined. The analysis must be based on an appropriate description of the blade's deformation geometry including elastic bending and twist. Built-in pretwist angles complicate the deformation process ant its definition. Relationships between the twist variables associated with different rotation sequences and corresponding forms of the transformation matrix are lasted. Relationships between the twist variables associated with first, the pretwist combined with the deformation twist are included. Many of the corresponding forms of the transformation matrix for the two cases are listed. It is shown that twist variables connected with the combined twist treatment are related to those where the pretwist is applied initially. A method to determine the relationships and some results are outlined. A procedure to evaluate the transformation matrix that eliminates the Eulerlike sequence altogether is demonstrated. The resulting form of the transformation matrix is unaffected by rotation sequence or pretwist treatment.

  5. Resistance to change and preference for variable versus fixed response sequences.

    PubMed

    Arantes, Joana; Berg, Mark E; Le, Dien; Grace, Randolph C

    2012-07-01

    In Experiment 1, 4 pigeons were trained on a multiple chain schedule in which the initial link was a variable-interval (VI) 20-s schedule signalled by a red or green center key, and terminal links required four responses made to the left (L) and/or right (R) keys. In the REPEAT component, signalled by red keylights, only LRLR terminal-link response sequences were reinforced, while in the VARY component, signalled by green keylights, terminal-link response sequences were reinforced if they satisfied a variability criterion. The reinforcer rate for both components was equated by adjusting the reinforcer probability for correct REPEAT sequences across sessions. Results showed that initial- and terminal-link responding in the VARY component was generally more resistant to prefeeding, extinction, and response-independent food than responding in the REPEAT component. In Experiment 2, the REPEAT and VARY contingencies were arranged as terminal links of a concurrent chain and the relative reinforcer rate was manipulated across conditions. For all pigeons, initial-link response allocation was biased toward the alternative associated with the VARY terminal link. These results replicate previous reports that operant variation is more resistant to change than operant repetition (Doughty & Lattal, 2001), and show that variation is preferred to repetition with reinforcer-related variables controlled. Behavioral momentum theory (Nevin & Grace, 2000) predicts the covariation of preference and resistance to change in Experiments 1 and 2, but does not explain why these aspects of behavior should depend on contingencies that require repetition or variation.

  6. Genetic variability of Echinococcus granulosus from the Tibetan plateau inferred by mitochondrial DNA sequences.

    PubMed

    Yan, Ning; Nie, Hua-Ming; Jiang, Zhong-Rong; Yang, Ai-Guo; Deng, Shi-Jin; Guo, Li; Yu, Hua; Yan, Yu-Bao; Tsering, Dawa; Kong, Wei-Shu; Wang, Ning; Wang, Jia-Hai; Xie, Yue; Fu, Yan; Yang, De-Ying; Wang, Shu-Xian; Gu, Xiao-Bin; Peng, Xue-Rong; Yang, Guang-You

    2013-09-01

    To analyse genetic variability and population structure, 84 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from various host species at different sites of the Tibetan plateau in China were sequenced for the whole mitochondrial nad1 (894 bp) and atp6 (513 bp) genes. The vast majority were classified as G1 genotype (n=82), and two samples from human patients in Sichuan province were identified as G3 genotype. Based on the concatenated sequences of nad1+atp6, 28 different haplotypes (NA1-NA28) were identified. A parsimonious network of the concatenated sequence haplotypes showed star-like features in the overall population, with NA1 as the major haplotype in the population networks. By AMOVA it was shown that variation of E. granulosus within the overall population was the main pattern of the total genetic variability. Neutrality indexes of the concatenated sequence (nad1+atp6) were computed by Tajima's D and Fu's Fs tests and showed high negative values for E. granulosus, indicating significant deviations from neutrality. FST and Nm values suggested that the populations were not genetically differentiated.

  7. 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.

  8. From Asia to Europe: mitochondrial DNA sequence variability in Bulgarians and Turks.

    PubMed

    Calafell, F; Underhill, P; Tolun, A; Angelicheva, D; Kalaydjieva, L

    1996-01-01

    Two hypervariable sequence segments in the control region of mitochondrial DNA were determined in samples of Bulgarians and Turks. The Turkish sample presented a higher degree of internal diversity, in terms of total number of variable nucleotides, as well as in the average pairwise nucleotide difference. Pairwise difference distributions were built for both samples, yielding smooth bell shapes in agreement with the Rogers and Harpending model. The Bulgarian and Turkish data were compared with several European and W. Asian Caucasoid populations (Basques, Tuscans, Sardinians, British, Middle Easterners and Indians). Mean pairwise differences suggest that a demographic expansion occurred sequentially in the Middle East, through Turkey, to the rest of Europe (Bulgaria included). Current mutation rate estimates date this expansion in times ranging between 50,000 and 100,000 years ago and, thus, would correspond to the arrival of anatomically modern humans in Europe. Sequence trees for segment I show that European and Middle Eastern sequences derived from the reference sequence. Coalescence times for segment I sequences agree with those predicted by pairwise distributions. Genetic trees were constructed between populations and revealed an extreme homogeneity between European samples.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. Sequence variability in three mitochondrial genes among four roundworm species from wild animals in China.

    PubMed

    Chang, Qiao-Cheng; Gao, Jun-Feng; Sheng, Zhong-Hua; Lou, Yan; Zheng, Xu; Wang, Chun-Ren

    2015-02-01

    Sequence variability in three mitochondrial DNA (mtDNA) regions, namely portions of cytochrome c oxidase subunit 1 (pcox1), NADH dehydrogenase subunit 1 (pnad1) and NADH dehydrogenase subunit 4 (pnad4), for Toxocara canis. Baylisacaris transfuga. Ascaris suum and Parascaris equorum from Canis lupus. Ursus thibetanus. Sus scrofa and Equus burchelli in China were examined. The lengths of the sequences of pcox1, pnad1 and pnad4 were 711 bp, 648 bp and 666 bp, respectively. No intra-species differences were detected in pcox1 for the four examined ascarid species, in pnad1 for T. canis. A. suum and P. equorum, and in pnad4 for B. transfuga and P. equorum. Sequence differences in pnad4 for six roundworm samples of T. canis and P. equorum were 0-0.1% and 0-0.3%, respectively, and were 0-0.3% in pnad1 for six roundworm samples isolate of B. transfuga. The inter-specific sequence differences among four species were 8.7-12.4% for pcox1, 13.9-17.7% for pnad1, and 14.0-25.7% for pnad4. Phylogenetic analyses suggested that the three mtDNA fragments could be used to identify ascarid species in families Ascaridiae and Toxocaridae.

  16. Resistance to change and preference for variable versus fixed response sequences.

    PubMed

    Arantes, Joana; Berg, Mark E; Le, Dien; Grace, Randolph C

    2012-07-01

    In Experiment 1, 4 pigeons were trained on a multiple chain schedule in which the initial link was a variable-interval (VI) 20-s schedule signalled by a red or green center key, and terminal links required four responses made to the left (L) and/or right (R) keys. In the REPEAT component, signalled by red keylights, only LRLR terminal-link response sequences were reinforced, while in the VARY component, signalled by green keylights, terminal-link response sequences were reinforced if they satisfied a variability criterion. The reinforcer rate for both components was equated by adjusting the reinforcer probability for correct REPEAT sequences across sessions. Results showed that initial- and terminal-link responding in the VARY component was generally more resistant to prefeeding, extinction, and response-independent food than responding in the REPEAT component. In Experiment 2, the REPEAT and VARY contingencies were arranged as terminal links of a concurrent chain and the relative reinforcer rate was manipulated across conditions. For all pigeons, initial-link response allocation was biased toward the alternative associated with the VARY terminal link. These results replicate previous reports that operant variation is more resistant to change than operant repetition (Doughty & Lattal, 2001), and show that variation is preferred to repetition with reinforcer-related variables controlled. Behavioral momentum theory (Nevin & Grace, 2000) predicts the covariation of preference and resistance to change in Experiments 1 and 2, but does not explain why these aspects of behavior should depend on contingencies that require repetition or variation. PMID:22851789

  17. 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

  18. Purification of a marsupial insulin: amino-acid sequence of insulin from the eastern grey kangaroo Macropus giganteus.

    PubMed

    Treacy, G B; Shaw, D C; Griffiths, M E; Jeffrey, P D

    1989-03-24

    Insulin has been purified from kangaroo pancreas by acidic ethanol extraction, diethyl ether precipitation and gel filtration. The amino-acid sequence of this, the first marsupial insulin to be studied, is reported. It differs from human insulin by only four amino-acid substitutions, all in regions of the molecule previously known to be variable. However, it should be noted that one of these, asparagine for threonine at A8, has not been reported before. Computer comparisons of all 43 insulin sequences reported to date with kangaroo insulin show it to be most closely related to a group of mammalian insulins (dog, pig, cow, human) known to be of high biological potency. The measurement of blood glucose lowering in the rabbit by kangaroo insulin is consistent with this conclusion. Comparisons of amino-acid sequences of other proteins with their kangaroo counterparts show a greater difference, in line with the time of divergence of marsupials. The limited differences observed in insulin and cytochrome c suggest that their structures need to be closely conserved in order to maintain function.

  19. 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

  20. Exome sequence analysis suggests genetic burden contributes to phenotypic variability and complex neuropathy

    PubMed Central

    Gonzaga-Jauregui, Claudia; Harel, Tamar; Gambin, Tomasz; Kousi, Maria; Griffin, Laurie B.; Francescatto, Ludmila; Ozes, Burcak; Karaca, Ender; Jhangiani, Shalini; Bainbridge, Matthew N.; Lawson, Kim S.; Pehlivan, Davut; Okamoto, Yuji; Withers, Marjorie; Mancias, Pedro; Slavotinek, Anne; Reitnauer, Pamela J; Goksungur, Meryem T.; Shy, Michael; Crawford, Thomas O.; Koenig, Michel; Willer, Jason; Flores, Brittany N.; Pediaditrakis, Igor; Us, Onder; Wiszniewski, Wojciech; Parman, Yesim; Antonellis, Anthony; Muzny, Donna M.; Katsanis, Nicholas; Battaloglu, Esra; Boerwinkle, Eric; Gibbs, Richard A.; Lupski, James R.

    2015-01-01

    Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ~45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy associated genes in subjects versus controls; confirmed in a second ethnically discrete neuropathy cohort, suggesting mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HMPVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity. PMID:26257172

  1. 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)

  2. Variability analysis of 'Persian' acid lime tree selections using agronomic and molecular markers.

    PubMed

    Santos, M G; Passos, O S; Soares Filho, W S; Girardi, E A; Gesteira, A S; Ferreira, C F

    2013-01-01

    'Persian' acid lime (PAL) is the most important triploid commercial citrus crop planted in the world. Little is known about the genetic variability of the selections used in Brazil. Therefore, 25 genotypes originating from the PAL, and three control species, Citrus sunki, C. limon, and C. aurantiifolia, were assessed using inter-simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) molecular markers and agronomic traits of the fruit. The dendrograms were designed using the mean Euclidean distance for the physicochemical attributes of the fruit (weight, length, diameter, peel color, peel thickness, number of seeds, juice yield, titratable acidity, soluble solids, and ratio) and the Jaccard distances using the data from the ISSR and IRAP molecular markers. In the physicochemical analysis, the genotypes were grouped according to species. The trait that contributed most to the diversity among accessions was the number of seeds. The 17 ISSR primers produced 69 polymorphic bands in the molecular analysis, and the seven IRAP primers generated 30 polymorphic bands. The markers detected polymorphisms within and among the PALs; two groups were formed within the PALs. PMID:24222236

  3. 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).

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 3DFlu: database of sequence and structural variability of the influenza hemagglutinin at population scale

    PubMed Central

    Mazzocco, Giovanni; Lazniewski, Michal; Migdał, Piotr; Szczepińska, Teresa; Radomski, Jan P.; Plewczynski, Dariusz

    2016-01-01

    The influenza virus type A (IVA) is an important pathogen which is able to cause annual epidemics and even pandemics. This fact is the consequence of the antigenic shifts and drifts capabilities of IVA, caused by the high mutation rate and the reassortment capabilities of the virus. The hemagglutinin (HA) protein constitutes the main IVA antigen and has a crucial role in the infection mechanism, being responsible for the recognition of host-specific sialic acid derivatives. Despite the relative abundance of HA sequence and serological studies, comparative structure-based analysis of HA are less investigated. The 3DFlu database contains well annotated HA representatives: 1192 models and 263 crystallographic structures. The relations between these proteins are defined using different metrics and are visualized as a network in the provided web interface. Moreover structural and sequence comparison of the proteins can be explored. Metadata information (e.g. protein identifier, IVA strain, year and location of infection) can enhance the exploration of the presented data. With our database researchers gain a useful tool for the exploration of high quality HA models, viewing and comparing changes in the HA viral subtypes at several information levels (sequence, structure, ESP). The complete and integrated view of those relations might be useful to determine the efficiency of transmission, pathogenicity and for the investigation of evolutionary tendencies of the influenza virus. Database URL: http://nucleus3d.cent.uw.edu.pl/influenza PMID:27694207

  10. 3DFlu: database of sequence and structural variability of the influenza hemagglutinin at population scale

    PubMed Central

    Mazzocco, Giovanni; Lazniewski, Michal; Migdał, Piotr; Szczepińska, Teresa; Radomski, Jan P.; Plewczynski, Dariusz

    2016-01-01

    The influenza virus type A (IVA) is an important pathogen which is able to cause annual epidemics and even pandemics. This fact is the consequence of the antigenic shifts and drifts capabilities of IVA, caused by the high mutation rate and the reassortment capabilities of the virus. The hemagglutinin (HA) protein constitutes the main IVA antigen and has a crucial role in the infection mechanism, being responsible for the recognition of host-specific sialic acid derivatives. Despite the relative abundance of HA sequence and serological studies, comparative structure-based analysis of HA are less investigated. The 3DFlu database contains well annotated HA representatives: 1192 models and 263 crystallographic structures. The relations between these proteins are defined using different metrics and are visualized as a network in the provided web interface. Moreover structural and sequence comparison of the proteins can be explored. Metadata information (e.g. protein identifier, IVA strain, year and location of infection) can enhance the exploration of the presented data. With our database researchers gain a useful tool for the exploration of high quality HA models, viewing and comparing changes in the HA viral subtypes at several information levels (sequence, structure, ESP). The complete and integrated view of those relations might be useful to determine the efficiency of transmission, pathogenicity and for the investigation of evolutionary tendencies of the influenza virus. Database URL: http://nucleus3d.cent.uw.edu.pl/influenza

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Variable Genome Sequences of the Murine Pneumotropic Virus (Polyomaviridae) Regulatory Region Isolated from an Infected Mouse Tissue Viral Suspension

    PubMed Central

    Libbey, Jane E.

    2016-01-01

    The murine pneumotropic virus genome, isolated from an infected murine tissue homogenate, was sequenced to completion. The lungs, liver, spleen, and kidneys were the source of the tissue homogenate in order to mirror the heterogeneity of the virus population in vivo. The regulatory region sequence was found to be highly variable. PMID:27231357

  16. Effects of "D"-Amphetamine and Ethanol on Variable and Repetitive Key-Peck Sequences in Pigeons

    ERIC Educational Resources Information Center

    Ward, Ryan D.; Bailey, Ericka M.; Odum, Amy L.

    2006-01-01

    This experiment assessed the effects of "d"-Amphetamine and ethanol on reinforced variable and repetitive key-peck sequences in pigeons. Pigeons responded on two keys under a multiple schedule of Repeat and Vary components. In the Repeat component, completion of a target sequence of right, right, left, left resulted in food. In the Vary component,…

  17. 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.

  18. GLC analysis of Indian rapeseed-mustard to study the variability of fatty acid composition.

    PubMed

    Kaushik, N; Agnihotri, A

    2000-12-01

    Rapeseed-mustard is one of the most economically important oilseed crops in India. Speciality oils having high amounts of a specific fatty acid are of immense importance for both nutritional and industrial purposes. Oil high in oleic acid has demand in commercial food-service applications due to a long shelf-life and cholesterol-reducing properties. Both linoleic and linolenic acids are essential fatty acids; however, less than 3% linolenic acid is preferred for oil stability. High erucic acid content is beneficial for the polymer industry, whereas low erucic acid is recommended for food purposes. Therefore, it is important to undertake systematic characterization of the available gene pool for its variable fatty acid profile to be utilized for specific purposes. In the present study the Indian rapeseed-mustard germplasm and some newly developed low-erucic-acid strains were analysed by GLC to study the fatty acid composition in these lines. The GLC analysis revealed that the rapeseed-mustard varieties being commonly grown in India are characterized by high erucic acid content (30-51%) in the oil with low levels of oleic acid (13-23%). However, from among the recently developed low-erucic-acid strains, several lines were identified with comparatively high oleic acid (60-70%), moderate to high linoleic acid (13-40%) and low linolenic acid (< 10%) contents. Work is in progress at TERI (New Delhi, India) to utilize these lines for development of strains with particular fatty acid compositions for specific purposes.

  19. The variability of PSV response spectra across a dense array deployed during the Northridge aftershock sequence

    USGS Publications Warehouse

    Field, E.H.; Hough, S.E.

    1997-01-01

    This study addresses the variability of pseudo-velocity response spectra across an array deployed on stiff soil in the San Fernando Valley during the Northridge (Mw 6.7) aftershock sequence. The separation between stations ranged from 0.5 to 5 km, and the aftershock magnitudes ranged from 2.3 to 4.0. We find that 95-percent of observed response spectra are within a factor of 1.9 to 2.6 of the network average. Statistically significant relative amplification factors were found for some of the sites, but the variability of observed response spectra is not significantly reduced by correcting for these effects. This implies that microzonation efforts on less than 5-km distance scales are not warranted at these types of sites. We also found a distance dependence for the response-spectral variability between neighboring sites. 95-percent are within a factor of ???2.3 at 0.5 km, increasing to 95-percent within a factor of ???4.2 at 5 km. No frequency dependence in these values could be resolved. Additional work is needed to examine the influence of other factors such as earthquake magnitude.

  20. Neisseria meningitidis antigen NMB0088: sequence variability, protein topology and vaccine potential.

    PubMed

    Sardiñas, Gretel; Yero, Daniel; Climent, Yanet; Caballero, Evelin; Cobas, Karem; Niebla, Olivia

    2009-02-01

    The significance of Neisseria meningitidis serogroup B membrane proteins as vaccine candidates is continually growing. Here, we studied different aspects of antigen NMB0088, a protein that is abundant in outer-membrane vesicle preparations and is thought to be a surface protein. The gene encoding protein NMB0088 was sequenced in a panel of 34 different meningococcal strains with clinical and epidemiological relevance. After this analysis, four variants of NMB0088 were identified; the variability was confined to three specific segments, designated VR1, VR2 and VR3. Secondary structure predictions, refined with alignment analysis and homology modelling using FadL of Escherichia coli, revealed that almost all the variable regions were located in extracellular loop domains. In addition, the NMB0088 antigen was expressed in E. coli and a procedure for obtaining purified recombinant NMB0088 is described. The humoral immune response elicited in BALB/c mice was measured by ELISA and Western blotting, while the functional activity of these antibodies was determined in a serum bactericidal assay and an animal protection model. After immunization in mice, the recombinant protein was capable of inducing a protective response when it was administered inserted into liposomes. According to our results, the recombinant NMB0088 protein may represent a novel antigen for a vaccine against meningococcal disease. However, results from the variability study should be considered for designing a cross-protective formulation in future studies.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Structural Conservation Predominates Over Sequence Variability in the Crown of HIV Type 1's V3 Loop

    PubMed Central

    Almond, David; Kimura, Tetsuya; Kong, XiangPeng; Swetnam, James; Zolla-Pazner, Susan

    2010-01-01

    Abstract The diversity of HIV-1 is a confounding problem for vaccine design, as the human immune response appears to favor poor or strain-specific responses to any given HIV-1 virus strain. A significant portion of this diversity is manifested as sequence variability in the loops of HIV-1's surface envelope glycoprotein. Here we show that the most variable sequence positions in the third variable (V3) loop crown cluster to a small zone on the surface of one face of the V3 loop ß-hairpin conformation. These results provide a novel visualization of the gp120 V3 loop, specifically demonstrating a surprising preponderance of conserved three-dimensional structure in a highly sequence-variable region. From a structural point of view, there appears to be less diversity in this region of the HIV-1 “principle neutralizing domain” than previously appreciated. PMID:20560796

  9. 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.

  10. De novo sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, reveal a variable genomic landscape.

    PubMed

    Tully, Benjamin J; Emerson, Joanne B; Andrade, Karen; Brocks, Jochen J; Allen, Eric E; Banfield, Jillian F; Heidelberg, Karla B

    2015-01-01

    Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters) for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.

  11. DNA sequence of immunoglobulin heavy chain variable region gene in thyroid lymphoma.

    PubMed

    Miwa, H; Takakuwa, T; Nakatsuka, S; Tomita, Y; Matsuzuka, F; Aozasa, K

    2001-10-01

    Patho-epidemiological studies have shown that thyroid lymphoma (TL) develops in thyroid affected by chronic lymphocytic thyroiditis (CLTH). CLTH is categorized as an organ-specific autoimmune disease, in which activated B-lymphocytes secrete a number of autoantibodies. Because antigenic stimulation might be involved in the pathogenesis of TL, the variable region in heavy chain (V(H)) genes was characterized in 13 cases with TL and 3 with CLTH. Clonal rearrangement of the V(H) gene was found in 11 cases of TL, and cloning study with sequencing of complimentary determining region (CDR) 3 revealed the presence of a major clone in 4. Three of the 4 cases used V(H) 3 gene, with the homologous germline gene of V3-30 in two cases and VH26 in one case. A biased usage of V(H) 3 and V(H) 4 genes with the homologous germline gene of VH26 in V(H) 3 gene was reported previously in cases with CLTH. A high level of somatic mutation (1-21%, average 12%) with non-random distribution of replacement and silent mutations was accumulated in all cases. The frequency of the occurrence of minor clones ranged from 29-44% per case, indicating the presence of on-going mutation. DNA sequencing of immunoglobulin V(H) gene suggests that TL develops among activated lymphoid cells in CLTH at the germinal center stage under antigen selection. PMID:11676854

  12. Genome Sequence Variability Predicts Drug Precautions and Withdrawals from the Market

    PubMed Central

    Baik, Su Youn; Lee, Soo Youn; Park, Chan Hee; Park, Paul J.; Kim, Ju Han

    2016-01-01

    Despite substantial premarket efforts, a significant portion of approved drugs has been withdrawn from the market for safety reasons. The deleterious impact of nonsynonymous substitutions predicted by the SIFT algorithm on structure and function of drug-related proteins was evaluated for 2504 personal genomes. Both withdrawn (n = 154) and precautionary (Beers criteria (n = 90), and US FDA pharmacogenomic biomarkers (n = 96)) drugs showed significantly lower genomic deleteriousness scores (P < 0.001) compared to others (n = 752). Furthermore, the rates of drug withdrawals and precautions correlated significantly with the deleteriousness scores of the drugs (P < 0.01); this trend was confirmed for all drugs included in the withdrawal and precaution lists by the United Nations, European Medicines Agency, DrugBank, Beers criteria, and US FDA. Our findings suggest that the person-to-person genome sequence variability is a strong independent predictor of drug withdrawals and precautions. We propose novel measures of drug safety based on personal genome sequence analysis. PMID:27690231

  13. 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.

  14. 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.

  15. Substantial Variability of Multiple Microbial Communities Collected at Similar Acidic Mine Water Outlets.

    PubMed

    Falteisek, Lukáš; Duchoslav, Vojtěch; Čepička, Ivan

    2016-07-01

    Deep sequencing of prokaryotic 16S rDNA regularly reveals thousands of microbial species thriving in many common habitats. It is still unknown how this huge microbial diversity, including many potentially competing organisms, may persist at a single site. One of plausible hypotheses is that a large number of spatially separated microcommunities exist within each complex habitat. Smaller subset of the species may exist in each microcommunity and actually interact with each other. We sampled two groups of microbial stalactites growing at a single acidic mine drainage outlet as a model of multiplicated, low-complexity microhabitat. Samples from six other sites were added for comparison. Both tRFLP and 16S rDNA pyrosequencing showed that microbial communities containing 6 to 51 species-level operational taxonomic units (OTU) inhabited all stalactites. Interestingly, most OTUs including the highly abundant ones unpredictably alternated regardless of physical and environmental distance of the stalactites. As a result, the communities clustered independently on sample site and other variables when using both phylogenetic dissimilarity and OTU abundance metrics. Interestingly, artificial communities generated by pooling the biota of several adjacent stalactites together clustered by the locality more strongly than when the stalactites were analyzed separately. The most probable interpretation is that each stalactite contains likely random selection from the pool of plausible species. Such degree of stochasticity in assembly of extremophilic microbial communities is significantly greater than commonly proposed and requires caution when interpreting microbial diversity.

  16. Selection of specific interactors from phage display library based on sea lamprey variable lymphocyte receptor sequences.

    PubMed

    Wezner-Ptasinska, Magdalena; Otlewski, Jacek

    2015-12-01

    Variable lymphocyte receptors (VLRs) are non-immunoglobulin components of adaptive immunity in jawless vertebrates. These proteins composed of leucine-rich repeat modules offer some advantages over antibodies in target binding and therefore are attractive candidates for biotechnological applications. In this paper we report the design and characterization of a phage display library based on a previously proposed dVLR scaffold containing six LRR modules [Wezner-Ptasinska et al., 2011]. Our library was designed based on a consensus approach in which the randomization scheme reflects the frequencies of amino acids naturally occurring in respective positions responsible for antigen recognition. We demonstrate general applicability of the scaffold by selecting dVLRs specific for lysozyme and S100A7 protein with KD values in the micromolar range. The dVLR library could be used as a convenient alternative to antibodies for effective isolation of high affinity binders.

  17. 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.

  18. 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/.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. What is the intrapatient variability of mycophenolic acid trough levels?

    PubMed

    Todorova, Ekaterina K; Huang, Shih-Han S; Kobrzynski, Marta C; Filler, Guido

    2015-11-01

    TDM of MPA, the active compound of MMF, is rarely used despite its substantial intra- and interpatient variability. Little is known about the utility of long-term MPA TDM. Data are expressed as mean (one standard deviation). All available data from 27 renal transplant recipients (mean age at transplantation: 7.7 [5.0] yr) with an average follow-up of 9.3 (4.6) yr were analyzed. MPA levels were measured using the EMIT. GFR was measured using cystatin C and eGFR was calculated using the Filler formula. Intrapatient CV of the trough level was calculated as the ratio of the mean divided by one standard deviation. Mean cystatin C eGFR was 56.9 (24.4) mL/min/1.73 m(2) . There was a weak but significant correlation between the MPA trough level and the AUC (Spearman r = 0.6592, p < 0.0001). A total of 1964 MPA trough levels (73 [45]/patient) were measured, as compared to 3462 Tac trough levels (144 [71]/patient). The average MPA trough level was 3.01 (1.26) mg/L and the average trough Tac level was 7.3 (1.8) ng/mL. Intrapatient CV was statistically higher (p = 0.00093) for MPA at 0.68 (0.29) when compared to Tac with a CV of 0.46 (0.12). CV did not correlate with eGFR. Intrapatient MPA trough level CV is significantly higher than for Tac, while CV for both MPA and Tac was high. MPA trough level monitoring may be a feasible monitoring option to improve patient exposure and possibly outcomes. PMID:26201386

  5. What is the intrapatient variability of mycophenolic acid trough levels?

    PubMed

    Todorova, Ekaterina K; Huang, Shih-Han S; Kobrzynski, Marta C; Filler, Guido

    2015-11-01

    TDM of MPA, the active compound of MMF, is rarely used despite its substantial intra- and interpatient variability. Little is known about the utility of long-term MPA TDM. Data are expressed as mean (one standard deviation). All available data from 27 renal transplant recipients (mean age at transplantation: 7.7 [5.0] yr) with an average follow-up of 9.3 (4.6) yr were analyzed. MPA levels were measured using the EMIT. GFR was measured using cystatin C and eGFR was calculated using the Filler formula. Intrapatient CV of the trough level was calculated as the ratio of the mean divided by one standard deviation. Mean cystatin C eGFR was 56.9 (24.4) mL/min/1.73 m(2) . There was a weak but significant correlation between the MPA trough level and the AUC (Spearman r = 0.6592, p < 0.0001). A total of 1964 MPA trough levels (73 [45]/patient) were measured, as compared to 3462 Tac trough levels (144 [71]/patient). The average MPA trough level was 3.01 (1.26) mg/L and the average trough Tac level was 7.3 (1.8) ng/mL. Intrapatient CV was statistically higher (p = 0.00093) for MPA at 0.68 (0.29) when compared to Tac with a CV of 0.46 (0.12). CV did not correlate with eGFR. Intrapatient MPA trough level CV is significantly higher than for Tac, while CV for both MPA and Tac was high. MPA trough level monitoring may be a feasible monitoring option to improve patient exposure and possibly outcomes.

  6. 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

  7. Variable Temperature Infrared Spectroscopy Investigations of Benzoic Acid Desorption from Sodium and Calcium Montmorillonite Clays.

    PubMed

    Nickels, Tara M; Ingram, Audrey L; Maraoulaite, Dalia K; White, Robert L

    2015-12-01

    Processes involved in thermal desorption of benzoic acid from sodium and calcium montmorillonite clays are investigated by using variable temperature diffuse reflection Fourier transform infrared spectroscopy (DRIFTS). By monitoring the temperature dependence of infrared absorbance bands while heating samples, subtle changes in molecular vibrations are detected and employed to characterize specific benzoic acid adsorption sites. Abrupt changes in benzoic acid adsorption site properties occur for both clay samples at about 125 °C. Difference spectra absorbance band frequency variations indicate that adsorbed benzoic acid interacts with interlayer cations through water bridges and that these interactions can be disrupted by the presence of organic anions, in particular, benzoate. PMID:26647147

  8. 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

  9. 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.

  10. Assessment of the nucleotide sequence variability in the bovine T-cell receptor alpha delta joining gene region.

    PubMed

    Fries, R; Ewald, D; Thaller, G; Buitkamp, J

    2001-05-01

    The sequence of 2,193 nucleotides from the bovine T-cell receptor alpha/delta joining gene region (TCRADJ) was determined and compared with the corresponding human and murine sequences. The identity was 75.3% for the comparison of the Bos taurus vs. the Homo sapiens sequence and 63.8% for the Bos taurus vs. the Mus musculus sequence. This comparison permitted the identification of the putatively functional elements within the bovine sequence. Direct sequencing of 2,110 nucleotides in nine animals revealed 12 variable sites. Estimates, based on direct sequencing in three Holstein Friesian animals, for the two measures of sequence variability, nucleotide polymorphism (u) and nucleotide diversity (p), were 0.00050 (60.00036) and 0.00077 (60.00056), respectively. The test statistic, Tajima's D, for the comparison of the two measures indicates that the difference between u and p is close to significance (P < 0.05), suggesting the possibility of selective forces acting on the studied genomic region. Allelic variation at 5 of the 12 variable sites was analysed in 359 animals (48 Anatolian Black, 56 Braunvieh, 115 Fleckvieh, 47 Holstein Friesian, 50 Simmental and 43 Pinzgauer) using the oligonucleotide ligation assay (OLA) in combination with the enzyme linked immunoabsorbant assay (ELISA). Nine unambiguous haplotypes could be derived based on animals with a maximum of one heterozygous site. Four to seven haplotypes were present in the different breeds. When taking into account the frequencies of the haplotypes in the different breeds, especially in Anatolian Black, an ancestral cattle population, we could establish the likely phylogenetic relationships of the haplotypes. Such haplotype trees are the basis for cladistic candidate gene analysis. Our study demonstrates that the systematic search of single nucleotide polymorphisms (SNPs) is useful for analysing all aspects of variability of a given genomic region.

  11. The functional importance of sequence versus expression variability of MHC alleles in parasite resistance.

    PubMed

    Axtner, Jan; Sommer, Simone

    2012-12-01

    Understanding selection processes driving the pronounced allelic polymorphism of the major histocompatibility complex (MHC) genes and its functional associations to parasite load have been the focus of many recent wildlife studies. Two main selection scenarios are currently debated which explain the susceptibility or resistance to parasite infections either by the effects of (1) specific MHC alleles which are selected frequency-dependent in space and time or (2) a heterozygote or divergent allele advantage. So far, most studies have focused only on structural variance in co-evolutionary processes although this might not be the only trait subject to natural selection. In the present study, we analysed structural variance stretching from exon1 through exon3 of MHC class II DRB genes as well as genotypic expression variance in relation to the gastrointestinal helminth prevalence and infection intensity in wild yellow-necked mice (Apodemus flavicollis). We found support for the functional importance of specific alleles both on the sequence and expression level. By resampling a previously investigated study population we identified specific MHC alleles affected by temporal shifts in parasite pressure and recorded associated changes in allele frequencies. The allele Apfl-DRB*23 was associated with resistance to infections by the oxyurid nematode Syphacia stroma and at the same time with susceptibility to cestode infection intensity. In line with our expectation, MHC mRNA transcript levels tended to be higher in cestode-infected animals carrying the allele Apfl-DRB*23. However, no support for a heterozygote or divergent allele advantage on the sequence or expression level was detected. The individual amino acid distance of genotypes did not explain individual differences in parasite loads and the genetic distance had no effect on MHC genotype expression. For ongoing studies on the functional importance of expression variance in parasite resistance, allele

  12. 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.

  13. 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

  14. 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.

  15. Characteristic of HIV-1 in V3 loop region based on seroreactivity and amino acid sequences in Thailand.

    PubMed

    Balachandra, Kruavon; Matsuo, Kazuhiro; Sutthent, Ruengpung; Hoisanka, Narin; Boonsarthorn, Naphasawan; Sawanpanyalert, Pathom; Warachit, Paijit; Yamazaki, Shudo; Honda, Mitsuo

    2002-06-01

    The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.

  16. IgH sequences in common variable immune deficiency reveal altered B cell development and selection**

    PubMed Central

    Roskin, Krishna M.; Simchoni, Noa; Liu, Yi; Lee, Ji-Yeun; Seo, Katie; Hoh, Ramona A.; Pham, Tho; Park, Joon H.; Furman, David; Dekker, Cornelia L.; Davis, Mark M.; James, Judith A.; Nadeau, Kari C.; Cunningham-Rundles, Charlotte; Boyd, Scott D.

    2015-01-01

    Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ∼1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity determining region 3 (CDR3). We observed decreased selection against antibodies with long CDR3 regions in memory repertoires and decreased V gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive both from decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. CVID patients also exhibited abnormal clonal expansion of unmutated B cells relative to controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B cell stage and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients. PMID:26311730

  17. 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.

  18. 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

  19. 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

  20. IgG variable region and VH CDR3 diversity in unimmunized mice analyzed by massively parallel sequencing.

    PubMed

    Lu, Jin; Panavas, Tadas; Thys, Kim; Aerssens, Jeroen; Naso, Michael; Fisher, Jamie; Rycyzyn, Michael; Sweet, Raymond W

    2014-02-01

    Most antigen-specific mouse antibodies have been derived by hybridoma technology, predominantly through use of the Balb/c strain. Much of the Balb/c germline repertoire of variable genes (V regions) is known. However, there is little information about the background expressed repertoire of IgG antibodies in mice, which reflects the baseline against which antigen-specific antibodies are generated through immunization. To assess this baseline repertoire, RNA was isolated from splenic B-cells enriched for expression of IgG from three mice. The RNA was individually amplified with three distinct PCR primer sets for comprehensive recovery of the heavy and light chain variable regions. Each PCR product was independently subjected to deep sequencing using 454 pyro-sequencing technology and analysed for redundancy, open reading frame, germline representation, and CDR3 sequence of the heavy chain variable region (VH CDR3) within and across the primer sets and mice. A highly skewed abundance of heavy and light chain variable gene usage was observed for all three primers in all three mice. While showing considerable overlap, there were differences among these profiles indicative of primer bias and animal-to-animal variation. VH CDR3 sequences were likewise highly skewed indicating that the heavy chain genes profiles substantially reflected individual antibodies. This observation was confirmed through analysis of randomly selected complete heavy chain variable sequences. However, there was very little redundancy in VH CDR3 sequences across the different mice. We conclude that the background IgG repertoire in young, unimmunized mice is highly skewed within individual mice and is diverse among them, a pattern similar to that observed in highly immunized mice.

  1. 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

  2. Increased sensitivity and variability of phytotoxicity responses in Arctic soils to a reference toxicant, boric acid.

    PubMed

    Anaka, Alison; Wickstrom, Mark; Siciliano, Steven Douglas

    2008-03-01

    Industrial and human activities in the Arctic regions may pose a risk to terrestrial Arctic ecosystem functions. One of the most common terrestrial toxicological end points, primary productivity, typically is assessed using a plant phytotoxicity test. Because of cryoturbation, a soil mixing process common in polar regions, we hypothesized that phytotoxicity test results in Arctic soils would be highly variable compared to other terrestrial ecosystems. The variability associated with phytotoxicity tests was evaluated using Environment Canada's standardized plant toxicity test in three cryoturbated soils from Canada's Arctic exposed to a reference toxicant, boric acid. Northern wheatgrass (Elymus lanceolatus) not only was more sensitive to toxicants in Arctic soils, its response to toxicants was more variable compared to that in temperate soils. The phytotoxicity of boric acid in cryosols was much greater than commonly reported in other soils, with a boric acid concentration of less than 150 microg/g soil needed to inhibit root and shoot growth by 20%. Large variability also was found in the phytotoxicity test results, with coefficients of variation for 10 samples ranging from 160 to 79%. The increased toxicity of boric acid in cryosols and variability in test response was not explained by soil properties. Based on our admittedly limited data set of three different Arctic soils, we recommend that more than 30 samples be taken from each control and potentially impacted area to accurately assess contaminant effects at sites in northern Canada. Such intensive sampling will insure that false-negative results for toxicant impacts in Arctic soils are minimized.

  3. 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.

  4. 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.

  5. Solid phase sequencing of biopolymers

    DOEpatents

    Cantor, Charles; Koster, Hubert

    2010-09-28

    This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified 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 probes 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 DNA or RNA 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 molecular weight analysis and identification of the target sequence.

  6. 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

  7. 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.

  8. 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

  9. Application of whole genome and RNA sequencing to investigate the genomic landscape of common variable immunodeficiency disorders.

    PubMed

    van Schouwenburg, Pauline A; Davenport, Emma E; Kienzler, Anne-Kathrin; Marwah, Ishita; Wright, Benjamin; Lucas, Mary; Malinauskas, Tomas; Martin, Hilary C; Lockstone, Helen E; Cazier, Jean-Baptiste; Chapel, Helen M; Knight, Julian C; Patel, Smita Y

    2015-10-01

    Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways. PMID:26122175

  10. 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.

  11. 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.

  12. 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.

  13. 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)

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Extremely acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening sequences in their 16S rRNA genes.

    PubMed

    Baker, Brett J; Hugenholtz, Philip; Dawson, Scott C; Banfield, Jillian F

    2003-09-01

    During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.

  9. Investigation of the gibberellic acid optimization with a statistical tool from Penicillium variable in batch reactor.

    PubMed

    Isa, Nur Kamilah Md; Mat Don, Mashitah

    2014-01-01

    The culture conditions for gibberellic acid (GA3) production by the fungus Penicillium variable (P. variable) was optimized using a statistical tool, response surface methodology (RSM). Interactions of culture conditions and optimization of the system were studied using Box-Behnken design (BBD) with three levels of three variables in a batch flask reactor. Experimentation showed that the model developed based on RSM and BBD had predicted GA3 production with R(2) = 0.987. The predicted GA3 production was optimum (31.57 mg GA3/kg substrate) when the culture conditions were at 7 days of incubation period, 21% v/w of inoculum size, and 2% v/w of olive oil concentration as a natural precursor. The results indicated that RSM and BBD methods were effective for optimizing the culture conditions of GA3 production by P. variable mycelia. PMID:24499362

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. [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.

  1. In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop.

    PubMed Central

    Donaldson, Y K; Bell, J E; Holmes, E C; Hughes, E S; Brown, H K; Simmonds, P

    1994-01-01

    The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences

  2. 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

  3. 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.

  4. 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

  5. [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

  6. 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.

  7. 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

  8. Diurnal variations in leaf fluorescence induction kinetics: variable fluorescence in crassulacean Acid metabolism plants.

    PubMed

    Everson, G; Chen, S S; Black, C C

    1983-06-01

    The variable fluorescence of leaves from Kalanchoë daigremontiana and pineapple, Ananas comosus, both CAM plants, was found to change over a 24-hour cycle and to exhibit high temperature-dependent maxima during the night period. The time course of the induced fluorescence was correlated with malic acid accumulation but not with other aspects of CAM such as with the nature of the decarboxylation pathway or with stomatal movements. The variable fluorescences of sunflower (Helianthus annuus L.) and corn (Zea mays L.) leaves were compared with the CAM plants diurnally; both plants also exhibit high fluorescence maxima during the night period. We conclude that the assembly of the photosystems in the light is a primary process in photosynthesis induction and may be influenced by other cellular metabolic processes, specifically in the case of CAM leaves by malic acid accumulation. PMID:16663024

  9. 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

  10. 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

  11. Amino acid sequence similarity of the VP7 protein of human rotavirus HCR3 to that of canine and feline rotaviruses.

    PubMed

    Li, B; Clark, H F; Gouvea, V

    1994-01-01

    The sequence of the VP7 gene of human rotavirus strain HCR3 was determined and its predicted amino acid sequence was compared with that of other rotavirus strains. The VP7 gene is 1062 nucleotides long and contains a single open reading frame of 981 nucleotides capable of encoding a protein of 326 amino acids. The VP7 amino acid sequence similarity of strain HCR3 to those of various human and animal G3 serotypes ranged from 88.7 to 99.4%, and from 60.4 to 88.3% to strains representing each of the other 13 G serotypes. Alignment of four variable regions [VR4, VR5(A), VR7(B) and VR8(C)] of HCR3 with those of G3 strains of different host species showed that HCR3 possesses a sequence almost identical to that of canine rotaviruses and feline rotavirus strain CAT97 in all four regions. A considerable divergence in regions VR4, VR7(B) and VR8(C) was found with strains of human, mouse, monkey, horse and rabbit rotaviruses. This observation together with results of our previous study on VP4 indicated that human rotavirus HCR3 is genetically more closely related to animal rotaviruses than to other human rotaviruses. PMID:8113730

  12. Solid phase sequencing of biopolymers

    SciTech Connect

    Cantor, Charles R.; Hubert, Koster

    2014-06-24

    This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified 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 probes 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. Probes may be affixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

  13. 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

  14. 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.

  15. First-Principles Investigation of Nanopore Sequencing Using Variable Voltage Bias on Graphene-Based Nanoribbons.

    PubMed

    McFarland, Hannah L; Ahmed, Towfiq; Zhu, Jian-Xin; Balatsky, Alexander V; Haraldsen, Jason T

    2015-07-01

    In this study, we examine the mechanism of nanopore-based DNA sequencing using a voltage bias across a graphene nanoribbon. Using density function theory and a nonequilibrium Green's function approach, we determine the transmission spectra and current profile for adenine, guanine, cytosine, thymine, and uracil as a function of bias voltage in an energy minimized configuration. Utilizing the transmission current, we provide a general methodology for the development of a three nanopore graphene-based device that can be used to distinguish between the various nucleobases for DNA/RNA sequencing. From our analysis, we deduce that it is possible to use different transverse currents across a multinanopore device to differentiate between nucleobases using various voltages of 0.5, 1.3, and 1.6 V. Overall, our goal is to improve nanopore design to further DNA/RNA nucleobase sequencing and biomolecule identification techniques.

  16. First-Principles Investigation of Nanopore Sequencing Using Variable Voltage Bias on Graphene-Based Nanoribbons.

    PubMed

    McFarland, Hannah L; Ahmed, Towfiq; Zhu, Jian-Xin; Balatsky, Alexander V; Haraldsen, Jason T

    2015-07-01

    In this study, we examine the mechanism of nanopore-based DNA sequencing using a voltage bias across a graphene nanoribbon. Using density function theory and a nonequilibrium Green's function approach, we determine the transmission spectra and current profile for adenine, guanine, cytosine, thymine, and uracil as a function of bias voltage in an energy minimized configuration. Utilizing the transmission current, we provide a general methodology for the development of a three nanopore graphene-based device that can be used to distinguish between the various nucleobases for DNA/RNA sequencing. From our analysis, we deduce that it is possible to use different transverse currents across a multinanopore device to differentiate between nucleobases using various voltages of 0.5, 1.3, and 1.6 V. Overall, our goal is to improve nanopore design to further DNA/RNA nucleobase sequencing and biomolecule identification techniques. PMID:26266743

  17. [Identification of new conserved and variable regions in the 16S rRNA gene of acetic acid bacteria and acetobacteraceae family].

    PubMed

    Chakravorty, S; Sarkar, S; Gachhui, R

    2015-01-01

    The Acetobacteraceae family of the class Alpha Proteobacteria is comprised of high sugar and acid tolerant bacteria. The Acetic Acid Bacteria are the economically most significant group of this family because of its association with food products like vinegar, wine etc. Acetobacteraceae are often hard to culture in laboratory conditions and they also maintain very low abundances in their natural habitats. Thus identification of the organisms in such environments is greatly dependent on modern tools of molecular biology which require a thorough knowledge of specific conserved gene sequences that may act as primers and or probes. Moreover unconserved domains in genes also become markers for differentiating closely related genera. In bacteria, the 16S rRNA gene is an ideal candidate for such conserved and variable domains. In order to study the conserved and variable domains of the 16S rRNA gene of Acetic Acid Bacteria and the Acetobacteraceae family, sequences from publicly available databases were aligned and compared. Near complete sequences of the gene were also obtained from Kombucha tea biofilm, a known Acetobacteraceae family habitat, in order to corroborate the domains obtained from the alignment studies. The study indicated that the degree of conservation in the gene is significantly higher among the Acetic Acid Bacteria than the whole Acetobacteraceae family. Moreover it was also observed that the previously described hypervariable regions V1, V3, V5, V6 and V7 were more or less conserved in the family and the spans of the variable regions are quite distinct as well.

  18. 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)

  19. Genetic variability among Schistosoma japonicum isolates from the Philippines, Japan and China revealed by sequence analysis of three mitochondrial genes.

    PubMed

    Chen, Fen; Li, Juan; Sugiyama, Hiromu; Zhou, Dong-Hui; Song, Hui-Qun; Zhao, Guang-Hui; Zhu, Xing-Quan

    2015-02-01

    The present study examined sequence variability in the mitochondrial (mt) protein-coding genes cytochrome b (cytb), NADH dehydrogenase subunits 2 and 6 (nad2 and nad6) among 24 isolates of Schistosoma japonicum from different endemic regions in the Philippines, Japan and China. The complete cytb, nad2 and nad6 genes were amplified and sequenced separately from individual schistosome. Sequence variations for isolates from the Philippines were 0-0.5% for cytb, 0-0.6% for nad2, and 0-0.9% for nad6. Variation was 0-0.5%, 0.1-0.8%, 0-0.7% for corresponding genes for schistosome samples from mainland China. For worms in Japan, genetic variations were 0-0.2%, 0.1-0.2% and 0 for the three genes, respectively. Sequence variations were 0-1.0%, 0-1.8% and 0-1.1% for cytb, nad2 and nad6, respectively, among schistosome isolates from different geographical strains in the Philippines, Japan and China. Of the three countries, lowest sequence variations were found between isolates from mainland China and the Philippines and highest were detected between Japan and the Philippines in three mtDNA genes. Phylogenetic analyses based on the combined sequences of cytb, nad2 and nad6 revealed that all isolates in the Philippines clustered together sistered to samples from Yunnan and Zhejiang provinces in China, while isolates from Yamanashi in Japan were in a solitary clade. These results demonstrated the usefulness of the combined three mtDNA sequences for studying genetic diversity and population structure among S. japonicum isolates from the Philippines, China and Japan.

  20. 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.

  1. Temporal variability in urinary levels of drinking water disinfection byproducts dichloroacetic acid and trichloroacetic acid among men

    SciTech Connect

    Wang, Yi-Xin; Zeng, Qiang; Wang, Le; Huang, Yue-Hui; Lu, Zhi-Wei; Wang, Peng; He, Meng-Jie; Huang, Xin; Lu, Wen-Qing

    2014-11-15

    Urinary haloacetic acids (HAAs), such as dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA), have been suggested as potential biomarkers of exposure to drinking water disinfection byproducts (DBPs). However, variable exposure to and the short elimination half-lives of these biomarkers can result in considerable variability in urinary measurements, leading to exposure misclassification. Here we examined the variability of DCAA and TCAA levels in the urine among eleven men who provided urine samples on 8 days over 3 months. The urinary concentrations of DCAA and TCAA were measured by gas chromatography coupled with electron capture detection. We calculated the intraclass correlation coefficients (ICCs) to characterize the within-person and between-person variances and computed the sensitivity and specificity to assess how well single or multiple urine collections accurately determined personal 3-month average DCAA and TCAA levels. The within-person variance was much higher than the between-person variance for all three sample types (spot, first morning, and 24-h urine samples) for DCAA (ICC=0.08–0.37) and TCAA (ICC=0.09–0.23), regardless of the sampling interval. A single-spot urinary sample predicted high (top 33%) 3-month average DCAA and TCAA levels with high specificity (0.79 and 0.78, respectively) but relatively low sensitivity (0.47 and 0.50, respectively). Collecting two or three urine samples from each participant improved the classification. The poor reproducibility of the measured urinary DCAA and TCAA concentrations indicate that a single measurement may not accurately reflect individual long-term exposure. Collection of multiple urine samples from one person is an option for reducing exposure classification errors in studies exploring the effects of DBP exposure on reproductive health. - Highlights: • We evaluated the variability of DCAA and TCAA levels in the urine among men. • Urinary DCAA and TCAA levels varied greatly over a 3-month

  2. 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.

  3. 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

  4. 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

  5. Draft Genome Sequences of 11 Salmonella enterica Strains with Variable Levels of Barotolerance.

    PubMed

    Ronholm, Jennifer; Petronella, Nicholas; Tamber, Sandeep

    2016-01-01

    The diversity of the genus Salmonella is reflected in the physiological adaptations used by its members in response to stressors such as high pressure. Here we report the draft whole genome sequences of 11 Salmonella enterica strains, five sensitive strains and six demonstrating high levels of pressure resistance. PMID:27660773

  6. Draft Genome Sequences of 11 Salmonella enterica Strains with Variable Levels of Barotolerance

    PubMed Central

    Ronholm, Jennifer; Petronella, Nicholas

    2016-01-01

    The diversity of the genus Salmonella is reflected in the physiological adaptations used by its members in response to stressors such as high pressure. Here we report the draft whole genome sequences of 11 Salmonella enterica strains, five sensitive strains and six demonstrating high levels of pressure resistance. PMID:27660773

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. PRE-MAIN SEQUENCE VARIABLES IN THE VMR-D: IDENTIFICATION OF T TAURI-LIKE ACCRETING PROTOSTARS THROUGH SPITZER-IRAC VARIABILITY

    SciTech Connect

    Giannini, T.; Lorenzetti, D.; De Luca, M.; Nisini, B.; Elia, D.; Strafella, F.; Fazio, G.; Marengo, M.; Smith, H. A. E-mail: dloren@oa-roma.inaf.i E-mail: deluca@oa-roma.inaf.i E-mail: eliad@le.infn.i E-mail: massimo.de.luca@lra.ens.f E-mail: hsmith@cfa.harvard.ed

    2009-10-10

    We present a study of the infrared variability of young stellar objects by means of two Spitzer-IRAC images of the Vela Molecular Cloud D (VMR-D) obtained in observations separated in time by about six months. By using the same space-born IR instrumentation, this study eliminates all the unwanted effects due to differences in sensitivity, confusion, saturation, calibration, and filter bandpasses, issues that are usually unavoidable when comparing catalogs obtained from different instruments. The VMR-D map covers about 1.5 deg{sup 2} of a site where star formation is actively ongoing. We are interested in accreting pre-main sequence variables whose luminosity variations are due to intermittent events of disk accretion (i.e., active T Tauri stars and EXor-type objects). The variable objects have been selected from a catalog of more than 170,000 sources detected at an S/N >= 5. We then searched the sample of variables for ones whose photometric properties such as IR excess, color-magnitude relationships, and spectral energy distribution, are as close as possible to those of known EXor's. Indeed, the latter are monitored in a more systematic way than T Tauri stars and the mechanisms that regulate the observed phenomenology are exactly the same. Hence, the modalities of the EXor behavior are adopted as driving criterion for selecting variables in general. We ultimately selected 19 bona fide candidates that constitute a well defined sample of new variable targets for further investigation (monitoring, spectroscopy). Out of these, 10 sources present a Spitzer MIPS 24 mum counterpart, and have been classified as three Class I, five flat spectrum, and two Class II objects, while the spectral energy distribution of the other nine sources is compatible with evolutionary phases older than Class I. This is consistent with what is known about the small sample of known EXor's, whose properties have driven the present selection and suggests that the accretion flaring or EXor stage

  15. Variable clinical manifestations of a glycine to glutamic acid substitution of the COL3A1 gene at residue 736

    SciTech Connect

    Pope, F.M.; Narcisi, P.; Richards, A.J.

    1994-09-01

    Glycine substitutions at the 3{prime} end of the COL3A1 gene generally produce a characteristic clinical phenotype including acrogeria and severe vascular fragility. Here we report a three generation British family in which the propositus presented with aneurysms of the groins. He, his mother, sister and elder daughter all had the external clinical phenotype of vascular EDS IV whilst another daughter and nephew were clinically normal. Cultured skin fibroblasts from the propositus and his clinically affected relatives poorly secreted normal and overmodified collagen III species. Normal components of secreted proteins predominated whilst overmodified molecules were prominent in intracellular material. Surprisingly the normal children also secreted less collagen type III than expected (though more than their clinically abnormal relatives). cDNA from bases 2671 to 3714 were amplified as four overlapping PCR fragments and analysed by DGGE. The region between 2671 and 3015 was heterozygous. Sequencing showed a mutation of glycine to glutamic acid at residue 736. This mutation created an extra Apa 1 restriction site which was suitable for family studies. These showed inheritance of the mutant gene by both vascular and non-vascular clinical phenotypes. This family therefore illustrates that replacement of glycine to glutamic acid at position 736 produces variable clinical and biochemical phenotypes ranging from easily recognizable vascular EDS IV with very poor collagen secretion to an EDS III-like picture and with less severe protein disturbance. The reasons for these differences are at present unexplained.

  16. Mutation-selection models of coding sequence evolution with site-heterogeneous amino acid fitness profiles.

    PubMed

    Rodrigue, Nicolas; Philippe, Hervé; Lartillot, Nicolas

    2010-03-01

    Modeling the interplay between mutation and selection at the molecular level is key to evolutionary studies. To this end, codon-based evolutionary models have been proposed as pertinent means of studying long-range evolutionary patterns and are widely used. However, these approaches have not yet consolidated results from amino acid level phylogenetic studies showing that selection acting on proteins displays strong site-specific effects, which translate into heterogeneous amino acid propensities across the columns of alignments; related codon-level studies have instead focused on either modeling a single selective context for all codon columns, or a separate selective context for each codon column, with the former strategy deemed too simplistic and the latter deemed overparameterized. Here, we integrate recent developments in nonparametric statistical approaches to propose a probabilistic model that accounts for the heterogeneity of amino acid fitness profiles across the coding positions of a gene. We apply the model to a dozen real protein-coding gene alignments and find it to produce biologically plausible inferences, for instance, as pertaining to site-specific amino acid constraints, as well as distributions of scaled selection coefficients. In their account of mutational features as well as the heterogeneous regimes of selection at the amino acid level, the modeling approaches studied here can form a backdrop for several extensions, accounting for other selective features, for variable population size, or for subtleties of mutational features, all with parameterizations couched within population-genetic theory. PMID:20176949

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Resistance to Change and Preference for Variable versus Fixed Response Sequences

    ERIC Educational Resources Information Center

    Arantes, Joana; Berg, Mark E.; Le, Dien; Grace, Randolph C.

    2012-01-01

    In Experiment 1, 4 pigeons were trained on a multiple chain schedule in which the initial link was a variable-interval (VI) 20-s schedule signalled by a red or green center key, and terminal links required four responses made to the left (L) and/or right (R) keys. In the REPEAT component, signalled by red keylights, only LRLR terminal-link…

  7. 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

  8. 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.

  9. 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.

  10. Molecular Dynamics Simulations of Silica-Filled Copolymers with Variable Sequence for Applications in Tire Treads

    NASA Astrophysics Data System (ADS)

    Trazkovich, Alex J.; Hall, Lisa M.

    We simulate a simple nanocomposite relevant to tire tread compounds consisting of a single spherical nanoparticle surrounded by coarse-grained polymer chains. The polymers are composed of two different monomer types, which have different interaction strengths with the nanoparticle. The monomer sequence can be varied to model different copolymer configurations. We study the polymer end-to-end vector autocorrelation functions to obtain relaxation times of adsorbed and bulk polymer, showing how the interphase is affected by the polymer type and the monomer-nanoparticle interaction strengths. An understanding of the effect of copolymer sequence on the range of the polymer interphase and the magnitude of the effect on chain dynamics is critical to tire tread material design since the primary polymer component of modern tire tread is styrene-butadiene rubber (SBR) copolymer, which may be synthesized in primarily random or in various blocky copolymer configurations. Macromolecular adsorption to and desorption from filler surfaces has a significant effect on hysteresis, and in tire treads, hysteresis must be controlled to optimize the tradeoff between traction and rolling resistance. Superior tire tread materials must have high hysteresis under the operating conditions of traction while maintaining low hysteresis under the operating conditions of rolling resistance. An opportunity exists to control hysteresis through the use of SBR with specific monomer sequences.

  11. 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.

  12. Three Ingredients for Improved Global Aftershock Forecasts: Tectonic Region, Time-Dependent Catalog Incompleteness, and Inter-Sequence Variability

    NASA Astrophysics Data System (ADS)

    Page, M. T.; Hardebeck, J.; Felzer, K. R.; Michael, A. J.; van der Elst, N.

    2015-12-01

    Following a large earthquake, seismic hazard can be orders of magnitude higher than the long-term average as a result of aftershock triggering. Due to this heightened hazard, there is a demand from emergency managers and the public for rapid, authoritative, and reliable aftershock forecasts. In the past, USGS aftershock forecasts following large, global earthquakes have been released on an ad-hoc basis with inconsistent methods, and in some cases, aftershock parameters adapted from California. To remedy this, we are currently developing an automated aftershock product that will generate more accurate forecasts based on the Reasenberg and Jones (Science, 1989) method. To better capture spatial variations in aftershock productivity and decay, we estimate regional aftershock parameters for sequences within the Garcia et al. (BSSA, 2012) tectonic regions. We find that regional variations for mean aftershock productivity exceed a factor of 10. The Reasenberg and Jones method combines modified-Omori aftershock decay, Utsu productivity scaling, and the Gutenberg-Richter magnitude distribution. We additionally account for a time-dependent magnitude of completeness following large events in the catalog. We generalize the Helmstetter et al. (2005) equation for short-term aftershock incompleteness and solve for incompleteness levels in the global NEIC catalog following large mainshocks. In addition to estimating average sequence parameters within regions, we quantify the inter-sequence parameter variability. This allows for a more complete quantification of the forecast uncertainties and Bayesian updating of the forecast as sequence-specific information becomes available.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Statistical Variability and Tokunaga Branching of Aftershock Sequences Utilizing BASS Model Simulations

    NASA Astrophysics Data System (ADS)

    Yoder, Mark R.; Van Aalsburg, Jordan; Turcotte, Donald L.; Abaimov, Sergey G.; Rundle, John B.

    2013-01-01

    Aftershock statistics provide a wealth of data that can be used to better understand earthquake physics. Aftershocks satisfy scale-invariant Gutenberg-Richter (GR) frequency-magnitude statistics. They also satisfy Omori's law for power-law seismicity rate decay and Båth's law for maximum-magnitude scaling. The branching aftershock sequence (BASS) model, which is the scale-invariant limit of the epidemic-type aftershock sequence model (ETAS), uses these scaling laws to generate synthetic aftershock sequences. One objective of this paper is to show that the branching process in these models satisfies Tokunaga branching statistics. Tokunaga branching statistics were originally developed for drainage networks and have been subsequently shown to be valid in many other applications associated with complex phenomena. Specifically, these are characteristic of a universality class in statistical physics associated with diffusion-limited aggregation. We first present a deterministic version of the BASS model and show that it satisfies the Tokunaga side-branching statistics. We then show that a fully stochastic BASS simulation gives similar results. We also study foreshock statistics using our BASS simulations. We show that the frequency-magnitude statistics in BASS simulations scale as the exponential of the magnitude difference between the mainshock and the foreshock, inverse GR scaling. We also show that the rate of foreshock occurrence in BASS simulations decays inversely with the time difference between foreshock and mainshock, an inverse Omori scaling. Both inverse scaling laws have been previously introduced empirically to explain observed foreshock statistics. Observations have demonstrated both of these scaling relations to be valid, consistent with our simulations. ETAS simulations, in general, do not generate Båth's law and do not generate inverse GR scaling.

  2. A massively parallel sequencing approach uncovers ancient origins and high genetic variability of endangered Przewalski's horses.

    PubMed

    Goto, Hiroki; Ryder, Oliver A; Fisher, Allison R; Schultz, Bryant; Kosakovsky Pond, Sergei L; Nekrutenko, Anton; Makova, Kateryna D

    2011-01-01

    The endangered Przewalski's horse is the closest relative of the domestic horse and is the only true wild horse species surviving today. The question of whether Przewalski's horse is the direct progenitor of domestic horse has been hotly debated. Studies of DNA diversity within Przewalski's horses have been sparse but are urgently needed to ensure their successful reintroduction to the wild. In an attempt to resolve the controversy surrounding the phylogenetic position and genetic diversity of Przewalski's horses, we used massively parallel sequencing technology to decipher the complete mitochondrial and partial nuclear genomes for all four surviving maternal lineages of Przewalski's horses. Unlike single-nucleotide polymorphism (SNP) typing usually affected by ascertainment bias, the present method is expected to be largely unbiased. Three mitochondrial haplotypes were discovered-two similar ones, haplotypes I/II, and one substantially divergent from the other two, haplotype III. Haplotypes I/II versus III did not cluster together on a phylogenetic tree, rejecting the monophyly of Przewalski's horse maternal lineages, and were estimated to split 0.117-0.186 Ma, significantly preceding horse domestication. In the phylogeny based on autosomal sequences, Przewalski's horses formed a monophyletic clade, separate from the Thoroughbred domestic horse lineage. Our results suggest that Przewalski's horses have ancient origins and are not the direct progenitors of domestic horses. The analysis of the vast amount of sequence data presented here suggests that Przewalski's and domestic horse lineages diverged at least 0.117 Ma but since then have retained ancestral genetic polymorphism and/or experienced gene flow.

  3. The use of variable temperature and magic-angle sample spinning in studies of fulvic acids

    USGS Publications Warehouse

    Earl, W.L.; Wershaw, R. L.; Thorn, K.A.

    1987-01-01

    Intensity distortions and poor signal to noise in the cross-polarization magic-angle sample spinning NMR of fulvic acids were investigated and attributed to molecular mobility in these ostensibly "solid" materials. We have shown that inefficiencies in cross polarization can be overcome by lowering the sample temperature to about -60??C. These difficulties can be generalized to many other synthetic and natural products. The use of variable temperature and cross-polarization intensity as a function of contact time can yield valuable qualitative information which can aid in the characterization of many materials. ?? 1987.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Hydrogen-isotopic variability in fatty acids from Yellowstone National Park hot spring microbial communities

    NASA Astrophysics Data System (ADS)

    Osburn, Magdalena R.; Sessions, Alex L.; Pepe-Ranney, Charles; Spear, John R.

    2011-09-01

    We report the abundances and hydrogen-isotopic compositions (D/H ratios) of fatty acids extracted from hot-spring microbial mats in Yellowstone National Park. The terrestrial hydrothermal environment provides a useful system for studying D/H fractionations because the numerous microbial communities in and around the springs are visually distinct, separable, and less complex than those in many other aquatic environments. D/H fractionations between lipids and water ranged from -374‰ to +41‰ and showed systematic variations between different types of microbial communities. Lipids produced by chemoautotrophic hyperthermophilic bacteria, such as icosenoic acid (20:1), generally exhibited the largest and most variable fractionations from water (-374‰ to -165‰). This was in contrast to lipids characteristic of heterotrophs, such as branched, odd chain-length fatty acids, which had the smallest fractionations (-163‰ to +41‰). Mats dominated by photoautotrophs exhibited intermediate fractionations similar in magnitude to those expressed by higher plants. These data support the hypothesis that variations in lipid D/H are strongly influenced by central metabolic pathways. Shifts in the isotopic compositions of individual fatty acids across known ecological boundaries show that the isotopic signature of specific metabolisms can be recognized in modern environmental samples, and potentially recorded in ancient ones. Considering all sampled springs, the total range in D/H ratios is similar to that observed in marine sediments, suggesting that the trends observed here are not exclusive to the hydrothermal environment.

  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. Beyond Junk-Variable Tandem Repeats as Facilitators of Rapid Evolution of Regulatory and Coding Sequences

    PubMed Central

    Gemayel, Rita; Cho, Janice; Boeynaems, Steven; Verstrepen, Kevin J.

    2012-01-01

    Copy Number Variations (CNVs) and Single Nucleotide Polymorphisms (SNPs) have been the major focus of most large-scale comparative genomics studies to date. Here, we discuss a third, largely ignored, type of genetic variation, namely changes in tandem repeat number. Historically, tandem repeats have been designated as non functional “junk” DNA, mostly as a result of their highly unstable nature. With the exception of tandem repeats involved in human neurodegenerative diseases, repeat variation was often believed to be neutral with no phenotypic consequences. Recent studies, however, have shown that as many as 10% to 20% of coding and regulatory sequences in eukaryotes contain an unstable repeat tract. Contrary to initial suggestions, tandem repeat variation can have useful phenotypic consequences. Examples include rapid variation in microbial cell surface, tuning of internal molecular clocks in flies and the dynamic morphological plasticity in mammals. As such, tandem repeats can be useful functional elements that facilitate evolvability and rapid adaptation. PMID:24704980

  13. Kernels for longitudinal data with variable sequence length and sampling intervals.

    PubMed

    Lu, Zhengdong; Leen, Todd K; Kaye, Jeffrey

    2011-09-01

    We develop several kernel methods for classification of longitudinal data and apply them to detect cognitive decline in the elderly. We first develop mixed-effects models, a type of hierarchical empirical Bayes generative models, for the time series. After demonstrating their utility in likelihood ratio classifiers (and the improvement over standard regression models for such classifiers), we develop novel Fisher kernels based on mixture of mixed-effects models and use them in support vector machine classifiers. The hierarchical generative model allows us to handle variations in sequence length and sampling interval gracefully. We also give nonparametric kernels not based on generative models, but rather on the reproducing kernel Hilbert space. We apply the methods to detecting cognitive decline from longitudinal clinical data on motor and neuropsychological tests. The likelihood ratio classifiers based on the neuropsychological tests perform better than than classifiers based on the motor behavior. Discriminant classifiers performed better than likelihood ratio classifiers for the motor behavior tests.

  14. Automatic Recognition of Element Classes and Boundaries in the Birdsong with Variable Sequences

    PubMed Central

    Okanoya, Kazuo

    2016-01-01

    Researches on sequential vocalization often require analysis of vocalizations in long continuous sounds. In such studies as developmental ones or studies across generations in which days or months of vocalizations must be analyzed, methods for automatic recognition would be strongly desired. Although methods for automatic speech recognition for application purposes have been intensively studied, blindly applying them for biological purposes may not be an optimal solution. This is because, unlike human speech recognition, analysis of sequential vocalizations often requires accurate extraction of timing information. In the present study we propose automated systems suitable for recognizing birdsong, one of the most intensively investigated sequential vocalizations, focusing on the three properties of the birdsong. First, a song is a sequence of vocal elements, called notes, which can be grouped into categories. Second, temporal structure of birdsong is precisely controlled, meaning that temporal information is important in song analysis. Finally, notes are produced according to certain probabilistic rules, which may facilitate the accurate song recognition. We divided the procedure of song recognition into three sub-steps: local classification, boundary detection, and global sequencing, each of which corresponds to each of the three properties of birdsong. We compared the performances of several different ways to arrange these three steps. As results, we demonstrated a hybrid model of a deep convolutional neural network and a hidden Markov model was effective. We propose suitable arrangements of methods according to whether accurate boundary detection is needed. Also we designed the new measure to jointly evaluate the accuracy of note classification and boundary detection. Our methods should be applicable, with small modification and tuning, to the songs in other species that hold the three properties of the sequential vocalization. PMID:27442240

  15. Automatic Recognition of Element Classes and Boundaries in the Birdsong with Variable Sequences.

    PubMed

    Koumura, Takuya; Okanoya, Kazuo

    2016-01-01

    Researches on sequential vocalization often require analysis of vocalizations in long continuous sounds. In such studies as developmental ones or studies across generations in which days or months of vocalizations must be analyzed, methods for automatic recognition would be strongly desired. Although methods for automatic speech recognition for application purposes have been intensively studied, blindly applying them for biological purposes may not be an optimal solution. This is because, unlike human speech recognition, analysis of sequential vocalizations often requires accurate extraction of timing information. In the present study we propose automated systems suitable for recognizing birdsong, one of the most intensively investigated sequential vocalizations, focusing on the three properties of the birdsong. First, a song is a sequence of vocal elements, called notes, which can be grouped into categories. Second, temporal structure of birdsong is precisely controlled, meaning that temporal information is important in song analysis. Finally, notes are produced according to certain probabilistic rules, which may facilitate the accurate song recognition. We divided the procedure of song recognition into three sub-steps: local classification, boundary detection, and global sequencing, each of which corresponds to each of the three properties of birdsong. We compared the performances of several different ways to arrange these three steps. As results, we demonstrated a hybrid model of a deep convolutional neural network and a hidden Markov model was effective. We propose suitable arrangements of methods according to whether accurate boundary detection is needed. Also we designed the new measure to jointly evaluate the accuracy of note classification and boundary detection. Our methods should be applicable, with small modification and tuning, to the songs in other species that hold the three properties of the sequential vocalization. PMID:27442240

  16. Massively parallel rRNA gene sequencing exacerbates the potential for biased community diversity comparisons due to variable library sizes

    SciTech Connect

    Gihring, Thomas; Green, Stefan; Schadt, Christopher Warren

    2011-01-01

    Technologies for massively parallel sequencing are revolutionizing microbial ecology and are vastly increasing the scale of ribosomal RNA (rRNA) gene studies. Although pyrosequencing has increased the breadth and depth of possible rRNA gene sampling, one drawback is that the number of reads obtained per sample is difficult to control. Pyrosequencing libraries typically vary widely in the number of sequences per sample, even within individual studies, and there is a need to revisit the behaviour of richness estimators and diversity indices with variable gene sequence library sizes. Multiple reports and review papers have demonstrated the bias in non-parametric richness estimators (e.g. Chao1 and ACE) and diversity indices when using clone libraries. However, we found that biased community comparisons are accumulating in the literature. Here we demonstrate the effects of sample size on Chao1, ACE, CatchAll, Shannon, Chao-Shen and Simpson's estimations specifically using pyrosequencing libraries. The need to equalize the number of reads being compared across libraries is reiterated, and investigators are directed towards available tools for making unbiased diversity comparisons.

  17. Evolution of the recombination signal sequences in the Ig heavy-chain variable region locus of mammals

    PubMed Central

    Hassanin, Alexandre; Golub, Rachel; Lewis, Susanna M.; Wu, Gillian E.

    2000-01-01

    The Ig and T cell receptor (TCR) loci have an exceptionally dynamic evolutionary history, but the mechanisms responsible remain a subject of speculation. Ig and TCR genes are unique in vertebrates in that they are assembled from V, D, and J segments by site-specific recombination in developing lymphocytes. Here we examine the extent to which the V(D)J recombination in germline cells may have been responsible for remodeling Ig and TCR loci in mammals by asking whether gene segments have evolved as a unit, or whether, instead, recombination signal sequences (RSSs) and coding sequences have different phylogenies. Four distinct types of RSS have been defined in the human Ig heavy-chain variable region (Vh) locus, namely H1, H2, H3, and H5, and no other RSS type has been detected in other mammalian species. There is a well-supported discrepancy between the evolutionary history of the RSSs as compared with the Vh coding sequences: the RSS type H2 of one Vh gene segment has clearly become replaced by a RSS type H3 during mammalian evolution, between 115 and 65 million years ago. Two general models might explain the RSS swap: the first involves an unequal crossing over, and the second implicates germline activation of V(D)J recombination. The Vh-H2/RSS-H3 recombination product has likely been selected during the evolution of mammals because it provides better V(D)J recombination efficiency. PMID:11027341

  18. Spatial and Temporal Variability of Macronutrients in a Lime-amended Acid Paddy Field

    NASA Astrophysics Data System (ADS)

    Vidal Vázquez, E.; Morales, L. A.; Paz González, A.

    2012-04-01

    Soil spatial variability is a natural occurring and or management induced feature that is important for site-specific management practices such as variable rate fertilization. Since rice paddy fields are flat and flooded, apparently they should be homogeneous and subsequently it could be thought that spatial variability in yields and soil attributes might be negligible. However, significant levels of variability in soil general properties, soil nutrients and rice yields have been observed even in small paddy fields. Describing spatial variability of within-field properties is a fundamental first step toward determining management strategies. The aim of this study was to analyze patterns of spatial variability in available macronutrients (NH4+-N, P and K) from an acid rice soil submitted to lime amendment. The experimental site was located at Corrientes province, Argentina. The climate is warm, subtropical with abundant rainfall the whole year round. The study soil was typic Plintacualf. Field trials were set up involving three treatments: control, without lime addition, plus two different dolomite doses of 625 and 1250 kg.ha-1. Before lime addition, soil pH was 3.7; organic matter content was 2.14 % and cation exchange capacity (CEC) was 21.7 Cmolc kg -1. Soil was sampled at three different stages, first before sowing in aerobic conditions and them two more times in anaerobiosis, i.e. by bunch formation and flowering. Ninety-six soil samples per treatment were taken during each of the three sampling periods. NH4+-N, P and K were routinely determined. Spatial variability was assessed through the analysis of semivariograms. Next, kriging maps were constructed and compared for successive sampling dates. The statistical variability of NH4+-N, P and K over the study period was low to medium, depending on treatment and sampling dates. Lime application produced a positive effect on the NH4+ availability at sowing time. Increased Olsen-P availability during sowing and

  19. Estimating Spatially Variable Parameters of the Epidemic Type Aftershock Sequence (ETAS) in California

    NASA Astrophysics Data System (ADS)

    Nandan, Shyam; Ouillon, Guy; Sornette, Didier; Wiemer, Stefan

    2016-04-01

    The ETAS model is widely employed to model the spatio-temporal distribution of earthquakes, generally using spatially invariant parameters, which is most likely a gross simplification considering the extremely heterogeneous structure of the Earth's crust. We propose an efficient method for the estimation of spatially varying parameters, using an expectation maximization (EM) algorithm and spatial Voronoi tessellations. We assume that each Voronoi cell is characterized by a set of eight constant ETAS parameters. For a given number of randomly distributed cells, Vi=1 to N, we jointly invert the ETAS parameters within each cell using an EM algorithm. This process is progressively repeated several times for a given N (which controls the complexity), which is itself increased incrementally. We use the Bayesian Information Criterion (BIC) to rank all the inverted models given their likelihood and complexity and select the top 1% models to compute the average model at any location. Using a synthetic catalog, we also check that the proposed method correctly inverts the known parameters. We apply the proposed method to earthquakes (M>=3) included in the ANSS catalog that occurred within the time period 1981-2016 in the spatial polygon defined by RELM/CSEP around California. The results indicate significant spatial variation of the ETAS parameters. Using these spatially variable estimates of ETAS parameters, we are better equipped to answer some important questions: (1) What is the seismic hazard (both long- and short-term) in a given region? (2) What kind of earthquakes dominate triggering? (3) are there regions where earthquakes are most likely preceded by foreshocks? Last but not the least, a possible correlation of the spatially varying ETAS parameters with spatially variable geophysical properties can lead to an improved understanding of the physics of earthquake triggering beside providing physical meaning to the parameters of the purely statistical ETAS model.

  20. Variable Number of Tandem Repeat Markers in the Genome Sequence of Mycosphaerella Fijiensis, the Causal Agent of Black Leaf Streak Disease of Banana (Musa spp.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycosphaerella fijiensis, the causal agent of banana leaf streak disease (commonly known as black Sigatoka), is the most devastating pathogen attacking bananas (Musa spp). Recently the whole genome sequence of M. fijiensis became available. This sequence was screened for the presence of Variable Num...

  1. HLA-F coding and regulatory segments variability determined by massively parallel sequencing procedures in a Brazilian population sample.

    PubMed

    Lima, Thálitta Hetamaro Ayala; Buttura, Renato Vidal; Donadi, Eduardo Antônio; Veiga-Castelli, Luciana Caricati; Mendes-Junior, Celso Teixeira; Castelli, Erick C

    2016-10-01

    Human Leucocyte Antigen F (HLA-F) is a non-classical HLA class I gene distinguished from its classical counterparts by low allelic polymorphism and distinctive expression patterns. Its exact function remains unknown. It is believed that HLA-F has tolerogenic and immune modulatory properties. Currently, there is little information regarding the HLA-F allelic variation among human populations and the available studies have evaluated only a fraction of the HLA-F gene segment and/or have searched for known alleles only. Here we present a strategy to evaluate the complete HLA-F variability including its 5' upstream, coding and 3' downstream segments by using massively parallel sequencing procedures. HLA-F variability was surveyed on 196 individuals from the Brazilian Southeast. The results indicate that the HLA-F gene is indeed conserved at the protein level, where thirty coding haplotypes or coding alleles were detected, encoding only four different HLA-F full-length protein molecules. Moreover, a same protein molecule is encoded by 82.45% of all coding alleles detected in this Brazilian population sample. However, the HLA-F nucleotide and haplotype variability is much higher than our current knowledge both in Brazilians and considering the 1000 Genomes Project data. This protein conservation is probably a consequence of the key role of HLA-F in the immune system physiology.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Microbiome characterization using SMRT sequencing on 16S rRNA genes across a range of amplicon sizes and variable region content

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sequence of variable regions along the 16S ribosomal RNA gene is often used to conduct metagenomic surveys of bacterial populations in specific habitats, because of the inter-species variability in these regions and because it is possible to design amplification primers in sections of the gene t...

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. Time Variability of the Dust Sublimation Zones in Pre-Main Sequence Disk Systems

    NASA Technical Reports Server (NTRS)

    Sitko, Michael L.; Carpenter, W. J.; Grady, C. A.; Russel, R. W.; Lynch, D. K.; Rudy, R. J.; Mazuk, S. M.; Venturini, C. C.; Kimes, R. L.; Beerman, L. C.; Ablordeppey, K. E.; Puetter, R. C.; Wisnewski, P.; Brafford, S. M.; Polomski, E. R.; Hammel, H. B.; Perry, R. B.; Wilde, J. L.

    2007-01-01

    The dust sublimation zone (DSZ) is the region of pre-main sequence (PMS) disks where dust grains most easily anneal, sublime, and condense out of the gas. Because of this, it is a location where crystalline material may be enhanced and redistributed throughout the rest of the disk. A decade-long program to monitor the thermal emission of the grains located in this region demonstrates that large changes in emitted flux occur in many systems. Changes in the thermal emission between 3 and 13.5 microns were observed in HD 31648 (MWC 480), HD 163296 (MWC 275), and DG Tau. This emission is consistent with it being produced at the DSZ, where the transition from a disk of gas to one of gas+dust occurs. In the case of DG Tau, the outbursts were accompanied by increased emission on the 10 micron silicate band on one occasion, while on another occasion it went into absorption. This requires lofting of the material above the disk into the line of sight. Such changes will affect the determination of the inner disk structure obtained through interferometry measurements, and this has been confirmed in the case of HD 163296. Cyclic variations in the heating of the DSZ will lead to the annealing of large grains, the sublimation of smaller grains, possibly followed by re-condensation as the zone enters a cooling phase. Lofting of dust above the disk plane, and outward acceleration by stellar winds and radiation pressure, can re-distribute the processed material to cooler regions of the disk, where cometesimals form. This processing is consistent with the detection of the preferential concentration of large crystalline grains in the inner few AU of PMS disks using interferometric spectroscopy with the VLTI.

  2. 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.

  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. 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.

  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. Detection limits of tidal-wetland sequences to identify variable rupture modes of megathrust earthquakes

    NASA Astrophysics Data System (ADS)

    Shennan, Ian; Garrett, Ed; Barlow, Natasha

    2016-10-01

    Recent paleoseismological studies question whether segment boundaries identified for 20th and 21st century great, >M8, earthquakes persist through multiple earthquake cycles or whether smaller segments with different boundaries rupture and cause significant hazards. The smaller segments may include some currently slipping rather than locked. In this review, we outline general principles regarding indicators of relative sea-level change in tidal wetlands and the conditions in which paleoseismic indicators must be distinct from those resulting from non-seismic processes. We present new evidence from sites across southcentral Alaska to illustrate different detection limits of paleoseismic indicators and consider alternative interpretations for marsh submergence and emergence. We compare predictions of coseismic uplift and subsidence derived from geophysical models of earthquakes with different rupture modes. The spatial patterns of agreement and misfits between model predictions and quantitative reconstructions of coseismic submergence and emergence suggest that no earthquake within the last 4000 years had a pattern of rupture the same as the Mw 9.2 Alaska earthquake in 1964. From the Alaska examples and research from other subduction zones we suggest that If we want to understand whether a megathrust ruptures in segments of variable length in different earthquakes, we need to be site-specific as to what sort of geological-based criteria eliminate the possibility of a particular rupture mode in different earthquakes. We conclude that coastal paleoseismological studies benefit from a methodological framework that employs rigorous evaluation of five essential criteria and a sixth which may be very robust but only occur at some sites: 1 - lateral extent of peat-mud or mud-peat couplets with sharp contacts; 2 - suddenness of submergence or emergence, and replicated within each site; 3 - amount of vertical motion, quantified with 95% error terms and replicated within each

  10. 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.

  11. 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.

  12. 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.

  13. Three ingredients for Improved global aftershock forecasts: Tectonic region, time-dependent catalog incompleteness, and inter-sequence variability

    USGS Publications Warehouse

    Page, Morgan T.; Van Der Elst, Nicholas; Hardebeck, Jeanne L.; Felzer, Karen; Michael, Andrew J.

    2016-01-01

    Following a large earthquake, seismic hazard can be orders of magnitude higher than the long‐term average as a result of aftershock triggering. Because of this heightened hazard, emergency managers and the public demand rapid, authoritative, and reliable aftershock forecasts. In the past, U.S. Geological Survey (USGS) aftershock forecasts following large global earthquakes have been released on an ad hoc basis with inconsistent methods, and in some cases aftershock parameters adapted from California. To remedy this, the USGS is currently developing an automated aftershock product based on the Reasenberg and Jones (1989) method that will generate more accurate forecasts. To better capture spatial variations in aftershock productivity and decay, we estimate regional aftershock parameters for sequences within the García et al. (2012) tectonic regions. We find that regional variations for mean aftershock productivity reach almost a factor of 10. We also develop a method to account for the time‐dependent magnitude of completeness following large events in the catalog. In addition to estimating average sequence parameters within regions, we develop an inverse method to estimate the intersequence parameter variability. This allows for a more complete quantification of the forecast uncertainties and Bayesian updating of the forecast as sequence‐specific information becomes available.

  14. Correlating multidimensional fetal heart rate variability analysis with acid-base balance at birth.

    PubMed

    Frasch, Martin G; Xu, Yawen; Stampalija, Tamara; Durosier, Lucien D; Herry, Christophe; Wang, Xiaogang; Casati, Daniela; Seely, Andrew Je; Alfirevic, Zarko; Gao, Xin; Ferrazzi, Enrico

    2014-12-01

    Fetal monitoring during labour currently fails to accurately detect acidemia. We developed a method to assess the multidimensional properties of fetal heart rate variability (fHRV) from trans-abdominal fetal electrocardiogram (fECG) during labour. We aimed to assess this novel bioinformatics approach for correlation between fHRV and neonatal pH or base excess (BE) at birth.We enrolled a prospective pilot cohort of uncomplicated singleton pregnancies at 38-42 weeks' gestation in Milan, Italy, and Liverpool, UK. Fetal monitoring was performed by standard cardiotocography. Simultaneously, with fECG (high sampling frequency) was recorded. To ensure clinician blinding, fECG information was not displayed. Data from the last 60 min preceding onset of second-stage labour were analyzed using clinically validated continuous individualized multiorgan variability analysis (CIMVA) software in 5 min overlapping windows. CIMVA allows simultaneous calculation of 101 fHRV measures across five fHRV signal analysis domains. We validated our mathematical prediction model internally with 80:20 cross-validation split, comparing results to cord pH and BE at birth.The cohort consisted of 60 women with neonatal pH values at birth ranging from 7.44 to 6.99 and BE from -0.3 to -18.7 mmol L(-1). Our model predicted pH from 30 fHRV measures (R(2) = 0.90, P < 0.001) and BE from 21 fHRV measures (R(2) = 0.77, P < 0.001).Novel bioinformatics approach (CIMVA) applied to fHRV derived from trans-abdominal fECG during labor correlated well with acid-base balance at birth. Further refinement and validation in larger cohorts are needed. These new measurements of fHRV might offer a new opportunity to predict fetal acid-base balance at birth. PMID:25407948

  15. 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.

  16. Reconstruction of cyclooxygenase evolution in animals suggests variable, lineage-specific duplications, and homologs with low sequence identity.

    PubMed

    Havird, Justin C; Kocot, Kevin M; Brannock, Pamela M; Cannon, Johanna T; Waits, Damien S; Weese, David A; Santos, Scott R; Halanych, Kenneth M

    2015-04-01

    Cyclooxygenase (COX) enzymatically converts arachidonic acid into prostaglandin G/H in animals and has importance during pregnancy, digestion, and other physiological functions in mammals. COX genes have mainly been described from vertebrates, where gene duplications are common, but few studies have examined COX in invertebrates. Given the increasing ease in generating genomic data, as well as recent, although incomplete descriptions of potential COX sequences in Mollusca, Crustacea, and Insecta, assessing COX evolution across Metazoa is now possible. Here, we recover 40 putative COX orthologs by searching publicly available genomic resources as well as ~250 novel invertebrate transcriptomic datasets. Results suggest the common ancestor of Cnidaria and Bilateria possessed a COX homolog similar to those of vertebrates, although such homologs were not found in poriferan and ctenophore genomes. COX was found in most crustaceans and the majority of molluscs examined, but only specific taxa/lineages within Cnidaria and Annelida. For example, all octocorallians appear to have COX, while no COX homologs were found in hexacorallian datasets. Most species examined had a single homolog, although species-specific COX duplications were found in members of Annelida, Mollusca, and Cnidaria. Additionally, COX genes were not found in Hemichordata, Echinodermata, or Platyhelminthes, and the few previously described COX genes in Insecta lacked appreciable sequence homology (although structural analyses suggest these may still be functional COX enzymes). This analysis provides a benchmark for identifying COX homologs in future genomic and transcriptomic datasets, and identifies lineages for future studies of COX. PMID:25758350

  17. Reconstruction of cyclooxygenase evolution in animals suggests variable, lineage-specific duplications, and homologs with low sequence identity.

    PubMed

    Havird, Justin C; Kocot, Kevin M; Brannock, Pamela M; Cannon, Johanna T; Waits, Damien S; Weese, David A; Santos, Scott R; Halanych, Kenneth M

    2015-04-01

    Cyclooxygenase (COX) enzymatically converts arachidonic acid into prostaglandin G/H in animals and has importance during pregnancy, digestion, and other physiological functions in mammals. COX genes have mainly been described from vertebrates, where gene duplications are common, but few studies have examined COX in invertebrates. Given the increasing ease in generating genomic data, as well as recent, although incomplete descriptions of potential COX sequences in Mollusca, Crustacea, and Insecta, assessing COX evolution across Metazoa is now possible. Here, we recover 40 putative COX orthologs by searching publicly available genomic resources as well as ~250 novel invertebrate transcriptomic datasets. Results suggest the common ancestor of Cnidaria and Bilateria possessed a COX homolog similar to those of vertebrates, although such homologs were not found in poriferan and ctenophore genomes. COX was found in most crustaceans and the majority of molluscs examined, but only specific taxa/lineages within Cnidaria and Annelida. For example, all octocorallians appear to have COX, while no COX homologs were found in hexacorallian datasets. Most species examined had a single homolog, although species-specific COX duplications were found in members of Annelida, Mollusca, and Cnidaria. Additionally, COX genes were not found in Hemichordata, Echinodermata, or Platyhelminthes, and the few previously described COX genes in Insecta lacked appreciable sequence homology (although structural analyses suggest these may still be functional COX enzymes). This analysis provides a benchmark for identifying COX homologs in future genomic and transcriptomic datasets, and identifies lineages for future studies of COX.

  18. Evidence for change in climate variability during the late-holocene inferred from a sequence of Lake Michigan dune ridges

    SciTech Connect

    Lichter, J. )

    1994-06-01

    The timing of ridge formation at a sequence of northern Lake Michigan foredune ridges was calibrated with the historical lake-level record and with climate records to reconstruct a history of climate-related lake-level variation. Foredune ridges are constructed during receding and low lake levels related to regional drought. Shore recession during high lake levels may promote eolian erosion which modifies the shore-parallel foredune ridges into parabolic dunes. A chronology of ridge formation over the last 2400 years indicates that parabolic dunes developed only during periods of frequent ridge formation and drought. Analysis of ridge formation during the historical record of lake-level variation suggest that this association results from increase variability in regional water balances as opposed to variation in sediment supply. Periods of high variability in regional water balances occurred between 380 BC and AD 20, AD 20, AD 20-300, AD 880-990, AD 1180-1280, and AD 1520-1650.

  19. Screening of variables influencing the clavulanic acid production by Streptomyces DAUFPE 3060 strain.

    PubMed

    Viana, D A; Carneiro-Cunha, M N; Araújo, J M; Barros-Neto, B; Lima-Filho, J L; Converti, A; Pessoa-Júnior, A; Porto, A L F

    2010-03-01

    Clavulanic acid (CA) is a beta-lactam antibiotic, which has a potent beta-lactamase inhibiting activity. The influence of five variables, namely pH (6.0, 6.4, and 6.8), temperature (28 degrees C, 30 degrees C, and 32 degrees C), agitation intensity (150, 200, and 250 rpm), glycerol concentration (5.0, 7.5, and 10 g/L) and soybean flour concentration (5.0, 12.5, and 20 g/L), on CA production by a new isolate of Streptomyces (DAUFPE 3060) was investigated in 250-mL Erlenmeyer flasks using a fractional factorial design. Temperature and soybean flour concentration were shown to be the two variables that exerted the most important effects on the production of CA at 95% confidence level. The highest CA concentration (494 mg/L) was obtained after 48 h at 150 rpm, 32 degrees C, pH 6.0, 5.0 g/L glycerol, and 20 g/L soybean flour concentrations. Under these conditions, the yields of biomass and product on consumed substrate were 0.26 g(X)/g(S) and 64.3 mg(P)/g(S), respectively. Fermentations performed in 3.0-L bench-scale fermenter allowed increasing the CA production by about 60%. PMID:19475517

  20. 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.

  1. The Baltic Sea: Geophysical and geochemical properties of Holocene sediment sequences as indicators of past environmental variability

    NASA Astrophysics Data System (ADS)

    Lenz, Conny; Reinholdsson, Maja; Zillén, Lovisa; Conley, Daniel J.; Snowball, Ian

    2010-05-01

    The Baltic Sea has undergone large environmental changes since the retreat of the Weischselian Ice-sheet. In the Late Glacial Period and the early Holocene these changes were most likely caused by natural environmental changes (i.e. changes in the morphology and depths of the Baltic basin and the sills). In more recent time anthropogenic impacts have become more important as a possible and likely cause for changes. During the whole Holocene period climate variability played an important role. However, the relative importance between humans and nature is largely unknown. Here we present the results of a combined geophysical and geochemical study on selected sediment sequences from the Baltic Sea within the two BONUS (Baltic Organisations Network For Funding Science) funded projects HYPER (HYPoxia mitigation for Baltic Sea Ecosystem Restoration) and Baltic GAS (GAS storage and effects of climate change and eutrophication). The over-all aim of these projects is to understand large-scale Baltic Sea ecosystem responses to environmental, climate and anthropogenic forcing. During two Baltic Sea research cruises in 2009 long sediment cores from 8 different locations were recovered. We present preliminary results from one site (LL19) located in the north central Baltic Proper at 169 m water depth. The Littorina Sea sediment record (i.e. the last c. 8000 years) is characterised by alternating periods of homogenised sediments (indicative of oxic conditions) and laminated sediments (indicative of hypoxic/anoxic conditions). Mineral magnetic properties illustrate clear changes between laminated and non-laminated sections of the core. The concentration of ferrimagnetic minerals, as revealed by initial magnetic susceptibility (χ) and saturation isothermal remanent magnetization (SIRM) is variable. The laminated sections in particular show high concentrations and to reveal the origin of the ferrimagnetic signal additional magnetic properties were measured, specifically the

  2. 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.

  3. 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.

  4. Sequence analysis and genetic variability of stearoyl CoA desaturase (SCD) gene in the Italian Mediterranean river buffalo.

    PubMed

    Pauciullo, A; Cosenza, G; D'Avino, A; Colimoro, L; Nicodemo, D; Coletta, A; Feligini, M; Marchitelli, C; Di Berardino, D; Ramunno, L

    2010-12-01

    Stearoyl-CoA desaturase (SCD) plays a key metabolic role by changing the saturated FA content of ruminant milk and meat. In this study we characterized for the first time the stearoyl-CoA desaturase (SCD) gene in river buffalo (Bubalus bubalis) and investigated its genetic variability. On a total of 78 buffaloes, 15 SNPs were detected and 6 of them were preliminarily genotyped. In particular, the g.133A>C SNP was found to create a new consensus site for the SP1 binding site, thus generating a new tandem repeat in the promoter region. A preliminary association study with the milk fatty acid content showed that the C allele significantly affects the total desaturation index (P<0.01). Linkage disequilibrium analysis allowed identification of 7 haplotypes and 4 tag SNPs. Such polymorphisms could represent useful genetic markers for association studies with fatty acid composition, but further studies are needed to evaluate their potential use to improve the nutritional quality of the dairy products. PMID:20691778

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Ultra high-throughput nucleic acid sequencing as a tool for virus discovery in the turkey gut.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, the use of the next generation of nucleic acid sequencing technology (i.e., 454 pyrosequencing, as developed by Roche/454 Life Sciences) has allowed an in-depth look at the uncultivated microorganisms present in complex environmental samples, including samples with agricultural importance....

  18. 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.

  19. A combined supplementation of vitamin B12 and omega-3 fatty acids across two generations improves cardiometabolic variables in rats.

    PubMed

    Khaire, Amrita; Rathod, Richa; Randhir, Karuna; Kale, Anvita; Joshi, Sadhana

    2016-09-14

    Our earlier studies indicate that micronutrients (vitamin B12, folic acid) and omega-3 fatty acids especially docosahexaenoic acid (DHA) are interlinked in one carbon cycle. The present study examines the effects of a sustained vitamin B12 deficiency/supplementation in the presence of omega-3 fatty acids across two generations on the pregnancy outcome and cardiometabolic profile [blood pressure, plasma lipid profile (cholesterol and triglycerides), plasma/liver fatty acid profile and hepatic lipid metabolism] in the second generation adult Wistar rat offspring. Two generations of animals were fed the following diets: control; vitamin B12 deficient; vitamin B12 supplemented; vitamin B12 deficient diet supplemented with omega-3 fatty acids; vitamin B12 and omega-3 fatty acid supplemented diets. Male offspring were sacrificed at 3 months of age. Vitamin B12 deficiency lowered the weight gain (p < 0.01) during pregnancy, increased systolic (p < 0.05) and diastolic (p < 0.01) blood pressure, and lowered the levels of plasma/liver DHA (p < 0.05 for both) but did not affect the lipid profile. Vitamin B12 supplementation showed weight gain, blood pressure and the fatty acid profile similar to the control. However, it increased (p < 0.05) the levels of plasma triglycerides. Omega-3 fatty acid supplementation to the vitamin B12 deficient group lowered the weight gain although the levels of cardiometabolic variables were comparable to the control. Omega-3 fatty acid supplementation in the presence of vitamin B12 improved the pregnancy outcome and all cardio-metabolic variables. Our study highlights the adverse effects of sustained vitamin B12 deficiency across two generations on the pregnancy outcome, fatty acid profile and blood pressure while a combined supplementation of vitamin B12 and omega-3 fatty acids is beneficial.

  20. A combined supplementation of vitamin B12 and omega-3 fatty acids across two generations improves cardiometabolic variables in rats.

    PubMed

    Khaire, Amrita; Rathod, Richa; Randhir, Karuna; Kale, Anvita; Joshi, Sadhana

    2016-09-14

    Our earlier studies indicate that micronutrients (vitamin B12, folic acid) and omega-3 fatty acids especially docosahexaenoic acid (DHA) are interlinked in one carbon cycle. The present study examines the effects of a sustained vitamin B12 deficiency/supplementation in the presence of omega-3 fatty acids across two generations on the pregnancy outcome and cardiometabolic profile [blood pressure, plasma lipid profile (cholesterol and triglycerides), plasma/liver fatty acid profile and hepatic lipid metabolism] in the second generation adult Wistar rat offspring. Two generations of animals were fed the following diets: control; vitamin B12 deficient; vitamin B12 supplemented; vitamin B12 deficient diet supplemented with omega-3 fatty acids; vitamin B12 and omega-3 fatty acid supplemented diets. Male offspring were sacrificed at 3 months of age. Vitamin B12 deficiency lowered the weight gain (p < 0.01) during pregnancy, increased systolic (p < 0.05) and diastolic (p < 0.01) blood pressure, and lowered the levels of plasma/liver DHA (p < 0.05 for both) but did not affect the lipid profile. Vitamin B12 supplementation showed weight gain, blood pressure and the fatty acid profile similar to the control. However, it increased (p < 0.05) the levels of plasma triglycerides. Omega-3 fatty acid supplementation to the vitamin B12 deficient group lowered the weight gain although the levels of cardiometabolic variables were comparable to the control. Omega-3 fatty acid supplementation in the presence of vitamin B12 improved the pregnancy outcome and all cardio-metabolic variables. Our study highlights the adverse effects of sustained vitamin B12 deficiency across two generations on the pregnancy outcome, fatty acid profile and blood pressure while a combined supplementation of vitamin B12 and omega-3 fatty acids is beneficial. PMID:27528436

  1. 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

  2. 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

  3. The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin.

    PubMed

    Elleman, T C

    1972-08-01

    1. The amino acid sequence of a protein from the reduced and carboxymethylated high-sulphur fraction of wool has been determined. 2. The sequence of this S-carboxymethylkerateine (SCMK-B2C) of 151 amino acid residues displays much internal homology and an unusual residue distribution. Thus a ten-residue sequence occurs four times near the N-terminus and five times near the C-terminus with few changes. These regions contain much of the molecule's half-cystine, whereas between them there is a region of 19 residues that are mainly small and devoid of cystine and proline. 3. Certain models of the wool fibre based on its mechanical and physical properties propose a matrix of small compact globular units linked together to form beaded chains. The unusual distribution of the component residues of protein SCMK-B2C suggests structures in the wool-fibre matrix compatible with certain features of the proposed models.

  4. Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores.

    PubMed

    Zahid, Osama K; Wang, Fanny; Ruzicka, Jan A; Taylor, Ethan W; Hall, Adam R

    2016-03-01

    The detection and quantification of short nucleic acid sequences has many potential applications in studying biological processes, monitoring disease initiation and progression, and evaluating environmental systems, but is challenging by nature. We present here an assay based on the solid-state nanopore platform for the identification of specific sequences in solution. We demonstrate that hybridization of a target nucleic acid with a synthetic probe molecule enables discrimination between duplex and single-stranded molecules with high efficacy. Our approach requires limited preparation of samples and yields an unambiguous translocation event rate enhancement that can be used to determine the presence and abundance of a single sequence within a background of nontarget oligonucleotides. PMID:26824296

  5. Sequence of cDNA for rat cystathionine gamma-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes.

    PubMed Central

    Erickson, P F; Maxwell, I H; Su, L J; Baumann, M; Glode, L M

    1990-01-01

    A cDNA clone for cystathionine gamma-lyase was isolated from a rat cDNA library in lambda gt11 by screening with a monospecific antiserum. The identity of this clone, containing 600 bp proximal to the 3'-end of the gene, was confirmed by positive hybridization selection. Northern-blot hybridization showed the expected higher abundance of the corresponding mRNA in liver than in brain. Two further cDNA clones from a plasmid pcD library were isolated by colony hybridization with the first clone and were found to contain inserts of 1600 and 1850 bp. One of these was confirmed as encoding cystathionine gamma-lyase by hybridization with two independent pools of oligodeoxynucleotides corresponding to partial amino acid sequence information for cystathionine gamma-lyase. The other clone (estimated to represent all but 8% of the 5'-end of the mRNA) was sequenced and its deduced amino acid sequence showed similarity to those of the Escherichia coli enzymes cystathionine beta-lyase and cystathionine gamma-synthase throughout its length, especially to that of the latter. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:2201285

  6. Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution

    NASA Technical Reports Server (NTRS)

    Eriksson, M.; Christensen, L.; Schmidt, J.; Haaima, G.; Orgel, L.; Nielsen, P. E.

    1998-01-01

    The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.

  7. The cDNA-derived amino acid sequence of hemoglobin II from Lucina pectinata.

    PubMed

    Torres-Mercado, Elineth; Renta, Jessicca Y; Rodríguez, Yolanda; López-Garriga, Juan; Cadilla, Carmen L

    2003-11-01

    Hemoglobin II from the clam Lucina pectinata is an oxygen-reactive protein with a unique structural organization in the heme pocket involving residues Gln65 (E7), Tyr30 (B10), Phe44 (CD1), and Phe69 (E11). We employed the reverse transcriptase-polymerase chain reaction (RT-PCR) and methods to synthesize various cDNA(HbII). An initial 300-bp cDNA clone was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbII-partial cDNA sequence were used to obtain the 5' and 3' ends of the cDNA by RACE. The length of the HbII cDNA, estimated from overlapping clones, was approximately 2114 bases. Northern blot analysis revealed that the mRNA size of HbII agrees with the estimated size using cDNA data. The coding region of the full-length HbII cDNA codes for 151 amino acids. The calculated molecular weight of HbII, including the heme group and acetylated N-terminal residue, is 17,654.07 Da.

  8. Assessing amino acid racemization variability in coral intra-crystalline protein for geochronological applications

    NASA Astrophysics Data System (ADS)

    Hendy, Erica J.; Tomiak, Peter J.; Collins, Matthew J.; Hellstrom, John; Tudhope, Alexander W.; Lough, Janice M.; Penkman, Kirsty E. H.

    2012-06-01

    Over 500 Free Amino Acid (FAA) and corresponding Total Hydrolysed Amino Acid (THAA) analyses were completed from eight independently-dated, multi-century coral cores of massive Porites sp. colonies. This dataset allows us to re-evaluate the application of amino acid racemization (AAR) for dating late Holocene coral material, 20 years after Goodfriend et al. (GCA56 (1992), 3847) first showed AAR had promise for developing chronologies in coral cores. This re-assessment incorporates recent method improvements, including measurement by RP-HPLC, new quality control approaches (e.g. sampling and sub-sampling protocols, statistically-based data screening criteria), and cleaning steps to isolate the intra-crystalline skeletal protein. We show that the removal of the extra-crystalline contaminants and matrix protein is the most critical step for reproducible results and recommend a protocol of bleaching samples in NaOCl for 48 h to maximise removal of open system proteins while minimising the induced racemization. We demonstrate that AAR follows closed system behaviour in the intra-crystalline fraction of the coral skeletal proteins. Our study is the first to assess the natural variability in intra-crystalline AAR between colonies, and we use coral cores taken from the Great Barrier Reef, Australia, and Jarvis Island in the equatorial Pacific to explore variability associated with different environmental conditions and thermal histories. Chronologies were developed from THAA Asx D/L, Ala D/L, Glx D/L and FAA Asx D/L for each core and least squares Monte Carlo modelling applied in order to quantify uncertainty of AAR age determinations and assess the level of dating resolution possible over the last 5 centuries. AAR within colonies follow consistent stratigraphic aging. However, there are systematic differences in rates between the colonies, which would preclude direct comparison from one colony to another for accurate age estimation. When AAR age models are developed from

  9. Assessing amino acid racemization variability in coral intra-crystalline protein for geochronological applications.

    PubMed

    Hendy, Erica J; Tomiak, Peter J; Collins, Matthew J; Hellstrom, John; Tudhope, Alexander W; Lough, Janice M; Penkman, Kirsty E H

    2012-06-01

    Over 500 Free Amino Acid (FAA) and corresponding Total Hydrolysed Amino Acid (THAA) analyses were completed from eight independently-dated, multi-century coral cores of massive Porites sp. colonies. This dataset allows us to re-evaluate the application of amino acid racemization (AAR) for dating late Holocene coral material, 20 years after Goodfriend et al. (GCA56 (1992), 3847) first showed AAR had promise for developing chronologies in coral cores. This re-assessment incorporates recent method improvements, including measurement by RP-HPLC, new quality control approaches (e.g. sampling and sub-sampling protocols, statistically-based data screening criteria), and cleaning steps to isolate the intra-crystalline skeletal protein. We show that the removal of the extra-crystalline contaminants and matrix protein is the most critical step for reproducible results and recommend a protocol of bleaching samples in NaOCl for 48 h to maximise removal of open system proteins while minimising the induced racemization. We demonstrate that AAR follows closed system behaviour in the intra-crystalline fraction of the coral skeletal proteins. Our study is the first to assess the natural variability in intra-crystalline AAR between colonies, and we use coral cores taken from the Great Barrier Reef, Australia, and Jarvis Island in the equatorial Pacific to explore variability associated with different environmental conditions and thermal histories. Chronologies were developed from THAA Asx D/L, Ala D/L, Glx D/L and FAA Asx D/L for each core and least squares Monte Carlo modelling applied in order to quantify uncertainty of AAR age determinations and assess the level of dating resolution possible over the last 5 centuries. AAR within colonies follow consistent stratigraphic aging. However, there are systematic differences in rates between the colonies, which would preclude direct comparison from one colony to another for accurate age estimation. When AAR age models are developed

  10. Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

    PubMed Central

    Devault, A; Lazure, C; Nault, C; Le Moual, H; Seidah, N G; Chrétien, M; Kahn, P; Powell, J; Mallet, J; Beaumont, A

    1987-01-01

    Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase. Images Fig. 1. Fig. 3. PMID:2440677

  11. Sources of individual variability: miRNAs that predispose to neuropathic pain identified using genome-wide sequencing

    PubMed Central

    2014-01-01

    Background We carried out a genome-wide study, using microRNA sequencing (miRNA-seq), aimed at identifying miRNAs in primary sensory neurons that are associated with neuropathic pain. Such scans usually yield long lists of transcripts regulated by nerve injury, but not necessarily related to pain. To overcome this we tried a novel search strategy: identification of transcripts regulated differentially by nerve injury in rat lines very similar except for a contrasting pain phenotype. Dorsal root ganglia (DRGs) L4 and 5 in the two lines were excised 3 days after spinal nerve ligation surgery (SNL) and small RNAs were extracted and sequenced. Results We identified 284 mature miRNA species expressed in rat DRGs, including several not previously reported, and 3340 unique small RNA sequences. Baseline expression of miRNA was nearly identical in the two rat lines, consistent with their shared genetic background. In both lines many miRNAs were nominally up- or down-regulated following SNL, but the change was similar across lines. Only 3 miRNAs that were expressed abundantly (rno-miR-30d-5p, rno-miR-125b-5p) or at moderate levels (rno-miR-379-5p) were differentially regulated. This makes them prime candidates as novel PNS determinants of neuropathic pain. The first two are known miRNA regulators of the expression of Tnf, Bdnf and Stat3, gene products intimately associated with neuropathic pain phenotype. A few non-miRNA, small noncoding RNAs (sncRNAs) were also differentially regulated. Conclusions Despite its genome-wide coverage, our search strategy yielded a remarkably short list of neuropathic pain-related miRNAs. As 2 of the 3 are validated regulators of important pro-nociceptive compounds, it is likely that they contribute to the orchestration of gene expression changes that determine individual variability in pain phenotype. Further research is required to determine whether some of the other known or predicted gene targets of these miRNAs, or of the differentially

  12. 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.

  13. Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium.

    PubMed

    He, J S; Ruettinger, R T; Liu, H M; Fulco, A J

    1989-12-22

    The gene encoding barbiturate-inducible cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH2-terminal sequence of P-450BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an Mr of 47,439. The sequence is most, but less than 27%, similar to that of P-450CAM from Pseudomonas putida, so that P-450BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH-P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987-10995). PMID:2597681

  14. Sample Prep, Workflow Automation and Nucleic Acid Fractionation for Next Generation Sequencing

    SciTech Connect

    Roskey, Mark

    2010-06-03

    Mark Roskey of Caliper LifeSciences discusses how the company's technologies fit into the next generation sequencing workflow on June 3, 2010 at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM

  15. Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

    PubMed

    Fellah, J S; Wiles, M V; Charlemagne, J; Schwager, J

    1992-10-01

    cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences. PMID:1382992

  16. Genomic Variability of Haemophilus influenzae Isolated from Mexican Children Determined by Using Enterobacterial Repetitive Intergenic Consensus Sequences and PCR

    PubMed Central

    Gomez-De-Leon, Patricia; Santos, Jose I.; Caballero, Javier; Gomez, Demostenes; Espinosa, Luz E.; Moreno, Isabel; Piñero, Daniel; Cravioto, Alejandro

    2000-01-01

    Genomic fingerprints from 92 capsulated and noncapsulated strains of Haemophilus influenzae from Mexican children with different diseases and healthy carriers were generated by PCR using the enterobacterial repetitive intergenic consensus (ERIC) sequences. A cluster analysis by the unweighted pair-group method with arithmetic averages based on the overall similarity as estimated from the characteristics of the genomic fingerprints, was conducted to group the strains. A total of 69 fingerprint patterns were detected in the H. influenzae strains. Isolates from patients with different diseases were represented by a variety of patterns, which clustered into two major groups. Of the 37 strains isolated from cases of meningitis, 24 shared patterns and were clustered into five groups within a similarity level of 1.0. One fragment of 1.25 kb was common to all meningitis strains. H. influenzae strains from healthy carriers presented fingerprint patterns different from those found in strains from sick children. Isolates from healthy individuals were more variable and were distributed differently from those from patients. The results show that ERIC-PCR provides a powerful tool for the determination of the distinctive pathogenicity potentials of H. influenzae strains and encourage its use for molecular epidemiology investigations. PMID:10878033

  17. Extremophiles in Mineral Sulphide Heaps: Some Bacterial Responses to Variable Temperature, Acidity and Solution Composition

    PubMed Central

    Watling, Helen R.; Shiers, Denis W.; Collinson, David M.

    2015-01-01

    In heap bioleaching, acidophilic extremophiles contribute to enhanced metal extraction from mineral sulphides through the oxidation of Fe(II) and/or reduced inorganic sulphur compounds (RISC), such as elemental sulphur or mineral sulphides, or the degradation of organic compounds derived from the ore, biota or reagents used during mineral processing. The impacts of variable solution acidity and composition, as well as temperature on the three microbiological functions have been examined for up to four bacterial species found in mineral sulphide heaps. The results indicate that bacteria adapt to sufficiently high metal concentrations (Cu, Ni, Co, Zn, As) to allow them to function in mineral sulphide heaps and, by engaging alternative metabolic pathways, to extend the solution pH range over which growth is sustained. Fluctuating temperatures during start up in sulphide heaps pose the greatest threat to efficient bacterial colonisation. The large masses of ores in bioleaching heaps mean that high temperatures arising from sulphide oxidation are hard to control initially, when the sulphide content of the ore is greatest. During that period, mesophilic and moderately thermophilic bacteria are markedly reduced in both numbers and activity. PMID:27682094

  18. Extremophiles in Mineral Sulphide Heaps: Some Bacterial Responses to Variable Temperature, Acidity and Solution Composition.

    PubMed

    Watling, Helen R; Shiers, Denis W; Collinson, David M

    2015-07-09

    In heap bioleaching, acidophilic extremophiles contribute to enhanced metal extraction from mineral sulphides through the oxidation of Fe(II) and/or reduced inorganic sulphur compounds (RISC), such as elemental sulphur or mineral sulphides, or the degradation of organic compounds derived from the ore, biota or reagents used during mineral processing. The impacts of variable solution acidity and composition, as well as temperature on the three microbiological functions have been examined for up to four bacterial species found in mineral sulphide heaps. The results indicate that bacteria adapt to sufficiently high metal concentrations (Cu, Ni, Co, Zn, As) to allow them to function in mineral sulphide heaps and, by engaging alternative metabolic pathways, to extend the solution pH range over which growth is sustained. Fluctuating temperatures during start up in sulphide heaps pose the greatest threat to efficient bacterial colonisation. The large masses of ores in bioleaching heaps mean that high temperatures arising from sulphide oxidation are hard to control initially, when the sulphide content of the ore is greatest. During that period, mesophilic and moderately thermophilic bacteria are markedly reduced in both numbers and activity.

  19. Extremophiles in Mineral Sulphide Heaps: Some Bacterial Responses to Variable Temperature, Acidity and Solution Composition

    PubMed Central

    Watling, Helen R.; Shiers, Denis W.; Collinson, David M.

    2015-01-01

    In heap bioleaching, acidophilic extremophiles contribute to enhanced metal extraction from mineral sulphides through the oxidation of Fe(II) and/or reduced inorganic sulphur compounds (RISC), such as elemental sulphur or mineral sulphides, or the degradation of organic compounds derived from the ore, biota or reagents used during mineral processing. The impacts of variable solution acidity and composition, as well as temperature on the three microbiological functions have been examined for up to four bacterial species found in mineral sulphide heaps. The results indicate that bacteria adapt to sufficiently high metal concentrations (Cu, Ni, Co, Zn, As) to allow them to function in mineral sulphide heaps and, by engaging alternative metabolic pathways, to extend the solution pH range over which growth is sustained. Fluctuating temperatures during start up in sulphide heaps pose the greatest threat to efficient bacterial colonisation. The large masses of ores in bioleaching heaps mean that high temperatures arising from sulphide oxidation are hard to control initially, when the sulphide content of the ore is greatest. During that period, mesophilic and moderately thermophilic bacteria are markedly reduced in both numbers and activity.

  20. Sequence Comparison and Phylogeny of Nucleotide Sequence of Coat Protein and Nucleic Acid Binding Protein of a Distinct Isolate of Shallot virus X from India.

    PubMed

    Majumder, S; Baranwal, V K

    2011-06-01

    Shallot virus X (ShVX), a type species in the genus Allexivirus of the family Alfaflexiviridae has been associated with shallot plants in India and other shallot growing countries like Russia, Germany, Netherland, and New Zealand. Coat protein (CP) and nucleic acid binding protein (NB) region of the virus was obtained by reverse transcriptase polymerase chain reaction from scales leaves of shallot bulbs. The partial cDNA contained two open reading frames encoding proteins of molecular weights of 28.66 and 14.18 kDa belonging to Flexi_CP super-family and viral NB super-family, respectively. The percent identity and phylogenetic analysis of amino acid sequences of CP and NB region of the virus associated with shallot indicated that it was a distinct isolate of ShVX.

  1. Sequence Comparison and Phylogeny of Nucleotide Sequence of Coat Protein and Nucleic Acid Binding Protein of a Distinct Isolate of Shallot virus X from India.

    PubMed

    Majumder, S; Baranwal, V K

    2011-06-01

    Shallot virus X (ShVX), a type species in the genus Allexivirus of the family Alfaflexiviridae has been associated with shallot plants in India and other shallot growing countries like Russia, Germany, Netherland, and New Zealand. Coat protein (CP) and nucleic acid binding protein (NB) region of the virus was obtained by reverse transcriptase polymerase chain reaction from scales leaves of shallot bulbs. The partial cDNA contained two open reading frames encoding proteins of molecular weights of 28.66 and 14.18 kDa belonging to Flexi_CP super-family and viral NB super-family, respectively. The percent identity and phylogenetic analysis of amino acid sequences of CP and NB region of the virus associated with shallot indicated that it was a distinct isolate of ShVX. PMID:23637504

  2. Gene structure and amino acid sequence of Latimeria chalumnae (coelacanth) myelin DM20: phylogenetic relation of the fish.

    PubMed

    Tohyama, Y; Kasama-Yoshida, H; Sakuma, M; Kobayashi, Y; Cao, Y; Hasegawa, M; Kojima, H; Tamai, Y; Tanokura, M; Kurihara, T

    1999-07-01

    The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 corresponding to exons 2-7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria. The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria. Southern blot analysis indicates that Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941-1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.

  3. Variability in coconut (Cocos nucifera L.) germplasm and hybrids for fatty acid profile of oil.

    PubMed

    Kumar, S Naresh

    2011-12-28

    Coconut oil, the main product of coconut fruit, is the richest source of glycerol and lauric acid and hence is called lauric oil. This paper reports the fatty acid profile of oil from 60 Talls, 14 Dwarfs, and 34 hybrids. These include collections from 13 countries covering a large coconut-growing area of the world, apart from the indigenous ones. Capillary gas chromatography analysis of oil indicated a wider variation for the fatty acid profile than earlier reported. Apart from this, for the first time other fatty acids such as behenic and lignoceric acids were detected. Oil from cultivars and hybrids of coconut has significantly differed, particularly for commercially important fatty acids such as lauric acid and unsaturated fatty acids. However, coconut oil seems to have a conserved fatty acid profile, mainly because of low unsaturated fatty acids, indicating the possibility of grouping cultivars on the basis of their fatty acid profiles. The cluster analysis based on fatty acid profile indicated grouping together of geographically and typically closely related cultivars. Cultivars with high concentrations of specific fatty acids can be of potential use for industrial exploitation, whereas those with high concentrations of short- and medium-chain fatty acids and unsaturated fatty acids are more suitable for human consumption. Cultivars and hybrids with high and low values for each of the fatty acids are also identified.

  4. Amino acid sequence and structural properties of protein p12, an African swine fever virus attachment protein.

    PubMed Central

    Alcamí, A; Angulo, A; López-Otín, C; Muñoz, M; Freije, J M; Carrascosa, A L; Viñuela, E

    1992-01-01

    The gene encoding the African swine fever virus protein p12, which is involved in virus attachment to the host cell, has been mapped and sequenced in the genome of the Vero-adapted virus strain BA71V. The determination of the N-terminal amino acid sequence and the hybridization of oligonucleotide probes derived from this sequence to cloned restriction fragments allowed the mapping of the gene in fragment EcoRI-O, located in the central region of the viral genome. The DNA sequence of an EcoRI-XbaI fragment showed an open reading frame which is predicted to encode a polypeptide of 61 amino acids. The expression of this open reading frame in rabbit reticulocyte lysates and in Escherichia coli gave rise to a 12-kDa polypeptide that was immunoprecipitated with a monoclonal antibody specific for protein p12. The hydrophilicity profile indicated the existence of a stretch of 22 hydrophobic residues in the central part that may anchor the protein in the virus envelope. Three forms of the protein with apparent molecular masses of 17, 12, and 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been observed, depending on the presence of 2-mercaptoethanol and alkylation with 4-vinylpyridine, indicating that disulfide bonds are responsible for the multimerization of the protein. This result was in agreement with the existence of a cysteine-rich domain in the C-terminal region of the predicted amino acid sequence. The protein was synthesized at late times of infection, and no posttranslational modifications such as glycosylation, phosphorylation, or fatty acid acylation were detected. Images PMID:1583732

  5. Amino acid sequence of an intracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells.

    PubMed

    Löffler, A; Glund, K; Irie, M

    1993-06-15

    The primary structure of an intracellular ribonuclease (RNase LX) from cultured tomato (Lycopersicon esculentum) cells has been determined. Previous studies have shown that the protein is located inside the tomato cells but outside the vacuoles and that its synthesis is induced after depleting the cells for phosphate [Löffler, A., Abel, S., Jost, W., Beintema, J. J., Glund, K. (1992) Plant Physiol. 98, 1472-1478]. Sequence analysis was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the protein. RNase LX consists of 213 amino acids and has a molecular mass of 24300 Da and an isoelectric point of 5.33. The enzyme contains 10 half-cystines and there are no potential N-glycosylation sites detectable in the sequence. RNase LX, as compared to an extracellular tomato RNase (RNase LE), which is also phosphate regulated and the amino acid sequence of which was recently established [Jost, W., Bak, H., Glund, K., Terpstra, P. & Beintema, J. J. (1991) Eur. J. Biochem. 198, 1-6] has 60% of all amino acids identical and in identical positions, revealing a high degree of similarity between both proteins. In contrast to RNase LE, RNase LX has a C-terminal extension of nine amino acids. The C-terminal tetrapeptide HDEF may be a retention signal of the protein in the endoplasmic reticulum. PMID:8319673

  6. Complete genome sequence of Enterococcus mundtii QU 25, an efficient L-(+)-lactic acid-producing bacterium.

    PubMed

    Shiwa, Yuh; Yanase, Hiroaki; Hirose, Yuu; Satomi, Shohei; Araya-Kojima, Tomoko; Watanabe, Satoru; Zendo, Takeshi; Chibazakura, Taku; Shimizu-Kadota, Mariko; Yoshikawa, Hirofumi; Sonomoto, Kenji

    2014-08-01

    Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce l-lactic acid. The use of this strain is highly desirable for economical l-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified-one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.

  7. Rational design of translational pausing without altering the amino acid sequence dramatically promotes soluble protein expression: a strategic demonstration.

    PubMed

    Chen, Wei; Jin, Jingjie; Gu, Wei; Wei, Bo; Lei, Yun; Xiong, Sheng; Zhang, Gong

    2014-11-10

    The production of many pharmaceutical and industrial proteins in prokaryotic hosts is hindered by the insolubility of industrial expression products resulting from misfolding. Even with a correct primary sequence, an improper translation elongation rate in a heterologous expression system is an important cause of misfolding. In silico analysis revealed that most of the endogenous Escherichia coli genes display translational pausing sites that promote correct folding, and almost 1/5 genes have pausing sites at the 3'-termini of their coding sequence. Therefore, we established a novel strategy to efficiently promote the expression of soluble and active proteins without altering the amino acid sequence or expression conditions. This strategy uses the rational design of translational pausing based on structural information solely through synonymous substitutions, i.e. no change on the amino acids sequence. We demonstrated this strategy on a promising antiviral candidate, Cyanovirin-N (CVN), which could not be efficiently expressed in any previously reported system. By introducing silent mutations, we increased the soluble expression level in E. coli by 2000-fold without altering the CVN protein sequence, and the specific activity was slightly higher for the optimized CVN than for the wild-type variant. This strategy introduces new possibilities for the production of bioactive recombinant proteins.

  8. Gastropod arginine kinases from Cellana grata and Aplysia kurodai. Isolation and cDNA-derived amino acid sequences.

    PubMed

    Suzuki, T; Inoue, N; Higashi, T; Mizobuchi, R; Sugimura, N; Yokouchi, K; Furukohri, T

    2000-12-01

    Arginine kinase (AK) was isolated from the radular muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. The denatured relative molecular mass values were estimated to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme from Aplysia gave a Km value of 0.6 mM for arginine and a Vmax value of 13 micromole Pi min(-1) mg protein(-1) for the forward reaction. These values are comparable to other molluscan AKs. The cDNAs encoding Cellana and Aplysia AKs were amplified by polymerase chain reaction, and the nucleotide sequences of 1,608 and 1,239 bp, respectively, were determined. The open reading frame for Cellana AK is 1044 nucleotides in length and encodes a protein with 347 amino acid residues, and that for A. kurodai is 1077 nucleotides and 354 residues. The cDNA-derived amino acid sequences were validated by chemical sequencing of internal lysyl endopeptidase peptides. The amino acid sequences of Cellana and Aplysia AKs showed the highest percent identity (66-73%) with those of the abalone Nordotis and turbanshell Battilus belonging to the same class Gastropoda. These AK sequences still have a strong homology (63-71%) with that of the chiton Liolophura (class Polyplacophora), which is believed to be one of the most primitive molluscs. On the other hand, these AK sequences are less homologous (55-57%) with that of the clam Pseudocardium (class Bivalvia), suggesting that the biological position of the class Polyplacophora should be reconsidered.

  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. Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum

    PubMed Central

    Sugiura, Mayumi; Harumoto, Terue

    2001-01-01

    Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2′-formylamino-5′-hydroxy-benzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20–30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone 1 activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns. PMID:11724922

  11. Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum.

    PubMed

    Sugiura, M; Harumoto, T

    2001-12-01

    Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2'-formylamino-5'-hydroxy-benzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20-30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone 1 activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns.

  12. Variable region sequences and idiotypic expression of a protective human immunoglobulin M antibody to capsular polysaccharides of Neisseria meningitidis group B and Escherichia coli K1.

    PubMed Central

    Azmi, F H; Lucas, A H; Raff, H V; Granoff, D M

    1994-01-01

    We determined the heavy (H)- and light (L)-chain variable (V) region nucleotide and translated amino acid sequences of the human immunoglobulin M(kappa) monoclonal antibody (MAb) 5E1, which is specific for the polysaccharide capsule of Escherichia coli K1 and Neisseria meningitidis group B (poly[alpha(2-->8)-N-acetylneuraminic acid]) and which is protective in animal models of infection. The 5E1 VH gene is a member of the VHIIIb family and is 97% homologous to the 9.1 germ line gene. The 5E1 VL gene is a member of the kappa I subgroup and is 98% homologous to the germ line gene, 15A, also known as KLO12. The VL and/or VH genes used by 5E1 are highly homologous to the V genes encoding antibodies to the Haemophilus influenzae type b polysaccharide and to antibodies reactive with self-antigens such as erythrocyte "i," DNA, and thyroid peroxidase. We also produced three murine anti-idiotype (Id) MAbs against 5E1. All three anti-Ids recognize a minor subset of antimeningococcal B polysaccharide antibodies present in serum from normal adults. Two of the anti-Ids define distinct Ids associated with antibodies having kappa I-15A V regions. These 15A-associated Ids are expressed by some heterologous human antimeningococcal B polysaccharide MAbs, and they also are independently expressed by two human MAbs that are specific for either the H. influenzae b polysaccharide or the i erythrocyte antigen and that utilize the kappa I-15A V region. Taken together, these data indicate that the 5E1 antibody uses V regions that recur in the human antibody repertoires to this polysaccharide and to structurally dissimilar polysaccharides and autoantigens. Thus, the poor immunogenicity of poly[alpha(2-->8)-N-acetylneuraminic acid] cannot be explained by the unavailability of certain critical VH and VL genes required for generation of antibody response. PMID:8168940

  13. Sequence of the cDNA and 5'-flanking region for human acid alpha-glucosidase, detection of an intron in the 5' untranslated leader sequence, definition of 18-bp polymorphisms, and differences with previous cDNA and amino acid sequences.

    PubMed

    Martiniuk, F; Mehler, M; Tzall, S; Meredith, G; Hirschhorn, R

    1990-03-01

    Acid maltase or acid alpha-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose and is deficient in glycogen storage disease type II. Previously, we isolated a partial cDNA (1.9 kb) for human GAA; we have now used this cDNA to isolate and determine sequence in longer cDNAs from four additional independent cDNA libraries. Primer extension studies indicated that the mRNA extended approximately 200 bp 5' of the cDNA sequence obtained. Therefore, we isolated a genomic fragment containing 5' cDNA sequences that overlapped the previous cDNA sequence and extended an additional 24 bp to an initiation codon within a Kozak consensus sequence. The sequence of the genomic clone revealed an intron-exon junction 32 bp 5' to the ATG, indicating that the 5' leader sequence was interrupted by an intron. The remaining 186 bp of 5' untranslated sequence was identified approximately 3 kb upstream. The promoter region upstream from the start site of transcription was GC rich and contained areas of homology to Sp1 binding sites but no identifiable CAAT or TATA box. The combined data gave a nucleotide sequence of 2,856 bp for the coding region from the ATG to a stop codon, predicting a protein of 952 amino acids. The 3' untranslated region contained 555 bp with a polyadenylation signal at 3,385 bp followed by 16 bp prior to a poly(A) tail. This sequence of the GAA coding region differs from that reported by Hoefsloot et al. (1988) in three areas that change a total of 42 amino acids. Direct determination of the amino acid sequence in one of these areas confirmed the nucleotide sequence reported here but also disagreed with the directly determined amino acid sequence reported by Hoefsloot et al. (1988). At two other areas, changes in base pairs predicted new restriction sites that were identified in cDNAs from several independent libraries. The amino acid changes in all three ares increased the homology to rabbit-human isomaltase. Therefore, we believe that our

  14. Sequence variability in HC-Pro genes of Korean Soybean mosaic virus isolates is associated with differences in gene silencing suppression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean mosaic virus (SMV), a member of the family Potyviridae, is an important viral pathogen affecting soybean production in Korea. The variability in helper component proteinase (HC-Pro) sequence and pathogenicity of SMV isolates from seven provinces of Korea was investigated and compared with th...

  15. Sequence variability in HC-Pro coding regions of Korean Soybean mosaic virus isolates is associated with differences in RNA silencing suppression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean mosaic virus (SMV), a member of the family Potyviridae, is an important viral pathogen affecting soybean production in Korea. The variability in helper component proteinase (HC-Pro) sequences and pathogenicity of SMV tissue samples from seven Korean provinces was investigated and compared wi...

  16. Holocene climate variability, vegetation dynamics and fire regime in the central Pyrenees: the Basa de la Mora sequence (NE Spain)

    NASA Astrophysics Data System (ADS)

    Pérez-Sanz, A.; González-Sampériz, P.; Moreno, A.; Valero-Garcés, B.; Gil-Romera, G.; Rieradevall, M.; Tarrats, P.; Lasheras-Álvarez, L.; Morellón, M.; Belmonte, A.; Sancho, C.; Sevilla-Callejo, M.; Navas, A.

    2013-08-01

    High resolution multiproxy data (pollen, sedimentology, geochemistry, chironomids and charcoal) from the Basa de la Mora (BSM) lake sequence (42° 32' N, 0° 19' E, 1914 m a.s.l.) show marked climate variability in the central southern Pyrenees throughout the Holocene. A robust age model based on 15 AMS radiocarbon dates underpins the first precise reconstruction of rapid climate changes during the Holocene from this area. During the Early Holocene, increased winter snowpack and high snowmelt during summer, as a consequence of high seasonality, led to higher lake levels, a chironomid community dominated by non-lacustrine taxa (Orthocladiinae) related to higher inlet streams, and a forested landscape with intense run-off processes in the watershed. From 9.8 to 8.1 cal ka BP, climate instability is inferred from rapid and intense forest shifts and high fluctuation in surface run-off. Shifts among conifers and mesophytes reveal at least four short-lived dry events at 9.7, 9.3, 8.8 and 8.3 cal ka BP. Between 8.1 and 5.7 cal ka BP a stable climate with higher precipitation favoured highest lake levels and forest expansion, with spread of mesophytes, withdrawal of conifers and intensification of fires, coinciding with the Holocene Climate Optimum. At 5.7 cal ka BP a major change leading to drier conditions contributed to a regional decline in mesophytes, expansion of pines and junipers, and a significant lake level drop. Despite drier conditions, fire activity dropped as consequence of biomass reduction. Two arid intervals occurred between 2.9 and 2.4 cal ka BP and at 1.2-0.7 cal ka BP (800-1300 AD). The latter coincides with the Medieval Climate Anomaly and is one of the most arid phases of the Holocene in BSM sequence. Anthropogenic disturbances were small until 700 AD, when human pressure over landscape intensified, with Olea cultivation in the lowlands and significant deforestation in highlands. Colder and unfavourable weather conditions during the second part of the

  17. 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

  18. A knowledge engineering approach to recognizing and extracting sequences of nucleic acids from scientific literature.

    PubMed

    García-Remesal, Miguel; Maojo, Victor; Crespo, José

    2010-01-01

    In this paper we present a knowledge engineering approach to automatically recognize and extract genetic sequences from scientific articles. To carry out this task, we use a preliminary recognizer based on a finite state machine to extract all candidate DNA/RNA sequences. The latter are then fed into a knowledge-based system that automatically discards false positives and refines noisy and incorrectly merged sequences. We created the knowledge base by manually analyzing different manuscripts containing genetic sequences. Our approach was evaluated using a test set of 211 full-text articles in PDF format containing 3134 genetic sequences. For such set, we achieved 87.76% precision and 97.70% recall respectively. This method can facilitate different research tasks. These include text mining, information extraction, and information retrieval research dealing with large collections of documents containing genetic sequences.

  19. A knowledge engineering approach to recognizing and extracting sequences of nucleic acids from scientific literature.

    PubMed

    García-Remesal, Miguel; Maojo, Victor; Crespo, José

    2010-01-01

    In this paper we present a knowledge engineering approach to automatically recognize and extract genetic sequences from scientific articles. To carry out this task, we use a preliminary recognizer based on a finite state machine to extract all candidate DNA/RNA sequences. The latter are then fed into a knowledge-based system that automatically discards false positives and refines noisy and incorrectly merged sequences. We created the knowledge base by manually analyzing different manuscripts containing genetic sequences. Our approach was evaluated using a test set of 211 full-text articles in PDF format containing 3134 genetic sequences. For such set, we achieved 87.76% precision and 97.70% recall respectively. This method can facilitate different research tasks. These include text mining, information extraction, and information retrieval research dealing with large collections of documents containing genetic sequences. PMID:21096556

  20. Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

    PubMed Central

    2009-01-01

    A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA. PMID:21637462

  1. Identification of novel rice low phytic acid mutations via TILLING by sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate or InsP6) accounts for 75-85% of the total phosphorus in seeds. Low phytic acid (lpa) mutants exhibit decreases in seed InsP6 with corresponding increases in inorganic P which, unlike phytic acid P, is readily utilized by humans and monogastric ...

  2. Complete amino acid sequence of an acidic, cardiotoxic phospholipase A2 from the venom of Ophiophagus hannah (King Cobra): a novel cobra venom enzyme with "pancreatic loop".

    PubMed

    Huang, M Z; Gopalakrishnakone, P; Chung, M C; Kini, R M

    1997-02-15

    A phospholipase A2 (OHV A-PLA2) from the venom of Ophiophagus hannah (King cobra) is an acidic protein exhibiting cardiotoxicity, myotoxicity, and antiplatelet activity. The complete amino acid sequence of OHV A-PLA2 has been determined using a combination of Edman degradation and mass spectrometric techniques. OHV A-PLA2 is composed of a single chain of 124 amino acid residues with 14 cysteines and a calculated molecular weight of 13719 Da. It contains the loop of residues (62-66) found in pancreatic PLA2s and hence belongs to class IB enzymes. This pancreatic loop is between two proline residues (Pro 59 and Pro 68) and contains several hydrophilic amino acids (Ser and Asp). This region has high degree of conformational flexibility and is on the surface of the molecule, and hence it may be a potential protein-protein interaction site. A relatively low sequence homology is found between OHV A-PLA2 and other known cardiotoxic PLA2s, and hence a contiguous segment could not be identified as a site responsible for the cardiotoxic activity.

  3. Evaluation of a novel food composition database that includes glutamine and other amino acids derived from gene sequencing data

    PubMed Central

    Lenders, CM; Liu, S; Wilmore, DW; Sampson, L; Dougherty, LW; Spiegelman, D; Willett, WC

    2011-01-01

    Objectives To determine the content of glutamine in major food proteins. Subjects/Methods We used a validated 131-food item food frequency questionnaire (FFQ) to identify the foods that contributed the most to protein intake among 70 356 women in the Nurses’ Health Study (NHS, 1984). The content of glutamine and other amino acids in foods was calculated based on protein fractions generated from gene sequencing methods (Swiss Institute of Bioinformatics) and compared with data from conventional (USDA) and modified biochemical (Khun) methods. Pearson correlation coefficients were used to compare the participants’ dietary intakes of amino acids by sequencing and USDA methods. Results The glutamine content varied from 0.01 to to 9.49 g/100 g of food and contributed from 1 to to 33% of total protein for all FFQ foods with protein. When comparing the sequencing and Kuhn’s methods, the proportion of glutamine in meat was 4.8 vs 4.4%. Among NHS participants, mean glutamine intake was 6.84 (s.d.=2.19) g/day and correlation coefficients for amino acid between intakes assessed by sequencing and USDA methods ranged from 0.94 to 0.99 for absolute intake, −0.08 to 0.90 after adjusting for 100 g of protein, and 0.88 to 0.99 after adjusting for 1000 kcal. The between-person coefficient of variation of energy-adjusted intake of glutamine was 16%. Conclusions These data suggest that (1) glutamine content can be estimated from gene sequencing methods and (2) there is a reasonably wide variation in energy-adjusted glutamine intake, allowing for exploration of glutamine consumption and disease. PMID:19756030

  4. The amino acid sequences of the cytochromes c553 from Porphyridium cruentum and Aphanizomenon flos-aquae.

    PubMed

    Sprinkle, J R; Hermodson, M; Krogmann, D W

    1986-01-01

    The amino acid sequences of cytochrome c553 from the eukaryotic red alga Porphyridium cruentum and from the prokaryotic cyanobacterium Aphanizomenon flos-aquae have been determined from the tryptic and cyanogen bromide peptides. The results indicate that a charged region of these proteins has evolved with special rapidity to accomodate a rapid evolution of a binding site in the P700 electron acceptor complex.

  5. First draft genome sequencing of indole acetic acid producing and plant growth promoting fungus Preussia sp. BSL10.

    PubMed

    Khan, Abdul Latif; Asaf, Sajjad; Khan, Abdur Rahim; Al-Harrasi, Ahmed; Al-Rawahi, Ahmed; Lee, In-Jung

    2016-05-10

    Preussia sp. BSL10, family Sporormiaceae, was actively producing phytohormone (indole-3-acetic acid) and extra-cellular enzymes (phosphatases and glucosidases). The fungus was also promoting the growth of arid-land tree-Boswellia sacra. Looking at such prospects of this fungus, we sequenced its draft genome for the first time. The Illumina based sequence analysis reveals an approximate genome size of 31.4Mbp for Preussia sp. BSL10. Based on ab initio gene prediction, total 32,312 coding sequences were annotated consisting of 11,967 coding genes, pseudogenes, and 221 tRNA genes. Furthermore, 321 carbohydrate-active enzymes were predicted and classified into many functional families. PMID:26995610

  6. The amino acid sequence of GTP:AMP phosphotransferase from beef-heart mitochondria. Extensive homology with cytosolic adenylate kinase.

    PubMed

    Wieland, B; Tomasselli, A G; Noda, L H; Frank, R; Schulz, G E

    1984-09-01

    The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three-dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.

  7. Fusion protein predicted amino acid sequence of the first US avian pneumovirus isolate and lack of heterogeneity among other US isolates.

    PubMed

    Seal, B S; Sellers, H S; Meinersmann, R J

    2000-02-01

    Avian pneumovirus (APV) was first isolated from turkeys in the west-central US following emergence of turkey rhinotracheitis (TRT) during 1996. Subsequently, several APV isolates were obtained from the north-central US. Matrix (M) and fusion (F) protein genes of these isolates were examined for sequence heterogeneity and compared with European APV subtypes A and B. Among US isolates the M gene shared greater than 98% nucleotide sequence identity with only one nonsynonymous change occurring in a single US isolate. Although the F gene among US APV isolates shared 98% nucleotide sequence identity, nine conserved substitutions were detected in the predicted amino acid sequence. The predicted amino acid sequence of the US APV isolate's F protein had 72% sequence identity to the F protein of APV subtype A and 71% sequence identity to the F protein of APV subtype B. This compares with 83% sequence identity between the APV subtype A and B predicted amino acid sequences of the F protein. The US isolates were phylogenetically distinguishable from their European counterparts based on F gene nucleotide or predicted amino acid sequences. Lack of sequence heterogeneity among US APV subtypes indicates these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the F protein among APV isolates supports classification of US isolates as a new APV subtype C.

  8. Amino acid sequence and glycosylation of functional unit RtH2-e from Rapana thomasiana (gastropod) hemocyanin.

    PubMed

    Stoeva, Stanka; Idakieva, Krasimira; Betzel, Christian; Genov, Nicolay; Voelter, Wolfgang

    2002-03-15

    The complete amino acid sequence of Rapana thomasiana hemocyanin functional unit RtH2-e was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin, and trypsin. The single-polypeptide chain of RtH2-e consists of 413 amino acid residues and contains two consensus sequences NXS/T (positions 11-19 and 127-129), potential sites for N-glycosylation. Monosaccharide analysis of RtH2-e revealed a carbohydrate content of about 1.1% and the presence of xylose, fucose, mannose, and N-acetylglucosamine, demonstrating that only N-linked carbohydrate chains of high-mannose type seem to be present. On basis of the monosaccharide composition and MALDI-MS analysis of native and PNGase-F-treated chymotryptic glycopeptide fragment of RtH2-e the oligosaccharide Man(5)GlcNAc(2), attached to Asn(127), is suggested. Multiple sequence alignments with other molluscan hemocyanin e functional units revealed an identity of 63% to the cephalopod Octopus dofleini and of 69% to the gastropod Haliotis tuberculata. The present results are discussed in view of the recently determined X-ray structure of the functional unit g of the O. dofleini hemocyanin. PMID:11888200

  9. Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor

    PubMed Central

    1988-01-01

    Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'- untranslated region of 2.2 kb. The open reading frame encodes a 1,245- residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells. PMID:3264556

  10. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family.

    PubMed

    Ito, Y; Yoshikawa, A; Hotani, T; Fukuda, S; Sugimura, K; Imoto, T

    1999-01-01

    Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features.

  11. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family.

    PubMed

    Ito, Y; Yoshikawa, A; Hotani, T; Fukuda, S; Sugimura, K; Imoto, T

    1999-01-01

    Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features. PMID:9914527

  12. Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance

    PubMed Central

    Baranzoni, Gian Marco; Reichenberger, Erin R.; Kim, Gwang-Hee; Breidt, Frederick; Kay, Kathryn; Oh, Deog-Hwan

    2016-01-01

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here. PMID:27469964

  13. Complete genome sequences of Escherichia coli O157:H7 strains SRCC 1675 and 28RC that vary in acid resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented....

  14. Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance.

    PubMed

    Baranzoni, Gian Marco; Fratamico, Pina M; Reichenberger, Erin R; Kim, Gwang-Hee; Breidt, Frederick; Kay, Kathryn; Oh, Deog-Hwan

    2016-01-01

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here. PMID:27469964

  15. Buffalo (Bubalus bubalis) interleukin-2: sequence analysis reveals high nucleotide and amino acid identity with interleukin-2 of cattle and other ruminants.

    PubMed

    Sreekumar, E; Premraj, A; Saravanakumar, M; Rasool, T J

    2002-08-01

    A 4400-bp genomic sequence and a 332-bp truncated cDNA sequence of the interleukin-2 (IL-2) gene of Indian water buffalo (Bubalus bubalis) were amplified by polymerase chain reaction and cloned. The coding sequence of the buffalo IL-2 gene was assembled from the 5' end of the genomic clone and the truncated cDNA clone. This sequence had 98.5% nucleotide identity and 98% amino acid identity with cattle IL-2. Three amino acid substitutions were observed at positions 63, 124 and 135. Comparison of the predicted protein structure of buffalo IL-2 with that of human and cattle IL-2 did not reveal significant differences. The putative amino acids responsible for IL-2 receptor binding were conserved in buffalo, cattle and human IL-2. The amino acid sequence of buffalo IL-2 also showed very high identity with that of other ruminants, indicating functional cross-reactivity.

  16. Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana.

    PubMed

    Uchikoba, T; Arima, K; Yonezawa, H; Shimada, M; Kaneda, M

    2000-10-18

    A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit enlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain. The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.

  17. Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3)

    PubMed Central

    1988-01-01

    Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion- promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+- binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins. PMID:2454931

  18. 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

  19. Sequence analysis of four acidic beta-crystallin subunits of amphibian lenses: phylogenetic comparison between beta- and gamma-crystallins.

    PubMed

    Lu, S F; Pan, F M; Chiou, S H

    1996-04-16

    beta-Crystallins composed of the most heterogeneous group of subunit chains among the three major crystallin families of vertebrates, i.e. alpha-, beta- and gamma-crystallins, are less well understood at the structural and functional levels than the other two. They comprise a multigene family with at least three basic (betaB1-3) and four acidic (betaA1-4) subunit polypeptides. In order to facilitate the determination of the primary sequences of all these ubiquitous crystallin subunits present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses. We report here a protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta-crystallin acidic subunit polypeptides by polymerase chain reaction (PCR). Four complete full-length reading frames with two each of 597 and 648 base pairs, which cover four deduced protein sequences of 198 (betaA1-1 and betaA1-2) and 215 (betaA3-1 and betaA3-2) amino acids including the universal initiating methionine, were revealed by nucleotide sequencing. They show about 96-98% sequence similarity among themselves and 76-80%, 80-83% to the homologous betaA1/A3 crystallins of bovine and human species respectively, revealing the close structural relationship among acidic subunits of all beta-crystallins even from remotely related species. In this study a phylogenetic comparison based on amino-acid sequences of various betaA1/A3 crystallins plus the major basic beta-crystallin (betaBp) and gamma-crystallin from different vertebrate species is made using a combination of distance matrix and approximate parsimony methods, which correctly groups these betaA crystallin chains together as one family distinct from basic beta-crystallins and gamma-crystallin and further corroborates the supposition that beta- and gamma-crystallins form a superfamily with a common ancestry.

  20. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

    PubMed Central

    Kim, Ki-Hyun; Nielsen, Peter E.; Glazer, Peter M.

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  1. Variable responses of different human cancer cells to the lichen compounds parietin, atranorin, usnic acid and gyrophoric acid.

    PubMed

    Bačkorová, M; Bačkor, M; Mikeš, J; Jendželovský, R; Fedoročko, P

    2011-02-01

    One of the ways for searching for potentially new anti-cancer drugs is the testing of various naturally synthesized compounds. Lichens are a source of unique chemical agents of which some have already been proved to be effective against various cancer in vitro models. Our study reports on the sensitivity of up to nine human cancer cell lines (A2780, HeLa, MCF-7, SK-BR-3, HT-29, HCT-116 p53(+/+), HCT-116 p53(-/-), HL-60 and Jurkat) to the anti-proliferative/cytotoxic effects of four typical secondary metabolites of lichens (parietin, atranorin, usnic acid and gyrophoric acid). Variations in the dynamics of tumour cell line populations were evaluated by the MTT, clonogenic and viability assays, cell proliferation and detachment, cell cycle transition and apoptotic nuclear morphology, thereby confirming their concentration- and time-dependent cytotoxicity. However, in comparison with parietin and gyrophoric acid, the suppression of viability and cell proliferation by usnic acid or atranorin was found to be more efficient at equitoxic doses and correlated more strongly with an increased number of floating cells or a higher apoptotic index. Moreover, the analysis of cell cycle distribution also revealed an accumulation of cells in S-phase. This study has confirmed a differential sensitivity of cancer cell lines to lichen secondary metabolites. PMID:20837130

  2. Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.

    PubMed Central

    Birktoft, J J; Fernley, R T; Bradshaw, R A; Banaszak, L J

    1982-01-01

    The amino acid sequence of porcine heart mitochondrial malate dehydrogenase (mMDH; L-malate: NAD+ oxidoreductase, EC 1.1.1.37) has been compared with the sequences of six different lactate dehydrogenases (LDH; L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and with the "x-ray" sequence of cytoplasmic malate dehydrogenase (sMDH). The main points are that (i) all three enzymes are homologous; (ii) invariant residues in the catalytic center of these enzymes include a histidine and an internally located aspartate that function as a proton relay system; (iii) numerous residues important to coenzyme binding are conserved, including several glycines and charged residues; and (iv) amino acid side chains present in the subunit interface common to the MDHs and LDHs appear to be better conserved than those in the protein interior. It is concluded that LDH, sMDH, and mMDH are derived from a common ancestral gene and probably have similar catalytic mechanisms. PMID:6959107

  3. Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage

    PubMed Central

    Ebhardt, H Alexander; Nan, Jie; Chaulk, Steven G; Fahlman, Richard P; Aebersold, Ruedi

    2014-01-01

    RATIONALE Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues. METHODS Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNAArg onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini. RESULTS We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue. CONCLUSIONS We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd. PMID:25380496

  4. Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu.

    PubMed

    Jenkins, C; Fuerst, J A

    2001-05-01

    Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions. PMID:11443344

  5. Late Glacial to Holocene environmental variabilities: A new multi-proxy paleolimnological study of sedimentary sequences from Como (northern Italy)

    NASA Astrophysics Data System (ADS)

    Höbig, N.; Martinelli, E.; Motella, S.; Michetti, A. M.; Livio, F.; Tinner, W.; Reicherter, K.; Castelletti, L.

    2012-04-01

    Lake Como (northern Italy) is the deepest Italian lake, reaching a depth of about 425 m. The lambda-shaped lake expands about 45 km in NE-SW direction. Southwards of the hydrologically closed western branch, two sediment cores of 70 m (S1) and 65 m length (S2) were taken in the year 2005 close to the cathedral of Como (Piazza Verdi). The drilling sites are located in the middle of the Southern Alps, some 300 m from the present-day lakeshore. The cores provide the first detailed Late Glacial to Holocene multi-proxy record for the Lake Como basin. Our research is aimed at investigating the environmental and geological evolution of the Insubria Region. The multi-proxy study of the stratigraphic sequences contain geophysical, geotechnical, sedimentological, paleobotanical, and radiocarbon analyses. They have been performed for core S1 and are still in progress on core S2. With this data the working group focuses on two main issues. The first topic is the reconstruction of the natural and anthropogenic processes controlling the ground subsidence in the Como urban area (e.g., Comerci et al., 2007) and another aim is to reconstruct vegetation and land-use dynamics. In particular, 150 samples of vegetal macroremains have been collected in the palustrine deposits along S1 core, down to 31,00 m. Below this depth (dated 14C 12,496 ± 55 yr BP - 15,050 - 14,250 cal yr BP), the amount of plant macroremains in the sediment drops dramatically. The taxonomic determination was carried out on more than 800 macroremains. They are represented by fragments of wood, leaves, needles, seeds, fruits, mosses and tiny charcoals (Motella, 2009, unpublished PhD Thesis). Picea/Larix, Pinus sp., Juniperus with Betula, found in the deeper levels (30.80 - 30.00 m), are the first arboreal taxa that colonized the shores of Lake Como, and show that the reforestation began in this area about 16,000 years ago. During the early Holocene (25.10 m) Abies alba expanded and further upwards the sequence

  6. Complete amino acid sequence of human plasma Zn-. cap alpha. /sub 2/-glycoprotein and its homology to histocompatibility antigens

    SciTech Connect

    Araki, T.; Gejyo, F.; Takagaki, K.; Haupt, H.; Schwick, H.G.; Buergi, W.; Marti, T.; Schaller, J.; Rickli, E.; Brossmer, R.

    1988-02-01

    In the present study the complete amino acid sequence of human plasma Zn-..cap alpha../sub 2/-glycoprotein was determined. This protein whose biological function is unknown consists of a single polypeptide chain of 276 amino acid residues including 8 tryptophan residues and has a pyroglutamyl residue at the amino terminus. The location of the two disulfide bonds in the polypeptide chain was also established. The three glycans, whose structure was elucidated with the aid of 500 MHz /sup 1/H NMR spectroscopy, were sialylated N-biantennas. The molecular weight calculated from the polypeptide and carbohydrate structure is 38,478, which is close to the reported value of approx. = 41,000 based on physicochemical measurements. The predicted secondary structure appeared to comprised of 23% ..cap alpha..-helix, 27% ..beta..-sheet, and 22% ..beta..-turns. The three N-glycans were found to be located in ..beta..-turn regions. An unexpected finding was made by computer analysis of the sequence data; this revealed that Zn-..cap alpha../sub 2/-glycoprotein is closely related to antigens of the major histocompatibility complex in amino acid sequence and in domain structure. There was an unusually high degree of sequence homology with the ..cap alpha.. chains of class I histocompatibility antigens. Moreover, this plasma protein was shown to be a member of the immunoglobulin gene superfamily. Zn-..cap alpha../sub 2/-glycoprotein appears to be truncated secretory major histocompatibility complex-related molecule, and it may have a role in the expression of the immune response.

  7. ENTPRISE: An Algorithm for Predicting Human Disease-Associated Amino Acid Substitutions from Sequence Entropy and Predicted Protein Structures

    PubMed Central

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2016-01-01

    The advance of next-generation sequencing technologies has made exome sequencing rapid and relatively inexpensive. A major application of exome sequencing is the identification of genetic variations likely to cause Mendelian diseases. This requires processing large amounts of sequence information and therefore computational approaches that can accurately and efficiently identify the subset of disease-associated variations are needed. The accuracy and high false positive rates of existing computational tools leave much room for improvement. Here, we develop a boosted tree regression machine-learning approach to predict human disease-associated amino acid variations by utilizing a comprehensive combination of protein sequence and structure features. On comparing our method, ENTPRISE, to the state-of-the-art methods SIFT, PolyPhen-2, MUTATIONASSESSOR, MUTATIONTASTER, FATHMM, ENTPRISE exhibits significant improvement. In particular, on a testing dataset consisting of only proteins with balanced disease-associated and neutral variations defined as having the ratio of neutral/disease-associated variations between 0.3 and 3, the Mathews Correlation Coefficient by ENTPRISE is 0.493 as compared to 0.432 by PPH2-HumVar, 0.406 by SIFT, 0.403 by MUTATIONASSESSOR, 0.402 by PPH2-HumDiv, 0.305 by MUTATIONTASTER, and 0.181 by FATHMM. ENTPRISE is then applied to nucleic acid binding proteins in the human proteome. Disease-associated predictions are shown to be highly correlated with the number of protein-protein interactions. Both these predictions and the ENTPRISE server are freely available for academic users as a web service at http://cssb.biology.gatech.edu/entprise/. PMID:26982818

  8. Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

    PubMed

    Danik, M; Chinn, A M; Lafeuillade, B; Keramidas, M; Aguesse-Germon, S; Penhoat, A; Chen, H; Mosher, D F; Chambaz, E M; Feige, J J

    1999-06-01

    Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex. PMID:10342868

  9. ENTPRISE: An Algorithm for Predicting Human Disease-Associated Amino Acid Substitutions from Sequence Entropy and Predicted Protein Structures.

    PubMed

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2016-01-01

    The advance of next-generation sequencing technologies has made exome sequencing rapid and relatively inexpensive. A major application of exome sequencing is the identification of genetic variations likely to cause Mendelian diseases. This requires processing large amounts of sequence information and therefore computational approaches that can accurately and efficiently identify the subset of disease-associated variations are needed. The accuracy and high false positive rates of existing computational tools leave much room for improvement. Here, we develop a boosted tree regression machine-learning approach to predict human disease-associated amino acid variations by utilizing a comprehensive combination of protein sequence and structure features. On comparing our method, ENTPRISE, to the state-of-the-art methods SIFT, PolyPhen-2, MUTATIONASSESSOR, MUTATIONTASTER, FATHMM, ENTPRISE exhibits significant improvement. In particular, on a testing dataset consisting of only proteins with balanced disease-associated and neutral variations defined as having the ratio of neutral/disease-associated variations between 0.3 and 3, the Mathews Correlation Coefficient by ENTPRISE is 0.493 as compared to 0.432 by PPH2-HumVar, 0.406 by SIFT, 0.403 by MUTATIONASSESSOR, 0.402 by PPH2-HumDiv, 0.305 by MUTATIONTASTER, and 0.181 by FATHMM. ENTPRISE is then applied to nucleic acid binding proteins in the human proteome. Disease-associated predictions are shown to be highly correlated with the number of protein-protein interactions. Both these predictions and the ENTPRISE server are freely available for academic users as a web service at http://cssb.biology.gatech.edu/entprise/.

  10. 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

  11. 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

  12. "De-novo" amino acid sequence elucidation of protein G'e by combined "Top-Down" and "Bottom-Up" mass spectrometry

    NASA Astrophysics Data System (ADS)

    Yefremova, Yelena; Al-Majdoub, Mahmoud; Opuni, Kwabena F. M.; Koy, Cornelia; Cui, Weidong; Yan, Yuetian; Gross, Michael L.; Glocker, Michael O.

    2015-03-01

    Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein Ǵ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α- N-gluconoylation and α- N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α- N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant ( K d ) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

  13. "De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

    PubMed

    Yefremova, Yelena; Al-Majdoub, Mahmoud; Opuni, Kwabena F M; Koy, Cornelia; Cui, Weidong; Yan, Yuetian; Gross, Michael L; Glocker, Michael O

    2015-03-01

    Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins. PMID:25560987

  14. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    PubMed

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/. PMID:26424080

  15. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms

    PubMed Central

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/ PMID:26424080

  16. JRC GMO-Amplicons: a collection of nucleic acid sequences related to genetically modified organisms.

    PubMed

    Petrillo, Mauro; Angers-Loustau, Alexandre; Henriksson, Peter; Bonfini, Laura; Patak, Alex; Kreysa, Joachim

    2015-01-01

    The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/.

  17. Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

    PubMed Central

    Rizwan, Muhammad; Rönnberg, Bengt; Cistjakovs, Maksims; Lundkvist, Åke; Pipkorn, Rudiger; Blomberg, Jonas

    2016-01-01

    Background: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides. Methods: We here report that peptides of 100 amino acids or longer (“megapeptides”), designed and synthesized for optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen. Results: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses. Conclusion: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays. PMID:27600087

  18. The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin

    PubMed Central

    Elleman, T. C.

    1972-01-01

    1. The amino acid sequence of a protein from the reduced and carboxymethylated high-sulphur fraction of wool has been determined. 2. The sequence of this S-carboxymethylkerateine (SCMK-B2C) of 151 amino acid residues displays much internal homology and an unusual residue distribution. Thus a ten-residue sequence occurs four times near the N-terminus and five times near the C-terminus with few changes. These regions contain much of the molecule's half-cystine, whereas between them there is a region of 19 residues that are mainly small and devoid of cystine and proline. 3. Certain models of the wool fibre based on its mechanical and physical properties propose a matrix of small compact globular units linked together to form beaded chains. The unusual distribution of the component residues of protein SCMK-B2C suggests structures in the wool-fibre matrix compatible with certain features of the proposed models. PMID:4678578

  19. Amino acid sequence coevolution in the insect bursicon ligand-receptor system.

    PubMed

    Hughes, Austin L

    2012-06-01

    The pattern of amino acid residue replacement in the components of the bursicon signaling system (involving the BURSα/BURSβ heterodimer and its receptor BURSrec) was reconstructed across a phylogeny of 17 insect species, in order to test for the co-occurrence of replacements at sets of individual sites. Sets of three or more branches with perfectly concordant changes occurred to a greater extent than expected by chance, given the observed level of amino acid change. The latter sites (SPC sites) were found to have distinctive characteristics: (1) the mean number of changes was significantly lower at SPC sites than that at other sites with multiple changes; (2) SPC sites had a significantly greater tendency toward parallel amino acid changes than other sites with multiple changes, but no greater tendency toward convergent changes; and (3) parallel changes tended to involve relatively similar amino acids, as indicated by relatively low mean chemical distances. The results implicated functional constraint, permitting only a limited subset of amino acids in a given site, as a major factor in causing both parallel amino acid replacement and coordinated amino acid changes in different sites of the same protein and of interacting proteins in this system.

  20. Sequence Polymorphism and Expression Variability of Crassostrea gigas Immune Related Genes Discriminate Two Oyster Lines Contrasted in Term of Resistance to Summer Mortalities

    PubMed Central

    Schmitt, Paulina; Santini, Adrien; Vergnes, Agnès; Degremont, Lionel; de Lorgeril, Julien

    2013-01-01

    Summer mortalities of Crassostreagigas are a major concern in oyster aquaculture. They are the result of a complex interaction between the host, pathogens and environmental factors. Oyster genetics have been identified as an essential determinant of oyster susceptibility to summer mortalities. As the capability of oysters to circumvent diseases depends in part on their immune defenses, we aimed to analyze the gene expression and sequence polymorphism of 42 immune related genes in two oyster lines selected for their “High” (H) and “Low” (L) survival to summer mortalities. Results showed that the variability of gene expression and the sequence polymorphism acting on particular genes could enable the discrimination between H and L oyster lines. Besides, a higher sequence polymorphism was observed on the L line affecting 11 of the 42 analyzed genes. By analyzing gene expression, sequence polymorphism and gene copy number of two antimicrobial peptide families (Cg-Defs and Cg-Prp), and an antimicrobial protein (Cg-BPI) on individual oysters, we showed that gene expression and/or sequence polymorphism could also discriminate H and L oyster lines. Finally, we observed a positive correlation between the gene expression and the gene copy number of antimicrobials and that sequence polymorphism could be encoded in the genome. Overall, this study gives new insights in the relationship between oyster immunity and divergent phenotypes, and discusses the potential implication of antimicrobial diversity in oyster survival to summer mortalities. PMID:24086661

  1. Characterization of DNA-binding sequences for CcaR in the cephamycin-clavulanic acid supercluster of Streptomyces clavuligerus.

    PubMed

    Santamarta, I; López-García, M T; Kurt, A; Nárdiz, N; Alvarez-Álvarez, R; Pérez-Redondo, R; Martín, J F; Liras, P

    2011-08-01

    RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

  2. The nucleotide sequence of HLA-B{sup *}2704 reveals a new amino acid substitution in exon 4 which is also present in HLA-B{sup *}2706

    SciTech Connect

    Rudwaleit, M.; Bowness, P.; Wordsworth, P.

    1996-12-31

    The HLA-B27 subtype HLA-B{sup *}2704 is virtually absent in Caucasians but common in Orientals, where it is associated with ankylosing spondylitis. The amino acid sequence of HLA-B{sup *}2704 has been established by peptide mapping and was shown to differ by two amino acids from HLA-B{sup *}2705, HLA-B{sup *}2704 is characterized by a serine for aspartic acid substitution at position 77 and glutamic acid for valine at position 152. To date, however, no nucleotide sequence confirming these changes at the DNA level has been published. 13 refs., 2 figs.

  3. The shikimate pathway: review of amino acid sequence, function and three-dimensional structures of the enzymes.

    PubMed

    Mir, Rafia; Jallu, Shais; Singh, T P

    2015-06-01

    The aromatic compounds such as aromatic amino acids, vitamin K and ubiquinone are important prerequisites for the metabolism of an organism. All organisms can synthesize these aromatic metabolites through shikimate pathway, except for mammals which are dependent on their diet for these compounds. The pathway converts phosphoenolpyruvate and erythrose 4-phosphate to chorismate through seven enzymatically catalyzed steps and chorismate serves as a precursor for the synthesis of variety of aromatic compounds. These enzymes have shown to play a vital role for the viability of microorganisms and thus are suggested to present attractive molecular targets for the design of novel antimicrobial drugs. This review focuses on the seven enzymes of the shikimate pathway, highlighting their primary sequences, functions and three-dimensional structures. The understanding of their active site amino acid maps, functions and three-dimensional structures will provide a framework on which the rational design of antimicrobial drugs would be based. Comparing the full length amino acid sequences and the X-ray crystal structures of these enzymes from bacteria, fungi and plant sources would contribute in designing a specific drug and/or in developing broad-spectrum compounds with efficacy against a variety of pathogens.

  4. Characterization of fatty acid-producing wastewater microbial communities using next generation sequencing technologies

    EPA Science Inventory

    While wastewater represents a viable source of bacterial biodiesel production, very little is known on the composition of these microbial communities. We studied the taxonomic diversity and succession of microbial communities in bioreactors accumulating fatty acids using 454-pyro...

  5. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of WIPO... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid...

  6. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of WIPO... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid...

  7. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of WIPO... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid...

  8. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of WIPO... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid...

  9. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of WIPO... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid...

  10. Complete amino acid sequence of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris.

    PubMed

    Lehman, L D; Jones, B N; Dwulet, F E; Bogardt, R A; Gurd, F R

    1980-10-21

    The complete primary structure of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the apomyoglobin at its two methionine residues with cyanogen bromide along with the four peptides resulting from the cleavage with trypsin of the citraconylated apomyoglobin at its three arginine residues. Further digestion of the central cyanogen bromide peptide with S. aureus strain V8 protease and the 1,2-cyclohexanedione-treated central cyanogen bromide peptide with trypsin enabled the determination of the remainder of the covalent structure. This myoglobin differs from the cetacean myoglobins determined to date at 12 to 17 positions. These large sequence differences reflect the distant taxonomic relationships between the goose-beaked whale and the other species of Cetacea the myoglobin sequences of which have previously been determined.

  11. Peptide mapping and amino acid sequencing of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24.

    PubMed

    Kim, S I; Ha, K S

    1997-10-31

    The partial amino acid sequences of two catechol 1,2-dioxygenases (CD I1 and CD I2) from Acinetobacter lwoffii K24 have been determined by analysis of peptides after cleavages with endopeptidase Lys-C, endopeptidase Glu-C, trypsin, and chemicals (cyanogen bromide and BNPS-skatole). They include 248 amino acid sequences (4 fragments) of CD I1 and 211 amino acid sequences (5 fragments) of CD I2. Two enzymes have more than 50% sequence homology with type I catechol 1,2-dioxygenases and less than 30% sequence homology with type II catechol 1,2-dioxygenases. Two enzymes have similar hydropathy profiles in the N-terminal region, suggesting that they have similar secondary structures. PMID:9387151

  12. Assessment of sequence variability in a p23 gene region within and among three genotypes of the Theileria orientalis complex from south-eastern Australia.

    PubMed

    Perera, Piyumali K; Gasser, Robin B; Jabbar, Abdul

    2015-03-01

    Oriental theileriosis is a tick-borne, protozoan disease of cattle caused by one or more genotypes of Theileria orientalis complex. In this study, we assessed sequence variability in a region of the 23kDa piroplasm membrane protein (p23) gene within and among three T. orientalis genotypes (designated buffeli, chitose and ikeda) in south-eastern Australia. Genomic DNA (n=100) was extracted from blood of infected cattle from various locations endemic for oriental theileriosis and tested by polymerase chain reaction (PCR)-coupled mutation scanning (single-strand conformation polymorphism (SSCP)) and targeted sequencing analysis. Eight distinct sequences represented all DNA samples, and three genotypes were found: buffeli (n=3), chitose (3) and ikeda (2). Nucleotide pairwise comparisons among these eight sequences revealed considerably higher variability among the genotypes (6.6-11.7%) than within them (0-1.9%), indicating that the p23 gene region allows the accurate identification of T. orientalis genotypes. In the future, we will combine this gene with other molecular markers to study the genetic structure of T. orientalis populations in Australasia, which will pave the way to establish a highly sensitive and specific PCR-based assay for genotypic diagnosis of infection and for assessing levels of parasitaemia in cattle.

  13. New Insights into Poly(Lactic-co-glycolic acid) Microstructure: Using Repeating Sequence Copolymers to Decipher Complex NMR and Thermal Behavior

    PubMed Central

    Stayshich, Ryan M.; Meyer, Tara Y.

    2012-01-01

    Sequence, which Nature uses to spectacular advantage, has not been fully exploited in synthetic copolymers. To investigate the effect of sequence and stereosequence on the physical properties of copolymers a family of complex isotactic, syndiotactic and atactic repeating sequence poly(lactic-co-glycolic acid) copolymers (RSC PLGAs) were prepared and their NMR and thermal behavior was studied. The unique suitability of polymers prepared from the bioassimilable lactic and glycolic acid monomers for biomedical applications makes them ideal candidates for this type of sequence engineering. Polymers with repeating units of LG, GLG and LLG (L = lactic, G = glycolic) with controlled and varied tacticities were synthesized by assembly of sequence specific, stereopure dimeric, trimeric and hexameric segmer units. Specifically labeled deuterated lactic and glycolic acid segmers were likewise prepared and polymerized. Molecular weights for the copolymers ranged from Mn = 12-40 kDa by size exclusion chromatography in THF. Although the effects of sequence-influenced solution conformation were visible in all resonances of the 1H and 13C NMR spectra, the diastereotopic methylene resonances in the 1H NMR (CDCl3) for the glycolic units of the copolymers proved most sensitive. An octad level of resolution, which corresponds to an astounding 31-atom distance between the most separated stereocenters, was observed in some mixed sequence polymers. Importantly, the level of sensitivity of a particular NMR resonance to small differences in sequence was found to depend on the sequence itself. Thermal properties were also correlated with sequence. PMID:20681726

  14. Effects of the amino acid sequence on thermal conduction through β-sheet crystals of natural silk protein.

    PubMed

    Zhang, Lin; Bai, Zhitong; Ban, Heng; Liu, Ling

    2015-11-21

    Recent experiments have discovered very different thermal conductivities between the spider silk and the silkworm silk. Decoding the molecular mechanisms underpinning the distinct thermal properties may guide the rational design of synthetic silk materials and other biomaterials for multifunctionality and tunable properties. However, such an understanding is lacking, mainly due to the complex structure and phonon physics associated with the silk materials. Here, using non-equilibrium molecular dynamics, we demonstrate that the amino acid sequence plays a key role in the thermal conduction process through β-sheets, essential building blocks of natural silks and a variety of other biomaterials. Three representative β-sheet types, i.e. poly-A, poly-(GA), and poly-G, are shown to have distinct structural features and phonon dynamics leading to different thermal conductivities. A fundamental understanding of the sequence effects may stimulate the design and engineering of polymers and biopolymers for desired thermal properties. PMID:26455593

  15. Amino acid sequence of myoglobin from the chiton Liolophura japonica and a phylogenetic tree for molluscan globins.

    PubMed

    Suzuki, T; Furukohri, T; Okamoto, S

    1993-02-01

    Myoglobin was isolated from the radular muscle of the chiton Liolophura japonica, a primitive archigastropodic mollusc. Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved in Liolophura myoglobin. The autoxidation rate at physiological conditions indicated that Liolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence of Liolophura myoglobin shows low homology (11-21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26-29%) with monomeric myoglobins from the gastropodic molluscs Aplysia, Dolabella, and Bursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively. Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clams Anadara, Scapharca, and Barbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiontharboring clams Calyptogena and Lucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.

  16. 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

  17. Draft Genome Sequence of Ustilago trichophora RK089, a Promising Malic Acid Producer.

    PubMed

    Zambanini, Thiemo; Buescher, Joerg M; Meurer, Guido; Wierckx, Nick; Blank, Lars M

    2016-01-01

    The basidiomycetous smut fungus Ustilago trichophora RK089 produces malate from glycerol. De novo genome sequencing revealed a 20.7-Mbp genome (301 gap-closed contigs, 246 scaffolds). A comparison to the genome of Ustilago maydis 521 revealed all essential genes for malate production from glycerol contributing to metabolic engineering for improving malate production. PMID:27469969

  18. Rapid Nucleic Acid Sequencing Methods--Alternative Approaches to Facilitating Learning.

    ERIC Educational Resources Information Center

    Bryce, Charles F. A.

    1982-01-01

    Because advanced students had difficulty in interpreting cleavage patterns obtained by gel electrophoresis related to rapid sequencing techniques for DNA and RNA, several formats were developed to aid in understanding this topic. Formats included print, print plus scrambled print, interactive computer-based instruction, and high-resolution…

  19. Isolation and amino acid sequence of crustacean hyperglycemic hormone precursor-related peptides.

    PubMed

    Tensen, C P; Verhoeven, A H; Gaus, G; Janssen, K P; Keller, R; Van Herp, F

    1991-01-01

    The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.

  20. Draft Genome Sequence of Ustilago trichophora RK089, a Promising Malic Acid Producer

    PubMed Central

    Zambanini, Thiemo; Buescher, Joerg M.; Meurer, Guido; Blank, Lars M.

    2016-01-01

    The basidiomycetous smut fungus Ustilago trichophora RK089 produces malate from glycerol. De novo genome sequencing revealed a 20.7-Mbp genome (301 gap-closed contigs, 246 scaffolds). A comparison to the genome of Ustilago maydis 521 revealed all essential genes for malate production from glycerol contributing to metabolic engineering for improving malate production. PMID:27469969

  1. Effect of production variables on the physicochemical characteristics of celecoxib-loaded stearic and alginic acids-based microparticles.

    PubMed

    Shunmugaperumal, Tamilvanan; Sharma, Deepak; Thakur, Ashutosh; Vinaykumar

    2013-12-01

    The objectives of the current investigation were (1) to prepare the microparticles based on stearic and alginic acids from an aqueous system by hot (melt) dispersion method, (2) to achieve a higher drug entrapment efficiency and process yield (%) by changing the production variables such as stirring speed, concentration of stabilizer in aqueous dispersion medium, volume of aqueous dispersion medium, and stirring time, and (3) to see whether or not a retardation in drug release profile was attained from the celecoxib-loaded stearic and alginic acids-based microparticles compared to that of the celecoxib alone. The addition of alginic acid into stearic acid produced spherical-shaped particles with an almost smooth surface. Higher drug entrapment efficiency and process yield (%) values were obtained when the microparticles were prepared at 1000 r/min using 0.1% w/v polyvinyl alcohol in 100 mL aqueous dispersion medium and 30 min stirring time. The in vitro dissolution study in 900 mL of 2% sodium lauryl sulfate (SLS) solution at 75 r/min, however, showed only around 10% retardation in drug release from microparticles compared to the drug release from pure celecoxib alone. This indicated that the gel-like network formed by the alginic acid around the microparticles could not prevent the drug leakage from the microparticles.

  2. The sequence of rat leukosialin (W3/13 antigen) reveals a molecule with O-linked glycosylation of one third of its extracellular amino acids.

    PubMed Central

    Killeen, N; Barclay, A N; Willis, A C; Williams, A F

    1987-01-01

    Leukosialin is one of the major glycoproteins of thymocytes and T lymphocytes and is notable for a very high content of O-linked carbohydrate structures. The full protein sequence for rat leukosialin as translated from cDNA clones is now reported. The molecule contains 371 amino acids with 224 residues outside the cell, one transmembrane sequence and 124 cytoplasmic residues. Data from the peptide sequence and carbohydrate composition suggest that one in three of the extracellular amino acids may be O-glycosylated with no N-linked glycosylation sites. The cDNA sequence contained a CpG rich region in the 3' coding sequence and a large 3' non-coding region which included tandem repeats of the sequence GGAT. Images Fig. 4. PMID:2965006

  3. Amino acid sequences of the alpha and beta chains of adult hemoglobin of the slender loris, Loris tardigradus.

    PubMed

    Maita, T; Goodman, M; Matsuda, G

    1978-08-01

    alpha and beta chains from adult hemoglobin of the slender loris (Loris tardigradus) were isolated by Amberlite CG-50 column chromatography. After S-aminoethylation, both chains were digested with trypsin and the amino acid sequences of the tryptic peptides obtained were analyzed. Further, the order of these tryptic peptides in each chain was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the slender loris thus obtained with those of adult hemoglobin of the slow loris, 4 amino acid substitutions in the alpha chains and 2 in the beta chains were recognized.

  4. An Interpretation of the Ancestral Codon from Miller’s Amino Acids and Nucleotide Correlations in Modern Coding Sequences

    PubMed Central

    Carels, Nicolas; de Leon, Miguel Ponce

    2015-01-01

    Purine bias, which is usually referred to as an “ancestral codon”, is known to result in short-range correlations between nucleotides in coding sequences, and it is common in all species. We demonstrate that RWY is a more appropriate pattern than the classical RNY, and purine bias (Rrr) is the product of a network of nucleotide compensations induced by functional constraints on the physicochemical properties of proteins. Through deductions from universal correlation properties, we also demonstrate that amino acids from Miller’s spark discharge experiment are compatible with functional primeval proteins at the dawn of living cell radiation on earth. These amino acids match the hydropathy and secondary structures of modern proteins. PMID:25922573

  5. A comparative study of fatty acid profile and formation of biofilm in Geobacillus gargensis exposed to variable abiotic stress.

    PubMed

    Al-Beloshei, Noor Essa; Al-Awadhi, Husain; Al-Khalaf, Rania A; Afzal, Mohammad

    2015-01-01

    Understanding bacterial fatty acid (FA) profile has a great taxonomic significance as well as clinical importance for diagnosis issues. Both the composition and nature of membrane FAs change under different nutritional, biotic and (or) abiotic stresses, and environmental stress. Bacteria produce both odd-carbon as well as branched-chain fatty acids (BCFAs). This study was designed to examine the effect of abiotic pressure, including salinity, temperature, pH, and oxinic stress on the growth, development, and FA profile in thermophilic Geobacillus gargensis. Under these stresses, 3 parametric ratios, 2-methyl fatty acids/3-methyl fatty acids (iso-/anteiso-FAs), BCFAs/straight-chain saturated fatty acids (SCSFA), and SCSFAs/straight-chain unsaturated fatty acids (SCUFA), in addition to total lipids affected by variable stresses were measured. Our results indicate that the ratio of total iso-/anteiso-FAs increased at the acidic pH range of 4.1-5.2 and decreased with increasing pH. The reverse was true for salt stress when iso-/anteiso-FAs ratio increased with salt concentration. The BCFAs/SCSFAs and SCSFAs/SCUFAs ratios increased at neutral and alkaline pH and high salt concentration, reduced incubation time, and comparatively high temperature (55-65 °C) of the growth medium. The bacterial total lipid percentage deceased with increasing salt concentration, incubation period, but it increased with temperature. The formation of extracellular polymeric substances was observed under all stress conditions and with the addition of sodium dodecyl sulfate (2 and 5 mmol/L) to the growth medium. The membrane phospholipid composition of the bacterium was analyzed by thin-layer chromatography.

  6. A comparative study of fatty acid profile and formation of biofilm in Geobacillus gargensis exposed to variable abiotic stress.

    PubMed

    Al-Beloshei, Noor Essa; Al-Awadhi, Husain; Al-Khalaf, Rania A; Afzal, Mohammad

    2015-01-01

    Understanding bacterial fatty acid (FA) profile has a great taxonomic significance as well as clinical importance for diagnosis issues. Both the composition and nature of membrane FAs change under different nutritional, biotic and (or) abiotic stresses, and environmental stress. Bacteria produce both odd-carbon as well as branched-chain fatty acids (BCFAs). This study was designed to examine the effect of abiotic pressure, including salinity, temperature, pH, and oxinic stress on the growth, development, and FA profile in thermophilic Geobacillus gargensis. Under these stresses, 3 parametric ratios, 2-methyl fatty acids/3-methyl fatty acids (iso-/anteiso-FAs), BCFAs/straight-chain saturated fatty acids (SCSFA), and SCSFAs/straight-chain unsaturated fatty acids (SCUFA), in addition to total lipids affected by variable stresses were measured. Our results indicate that the ratio of total iso-/anteiso-FAs increased at the acidic pH range of 4.1-5.2 and decreased with increasing pH. The reverse was true for salt stress when iso-/anteiso-FAs ratio increased with salt concentration. The BCFAs/SCSFAs and SCSFAs/SCUFAs ratios increased at neutral and alkaline pH and high salt concentration, reduced incubation time, and comparatively high temperature (55-65 °C) of the growth medium. The bacterial total lipid percentage deceased with increasing salt concentration, incubation period, but it increased with temperature. The formation of extracellular polymeric substances was observed under all stress conditions and with the addition of sodium dodecyl sulfate (2 and 5 mmol/L) to the growth medium. The membrane phospholipid composition of the bacterium was analyzed by thin-layer chromatography. PMID:25496258

  7. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... submissions in computer readable form. (a) The computer readable form required by § 1.821(e) shall meet the following requirements: (1) The computer readable form shall contain a single “Sequence Listing” as either...

  8. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... submissions in computer readable form. (a) The computer readable form required by § 1.821(e) shall meet the following requirements: (1) The computer readable form shall contain a single “Sequence Listing” as either...

  9. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... submissions in computer readable form. (a) The computer readable form required by § 1.821(e) shall meet the following requirements: (1) The computer readable form shall contain a single “Sequence Listing” as either...

  10. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... submissions in computer readable form. (a) The computer readable form required by § 1.821(e) shall meet the following requirements: (1) The computer readable form shall contain a single “Sequence Listing” as either...

  11. 37 CFR 1.824 - Form and format for nucleotide and/or amino acid sequence submissions in computer readable form.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... nucleotide and/or amino acid sequence submissions in computer readable form. 1.824 Section 1.824 Patents... submissions in computer readable form. (a) The computer readable form required by § 1.821(e) shall meet the following requirements: (1) The computer readable form shall contain a single “Sequence Listing” as either...

  12. DNA sequence variability of IGHG3 alleles associated to the main G3m haplotypes in human populations.

    PubMed

    Dard, P; Lefranc, M P; Osipova, L; Sanchez-Mazas, A

    2001-10-01

    The present study investigates the molecular basis of the G3m polymorphism expressed by the heavy constant domains of human immunoglobulins gamma 3 chains. By using a new protocol allowing the specific cloning of IGHG3 genes, a total of 51 full-length IGHG3 genomic sequences (about 2 kb) isolated from African, Siberian, West Asian and European population samples were sequenced. IGHG3 sequences were assigned precise G3m haplotypes on the basis of specific associations between G3m allotypes and IGHG3 RFLPs. Specific DNA substitutions involved in the expression of G3m(5), G3m(6), G3m(15), G3m(16), G3m(21), G3m(24) and G3m(28) allotypes were then deduced, elucidating almost completely the determination of the G3m polymorphism at the DNA level. The molecular evolution of G3m haplotypes was investigated by a maximum likelihood phylogeny of IGHG3 sequences. Sequence clusters are shown to be G3m haplotype-specific, corroborating the Gm molecular model deduced from serology, and showing that populations differentiation is much more recent than G3m haplotypes differentiation. The widely distributed G3m(5,10,11,13,14) haplotype is likely to be ancestral to the other G3m haplotypes presently found at high frequencies in different continental areas.

  13. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

    SciTech Connect

    Xie, Gary; Dalin, Eileen; Tice, Hope; Chertkov, Olga; Land, Miriam L

    2011-01-01

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 C and pH 5.0 and fer-ments 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 hemi-cellulose. This bacterium is also considered as a potential probiotic. Complete genome squence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

  14. Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

    SciTech Connect

    Rhee, Mun Su; Moritz, Brelan E.; Xie, Gary; Glavina Del Rio, Tijana; Dalin, Eileen; Tice, Hope; Bruce, David; Goodwin, Lynne A.; Chertkov, Olga; Brettin, Thomas S; Han, Cliff; Detter, J. Chris; Pitluck, Sam; Land, Miriam L; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O.; Shanmugam, Keelnathan T.

    2011-01-01

    Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 C and pH 5.0 and fer- ments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this spo- rogenic lactic acid bacterium to grow at 50-55 C and pH 5.0 makes this organism an attrac- tive 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 hemi- cellulose. This bacterium is also considered as a potential probiotic. Complete genome se- quence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

  15. Detection of peptidic sequences in the ancient acidic sediments of Río Tinto, Spain.

    PubMed

    Colín-García, María; Kanawati, Basem; Harir, Mourad; Schmitt-Kopplin, Phillippe; Amils, Ricardo; Parro, Victor; García, Miriam; Fernández-Remolar, David

    2011-12-01

    Biomarkers are molecules that are produced by or can be associated with biological activities. They can be used as tracers that give us an idea of the ancient biological communities that produced them, the paleoenvironmental conditions where they lived, or the mechanism involved in their transformation and preservation. As a consequence, the preservation potential of molecules over time depends largely on their nature, but also on the conditions of the environment, which controls the decomposition kinetics. In this context, proteins and nucleic acids, which are biomolecules bearing biological information, are among the most labile molecules. In this research, we report the presence of short-chained peptides obtained from extracts of ferruginous sedimentary deposits that have been produced under the acidic and oxidizing solutions of Río Tinto, Spain. These preliminary results go against the paradigmatic idea that considers the acidic and oxidizing environments inappropriate for the preservation of molecular information.

  16. Contemporaneous deposition of phyllosilicates and sulfates: Using Australian acidic saline lake deposits to describe geochemical variability on Mars

    USGS Publications Warehouse

    Baldridge, A.M.; Hook, S.J.; Crowley, J.K.; Marion, G.M.; Kargel, J.S.; Michalski, J.L.; Thomson, B.J.; de Souza, Filho C.R.; Bridges, N.T.; Brown, A.J.

    2009-01-01

    Studies of the origin of the Martian sulfate and phyllosilicate deposits have led to the hypothesis that there was a marked, global-scale change in the Mars environment from circum-neutral pH aqueous alteration in the Noachian to an acidic evaporitic system in the late Noachian to Hesperian. However, terrestrial studies suggest that two different geochemical systems need not be invoked to explain such geochemical variation.Western Australian acidic playa lakes have large pH differences separated vertically and laterally by only a few tens of meters, demonstrating how highly variable chemistries can coexist over short distances in natural environments. We suggest diverse and variable Martian aqueous environments where the coetaneous formation of phyllosilicates and sulfates at the Australian sites are analogs for regions where phyllosilicates and sulfates coexist on Mars. In these systems, Fe and alkali earth phyllosilicates represent deep facies associated with upwelling neutral to alkaline groundwater, whereas aluminous phyllosilicates and sulfates represent near-surface evaporitic facies formed from more acidic brines. Copyright 2009 by the American Geophysical Union.

  17. High Sequence Variability of the ppE18 Gene of Clinical Mycobacterium tuberculosis Complex Strains Potentially Impacts Effectivity of Vaccine Candidate M72/AS01E.

    PubMed

    Homolka, Susanne; Ubben, Tanja; Niemann, Stefan

    2016-01-01

    The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies.

  18. Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

    SciTech Connect

    Safford, R.; de Silva, J.; Lucas, C.; Windust, J.H.C.; Shedden, J.; James, C.M.; Sidebottom, C.M.; Slabas, A.R.; Tombs, M.P.; Hughes, S.G.

    1987-03-10

    Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from approx. 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH.

  19. Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Abscisic Acid-Producing Botrytis cinerea

    PubMed Central

    Gong, Tao; Shu, Dan; Yang, Jie; Ding, Zhong-Tao; Tan, Hong

    2014-01-01

    Botrytis cinerea is a model species with great importance as a pathogen of plants and has become used for biotechnological production of ABA. The ABA cluster of B. cinerea is composed of an open reading frame without significant similarities (bcaba3), followed by the genes (bcaba1 and bcaba2) encoding P450 monooxygenases and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). In B. cinerea ATCC58025, targeted inactivation of the genes in the cluster suggested at least three genes responsible for the hydroxylation at carbon atom C-1' and C-4' or oxidation at C-4' of ABA. Our group has identified an ABA-overproducing strain, B. cinerea TB-3-H8. To differentiate TB-3-H8 from other B. cinerea strains with the functional ABA cluster, the DNA sequence of the 12.11-kb region containing the cluster of B. cinerea TB-3-H8 was determined. Full-length cDNAs were also isolated for bcaba1, bcaba2, bcaba3 and bcaba4 from B. cinerea TB-3-H8. Sequence comparison of the four genes and their flanking regions respectively derived from B. cinerea TB-3-H8, B05.10 and T4 revealed that major variations were located in intergenic sequences. In B. cinerea TB-3-H8, the expression profiles of the four function genes under ABA high-yield conditions were also analyzed by real-time PCR. PMID:25268614

  20. Complete Genome Sequence of Moraxella osloensis Strain KMC41, a Producer of 4-Methyl-3-Hexenoic Acid, a Major Malodor Compound in Laundry.

    PubMed

    Goto, Takatsugu; Hirakawa, Hideki; Morita, Yuji; Tomida, Junko; Sato, Jun; Matsumura, Yuta; Mitani, Asako; Niwano, Yu; Takeuchi, Kohei; Kubota, Hiromi; Kawamura, Yoshiaki

    2016-01-01

    We report the complete genome sequence of Moraxella osloensis strain KMC41, isolated from laundry with malodor. The KMC41 genome comprises a 2,445,556-bp chromosome and three plasmids. A fatty acid desaturase and at least four β-oxidation-related genes putatively associated with 4-methyl-3-hexenoic acid generation were detected in the KMC41 chromosome. PMID:27445387

  1. L-Rhamnose-binding lectin from eggs of the Echinometra lucunter: Amino acid sequence and molecular modeling.

    PubMed

    Carneiro, Rômulo Farias; Teixeira, Claudener Souza; de Melo, Arthur Alves; de Almeida, Alexandra Sampaio; Cavada, Benildo Sousa; de Sousa, Oscarina Viana; da Rocha, Bruno Anderson Matias; Nagano, Celso Shiniti; Sampaio, Alexandre Holanda

    2015-01-01

    An L-rhamnose-binding lectin named ELEL was isolated from eggs of the rock boring sea urchin Echinometra lucunter by affinity chromatography on lactosyl-agarose. ELEL is a homodimer linked by a disulfide bond with subunits of 11 kDa each. The new lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as L-rhamnose, melibiose, galactose and lactose. The amino acid sequence of ELEL was determined by tandem mass spectrometry. The ELEL subunit has 103 amino acids, including nine cysteine residues involved in four conserved intrachain disulfide bonds and one interchain disulfide bond. The full sequence of ELEL presents conserved motifs commonly found in rhamnose-binding lectins, including YGR, DPC and KYL. A three-dimensional model of ELEL was created, and molecular docking revealed favorable binding energies for interactions between ELEL and rhamnose, melibiose and Gb3 (Galα1-4Galβ1-4Glcβ1-Cer). Furthermore, ELEL was able to agglutinate Gram-positive bacterial cells, suggesting its ability to recognize pathogens.

  2. Amino acid sequence and some properties of lectin-D from the roots of pokeweed (Phytolacca americana).

    PubMed

    Yamaguchi, K; Mori, A; Funatsu, G

    1996-08-01

    Two pokeweed lectins, designated PL-D1 and PL-D2, have been isolated from the roots of pokeweed (Phytolacca americana) using chitin affinity column chromatography followed by gel filtration on a Sephacryl S-200 column and fast protein liquid chromatography on a Mono-Q column, and their amino acid sequences have been analyzed. PL-D1 consists of 84 amino acid residues and has a molecular mass of 9317, while PL-D2 has an identical sequence with PL-D1 except lack of the C-terminal Leu-Thr. PL-D is composed of two chitin-binding domains, A and B, with 50% homology with each other. Both PL-Ds did not agglutinate native rabbit erythrocytes, but showed about 0.1% of the agglutinating activity of wheat germ agglutinin toward trypsin-treated erythrocytes. In the presence of beta (1-->4) linked oligomers of N-acetyl-D-glucosamine, which inhibit the hemagglutination, PL-D1 had an ultraviolet-difference spectrum with maxima at 292-294 nm and 284-285 nm, attributed to the red shift of the tryptophan residue, suggesting the location of tryptophan residue(s) at or near saccharide-binding site of PL-D1.

  3. Phylogenetic analysis of dicyemid mesozoans (phylum Dicyemida) from innexin amino acid sequences: dicyemids are not related to Platyhelminthes.

    PubMed

    Suzuki, Takahito G; Ogino, Kazutoyo; Tsuneki, Kazuhiko; Furuya, Hidetaka

    2010-06-01

    Dicyemid mesozoans are endoparasites, or endosymbionts, found only in the renal sac of benthic cephalopod molluscs. The body organization of dicyemids is very simple, consisting of usually 10 to 40 cells, with neither body cavities nor differentiated organs. Dicyemids were considered as primitive animals, and the out-group of all metazoans, or as occupying a basal position of lophotrochozoans close to flatworms. We cloned cDNAs encoding for the gap junction component proteins, innexin, from the dicyemids. Its expression pattern was observed by whole-mount in situ hybridization. In adult individuals, the innexin was expressed in calottes, infusorigens, and infusoriform embryos. The unique temporal pattern was observed in the developing infusoriform embryos. Innexin amino acid sequences had taxon-specific indels which enabled identification of the 3 major protostome lineages, i.e., 2 ecdysozoans (arthropods and nematodes) and the lophotrochozoans. The dicyemids show typical, lophotrochozoan-type indels. In addition, the Bayesian and maximum likelihood trees based on the innexin amino acid sequences suggested dicyemids to be more closely related to the higher lophotrochozoans than to the flatworms. Flatworms were the sister group, or consistently basal, to the other lophotrochozoan clade that included dicyemids, annelids, molluscs, and brachiopods.

  4. The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

    PubMed

    Negreiros, A N; Carvalho, M M; Xavier Filho, J; Blanco-Labra, A; Shewry, P R; Richardson, M

    1991-01-01

    The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin. PMID:1367792

  5. Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2232 from Karijini National Park, Australia

    PubMed Central

    Walker, Robert; Watkin, Elizabeth; Tian, Rui; Bräu, Lambert; O’Hara, Graham; Goodwin, Lynne; Han, James; Reddy, Tatiparthi; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2013-01-01

    Burkholderia sp. strain WSM2232 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod that was trapped in 2001 from acidic soil collected from Karijini National Park (Australia) using Gastrolobium capitatum as a host. WSM2232 was effective in nitrogen fixation with G. capitatum but subsequently lost symbiotic competence during long-term storage. Here we describe the features of Burkholderia sp. strain WSM2232, together with genome sequence information and its annotation. The 7,208,311 bp standard-draft genome is arranged into 72 scaffolds of 72 contigs containing 6,322 protein-coding genes and 61 RNA-only encoding genes. The loss of symbiotic capability can now be attributed to the loss of nodulation and nitrogen fixation genes from the genome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:25197442

  6. Fatty acid, flavonol, and mineral composition variability among seven macrotyloma uniflorum (Lam.) verdc. accessions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Horse gram [Macrotyloma uniflorum (Lam.) Verdc.] seeds containing high concentrations of fatty acids, flavonols and minerals will provide government, public and private organizations with a nutritious and healthy food for use by malnourished and food deprived people worldwide. Seeds from seven horse...

  7. [Fatty acid composition variability of rapeseed oil: classical selection and biotechnology].

    PubMed

    Sakhno, L A

    2010-01-01

    The problems and achievements in the rapeseed Brassica napus L. var. oleifera breeding directed on the change of fatty acid composition in seed oil with the use of traditional and genetic engineering approaches are analyzed. It is noticed that the combination of biotechnological workings out and methods of classical breeding is the optimum for the further improvement of rapeseed oil composition.

  8. Oil and fatty acid diversity in genetically variable clones of Moringa oleifera from India.

    PubMed

    Banerji, R; Bajpai, Aruna; Verma, S C

    2009-01-01

    The physico-chemical properties of oil from Moringa oleifera seeds from India were determined in the present study. The petroleum ether extracted oil ranged from 27.83 - 45.07% on kernel basis and 15.1-28.4% on whole seed basis in 20 different clones. Leaves and pods showed a good source of vitamin C. Oleic acid (C18:1) has been found to be the major fatty acid being 78.91-85.52% as compared to olive oil, which is considered to be richest source of oleic acid. All the clones from India did not show any presence of behenic acid (C 22:0). The oil was also found to contain high levels of beta-sitosterol ranged from 42.29-47.94% stigmasterol from 13.66-16.61%, campesterol from 12.53-16.63%. The gamma- and delta-tocopherol were found to be in the range of 128.0-146.95, 51.88-63.5 and 55.23-63.84 mg/kg, respectively.

  9. Oil and fatty acid diversity in genetically variable clones of Moringa oleifera from India.

    PubMed

    Banerji, R; Bajpai, Aruna; Verma, S C

    2009-01-01

    The physico-chemical properties of oil from Moringa oleifera seeds from India were determined in the present study. The petroleum ether extracted oil ranged from 27.83 - 45.07% on kernel basis and 15.1-28.4% on whole seed basis in 20 different clones. Leaves and pods showed a good source of vitamin C. Oleic acid (C18:1) has been found to be the major fatty acid being 78.91-85.52% as compared to olive oil, which is considered to be richest source of oleic acid. All the clones from India did not show any presence of behenic acid (C 22:0). The oil was also found to contain high levels of beta-sitosterol ranged from 42.29-47.94% stigmasterol from 13.66-16.61%, campesterol from 12.53-16.63%. The gamma- and delta-tocopherol were found to be in the range of 128.0-146.95, 51.88-63.5 and 55.23-63.84 mg/kg, respectively. PMID:19075502

  10. Combined effects of independent variables on yield and protein content of pectin extracted from sugar beet pulp by citric acid.

    PubMed

    Li, De-Qiang; Du, Guang-Ming; Jing, Wei-Wen; Li, Jun-Fang; Yan, Jia-Yu; Liu, Zhi-Yong

    2015-09-20

    The extraction of pectin from sugar beet pulp by citric acid was carried out under different conditions using Box-Behnken design for four independent variables (pH, temperature, time and liquid to solid ratio). The yield of sugar beet pulp pectin ranged from 6.3% to 23.0%, and the content of protein from 1.5% to 4.5%. All independent variables significantly affected the yield, and all variables except liquid to solid ratio significantly affected the protein content. The yield increased as decreasing pH of extracting solution, extending time and advancing temperature, and an opposite relationship of effects between variables and content of protein was obtained. The chemical composition of collected samples was determined. Moreover, from the results of emulsifying properties study, the extracted pectin from sugar beet pulp could prepare steady oil-in-water emulsions. Therefore, it was inferred that the extraction conditions could influence yield and protein content, resulting in different emulsifying property.

  11. Variability of oil content and of major fatty acid composition in almond (Prunus amygdalus Batsch) and its relationship with kernel quality.

    PubMed

    Kodad, O; Socias I Company, R

    2008-06-11

    Oil content and fatty acid composition were determined for two years in the kernel oil of eight cultivars and 47 advanced self-compatible almond genotypes developed in an almond breeding program. Considerable variation between genotypes was found for all parameters. Oil content ranged from 48% to 67% of the total kernel dry weight but was consistent over the two years. Fatty acid composition was also very variable, with significant differences between genotypes, even in genotypes of the same progeny. Oleic acid, ranging from 63% to 78%, and linoleic acid, ranging from 12% to 27%, were the major fatty acids, showing higher values in some selections than in their parents. The large variability observed for all fatty acids and the presence of selections with higher oil and fatty acid contents than the commercial cultivars represents a very promising base to obtain new almond cultivars with oil of higher quality, satisfying the industrial and consumer sectors.

  12. Immunoglobulin variable gene segment V{sub H}81X of the mouse is embedded in L1 transposon sequences

    SciTech Connect

    Bachl, J.; Defranoux, N.; Wabl, M.

    1995-01-11

    L1 elements are widely distributed over the mammalian genome, but the question of their biological significance is still open. The mouse heavy (H) chain variable region V{sub H}81X is overrepresented in the pre-B-cell repertoire; the significance of this is controversial, and V{sub H}81X has been the subject of much research. Here we present data showing that the mouse H chain variable region X{sub H}81X is embedded in the remnants of a LINE-1 element.

  13. Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

    PubMed

    Aramori, I; Fukagawa, M; Tsumura, M; Iwami, M; Ono, H; Kojo, H; Kohsaka, M; Ueda, Y; Imanaka, H

    1991-12-01

    A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis. PMID:1744041

  14. Amino acid sequence and posttranslational modifications of human factor VII sub a from plasma and transfected baby hamster kidney cells

    SciTech Connect

    Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U. )

    1988-10-04

    Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII{sub a}, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca{sup 2+} and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII{sub a} molecule, namely, 10 {gamma}-carboxylated, N-terminally located glutamic acid residues, 1 {beta}-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII{sub a} as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII{sub a}. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII{sub a} was found to be identical with human factor VII{sub a}. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII{sub a}. In the recombinant factor VII{sub a}, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII{sub a} and human plasma factor VII{sub a}. These results show that factor VII{sub a} as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII{sub a} and that this cell line thus might represent an alternative source for human factor VII{sub a}.

  15. Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins.

    PubMed

    Katayama, K; Ericsson, L H; Enfield, D L; Walsh, K A; Neurath, H; Davie, E W; Titani, K

    1979-10-01

    The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds. Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in gamma-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases.

  16. HLA-E coding and 3' untranslated region variability determined by next-generation sequencing in two West-African population samples.

    PubMed

    Castelli, Erick C; Mendes-Junior, Celso T; Sabbagh, Audrey; Porto, Iane O P; Garcia, André; Ramalho, Jaqueline; Lima, Thálitta H A; Massaro, Juliana D; Dias, Fabrício C; Collares, Cristhianna V A; Jamonneau, Vincent; Bucheton, Bruno; Camara, Mamadou; Donadi, Eduardo A

    2015-12-01

    HLA-E is a non-classical Human Leucocyte Antigen class I gene with immunomodulatory properties. Whereas HLA-E expression usually occurs at low levels, it is widely distributed amongst human tissues, has the ability to bind self and non-self antigens and to interact with NK cells and T lymphocytes, being important for immunosurveillance and also for fighting against infections. HLA-E is usually the most conserved locus among all class I genes. However, most of the previous studies evaluating HLA-E variability sequenced only a few exons or genotyped known polymorphisms. Here we report a strategy to evaluate HLA-E variability by next-generation sequencing (NGS) that might be used to other HLA loci and present the HLA-E haplotype diversity considering the segment encoding the entire HLA-E mRNA (including 5'UTR, introns and the 3'UTR) in two African population samples, Susu from Guinea-Conakry and Lobi from Burkina Faso. Our results indicate that (a) the HLA-E gene is indeed conserved, encoding mainly two different protein molecules; (b) Africans do present several unknown HLA-E alleles presenting synonymous mutations; (c) the HLA-E 3'UTR is quite polymorphic and (d) haplotypes in the HLA-E 3'UTR are in close association with HLA-E coding alleles. NGS has proved to be an important tool on data generation for future studies evaluating variability in non-classical MHC genes.

  17. Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

    PubMed Central

    Valdes-Rodriguez, S; Segura-Nieto, M; Chagolla-Lopez, A; Verver y Vargas-Cortina, A; Martinez-Gallardo, N; Blanco-Labra, A

    1993-01-01

    A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima. PMID:8290633

  18. 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.

  19. The nucleotide sequences of some large ribonuclease T1 products from bacteriophage R17 ribonucleic acid

    PubMed Central

    Jeppesen, Peter G. N.

    1971-01-01

    A method of `fingerprinting' high-molecular-weight 32P-labelled RNA species, using a two-dimensional thin-layer-chromatographic separation of ribonuclease T1 digestion products, has been applied to RNA from the Escherichia coli bacteriophage R17. The `fingerprinting' technique, besides giving a unique pattern that can be used as a characterization of the RNA, has made it possible to isolate a number of the larger oligonucleotides and to determine their nucleotide sequences. ImagesPLATE 1 PMID:5158505

  20. Comparison of the complete sequences of three different isolates of Pepino mosaic virus: size variability of the TGBp3 protein between tomato and L. peruvianum isolates.

    PubMed

    López, C; Soler, S; Nuez, F

    2005-03-01

    The complete nucleotide sequence of the genomes of two Spanish isolates (LE-2000 and LE-2002) from tomato and one Peruvian isolate (LP-2001) from Lycopersicon peruvianum of the Pepino mosaic virus (PepMV) were determined. The tomato isolates share identities higher than 99%, while the genome of LP-2001 had mean nucleotide identities of 95.6% to 96.0% with tomato isolates. The predicted amino acid sequences showed similarities ranging between 95.2% and 100% with TGBp3 and TGBp2 and CP proteins, respectively. In LP-2001 two main differences were found with respect to the tomato isolates; (i) the 5' untranslated region (UTR) was 2 nt shorter by deletion at position 12-13 and it had some polymorphims at the putative promoter sequence reported for PepMV tomato isolates and other potexviruses, which could be functionally significant for RNA replication, and (ii) the TGBp3 protein had two extra amino acids in the C-terminal region.