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Sample records for acid sequences determined

  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. N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis.

    PubMed

    Chang, Elizabeth; Pourmal, Sergei; Zhou, Chun; Kumar, Rupesh; Teplova, Marianna; Pavletich, Nikola P; Marians, Kenneth J; Erdjument-Bromage, Hediye

    2016-07-01

    In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods. PMID:27006647

  3. N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis

    PubMed Central

    Chang, Elizabeth; Pourmal, Sergei; Zhou, Chun; Kumar, Rupesh; Teplova, Marianna; Pavletich, Nikola P.; Marians, Kenneth J.

    2016-01-01

    In recent history, alternative approaches to Edman sequencing have been investigated, and to this end, the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group (PSRG) initiated studies in 2014 and 2015, looking into bottom-up and top-down N-terminal (Nt) dimethyl derivatization of standard quantities of intact proteins with the aim to determine Nt sequence information. We have expanded this initiative and used low picomole amounts of myoglobin to determine the efficiency of Nt-dimethylation. Application of this approach on protein domains, generated by limited proteolysis of overexpressed proteins, confirms that it is a universal labeling technique and is very sensitive when compared with Edman sequencing. Finally, we compared Edman sequencing and Nt-dimethylation of the same polypeptide fragments; results confirm that there is agreement in the identity of the Nt amino acid sequence between these 2 methods. PMID:27006647

  4. Composition for nucleic acid sequencing

    DOEpatents

    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.

  5. Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA.

    PubMed

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1988-09-01

    We report the isolation of cDNA clones for a Plasmodium falciparum gene that encodes the complete amino acid sequence of a previously identified exported blood stage antigen. The Mr of this antigen protein had been determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis, by different workers, to be 113,000, 126,000, and 140,000. We show, by cDNA nucleotide sequence analysis, that this antigen gene encodes a 989 amino acid protein (111 kDa) that contains a potential signal peptide, but not a membrane anchor domain. In the FCR3 strain the serine content of the protein was 11%, of which 57% of the serine residues were localized within a 201 amino acid sequence that included 35 consecutive serine residues. The protein also contained three possible N-linked glycosylation sites and numerous possible O-linked glycosylation sites. The mRNA was abundant during late trophozoite-schizont parasite stages. We propose to identity this antigen, which had been called p126, by the acronym SERA, serine-repeat antigen, based on its complete structure. The usefulness of the cloned cDNA as a source of a possible malaria vaccine is considered in view of the previously demonstrated ability of the antigen to induce parasite-inhibitory antibodies and a protective immune response in Saimiri monkeys. PMID:2847041

  6. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    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

  7. 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.

  8. 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.

  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. High speed nucleic acid sequencing

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

  11. Amino-Acid Sequence of Porcine Pepsin

    PubMed Central

    Tang, J.; Sepulveda, P.; Marciniszyn, J.; Chen, K. C. S.; Huang, W-Y.; Tao, N.; Liu, D.; Lanier, J. P.

    1973-01-01

    As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it was not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra amino-acid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen. PMID:4587252

  12. Chip-based sequencing nucleic acids

    DOEpatents

    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.

  13. 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.

  14. Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination.

    PubMed Central

    Chen, Y C; Peoples, O P; Walsh, C T

    1988-01-01

    The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli. Images PMID:3338974

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Amino acid sequence of Salmonella typhimurium branched-chain amino acid aminotransferase.

    PubMed

    Feild, M J; Nguyen, D C; Armstrong, F B

    1989-06-13

    The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase (transaminase B, EC 2.6.1.42) of Salmonella typhimurium was determined. An Escherichia coli recombinant containing the ilvGEDAY gene cluster of Salmonella was used as the source of the hexameric enzyme. The peptide fragments used for sequencing were generated by treatment with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. The enzyme subunit contains 308 residues and has a molecular weight of 33,920. To determine the coenzyme-binding site, the pyridoxal 5-phosphate containing enzyme was treated with tritiated sodium borohydride prior to trypsin digestion. Peptide map comparisons with an apoenzyme tryptic digest and monitoring radioactivity incorporation allowed identification of the pyridoxylated peptide, which was then isolated and sequenced. The coenzyme-binding site is the lysyl residue at position 159. The amino acid sequence of Salmonella transaminase B is 97.4% identical with that of Escherichia coli, differing in only eight amino acid positions. Sequence comparisons of transaminase B to other known aminotransferase sequences revealed limited sequence similarity (24-33%) when conserved amino acid substitutions are allowed and alignments were forced to occur on the coenzyme-binding site. PMID:2669973

  20. 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

  1. 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.

  2. 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.

  3. Amino acid sequence of bovine heart coupling factor 6.

    PubMed Central

    Fang, J K; Jacobs, J W; Kanner, B I; Racker, E; Bradshaw, R A

    1984-01-01

    The amino acid sequence of bovine heart mitochondrial coupling factor 6 (F6) has been determined by automated Edman degradation of the whole protein and derived peptides. Preparations based on heat precipitation and ethanol extraction showed allotypic variation at three positions while material further purified by HPLC yielded only one sequence that also differed by a Phe-Thr replacement at residue 62. The mature protein contains 76 amino acids with a calculated molecular weight of 9006 and a pI of approximately equal to 5, in good agreement with experimentally measured values. The charged amino acids are mainly clustered at the termini and in one section in the middle; these three polar segments are separated by two segments relatively rich in nonpolar residues. Chou-Fasman analysis suggests three stretches of alpha-helix coinciding (or within) the high-charge-density sequences with a single beta-turn at the first polar-nonpolar junction. Comparison of the F6 sequence with those of other proteins did not reveal any homologous structures. PMID:6149548

  4. Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

    PubMed Central

    Fukushima, J; Yamamoto, S; Morihara, K; Atsumi, Y; Takeuchi, H; Kawamoto, S; Okuda, K

    1989-01-01

    The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin. PMID:2493453

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

    PubMed

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

    1988-05-15

    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

  6. 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 sequence disclosures must include a copy of the sequence listing in accordance with the requirements in 37 CFR...

  7. Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

    PubMed

    Shioiri, Tatsumasa; Tsuchimoto, Jun; Watanabe, Hideto; Sugiura, Nobuo

    2016-06-01

    Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains. PMID:26791444

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Amino acid sequence of the nonsecretory ribonuclease of human urine.

    PubMed

    Beintema, J J; Hofsteenge, J; Iwama, M; Morita, T; Ohgi, K; Irie, M; Sugiyama, R H; Schieven, G L; Dekker, C A; Glitz, D G

    1988-06-14

    The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3166997

  13. 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.

  14. Analysis and Annotation of Nucleic Acid Sequence

    SciTech Connect

    David J. States

    1998-08-01

    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.

  15. Effect of temperature and solvent composition on acid dissociation equilibria, I: Sequenced (s)(s)pKa determination of compounds commonly used as buffers in high performance liquid chromatography coupled to mass spectroscopy detection.

    PubMed

    Padró, Juan M; Acquaviva, Agustín; Tascon, Marcos; Gagliardi, Leonardo G; Castells, Cecilia B

    2012-05-01

    A new automated and rapid potentiometric method for determining the effect of organic-solvent composition on pK(a) has been developed. It is based on the measurements of pH values of buffer solutions of variable solvent compositions using a combined glass electrode. Additions of small volumes of one precisely thermostated solution into another, both containing exactly the same analytical concentrations of the buffer components, can produce continuous changes in the solvent composition. Two sequences of potential measurements, one of increasing and the other of decreasing solvent content, are sufficient to obtain the pK(a) values of the acidic compound within the complete solvent-composition range in about 2h. The experimental design, procedures, and calculations needed to convert the measured pH into the thermodynamic pK(a) values are thoroughly discussed. This rapid and automated method allows the systematic study of the effect of solvent compositions and temperatures on the pK(a). It has been applied to study the dissociation constants of two monoprotic acids: formic acid and triethylamine:HCl in acetonitrile/water mixtures within the range from 0 to 90% (v/v) at temperatures between 20°C and 60°C. These volatile compounds are frequently used to control the pH of the mobile phase in HPLC, especially in methods coupled to mass-spectrometry detection. The obtained pK(a) values are in excellent agreement with those previously reported. The results were fitted to empirical functions between pK(a) and temperature and composition. These equations, which can be used to estimate the pK(a) of these substances at any composition and temperature, would be highly useful in practical work during chromatographic method development. PMID:22502616

  16. The Chinese hamster Alu-equivalent sequence: a conserved highly repetitious, interspersed deoxyribonucleic acid sequence in mammals has a structure suggestive of a transposable element.

    PubMed Central

    Haynes, S R; Toomey, T P; Leinwand, L; Jelinek, W R

    1981-01-01

    A consensus sequence has been determined for a major interspersed deoxyribonucleic acid repeat in the genome of Chinese hamster ovary cells (CHO cells). This sequence is extensively homologous to (i) the human Alu sequence (P. L. Deininger et al., J. Mol. Biol., in press), (ii) the mouse B1 interspersed repetitious sequence (Krayev et al., Nucleic Acids Res. 8:1201-1215, 1980) (iii) an interspersed repetitious sequence from African green monkey deoxyribonucleic acid (Dhruva et al., Proc. Natl. Acad. Sci. U.S.A. 77:4514-4518, 1980) and (iv) the CHO and mouse 4.5S ribonucleic acid (this report; F. Harada and N. Kato, Nucleic Acids Res. 8:1273-1285, 1980). Because the CHO consensus sequence shows significant homology to the human Alu sequence it is termed the CHO Alu-equivalent sequence. A conserved structure surrounding CHO Alu-equivalent family members can be recognized. It is similar to that surrounding the human Alu and the mouse B1 sequences, and is represented as follows: direct repeat-CHO-Alu-A-rich sequence-direct repeat. A composite interspersed repetitious sequence has been identified. Its structure is represented as follows: direct repeat-residue 47 to 107 of CHO-Alu-non-Alu repetitious sequence-A-rich sequence-direct repeat. Because the Alu flanking sequences resemble those that flank known transposable elements, we think it likely that the Alu sequence dispersed throughout the mammalian genome by transposition. Images PMID:9279371

  17. 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

  18. Detecting frame shifts by amino acid sequence comparison.

    PubMed

    Claverie, J M

    1993-12-20

    Various amino acid substitution scoring matrices are used in conjunction with local alignments programs to detect regions of similarity and infer potential common ancestry between proteins. The usual scoring schemes derive from the implicit hypothesis that related proteins evolve from a common ancestor by the accumulation of point mutations and that amino acids tend to be progressively substituted by others with similar properties. However, other frequent single mutation events, like nucleotide insertion or deletion and gene inversion, change the translation reading frame and cause previously encoded amino acid sequences to become unrecognizable at once. Here, I derive five new types of scoring matrix, each capable of detecting a specific frame shift (deletion, insertion and inversion in 3 frames) and use them with a regular local alignments program to detect amino acid sequences that may have derived from alternative reading frames of the same nucleotide sequence. Frame shifts are inferred from the sole comparison of the protein sequences. The five scoring matrices were used with the BLASTP program to compare all the protein sequences in the Swissprot database. Surprisingly, the searches revealed hundreds of highly significant frame shift matches, of which many are likely to represent sequencing errors. Others provide some evidence that frame shift mutations might be used in protein evolution as a way to create new amino acid sequences from pre-existing coding regions. PMID:7903399

  19. 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

  20. Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences.

    PubMed Central

    Bellini, W J; Englund, G; Richardson, C D; Rozenblatt, S; Lazzarini, R A

    1986-01-01

    The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer. Images PMID:3754588

  1. Determining orientation and direction of DNA sequences

    DOEpatents

    Goodwin, Edwin H.; Meyne, Julianne

    2000-01-01

    Determining orientation and direction of DNA sequences. A method by which fluorescence in situ hybridization can be made strand specific is described. Cell cultures are grown in a medium containing a halogenated nucleotide. The analog is partially incorporated in one DNA strand of each chromatid. This substitution takes place in opposite strands of the two sister chromatids. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand nicks. These nicks enable the substituted strand to be denatured and solubilized by heat, treatment with high or low pH aqueous solutions, or by immersing the strands in 2.times.SSC (0.3M NaCl+0.03M sodium citrate), to name three procedures. It is unnecessary to enzymatically digest the strands using Exo III or another exonuclease in order to excise and solubilize nucleotides starting at the sites of the nicks. The denaturing/solubilizing process removes most of the substituted strand while leaving the prereplication strand largely intact. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present.

  2. 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

  3. 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

  4. 37 CFR 1.821 - Nucleotide and/or amino acid sequence disclosures in patent applications.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... acids are not intended to be embraced by this definition. Any amino acid sequence that contains post-translationally modified amino acids may be described as the amino acid sequence that is initially translated... sequence of four or more amino acids or an unbranched sequence of ten or more nucleotides....

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

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... acids are not intended to be embraced by this definition. Any amino acid sequence that contains post-translationally modified amino acids may be described as the amino acid sequence that is initially translated... sequence of four or more amino acids or an unbranched sequence of ten or more nucleotides....

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

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... acids are not intended to be embraced by this definition. Any amino acid sequence that contains post-translationally modified amino acids may be described as the amino acid sequence that is initially translated... sequence of four or more amino acids or an unbranched sequence of ten or more nucleotides....

  7. A method to find palindromes in nucleic acid sequences.

    PubMed

    Anjana, Ramnath; Shankar, Mani; Vaishnavi, Marthandan Kirti; Sekar, Kanagaraj

    2013-01-01

    Various types of sequences in the human genome are known to play important roles in different aspects of genomic functioning. Among these sequences, palindromic nucleic acid sequences are one such type that have been studied in detail and found to influence a wide variety of genomic characteristics. For a nucleotide sequence to be considered as a palindrome, its complementary strand must read the same in the opposite direction. For example, both the strands i.e the strand going from 5' to 3' and its complementary strand from 3' to 5' must be complementary. A typical nucleotide palindromic sequence would be TATA (5' to 3') and its complimentary sequence from 3' to 5' would be ATAT. Thus, a new method has been developed using dynamic programming to fetch the palindromic nucleic acid sequences. The new method uses less memory and thereby it increases the overall speed and efficiency. The proposed method has been tested using the bacterial (3891 KB bases) and human chromosomal sequences (Chr-18: 74366 kb and Chr-Y: 25554 kb) and the computation time for finding the palindromic sequences is in milli seconds. PMID:23515654

  8. Zygosity determination of multiple pregnancy by deoxyribonucleic acid fingerprints.

    PubMed

    Azuma, C; Kamiura, S; Nobunaga, T; Negoro, T; Saji, F; Tanizawa, O

    1989-03-01

    We used a new method of deoxyribonucleic acid analysis to determine zygosity in multiple pregnancies. This method uses a minisatellite core probe, requires only a small amount of deoxyribonucleic acid, and detects the restriction fragment length polymorphisms that are a result of allelic differences in the number of tandem repeats that contain the core sequence. Southern blot hybridization showed an individual-specific deoxyribonucleic acid fingerprint and each polymorphic band in the sibling could be identified within one (but not both) of the parents. Identical deoxyribonucleic acid fingerprints among the siblings of multiple pregnancy indicate they must be monozygotic. This method is sufficiently reliable and rapid so the determination of zygosity in multiple pregnancy can be made the same day the fetal deoxyribonucleic acid is made available. PMID:2564742

  9. Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences.

    PubMed

    Navon, Sharon Penias; Kornberg, Guy; Chen, Jin; Schwartzman, Tali; Tsai, Albert; Puglisi, Elisabetta Viani; Puglisi, Joseph D; Adir, Noam

    2016-06-28

    Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel. PMID:27307442

  10. Amino Acid Sequence of Anionic Peroxidase from the Windmill Palm Tree Trachycarpus fortunei

    PubMed Central

    2015-01-01

    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. PMID:25383699

  11. Complete Genome Sequence of the WHO International Standard for HIV-2 RNA Determined by Deep Sequencing

    PubMed Central

    Ham, Claire; Morris, Clare

    2016-01-01

    The World Health Organization (WHO) International Standard for HIV-2 RNA nucleic acid assays was characterized by complete genome deep sequencing. The entire coding sequence and flanking long terminal repeats (LTRs), including minority species, were assigned subtype A. This information will aid design, development, and evaluation of HIV-2 RNA amplification assays. PMID:26847885

  12. On Quantum Algorithm for Multiple Alignment of Amino Acid Sequences

    NASA Astrophysics Data System (ADS)

    Iriyama, Satoshi; Ohya, Masanori

    2009-02-01

    The alignment of genome sequences or amino acid sequences is one of fundamental operations for the study of life. Usual computational complexity for the multiple alignment of N sequences with common length L by dynamic programming is O(LN). This alignment is considered as one of the NP problems, so that it is desirable to find a nice algorithm of the multiple alignment. Thus in this paper we propose the quantum algorithm for the multiple alignment based on the works12,1,2 in which the NP complete problem was shown to be the P problem by means of quantum algorithm and chaos information dynamics.

  13. Protein chemotaxonomy. XIII. Amino acid sequence of ferredoxin from Panax ginseng.

    PubMed

    Mino, Yoshiki

    2006-08-01

    The complete amino acid sequence of [2Fe-2S] ferredoxin from Panax ginseng (Araliaceae) has been determined by automated Edman degradation of the entire S-carboxymethylcysteinyl protein and of the peptides obtained by enzymatic digestion. This ferredoxin has a unique amino acid sequence, which includes an insertion of Tyr at the 3rd position from the amino-terminus and a deletion of two amino acid residues at the carboxyl terminus. This ferredoxin had 18 differences in its amino acid sequence compared to that of Petroselinum sativum (Umbelliferae). In contrast, 23-33 differences were observed compared to other dicotyledonous plants. This suggests that Panax ginseng is related taxonomically to umbelliferous plants. PMID:16880642

  14. 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

  15. 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

  16. Two distinct ferredoxins from Rhodobacter capsulatus: complete amino acid sequences and molecular evolution.

    PubMed

    Saeki, K; Suetsugu, Y; Yao, Y; Horio, T; Marrs, B L; Matsubara, H

    1990-09-01

    Two distinct ferredoxins were purified from Rhodobacter capsulatus SB1003. Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation. Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6,728 and 12,549 excluding iron and sulfur atoms. Both contained two Cys clusters in their amino acid sequences. The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxxCP, in common with the ferredoxins found in Clostridia. The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met. The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristics feature of Azotobacter-[3Fe-4S] [4Fe-4S]-ferredoxin. Ferredoxin II was also similar to Azotobacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type. The evolutionary relationship of the two together with a putative one recently found to be encoded in nifENXQ region in this bacterium [Moreno-Vivian et al. (1989) J. Bacteriol. 171, 2591-2598] is discussed. PMID:2277040

  17. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor. alpha. subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    SciTech Connect

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M. )

    1991-05-21

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) {alpha}subunit forms a binding site for {alpha}-bungarotoxin ({alpha}-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR {alpha}1 subunits were tested for their ability to bind {sup 125}I-{alpha}-BTX, and differences in {alpha}-BTX affinity were determined by using solution (IC{sub 50}s) and solid-phase (K{sub d}s) assays. Panels of overlapping peptides corresponding to the complete {alpha}1 subunit of mouse and human were also tested for {alpha}-BTX binding, but other sequence segments forming the {alpha}-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased {alpha}-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/{alpha}1-subunit interface a vicinal disulfide bound was not required for {alpha}-BTX binding.

  18. 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.

  19. 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.

  20. The amino-acid sequence of leghemoglobin component a from Phaseolus vulgaris (kidney bean).

    PubMed

    Lehtovaara, P; Ellfolk, N

    1975-06-01

    1. Leghemoglobin component a from Phaseolus vulgaris (kidney bean) was digested with trypsin; 15 tryptic peptides and free lysine were purified and the amino acid sequences of the peptides determined. 2. The internal order of the tryptic peptides was determined by the bridge peptides obtained from the thermolytic digest and the dilute acid hydrolyzate of kidney bean leghemoglobin a; 12 thermolytic peptides and two acid hydrolysis peptides were purified and the sequences were partially or completely determined. 3. The complete amino acid sequence of kidney bean leghemoglobin a is compared to that of leghemoglobin a from soybean (Glycine max) and to some animal globins. As regards sequence, the kidney bean globin has 79% identity with the soybean globin and 21% identity with human hemoglobin gamma-chain. Seven of the 14 amino acid residues common to most globins are found in the kidney bean globin. Trp-15 and Tyr-145 are evolutionarily conserved in this globin, which confirms the concept of a common origin of animal and plant globins. PMID:809270

  1. Individual and simultaneous determination of uric acid and ascorbic acid by flow injection analysis.

    PubMed

    Almuaibed, A M; Townshend, A

    1992-11-01

    Flow injection methods for the individual and simultaneous determination of ascorbic acid and uric acid are proposed. A spectrophotometer and a miniamperometric detector are connected in sequence. The calibration graphs for uric acid obtained by measuring its absorbance at 293 nm and its current at +0.6 V are linear up to at least 80 and 70 mug/ml, respectively, with an rsd (n = 10) of 1 % for both methods at mid-range concentrations. The calibration graph for ascorbic acid with amperometric detection is linear up to 80 mg/l. with an rsd (n = 10) of 0.8% at 30 mg/l. The simultaneous determination of uric acid and ascorbic acid is based on measurement of the absorbance of uric acid at 393 nm and amperometric determination of both analytes at +0.6 V. The average relative errors of the analysis of binary mixtures of uric acid and ascorbic acid are 2.2 and 4.2%, respectively. PMID:18965554

  2. Complete amino acid sequence of the Mu heavy chain of a human IgM immunoglobulin.

    PubMed

    Putnam, F W; Florent, G; Paul, C; Shinoda, T; Shimizu, A

    1973-10-19

    The amino acid sequence of the micro, chain of a human IgM immunoglobulin, including the location of all disulfide bridges and oligosaccharides, has been determined. The homology of the constant regions of immunoglobulin micro, gamma, alpha, and epsilon heavy chains reveals evolutionary relationships and suggests that two genes code for each heavy chain. PMID:4742735

  3. Partial amino acid sequence of fructose-1,6-bisphosphatase from the blue-green algae Synechococcus leopoliensis.

    PubMed

    Marcus, F; Latshaw, S P; Steup, M; Gerbling, K P

    1989-08-01

    Purified fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus leopoliensis was S-carboxymethylated and cleaved with trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography and the amino acid sequence of six of the purified peptides was determined by gas-phase microsequencing. The results revealed sequence homology with other fructose-1,6-bisphosphatases. The obtained sequence data provides information required for the design of oligonucleotide hybridization probes to screen existing libraries of cyanobacterial DNA. The determination of the amino acid sequence of cyanobacterial proteins may yield important information with respect to the endosymbiotic theory of evolution. PMID:2550924

  4. Complete Genome Sequence of a Reference Stock of Simian Immunodeficiency Virus RNA (SIVmac251/32H/L28) Determined by Deep Sequencing

    PubMed Central

    Jenkins, Adrian; Ham, Claire; Almond, Neil

    2016-01-01

    A reference preparation for simian immunodeficiency virus (SIV) RNA nucleic acid assays was characterized by complete genome deep sequencing. The entire coding sequence and flanking long terminal repeats, including minority species, were determined. This information will inform SIV research investigations and aid evaluation and development of amplification assays for SIV RNA quantification. PMID:27231355

  5. Complete Genome Sequence of a Reference Stock of Simian Immunodeficiency Virus RNA (SIVmac251/32H/L28) Determined by Deep Sequencing.

    PubMed

    Jenkins, Adrian; Ham, Claire; Almond, Neil; Berry, Neil

    2016-01-01

    A reference preparation for simian immunodeficiency virus (SIV) RNA nucleic acid assays was characterized by complete genome deep sequencing. The entire coding sequence and flanking long terminal repeats, including minority species, were determined. This information will inform SIV research investigations and aid evaluation and development of amplification assays for SIV RNA quantification. PMID:27231355

  6. 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.

  7. Spectrofluorimetric determination of ellagic acid in brandy.

    PubMed

    Sádecká, Jana; Tóthová, Jana

    2012-12-01

    Two spectrofluorimetric methods have been developed for the rapid determination of ellagic acid. The first method is based on the complex formation between ellagic acid and borax in methanol solution. The fluorescence of the complex is monitored at an emission wavelength of 456nm with excitation at 383nm. Linear calibration curve was obtained from 2.5×10(-8) to 7.5×10(-7)molL(-1) and the limit of determination was 4×10(-9)molL(-1). The second method is based on the complex formation between ellagic acid and boric acid in ethanol solution. The fluorescence of the complex is monitored at an emission wavelength of 447nm with excitation at 387nm. Linear calibration curve was obtained from 1.25×10(-7) to 1.00×10(-6)molL(-1) and the limit of determination was 7×10(-9)molL(-1). The methods were successfully applied for the determination of ellagic acid in brandy samples. The results were found to be in good agreement with those obtained by HPLC method. PMID:22953802

  8. Amino acid sequence of the Amur tiger prion protein.

    PubMed

    Wu, Changde; Pang, Wanyong; Zhao, Deming

    2006-10-01

    Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank. PMID:16780982

  9. DNA Sequence Determination by Hybridization: A Strategy for Efficient Large-Scale Sequencing

    NASA Astrophysics Data System (ADS)

    Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoddy, J.; Funkhouser, W. K.; Koop, B.; Hood, L.; Crkvenjakov, R.

    1993-06-01

    The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.

  10. DNA sequence determination by hybridization: A strategy for efficient large-scale sequencing

    SciTech Connect

    Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoody, J.; Crkvenjakov, R. ); Funkhouser, W.K.; Koop, B.; Hood, L. )

    1993-06-11

    The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project. 22 refs., 3 figs.

  11. Amino acid sequence of a vitamin K-dependent Ca2+-binding peptide from bovine prothrombin.

    PubMed

    Howard, J B; Fausch, M D

    1975-08-10

    The amino acid sequence of a 31-residue peptide from bovine prothrombin has been determined. This peptide has been shown to contain the vitamin K-dependent modification required for Ca2+ binding (Nelsestuen, G. L., and Suttie, J. W. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 3366-3370) and the modified amino acid, gamma-carboxyglutamic acid (Nelsestuen, G. L., Zytkovicz, T., and Howard, J. B. (1974) J. Biol. Chem. 249, 6347-6350). The peptide was shown to correspond to residues 12 to 42 of prothrombin. PMID:807581

  12. Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

    PubMed Central

    Miller, Janet C.; Waley, S. G.

    1971-01-01

    1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes. PMID:5165707

  13. Bioluminescent determination of free fatty acids.

    PubMed

    Kather, H; Wieland, E

    1984-08-01

    A simple, highly specific, and sensitive bioluminescent method for determination of free fatty acids in unextracted plasma or serum has been developed. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The pyrophosphate formed is used to phosphorylate fructose 6-phosphate in a reaction catalyzed by the enzyme pyrophosphate-fructose-6-phosphate phosphotransferase (EC 4.1.2.13). The triosephosphates produced from fructose 1,6-bisphosphate by aldolase are oxidized by NAD in the presence of arsenate to 3-phosphoglycerate. The NADH is detected via the bacterial NADH-linked luciferase system. Excellent agreement has been obtained by comparison with accepted methods. In addition, for the determination of serum free fatty acids, the method is particularly applicable for following lipolysis of isolated adipocytes. PMID:6486422

  14. Protein location prediction using atomic composition and global features of the amino acid sequence

    SciTech Connect

    Cherian, Betsy Sheena; Nair, Achuthsankar S.

    2010-01-22

    Subcellular location of protein is constructive information in determining its function, screening for drug candidates, vaccine design, annotation of gene products and in selecting relevant proteins for further studies. Computational prediction of subcellular localization deals with predicting the location of a protein from its amino acid sequence. For a computational localization prediction method to be more accurate, it should exploit all possible relevant biological features that contribute to the subcellular localization. In this work, we extracted the biological features from the full length protein sequence to incorporate more biological information. A new biological feature, distribution of atomic composition is effectively used with, multiple physiochemical properties, amino acid composition, three part amino acid composition, and sequence similarity for predicting the subcellular location of the protein. Support Vector Machines are designed for four modules and prediction is made by a weighted voting system. Our system makes prediction with an accuracy of 100, 82.47, 88.81 for self-consistency test, jackknife test and independent data test respectively. Our results provide evidence that the prediction based on the biological features derived from the full length amino acid sequence gives better accuracy than those derived from N-terminal alone. Considering the features as a distribution within the entire sequence will bring out underlying property distribution to a greater detail to enhance the prediction accuracy.

  15. 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.

  16. Designing amino acids to determine the local conformations of peptides.

    PubMed Central

    Burgess, A W

    1994-01-01

    The local conformations of proteins and peptides are determined by the amino acid sequence. However, the 20 amino acids encoded by the genome allow the peptide backbone to fold into many conformations, so that even for a small peptide it becomes very difficult to predict the three-dimensional structure. By using empirical conformational energy calculations, a set of amino acids has been designed that would be expected to constrain the conformation of a peptide or a protein to one or two local minima. Most of these amino acids are based on asymmetric substitutions at the C alpha atom of each residue. The H alpha atom of alanine was replaced by various groups: -OCH3, -NCH3, -SCH3, -CONH2, -CONHCH3, -CON(CH3)2, -NH.CO.CH3, -phenyl, or -o-(OCH3)phenyl. Several of these new amino acids are predicted to fold into unique peptide conformations such as right-handed alpha-helical, left-handed alpha-helical, or extended. In an attempt to produce an amino acid that favored the C(eq)7 conformation (torsion angles: phi = -70 degrees and psi = +70 degrees), an extra amide group was added to the C beta atom of the asparagine side chain. Conformationally restricted amino acids of this type could prove useful for developing new peptide pharmaceuticals, catalysts, or polymers. Images PMID:8146170

  17. Partial N-terminal sequence analysis of human class II molecules expressing the DQw3 determinant.

    PubMed

    Obata, F; Endo, T; Yoshii, M; Otani, F; Igarashi, M; Takenouchi, T; Ikeda, H; Ogasawara, K; Kasahara, M; Wakisaka, A

    1985-09-01

    HLA-DQ molecules were isolated from DRw9-homozygous and DR4-homozygous cell lines by using a monoclonal antibody HU-18, which recognizes class II molecules carrying the conventional DQw3 determinant. The partial N-terminal sequence analysis of the DQw3 molecules revealed that they have sequences homologous to those of murine I-A molecules. Within the limits of our sequence analysis, the DQw3 molecules from the two cell lines are identical to each other in both the alpha and beta chains. The DQ alpha as well as DQ beta chains were found to have amino acid substitutions when compared to other I-A-like molecules whose sequences have been reported. These differences may contribute to the DQw supertypic specificity. The polymorphic nature of DQ molecules is in marked contrast to that of DR molecules where DR alpha chains are highly conserved while DR beta chains have easily detectable amino acid substitutions. PMID:2411700

  18. Correlation between fibroin amino acid sequence and physical silk properties.

    PubMed

    Fedic, Robert; Zurovec, Michal; Sehnal, Frantisek

    2003-09-12

    The fiber properties of lepidopteran silk depend on the amino acid repeats that interact during H-fibroin polymerization. The aim of our research was to relate repeat composition to insect biology and fiber strength. Representative regions of the H-fibroin genes were sequenced and analyzed in three pyralid species: wax moth (Galleria mellonella), European flour moth (Ephestia kuehniella), and Indian meal moth (Plodia interpunctella). The amino acid repeats are species-specific, evidently a diversification of an ancestral region of 43 residues, and include three types of regularly dispersed motifs: modifications of GSSAASAA sequence, stretches of tripeptides GXZ where X and Z represent bulky residues, and sequences similar to PVIVIEE. No concatenations of GX dipeptide or alanine, which are typical for Bombyx silkworms and Antheraea silk moths, respectively, were found. Despite different repeat structure, the silks of G. mellonella and E. kuehniella exhibit similar tensile strength as the Bombyx and Antheraea silks. We suggest that in these latter two species, variations in the repeat length obstruct repeat alignment, but sufficiently long stretches of iterated residues get superposed to interact. In the pyralid H-fibroins, interactions of the widely separated and diverse motifs depend on the precision of repeat matching; silk is strong in G. mellonella and E. kuehniella, with 2-3 types of long homogeneous repeats, and nearly 10 times weaker in P. interpunctella, with seven types of shorter erratic repeats. The high proportion of large amino acids in the H-fibroin of pyralids has probably evolved in connection with the spinning habit of caterpillars that live in protective silk tubes and spin continuously, enlarging the tubes on one end and partly devouring the other one. The silk serves as a depot of energetically rich and essential amino acids that may be scarce in the diet. PMID:12816957

  19. New monoclonal antibodies to the Ebola virus glycoprotein: Identification and analysis of the amino acid sequence of the variable domains.

    PubMed

    Panina, A A; Aliev, T K; Shemchukova, O B; Dement'yeva, I G; Varlamov, N E; Pozdnyakova, L P; Bokov, M N; Dolgikh, D A; Sveshnikov, P G; Kirpichnikov, M P

    2016-03-01

    We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained. PMID:27193713

  20. 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.

  1. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    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.

  2. The amino acid sequence of rabbit muscle triose phosphate isomerase.

    PubMed Central

    Corran, P H; Waley, S G

    1975-01-01

    The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1975) 145, 5. PMID:1171682

  3. The amino acid sequence of chymopapain from Carica papaya.

    PubMed Central

    Watson, D C; Yaguchi, M; Lynn, K R

    1990-01-01

    Chymopapain is a polypeptide of 218 amino acid residues. It has considerable structural similarity with papain and papaya proteinase omega, including conservation of the catalytic site and of the disulphide bonding. Chymopapain is like papaya proteinase omega in carrying four extra residues between papain positions 168 and 169, but differs from both papaya proteinases in the composition of its S2 subsite, as well as in having a second thiol group, Cys-117. Some evidence for the amino acid sequence of chymopapain has been deposited as Supplementary Publication SUP 50153 (12 pages) at the British Library Document Supply Centre, Boston Spa., Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1990) 265, 5. The information comprises Supplement Tables 1-4, which contain, in order, amino acid compositions of peptides from tryptic, peptic, CNBr and mild acid cleavages, Supplement Fig. 1, showing re-fractionation of selected peaks from Fig. 2 of the main paper. Supplement Fig. 2, showing cation-exchange chromatography of the earliest-eluted peak of Fig. 3 of the main paper, Supplement Fig. 3, showing reverse-phase h.p.l.c. of the later-eluted peak from Fig. 3 of the main paper, and Supplement Fig. 4, showing the separation of peptides after mild acid hydrolysis of CNBr-cleavage fragment CB3. PMID:2106878

  4. The amino acid sequence of chymopapain from Carica papaya.

    PubMed

    Watson, D C; Yaguchi, M; Lynn, K R

    1990-02-15

    Chymopapain is a polypeptide of 218 amino acid residues. It has considerable structural similarity with papain and papaya proteinase omega, including conservation of the catalytic site and of the disulphide bonding. Chymopapain is like papaya proteinase omega in carrying four extra residues between papain positions 168 and 169, but differs from both papaya proteinases in the composition of its S2 subsite, as well as in having a second thiol group, Cys-117. Some evidence for the amino acid sequence of chymopapain has been deposited as Supplementary Publication SUP 50153 (12 pages) at the British Library Document Supply Centre, Boston Spa., Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1990) 265, 5. The information comprises Supplement Tables 1-4, which contain, in order, amino acid compositions of peptides from tryptic, peptic, CNBr and mild acid cleavages, Supplement Fig. 1, showing re-fractionation of selected peaks from Fig. 2 of the main paper. Supplement Fig. 2, showing cation-exchange chromatography of the earliest-eluted peak of Fig. 3 of the main paper, Supplement Fig. 3, showing reverse-phase h.p.l.c. of the later-eluted peak from Fig. 3 of the main paper, and Supplement Fig. 4, showing the separation of peptides after mild acid hydrolysis of CNBr-cleavage fragment CB3. PMID:2106878

  5. The amino acid sequence of cytochrome c-555 from the methane-oxidizing bacterium Methylococcus capsulatus.

    PubMed Central

    Ambler, R P; Dalton, H; Meyer, T E; Bartsch, R G; Kamen, M D

    1986-01-01

    The amino acid sequence of the cytochrome c-555 from the obligate methanotroph Methylococcus capsulatus strain Bath (N.C.I.B. 11132) was determined. It is a single polypeptide chain of 96 residues, binding a haem group through the cysteine residues at positions 19 and 22, and the only methionine residue is a position 59. The sequence does not closely resemble that of any other cytochrome c that has yet been characterized. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50131 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment. PMID:3006666

  6. Salmonella Serotype Determination Utilizing High-Throughput Genome Sequencing Data

    PubMed Central

    Zhang, Shaokang; Yin, Yanlong; Jones, Marcus B.; Zhang, Zhenzhen; Deatherage Kaiser, Brooke L.; Dinsmore, Blake A.; Fitzgerald, Collette; Fields, Patricia I.

    2015-01-01

    Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1–3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization. PMID:25762776

  7. 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

  8. Amino acid sequence prerequisites for the formation of cn ions.

    PubMed

    Downard, K M; Biemann, K

    1993-11-01

    Ammo acid sequence prerequisites are described for the formation of c, ions observed in high-energy collision-induced decomposition spectra of peptides. It is shown that the formation of cn ions is promoted by the nature of the amino acid C-terminal to the cleavage site. A propensity for cn cleavage preceding threonine, and to a lesser extent tryptophan, lysine, and serine, is demonstrated where fragmentation is directed N-terminally at these residues. In addition, the nature of the residue N-terminal to the cleavage site is shown to have little effect on cn ion formation. A mechanism for cn ion formation is proposed and its applicability to the results observed is discussed. PMID:24227531

  9. Ultrasensitive nucleic acid sequence detection by single-molecule electrophoresis

    SciTech Connect

    Castro, A; Shera, E.B.

    1996-09-01

    This is the final report of a one-year laboratory-directed research and development project at Los Alamos National Laboratory. There has been considerable interest in the development of very sensitive clinical diagnostic techniques over the last few years. Many pathogenic agents are often present in extremely small concentrations in clinical samples, especially at the initial stages of infection, making their detection very difficult. This project sought to develop a new technique for the detection and accurate quantification of specific bacterial and viral nucleic acid sequences in clinical samples. The scheme involved the use of novel hybridization probes for the detection of nucleic acids combined with our recently developed technique of single-molecule electrophoresis. This project is directly relevant to the DOE`s Defense Programs strategic directions in the area of biological warfare counter-proliferation.

  10. Folic Acid Determination Using Electrochemical Sensors

    PubMed Central

    Mirmoghtadaie, Leila; Shamaeizadeh, Nahal; Mirzanasiri, Nooshin

    2015-01-01

    Folic acid (FA) is a water soluble vitamin that exists in many natural species. The lack of FA causes some deficiencies in the human body, so finding a simple and sensitive method for determining the FA is important. One of the modern techniques which overcome the disadvantages of conventional determination methods is the sensors. Possibility of miniaturization, the development of microfabricated electrochemical (EC) sensors has resulted in high sensitivity, portability, improved performance and spatial resolution, low power consumption, and the opportunity for integration with other technologies made Micro-Electrical-Mechanical Systems-based EC sensors suitable to identify low concentration analytes and microorganisms in a variety of mediums. PMID:26605021

  11. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing

    PubMed Central

    Green, Richard E.; Malaspinas, Anna-Sapfo; Krause, Johannes; Briggs, Adrian W.; Johnson, Philip L. F.; Uhler, Caroline; Meyer, Matthias; Good, Jeffrey M.; Maricic, Tomislav; Stenzel, Udo; Prüfer, Kay; Siebauer, Michael; Burbano, Hernán A.; Ronan, Michael; Rothberg, Jonathan M.; Egholm, Michael; Rudan, Pavao; Brajković, Dejana; Kućan, Željko; Gušić, Ivan; Wikström, Mårten; Laakkonen, Liisa; Kelso, Janet; Slatkin, Montgomery; Pääbo, Svante

    2008-01-01

    Summary A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000-year-old Neandertal individual using 8,341 mtDNA sequences identified among 4.8 Gb of DNA generated from ~0.3 grams of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs and allows an estimate of the divergence date between the two mtDNA lineages of 660,000±140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared to other primate lineages suggesting that the effective population size of Neandertals was small. PMID:18692465

  12. Titrimetric determination of ascorbic acid using chloranil.

    PubMed

    Verma, K K; Jain, A; Rawat, R

    1984-01-01

    Ascorbic acid is oxidized and quantitatively titrated with chloranil (2,3,5,6-tetrachloro-1,4-benzoquinone) in the presence of hexamethylenetetramine in acetone-water; the end point is determined visually by the appearance of a golden yellow color. Colored solutions are assayed by setting the initial absorbance at 451 nm to zero or the minimum, titrating with chloranil solution, and measuring absorbance after each increment of titrant. A plot of the volume of chloranil added against the absorbance gives a straight line with the volume intercept as the end point. Interference by the thiol group of cysteine, glutathione, etc., is avoided by masking with acrylamide; interference by iron(II) is masked with ammonium thiocyanate and sodium potassium tartrate. Hydrogen sulfite and thiourea (which do not interfere) are added as antioxidants during extraction of ascorbic acid from drugs and fruits. PMID:6725194

  13. Determining Word Sequence Variation Patterns in Clinical Documents using Multiple Sequence Alignment

    PubMed Central

    Meng, Frank; Morioka, Craig A.; El-Saden, Suzie

    2011-01-01

    Sentences and phrases that represent a certain meaning often exhibit patterns of variation where they differ from a basic structural form by one or two words. We present an algorithm that utilizes multiple sequence alignments (MSAs) to generate a representation of groups of phrases that possess the same semantic meaning but also share in common the same basic word sequence structure. The MSA enables the determination not only of the words that compose the basic word sequence, but also of the locations within the structure that exhibit variation. The algorithm can be utilized to generate patterns of text sequences that can be used as the basis for a pattern-based classifier, as a starting point to bootstrap the pattern building process for a regular expression-based classifiers, or serve to reveal the variation characteristics of sentences and phrases within a particular domain. PMID:22195152

  14. Nucleotide and predicted amino acid sequences of cloned human and mouse preprocathepsin B cDNAs.

    PubMed Central

    Chan, S J; San Segundo, B; McCormick, M B; Steiner, D F

    1986-01-01

    Cathepsin B is a lysosomal thiol proteinase that may have additional extralysosomal functions. To further our investigations on the structure, mode of biosynthesis, and intracellular sorting of this enzyme, we have determined the complete coding sequences for human and mouse preprocathepsin B by using cDNA clones isolated from human hepatoma and kidney phage libraries. The nucleotide sequences predict that the primary structure of preprocathepsin B contains 339 amino acids organized as follows: a 17-residue NH2-terminal prepeptide sequence followed by a 62-residue propeptide region, 254 residues in mature (single chain) cathepsin B, and a 6-residue extension at the COOH terminus. A comparison of procathepsin B sequences from three species (human, mouse, and rat) reveals that the homology between the propeptides is relatively conserved with a minimum of 68% sequence identity. In particular, two conserved sequences in the propeptide that may be functionally significant include a potential glycosylation site and the presence of a single cysteine at position 59. Comparative analysis of the three sequences also suggests that processing of procathepsin B is a multistep process, during which enzymatically active intermediate forms may be generated. The availability of the cDNA clones will facilitate the identification of possible active or inactive intermediate processive forms as well as studies on the transcriptional regulation of the cathepsin B gene. PMID:3463996

  15. Complete genomic sequence of barley (Hordeum vulgare) endornavirus (HvEV) determined by next-generation sequencing.

    PubMed

    Candresse, Thierry; Marais, Armelle; Sorrentino, Roberto; Faure, Chantal; Theil, Sébastien; Cadot, Valérie; Rolland, Mathieu; Villemot, Julie; Rabenstein, Frank

    2016-03-01

    Endornaviruses are unusual plant-, fungus- and oomycete-infecting viruses with a large, ca 14- to 17-kb linear double-stranded RNA (dsRNA) genome and a persistent lifestyle. The complete genome sequence of an endornavirus from the barley (Hordeum vulgare) Nerz variety was determined from paired Illumina MySeq reads derived from purified dsRNAs. The genome is 14,243 nt long, with 5' and 3' non-coding regions of 207 and 47 nt, respectively. It encodes a single large protein of 4663 amino acids that carries conserved motifs for a methyltransferase, a helicase and an RNA-dependent RNA polymerase. The sequence of Hordeum vulgare endornavirus (HvEV) carries all the hallmarks of a typical member of the genus Endornavirus, with the exception of an UDP-glycosyltransferase motif observed in many, but not all, endornaviral genomes. PMID:26666441

  16. Determination of benzoic acid, chlorobenzoic acids and chlorendic acid in water

    SciTech Connect

    Dietz, E.A.; Cortellucci, N.J.; Singley, K.F. )

    1993-01-01

    To characterize and conduct treatment studies of a landfill leachate an analysis procedure was required to determine concentrations of benzoic acid, the three isomers of chlorobenzoic acid and chlorendic acid. The title compounds were isolated from acidified (pH 1) water by extraction with methyl t-butyl ether. Analytes were concentrated by back-extracting the ether with 0.1 N sodium hydroxide which was separated and acidified. This solution was analyzed by C[sub 18] reversed-phase HPLC with water/acetonitrile/acetic acid eluent and UV detection at 222 nm. The method has detection limits of 200 [mu]g/L for chlorendic acid and 100 [mu]g/L for benzoic acid and each isomer of chlorobenzoic acid. Validation studies with water which was fortified with the analytes at concentrations ranging from one to ten times detection limits resulted in average recoveries of >95%.

  17. Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison.

    PubMed

    Johnson, A M; Fielke, R; Lumb, R; Baverstock, P R

    1990-04-01

    Reverse transcription of total cellular RNA was used to obtain a partial sequence of the small subunit ribosomal RNA of Cryptosporidium, a protist currently placed in the phylum Apicomplexa. The semi-conserved regions were aligned with homologous sequences in a range of other eukaryotes, and the evolutionary relationships of Cryptosporidium were determined by two different methods of phylogenetic analysis. The prokaryotes Escherichia coli and Halobacterium cuti were included as outgroups. The results do not show an especially close relationship of Cryptosporidium to other members of the phylum Apicomplexa. PMID:2332273

  18. 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.

  19. Human liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells.

    PubMed Central

    Tani, K; Fujii, H; Nagata, S; Miwa, S

    1988-01-01

    Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes. Among these isozymes, abnormalities of liver (L)-type PK is considered to be associated with hereditary nonspherocytic hemolytic anemia in humans. We isolated and determined the full-length sequence of human L-type PK cDNA. The cDNA contains 1629 base pairs encoding 543 amino acids, 68 base pairs of 5'-noncoding sequence, and 734 base pairs of 3'-noncoding sequence. The similarity between human and rat L-type PK was 86.9% at the nucleotide sequence level and 92.4% at the amino acid sequence level. The full-length L-type PK cDNA was placed under the promoter of simian virus 40 and introduced into monkey COS cells. Human L-type PK activity was detected in the extract of COS cells by the classical PK electrophoresis method. Images PMID:3126495

  20. Human liver type pyruvate kinase: Complete amino acid sequence and the expression in mammalian cells

    SciTech Connect

    Tani, Kenzaburo; Nagata, Shigekazu ); Fujii, Hisaichi ); Miwa, Shiro )

    1988-03-01

    Pyruvate kinase (PK) has four isozymes (L, R, M{sub 1}, M{sub 2}) that are encoded by two different genes. Among these isozymes, abnormalities of liver (L)-type PK is considered to be associated with hereditary nonspherocytic hemolytic anemia in humans. The authors isolated and determined the full-length sequence of human L-type PK cDNA. The cDNA contains 1,629 base pairs encoding 543 amino acids, 68 base pairs of 5{prime}-noncoding sequence, and 734 base pairs of 3{prime}-noncoding sequence. The similarity between human and rat L-type PK was 86.9% at the nucleotide sequence level and 92.4% at the amino acid sequence level. The full-length L-type PK cDNA was placed under the promoter of simian virus 40 and introduced into monkey COS cells. Human L-type PK activity was detected in the extract of COS cells by the classical PK electrophoresis method.

  1. Nucleotide sequence of the phosphoglycerate kinase gene from the extreme thermophile Thermus thermophilus. Comparison of the deduced amino acid sequence with that of the mesophilic yeast phosphoglycerate kinase.

    PubMed Central

    Bowen, D; Littlechild, J A; Fothergill, J E; Watson, H C; Hall, L

    1988-01-01

    Using oligonucleotide probes derived from amino acid sequencing information, the structural gene for phosphoglycerate kinase from the extreme thermophile, Thermus thermophilus, was cloned in Escherichia coli and its complete nucleotide sequence determined. The gene consists of an open reading frame corresponding to a protein of 390 amino acid residues (calculated Mr 41,791) with an extreme bias for G or C (93.1%) in the codon third base position. Comparison of the deduced amino acid sequence with that of the corresponding mesophilic yeast enzyme indicated a number of significant differences. These are discussed in terms of the unusual codon bias and their possible role in enhanced protein thermal stability. Images Fig. 1. PMID:3052437

  2. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in the sequence. (4) The enumeration of amino acids may start at the first amino acid of the first..., counting backwards starting with the amino acid next to number 1. Otherwise, the enumeration of amino acids... sequence every 5 amino acids. The enumeration method for amino acid sequences that is set forth......

  3. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in the sequence. (4) The enumeration of amino acids may start at the first amino acid of the first..., counting backwards starting with the amino acid next to number 1. Otherwise, the enumeration of amino acids... sequence every 5 amino acids. The enumeration method for amino acid sequences that is set forth......

  4. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in the sequence. (4) The enumeration of amino acids may start at the first amino acid of the first..., counting backwards starting with the amino acid next to number 1. Otherwise, the enumeration of amino acids... sequence every 5 amino acids. The enumeration method for amino acid sequences that is set forth......

  5. Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

    PubMed Central

    Gerbl-Rieger, S; Peters, J; Kellermann, J; Lottspeich, F; Baumeister, W

    1991-01-01

    The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins. PMID:1848840

  6. 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

  7. The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena.

    PubMed Central

    Sato, S; Uchida, T

    1975-01-01

    1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined, 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes; Tyr-Pro-His-Gln-Tyr (38-42) in RNAase U2 and Tyr-Pro-His-Lys-Tyr (38-42) in RNAase T1, Glu-Phe-Pro-Leu-Val (61-65) in RNAase U2 and Glu-Trp-Pro-Ile-Leu (58-62) in RNAase T1, Asp-Arg-Val-Ile-Tyr-Gln (83-88) in RNAase U2 and Asp-Arg-Val-Phe-Asn (76-81) in RNAase T1 and Val-Thr-His-Thr-Gly-Ala (98-103) in RNAase U2 and Ile-Thr-His-Thr-Gly-Ala (90-95) in RNAase T1. All of the amino acid residues, histidine-40, glutamate-58, arginine-77 and histidine-92, which were found to play a crucial role in the biological activity of RNAase T1, were included in the regions cited here. 3. Detailed evidence for the amino acid sequence of the sequence of the proteins has been deposited as Supplementary Publication SUP 50041 (33 PAGES) AT THE British Library (Lending Division)(formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5. PMID:1156364

  8. DNA sequence representation by trianders and determinative degree of nucleotides

    PubMed Central

    Duplij, Diana; Duplij, Steven

    2005-01-01

    A new version of DNA walks, where nucleotides are regarded unequal in their contribution to a walk is introduced, which allows us to study thoroughly the “fine structure” of nucleotide sequences. The approach is based on the assumption that nucleotides have an inner abstract characteristic, the determinative degree, which reflects genetic code phenomenological properties and is adjusted to nucleotides physical properties. We consider each codon position independently, which gives three separate walks characterized by different angles and lengths, and that such an object is called triander which reflects the “strength” of branch. A general method for identifying DNA sequence “by triander” which can be treated as a unique “genogram” (or “gene passport”) is proposed. The two- and three-dimensional trianders are considered. The difference of sequences fine structure in genes and the intergenic space is shown. A clear triplet signal in coding sequences was found which is absent in the intergenic space and is independent from the sequence length. This paper presents the topological classification of trianders which can allow us to provide a detailed working out signatures of functionally different genomic regions. PMID:16052707

  9. Comparative In silico Study of Sex-Determining Region Y (SRY) Protein Sequences Involved in Sex-Determining

    PubMed Central

    Vakili Azghandi, Masoume; Nasiri, Mohammadreza; Shamsa, Ali; Jalali, Mohsen; Shariati, Mohammad Mahdi

    2016-01-01

    Background: The SRY gene (SRY) provides instructions for making a transcription factor called the sex-determining region Y protein. The sex-determining region Y protein causes a fetus to develop as a male. In this study, SRY of 15 spices included of human, chimpanzee, dog, pig, rat, cattle, buffalo, goat, sheep, horse, zebra, frog, urial, dolphin and killer whale were used for determine of bioinformatic differences. Methods: Nucleotide sequences of SRY were retrieved from the NCBI databank. Bioinformatic analysis of SRY is done by CLC Main Workbench version 5.5 and ClustalW (http:/www.ebi.ac.uk/clustalw/) and MEGA6 softwares. Results: The multiple sequence alignment results indicated that SRY protein sequences from Orcinus orca (killer whale) and Tursiopsaduncus (dolphin) have least genetic distance of 0.33 in these 15 species and are 99.67% identical at the amino acid level. Homosapiens and Pantroglodytes (chimpanzee) have the next lowest genetic distance of 1.35 and are 98.65% identical at the amino acid level. Conclusion: These findings indicate that the SRY proteins are conserved in the 15 species, and their evolutionary relationships are similar. PMID:27536700

  10. Determination of free fatty acids in beer.

    PubMed

    Bravi, Elisabetta; Marconi, Ombretta; Sileoni, Valeria; Perretti, Giuseppe

    2017-01-15

    Free fatty acids (FFA) content of beer affects the ability to form a stable head of foam and plays an important role in beer staling. Moreover, the presence of saturated FAs is related sometimes to gushing problems in beer. The aim of this research was to validate an analytical method for the determination of FFAs in beer. The extraction of FFAs in beer was achieved via Liquid-Liquid Cartridge Extraction (LLCE), the FFAs extract was purified by Solid Phase Extraction (SPE), methylated by boron trifluoride in methanol, and injected into GC-FID system. The performance criteria demonstrate that this method is suitable for the analysis of medium and long chain FFAs in beer. The proposed method was tested on four experimental beers. PMID:27542484

  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. Peptide sequencing by using a combination of partial acid hydrolysis and fast-atom-bombardment mass spectrometry.

    PubMed Central

    De Angelis, F; Botta, M; Ceccarelli, S; Nicoletti, R

    1986-01-01

    To overcome the limit of the intensity of ions carrying sequence information in structural determinations of peptides by fast-atom-bombardment m.s., we have developed a method that consists in taking spectra of the peptide acid hydrolysates at different hydrolysis times. Peaks correspond to the oligomers arising from the peptide partial hydrolysis. The sequence can then be identified from the structurally overlapping fragments. PMID:2428356

  13. Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin.

    PubMed

    Kingston, I B; Kingston, B L; Putnam, F W

    1979-04-01

    The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication. PMID:287005

  14. 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

  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. 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

  17. Determining physical constraints in transcriptional initiationcomplexes using DNA sequence analysis

    SciTech Connect

    Shultzaberger, Ryan K.; Chiang, Derek Y.; Moses, Alan M.; Eisen,Michael B.

    2007-07-01

    Eukaryotic gene expression is often under the control ofcooperatively acting transcription factors whose binding is limited bystructural constraints. By determining these structural constraints, wecan understand the "rules" that define functional cooperativity.Conversely, by understanding the rules of binding, we can inferstructural characteristics. We have developed an information theory basedmethod for approximating the physical limitations of cooperativeinteractions by comparing sequence analysis to microarray expressiondata. When applied to the coordinated binding of the sulfur amino acidregulatory protein Met4 by Cbf1 and Met31, we were able to create acombinatorial model that can correctly identify Met4 regulatedgenes.

  18. 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.

  19. Complete genome sequence of Lactobacillus plantarum ZS2058, a probiotic strain with high conjugated linoleic acid production ability.

    PubMed

    Yang, Bo; Chen, Haiqin; Tian, Fengwei; Zhao, Jianxin; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Wei

    2015-11-20

    Lactobacillus plantarum ZS2058 was isolated from sauerkraut and identified to synthesize the beneficial metabolite conjugated linoleic acid. The genome contains a 319,7363-bp chromosome and three plasmids. The sequence will facilitate identification and characterization of the genetic determinants for its putative biological benefits. PMID:26439428

  20. Impact significance determination--Basic considerations and a sequenced approach

    SciTech Connect

    Canter, L.W.; Canty, G.A. . Environmental and Ground Water Inst.)

    1993-09-01

    Determination of the significance of anticipated impacts of proposed projects is a key component in the overall environmental impact assessment (EIA) process. Definitions of significance and/or significant impacts are now included in the EIA guidelines or regulations of many countries and international organizations. Where possible in an EIA study, it is desirable to identify and/or establish the significance determination criteria prior to actual study conduction. This paper summarizes some findings of a survey of such definitions resulting from American, European, and other international experiences; both generic definitions and substantive area definitions are highlighted. Traditional perspectives on significance determination have involved institutional, technical, and public interest considerations. A sequenced approach for impact significance determination is described, with this approach organized around ten groups of issues or questions. Finally, the uses of significance criteria can be noted; included in such uses are: (1) determining if an environmental impact statement (EIS) will be required, or if an environmental assessment/finding of no significant impact (EA/FONSI) will suffice; (2) identifying the impacts that should be mitigated; (3) planning a baseline and/or post-EIS environmental monitoring program; and (4) documenting the interpretive rationale used in the conduction of the environmental impact study.

  1. 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.

  2. Analytical method for determination of benzene-arsenic acids

    SciTech Connect

    Mitchell, G.L.; Bayse, G.S.

    1988-01-01

    A sensitive analytical method has been modified for use in determination of several benzenearsonic acids, including arsanilic acid (p-aminobenzenearsonic acid), Roxarsone (3-nitro-4-hydroxybenzenearsonic acid), and p-ureidobenzene arsonic acid. Controlled acid hydrolysis of these compounds produces a quantitative yield of arsenate, which is measured colorimetrically as the molybdenum blue complex at 865 nm. The method obeys Beer's Law over the micromolar concentration range. These benzenearsonic acids are routinely used as feed additives in poultry and swine. This method should be useful in assessing tissue levels of the arsenicals in appropriate extracts.

  3. 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

  4. A new technique to determine organic and inorganic acid contamination.

    PubMed

    Vo, Evanly

    2002-01-01

    A new acid indicator pad was developed for the detection of acid breakthrough of gloves and chemical protective clothing. The pad carries a reagent which responds to acid contaminant by producing a color change. The pad was used to detect both organic and inorganic acids permeating through glove materials using the modified ASTM F-739 and direct permeability testing procedures. Breakthrough times for each type of glove were determined, and found to range from 4 min to > 4 h for propionic acid, from 3 min to > 4 h for acrylic acid, and from 26 min to > 4 h for HCl. A quantification was performed for propionic and acrylic acids following solvent desorption and gas chromatography. Both acids exhibited > 99% adsorption [the acid and its reactivity (the acid reacted with an indicator to contribute the color change)] on the pads at a spiking level of 1.8 microL for each acid. Acid recovery during quantification was calculated for each acid, ranging from 52-72% (RSD < or = 4.0%) for both acids over the spiking range 0.2-1.8 microL. The quantitative mass of the acids on the pads at the time of breakthrough detection ranged from 260-282 and 270-296 microg cm(-2) for propionic acid and acrylic acid, respectively. The new colorimetric indicator pad should be useful in detecting and collecting acid permeation samples through gloves and chemical protective clothing in both laboratory and field studies, for quantitative analysis. PMID:11827389

  5. Arterial Blood Carbonic Acid Inversely Determines Lactic and Organic Acids

    PubMed Central

    Aiken, Christopher Geoffrey Alexander

    2013-01-01

    Objective: To establish that arterial blood carbonic acid varies inversely with lactic acid in accordance with bicarbonate exchanging for lactate across cell membranes through the anion exchange mechanism to maintain the Gibbs-Donnan equilibrium. Study Design: Over 5 years, lactate was measured on all blood gases taken from neonatal admissions, as well as organic acid whenever electrolytes were required. Results: Arterial blood gases from 63 infants given high calcium TPN were analyzed. Twenty two needed continuous positive airways pressure (CPAP) only and 31 intermittent positive pressure ventilation (IPPV) and surfactant followed by CPAP to treat respiratory distress syndrome in 51 and meconium aspiration syndrome in 2. All survived and were free of infection. Excluded gases were those with high and falling lactate soon after delivery representing perinatal asphyxia, and those on dexamethasone. Strong inverse relations between carbonic and lactic acids were found at all gestational ages and, independent of glomerular filtration, between carbonic and organic acids. Lactate (mmol/L) = 62.53 X PCO2 -0.96(mmHg) r2 0.315, n 1232, p <0.001. Sixty divided by PCO2 is a convenient measure of physiological lactate at any given PCO2. In the first week, 9.13 ± 2.57% of arterial gases from infants on IPPV had lactates above 120/PCO2, significantly more than 4.74 ± 2.73% on CPAP (p<0.05) and 2.47 ± 2.39% on no support. Conclusion: Changes in arterial blood carbonic acid cause immediate inverse changes in lactic acid, because their anions interchange across cell membranes according to the Gibbs –Donnan equilibrium. Increasing PCO2 from 40 to 120 mmHg decreased lactate from 1.5 mmol/L to 0.5 mmol/L, so that the sum of carbonic and lactic acids increased from 2.72 mmol/L to only 4.17 mmol/L. This helps explain the neuroprotective effect of hypercapnoea and highlights the importance of avoiding any degree of hypocapnoea in infants on IPPV. PMID:24392387

  6. Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid.

    PubMed Central

    Mohn, W W

    1995-01-01

    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) PMID:7793937

  7. 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.

  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. 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.

  10. 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.

  11. [Nucleotide sequence determination of yeast mitochondrial phenylalanine-tRNA].

    PubMed

    Martin, R; Sibler, A P; Schneller, J M; Keith, G; Stahl, A J; Dirheimer, G

    1978-10-01

    The primary structure of mitochondrial tRNAPhe from Saccharomyces cerevisiae, purified by two-dimensional polyacrylamide gel electrophoresis, was determined using, standard procedures on in vivo 32P-labeled tRNA, as well as the new 5'-end postlabeling techniques. We propose a cloverleaf model which allows for tertiary interaction between cytosine in position 46 and guanine in position 15 and maximizes base pairing in the psi C stem, thus excluding the uracile in position 50 from base pairing in the psi C stem. Comparison of the primary structure of this tRNA with all other known procaryotic, chloroplastic or cytoplasmic tRNAsPhe sequences does not lead to any conclusion about the endosymbiotic theory of mitochondria evolution. PMID:103657

  12. 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

  13. Partial amino acid sequence of human factor D:homology with serine proteases.

    PubMed Central

    Volanakis, J E; Bhown, A; Bennett, J C; Mole, J E

    1980-01-01

    Human factor D purified to homogeneity by a modified procedure was subjected to NH2-terminal amino acid sequence analysis by using a modified automated Beckman sequencer. We identified 48 of the first 57 NH2-terminal amino acids in a single sequencer run, using microgram quantities of factor D. The deduced amino acid sequence represents approximately 25% of the primary structure of factor D. This extended NH2-terminal amino acid sequence of factor D was compared to that of other trypsin-related serine proteases. By visual inspection, strong homologies (33--50% identity) were observed with all the serine proteases included in the comparison. Interestingly, factor D showed a higher degree of homology to serine proteases of pancreatic origin than to those of serum origin. Images PMID:6987665

  14. Bile acid sulfotransferase I from rat liver sulfates bile acids and 3-hydroxy steroids: purification, N-terminal amino acid sequence, and kinetic properties.

    PubMed

    Barnes, S; Buchina, E S; King, R J; McBurnett, T; Taylor, K B

    1989-04-01

    A bile acid:3'phosphoadenosine-5'phosphosulfate:sulfotransferase (BAST I) from adult female rat liver cytosol has been purified 157-fold by a two-step isolation procedure. The N-terminal amino acid sequence of the 30,000 subunit has been determined for the first 35 residues. The Vmax of purified BAST I is 18.7 nmol/min per mg protein with N-(3-hydroxy-5 beta-cholanoyl)glycine (glycolithocholic acid) as substrate, comparable to that of the corresponding purified human BAST (Chen, L-J., and I. H. Segel, 1985. Arch. Biochem. Biophys. 241: 371-379). BAST I activity has a broad pH optimum from 5.5-7.5. Although maximum activity occurs with 5 mM MgCl2, Mg2+ is not essential for BAST I activity. The greatest sulfotransferase activity and the highest substrate affinity is observed with bile acids or steroids that have a steroid nucleus containing a 3 beta-hydroxy group and a 5-6 double bond or a trans A-B ring junction. These substrates have normal hyperbolic initial velocity curves with substrate inhibition occurring above 5 microM. Of the saturated 5 beta-bile acids, those with a single 3-hydroxy group are the most active. The addition of a second hydroxy group at the 6- or 7-position eliminates more than 99% of the activity. In contrast, 3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) is an excellent substrate. The initial velocity curves for glycolithocholic and deoxycholic acid conjugates are sigmoidal rather than hyperbolic, suggestive of an allosteric effect. Maximum activity is observed at 80 microM for glycolithocholic acid. All substrates, bile acids and steroids, are inhibited by the 5 beta-bile acid, 3-keto-5 beta-cholanoic acid. The data suggest that BAST I is the same protein as hydrosteroid sulfotransferase 2 (Marcus, C. J., et al. 1980. Anal. Biochem. 107: 296-304). PMID:2754334

  15. Amino acid sequence of Japanese quail (Coturnix japonica) and northern bobwhite (Colinus virginianus) myoglobin.

    PubMed

    Goodson, John; Beckstead, Robert B; Payne, Jason; Singh, Rakesh K; Mohan, Anand

    2015-08-15

    Myoglobin has an important physiological role in vertebrates, and as the primary sarcoplasmic pigment in meat, influences quality perception and consumer acceptability. In this study, the amino acid sequences of Japanese quail and northern bobwhite myoglobin were deduced by cDNA cloning of the coding sequence from mRNA. Japanese quail myoglobin was isolated from quail cardiac muscles, purified using ammonium sulphate precipitation and gel-filtration, and subjected to multiple enzymatic digestions. Mass spectrometry corroborated the deduced protein amino acid sequence at the protein level. Sequence analysis revealed both species' myoglobin structures consist of 153 amino acids, differing at only three positions. When compared with chicken myoglobin, Japanese quail showed 98% sequence identity, and northern bobwhite 97% sequence identity. The myoglobin in both quail species contained eight histidine residues instead of the nine present in chicken and turkey. PMID:25794748

  16. Nitric acid uptake by sulfuric acid solutions under stratospheric conditions - Determination of Henry's Law solubility

    NASA Technical Reports Server (NTRS)

    Reihs, Christa M.; Golden, David M.; Tolbert, Margaret A.

    1990-01-01

    The uptake of nitric acid by sulfuric acid solutions representative of stratospheric particulate at low temperatures was measured to determine the solubility of nitric acid in sulfuric acid solutions as a function of H2SO4 concentration and solution temperature. Solubilities are reported for sulfuric acid solutions ranging from 58 to 87 wt pct H2SO4 over a temperature range from 188 to 240 K, showing that, in general, the solubility of nitric acid increases with decreasing sulfuric acid concentration and with decreasing temperature. The measured solubilities indicate that nitric acid in the global stratosphere will be found predominantly in the gas phase.

  17. 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.

  18. 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.

  19. Spectrophotometric determination of tranexamic acid with chloranil.

    PubMed

    Wahbi, A A; Lotfi, E A; Aboul-Enein, H Y

    1984-01-01

    Tranexamic acid is reacted with aqueous alcoholic chloranil, buffered at pH 9, to give a complex with maximum absorption at 346 nm and with an apparent molar absorptivity of 15.7 x 10(3) l.mole(-1).cm(-1). A(max) is linearly related to concentration over the range 2-10 mug ml . When applied to tablets labelled as containing 500 mg each, the mean found was 496 +/- 4 mg. The results were comparable with those of the traditional formol titration method for amino-acids. PMID:18963530

  20. 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

  1. The amino acid sequence of protein CM-3 from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J

    1985-01-01

    Protein CM-3 from Dendroaspis polylepis polylepis venom was purified by gel filtration and ion exchange chromatography. It comprises 65 amino acids including eight half-cystines. The complete amino acid sequence of protein CM-3 has been elucidated. The sequence (residues 1-50) resembles that of the N-terminal sequence of the subunits of a synergistic type protein and residues 51-65 that of the C-terminal sequence of an angusticeps type protein. Mixtures of protein CM-3 and angusticeps type proteins showed no apparent synergistic effect, in that their toxicity in combination was no greater than the sum of their individual toxicities. PMID:4029488

  2. 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.

  3. Determination of the presence of hyaluronic acid in preparations containing amino acids: the molecular weight characterization.

    PubMed

    Bellomaria, A; Nepravishta, R; Mazzanti, U; Marchetti, M; Piccioli, P; Paci, M

    2014-10-15

    Several pharmaceutical preparations contain hyaluronic acid in the presence of a large variety of low molecular weight charged molecules like amino acids. In these mixtures, it is particularly difficult to determine the concentration and the molecular weight of the hyaluronic acid fragments. In fact zwitterionic compounds in high concentration behave by masking the hyaluronic acid due to the electrostatic interactions between amino acids and hyaluronic acid. In such conditions the common colorimetric test of the hyaluronic acid determination appears ineffective and in the (1)H NMR spectra the peaks of the polymer disappear completely. By a simple separation procedure the presence of hyaluronic acid was revealed by the DMAB test and (1)H NMR while its average molecular weight in the final product was determined by DOSY NMR spectroscopy alone. The latter determination is very important due to the healthy effects of some sizes of this polymer's fragments. PMID:25078662

  4. Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution.

    PubMed

    Ruepp, A; Müller, H N; Lottspeich, F; Soppa, J

    1995-03-01

    Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis. PMID:7868583

  5. "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.

  6. "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

  7. Determination of fumaric acid, maleic acid, and phthalic acid in groundwater and soil

    SciTech Connect

    Dietz, E.A.; Singley, K.F. . Technology Center)

    1994-01-01

    When present at > 1 [mu]g/mL, each title compound was determined in groundwater by ion-exclusion chromatography after sample acidification and filtration. For groundwater with one or all analyte concentrations of < 1 [mu]g/mL, the acid anions were first concentrated from a 100-mL sample using a quaternary amine anion-exchange cartridge. The acids were recovered by eluting the cartridge with 1 mL of N H[sub 2]SO[sub 4] and 2-mL deionized water washes; this solution then was examined by anion-exclusion chromatography. Analytes were monitored with a UV detector operated at 200 nm. The analysis procedures for groundwater were validated with solutions which were fortified with from 50 ng/mL to 200 [mu]g/mL of each analyte; recoveries ranged from 90 to 110%. The soil method was validated using fortified samples which contained each acid at concentrations of from 5 to 160 [mu]g/g. Recovery values were between 81 and 120%. For samples exhibiting minimal detector response from compounds other than the acids of interest, 100-[mu]L injection volumes provided an estimated detection limit of 1 [mu]g/g for soil and 10 ng/mL for groundwater.

  8. 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

  9. Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland

    SciTech Connect

    Randhawa, Z.I.; Smith, S.

    1987-03-10

    The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of (/sup 14/C)-labelled peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated M/sub r/ of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.

  10. The complete amino acid sequence of the A-chain of human plasma alpha 2HS-glycoprotein.

    PubMed

    Yoshioka, Y; Gejyo, F; Marti, T; Rickli, E E; Bürgi, W; Offner, G D; Troxler, R F; Schmid, K

    1986-02-01

    Normal human plasma alpha 2HS-glycoprotein has earlier been shown to be comprised of two polypeptide chains. Recently, the amino acid and carbohydrate sequences of the short chain were elucidated (Gejyo, F., Chang, J.-L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R.F., van Halbeck, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 4966-4971). In the present study, the amino acid sequence of the long chain of this protein, designated A-chain, was determined and found to consist of 282 amino acid residues. Twenty-four amino acid doublets were found; the most abundant of these are Pro-Pro and Ala-Ala which each occur five times. Of particular interest is the presence of three Gly-X-Pro and one Gly-Pro-X sequences that are characteristic of the repeating sequences of collagens. Chou-Fasman evaluation of the secondary structure suggested that the A-chain contains 29% alpha-helix, 24% beta-pleated sheet, and 26% reverse turns and, thus, approximately 80% of the polypeptide chain may display ordered structure. Four glycosylation sites were identified. The two N-glycosidic oligosaccharides were found in the center region (residues 138 and 158), whereas the two O-glycosidic heterosaccharides, both linked to threonine (residues 238 and 252), occur within the carboxyl-terminal region. The N-glycans are linked to Asn residues in beta-turns, while the O-glycans are located in short random segments. Comparison of the sequence of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank demonstrated that the A-chain is not significantly related to any known proteins. However, the proline-rich carboxyl-terminal region of the A-chain displays some sequence similarity to collagens and the collagen-like domains of complement subcomponent C1q. PMID:3944104

  11. Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase.

    PubMed

    Taillon, B E; Little, R; Lawther, R P

    1988-03-31

    The nucleotide sequence of the gene, ilvA, for biosynthetic threonine deaminase (Tda) from Salmonella typhimurium was determined. The deduced amino acid sequence was compared with the deduced amino acid sequences of the biosynthetic Tda from Escherichia coli K-12 (ilvA) and Saccharomyces cerevisiae (ILV1) and the biodegradative Tda from E. coli K-12 (tdc). The comparison indicated the presence of two types of blocks of homologous amino acids. The first type of homology is in the N-terminal portion of all four isozymes of Tda and probably indicates amino acids involved in catalysis. The second type of homology is found in the C-terminal portion of the three biosynthetic isozymes and presumably is involved in either (i) the binding or interaction of the allosteric effector isoleucine with the enzyme, or (ii) subunit interactions. The sites of amino acid changes of two E. coli K-12 ilvA alleles with altered response to isoleucine are consistent with the conclusion that the C-terminal portion of biosynthetic Tda is involved in allosteric regulation. PMID:3290055

  12. Accuracy of sequence alignment and fold assessment using reduced amino acid alphabets.

    PubMed

    Melo, Francisco; Marti-Renom, Marc A

    2006-06-01

    Reduced or simplified amino acid alphabets group the 20 naturally occurring amino acids into a smaller number of representative protein residues. To date, several reduced amino acid alphabets have been proposed, which have been derived and optimized by a variety of methods. The resulting reduced amino acid alphabets have been applied to pattern recognition, generation of consensus sequences from multiple alignments, protein folding, and protein structure prediction. In this work, amino acid substitution matrices and statistical potentials were derived based on several reduced amino acid alphabets and their performance assessed in a large benchmark for the tasks of sequence alignment and fold assessment of protein structure models, using as a reference frame the standard alphabet of 20 amino acids. The results showed that a large reduction in the total number of residue types does not necessarily translate into a significant loss of discriminative power for sequence alignment and fold assessment. Therefore, some definitions of a few residue types are able to encode most of the relevant sequence/structure information that is present in the 20 standard amino acids. Based on these results, we suggest that the use of reduced amino acid alphabets may allow to increasing the accuracy of current substitution matrices and statistical potentials for the prediction of protein structure of remote homologs. PMID:16506243

  13. Retroviral DNA Sequences as a Means for Determining Ancient Diets

    PubMed Central

    Rivera-Perez, Jessica I.; Cano, Raul J.; Narganes-Storde, Yvonne; Chanlatte-Baik, Luis; Toranzos, Gary A.

    2015-01-01

    For ages, specialists from varying fields have studied the diets of the primeval inhabitants of our planet, detecting diet remains in archaeological specimens using a range of morphological and biochemical methods. As of recent, metagenomic ancient DNA studies have allowed for the comparison of the fecal and gut microbiomes associated to archaeological specimens from various regions of the world; however the complex dynamics represented in those microbial communities still remain unclear. Theoretically, similar to eukaryote DNA the presence of genes from key microbes or enzymes, as well as the presence of DNA from viruses specific to key organisms, may suggest the ingestion of specific diet components. In this study we demonstrate that ancient virus DNA obtained from coprolites also provides information reconstructing the host’s diet, as inferred from sequences obtained from pre-Columbian coprolites. This depicts a novel and reliable approach to determine new components as well as validate the previously suggested diets of extinct cultures and animals. Furthermore, to our knowledge this represents the first description of the eukaryotic viral diversity found in paleofaeces belonging to pre-Columbian cultures. PMID:26660678

  14. Retroviral DNA Sequences as a Means for Determining Ancient Diets.

    PubMed

    Rivera-Perez, Jessica I; Cano, Raul J; Narganes-Storde, Yvonne; Chanlatte-Baik, Luis; Toranzos, Gary A

    2015-01-01

    For ages, specialists from varying fields have studied the diets of the primeval inhabitants of our planet, detecting diet remains in archaeological specimens using a range of morphological and biochemical methods. As of recent, metagenomic ancient DNA studies have allowed for the comparison of the fecal and gut microbiomes associated to archaeological specimens from various regions of the world; however the complex dynamics represented in those microbial communities still remain unclear. Theoretically, similar to eukaryote DNA the presence of genes from key microbes or enzymes, as well as the presence of DNA from viruses specific to key organisms, may suggest the ingestion of specific diet components. In this study we demonstrate that ancient virus DNA obtained from coprolites also provides information reconstructing the host's diet, as inferred from sequences obtained from pre-Columbian coprolites. This depicts a novel and reliable approach to determine new components as well as validate the previously suggested diets of extinct cultures and animals. Furthermore, to our knowledge this represents the first description of the eukaryotic viral diversity found in paleofaeces belonging to pre-Columbian cultures. PMID:26660678

  15. Antibiotic Selection Pressure Determination through Sequence-Based Metagenomics

    PubMed Central

    El-Hadidi, Mohamed; Huson, Daniel H.; Schütz, Monika; Weidenmaier, Christopher; Autenrieth, Ingo B.; Peter, Silke

    2015-01-01

    The human gut forms a dynamic reservoir of antibiotic resistance genes (ARGs). Treatment with antimicrobial agents has a significant impact on the intestinal resistome and leads to enhanced horizontal transfer and selection of resistance. We have monitored the development of intestinal ARGs over a 6-day course of ciprofloxacin (Cp) treatment in two healthy individuals by using sequenced-based metagenomics and different ARG quantification methods. Fixed- and random-effect models were applied to determine the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Among various shifts in the composition of the intestinal resistome, we found in one individual a strong positive selection for class D beta-lactamases which were partly located on a mobile genetic element. Furthermore, a trend to a negative selection has been observed with class A beta-lactamases (−2.66 hits per million sample reads/defined daily dose; P = 0.06). By 4 weeks after the end of treatment, the composition of ARGs returned toward their initial state but to a different degree in both subjects. We present here a novel analysis algorithm for the determination of antibiotic selection pressure which can be applied in clinical settings to compare therapeutic regimens regarding their effect on the intestinal resistome. This information is of critical importance for clinicians to choose antimicrobial agents with a low selective force on their patients' intestinal ARGs, likely resulting in a diminished spread of resistance and a reduced burden of hospital-acquired infections with multidrug-resistant pathogens. PMID:26369961

  16. Characterization of mouse cellular deoxyribonucleic acid homologous to Abelson murine leukemia virus-specific sequences.

    PubMed Central

    Dale, B; Ozanne, B

    1981-01-01

    The genome of Abelson murine leukemia virus (A-MuLV) consists of sequences derived from both BALB/c mouse deoxyribonucleic acid and the genome of Moloney murine leukemia virus. Using deoxyribonucleic acid linear intermediates as a source of retroviral deoxyribonucleic acid, we isolated a recombinant plasmid which contained 1.9 kilobases of the 3.5-kilobase mouse-derived sequences found in A-MuLV (A-MuLV-specific sequences). We used this clone, designated pSA-17, as a probe restriction enzyme and Southern blot analyses to examine the arrangement of homologous sequences in BALB/c deoxyribonucleic acid (endogenous Abelson sequences). The endogenous Abelson sequences within the mouse genome were interrupted by noncoding regions, suggesting that a rearrangement of the cell sequences was required to produce the sequence found in the virus. Endogenous Abelson sequences were arranged similarly in mice that were susceptible to A-MuLV tumors and in mice that were resistant to A-MuLV tumors. An examination of three BALB/c plasmacytomas and a BALB/c early B-cell tumor likewise revealed no alteration in the arrangement of the endogenous Abelson sequences. Homology to pSA-17 was also observed in deoxyribonucleic acids prepared from rat, hamster, chicken, and human cells. An isolate of A-MuLV which encoded a 160,000-dalton transforming protein (P160) contained 700 more base pairs of mouse sequences than the standard A-MuLV isolate, which encoded a 120,000-dalton transforming protein (P120). Images PMID:9279386

  17. 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

  18. Detection and sequence determination of a new variant beta-lactoglobulin II from donkey.

    PubMed

    Cunsolo, Vincenzo; Costa, Alessia; Saletti, Rosaria; Muccilli, Vera; Foti, Salvatore

    2007-01-01

    The sequence determination of a new variant of beta-LG II, detected as a minor component by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) analysis of the whey fraction from a milk sample taken from an individual donkey belonging to the 'Ragusana' species of eastern Sicily, is reported. Direct RP-HPLC/ESI-MS analysis of the whey fraction from this milk sample allowed the identification of a new variant of beta-LG II, based on the determination of the M(r) of the intact protein. The new protein, with an experimentally determined M(r) of 18311 Da, was detected as a minor component in the whey fraction investigated. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)MS and RP-HPLC/ESI-MS/MS analyses of the tryptic digest of the new protein demonstrate that it presents two amino acid substitutions with respect to the sequence of beta-LG II A, namely a substitution Pro-->Cys at position 110, and a substitution Asp-->Gly at position 162. The disulfide bonds between the four cysteines, not directly determined in donkey's and horse's beta-LG II, were shown to occur between Cys(106)-Cys(120) and Cys(66)-Cys(161), as in other mammalian beta-LGs. The new beta-LG II variant from donkey was named D. PMID:17377935

  19. 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.

  20. 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

  1. Complete Genome Sequence of Enterococcus mundtii QU 25, an Efficient l-(+)-Lactic Acid-Producing Bacterium

    PubMed Central

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

    2014-01-01

    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. PMID:24568933

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

    PubMed

    Pinto, Ameet J; Sharp, Jonathan O; Yoder, Michael J; Almstrand, Robert

    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

  3. 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

  4. Determination of Acidity Constants by Gradient Flow-Injection Titration

    ERIC Educational Resources Information Center

    Conceicao, Antonio C. L.; Minas da Piedade, Manuel E.

    2006-01-01

    A three-hour laboratory experiment, designed for an advanced undergraduate course in instrumental analysis that illustrates the application of the gradient chamber flow-injection titration (GCFIT) method with spectrophotometric detection to determine acidity constants is presented. The procedure involves the use of an acid-base indicator to obtain…

  5. 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

  6. The amino acid sequence of protein SCMK-B2A from the high-sulphur fraction of wool keratin

    PubMed Central

    Elleman, T. C.

    1972-01-01

    1. The amino acid sequence of protein SCMK-B2A, a reduced and S-carboxymethylated protein from the high-sulphur fraction of wool, has been determined. 2. This protein of 171 amino acid residues displays both a high degree of internal homology and extensive external homology with other members of the SCMK-B2 group of proteins. 3. Evidence is presented which suggests that the SCMK-B2 group of proteins are produced by separate non-allelic genes. ImagesPLATE 1 PMID:4679226

  7. Evaluation of ultrafiltration for determining molecular weight of fulvic acid

    USGS Publications Warehouse

    Aiken, G.R.

    1984-01-01

    Two commonly used ultrafiltration membranes are evaluated for the determination of molecular weights of humic substances. Polyacrylic acids of Mr 2000 and 5000 and two well-characterized fulvic acids are used as standards. Molecular size characteristics of standards, as determined by small-angle X-ray scattering, are presented. Great care in evaluating molecular weight data obtained by ultrafiltration is needed because of broad nominal cutoffs and membrane-solute interactions.

  8. 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.

  9. N-terminal sequence of amino acids and some properties of an acid-stable alpha-amylase from citric acid-koji (Aspergillus usamii var.).

    PubMed

    Suganuma, T; Tahara, N; Kitahara, K; Nagahama, T; Inuzuka, K

    1996-01-01

    An acid-stable alpha-amylase (AA) was purified from an acidic extract of citric acid-koji (A. usamii var.). The N-terminal sequence of the first 20 amino acids of the enzyme was identical with that of AA from A. niger, but the two enzymes differed in molecular weight. HPLC analysis for identifying the anomers of products indicated that the AA hydrolyzed maltopentaose (G5) at the third glycoside bond predominantly, which differed from Taka-amylase A and the neutral alpha-amylase (NA) from the citric acid-koji. PMID:8824843

  10. Sequence Determination from Overlapping Fragments: A Simple Model of Whole-Genome Shotgun Sequencing

    NASA Astrophysics Data System (ADS)

    Derrida, Bernard; Fink, Thomas M.

    2002-02-01

    Assembling fragments randomly sampled from along a sequence is the basis of whole-genome shotgun sequencing, a technique used to map the DNA of the human and other genomes. We calculate the probability that a random sequence can be recovered from a collection of overlapping fragments. We provide an exact solution for an infinite alphabet and in the case of constant overlaps. For the general problem we apply two assembly strategies and give the probability that the assembly puzzle can be solved in the limit of infinitely many fragments.

  11. cDNA cloning and structural characterization of a lectin from the mussel Crenomytilus grayanus with a unique amino acid sequence and antibacterial activity.

    PubMed

    Kovalchuk, Svetlana N; Chikalovets, Irina V; Chernikov, Oleg V; Molchanova, Valentina I; Li, Wei; Rasskazov, Valery A; Lukyanov, Pavel A

    2013-10-01

    An amino acid sequence of GalNAc/Gal-specific lectin from the mussel Crenomytilus grayanus (CGL) was determined by cDNA sequencing. CGL consists of 150 amino acid residues, contains three tandem repeats with high sequence similarities to each other (up to 73%) and does not belong to any known lectins family. According to circular dichroism results CGL is a β/α-protein with the predominance of β-structure. CGL was predicted to adopt a ß-trefoil fold. The lectin exhibits antibacterial activity and might be involved in the recognition and clearance of bacterial pathogens in the shellfish. PMID:23886951

  12. Snake venom toxins. The amino acid sequence of toxin Vi2, a homologue of pancreatic trypsin inhibitor, from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Strydom, D J

    1977-04-25

    The amino acid sequence of venom component Vi2, a protein of low toxicity from Dendroaspis polylepis polylepis venom was determined by automatic sequence analysis in combination with sequence studies on tryptic peptides. This protein, the most retarded fraction of this venom on a cation-exchange resin, is a homologue of bovine pancreatic trypsin inhibitor consisting of a single chain of 57 amino acid residues containing six half-cystine residues. The active site lysyl residue of bovine trypsin inhibitor is conserved in Vi2 although large differences are found in the rest of the molecule. PMID:857902

  13. Determination of the acidic sites of purified single-walled carbon nanotubes by acid base titration

    NASA Astrophysics Data System (ADS)

    Hu, H.; Bhowmik, P.; Zhao, B.; Hamon, M. A.; Itkis, M. E.; Haddon, R. C.

    2001-09-01

    We report the measurement of the acidic sites in three different samples of commercially available full-length purified single-walled carbon nanotubes (SWNTs) - as obtained from CarboLex (CLI), Carbon Solutions (CSI) and Tubes@Rice (TAR) - by simple acid-base titration methods. Titration of the purified SWNTs with NaOH and NaHCO 3 solutions was used to determine the total percentage of acidic sites and carboxylic acid groups, respectively. The total percentage of acidic sites in full length purified SWNTs from TAR, CLI and CSI are about 1-3%.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

    PubMed Central

    Chintapalli, Sree V.; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L.; van Rossum, Damian B.; Anishkin, Andriy; Adams, Sean H.

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  19. Molecular dynamic simulations reveal the structural determinants of Fatty Acid binding to oxy-myoglobin.

    PubMed

    Chintapalli, Sree V; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L; van Rossum, Damian B; Anishkin, Andriy; Adams, Sean H

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a "U" shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  20. Ultrasensitive determination of DNA sequences by flow injection chemiluminescence using silver ions as labels.

    PubMed

    Zheng, Lichun; Liu, Xiuhui; Zhou, Min; Ma, Yongjun; Wu, Guofan; Lu, Xiaoquan

    2014-10-27

    We presented a new strategy for ultrasensitive detection of DNA sequences based on the novel detection probe which was labeled with Ag(+) using metallothionein (MT) as a bridge. The assay relied on a sandwich-type DNA hybridization in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on Fe3O4@Au composite magnetic nanoparticles (MNPs), and then the Ag(+)-modified detection probes were used to monitor the presence of the specific DNA targets. After being anchored on the hybrids, Ag(+) was released down through acidic treatment and sensitively determined by a coupling flow injection-chemiluminescent reaction system (Ag(+)-Mn(2+)-K2S2O8-H3PO4-luminol) (FI-CL). The experiment results showed that the CL intensities increased linearly with the concentrations of DNA targets in the range from 10 to 500 pmol L(-1) with a detection limit of 3.3 pmol L(-1). The high sensitivity in this work may be ascribed to the high molar ratio of Ag(+)-MT, the sensitive determination of Ag(+) by the coupling FI-CL reaction system and the perfect magnetic separation based on Fe3O4@Au composite MNPs. Moreover, the proposed strategy exhibited excellent selectivity against the mismatched DNA sequences and could be applied to real samples analysis. PMID:25263118

  1. Sequence of arrival determines plant-mediated interactions between herbivores

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Summary 1. Induced changes in plant quality are important factors mediating indirect interactions between herbivores. Although the sequence of attack has been shown to influence plant responses, little is known about how it may affect the outcome of insect-plant-insect interactions. 2. We therefore...

  2. 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

  3. Characterization of N-glycosylation and amino acid sequence features of immunoglobulins from swine.

    PubMed

    Lopez, Paul G; Girard, Lauren; Buist, Marjorie; de Oliveira, Andrey Giovanni Gomes; Bodnar, Edward; Salama, Apolline; Soulillou, Jean-Paul; Perreault, Hélène

    2016-02-01

    The primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to β-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immunogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features. PMID:26586247

  4. 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. PMID:11281267

  5. [Spectrophotometric determination of aromatic amino compounds with J-acid].

    PubMed

    Yin, Xiao-hang; Shi, Wen-jian; Shen, Xin; Ma, Jun-tao; Li, Liang

    2015-01-01

    The problems such as chromogenic reaction selectivity, reaction rate, sensitivity and water-solubility of azo compounds were considered. The molecular structures of coupling components were theoretically designed and screened in the present research The reaction conditions and methods of chromogenic reaction were investigated. J-Acid (2-amino-5-naphthol-7-sulfonic acid) as a coupling reagent to determine aromatic amino compounds was established. In the presence of potassium bromide, at room temperature, nitrite reacted with aromatic amino compounds in the medium of thin hydrochloric acid. Then diazonium salt reacted with J-Acid in the aqueous solution of sodium carbonate, forming coloured azo dye, which had a maximum adsorption at 480 nm. The molar adsorption coeffcients of aniline, 4-aminobenzene sulfonic acid and 1-naphthylamine were 3. 95 X 10(4), 3. 24 X 10(4) and 3. 91 X 10(4) L . mol-1 . cm-1 , respectively. Experimental results showed that common coexisting ions on the surface water did not affect the results of determination. J-Acid of spectrophotometry was used to determine the samples of Shanghai Fu Xing Dao canal. Meanwhile, recovery experiments by standard addition method were done. Experiment results showed that the recoveries of aniline were in the range of 98. 5%-102. 1%, and RSD was 2. 08%. J-Acid is a common organic reagent. It is soluble in water and low volatile, and its toxicity is much lower than N-ethylenediamine. spectrophotometric determination of aromatic amino compounds by J-Acid has the advantage of high sensitivity, good selectivity, simple rapid operation and accurate results, and thus it can be used for the determination of trace aromatic amino compounds in the environmental water. PMID:25993847

  6. 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.

  7. Kinetic-spectrophotometric determination of ascorbic acid by inhibition of the hydrochloric acid-bromate reaction

    NASA Astrophysics Data System (ADS)

    Ensafi, Ali A.; Rezaei, B.; Movahedinia, H.

    2002-10-01

    A new analytical method was developed for the determination of ascorbic acid in fruit juice and pharmaceuticals. The method is based on its inhibition effect on the reaction between hydrochloric acid and bromate. The decolourisation of Methyl Orange by the reaction products was used to monitor the reaction spectrophotometrically at 510 nm. The linearity range of the calibration graph depends on bromate concentration. The variable affecting the rate of the reaction was investigated. The method is simple, rapid, relatively sensitive and precise. The limit of detection is 7.6×10 -6 M and calibration rang is 8×10 -6-1.2×10 -3 M ascorbic acid. The relative standard deviation of seven replication determinations of 8×10 -6 and 2×10 -5 M ascorbic acid was 2.8 and 1.7%, respectively. The influence of potential interfering substance was studied. The method was successfully applied for the determination of ascorbic acid in pharmaceuticals.

  8. 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

  9. Automated mass spectrometric sequence determination of cyclic peptide library members.

    PubMed

    Redman, James E; Wilcoxen, Keith M; Ghadiri, M Reza

    2003-01-01

    Cyclic peptides have come under scrutiny as potential antimicrobial therapeutic agents. Combinatorial split-and-pool synthesis of cyclic peptides can afford single compound per well libraries for antimicrobial screening, new lead identification, and construction of quantitative structure-activity relationships (QSAR). Here, we report a new sequencing protocol for rapid identification of the members of a cyclic peptide library based on automated computer analysis of mass spectra, obviating the need for library encoding/decoding strategies. Furthermore, the software readily integrates with common spreadsheet and database packages to facilitate data visualization and archiving. The utility of the new MS-sequencing approach is demonstrated using sonic spray ionization ion trap MS and MS/MS spectrometry on a single compound per bead cyclic peptide library and validated with individually synthesized pure cyclic D,L-alpha-peptides. PMID:12523832

  10. Determinants of the rate of protein sequence evolution

    PubMed Central

    Zhang, Jianzhi; Yang, Jian-Rong

    2015-01-01

    The rate and mechanism of protein sequence evolution have been central questions in evolutionary biology since the 1960s. Although the rate of protein sequence evolution depends primarily on the level of functional constraint, exactly what constitutes functional constraint has remained unclear. The increasing availability of genomic data has allowed for much needed empirical examinations on the nature of functional constraint. These studies found that the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance. A combination of theoretical and empirical analyses have identified multiple mechanisms behind these observations and demonstrated a prominent role that selection against errors in molecular and cellular processes plays in protein evolution. PMID:26055156

  11. Sequence determinants of improved CRISPR sgRNA design

    PubMed Central

    Xu, Han; Xiao, Tengfei; Chen, Chen-Hao; Li, Wei; Meyer, Clifford A.; Wu, Qiu; Wu, Di; Cong, Le; Zhang, Feng; Liu, Jun S.; Brown, Myles; Liu, X. Shirley

    2015-01-01

    The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies. PMID:26063738

  12. A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries

    PubMed Central

    Volles, Michael J.; Lansbury, Peter T.

    2005-01-01

    A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 109 sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an

  13. Canine preprorelaxin: nucleic acid sequence and localization within the canine placenta.

    PubMed

    Klonisch, T; Hombach-Klonisch, S; Froehlich, C; Kauffold, J; Steger, K; Steinetz, B G; Fischer, B

    1999-03-01

    Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E-->D and L-->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta. PMID:10026098

  14. Ab initio detection of fuzzy amino acid tandem repeats in protein sequences

    PubMed Central

    2012-01-01

    Background Tandem repetitions within protein amino acid sequences often correspond to regular secondary structures and form multi-repeat 3D assemblies of varied size and function. Developing internal repetitions is one of the evolutionary mechanisms that proteins employ to adapt their structure and function under evolutionary pressure. While there is keen interest in understanding such phenomena, detection of repeating structures based only on sequence analysis is considered an arduous task, since structure and function is often preserved even under considerable sequence divergence (fuzzy tandem repeats). Results In this paper we present PTRStalker, a new algorithm for ab-initio detection of fuzzy tandem repeats in protein amino acid sequences. In the reported results we show that by feeding PTRStalker with amino acid sequences from the UniProtKB/Swiss-Prot database we detect novel tandemly repeated structures not captured by other state-of-the-art tools. Experiments with membrane proteins indicate that PTRStalker can detect global symmetries in the primary structure which are then reflected in the tertiary structure. Conclusions PTRStalker is able to detect fuzzy tandem repeating structures in protein sequences, with performance beyond the current state-of-the art. Such a tool may be a valuable support to investigating protein structural properties when tertiary X-ray data is not available. PMID:22536906

  15. Multimodal phylogeny for taxonomy: integrating information from nucleotide and amino acid sequences.

    PubMed

    Bicego, Manuele; Dellaglio, Franco; Felis, Giovanna E

    2007-10-01

    The crucial role played by the analysis of microbial diversity in biotechnology-based innovations has increased the interest in the microbial taxonomy research area. Phylogenetic sequence analyses have contributed significantly to the advances in this field, also in the view of the large amount of sequence data collected in recent years. Phylogenetic analyses could be realized on the basis of protein-encoding nucleotide sequences or encoded amino acid molecules: these two mechanisms present different peculiarities, still starting from two alternative representations of the same information. This complementarity could be exploited to achieve a multimodal phylogenetic scheme that is able to integrate gene and protein information in order to realize a single final tree. This aspect has been poorly addressed in the literature. In this paper, we propose to integrate the two phylogenetic analyses using basic schemes derived from the multimodality fusion theory (or multiclassifier systems theory), a well-founded and rigorous branch for which its powerfulness has already been demonstrated in other pattern recognition contexts. The proposed approach could be applied to distance matrix-based phylogenetic techniques (like neighbor joining), resulting in a smart and fast method. The proposed methodology has been tested in a real case involving sequences of some species of lactic acid bacteria. With this dataset, both nucleotide sequence- and amino acid sequence-based phylogenetic analyses present some drawbacks, which are overcome with the multimodal analysis. PMID:17933011

  16. 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

  17. A classification of glycosyl hydrolases based on amino acid sequence similarities.

    PubMed Central

    Henrissat, B

    1991-01-01

    The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised. PMID:1747104

  18. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.

    PubMed Central

    Henrissat, B; Bairoch, A

    1993-01-01

    301 glycosyl hydrolases and related enzymes corresponding to 39 EC entries of the I.U.B. classification system have been classified into 35 families on the basis of amino-acid-sequence similarities [Henrissat (1991) Biochem. J. 280, 309-316]. Approximately half of the families were found to be monospecific (containing only one EC number), whereas the other half were found to be polyspecific (containing at least two EC numbers). A > 60% increase in sequence data for glycosyl hydrolases (181 additional enzymes or enzyme domains sequences have since become available) allowed us to update the classification not only by the addition of more members to already identified families, but also by the finding of ten new families. On the basis of a comparison of 482 sequences corresponding to 52 EC entries, 45 families, out of which 22 are polyspecific, can now be defined. This classification has been implemented in the SWISS-PROT protein sequence data bank. PMID:8352747

  19. Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.

    PubMed

    Orum, H; Nielsen, P E; Jørgensen, M; Larsson, C; Stanley, C; Koch, T

    1995-09-01

    Using an oligohistidine peptide nucleic acids (oligohistidine-PNA) chimera, we have developed a rapid hybrid selection method that allows efficient, sequence-specific purification of a target nucleic acid. The method exploits two fundamental features of PNA. First, that PNA binds with high affinity and specificity to its complementary nucleic acid. Second, that amino acids are easily attached to the PNA oligomer during synthesis. We show that a (His)6-PNA chimera exhibits strong binding to chelated Ni2+ ions without compromising its native PNA hybridization properties. We further show that these characteristics allow the (His)6-PNA/DNA complex to be purified by the well-established method of metal ion affinity chromatography using a Ni(2+)-NTA (nitrilotriactic acid) resin. Specificity and efficiency are the touchstones of any nucleic acid purification scheme. We show that the specificity of the (His)6-PNA selection approach is such that oligonucleotides differing by only a single nucleotide can be selectively purified. We also show that large RNAs (2224 nucleotides) can be captured with high efficiency by using multiple (His)6-PNA probes. PNA can hybridize to nucleic acids in low-salt concentrations that destabilize native nucleic acid structures. We demonstrate that this property of PNA can be utilized to purify an oligonucleotide in which the target sequence forms part of an intramolecular stem/loop structure. PMID:7495562

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

    PubMed

    Kim, Y; Lee, J; Lee, C

    2008-01-01

    Genetic variability might contribute to species specificity of prion diseases in various organisms. In this study, structures of the prion protein gene (PRNP) and its amino acids were compared among species of which sequence data were available. Comparisons of PRNP DNA sequences among 12 species including human, chimpanzee, monkey, bovine, ovine, dog, mouse, rat, wallaby, opossum, chicken and zebrafish allowed us to identify candidate regulatory regions in intron 1 and 3'-untranslated region (UTR) in addition to the coding region. Highly conserved putative binding sites for transcription factors, such as heat shock factor 2 (HSF2) and myocite enhancer factor 2 (MEF2), were discovered in the intron 1. In 3'-UTR, the functional sequence (ATTAAA) for nucleus-specific polyadenylation was found in all the analysed species. The functional sequence (TTTTTAT) for maturation-specific polyadenylation was identically observed only in ovine, and one or two nucleotide mismatches in the other species. A comparison of the amino acid sequences in 53 species revealed a large sequence identity. Especially the octapeptide repeat region was observed in all the species but frog and zebrafish. Functional changes and susceptibility to prion diseases with various isoforms of prion protein could be caused by numeric variability and conformational changes discovered in the repeat sequences. PMID:18397498

  1. 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

  2. Using isotope dilution mass spectrometry to determine aqueous trichloroacetic acid

    SciTech Connect

    Norwood, D.L.; Christman, R.F.; Johnson, J.D.; Hass, J.R.

    1986-01-01

    The development, verification, and application of a method based on isotope-dilution gas chromatography-mass spectrometry to determine aqueous trichloroacetic acid (TCAA) at the micrograms per litre level are described. The simultaneous determination of aqueous chloroform is also demonstrated. Trichloroacetic acid is shown to be a significant by-product of the chlorination of raw waters in the laboratory and to constitute a large fraction of the total organic halide (TOX) formed. Analysis of finished-water samples indicated that TCAA, like trihalomethanes is ubiquitous. Positive correlations exist between the levels of TCAA in laboratory-chlorinated raw waters and in finished waters and measured TOX.

  3. cDNA-derived amino-acid sequence of a land turtle (Geochelone carbonaria) beta-chain hemoglobin.

    PubMed

    Bordin, S; Meza, A N; Saad, S T; Ogo, S H; Costa, F F

    1997-06-01

    The cDNA sequence encoding the turtle Geochelone carbonaria beta-chain was determinated. The isolation of hemoglobin mRNA was based on degenerate primers' PCR in combination with 5'- and 3'-RACE protocol. The full length cDNA is 615 bp with the ATG start codon at position 53 and TGA stop codon at position 495; The AATAAA polyadenylation signal is found at position 599. The deduced polypeptyde contains 146 amino-acid residues. The predicted amino acid sequence shares 83% identity with the beta-globin of a related specie, the aquatic turtle C. p. belli. Otherwise, identity is higher when compared with chicken beta-Hb (80%) than with other reptilian orders (Squamata, 69%, and Crocodilia, 61%). Compared with human HbA, there is 67% identity, and at least three amino acid substitutions could be of some functional significance (Glu43 beta-->Ser, His116 beta-->Thr and His143 beta-->Leu). To our knowledge this represents the first cDNA sequence of a reptile globin gene described. PMID:9238523

  4. Determining 3-D motion and structure from image sequences

    NASA Technical Reports Server (NTRS)

    Huang, T. S.

    1982-01-01

    A method of determining three-dimensional motion and structure from two image frames is presented. The method requires eight point correspondences between the two frames, from which motion and structure parameters are determined by solving a set of eight linear equations and a singular value decomposition of a 3x3 matrix. It is shown that the solution thus obtained is unique.

  5. Determinants of congruency sequence effects without learning and memory confounds.

    PubMed

    Weissman, Daniel H; Jiang, Jiefeng; Egner, Tobias

    2014-10-01

    A common finding in distracter interference (e.g., Flanker) tasks is that the difference in mean reaction time (RT) between incongruent and congruent trials-the congruency effect-is smaller when the previous trial was incongruent relative to congruent. Over the past 2 decades, 2 main accounts of this congruency sequence effect (CSE) have been proposed. One posits that the CSE indexes trial-by-trial adjustments of cognitive control, which are triggered by expectation, response conflict, negative affect, or response suppression. The other holds that the CSE indexes feature integration and/or contingency learning processes that are confounded with congruency sequence in most studies. In 3 online experiments involving over 450 participants, we observed CSEs without such confounds when 2 preconditions were met: (a) stimulus-response translation could be completed more rapidly for the distracter than for the target and (b) the distracter and target appeared at the same location. We also found that CSE magnitude did not vary consistently with the size of the congruency effect. These findings reveal that CSEs can be observed in the absence of feature integration and contingency learning confounds, but impose important new constraints on certain cognitive control accounts of this phenomenon. PMID:25089574

  6. Potentiometric determination of free acidity in presence of hydrolysable ions and a sequential determination of hydrazine.

    PubMed

    Ganesh, S; Khan, Fahmida; Ahmed, M K; Pandey, S K

    2011-08-15

    A simple potentiometric method for the determination of free acidity in presence of hydrolysable ions and sequential determination of hydrazine is developed and described. Both free acid and hydrazine are estimated from the same aliquot. In this method, free acid is titrated with standard sodium carbonate solution after the metal ions in solutions are masked with EDTA. Once the end point for the free acid is determined at pH 3.0, an aliquot of formaldehyde is added to liberate the acid equivalent to hydrazine which is then titrated with the same standard sodium carbonate solution using an automatic titration system. The described method is simple, accurate and reproducible. This method is especially applicable to all ranges of nitric acid and heavy metal ion concentration relevant to Purex process used for nuclear fuel reprocessing. The overall recovery of nitric acid is 98.9% with 1.2% relative standard deviation. Hydrazine content has also been determined in the same aliquot with a recovery of nitric acid is 99% with 2% relative standard deviation. The major advantage of the method is that generation of corrosive analytical wastes containing oxalate or sulphate is avoided. Valuable metals like uranium and plutonium can easily be recovered from analytical waste before final disposal. PMID:21726724

  7. 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

  8. Draft Genome Sequence of Perfluorooctane Acid-Degrading Bacterium Pseudomonas parafulva YAB-1

    PubMed Central

    Tang, Chongjian; Peng, Qingjing; Peng, Qingzhong

    2015-01-01

    Pseudomonas parafulva YAB-1, isolated from perfluorinated compound-contaminated soil, has the ability to degrade perfluorooctane acid (PFOA) compound. Here, we report the draft genome sequence and annotation of the PFOA-degrading bacterium P. parafulva YAB-1. The data provide the basis to investigate the molecular mechanism of PFOA metabolism. PMID:26337877

  9. Cloning and nucleotide sequencing of genes for three small, acid-soluble proteins from Bacillus subtilis spores.

    PubMed Central

    Connors, M J; Mason, J M; Setlow, P

    1986-01-01

    Three Bacillus subtilis genes (termed sspA, sspB, and sspD) which code for small, acid-soluble spore proteins (SASPs) have been cloned, and their complete nucleotide sequence has been determined. The amino acid sequences of the SASPs coded for by these genes are similar to each other and to those of the SASP-1 of B. subtilis (coded for by the sspC gene) and the SASP-A/C family of B. megaterium. The sspA and sspB genes are expressed only in sporulation, in parallel with each other and with the sspC gene. Two regions upstream of the postulated transcription start sites for the sspA and B genes have significant homology with the analogous regions of the sspC gene and the SASP-A/C gene family. Purification of two of the three major B, subtilis SASPs (alpha and beta) and determination of their amino-terminal sequences indicated that the sspA gene codes for SASP-alpha and that the sspB gene codes for SASP-beta. This was confirmed by the introduction of deletion mutations into the cloned sspA and sspB genes and transfer of these deletions into the B. subtilis chromosome with concomitant loss of the wild-type gene. Images PMID:3009398

  10. Determination of ursolic and oleanolic acid in Sambuci fructus.

    PubMed

    Gleńsk, Michał; Gliński, Jan A; Włodarczyk, Maciej; Stefanowicz, Piotr

    2014-12-01

    Elderberries are used in the preparation of pie, jelly, punch, wine, or liqueur, as well as in many herbal remedies and food supplements. Elderberry products may provide diaphoretic, diuretic, antioxidant, and immunostimulant activities that offer protection against cold and flu. Herein, we report for the first time the qualitative and quantitative evaluation of two isomeric triterpenoids isolated from Sambuci fructus. The analysis revealed that ursolic acid and oleanolic acid are present in Sambuci fructus. The average concentration of ursolic acid was ca. three times higher than the concentration of oleanolic acid. The triterpenoids were detected and quantified using chromatographic methods such as TLC and HPLC. Spectroscopic techniques, including HR-MS and 2D-NMR, allowed unequivocal structure determination. PMID:25491337

  11. Sequence Determinants of Circadian Gene Expression Phase in Cyanobacteria

    PubMed Central

    Vijayan, Vikram

    2013-01-01

    The cyanobacterium Synechococcus elongatus PCC 7942 exhibits global biphasic circadian oscillations in gene expression under constant-light conditions. Class I genes are maximally expressed in the subjective dusk, whereas class II genes are maximally expressed in the subjective dawn. Here, we identify sequence features that encode the phase of circadian gene expression. We find that, for multiple genes, an ∼70-nucleotide promoter fragment is sufficient to specify class I or II phase. We demonstrate that the gene expression phase can be changed by random mutagenesis and that a single-nucleotide substitution is sufficient to change the phase. Our study provides insight into how the gene expression phase is encoded in the cyanobacterial genome. PMID:23204469

  12. Homogentisic acid interference in routine urine creatinine determination.

    PubMed

    Loken, Perry R; Magera, Mark J; Introne, Wendy; Tortorelli, Silvia; Gavrilov, Dimitar; Oglesbee, Devin; Rinaldo, Piero; Matern, Dietrich; Raymond, Kimiyo

    2010-05-01

    We report the artifactual elevation of homogentisic acid (HGA) in urine from alkaptonuric patients after replacing the creatinine method (Jaffe reaction) in our laboratory with an automated enzymatic method. Samples with elevated HGA by GC-MS had lower creatinine values as determined by the enzymatic method than by the Jaffe reaction. The low creatinine values were due to interference by HGA in the enzymatic method. The enzymatic method is unsuitable for creatinine determination in urine of patients with alkaptonuria. PMID:20138792

  13. 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.

  14. Use of a structural alphabet to find compatible folds for amino acid sequences

    PubMed Central

    Mahajan, Swapnil; de Brevern, Alexandre G; Sanejouand, Yves-Henri; Srinivasan, Narayanaswamy; Offmann, Bernard

    2015-01-01

    The structural annotation of proteins with no detectable homologs of known 3D structure identified using sequence-search methods is a major challenge today. We propose an original method that computes the conditional probabilities for the amino-acid sequence of a protein to fit to known protein 3D structures using a structural alphabet, known as “Protein Blocks” (PBs). PBs constitute a library of 16 local structural prototypes that approximate every part of protein backbone structures. It is used to encode 3D protein structures into 1D PB sequences and to capture sequence to structure relationships. Our method relies on amino acid occurrence matrices, one for each PB, to score global and local threading of query amino acid sequences to protein folds encoded into PB sequences. It does not use any information from residue contacts or sequence-search methods or explicit incorporation of hydrophobic effect. The performance of the method was assessed with independent test datasets derived from SCOP 1.75A. With a Z-score cutoff that achieved 95% specificity (i.e., less than 5% false positives), global and local threading showed sensitivity of 64.1% and 34.2%, respectively. We further tested its performance on 57 difficult CASP10 targets that had no known homologs in PDB: 38 compatible templates were identified by our approach and 66% of these hits yielded correctly predicted structures. This method scales-up well and offers promising perspectives for structural annotations at genomic level. It has been implemented in the form of a web-server that is freely available at http://www.bo-protscience.fr/forsa. PMID:25297700

  15. Use of a structural alphabet to find compatible folds for amino acid sequences.

    PubMed

    Mahajan, Swapnil; de Brevern, Alexandre G; Sanejouand, Yves-Henri; Srinivasan, Narayanaswamy; Offmann, Bernard

    2015-01-01

    The structural annotation of proteins with no detectable homologs of known 3D structure identified using sequence-search methods is a major challenge today. We propose an original method that computes the conditional probabilities for the amino-acid sequence of a protein to fit to known protein 3D structures using a structural alphabet, known as "Protein Blocks" (PBs). PBs constitute a library of 16 local structural prototypes that approximate every part of protein backbone structures. It is used to encode 3D protein structures into 1D PB sequences and to capture sequence to structure relationships. Our method relies on amino acid occurrence matrices, one for each PB, to score global and local threading of query amino acid sequences to protein folds encoded into PB sequences. It does not use any information from residue contacts or sequence-search methods or explicit incorporation of hydrophobic effect. The performance of the method was assessed with independent test datasets derived from SCOP 1.75A. With a Z-score cutoff that achieved 95% specificity (i.e., less than 5% false positives), global and local threading showed sensitivity of 64.1% and 34.2%, respectively. We further tested its performance on 57 difficult CASP10 targets that had no known homologs in PDB: 38 compatible templates were identified by our approach and 66% of these hits yielded correctly predicted structures. This method scales-up well and offers promising perspectives for structural annotations at genomic level. It has been implemented in the form of a web-server that is freely available at http://www.bo-protscience.fr/forsa. PMID:25297700

  16. Characterization and cDNA sequence of Bothriechis schlegeliil-amino acid oxidase with antibacterial activity.

    PubMed

    Vargas Muñoz, Leidy Johana; Estrada-Gomez, Sebastian; Núñez, Vitelbina; Sanz, Libia; Calvete, Juan J

    2014-08-01

    Snake venoms are complex mixtures of proteins including l-amino acid oxidase (lAAO). A lAAO (named BslAAO) with a mass of 56kDa and a theoretical Ip of 5.79, was purified from Bothriechis schlegelii venom through size-exclusion, ion exchange and affinity chromatography. The entire protein sequence of 498 amino acids, was determined from cDNA using reverse-transcribed mRNA isolated from venom gland. The enzyme showed dose-dependent inhibition of bacterial growth. BslAAO showed inhibitory effect against S. aureus with a MIC of 4μg/mL and a MBC of 8μg/mL. Against Acinetobacter baumannii, showed a MIC of 2μg/mL and MBC of 4μg/mL, No effect was observed in Escherichia coli. This antibacterial activity was inhibited by catalase, indicating that antimicrobial activity was due to H2O2 production. BslAAO did not show any cytotoxic activity toward mouse myoblast cell line C2C12 or peripheral blood mononuclear cells. The enzyme oxidated l-Leu, with a Km of 16.37μM and a Vmax of 0.39μM/min. Snake venoms lAAOs, are potential frames of different therapeutics molecules since these enzymes exhibit low MICs and MBCs and show to be harmless to human cells due to microorganisms being generally several fold more sensitive to reactive oxygen species than human tissues. PMID:24875315

  17. Software scripts for quality checking of high-throughput nucleic acid sequencers.

    PubMed

    Lazo, G R; Tong, J; Miller, R; Hsia, C; Rausch, C; Kang, Y; Anderson, O D

    2001-06-01

    We have developed a graphical interface to allow the researcher to view and assess the quality of sequencing results using a series of program scripts developed to process data generated by automated sequencers. The scripts are written in Perl programming language and are executable under the cgibin directory of a Web server environment. The scripts direct nucleic acid sequencing trace file data output from automated sequencers to be analyzed by the phred molecular biology program and are displayed as graphical hypertext mark-up language (HTML) pages. The scripts are mainly designed to handle 96-well microtiter dish samples, but the scripts are also able to read data from 384-well microtiter dishes 96 samples at a time. The scripts may be customized for different laboratory environments and computer configurations. Web links to the sources and discussion page are provided. PMID:11414222

  18. Sequence and phosphorylation level determination of two donkey beta-caseins by mass spectrometry.

    PubMed

    Cunsolo, Vincenzo; Cairone, Elisa; Saletti, Rosaria; Muccilli, Vera; Foti, Salvatore

    2009-07-01

    Two coeluting components, with experimentally measured M(r) values of 25529 and 24606 Da, were identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analysis, the two proteins were identified as donkey beta-CNs and their sequences characterized completely, using the two known beta-CNs from mare as references. The two donkey beta-CNs, showing a mass difference of 923 Da, differ by the presence of the domain E(27)SITHINK(34) in the full-length component (M(r) 25529 Da). In comparison with the mare's beta-CNs used as reference, they present nine amino acid substitutions: L-->S(37), R-->H(52), S-->N(81), P-->V(84), L-->V(91), R-->Q(203), P-->L/I(206), L-->F(210) and A-->P(219). Together, these substitutions account for the increase of 18 Da in the M(r) of the donkey beta-CNs with respect to the counterparts from the mare. The molecular mass determination by ESI-MS for the phosphorylated proteins showed that the full-length component was composed of highly multi-phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's beta-CNs, the full-length (226 amino acids) beta-CN was termed variant A, whereas the shorter (218 amino acids) beta-CN was termed variant A(Delta5). PMID:19462407

  19. Various instrumental approaches for determination of organic acids in wines.

    PubMed

    Zeravik, Jiri; Fohlerova, Zdenka; Milovanovic, Miodrag; Kubesa, Ondrej; Zeisbergerova, Marta; Lacina, Karel; Petrovic, Aleksandar; Glatz, Zdenek; Skladal, Petr

    2016-03-01

    Biosensors based on lactate oxidase, sarcosine oxidase and mixture of fumarase and sarcosine oxidase were used for monitoring of organic acids in wine samples. Additionally, tartaric acid was determined by modified colorimetric method based on formation of the vanadate-tartrate complex. The above mentioned methods were used for the analysis of 31 wine samples and obtained data were compared with the results from capillary electrophoresis as a basic standard method. This comparison showed a certain degree of correlation between biosensors and capillary electrophoresis. The provided information pointed to the potential uses of biosensors in the field of winemaking. PMID:26471576

  20. The outer capsid protein VP4 of equine rotavirus strain H-2 represents a unique VP4 type by amino acid sequence analysis.

    PubMed

    Hardy, M E; Gorziglia, M; Woode, G N

    1993-03-01

    The nucleotide and deduced amino acid sequence of G serotype 3 equine rotavirus strain H-2 was determined. A predicted 776-amino-acid H-2 VP4 shows less than or equal to 85.3% identity to other rotavirus VP4 types sequenced to date and thus represents a new P serotype. A PCR-generated probe derived from a cDNA clone of H-2 gene 4 hybridized to gene 4 of several tissue-culture-adapted equine rotavirus isolates, demonstrating that the gene 4 allele present in the H-2 strain is present in the equine rotavirus population. PMID:8382410

  1. 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

  2. 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

  3. 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,

    2013-01-25

    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.

  4. Fatty acid profile and Unigene-derived simple sequence repeat markers in tung tree (Vernicia fordii)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 se...

  5. 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.

  6. Complete nucleic acid sequence of Penaeus stylirostris densovirus (PstDNV) from India.

    PubMed

    Rai, Praveen; Safeena, Muhammed P; Karunasagar, Iddya; Karunasagar, Indrani

    2011-06-01

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp, recently been classified as Penaeus stylirostris densovirus (PstDNV). The complete nucleic acid sequence of PstDNV from India was obtained by cloning and sequencing of different DNA fragment of the virus. The genome organisation of PstDNV revealed that there were three major coding domains: a left ORF (NS1) of 2001 bp, a mid ORF (NS2) of 1092 bp and a right ORF (VP) of 990 bp. The complete genome and amino acid sequences of three proteins viz., NS1, NS2 and VP were compared with the genomes of the virus reported from Hawaii, China and Mexico and with partial sequence available from isolates from different regions. The phylogenetic analysis of shrimp, insect and vertebrate parvovirus sequences showed that the Indian PstDNV isolate is phylogenetically more closely related to one of the three isolates from Taiwan (AY355307), and two isolates (AY362547 and AY102034) from Thailand. PMID:21402111

  7. Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. I. Amino acid sequence of the cyanogen bromide peptides.

    PubMed

    Kingston, I B; Kingston, B L; Putnam, F W

    1980-04-10

    A histidine-rich fragment, Cp F5, with a molecular weight of 18,650 was isolated from human ceruloplasmin. It consists of 159 amino acids and contains a possible copper-binding site. The sequence of the first 18 NH2-terminal residues of Cp F5 was determined by automated Edman degradation. Cp F5 was cleaved by cyanogen bromide to produce nine fragments of from 2 to 63 residues. The amino acid sequence of all of the cyanogen bromide fragments was investigated using automated and manual Edman degradation, the fragments being digested with trypsin, chymotrypsin, thermolysin, staphylococcal protease, and pepsin as appropriate. The results, in conjunction with the data on the tryptic peptides reported in the accompanying paper (Kingston, I.B., Kingston, B.L., and Putnam, F.L. (1980) J. Biol. Chem. 255, 2886-2896), establish the complete amino acid sequence of Cp F5. PMID:6987229

  8. Molecular cytogenetics by polymerase catalyzed amplification or in situ labelling of specific nucleic acid sequences

    SciTech Connect

    Bolund, L.; Brandt, C.; Hindkjaer, J.; Koch, J.; Koelvraa, S.; Pedersen, S. )

    1993-01-01

    The Polymerase Chain Reaction (PCR) can be performed on isolated cells or chromosomes and the product can be analyzed by DNA technology or by FISH to test metaphases. The authors have good experiences analyzing aberrant chromosomes by FACS sorting, PCR with degenerated primers and painting of test metaphases with the PCR product. They also utilize polymerases for PRimed IN Situ labelling (PRINS) of specific nucleic acid sequences. In PRINS oligonucleotides are hybridized to their target sequences and labeled nucleotides are incorporated at the site of hybridization with the oligonucleotide as primer. PRINS may eventually allow the study of individual genes, gene expression and even somatic mutations (in mRNA) in single cells.

  9. 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.

  10. Nucleotide sequence of the fadR gene, a multifunctional regulator of fatty acid metabolism in Escherichia coli.

    PubMed Central

    DiRusso, C C

    1988-01-01

    The Escherichia coli fadR gene is a multifunctional regulator of fatty acid and acetate metabolism. In the present work the nucleotide sequence of the 1.3 kb DNA fragment which encodes FadR has been determined. The coding sequence of the fadR gene is 714 nucleotides long and is preceded by a typical E. coli ribosome binding site and is followed by a sequence predicted to be sufficient for factor-independent chain termination. Primer extension experiments demonstrated that the transcription of the fadR gene initiates with an adenine nucleotide 33 nucleotides upstream from the predicted start of translation. The derived fadR peptide has a calculated molecular weight of 26,972. This is in reasonable agreement with the apparent molecular weight of 29,000 previously estimated on the basis of maxi-cell analysis of plasmid encoded proteins. There is a segment of twenty amino acids within the predicted peptide which resembles the DNA recognition and binding site of many transcriptional regulatory proteins. Images PMID:2843809

  11. 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

  12. DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

    PubMed Central

    Mizuuchi, M; Weisberg, R A; Mizuuchi, K

    1986-01-01

    We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth. PMID:3012481

  13. Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering. Combinatorial library determination of optimal sequences.

    PubMed

    Krezel, Artur; Kopera, Edyta; Protas, Anna Maria; Poznański, Jarosław; Wysłouch-Cieszyńska, Aleksandra; Bal, Wojciech

    2010-03-17

    Previously we demonstrated for several examples that peptides having a general internal sequence R(N)-Yaa-Ser/Thr-Xaa-His-Zaa-R(C) (Yaa = Glu or Ala, Xaa = Ala or His, Zaa = Lys, R(N) and R(C) = any N- and C-terminal amino acid sequence) were hydrolyzed specifically at the Yaa-Ser/Thr peptide bond in the presence of Ni(II) ions at alkaline pH (Krezel, A., Mylonas, M., Kopera, E. and Bal, E. Acta Biochim. Polon. 2006, 53, 721-727 and references therein). Hereby we report the synthesis of a combinatorial library of CH(3)CO-Gly-Ala-(Ser/Thr)-Xaa-His-Zaa-Lys-Phe-Leu-NH(2) peptides, where Xaa residues included 17 common alpha-amino acids (except Asp, Glu, and Cys) and Zaa residues included 19 common alpha-amino acids (except Cys). The Ni(II)-dependent hydrolysis at 37 and 45 degrees C of batches of combinatorial peptide mixtures randomized at Zaa was monitored by MALDI-TOF mass spectrometry. The correctness of library-based predictions was confirmed by accurate measurements of hydrolysis rates of seven selected peptides using HPLC. The hydrolysis was strictly limited to the Ala-Ser/Thr bond in all library and individual peptide experiments. The effects of individual residues on hydrolysis rates were quantified and correlated with physical properties of their side chains according to a model of independent contributions of Xaa and Zaa residues. The principal component analysis calculations demonstrated partial molar side chain volume and the free energy of amino acid vaporization for both Xaa and Zaa residues and the amine pK(a) for Zaa residues to be the most significant empirical parameters influencing the hydrolysis rate. Therefore, efficient hydrolysis required bulky and hydrophobic residues at both variable positions Xaa and Zaa, which contributed independently to the hydrolysis rate. This relationship between the peptide sequence and the hydrolysis rate provides a basis for further research, aimed at the elucidation of the reaction mechanism and biotechnological

  14. Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen

    SciTech Connect

    Eaton, D.L.; Fless, G.M.; Kohr, W.J.; McLean, J.W.; Xu, Q.T.; Miller, C.G.; Lawn, R.M.; Scanu, A.M.

    1987-05-01

    Apolipoprotein(a) (apo(a)) is a glycoprotein with M/sub r/ approx. 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from the two tryptic fragments shows homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.

  15. Determining mutant spectra of three RNA viral samples using ultra-deep sequencing

    SciTech Connect

    Chen, H

    2012-06-06

    RNA viruses have extremely high mutation rates that enable the virus to adapt to new host environments and even jump from one species to another. As part of a viral transmission study, three viral samples collected from naturally infected animals were sequenced using Illumina paired-end technology at ultra-deep coverage. In order to determine the mutant spectra within the viral quasispecies, it is critical to understand the sequencing error rates and control for false positive calls of viral variants (point mutantations). I will estimate the sequencing error rate from two control sequences and characterize the mutant spectra in the natural samples with this error rate.

  16. Flow-injection chemiluminescence determination of chlorinated isocyanuric acids.

    PubMed

    Safavi, Afsaneh; Karimi, Mohammad Ali

    2003-02-01

    A rapid and sensitive flow-injection chemiluminescence method is described for the determination of dichloro- and trichloroisocyanuric acids based on the chemiluminescence produced during their reaction with luminol in alkaline medium. The effects of analytical and flow-injection variables on these chemiluminescence systems and determination of both oxidants are discussed. The optimized method yielded 3sigma detection limits of 8x10(-8) and 5x10(-8) mol L(-1) for the sodium dichloroisocyanurate and trichloroisocyanuric acid, respectively. The optimum conditions were found to be as follows: NaOH, 1x10(-1) mol L(-1); luminol, 5x10(-3) mol L(-1); KI, 2x10(-3) mol L(-1) and flow rate, 3.5 mL min(-1). PMID:12589508

  17. 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

  18. Amino acid sequences of peptides from a tryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

    PubMed Central

    Corfield, M. C.; Fletcher, J. C.; Robson, A.

    1967-01-01

    1. A tryptic digest of the protein fraction U.S.3 from oxidized wool has been separated into 32 peptide fractions by cation-exchange resin chromatography. 2. Most of these fractions have been resolved into their component peptides by a combination of the techniques of cation-exchange resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid compositions of 58 of the peptides in the digest present in the largest amounts have been determined. 4. The amino acid sequences of 38 of these have been completely elucidated and those of six others partially derived. 5. These findings indicate that the parent protein in wool from which the protein fraction U.S.3 is derived has a minimum molecular weight of 74000. 6. The structures of wool proteins are discussed in the light of the peptide sequences determined, and, in particular, of those sequences in fraction U.S.3 that could not be elucidated. PMID:16742497

  19. 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

  20. Determination of haploid DNA sequences in humans: application to the glucocerebrosidase pseudogene.

    PubMed

    Martínez-Arias, Rosa; Bertranpetit, Jaume; Comas, David

    2002-02-01

    Variation analyses in the human genome at the sequence level, especially human genetic population analysis and genetic epidemiology, are hampered by the difficulty to ascertain haplotypes on autosomal regions. We have designed a new methodological approach to obtain autosomal haploid sequences from diploid organisms. First, genotypes are unambiguously determined through long-range PCR and diploid DNA sequencing. Second, cloning the whole PCR-amplified segment and sequencing a single clone for those fragments that presented a heterozygous position discern the allelic phase. The second allele is deduced from the genotype, and the phase reconfirmed by sequencing a second clone. A hundred human chromosomes were analysed for a 5.4 kb encompassing the glucocerebrosidase pseudogene on human chromosome 1. Haplotypes were unambiguously ascertained for all samples. The manner to combine the used techniques makes this approach a novelty. Haploid sequences from diploid organisms are obtained in a less time consuming and more accurate manner than in other used procedures. PMID:12180141

  1. 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

  2. Self-sequencing of amino acids and origins of polyfunctional protocells.

    PubMed

    Fox, S W

    1984-01-01

    The primal role of the origins of proteins in molecular evolution is discussed. On the basis of this premise, the significance of the experimentally established self-sequencing of amino acids under simulated geological conditions is explained as due to the fact that the products are highly nonrandom and accordingly contain many kinds of information. When such thermal proteins are aggregated into laboratory protocells, an action that occurs readily, the resultant protocells also contain many kinds of information. Residue-by-residue order, enzymic activities, and lipid quality accordingly occur within each preparation of proteinoid (thermal protein). In this paper are reviewed briefly the phenomenon of self-sequencing of amino acids, its relationship to evolutionary processes, other significance of such self-ordering, and the experimental evidence for original polyfunctional protocells. PMID:6462684

  3. Self-Sequencing of Amino Acids and Origins of Polyfunctional Protocells

    NASA Astrophysics Data System (ADS)

    Fox, Sidney W.

    1984-12-01

    The primal role of the origins of proteins in molecular evolution is discussed. On the basis of this premise, the significance of the experimentally established self-sequencing of amino acids under simulated geological conditions is explained as due to the fact that the products are highly nonrandom and accordingly contain many kinds of information. When such thermal proteins are aggregated into laboratory protocells, an action that occurs readily, the resultant protocells also contain many kinds of information. Residue-by-residue order, enzymic activities, and lipid quality accordingly occur within each preparation of proteinoid (thermal protein). In this paper are reviewed briefly the phenomenon of self-sequencing of amino acids, its relationship to evolutionary processes, other significance of such self-ordering, and the experimental evidence for original polyfunctional protocells.

  4. Using Chou's pseudo amino acid composition to predict protein quaternary structure: a sequence-segmented PseAAC approach.

    PubMed

    Zhang, Shao-Wu; Chen, Wei; Yang, Feng; Pan, Quan

    2008-10-01

    In the protein universe, many proteins are composed of two or more polypeptide chains, generally referred to as subunits, which associate through noncovalent interactions and, occasionally, disulfide bonds to form protein quaternary structures. It has long been known that the functions of proteins are closely related to their quaternary structures; some examples include enzymes, hemoglobin, DNA polymerase, and ion channels. However, it is extremely labor-expensive and even impossible to quickly determine the structures of hundreds of thousands of protein sequences solely from experiments. Since the number of protein sequences entering databanks is increasing rapidly, it is highly desirable to develop computational methods for classifying the quaternary structures of proteins from their primary sequences. Since the concept of Chou's pseudo amino acid composition (PseAAC) was introduced, a variety of approaches, such as residue conservation scores, von Neumann entropy, multiscale energy, autocorrelation function, moment descriptors, and cellular automata, have been utilized to formulate the PseAAC for predicting different attributes of proteins. Here, in a different approach, a sequence-segmented PseAAC is introduced to represent protein samples. Meanwhile, multiclass SVM classifier modules were adopted to classify protein quaternary structures. As a demonstration, the dataset constructed by Chou and Cai [(2003) Proteins 53:282-289] was adopted as a benchmark dataset. The overall jackknife success rates thus obtained were 88.2-89.1%, indicating that the new approach is quite promising for predicting protein quaternary structure. PMID:18427713

  5. Sequence of morphological transitions in two-dimensional pattern growth from aqueous ascorbic Acid solutions.

    PubMed

    Paranjpe, A S

    2002-08-12

    A sequence of morphological transitions in two-dimensional dehydration patterns of aqueous solutions of ascorbic acid is observed with humidity as a control parameter. Change in morphology occurs due to humidity induced variation in the concentration of the metastable supersaturated solution phase formed after initial solvent evaporation. As percent humidity is varied from 40 to 80, patterns change from compact circular --> radial --> density modulated radial (a new morphology) --> density modulated circular --> density modulated dendritic (a new morphology) --> dense branching. PMID:12190528

  6. 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.

  7. Snake venom. The amino acid sequence of protein A from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J; Strydom, D J

    1980-12-01

    Protein A from Dendroaspis polylepis polylepis venom comprises 81 amino acids, including ten half-cystine residues. The complete primary structures of protein A and its variant A' were elucidated. The sequences of proteins A and A', which differ in a single position, show no homology with various neurotoxins and non-neurotoxic proteins and represent a new type of elapid venom protein. PMID:7461607

  8. Identification and Sequence of a tet(M) Tetracycline Resistance Determinant Homologue in Clinical Isolates of Escherichia coli

    PubMed Central

    Jones, C. Hal; Tuckman, Margareta; Murphy, Ellen; Bradford, Patricia A.

    2006-01-01

    The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host. PMID:17015654

  9. Easy modification of glassy carbon electrode for simultaneous determination of ascorbic acid, dopamine and uric acid.

    PubMed

    Thiagarajan, Soundappan; Tsai, Tsung-Hsuan; Chen, Shen-Ming

    2009-04-15

    A glassy carbon electrode (GCE) has been modified by electrochemical oxidation in mild acidic media (0.1 mol l(-1) H(2)SO(4)) and could be applied for individual and simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA). Oxidized GCE shows a single redox couple (E(0)'=-2.5 mV) which is based on the formation functional groups during the electrochemical pretreatment process. Proposed GCE successfully decreases the over potentials for the oxidation process of these species (AA, DA and UA) comparing with bare GCE. The oxidized GCE has its own simplicity, stability, high sensitivity and possesses the potential for simultaneous determination of AA, DA and UA. PMID:19162467

  10. Characterization of the microbial acid mine drainage microbial community using culturing and direct sequencing techniques.

    PubMed

    Auld, Ryan R; Myre, Maxine; Mykytczuk, Nadia C S; Leduc, Leo G; Merritt, Thomas J S

    2013-05-01

    We characterized the bacterial community from an AMD tailings pond using both classical culturing and modern direct sequencing techniques and compared the two methods. Acid mine drainage (AMD) is produced by the environmental and microbial oxidation of minerals dissolved from mining waste. Surprisingly, we know little about the microbial communities associated with AMD, despite the fundamental ecological roles of these organisms and large-scale economic impact of these waste sites. AMD microbial communities have classically been characterized by laboratory culturing-based techniques and more recently by direct sequencing of marker gene sequences, primarily the 16S rRNA gene. In our comparison of the techniques, we find that their results are complementary, overall indicating very similar community structure with similar dominant species, but with each method identifying some species that were missed by the other. We were able to culture the majority of species that our direct sequencing results indicated were present, primarily species within the Acidithiobacillus and Acidiphilium genera, although estimates of relative species abundance were only obtained from direct sequencing. Interestingly, our culture-based methods recovered four species that had been overlooked from our sequencing results because of the rarity of the marker gene sequences, likely members of the rare biosphere. Further, direct sequencing indicated that a single genus, completely missed in our culture-based study, Legionella, was a dominant member of the microbial community. Our results suggest that while either method does a reasonable job of identifying the dominant members of the AMD microbial community, together the methods combine to give a more complete picture of the true diversity of this environment. PMID:23485423

  11. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... approved by the Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51... base or modified or unusual amino acid may be presented in a given sequence as the corresponding unmodified base or amino acid if the modified base or modified or unusual amino acid is one of those...

  12. 37 CFR 1.822 - Symbols and format to be used for nucleotide and/or amino acid sequence data.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... approved by the Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51... base or modified or unusual amino acid may be presented in a given sequence as the corresponding unmodified base or amino acid if the modified base or modified or unusual amino acid is one of those...

  13. Nanopore Analysis of Nucleic Acids: Single-Molecule Studies of Molecular Dynamics, Structure, and Base Sequence

    NASA Astrophysics Data System (ADS)

    Olasagasti, Felix; Deamer, David W.

    Nucleic acids are linear polynucleotides in which each base is covalently linked to a pentose sugar and a phosphate group carrying a negative charge. If a pore having roughly the crosssectional diameter of a single-stranded nucleic acid is embedded in a thin membrane and a voltage of 100 mV or more is applied, individual nucleic acids in solution can be captured by the electrical field in the pore and translocated through by single-molecule electrophoresis. The dimensions of the pore cannot accommodate anything larger than a single strand, so each base in the molecule passes through the pore in strict linear sequence. The nucleic acid strand occupies a large fraction of the pore's volume during translocation and therefore produces a transient blockade of the ionic current created by the applied voltage. If it could be demonstrated that each nucleotide in the polymer produced a characteristic modulation of the ionic current during its passage through the nanopore, the sequence of current modulations would reflect the sequence of bases in the polymer. According to this basic concept, nanopores are analogous to a Coulter counter that detects nanoscopic molecules rather than microscopic [1,2]. However, the advantage of nanopores is that individual macromolecules can be characterized because different chemical and physical properties affect their passage through the pore. Because macromolecules can be captured in the pore as well as translocated, the nanopore can be used to detect individual functional complexes that form between a nucleic acid and an enzyme. No other technique has this capability.

  14. Cloning and nucleotide sequence determination of the Clostridium pasteurianum ferredoxin gene.

    PubMed Central

    Graves, M C; Mullenbach, G T; Rabinowitz, J C

    1985-01-01

    We have constructed a library of Clostridium pasteurianum DNA cloned in the plasmid pBR322. Based on the known amino acid sequence for C. pasteurianum ferredoxin, a 64-fold degenerate heptadecanucleotide pool was synthesized. This mixed probe hybridized to two clones which were shown to contain greater than 6 kilobase pairs of the same genomic DNA. Sequence analysis of a common Sau3A1 0.6-kilobase-pair fragment revealed that it contains the information for the apoferredoxin structural gene. According to the DNA sequence, the only post-translational processing of this small apoprotein is the hydrolysis of the initiator methionine. Putative transcription and translation start and stop signals are present within the sequence. Images PMID:3856844

  15. [Determination of body fluid based on analysis of nucleic acids].

    PubMed

    Korabečná, Marie

    2015-01-01

    Recent methodological approaches of molecular genetics allow isolation of nucleic acids (DNA and RNA) from negligible forensic samples. Analysis of these molecules may be used not only for individual identification based on DNA profiling but also for the detection of origin of the body fluid which (alone or in mixture with other body fluids) forms the examined biological trace. Such an examination can contribute to the evaluation of procedural, technical and tactical value of the trace. Molecular genetic approaches discussed in the review offer new possibilities in comparison with traditional spectrum of chemical, immunological and spectroscopic tests especially with regard to the interpretation of mixtures of biological fluids and to the confirmatory character of the tests. Approaches based on reverse transcription of tissue specific mRNA and their subsequent polymerase chain reaction (PCR) and fragmentation analysis are applicable on samples containing minimal amounts of biological material. Methods for body fluid discrimination based on examination of microRNA in samples provided so far confusing results therefore further development in this field is needed. The examination of tissue specific methylation of nucleotides in selected gene sequences seems to represent a promising enrichment of the methodological spectrum. The detection of DNA sequences of tissue related bacteria has been established and it provides satisfactory results mainly in combination with above mentioned methodological approaches. PMID:26419517

  16. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  17. Sequence determinants of thermodynamic stability in a WW domain—An all-β-sheet protein

    PubMed Central

    Jäger, Marcus; Dendle, Maria; Kelly, Jeffery W

    2009-01-01

    The stabilities of 66 sequence variants of the human Pin1 WW domain have been determined by equilibrium thermal denaturation experiments. All 34 residues composing the hPin1 WW three-stranded β-sheet structure could be replaced one at a time with at least one different natural or non-natural amino acid residue without leading to an unfolded protein. Alanine substitutions at only four positions within the hPin1 WW domain lead to a partially or completely unfolded protein—in the absence of a physiological ligand. The side chains of these four residues form a conserved, partially solvent-inaccessible, continuous hydrophobic minicore comprising the N- and C-termini. Ala mutations at five other residues, three of which constitute the ligand binding patch on the concave side of the β-sheet, significantly destabilize the hPin1 WW domain without leading to an unfolded protein. The remaining mutations affect protein stability only slightly, suggesting that only a small subset of side chain interactions within the hPin1 WW domain are mandatory for acquiring and maintaining a stable, cooperatively folded β-sheet structure. PMID:19565466

  18. Fluorometric Determination of Deoxyribonucleic Acid in Bacteria with Ethidium Bromide

    PubMed Central

    Donkersloot, J. A.; Robrish, S. A.; Krichevsky, M. I.

    1972-01-01

    A simple, sensitive, and rapid method is presented for the determination of deoxyribonucleic acid (DNA) in both gram-positive and gram-negative bacteria. It is based upon the fluorometric determination of DNA with ethidium bromide after alkaline digestion of the bacteria to hydrolyze the interfering ribonucleic acid. The assay takes less than 2 hr. Its sensitivity is at least 0.2 μg of DNA in a final solution of 4 ml and it uses commonly available filter or double monochromator fluorometers. Judicious choice of light source and filters allows an additional 10-fold increase in sensitivity with a filter fluorometer. Turbidity caused by bacteria or insoluble polysaccharides does not interfere with the fluorescence measurements. There was no significant difference between the results obtained with this method and those obtained with the indole and diphenylamine methods when these assays were applied to Escherichia coli and sucrose- or glucose-grown Streptococcus mutans. The method was also tested by determining the specific growth rate of E. coli. This new procedure should be especially useful for the determination of bacterial DNA in dilute suspensions and for the estimation of bacterial growth or DNA replication where more conventional methods are not applicable or sensitive enough. PMID:4561101

  19. The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes.

    PubMed

    Araki, T; Kuramoto, M; Torikata, T

    1990-09-01

    The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group. PMID:1368578

  20. Simultaneous determination of salicylic acid and salicylamide in biological fluids

    NASA Astrophysics Data System (ADS)

    Murillo Pulgarín, J. A.; Alañón Molina, A.; Sánchez-Ferrer Robles, I.

    2011-09-01

    A new methodology for the simultaneous determination of salicylic acid and salicylamide in biological fluids is proposed. The strong overlapping of the fluorescence spectra of both analytes makes impossible the conventional fluorimetric determination. For that reason, the use of fluorescence decay curves to resolve mixtures of analytes is proposed; this is a novel technique that provides the benefits in selectivity and sensitivity of the fluorescence decay curves. In order to assess the goodness of the proposed method, a prediction set of synthetic samples were analyzed obtaining recuperation percentages between 98.2 and 104.6%. Finally, a study of the detection limits was done using a new criterion resulting in values for the detection limits of 8.2 and 11.6 μg L -1 for salicylic acid and salicylamide respectively. The validity of the method was tested in human serum and human urine spiked with aliquots of the analytes. Recoveries obtained were 96.2 and 94.5% for salicylic acid and salicylamide respectively.

  1. Low levels of haptoglobin and putative amino acid sequence in Taiwanese Lanyu miniature pigs.

    PubMed

    Yueh, Sunny C H; Wang, Yao Horng; Lin, Kuan Yu; Tseng, Chi Feng; Chu, Hsien Pin; Chen, Kuen Jaw; Wang, Shih Sheng; Lai, I Hsiang; Mao, Simon J T

    2008-04-01

    Porcine haptoglobin (Hp) is an acute phase protein. Its plasma level increases significantly during inflammation and infection. One of the main functions of Hp is to bind free hemoglobin (Hb) and inhibit its oxidative activity. In the present report, we studied the Hp phenotype of Taiwanese Lanyu miniature pigs (TLY minipigs; n=43) and found their Hp structure to be a homodimer (beta-alpha-alpha-beta) similar to human Hp 1-1. Interestingly, Western blot and high performance liquid chromatographic (HPLC) analysis showed that 25% of the TLY minipigs possessed low or no plasma Hp level (<0.05 mg/ml). The Hp cDNA of these TLY minipigs was then cloned, and the translated amino acid sequence was analyzed. No sequences were found to be deficient; they showed a 99.7% identity with domestic pigs (NP_999165). The mean overall Hp level of the TLY minipigs (0.21 +/- 0.25 mg/ml; n=43) determined by enzyme-linked immunosorbent assay (ELISA) was markedly lower than that of domestic pigs (0.78 +/- 0.45 mg/ml; p<0.001), while 25% of the TLY minipigs had an Hp level that was extremely low (<0.05 mg/ml). In addition, the initial recovery rate (first 40 min) in the circulation of infused fluorescein isothiocyanate (FITC)-Hb was significantly higher in the TLY minipigs with extremely low Hp levels than those with high levels. This data suggests that the low concentration of Hp-Hb complex is responsible for the higher recovery rate of Hb in the circulation. TLY minipigs have been used as an experimental model for cardiovascular diseases; whether they can be used as a model for inflammatory diseases, with Hp as a marker, remains a topic of interest. However, since the Hp level varies significantly among individual TLY minipigs, it is necessary to prescreen the Hp levels of the animals to minimize variation in the experimental baseline. The present study may provide a reference value for future use of the TLY minipig as an animal model for inflammation-associated diseases. PMID:18460833

  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. Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin (Crocodylus siamensis).

    PubMed

    Srihongthong, Saowaluck; Pakdeesuwan, Anawat; Daduang, Sakda; Araki, Tomohiro; Dhiravisit, Apisak; Thammasirirak, Sompong

    2012-08-01

    Hemoglobin, α-chain, β-chain and fragmented hemoglobin of Crocodylus siamensis demonstrated both antibacterial and antioxidant activities. Antibacterial and antioxidant properties of the hemoglobin did not depend on the heme structure but could result from the compositions of amino acid residues and structures present in their primary structure. Furthermore, thirteen purified active peptides were obtained by RP-HPLC analyses, corresponding to fragments in the α-globin chain and the β-globin chain which are mostly located at the N-terminal and C-terminal parts. These active peptides operate on the bacterial cell membrane. The globin chains of Crocodylus siamensis showed similar amino acids to the sequences of Crocodylus niloticus. The novel amino acid substitutions of α-chain and β-chain are not associated with the heme binding site or the bicarbonate ion binding site, but could be important through their interactions with membranes of bacteria. PMID:22648692

  4. [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

  5. How Structural and Physicochemical Determinants Shape Sequence Constraints in a Functional Enzyme

    PubMed Central

    Abriata, Luciano A.; Palzkill, Timothy; Dal Peraro, Matteo

    2015-01-01

    The need for interfacing structural biology and biophysics to molecular evolution is being increasingly recognized. One part of the big problem is to understand how physics and chemistry shape the sequence space available to functional proteins, while satisfying the needs of biology. Here we present a quantitative, structure-based analysis of a high-resolution map describing the tolerance to all substitutions in all positions of a functional enzyme, namely a TEM lactamase previously studied through deep sequencing of mutants growing in competition experiments with selection against ampicillin. Substitutions are rarely observed within 7 Å of the active site, a stringency that is relaxed slowly and extends up to 15–20 Å, with buried residues being especially sensitive. Substitution patterns in over one third of the residues can be quantitatively modeled by monotonic dependencies on amino acid descriptors and predictions of changes in folding stability. Amino acid volume and steric hindrance shape constraints on the protein core; hydrophobicity and solubility shape constraints on hydrophobic clusters underneath the surface, and on salt bridges and polar networks at the protein surface together with charge and hydrogen bonding capacity. Amino acid solubility, flexibility and conformational descriptors also provide additional constraints at many locations. These findings provide fundamental insights into the chemistry underlying protein evolution and design, by quantitating links between sequence and different protein traits, illuminating subtle and unexpected sequence-trait relationships and pinpointing what traits are sacrificed upon gain-of-function mutation. PMID:25706742

  6. [Determination of dimethylbenzoic acid isomers in urine by gas chromatography].

    PubMed

    Kostrzewski, P; Wiaderna-Brycht, A; Czerski, B

    1994-01-01

    Trimethylobenzene (TMB) is a main ingredient of many organic solvents used in industry. In Farbasol (Polish trade name of the solvent) TMB occurs as a mixture of three isomers: pseudocumene (1, 2, 4-TMB) 30%; mesitylene (1, 3, 5-TMB) 15%; hemimellitene (1,2,3-TMB) 5%. As it is known in human organism, TMB is metabolized mainly to dimethylbenzoic (DMBA) and dimethylhippuric (DMHA) acids, and some authors suggest, that the acids excreted in urine can be biological indicators of exposure to TMB. This study was aimed at developing the method of determination of DMBA isomers in urine. Biological material was hydrolyzed with sodium hydroxide and next extracted with diethyl ether. DMBA concentration in urine was determined by gas chromatography using a variant of quantitative analysis with internal standard (5-methyl-2-isopropylphenol, thymol). Analytical parameters of the developed method of determination of DMBA isomers in urine such as linearity, precision, reproducibility, stability (192 days, when urine samples stored at-18 degrees C), detectability limit (400 micrograms/dm3) have been fully compatible with the requirements of biological monitoring. In order to confirm the presence of DMBA isomers in urine, four volunteers were exposed (8 hours) to Farbasol in toxicological chamber. The TMB concentration in the air, determined by means of gas chromatograph (HP 5890), amounted to 100 mg/m3 (MAC value in Poland). In urine samples collected 2,3-; 2,4-; 2,5-; 2,6-; 3,4-; 3,5-dimethylbenzoic acids were identified by means of GC/MSD.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8170375

  7. Intrageneric relationships of Enterococci as determined by reverse transcriptase sequencing of small-subunit rRNA.

    PubMed

    Williams, A M; Rodrigues, U M; Collins, M D

    1991-01-01

    The 16S ribosomal ribonucleic acid (rRNA) sequences of eleven Enterococcus species were determined by reverse transcription in an attempt to clarify their intrageneric relationships. Comparative analysis of the sequence data revealed the presence of several species groups within the genus. The species E. avium, E. malodoratus, E. pseudoavium and E. raffinosus formed a distinct group as did E. durans, E. faecium, E. hirae and E. mundtii and the pair of species E. casseliflavus and E. gallinarum. Of the remaining species, E. cecorum, E. columbae, E. faecalis and E. saccharolyticus formed distinct lines of descent within the genus, whereas E. solitarius displayed a closer affinity with Tetragenococcus halophilus than with other enterococcal species. PMID:1712504

  8. Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids.

    PubMed

    Tanaka, Junko; Doi, Nobuhide; Takashima, Hideaki; Yanagawa, Hiroshi

    2010-04-01

    Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279-284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids. PMID:20162614

  9. Identification of the amino acid sequence that targets peroxiredoxin 6 to lysosome-like structures of lung epithelial cells.

    PubMed

    Sorokina, Elena M; Feinstein, Sheldon I; Milovanova, Tatyana N; Fisher, Aron B

    2009-11-01

    Peroxiredoxin 6 (Prdx6), an enzyme with glutathione peroxidase and PLA2 (aiPLA2) activities, is highly expressed in respiratory epithelium, where it participates in phospholipid turnover and antioxidant defense. Prdx6 has been localized by immunocytochemistry and subcellular fractionation to acidic organelles (lung lamellar bodies and lysosomes) and cytosol. On the basis of their pH optima, we have postulated that protein subcellular localization determines the balance between the two activities of Prdx6. Using green fluorescent protein-labeled protein expression in alveolar epithelial cell lines, we showed Prdx6 localization to organellar structures resembling lamellar bodies in mouse lung epithelial (MLE-12) cells and lysosomes in A549 cells. Localization within lamellar bodies/lysosomes was in the luminal compartment. Targeting to lysosome-like organelles was abolished by the deletion of amino acids 31-40 from the Prdx6 NH2-terminal region; deletion of the COOH-terminal region had no effect. A green fluorescent protein-labeled peptide containing only amino acids 31-40 showed lysosomal targeting that was abolished by mutation of S32 or G34 within the peptide. Studies with mutated protein indicated that lipid binding was not necessary for Prdx6 targeting. This peptide sequence has no homology to known organellar targeting motifs. These studies indicate that the localization of Prdx6 in acidic organelles and consequent PLA2 activity depend on a novel 10-aa peptide located at positions 31-40 of the protein. PMID:19700648

  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. ION-EXCLUSION CHROMATOGRAPHIC DETERMINATION OF CARBOXYLIC ACIDS USED TO SUPPORT THE MICROBIALLY MEDIATED REDUCTIVE DECHLORINATION OF TETRACHLOROETHENE

    EPA Science Inventory

    An analytical method was developed for the determination of lactic acid, formic acid, acetic acid, propionic acid, and butyric acid in environmental microcosm samples using ion-exclusion chromatography. The chromatographic behavior of various eluents was studied to determine the ...

  12. A new technique for determining the distribution of N7-methylguanine using an automated DNA sequencer.

    PubMed

    Shoukry, S; Anderson, M W; Glickman, B W

    1991-11-01

    We have developed a method to determine rapidly the sequence specificity of DNA alkylation resulting from chemical treatment. The utility of this approach is demonstrated here in a study of the sequence specificity of alkylation by dimethylsulphate (DMS). The method is independent of the sequence chosen and makes use of the polymerase chain reaction (PCR) to generate a fluorescently labelled DNA target. In this study, a 302 bp segment of the Escherichia coli lacI gene was amplified and the product purified by liquid chromatography on a Mono Q column. This DNA was alkylated with DMS and treated with hot piperidine to produce single-strand breaks at sites of N7 alkylation. The distribution of the break points, and hence the position and extent of alkylation, were determined on an Applied Biosystems 370A automated DNA sequencer. PMID:1682064

  13. A method for accurate determination of terminal sequences of viral genomic RNA.

    PubMed

    Weng, Z; Xiong, Z

    1995-09-01

    A combination of ligation-anchored PCR and anchored cDNA cloning techniques were used to clone the termini of the saguaro cactus virus (SCV) RNA genome. The terminal sequences of the viral genome were subsequently determined from the clones. The 5' terminus was cloned by ligation-anchored PCR, whereas the 3' terminus was obtained by a technique we term anchored cDNA cloning. In anchored cDNA cloning, an anchor oligonucleotide was prepared by phosphorylation at the 5' end, followed by addition of a dideoxynucleotide at the 3' end to block the free hydroxyl group. The 5' end of the anchor was subsequently ligated to the 3' end of SCV RNA. The anchor-ligated, chimerical viral RNA was then reverse-transcribed into cDNA using a primer complementary to the anchor. The cDNA containing the complete 3'-terminal sequence was converted into ds-cDNA, cloned, and sequenced. Two restriction sites, one within the viral sequence and one within the primer sequence, were used to facilitate cloning. The combination of these techniques proved to be an easy and accurate way to determine the terminal sequences of SCV RNA genome and should be applicable to any other RNA molecules with unknown terminal sequences. PMID:9132274

  14. A Study on Amino Acids: Synthesis of Alpha-Aminophenylacetic Acid (Phenylglycine) and Determination of its Isoelectric Point.

    ERIC Educational Resources Information Center

    Barrelle, M.; And Others

    1983-01-01

    Background information and procedures are provided for an experimental study on aminophenylacetic acid (phenylglycine). These include physical chemistry (determination of isoelectric point by pH measurement) and organic chemistry (synthesis of an amino acid in racemic form) experiments. (JN)

  15. 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

  16. Green chromatography determination of fatty acid methyl esters in biodiesel.

    PubMed

    Mayo, Carlos Molina; Alayón, Andrea Brito; García Rodríguez, María Teresa; Jiménez Abizanda, Ana Isabel; Moreno, Francisco Jiménez

    2015-01-01

    This work proposes a green, simple and rapid chromatographic methodology for separation and determination of a group of 13 fatty acids methyl esters (FAMEs) by using a capillary gas chromatography with a flame ionization detector. The method was successfully applied for the determination of FAMEs in biodiesel samples from commercial and waste cooking oils, synthesized by homogeneous catalysis. Detection and quantification limits were in the μg L(-1) level. Direct injection of sample solution was compared with solid-phase extraction and solid-phase microextraction procedures, giving similar results. The lower analysis time represent considerable improvement compared with other papers. The described methodology is especially suitable for process control applications. The samples analysed showed total contents of FAMEs higher than 96.5%, which verifies the European regulations. PMID:25666201

  17. Advances in the Determination of Nucleic Acid Conformational Ensembles

    NASA Astrophysics Data System (ADS)

    Salmon, Loïc; Yang, Shan; Al-Hashimi, Hashim M.

    2014-04-01

    Conformational changes in nucleic acids play a key role in the way genetic information is stored, transferred, and processed in living cells. Here, we describe new approaches that employ a broad range of experimental data, including NMR-derived chemical shifts and residual dipolar couplings, small-angle X-ray scattering, and computational approaches such as molecular dynamics simulations to determine ensembles of DNA and RNA at atomic resolution. We review the complementary information that can be obtained from diverse sets of data and the various methods that have been developed to combine these data with computational methods to construct ensembles and assess their uncertainty. We conclude by surveying RNA and DNA ensembles determined using these methods, highlighting the unique physical and functional insights obtained so far.

  18. Novel method for PIK3CA mutation analysis: locked nucleic acid--PCR sequencing.

    PubMed

    Ang, Daphne; O'Gara, Rebecca; Schilling, Amy; Beadling, Carol; Warrick, Andrea; Troxell, Megan L; Corless, Christopher L

    2013-05-01

    Somatic mutations in PIK3CA are commonly seen in invasive breast cancer and several other carcinomas, occurring in three hotspots: codons 542 and 545 of exon 9 and in codon 1047 of exon 20. We designed a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant PIK3CA DNA with attention to avoiding amplification of a pseudogene on chromosome 22 that has >95% homology to exon 9 of PIK3CA. We tested 60 FFPE breast DNA samples with known PIK3CA mutation status (48 cases had one or more PIK3CA mutations, and 12 were wild type) as identified by PCR-mass spectrometry. PIK3CA exons 9 and 20 were amplified in the presence or absence of LNA-oligonucleotides designed to bind to the wild-type sequences for codons 542, 545, and 1047, and partially suppress their amplification. LNA-PCR sequencing confirmed all 51 PIK3CA mutations; however, the mutation detection rate by standard Sanger sequencing was only 69% (35 of 51). Of the 12 PIK3CA wild-type cases, LNA-PCR sequencing detected three additional H1047R mutations in "normal" breast tissue and one E545K in usual ductal hyperplasia. Histopathological review of these three normal breast specimens showed columnar cell change in two (both with known H1047R mutations) and apocrine metaplasia in one. The novel LNA-PCR shows higher sensitivity than standard Sanger sequencing and did not amplify the known pseudogene. PMID:23541593

  19. 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

  20. 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

  1. Unconventional amino acid sequence of the sun anemone (Stoichactis helianthus) polypeptide neurotoxin

    SciTech Connect

    Kem, W.; Dunn, B.; Parten, B.; Pennington, M.; Price, D.

    1986-05-01

    A 5000 dalton polypeptide neurotoxin (Sh-NI) purified by G50 Sephadex, P-cellulose, and SP-Sephadex chromatography was homogeneous by isoelectric focusing. Sh-NI was highly toxic to crayfish (LD/sub 50/ 0.6 ..mu..g/kg) but without effect upon mice at 15,000 ..mu..g/kg (i.p. injection). The reduced, /sup 3/H-carboxymethylated toxin and its fragments were subjected to automatic Edman degradation and the resulting PTH-amino acids were identified by HPLC, back hydrolysis, and scintillation counting. Peptides resulting from proteolytic (clostripain, staphylococcal protease) and chemical (tryptophan) cleavage were sequenced. The sequence is: AACKCDDEGPDIRTAPLTGTVDLGSCNAGWEKCASYYTIIADCCRKKK. This sequence differs considerably from the homologous Anemonia and Anthopleura toxins; many of the identical residues (6 half-cystines, G9, P10, R13, G19, G29, W30) are probably critical for folding rather than receptor recognition. However, the Sh-NI sequence closely resembles Radioanthus macrodactylus neurotoxin III and r. paumotensis II. The authors propose that Sh-NI and related Radioanthus toxins act upon a different site on the sodium channel.

  2. Cloning, sequence analysis and crystal structure determination of a miraculin-like protein from Murraya koenigii.

    PubMed

    Gahloth, Deepankar; Selvakumar, Purushotham; Shee, Chandan; Kumar, Pravindra; Sharma, Ashwani Kumar

    2010-02-01

    Earlier, the purification of a 21.4kDa protein with trypsin inhibitory activity from seeds of Murraya koenigii has been reported. The present study, based on the amino acid sequence deduced from both cDNA and genomic DNA, establishes it to be a miraculin-like protein and provides crystal structure at 2.9A resolution. The mature protein consists of 190 amino acid residues with seven cysteines arranged in three disulfide bridges. The amino acid sequence showed maximum homology and formed a distinct cluster with miraculin-like proteins, a soybean Kunitz super family member, in phylogenetic analyses. The major differences in sequence were observed at primary and secondary specificity sites in the reactive loop when compared to classical Kunitz family members. The crystal structure analysis showed that the protein is made of twelve antiparallel beta-strands, loops connecting beta-strands and two short helices. Despite similar overall fold, it showed significant differences from classical Kunitz trypsin inhibitors. PMID:19914199

  3. General Analytical Procedure for Determination of Acidity Parameters of Weak Acids and Bases

    PubMed Central

    Pilarski, Bogusław; Kaliszan, Roman; Wyrzykowski, Dariusz; Młodzianowski, Janusz; Balińska, Agata

    2015-01-01

    The paper presents a new convenient, inexpensive, and reagent-saving general methodology for the determination of pKa values for components of the mixture of diverse chemical classes weak organic acids and bases in water solution, without the need to separate individual analytes. The data obtained from simple pH-metric microtitrations are numerically processed into reliable pKa values for each component of the mixture. Excellent agreement has been obtained between the determined pKa values and the reference literature data for compounds studied. PMID:25692072

  4. Isolation, Identification, and Sequencing of a Goose-Derived Newcastle Disease Virus and Determination of Its Pathogenicity.

    PubMed

    Chen, Xiao-Qing; Li, Zi-Bing; Hu, Gui-Xue; Gu, Song-Zhi; Zhang, Shuang; Ying, Ying; Gao, Feng-Shan

    2015-06-01

    In August 2010, geese in the Meihekou area of Jilin province in China were found to be infected by a pathogen that caused a disease similar to Newcastle disease. To determine the causative agent of the infections, a virus was isolated from liver tissues of infected geese, followed by a pathogenicity determination. The isolated virus was named NDV/White Goose/China/Jilin(Meihekou)/MHK-1/2010. Specific primers were designed to amplify the whole genome of the MHK-1 virus, followed by sequencing and splicing of the entire genome. Sequencing and phylogenetic analysis of MHK-1 showed that the isolate was a virulent strain of Newcastle disease virus. The MHK-1 genome is 15,192 nucleotides long, and it belongs to the class II branch of Newcastle disease viruses, as evidenced by the amino acid sequence (112R-R-Q-K-R-F117) of the F protein. The hemagglutinin titer was 1:128 to 1:512. The chicken embryo mean death time, the intracerebral pathogenicity index, and the median lethal dose of chicken embryos of MHK-1 were 43 hr, 1.63, and 10(9)/ml, respectively, which revealed that the newly isolated MHK-1 strain is strongly pathogenic to geese. PMID:26473673

  5. Detection of Nucleic Acids with Graphene Nanopores: Ab Initio Characterization of a Novel Sequencing Device

    NASA Astrophysics Data System (ADS)

    Nelson, Tammie; Zhang, Bo; Prezhdo, Oleg

    2010-03-01

    We report an ab initio study of the interaction of two nucleobases, cytosine and adenine, with a novel graphene nanopore device for detecting the base sequence of a single-stranded nucleic acid (ssDNA or RNA). The nucleobases were inserted into a pore in a graphene nanoribbon, and the electrical current and conductance spectra were calculated as functions of voltage applied across the nanoribbon. The conductance spectra and charge densities were analyzed in the presence of each nucleobase in the graphene nanopore. The results indicate that, due to significant differences in the conductance spectra, the proposed device has adequate sensitivity to discriminate between different nucleotides. Moreover, we show that the nucleotide conductance spectra is not affected by its orientation inside the graphene nanopore. The proposed technique may be extremely useful for real applications in developing ultrafast, low cost DNA sequencing methods.

  6. CodingMotif: exact determination of overrepresented nucleotide motifs in coding sequences

    PubMed Central

    2012-01-01

    Background It has been increasingly appreciated that coding sequences harbor regulatory sequence motifs in addition to encoding for protein. These sequence motifs are expected to be overrepresented in nucleotide sequences bound by a common protein or small RNA. However, detecting overrepresented motifs has been difficult because of interference by constraints at the protein level. Sampling-based approaches to solve this problem based on codon-shuffling have been limited to exploring only an infinitesimal fraction of the sequence space and by their use of parametric approximations. Results We present a novel O(N(log N)2)-time algorithm, CodingMotif, to identify nucleotide-level motifs of unusual copy number in protein-coding regions. Using a new dynamic programming algorithm we are able to exhaustively calculate the distribution of the number of occurrences of a motif over all possible coding sequences that encode the same amino acid sequence, given a background model for codon usage and dinucleotide biases. Our method takes advantage of the sparseness of loci where a given motif can occur, greatly speeding up the required convolution calculations. Knowledge of the distribution allows one to assess the exact non-parametric p-value of whether a given motif is over- or under- represented. We demonstrate that our method identifies known functional motifs more accurately than sampling and parametric-based approaches in a variety of coding datasets of various size, including ChIP-seq data for the transcription factors NRSF and GABP. Conclusions CodingMotif provides a theoretically and empirically-demonstrated advance for the detection of motifs overrepresented in coding sequences. We expect CodingMotif to be useful for identifying motifs in functional genomic datasets such as DNA-protein binding, RNA-protein binding, or microRNA-RNA binding within coding regions. A software implementation is available at http://bioinformatics.bc.edu/chuanglab/codingmotif.tar PMID

  7. 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.

  8. Typhoidal Salmonellae: Use of Multi-Locus Sequence Typing to Determine Population Structure.

    PubMed

    Sharma, Priyanka; Dahiya, Sushila; Balaji, Veeraraghavan; Kanga, Anil; Panda, Preetilata; Das, Rashna; Dhanraju, Anbumani; Mendiratta, Deepak Kumar; Sood, Seema; Das, Bimal Kumar; Kapil, Arti

    2016-01-01

    Enteric fever is an invasive infection predominantly caused by Salmonella enterica serovars Typhi and Paratyphi A. The pathogens have evolved from other nontyphoidal salmonellaeto become invasive and host restricted. Emergence of antimicrobial resistance in typhoidal salmonellae in some countries is a major therapeutic concern as the travelers returning from endemic countries carry resistant strains to non endemic areas. In order to understand the epidemiology and to design disease control strategies molecular typing of the pathogen is very important. We performed Multilocus Sequence Typing (MLST) of 251 S. Typhi and 18 S. Paratyphi strains isolated from enteric fever patients from seven centers across India during 2010-2013to determine the population structure and prevalence of MLST sequence types in India. MLST analysis revealed the presence of five sequence types (STs) of typhoidal salmonellae in India namely ST1, ST2 and ST3 for S. Typhi and ST85 and ST129 for S. Paratyphi A.S. Typhi strains showed monophyletic lineage and clustered in to 3 Sequence Types-ST1, ST2 and ST3 and S. Paratyphi A isolates segregated in two sequence types ST85 and ST129 respectively. No association was found between antimicrobial susceptibility and sequence types. This study found ST1 as the most prevalent sequence type of S. Typhi in India followed by ST2, which is in concordance with previous studies and MLST database. In addition a rare sequence type ST3 has been found which is reported for the first time from the Indian subcontinent. Amongst S. Paratyphi A, the most common sequence type is ST129 as also reported from other parts of world. This distribution and prevalence suggest the common spread of the sequence types across the globe and these findings can help in understanding the disease distribution. PMID:27618626

  9. Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

    PubMed

    Watanabe, Kenshi; Ohno, Makoto; Taguchi, Masahiro; Kawamoto, Seiji; Ono, Kazuhisa; Aki, Tsunehiro

    2016-01-01

    Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids. PMID:26590171

  10. Comparison of bile acid synthesis determined by isotope dilution versus fecal acidic sterol output in human subjects

    SciTech Connect

    Duane, W.C.; Holloway, D.E.; Hutton, S.W.; Corcoran, P.J.; Haas, N.A.

    1982-05-01

    Fecal acidic sterol output has been found to be much lower than bile acid synthesis determined by isotope dilution. Because of this confusing discrepancy, we compared these 2 measurements done simultaneously on 13 occasions in 5 normal volunteers. In contrast to previous findings, bile acid synthesis by the Lindstedt isotope dilution method averaged 16.3% lower than synthesis simultaneously determined by fecal acidic sterol output (95% confidence limit for the difference - 22.2 to -10.4%). When one-sample determinations of bile acid pools were substituted for Lindstedt pools, bile acid synthesis by isotope dilution averaged 5.6% higher than synthesis by fecal acidic sterol output (95% confidence limits -4.9 to 16.1%). These data indicate that the 2 methods yield values in reasonably close agreement with one another. If anything, fecal acidic sterol outputs are slightly higher than synthesis by isotope dilution.

  11. Amino-terminal amino acid sequence of the major structural polypeptides of avian retroviruses: sequence homology between reticuloendotheliosis virus p30 and p30s of mammalian retroviruses.

    PubMed Central

    Hunter, E; Bhown, A S; Bennett, J C

    1978-01-01

    The major structural polypeptides, p30 of reticuloendotheliosis virus (REV) (strain T) and p27 of avian sarcoma virus B77, have been compared with regard to amino acid composition. NH2-terminal amino acid sequence, and immunological crossreactions. The amino acid composition of the two polypeptides is distinct, and a comparison of the first 30 NH2-terminal amino acids of REV p30 with that for the first 25 of B77 p27 yields only three homologous residues. In competition radioimmunoassays the polypeptides show no crossreactivity. A comparison of the amino acid composition and NH2-terminal amino acid sequence of REV p30 with those reported for several mammalian retrovirus p30s shows remarkable similarities. Both REV and mammalian p30s contain a large number of polar residues in their amino acid composition and show approximately 40% homology in the first 30 NH2-terminal amino acids. No crossreactivity could be observed, however, in competition radioimmunoassays between Rauscher murine leukemia virus p30 and that of REV. The observations reported here suggest a close evolutionary relationship between REV and the mammalian retroviruses. Images PMID:208072

  12. Draft Genome Sequence of Cupriavidus sp. Strain SK-3, a 4-Chlorobiphenyl- and 4-Clorobenzoic Acid-Degrading Bacterium

    PubMed Central

    Vilo, Claudia; Benedik, Michael J.; Ilori, Matthew

    2014-01-01

    We report the draft genome sequence of Cupriavidus sp. strain SK-3, which can use 4-chlorobiphenyl and 4-clorobenzoic acid as the sole carbon source for growth. The draft genome sequence allowed the study of the polychlorinated biphenyl degradation mechanism and the recharacterization of the strain SK-3 as a Cupriavidus species. PMID:24994805

  13. 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

  14. Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis subsp. lactis TOMSC161, Isolated from a Nonscalded Curd Pressed Cheese

    PubMed Central

    Velly, H.; Abraham, A.-L.; Loux, V.; Delacroix-Buchet, A.; Fonseca, F.; Bouix, M.

    2014-01-01

    Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation. PMID:25377704

  15. Draft Genome Sequence of Bacillus subtilis subsp. natto Strain CGMCC 2108, a High Producer of Poly-γ-Glutamic Acid.

    PubMed

    Tan, Siyuan; Meng, Yonghong; 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

  16. 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. PMID:27261456

  17. Formation Sequences of Iron Minerals in the Acidic Alteration Products and Variation of Hydrothermal Fluid Conditions

    NASA Astrophysics Data System (ADS)

    Isobe, H.; Yoshizawa, M.

    2008-12-01

    Iron minerals have important role in environmental issues not only on the Earth but also other terrestrial planets. Iron mineral species related to alteration products of primary minerals with surface or subsurface fluids are characterized by temperature, acidity and redox conditions of the fluids. We can see various iron- bearing alteration products in alteration products around fumaroles in geothermal/volcanic areas. In this study, zonal structures of iron minerals in alteration products of the geothermal area are observed to elucidate temporal and spatial variation of hydrothermal fluids. Alteration of the pyroxene-amphibole andesite of Garan-dake volcano, Oita, Japan occurs by the acidic hydrothermal fluid to form cristobalite leaching out elements other than Si. Hand specimens with unaltered or weakly altered core and cristobalite crust show various sequences of layers. XRD analysis revealed that the alteration degree is represented by abundance of cristobalite. Intermediately altered layers are characterized by occurrence including alunite, pyrite, kaolinite, goethite and hematite. A specimen with reddish brown core surrounded by cristobalite-rich white crust has brown colored layers at the boundary of core and the crust. Reddish core is characterized by occurrence of crystalline hematite by XRD. Another hand specimen has light gray core, which represents reduced conditions, and white cristobalite crust with light brown and reddish brown layers of ferric iron minerals between the core and the crust. On the other hand, hornblende crystals, typical ferrous iron-bearing mineral of the host rock, are well preserved in some samples with strongly decolorized cristobalite-rich groundmass. Hydrothermal alteration experiments of iron-rich basaltic material shows iron mineral species depend on acidity and temperature of the fluid. Oxidation states of the iron-bearing mineral species are strongly influenced by the acidity and redox conditions. Variations of alteration

  18. 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

  19. 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

  20. Sequence variation determining stereochemistry of a Δ11 desaturase active in moth sex pheromone biosynthesis.

    PubMed

    Ding, Bao-Jian; Carraher, Colm; Löfstedt, Christer

    2016-07-01

    A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution. PMID:27163509

  1. RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids

    PubMed Central

    Sample, Paul J.; Gaston, Kirk W.; Alfonzo, Juan D.; Limbach, Patrick A.

    2015-01-01

    Ribosomal ribonucleic acid (RNA), transfer RNA and other biological or synthetic RNA polymers can contain nucleotides that have been modified by the addition of chemical groups. Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers. Mass spectrometry (MS) has become the conventional approach for determining the nucleotide composition, modification status and sequence of modified RNAs. Modified RNAs are analyzed by MS using collision-induced dissociation tandem mass spectrometry (CID MS/MS), which produces a complex dataset of oligomeric fragments that must be interpreted to identify and place modified nucleosides within the RNA sequence. Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers. There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined ‘variable sequencing’, which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing. PMID:25820423

  2. Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays

    PubMed Central

    Matějková, Petra; Strouhal, Michal; Šmajs, David; Norris, Steven J; Palzkill, Timothy; Petrosino, Joseph F; Sodergren, Erica; Norton, Jason E; Singh, Jaz; Richmond, Todd A; Molla, Michael N; Albert, Thomas J; Weinstock, George M

    2008-01-01

    Background Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently. Results The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT) sequencing and the sequence was confirmed by whole genome fingerprinting (WGF). When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions), 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome. Conclusion The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism. PMID:18482458

  3. 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

  4. [Determination of docosahexaenoic acid in milk powder by gas chromatography using acid hydrolysis].

    PubMed

    Shao, Shiping; Xiang, Dapeng; Li, Shuang; Xi, Xinglin; Chen, Wenrui

    2015-11-01

    A method to determine docosahexenoic acid (DHA) in milk powder by gas chromatography was established. The milk powder samples were hydrolyzed with hydrochloric acid, extracted to get total fatty acids by Soxhlet extractor, then esterified with potassium hydroxide methanol solution to form methyl esters, and treated with sodium hydrogen sulfate. The optimal experiment conditions were obtained from orthogonal experiment L9(3(3)) which performed with three factors and three levels, and it requires the reaction performed with 1 mol/L potassium hydroxide solution at 25 degrees C for 5 min. The derivative treated with sodium hydrogen sulfate was separated on a column of SP-2560 (100 m x 0.25 mm x 0.20 μm), and determined in 55 min by temperature programming-gas chromatography. Good linearity was obtained in the range 5.0-300 mg/L with the correlation coefficient of 0.999 9. The relative standard deviations (RSDs) were 3.4%, 1.2% and 1.1% for the seven repeated experiments of 10, 50 and 100 mg/L of DHA, respectively. The limit of detection was 2 mg/kg, and the recoveries of DHA were in the range of 90.4%-93.5%. The results are satisfactory through the tests of practical samples. PMID:26939370

  5. Proteus mirabilis urease: nucleotide sequence determination and comparison with jack bean urease.

    PubMed

    Jones, B D; Mobley, H L

    1989-12-01

    Proteus mirabilis, a common cause of urinary tract infection, produces a potent urease that hydrolyzes urea to NH3 and CO2, initiating kidney stone formation. Urease genes, which were localized to a 7.6-kilobase-pair region of DNA, were sequenced by using the dideoxy method. Six open reading frames were found within a region of 4,952 base pairs which were predicted to encode polypeptides of 31.0 (ureD), 11.0 (ureA), 12.2 (ureB), 61.0 (ureC), 17.9 (ureE), and 23.0 (ureF) kilodaltons (kDa). Each open reading frame was preceded by a ribosome-binding site, with the exception of ureE. Putative promoterlike sequences were identified upstream of ureD, ureA, and ureF. Possible termination sites were found downstream of ureD, ureC, and ureF. Structural subunits of the enzyme were encoded by ureA, ureB, and ureC and were translated from a single transcript in the order of 11.0, 12.2, and 61.0 kDa. When the deduced amino acid sequences of the P. mirabilis urease subunits were compared with the amino acid sequence of the jack bean urease, significant amino acid similarity was observed (58% exact matches; 73% exact plus conservative replacements). The 11.0-kDa polypeptide aligned with the N-terminal residues of the plant enzyme, the 12.2-kDa polypeptide lined up with internal residues, and the 61.0-kDa polypeptide matched with the C-terminal residues, suggesting an evolutionary relationship of the urease genes of jack bean and P. mirabilis. PMID:2687233

  6. 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

  7. Determination of free fatty acids in beer wort.

    PubMed

    Bravi, Elisabetta; Benedetti, Paolo; Marconi, Ombretta; Perretti, Giuseppe

    2014-05-15

    The importance of free fatty acids (FFAs) in wort has been known for a long time because of their influence on beer quality and yeast metabolism. Lipids have a beneficial effect on yeast growth during fermentation as well as negative effects on beer quality. Lipids content of beer affects the ability to form a stable head of foam and plays an important role in beer staling. Moreover, the ratio of unsaturated and saturated fatty acids seems to be related to gushing problems. A novel, simple, and reliable procedure for quantitative analysis of FFAs in wort was developed and validated. The determination of FFAs in wort was achieved via liquid-liquid cartridge extraction, purification of FFA fraction by solid phase extraction, boron trifluoride in methanol methylation, and injection into GC-FID system. The proposed method has high accuracy (<0.3%, expressed as the bias), high precision (<1.2%, RSD), and recoveries ranging from 74% to 98%. The method was tested on two different wort samples (9° and 12° Plato). PMID:24423546

  8. Solid phase sequencing of biopolymers

    DOEpatents

    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.

  9. Swfoldrate: predicting protein folding rates from amino acid sequence with sliding window method.

    PubMed

    Cheng, Xiang; Xiao, Xuan; Wu, Zhi-cheng; Wang, Pu; Lin, Wei-zhong

    2013-01-01

    Protein folding is the process by which a protein processes from its denatured state to its specific biologically active conformation. Understanding the relationship between sequences and the folding rates of proteins remains an important challenge. Most previous methods of predicting protein folding rate require the tertiary structure of a protein as an input. In this study, the long-range and short-range contact in protein were used to derive extended version of the pseudo amino acid composition based on sliding window method. This method is capable of predicting the protein folding rates just from the amino acid sequence without the aid of any structural class information. We systematically studied the contributions of individual features to folding rate prediction. The optimal feature selection procedures are adopted by means of combining the forward feature selection and sequential backward selection method. Using the jackknife cross validation test, the method was demonstrated on the large dataset. The predictor was achieved on the basis of multitudinous physicochemical features and statistical features from protein using nonlinear support vector machine (SVM) regression model, the method obtained an excellent agreement between predicted and experimentally observed folding rates of proteins. The correlation coefficient is 0.9313 and the standard error is 2.2692. The prediction server is freely available at http://www.jci-bioinfo.cn/swfrate/input.jsp. PMID:22933332

  10. 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. PMID:27010507

  11. The amino acid sequences and activities of synergistic hemolysins from Staphylococcus cohnii.

    PubMed

    Mak, Pawel; Maszewska, Agnieszka; Rozalska, Malgorzata

    2008-10-01

    Staphylococcus cohnii ssp. cohnii and S. cohnii ssp. urealyticus are a coagulase-negative staphylococci considered for a long time as unable to cause infections. This situation changed recently and pathogenic strains of these bacteria were isolated from hospital environments, patients and medical staff. Most of the isolated strains were resistant to many antibiotics. The present work describes isolation and characterization of several synergistic peptide hemolysins produced by these bacteria and acting as virulence factors responsible for hemolytic and cytotoxic activities. Amino acid sequences of respective hemolysins from S. cohnii ssp. cohnii (named as H1C, H2C and H3C) and S. cohnii ssp. urealyticus (H1U, H2U and H3U) were identical. Peptides H1 and H3 possessed significant amino acid homology to three synergistic hemolysins secreted by Staphylococcus lugdunensis and to putative antibacterial peptide produced by Staphylococcus saprophyticus ssp. saprophyticus. On the other hand, hemolysin H2 had a unique sequence. All isolated peptides lysed red cells from different mammalian species and exerted a cytotoxic effect on human fibroblasts. PMID:18752624

  12. Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae

    PubMed Central

    Huang, Youhua; Huang, Xiaohong; Liu, Hong; Gong, Jie; Ouyang, Zhengliang; Cui, Huachun; Cao, Jianhao; Zhao, Yingtao; Wang, Xiujie; Jiang, Yulin; Qin, Qiwei

    2009-01-01

    Background Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis. Results We determined the complete nucleotide sequence of the STIV genome using 454 Life Science sequencing technology. The STIV genome is 105 890 bp in length with a base composition of 55.1% G+C. Computer assisted analysis revealed that the STIV genome contains 105 potential open reading frames (ORFs), which encode polypeptides ranging from 40 to 1,294 amino acids and 20 microRNA candidates. Among the putative proteins, 20 share homology with the ancestral proteins of the nuclear and cytoplasmic large DNA viruses (NCLDVs). Comparative genomic analysis showed that STIV has the highest degree of sequence conservation and a colinear arrangement of genes with frog virus 3 (FV3), followed by Tiger frog virus (TFV), Ambystoma tigrinum virus (ATV), Singapore grouper iridovirus (SGIV), Grouper iridovirus (GIV) and other iridovirus isolates. Phylogenetic analysis based on conserved core genes and complete genome sequence of STIV with other virus genomes was performed. Moreover, analysis of the gene gain-and-loss events in the family Iridoviridae suggested that the genes encoded by iridoviruses have evolved for favoring adaptation to different natural host species. Conclusion This study has provided the complete genome sequence of STIV. Phylogenetic analysis suggested that STIV and FV3 are strains of the same viral species belonging to the Ranavirus genus in

  13. Evolution of alpha-lactalbumins. The complete amino acid sequence of the alpha-lactalbumin from a marsupial (Macropus rufogriseus) and corrections to regions of sequence in bovine and goat alpha-lactalbumins.

    PubMed

    Shewale, J G; Sinha, S K; Brew, K

    1984-04-25

    alpha-Lactalbumin was purified from a whey protein fraction of the milk of the red-necked wallaby (Macropus rufogriseus). The complete amino acid sequence was determined from the results of automatic sequenator analyses of the intact protein, the three cyanogen bromide fragments, and of peptides generated from the larger, COOH-terminal CNBr fragment by digestion with trypsin or staphylococcal protease. This is the first sequence to be determined of an alpha-lactalbumin from a marsupial and differs from known eutherian alpha-lactalbumins in size and locations of deletions in alignments with the homologous type c lysozymes, as well as in having amino acid substitutions at 8 sites that are invariant in known eutherian proteins. Some corrections are also reported for two regions of sequence in both bovine and goat alpha-lactalbumins. The new and previously published information on alpha-lactalbumin sequences is analyzed in relation to the evolutionary history of the alpha-lactalbumin line as well as the relationship of structure to function in these proteins. PMID:6715332

  14. A Teaching and Learning Sequence about the Interplay of Chance and Determinism in Nonlinear Systems

    ERIC Educational Resources Information Center

    Stavrou, D.; Duit, R.; Komorek, M.

    2008-01-01

    A teaching and learning sequence aimed at introducing upper secondary school students to the interplay between chance and determinism in nonlinear systems is presented. Three experiments concerning nonlinear systems (deterministic chaos, self-organization and fractals) and one experiment concerning linear systems are introduced. Thirty upper…

  15. Simultaneous Determination of Salicylic Acid, Jasmonic Acid, Methyl Salicylate, and Methyl Jasmonate from Ulmus pumila Leaves by GC-MS

    PubMed Central

    Huang, Zhi-hong; Wang, Zhi-li; Shi, Bao-lin; Wei, Dong; Chen, Jian-xin; Wang, Su-li; Gao, Bao-jia

    2015-01-01

    Salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate are important phytohormones and defensive signaling compounds, so it is of great importance to determine their levels rapidly and accurately. The study uses Ulmus pumila leaves infected by Tetraneura akinire Sasaki at different stages as materials; after extraction with 80% methanol and ethyl acetate and purification with primary secondary amine (PSA) and graphitized carbon blacks (GCB), the contents of signal compounds salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate were determined by GC-MS. The results showed that the level of salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate increased remarkably in U. pumila once infected by T. akinire Sasaki, but the maximums of these four compounds occurred at different times. Salicylic acid level reached the highest at the early stage, and jasmonic acid level went to the maximum in the middle stage; by contrast, change of content of methyl salicylate and methyl jasmonate was the quite opposite. PMID:26457083

  16. Simultaneous Determination of Salicylic Acid, Jasmonic Acid, Methyl Salicylate, and Methyl Jasmonate from Ulmus pumila Leaves by GC-MS.

    PubMed

    Huang, Zhi-Hong; Wang, Zhi-Li; Shi, Bao-Lin; Wei, Dong; Chen, Jian-Xin; Wang, Su-Li; Gao, Bao-Jia

    2015-01-01

    Salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate are important phytohormones and defensive signaling compounds, so it is of great importance to determine their levels rapidly and accurately. The study uses Ulmus pumila leaves infected by Tetraneura akinire Sasaki at different stages as materials; after extraction with 80% methanol and ethyl acetate and purification with primary secondary amine (PSA) and graphitized carbon blacks (GCB), the contents of signal compounds salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate were determined by GC-MS. The results showed that the level of salicylic acid, jasmonic acid, methyl salicylate, and methyl jasmonate increased remarkably in U. pumila once infected by T. akinire Sasaki, but the maximums of these four compounds occurred at different times. Salicylic acid level reached the highest at the early stage, and jasmonic acid level went to the maximum in the middle stage; by contrast, change of content of methyl salicylate and methyl jasmonate was the quite opposite. PMID:26457083

  17. Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence

    PubMed Central

    2010-01-01

    Background Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. Results C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. Conclusions Analysis of the C. sticklandii genome and

  18. 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

  19. Isolation and amino acid sequences of squirrel monkey (Saimiri sciurea) insulin and glucagon

    SciTech Connect

    Yu, Jinghua ); Eng, J.; Yalow, R.S. City Univ. of New York, NY )

    1990-12-01

    It was reported two decades ago that insulin was not detectable in the glucose-stimulated state in Saimiri sciurea, the New World squirrel monkey, by a radioimmunoassay system developed with guinea pig anti-pork insulin antibody and labeled park insulin. With the same system, reasonable levels were observed in rhesus monkeys and chimpanzees. This suggested that New World monkeys, like the New World hystricomorph rodents such as the guinea pig and the coypu, might have insulins whose sequences differ markedly from those of Old World mammals. In this report the authors describe the purification and amino acid sequences of squirrel monkey insulin and glucagon. They demonstrate that the substitutions at B29, B27, A2, A4, and A17 of squirrel monkey insulin are identical with those previously found in another New World primate, the owl monkey (Aotus trivirgatus). The immunologic cross-reactivity of this insulin in their immunoassay system is only a few percent of that of human insulin. It appears that the peptides of the New World monkeys have diverged less from those of the Old World mammals than have those of the New World hystricomorph rodents. The striking improvements in peptide purification and sequencing have the potential for adding new information concerning the evolutionary divergence of species.

  20. Binding site discovery from nucleic acid sequences by discriminative learning of hidden Markov models

    PubMed Central

    Maaskola, Jonas; Rajewsky, Nikolaus

    2014-01-01

    We present a discriminative learning method for pattern discovery of binding sites in nucleic acid sequences based on hidden Markov models. Sets of positive and negative example sequences are mined for sequence motifs whose occurrence frequency varies between the sets. The method offers several objective functions, but we concentrate on mutual information of condition and motif occurrence. We perform a systematic comparison of our method and numerous published motif-finding tools. Our method achieves the highest motif discovery performance, while being faster than most published methods. We present case studies of data from various technologies, including ChIP-Seq, RIP-Chip and PAR-CLIP, of embryonic stem cell transcription factors and of RNA-binding proteins, demonstrating practicality and utility of the method. For the alternative splicing factor RBM10, our analysis finds motifs known to be splicing-relevant. The motif discovery method is implemented in the free software package Discrover. It is applicable to genome- and transcriptome-scale data, makes use of available repeat experiments and aside from binary contrasts also more complex data configurations can be utilized. PMID:25389269

  1. Dispersion functions and factors that determine resolution for DNA sequencing by gel electrophoresis

    SciTech Connect

    Sutherland, J.C.; Reynolds, K.J.; Fisk, D.J.

    1996-04-01

    The number of bases that can be read in a single run by a DNA sequencing instrument that detects fluorophore labeled DNA arriving at a ``finish-line`` located a fixed distance from the starting wells is influenced by numerous parameters. Strategies for improving the length-of-read of a DNA sequencer can be based on quantitative models of the separation of DNA by gel electrophoresis. The dispersion function of the electrophoretic system--the relationship between molecular contour length and time of arrival at the detector--is useful in characterizing the performance of a DNA sequencer. We adapted analytical representations of dispersion functions, originally developed for snapshot imaging of DNA gels, (samples electrophoresed for constant time), to finish-line imaging, and demonstrated that a logistic-type function with non-integral exponent is required to describe the experimental data. We use this dispersion function to determine the resolution length and resolving power of a LI-COR DNA sequencing system and a custom built capillary gel electrophoresis system, and discuss the factors that presently limit the number of bases that can be determined reliably in a single sequencing run.

  2. Immunoreactivity of polyclonal antibodies generated against the carboxy terminus of the predicted amino acid sequence of the Huntington disease gene

    SciTech Connect

    Alkatib, G.; Graham, R.; Pelmear-Telenius, A.

    1994-09-01

    A cDNA fragment spanning the 3{prime}-end of the Huntington disease gene (from 8052 to 9252) was cloned into a prokaryotic expression vector containing the E. Coli lac promoter and a portion of the coding sequence for {beta}-galactosidase. The truncated {beta}-galactosidase gene was cleaved with BamHl and fused in frame to the BamHl fragment of the Huntington disease gene 3{prime}-end. Expression analysis of proteins made in E. Coli revealed that 20-30% of the total cellular proteins was represented by the {beta}-galactosidase-huntingtin fusion protein. The identity of the Huntington disease protein amino acid sequences was confirmed by protein sequence analysis. Affinity chromatography was used to purify large quantities of the fusion protein from bacterial cell lysates. Affinity-purified proteins were used to immunize New Zealand white rabbits for antibody production. The generated polyclonal antibodies were used to immunoprecipitate the Huntington disease gene product expressed in a neuroblastoma cell line. In this cell line the antibodies precipitated two protein bands of apparent gel migrations of 200 and 150 kd which together, correspond to the calculated molecular weight of the Huntington disease gene product (350 kd). Immunoblotting experiments revealed the presence of a large precursor protein in the range of 350-750 kd which is in agreement with the predicted molecular weight of the protein without post-translational modifications. These results indicate that the huntingtin protein is cleaved into two subunits in this neuroblastoma cell line and implicate that cleavage of a large precursor protein may contribute to its biological activity. Experiments are ongoing to determine the precursor-product relationship and to examine the synthesis of the huntingtin protein in freshly isolated rat brains, and to determine cellular and subcellular distribution of the gene product.

  3. Nucleotide sequences and characterization of liv genes encoding components of the high-affinity branched-chain amino acid transport system in Salmonella typhimurium.

    PubMed

    Matsubara, K; Ohnishi, K; Kiritani, K

    1992-07-01

    A 7.6-kb fragment of Salmonella typhimurium LT2 containing the liv gene cluster, which specifies the high-affinity branched-chain amino acid transport system (LIV-I), has been isolated. The upstream region contains the livB and livC genes encoding the leucine-isoleucine-valine-threonine and leucine-specific binding proteins, respectively. In this study, the nucleotide sequence of the 4-kb downstream segment was determined and found to contain four reading frames, designated as livA, livE, livF, and livG, that encode putative membrane-associated proteins. The livA and livE genes encode hydrophobic proteins composed of 308 and 425 amino acid residues, respectively. The livF and livG genes encode hydrophilic proteins of 255 and 237 amino acids, respectively; both the proteins contain consensus amino acid sequences found in proteins with ATP-binding sites. These four genes linked together have a potential rho-independent transcriptional terminator adjacent to the 3'-end of livG. No promoter sequence was found in the immediate upstream region of the livAEFG cluster. The livA, livE, livF, and livG gene products were identified as proteins with apparent M(r)s of 25,500, 34,500, 28,000, and 26,000, respectively, by SDS-polyacryl-amide gel electrophoresis. The deduced amino acid sequences of these four proteins showed strong homology to those of the corresponding membrane-associated proteins required for the high-affinity branched-chain amino acid transport systems from both Escherichia coli and Pseudomonas aeruginosa. PMID:1429514

  4. Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation.

    PubMed Central

    Macke, J P; Hu, N; Hu, S; Bailey, M; King, V L; Brown, T; Hamer, D; Nathans, J

    1993-01-01

    To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, we have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser205-to-arg and glu793-to-asp, the biological significance of which is unknown. Images Figure 2 PMID:8213813

  5. Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation

    SciTech Connect

    Macke, J.P.; Nathans, J.; King, V.L. ); Hu, N.; Hu, S.; Hamer, D.; Bailey, M. ); Brown, T. )

    1993-10-01

    To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, the authors have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser[sup 205] -to-arg and glu[sup 793]-to-asp, the biological significance of which is unknown. 32 refs., 2 figs., 2 tabs.

  6. Complete cDNA and deduced amino acid sequence of the chaperonin containing T-complex polypeptide 1 (CCT) delta subunit from Aedes triseriatus mosquitoes.

    PubMed

    Blitvich, B J; Rayms-Keller, A; Blair, C D; Beaty, B J

    2001-01-01

    The chaperonin containing t-complex polypeptide 1 (CCT) assists in the ATP-dependent folding and assembly of newly translated actin and tubulin in the eukaryotic cytosol. CCT is composed of eight different subunits, each encoded by an independent gene. In this report, we used RT-PCR amplification and 5'- and 3'-rapid amplification of cDNA ends (RACE) to determine the complete cDNA sequence of the CCT delta subunit from Aedes triseriatus mosquitoes. The CCT delta cDNA is 1936 nucleotides in length and encodes a putative 533 amino acid protein with a calculated molecular mass of 57,179 daltons and pI of 7.15. Hydrophobic residues comprise 39.8% of the amino acid sequence and putative motifs for ATP-binding and ATPase-activity are present. The amino acid sequence displays strong sequence similarity to Drosophila melanogaster (92%), human (85%), puffer fish (84%) and mouse (84%) counterparts. CCT delta mRNA was detected in both biosynthetically active (embryonating) and dormant (diapausing) Ae. triseriatus embryos by RT-PCR analysis. PMID:11762197

  7. Novel graphene flowers modified carbon fibers for simultaneous determination of ascorbic acid, dopamine and uric acid.

    PubMed

    Du, Jiao; Yue, Ruirui; Ren, Fangfang; Yao, Zhangquan; Jiang, Fengxing; Yang, Ping; Du, Yukou

    2014-03-15

    A novel and sensitive carbon fiber electrode (CFE) modified by graphene flowers was prepared and used to simultaneously determine ascorbic acid (AA), dopamine (DA) and uric acid (UA). SEM images showed that beautiful and layer-petal graphene flowers homogeneously bloomed on the surface of CFE. Moreover, sharp and obvious oxidation peaks were found at the obtained electrode when compared with CFE and glassy carbon electrode (GCE) for the oxidation of AA, DA and UA. Also, the linear calibration plots for AA, DA and UA were observed, respectively, in the ranges of 45.4-1489.23 μM, 0.7-45.21 μM and 3.78-183.87 μM in the individual detection of each component. By simultaneously changing the concentrations of AA, DA and UA, their oxidation peaks appeared at -0.05 V, 0.16 V and 2.6 V, and the good linear responses ranges were 73.52-2305.53 μM, 1.36-125.69 μM and 3.98-371.49 μM, respectively. In addition, the obtained electrode showed satisfactory results when applied to the determination of AA, DA and UA in urine and serum samples. PMID:24140872

  8. Determination and analysis of the complete mitochondrial genome sequence of Taoyuan chicken.

    PubMed

    Liu, Li-Li; Xie, Hong-Bing; Yu, Qi-Fang; He, Shao-Ping; He, Jian-Hua

    2016-01-01

    Taoyuan chicken is excellent native breeds in China. This study firstly determined the complete mitochondrial genome sequence of Taoyuan chicken using PCR-based amplification and Sanger sequencing. The characteristic of the entire mitochondrial genome was analyzed in detail, with the base composition of 30.26% A, 23.79% T, 32.44% C, 13.50% G in the Taoyuan chicken (16,784 bp in length). It contained 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and a major non-coding control region (D-loop region). The complete mitochondrial genome sequence of Taoyuan chicken will be useful for the phylogenetics of poultry, and be available as basic data for the genetics and breeding. PMID:24617480

  9. [Determination of hepatitis B virus genotypes by DNA sequence analysis in patients from Ankara, Turkey].

    PubMed

    Külah, Canan; Cirak, Meltem Yalinay

    2010-04-01

    Hepatitis B virus (HBV) genotypes vary depending on the geographical region. The HBV genotype determined in Turkey has been genotype D which is found as the homogenously disseminated single genotype. The aim of this study was to determine HBV genotypes in a group of HBV infected patients who were admitted to a university hospital in Ankara, Turkey. Serum samples from HBsAg positive and anti-HBs negative 84 (52 male, 32 female) patients with HBV infection were included into the study. Anti-HBc was positive in 95.2%, HBeAg was positive in 47.6% and anti-HBe was positive in 11.9% of the patients. Mean HBV-DNA levels of the patients were 5.7 x 10(7) +/- 4.6 x 10(7) IU/ml; mean ALT levels were 131 +/- 171 IU/ml and mean AST levels were 98 +/- 170 IU/ml. HBV-DNA was extracted from serum by the phenol-chloroform method and PCR was performed to amplify the S gene region of HBV-DNA. Cycle sequencing of PCR products was performed by a commercial "Cy5/Cy5.5 Dye Primer Cycle Sequencing Kit" (Visible Genetics, Canada) based on dideoxy chain termination method. The sequences were read and analyzed in an automated fluorescence-based DNA-sequencing system (Long-Read Tower System, Visible Genetics, Canada). The nucleotide sequences of the patient samples were compared with the previously reported sequences in gene bank for each genotype. According to the comparative analysis of S-sequences of all patient samples with the published sequences of the genotypes in gene bank, all of the 84 hepatitis B strains (100%) were shown to be related to D genotypic group, subtype ayw. A phylogenetic analysis was performed and phylogenetic trees were constructed using programs in the PHYLIP phylogeny inference package. The patient samples clustered within the genotypic group D. According to these results, the main HBV genotype in our patients was genotype D in accordance with the previous molecular epidemiologic information on HBV in this geographic area. HBV genotype determination may help to

  10. Analysis of the full-length genome sequence of papaya lethal yellowing virus (PLYV), determined by deep sequencing, confirms its classification in the genus Sobemovirus.

    PubMed

    Pereira, Alvaro J; Alfenas-Zerbini, Poliane; Cascardo, Renan S; Andrade, Eduardo C; Murilo Zerbini, F

    2012-10-01

    Papaya lethal yellowing virus (PLYV) causes an economically important disease in papayas in northeastern Brazil. Based on biological and molecular properties, PLYV has been tentatively assigned to the genus Sobemovirus. We report the sequence of the full-length genome of a PLYV isolate from Brazil, determined by deep sequencing. The PLYV genome is 4,145 nt long and contains four ORFs, with an arrangement identical to that of sobemoviruses. The polyprotein and CP display significant sequence identity with the corresponding proteins of other sobemoviruses. Pairwise comparisons and phylogenetic analysis based on complete nucleotide sequences confirm the classification of PLYV in the genus Sobemovirus. PMID:22743825

  11. Full Genome Virus Detection in Fecal Samples Using Sensitive Nucleic Acid Preparation, Deep Sequencing, and a Novel Iterative Sequence Classification Algorithm

    PubMed Central

    Cotten, Matthew; Oude Munnink, Bas; Canuti, Marta; Deijs, Martin; Watson, Simon J.; Kellam, Paul; van der Hoek, Lia

    2014-01-01

    We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis. PMID:24695106

  12. Determining the sulfuric acid fog concentration in coke oven gas

    SciTech Connect

    Zin'kovskaya, S.I.; Okhrimenko, E.L.; Sobko, L.V.

    1982-11-06

    A volumetric method for the analysis of sulfuric acid aerosols at levels of acid greater (25-40 g/m/sup 3/) than those (1 g/m/sup 3/) analyzable by current methods is described. Coke oven gas after acid scrubbing and electrofiltration is passed through a Schott filter (pressure drop 100 mm Hg), the sulfuric acid aerosol being condensed on the filter which is washed with water and the washings filtered with NaOH (0.01 N after electrofilter, 1.0 N after the acid towers) to methyl orange end point. The error is +/- 2%.

  13. Quantitative Determination of Citric and Ascorbic Acid in Powdered Drink Mixes

    ERIC Educational Resources Information Center

    Sigmann, Samuella B.; Wheeler, Dale E.

    2004-01-01

    A procedure by which the reactions are used to quantitatively determine the amount of total acid, the amount of total ascorbic acid and the amount of citric acid in a given sample of powdered drink mix, are described. A safe, reliable and low-cost quantitative method to analyze consumer product for acid content is provided.

  14. 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.

  15. The amino acid sequence of a cereal Bowman-Birk type trypsin inhibitor from seeds of Jobs' tears (Coix lachryma-jobi L.).

    PubMed

    Ary, M B; Shewry, P R; Richardson, M

    1988-02-29

    The major trypsin inhibitor from seeds of Jobs' tears (Coix lachryma-jobi) was purified by heat treatment, fractional precipitation with (NH4)2SO4, ion-exchange chromatography on DEAE-Sepharose, gel-filtration on Sephadex G-75 and preparative reverse-phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with trypsin, chymotrypsin and the S. aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong homology with a number of Bowman-Birk inhibitors from legume seeds and similar proteins recently isolated from wheat and rice. PMID:3162215

  16. Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus.

    PubMed

    Franke, I H; Fegan, M; Hayward, A C; Sly, L I

    1998-01-01

    The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers. The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense. The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences. Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet. diazotrophicus from environmental samples with nifH gene-based primers. PMID:9489028

  17. The amino acid alphabet and the architecture of the protein sequence-structure map. I. Binary alphabets.

    PubMed

    Ferrada, Evandro

    2014-12-01

    The correspondence between protein sequences and structures, or sequence-structure map, relates to fundamental aspects of structural, evolutionary and synthetic biology. The specifics of the mapping, such as the fraction of accessible sequences and structures, or the sequences' ability to fold fast, are dictated by the type of interactions between the monomers that compose the sequences. The set of possible interactions between monomers is encapsulated by the potential energy function. In this study, I explore the impact of the relative forces of the potential on the architecture of the sequence-structure map. My observations rely on simple exact models of proteins and random samples of the space of potential energy functions of binary alphabets. I adopt a graph perspective and study the distribution of viable sequences and the structures they produce, as networks of sequences connected by point mutations. I observe that the relative proportion of attractive, neutral and repulsive forces defines types of potentials, that induce sequence-structure maps of vastly different architectures. I characterize the properties underlying these differences and relate them to the structure of the potential. Among these properties are the expected number and relative distribution of sequences associated to specific structures and the diversity of structures as a function of sequence divergence. I study the types of binary potentials observed in natural amino acids and show that there is a strong bias towards only some types of potentials, a bias that seems to characterize the folding code of natural proteins. I discuss implications of these observations for the architecture of the sequence-structure map of natural proteins, the construction of random libraries of peptides, and the early evolution of the natural amino acid alphabet. PMID:25473967

  18. The Amino Acid Alphabet and the Architecture of the Protein Sequence-Structure Map. I. Binary Alphabets

    PubMed Central

    Ferrada, Evandro

    2014-01-01

    The correspondence between protein sequences and structures, or sequence-structure map, relates to fundamental aspects of structural, evolutionary and synthetic biology. The specifics of the mapping, such as the fraction of accessible sequences and structures, or the sequences' ability to fold fast, are dictated by the type of interactions between the monomers that compose the sequences. The set of possible interactions between monomers is encapsulated by the potential energy function. In this study, I explore the impact of the relative forces of the potential on the architecture of the sequence-structure map. My observations rely on simple exact models of proteins and random samples of the space of potential energy functions of binary alphabets. I adopt a graph perspective and study the distribution of viable sequences and the structures they produce, as networks of sequences connected by point mutations. I observe that the relative proportion of attractive, neutral and repulsive forces defines types of potentials, that induce sequence-structure maps of vastly different architectures. I characterize the properties underlying these differences and relate them to the structure of the potential. Among these properties are the expected number and relative distribution of sequences associated to specific structures and the diversity of structures as a function of sequence divergence. I study the types of binary potentials observed in natural amino acids and show that there is a strong bias towards only some types of potentials, a bias that seems to characterize the folding code of natural proteins. I discuss implications of these observations for the architecture of the sequence-structure map of natural proteins, the construction of random libraries of peptides, and the early evolution of the natural amino acid alphabet. PMID:25473967

  19. 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

  20. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    PubMed Central

    Price, S R; Nightingale, M; Tsai, S C; Williamson, K C; Adamik, R; Chen, H C; Moss, J; Vaughan, M

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on the partial amino acid sequence of one of the soluble ARFs from bovine brain. Comparison of the deduced amino acid sequence of lambda ARF2B with sequences of peptides from the ARF protein (total of 60 amino acids) revealed only two differences. Whether these are cloning artifacts or reflect the existence of more than one ARF protein remains to be determined. Deduced amino acid sequences of ARF, Go alpha (the alpha subunit of a G protein that may be involved in regulation of ion fluxes), and c-Ha-ras gene product p21 show similarities in regions believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF apparently lacks a site analogous to that ADP-ribosylated by choleragen in G-protein alpha subunits. Although both the ARF proteins and the alpha subunits bind guanine nucleotides and serve as choleragen substrates, they must interact with the toxin A1 peptide in different ways. In addition to serving as an ADP-ribose acceptor, ARF interacts with the toxin in a manner that modifies its catalytic properties. PMID:3135549

  1. Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yean, Chan Yean

    2012-01-01

    In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis. PMID:22705404

  2. Noninvasive Antenatal Determination of Fetal Blood Group Using Next-Generation Sequencing.

    PubMed

    Rieneck, Klaus; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

    2016-01-01

    Hemolytic disease of the fetus and newborn (HDFN) is a condition characterized by a decreased lifespan of fetal red blood cells caused by maternally produced allospecific antibodies transferred to the fetus during pregnancy. The antibodies bind to the corresponding blood group antigens on fetal red blood cells and induce hemolysis. Cell-free DNA derived from the conceptus circulates in maternal blood. Using next-generation sequencing (NGS), it can be determined if this cell-free fetal DNA encodes the corresponding blood group antigen that is the target of the maternal allospecific antibodies. This determination carries no risk to the fetus. It is important to determine if the fetus is at risk of hemolysis to enable timely intervention. Many tests for blood groups are based solely on the presence or absence of a single nucleotide polymorphism (SNP). Antenatal determination of fetal blood group by NGS analysis holds advantages over polymerase chain reaction (PCR) determination based on allele specific amplification. PMID:26511760

  3. Spec-seq: determining protein–DNA-binding specificity by sequencing

    PubMed Central

    Zuo, Zheng; Chang, Yiming Kenny

    2015-01-01

    The specificity of protein–DNA interactions can be determined directly by sequencing the bound and unbound fractions in a standard binding reaction. The procedure is easy and inexpensive, and the accuracy can be high for thousands of sequences assayed in parallel. From the measurements, simple models of specificity, such as position weight matrices, can be assessed for their accuracy and more complex models developed if useful. Those may provide more accurate predictions of in vivo binding sites and can help us to understand the details of recognition. As an example, we demonstrate new information gained about the binding of lac repressor. One can apply the same method to combinations of factors that bind simultaneously to a single DNA and determine both the specificity of the individual factors and the cooperativity between them. PMID:25362070

  4. Spec-seq: determining protein-DNA-binding specificity by sequencing.

    PubMed

    Stormo, Gary D; Zuo, Zheng; Chang, Yiming Kenny

    2015-01-01

    The specificity of protein-DNA interactions can be determined directly by sequencing the bound and unbound fractions in a standard binding reaction. The procedure is easy and inexpensive, and the accuracy can be high for thousands of sequences assayed in parallel. From the measurements, simple models of specificity, such as position weight matrices, can be assessed for their accuracy and more complex models developed if useful. Those may provide more accurate predictions of in vivo binding sites and can help us to understand the details of recognition. As an example, we demonstrate new information gained about the binding of lac repressor. One can apply the same method to combinations of factors that bind simultaneously to a single DNA and determine both the specificity of the individual factors and the cooperativity between them. PMID:25362070

  5. Determination of the sequence of intersecting lines using Focused Ion Beam/Scanning Electron Microscope.

    PubMed

    Kim, Jiye; Kim, MinJung; An, JinWook; Kim, Yunje

    2016-05-01

    The aim of this study was to verify that the combination of focused ion beam (FIB) and scanning electron microscope/energy-dispersive X-ray (SEM/EDX) could be applied to determine the sequence of line crossings. The samples were transferred into FIB/SEM for FIB milling and an imaging operation. EDX was able to explore the chemical components and the corresponding elemental distribution in the intersection. The technique was successful in determining the sequence of heterogeneous line intersections produced using gel pens and red sealing ink with highest success rate (100% correctness). These observations show that the FIB/SEM was the appropriate instrument for an overall examination of document. PMID:27122423

  6. 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.

  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. Bacterial community compositions in sediment polluted by perfluoroalkyl acids (PFAAs) using Illumina high-throughput sequencing.

    PubMed

    Sun, Yajun; Wang, Tieyu; Peng, Xiawei; Wang, Pei; Lu, Yonglong

    2016-06-01

    The characterization of bacterial community compositions and the change in perfluoroalkyl acids (PFAAs) along a natural river distribution system were explored in the present study. Illumina high-throughput sequencing was used to explore bacterial community diversity and structure in sediment polluted by PFAAs from the Xiaoqing River, the area with concentrated fluorochemical facilities in China. The concentration of PFAAs was in the range of 8.44-465.60 ng/g dry weight (dw) in sediment. Perfluorooctanoic acid (PFOA) was the dominant PFAA in all samples, which accounted for 94.2 % of total PFAAs. High-level PFOA could lead to an obvious increase in relative abundance of Proteobacteria, ε-Proteobacteria, Thiobacillus, and Sulfurimonas and the decrease in relative abundance of other bacteria. Redundancy analysis revealed that PFOA played an important role in the formation of bacterial community, and PFOA at higher concentration could reduce the diversity of bacterial community. When the concentration of PFOA was below 100 ng/g dw in sediment, no significant effect on microbial community structure was observed. Thiobacillus and Sulfurimonas were positively correlated with the concentration of PFOA, suggesting that both genera were resistant to PFOA contamination. PMID:26780047

  9. 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. PMID:26773170

  10. Gallic Acid: Review of the Methods of Determination and Quantification.

    PubMed

    Fernandes, Felipe Hugo Alencar; Salgado, Hérida Regina Nunes

    2016-05-01

    Gallic acid (3,4,5 trihydroxybenzoic acid) is a secondary metabolite present in most plants. This metabolite is known to exhibit a range of bioactivities including antioxidant, antimicrobial, anti-inflammatory, and anticancer. There are various methods to analyze gallic acid including spectrometry, chromatography, and capillary electrophoresis, among others. They have been developed to identify and quantify this active ingredient in most biological matrices. The aim of this article is to review the available information on analytical methods for gallic acid, as well as presenting the advantages and limitations of each technique. PMID:26440222

  11. Soil surface acidity plays a determining role in the atmospheric-terrestrial exchange of nitrous acid

    PubMed Central

    Donaldson, Melissa A.; Bish, David L.; Raff, Jonathan D.

    2014-01-01

    Nitrous acid (HONO) is an important hydroxyl (OH) radical source that is formed on both ground and aerosol surfaces in the well-mixed boundary layer. Recent studies report the release of HONO from nonacidic soils, although it is unclear how soil that is more basic than the pKa of HONO (∼3) is capable of protonating soil nitrite to serve as an atmospheric HONO source. Here, we used a coated-wall flow tube and chemical ionization mass spectrometry (CIMS) to study the pH dependence of HONO uptake onto agricultural soil and model substrates under atmospherically relevant conditions (1 atm and 30% relative humidity). Experiments measuring the evolution of HONO from pH-adjusted surfaces treated with nitrite and potentiometric titrations of the substrates show, to our knowledge for the first time, that surface acidity rather than bulk aqueous pH determines HONO uptake and desorption efficiency on soil, in a process controlled by amphoteric aluminum and iron (hydr)oxides present. The results have important implications for predicting when soil nitrite, whether microbially derived or atmospherically deposited, will act as a net source or sink of atmospheric HONO. This process represents an unrecognized mechanism of HONO release from soil that will contribute to HONO emissions throughout the day. PMID:25512517

  12. Soil surface acidity plays a determining role in the atmospheric-terrestrial exchange of nitrous acid.

    PubMed

    Donaldson, Melissa A; Bish, David L; Raff, Jonathan D

    2014-12-30

    Nitrous acid (HONO) is an important hydroxyl (OH) radical source that is formed on both ground and aerosol surfaces in the well-mixed boundary layer. Recent studies report the release of HONO from nonacidic soils, although it is unclear how soil that is more basic than the pKa of HONO (∼ 3) is capable of protonating soil nitrite to serve as an atmospheric HONO source. Here, we used a coated-wall flow tube and chemical ionization mass spectrometry (CIMS) to study the pH dependence of HONO uptake onto agricultural soil and model substrates under atmospherically relevant conditions (1 atm and 30% relative humidity). Experiments measuring the evolution of HONO from pH-adjusted surfaces treated with nitrite and potentiometric titrations of the substrates show, to our knowledge for the first time, that surface acidity rather than bulk aqueous pH determines HONO uptake and desorption efficiency on soil, in a process controlled by amphoteric aluminum and iron (hydr)oxides present. The results have important implications for predicting when soil nitrite, whether microbially derived or atmospherically deposited, will act as a net source or sink of atmospheric HONO. This process represents an unrecognized mechanism of HONO release from soil that will contribute to HONO emissions throughout the day. PMID:25512517

  13. Sequence and chromatin determinants of cell-type-specific transcription factor binding.

    PubMed

    Arvey, Aaron; Agius, Phaedra; Noble, William Stafford; Leslie, Christina

    2012-09-01

    Gene regulatory programs in distinct cell types are maintained in large part through the cell-type-specific binding of transcription factors (TFs). The determinants of TF binding include direct DNA sequence preferences, DNA sequence preferences of cofactors, and the local cell-dependent chromatin context. To explore the contribution of DNA sequence signal, histone modifications, and DNase accessibility to cell-type-specific binding, we analyzed 286 ChIP-seq experiments performed by the ENCODE Consortium. This analysis included experiments for 67 transcriptional regulators, 15 of which were profiled in both the GM12878 (lymphoblastoid) and K562 (erythroleukemic) human hematopoietic cell lines. To model TF-bound regions, we trained support vector machines (SVMs) that use flexible k-mer patterns to capture DNA sequence signals more accurately than traditional motif approaches. In addition, we trained SVM spatial chromatin signatures to model local histone modifications and DNase accessibility, obtaining significantly more accurate TF occupancy predictions than simpler approaches. Consistent with previous studies, we find that DNase accessibility can explain cell-line-specific binding for many factors. However, we also find that of the 10 factors with prominent cell-type-specific binding patterns, four display distinct cell-type-specific DNA sequence preferences according to our models. Moreover, for two factors we identify cell-specific binding sites that are accessible in both cell types but bound only in one. For these sites, cell-type-specific sequence models, rather than DNase accessibility, are better able to explain differential binding. Our results suggest that using a single motif for each TF and filtering for chromatin accessible loci is not always sufficient to accurately account for cell-type-specific binding profiles. PMID:22955984

  14. Purification and partial amino acid sequence of the chloroplast cytochrome b-559.

    PubMed

    Widger, W R; Cramer, W A; Hermodson, M; Meyer, D; Gullifor, M

    1984-03-25

    The hydrophobic cytochrome b-559, purified from unstacked, ethanol-washed spinach thylakoid membranes, using extraction with 2% Triton X-100 in 4 M urea and three chromatographic steps in the presence of protease inhibitors, has a dominant band on sodium dodecyl sulfate-urea gels corresponding to Mr = 10,000. The yield of this preparation is 30-50% (5-10 mg) starting with 600 mg of chlorophyll. The heme content yields a calculated molecular weight of no more than 17,500/heme, and perhaps somewhat smaller after correction for impurities. The Mr = 10,000 band is stained by the tetramethylbenzidine-H2O2 heme reagent on lithium dodecyl sulfate gels run at 0 degrees C. The Mr = 10,000 protein, further separated by high performance liquid chromatography, contains a unique NH2 terminus that is not blocked, and the amino acid sequence for the first 27 residues is NH2-Ser-Gly-Ser-Thr-Gly-Glu-Arg-Ser-Phe-Ala-Asp-Ile-Ile-Thr-Ser-Ile-Arg-Tyr-Trp -Val-Ile-X-Ser-Ile-Thr-Ile-Pro. . . COOH. Approximately 55% of the amino acids are hydrophobic, based on amino acid analysis of the Mr = 10,000 peptide, which also indicated the presence of at least one histidine. Only one cytochrome b-559 component could be identified, whose yield indicated that it arises from a single b-559 protein in chloroplasts corresponding to the in situ high potential cytochrome of the chloroplast photosystem II. PMID:6706983

  15. 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. PMID:27244455

  16. Determination of Precise Pre-Main-Sequence Stellar Properties through Stellar and Disk Orbital Dynamics

    NASA Astrophysics Data System (ADS)

    Stassun, Keivan

    2016-05-01

    We summarize the current state-of-the-art in the measurement of direct, precise stellar masses at pre-main-sequence ages through the analysis of eclipsing binary orbits and circumstellar disk dynamics. We highlight two key issues: (1) The masses determined from disk dynamics require more precise distance determinations that should become available from Gaia soon, and (2) many eclipsing binaries appear disturbed by the presence of tertiary companions that inject heat into and puff up one or both of the inner binary stars, however the dynamical mechanism by which orbital energy is injected as heat remains unknown.

  17. 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.

  18. Determination of the absolute configuration of sialic acids in gangliosides from the sea cucumber Cucumaria echinata.

    PubMed

    Kisa, Fumiaki; Yamada, Koji; Miyamoto, Tomofumi; Inagaki, Masanori; Higuchi, Ryuichi

    2007-07-01

    Enantiomeric pairs of sialic acid, D- and L-NeuAc (N-acetylneuraminic acid), were converted to D- and L-arabinose, respectively, by chemical degradation. Using this method, the absolute configuration of the sialic acid residues, NeuAc and NeuGc (N-glycolylneuraminic acid), in the gangliosides from the sea cucumber Cucumaria echinata was determined to be the D-form. Although naturally occurring sialic acids have been believed to be the D-form on the basis of biosynthetic evidence, this is the first report of the determination of the absolute configuration of the sialic acid residues in gangliosides using chemical methods. PMID:17603199

  19. VOLTAMMETRIC METHODS FOR DETERMINATION OF METAL BINDING BY FULVIC ACID

    EPA Science Inventory

    The use of anodic stripping voltammetry (ASV) and differential pulse polarography (DPP) for the measurement of the concentrations of aquo ions in the presence of fulvic acid, and the subsequent use of these data for estimation of the metal--fulvic acid conditional stability const...

  20. Amino acid determination in some edible Mexican insects.

    PubMed

    Ladrón de Guevara, O; Padilla, P; García, L; Pino, J M; Ramos-Elorduy, J

    1995-06-01

    The amino acid contents of edible insects from different provinces of Mexico and reference proteins were analysed by reversed-phase high-performance liquid chromatography and ion exchange chromatography. The insect amino acid contents were higher than the adult requirements indicated by the WHO/FAO pattern. PMID:24178816

  1. Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions

    PubMed Central

    Chen, Zhangguo; Eder, Maxwell D.; Elos, Mihret T.; Viboolsittiseri, Sawanee S.; Chen, Xiaomi

    2016-01-01

    Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sμ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity. PMID:26810227

  2. Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions.

    PubMed

    Chen, Zhangguo; Eder, Maxwell D; Elos, Mihret T; Viboolsittiseri, Sawanee S; Chen, Xiaomi; Wang, Jing H

    2016-03-01

    Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sμ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity. PMID:26810227

  3. Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries

    PubMed Central

    Goodman, Andrew L.; Wu, Meng; Gordon, Jeffrey I.

    2012-01-01

    Insertion Sequencing (INSeq) is a method for determining the insertion site and relative abundance of large numbers of transposon mutants in a mixed population of isogenic mutants of a sequenced microbial species. INSeq is based on a modified mariner transposon containing MmeI sites at its ends, allowing cleavage at chromosomal sites 16–17bp from the inserted transposon. Genomic regions adjacent to the transposons are amplified by linear PCR with a biotinylated primer. Products are bound to magnetic beads, digested with MmeI, and barcoded with sample-specific linkers appended to each restriction fragment. After limited PCR amplification, fragments are sequenced using a high-throughput instrument. The sequence of each read can be used to map the location of a transposon in the genome. Read count measures the relative abundance of that mutant in the population. Solid-phase library preparation makes this protocol rapid (18h), easy to scale-up, amenable to automation, and useful for a variety of samples. A protocol for characterizing libraries of transposon mutant strains clonally arrayed in multi-well format is provided. PMID:22094732

  4. Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin.

    PubMed

    Yamashita, H; Theerasilp, S; Aiuchi, T; Nakaya, K; Nakamura, Y; Kurihara, Y

    1990-09-15

    A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed. PMID:2394746

  5. The determination of the DNA sequence specificity of bleomycin-induced abasic sites.

    PubMed

    Chen, Jon K; Murray, Vincent

    2016-06-01

    The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, was determined with high precision in purified plasmid DNA using an improved technique. This improved technique involved the labelling of the 5'- and 3'-ends of DNA with different fluorescent tags, followed by simultaneous cleavage by bleomycin and capillary electrophoresis with laser-induced fluorescence. This permitted the determination of bleomycin cleavage specificity with high accuracy since end-label bias was greatly reduced. Bleomycin produces single- and double-strand breaks, abasic sites and other base damage in DNA. This high-precision method was utilised to elucidate, for the first time, the DNA sequence specificity of bleomycin-induced DNA damage at abasic sites. This was accomplished using endonuclease IV that cleaves DNA at abasic sites after bleomycin damage. It was found that bleomycin-induced abasic sites formed at 5'-GC and 5'-GT sites while bleomycin-induced phosphodiester strand breaks formed mainly at 5'-GT dinucleotides. Since bleomycin-induced abasic sites are produced in the absence of molecular oxygen, this difference in DNA sequence specificity could be important in hypoxic tumour cells. PMID:26940956

  6. Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.

    PubMed Central

    Rubin, H; Salem, J S; Li, L S; Yang, F D; Mama, S; Wang, Z M; Fisher, A; Hamann, C S; Cooperman, B S

    1993-01-01

    Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase. The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfR1) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle. Images Fig. 2 Fig. 4 Fig. 5 PMID:8415692

  7. Spectrophotometric determination of norepinephrine with sodium iodate and determination of its acidity constants

    NASA Astrophysics Data System (ADS)

    Hashem, E. Y.; Youssef, A. K.

    2013-05-01

    A spectrophotometric method is proposed for the determination of norepinephrine (NE) and its bitartrate salts. The method was based on the development of a red color (λmax = 495 nm) with sodium iodate in aqueous alcoholic medium at pH 5. The color was stable for at least 4 hrs. The molar reacting ratio of NE to sodium iodate was 1:4. A linear relationship was obtained between the absorption intensity and NE concentration in the range of 3.384-37.224 μg/ml with detection limit of 0.067 μg/ml and correlation coefficient of 0.9972. The present work facilitated the determination of the three acidity constants, 7.564 ± 0.02, 9.036 ± 0.034, and 10.761 ± 0.023. The reaction mechanism was also described. The proposed method was successfully applied for the determination of NE in pharmaceutical formulations. Results for analysis of bulk drugs and injections agree with those of official methods.

  8. The determination of the helium abundance in main-sequence B stars

    NASA Technical Reports Server (NTRS)

    Wolff, S. C.; Heasley, J. N.

    1985-01-01

    It is pointed out that the abundances of deuterium, helium, and lithium provide fundamental constraints on cosmological models. The central question is related to the compatibility of the observed abundances with big bang models of primordial nucleosynthesis, taking into account a modification by subsequent nuclear processing in stars or a modification of standard models. The present paper has the objective to assess critically the feasibility of deriving accurate helium abundances from measurements of the photospheric lines in main-sequence B-type stars. A method is established for assigning atmospheric parameters to main-sequence stars with spectral types in the range (approximately) B1-B5. It is found that an analysis of stars in distant anticenter H II regions and clusters offer an alternative method which seems capable of determining relative abundances with more than the requisite accuracy.

  9. The developmental transcriptome landscape of bovine skeletal muscle defined by Ribo-Zero ribonucleic acid sequencing.

    PubMed

    Sun, X; Li, M; Sun, Y; Cai, H; Li, R; Wei, X; Lan, X; Huang, Y; Lei, C; Chen, H

    2015-12-01

    Ribonucleic acid sequencing (RNA-Seq) libraries are normally prepared with oligo(dT) selection of poly(A)+ mRNA, but it depends on intact total RNA samples. Recent studies have described Ribo-Zero technology, a novel method that can capture both poly(A)+ and poly(A)- transcripts from intact or fragmented RNA samples. We report here the first application of Ribo-Zero RNA-Seq for the analysis of the bovine embryonic, neonatal, and adult skeletal muscle whole transcriptome at an unprecedented depth. Overall, 19,893 genes were found to be expressed, with a high correlation of expression levels between the calf and the adult. Hundreds of genes were found to be highly expressed in the embryo and decreased at least 10-fold after birth, indicating their potential roles in embryonic muscle development. In addition, we present for the first time the analysis of global transcript isoform discovery in bovine skeletal muscle and identified 36,694 transcript isoforms. Transcriptomic data were also analyzed to unravel sequence variations; 185,036 putative SNP and 12,428 putative short insertions-deletions (InDel) were detected. Specifically, many stop-gain, stop-loss, and frameshift mutations were identified that probably change the relative protein production and sequentially affect the gene function. Notably, the numbers of stage-specific transcripts, alternative splicing events, SNP, and InDel were greater in the embryo than in the calf and the adult, suggesting that gene expression is most active in the embryo. The resulting view of the transcriptome at a single-base resolution greatly enhances the comprehensive transcript catalog and uncovers the global trends in gene expression during bovine skeletal muscle development. PMID:26641174

  10. Incorporating substrate sequence motifs and spatial amino acid composition to identify kinase-specific phosphorylation sites on protein three-dimensional structures

    PubMed Central

    2013-01-01

    Background Protein phosphorylation catalyzed by kinases plays crucial regulatory roles in cellular processes. Given the high-throughput mass spectrometry-based experiments, the desire to annotate the catalytic kinases for in vivo phosphorylation sites has motivated. Thus, a variety of computational methods have been developed for performing a large-scale prediction of kinase-specific phosphorylation sites. However, most of the proposed methods solely rely on the local amino acid sequences surrounding the phosphorylation sites. An increasing number of three-dimensional structures make it possible to physically investigate the structural environment of phosphorylation sites. Results In this work, all of the experimental phosphorylation sites are mapped to the protein entries of Protein Data Bank by sequence identity. It resulted in a total of 4508 phosphorylation sites containing the protein three-dimensional (3D) structures. To identify phosphorylation sites on protein 3D structures, this work incorporates support vector machines (SVMs) with the information of linear motifs and spatial amino acid composition, which is determined for each kinase group by calculating the relative frequencies of 20 amino acid types within a specific radial distance from central phosphorylated amino acid residue. After the cross-validation evaluation, most of the kinase-specific models trained with the consideration of structural information outperform the models considering only the sequence information. Furthermore, the independent testing set which is not included in training set has demonstrated that the proposed method could provide a comparable performance to other popular tools. Conclusion The proposed method is shown to be capable of predicting kinase-specific phosphorylation sites on 3D structures and has been implemented as a web server which is freely accessible at http://csb.cse.yzu.edu.tw/PhosK3D/. Due to the difficulty of identifying the kinase-specific phosphorylation

  11. Determining the rheology of active lava flows from photogrammetric image sequence processing

    NASA Astrophysics Data System (ADS)

    James, M. R.; Robson, S.; Pinkerton, H.

    2010-12-01

    We describe a photogrammetric approach used to determine the rheological properties of active lava flows based on stereo image sequences. Bulk rheological properties can be estimated from measurements of flow slope, velocity and dimensions and so, at flow-fronts, they can be calculated from sequential digital elevation models (DEMs) acquired as the flow advances over new ground. For useful flow parameters to be extracted, DEMs may need to be obtained at approximately minute intervals, over durations of up to multiple hours. To deliver such data, we use oblique stereo pair sequences captured by digital SLR cameras and a semi-automated DEM-generation pipeline. Although similar data could be acquired with a terrestrial laser scanner, with deployments in remote and hazardous regions the photogrammetric approach offers significant logistical advantages in terms of reduced equipment cost, bulk, weight and power requirements. We describe the application of the technique to an active lava flow on Mount Etna, Sicily, in 2006. Image sequences were acquired from two tripod-mounted cameras over a period of ~3 hours, as the flow-front advanced ~15 m. Photogrammetric control was provided by 11 targets placed in the scene, with their coordinates determined by dGPS. The cameras were synchronised by a shutter release cable and triggered by an external timer (intervalometer). Image pairs were obtained every minute with DEMs extraction carried out on every fourth epoch; 57 DEMs, with a 0.25-m resolution, were generated. We describe the challenges associated with data collection in this remote environment and the techniques required to automate the photogrammetric analysis and sequence-DEM generation.

  12. Sequence determination of a new parrot bornavirus-5 strain in Japan: implications of clade-specific sequence diversity in the regions interacting with host factors.

    PubMed

    Komorizono, Ryo; Makino, Akiko; Horie, Masayuki; Honda, Tomoyuki; Tomonaga, Keizo

    2016-06-01

    In this study, the genome sequence of a new parrot bornavirus-5 (PaBV-5) detected in Eclectus roratus was determined. Phylogenetic analysis showed that the genus Bornavirus is divided into three major clades and that PaBV-5 belongs to clade 2, which contains avian viruses that exhibit infectivity to mammalian cells. Sequence comparisons of the regions known to interact with host factors indicated that the clade 2 avian viruses possess sequences intermediate between the clade 1 mammalian viruses and the clade 3 avian viruses, suggesting that the identified regions might contribute to the differences in virological properties between the three clades. PMID:27166599

  13. 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.

  14. [Determination of organic acids in rice wine by ion-exclusion chromatography].

    PubMed

    Lin, Xiaojie; Wei, Wei; He, Zhigang; Lin, Xiaozi

    2014-03-01

    An ion-exclusion chromatographic method for the simultaneous determination of organic acids in rice wine was developed. An IC-Pak Ion Exclusion column (300 mm x 7.8 mm, 7 microm) was used at 50 degrees C. The mobile phases were H2SO4 (phase A) and acetonitrile (phase B) (98:2, v/v) at a flow rate of 0.5 mL/min. The gradient elution program was as follows: 0-40 min, 0.01 mol/L H2SO4 to 0.02 mol/L H2SO4; 40-50 min, 0.01 mol/L H2SO4. The injection volume was 10 microL. The detection wavelength was set at 210 nm. The results showed that oxalic acid, maleic acid, citric acid, tartaric acid, malic acid, ascorbic acid, succinic acid, lactic, fumaric acid, acetic acid, propionic acid, isobutyric acid and butyric acid were completely separated and determined in 30 min. The linear correlation coefficients were above 0.999 7 in the range of 0.001- 1.000 g/L. Under the optimized conditions, the recoveries of organic acids in rice wine were in the range of 93.4% - 103.8% with the relative standard deviations (RSDs, n = 5) of 0.1% - 1.5%. This method is feasible, convenient, fast, accurate and applicable for the quantitative analysis of the organic acids in rice wine. PMID:24984473

  15. Characterization of translational inhibitors from Phytolacca americana. Amino-terminal sequence determination and antibody-inhibitor conjugates.

    PubMed

    Bjorn, M J; Larrick, J; Piatak, M; Wilson, K J

    1984-10-23

    Two translational inhibitors (pokeweed antiviral protein and pokeweed antiviral protein II) isolated from the leaves of the pokeweed plant, Phytolacca americana, were characterized as to their behavior during reverse-phase HPLC and their amino-terminal sequences. Alignment of the sequences demonstrated that a substantial degree of homology was present (10 of 29 identical residues). Pokeweed antiviral protein was shown by reverse-phase chromatography to be composed of at least two components, pokeweed antiviral proteina and pokeweed antiviral proteinb, which comigrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, shared identical N-terminal amino-acid sequences through residue 31, and had similar specific activities in a cell-free translation inhibition assay. Pokeweed antiviral protein II was covalently coupled to a monoclonal antibody that recognizes the transferrin receptor (anti-transferrin receptor). The disulfide-linked conjugate inhibited protein synthesis in the human breast tumor cell line MCF-7, whereas anti-transferrin receptor, pokeweed antiviral protein II, or an immunotoxin composed of an irrelevant antiserum and pokeweed antiviral protein II, were nontoxic. The inhibitory dose 50% of anti-transferrin receptor-pokeweed antiviral protein II for MCF-7 cells was 0.7 nM, whereas the corresponding ricin A chain conjugate (anti-transferrin receptor-ricin A chain) was more potent with a inhibitory dose 50% of 0.1 nM. Pokeweed antiviral protein II can be added to the growing list of translation inhibitors that are effective as components of immunotoxins in vitro. Additional studies will be needed to determine whether pokeweed antiviral protein II immunotoxins provide advantageous properties for in vivo applications. PMID:6091760

  16. 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

  17. Genome Sequence of Schizochytrium sp. CCTCC M209059, an Effective Producer of Docosahexaenoic Acid-Rich Lipids

    PubMed Central

    Ji, Xiao-Jun; Mo, Kai-Qiang; Ren, Lu-Jing; Li, Gan-Lu; Huang, Jian-Zhong

    2015-01-01

    Schizochytrium is an effective species for producing omega-3 docosahexaenoic acid (DHA). Here, we report a genome sequence of Schizochytrium sp. CCTCC M209059, which has a genome size of 39.09 Mb. It will provide the genomic basis for further insights into the metabolic and regulatory mechanisms underlying the DHA formation. PMID:26251485

  18. Evolutionary Distance of Amino Acid Sequence Orthologs across Macaque Subspecies: Identifying Candidate Genes for SIV Resistance in Chinese Rhesus Macaques

    PubMed Central

    Ross, Cody T.; Roodgar, Morteza; Smith, David Glenn

    2015-01-01

    We use the Reciprocal Smallest Distance (RSD) algorithm to identify amino acid sequence orthologs in the Chinese and Indian rhesus macaque draft sequences and estimate the evolutionary distance between such orthologs. We then use GOanna to map gene function annotations and human gene identifiers to the rhesus macaque amino acid sequences. We conclude methodologically by cross-tabulating a list of amino acid orthologs with large divergence scores with a list of genes known to be involved in SIV or HIV pathogenesis. We find that many of the amino acid sequences with large evolutionary divergence scores, as calculated by the RSD algorithm, have been shown to be related to HIV pathogenesis in previous laboratory studies. Four of the strongest candidate genes for SIVmac resistance in Chinese rhesus macaques identified in this study are CDK9, CXCL12, TRIM21, and TRIM32. Additionally, ANKRD30A, CTSZ, GORASP2, GTF2H1, IL13RA1, MUC16, NMDAR1, Notch1, NT5M, PDCD5, RAD50, and TM9SF2 were identified as possible candidates, among others. We failed to find many laboratory experiments contrasting the effects of Indian and Chinese orthologs at these sites on SIVmac pathogenesis, but future comparative studies might hold fertile ground for research into the biological mechanisms underlying innate resistance to SIVmac in Chinese rhesus macaques. PMID:25884674

  19. Evolutionary distance of amino acid sequence orthologs across macaque subspecies: identifying candidate genes for SIV resistance in Chinese rhesus macaques.

    PubMed

    Ross, Cody T; Roodgar, Morteza; Smith, David Glenn

    2015-01-01

    We use the Reciprocal Smallest Distance (RSD) algorithm to identify amino acid sequence orthologs in the Chinese and Indian rhesus macaque draft sequences and estimate the evolutionary distance between such orthologs. We then use GOanna to map gene function annotations and human gene identifiers to the rhesus macaque amino acid sequences. We conclude methodologically by cross-tabulating a list of amino acid orthologs with large divergence scores with a list of genes known to be involved in SIV or HIV pathogenesis. We find that many of the amino acid sequences with large evolutionary divergence scores, as calculated by the RSD algorithm, have been shown to be related to HIV pathogenesis in previous laboratory studies. Four of the strongest candidate genes for SIVmac resistance in Chinese rhesus macaques identified in this study are CDK9, CXCL12, TRIM21, and TRIM32. Additionally, ANKRD30A, CTSZ, GORASP2, GTF2H1, IL13RA1, MUC16, NMDAR1, Notch1, NT5M, PDCD5, RAD50, and TM9SF2 were identified as possible candidates, among others. We failed to find many laboratory experiments contrasting the effects of Indian and Chinese orthologs at these sites on SIVmac pathogenesis, but future comparative studies might hold fertile ground for research into the biological mechanisms underlying innate resistance to SIVmac in Chinese rhesus macaques. PMID:25884674

  20. 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

  1. Draft Genome Sequence of Cutaneotrichosporon curvatus DSM 101032 (Formerly Cryptococcus curvatus), an Oleaginous Yeast Producing Polyunsaturated Fatty Acids.

    PubMed

    Hofmeyer, Thomas; Hackenschmidt, Silke; Nadler, Florian; Thürmer, Andrea; Daniel, Rolf; Kabisch, Johannes

    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

  2. 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

  3. Draft Genome Sequence of Acetobacter tropicalis Type Strain NBRC16470, a Producer of Optically Pure d-Glyceric Acid.

    PubMed

    Koike, Hideaki; Sato, Shun; Morita, Tomotake; Fukuoka, Tokuma; Habe, Hiroshi

    2014-01-01

    Here we report the 3.7-Mb draft genome sequence of Acetobacter tropicalis NBRC16470(T), 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

  4. 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

  5. Draft Genome Sequence of Burkholderia stabilis LA20W, a Trehalose Producer That Uses Levulinic Acid as a Substrate.

    PubMed

    Sato, Yuya; Koike, Hideaki; Kondo, Susumu; Hori, Tomoyuki; Kanno, Manabu; Kimura, Nobutada; Morita, Tomotake; Kirimura, Kohtaro; Habe, Hiroshi

    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

  6. 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

  7. 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....

  8. 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

  9. Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata.

    PubMed

    Hatakeyama, Tomomitsu; Matsuo, Noriaki; Shiba, Kouhei; Nishinohara, Shoichi; Yamasaki, Nobuyuki; Sugawara, Hajime; Aoyagi, Haruhiko

    2002-01-01

    CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment. PMID:11866098

  10. Using Conductivity Measurements to Determine the Identities and Concentrations of Unknown Acids: An Inquiry Laboratory Experiment

    ERIC Educational Resources Information Center

    Smith, K. Christopher; Garza, Ariana

    2015-01-01

    This paper describes a student designed experiment using titrations involving conductivity measurements to identify unknown acids as being either HCl or H[subscript 2]SO[subscript 4], and to determine the concentrations of the acids, thereby improving the utility of standard acid-base titrations. Using an inquiry context, students gain experience…

  11. Determination of permethrin resistance allele frequency of human head louse populations by quantitative sequencing.

    PubMed

    Kwon, Deok Ho; Yoon, Kyong Sup; Strycharz, Joseph P; Clark, J Marshall; Lee, Si Hyeock

    2008-09-01

    A quantitative sequencing (QS) protocol that detects the frequencies of sodium channel mutations (M815I, T917I, and L920F) responsible for knockdown resistance in permethrin-resistant head lice (Pediculus humanus capitis De Geer) was tested as a population genotyping method for use as a preliminary resistance monitoring tool. Genomic DNA fragments of the sodium channel a-subunit gene that encompass the three mutation sites were polymerase chain reaction (PCR)-1 amplified from individual head lice with either resistant or susceptible genotypes, and combined in various ratios to generate standard DNA template mixtures for QS. After sequencing, the signal ratios between resistant and susceptible nucleotides were calculated and plotted against the corresponding resistance allele frequencies. Quadratic regression coefficients of the plots were close to 1, demonstrating that the signal ratios are highly correlated with the resistance allele frequencies. Resistance allele frequencies predicted by QS, using either "pooled DNA" (DNA extracted from individual louse specimens and pooled) or "pooled specimen DNA" (DNA simultaneously extracted from multiple louse specimens), agreed well with those determined by individual sequencing, confirming the reliability and accuracy of QS as a population genotyping method and validating our approach of using the pooled specimen DNA as the DNA template for QS. Our protocol for QS was determined to be highly reliable for the prediction of resistance allele frequencies higher than approximately 7.4% at the 95% confidence level. According to the resistance allele frequencies determined by QS, pyrethroid resistance varies substantially among different geographical regions, emphasizing the importance of early resistance detection and proper management strategies. PMID:18826035

  12. 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

  13. Sequence of subunit c of the Na(+)-translocating F1F0 ATPase of Acetobacterium woodii: proposal for determinants of Na+ specificity as revealed by sequence comparisons.

    PubMed

    Rahlfs, S; Müller, V

    1997-03-10

    A 3.2 kb EcoRI fragment carrying genes for Na(+)-F1F0 ATPase was cloned from chromosomal DNA of Acetobacterium woodii. DNA sequence analysis revealed the presence of an open reading frame which was identified by data base searches and comparison with the experimentally derived N-terminal amino acid sequence to code for subunit c of Na(+)-F1F0 ATPase. A comparison of the primary sequences of the two well established Na(+)-translocating F1F0 ATPases from Acetobacterium woodii and Propionigenium modestum with H(+)-translocating enzymes indicates the length of the C-terminus as well as specific residues located in the cytoplasmic membrane to be important for Na+ transport. PMID:9119076

  14. Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

    PubMed Central

    Elango, N; Coligan, J E; Jambou, R C; Venkatesan, S

    1986-01-01

    The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5. Images PMID:3003381

  15. 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

  16. Identification of G and P genotype-specific motifs in the predicted VP7 and VP4 amino acid sequences.

    PubMed

    Ma, Yongping

    2015-12-01

    Equine rotavirus (ERV) strain L338 (G13P[18]) has a unique G and P genotype. However, the evolutionary relationship of L338 with other ERVs is still unknown. Here whole genome analysis of the L338 ERV strain was independently performed. Its genotype constellations were determined as G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11, confirming previous genotype assignments. The L338 strain only shared the P[18] and I6 genotypes with other ERVs. The nucleotide sequences of the other 9 RNA segments were different from those of cogent genes of all other group A rotavirus (RVA) strains including ERVs and formed unique phylogenetic lineages. The L338 evolutionary footprints were tentatively identified in both VP7 and VP4 amino acid sequences: two regions were found in VP7 and twelve in VP4. The conserved regions shared between L338 and other group A rotavirus strains (RVAs) indicated that L338 was more closely related genomically to animal and human RVAs other than ERVs, suggesting that L338 may not be an endogenous equine RV but have emerged as an interspecies reassortant with other RVA strains. Furthermore, genotype-specific motifs of all 27 G and 37 P types were identified in regions 7-1a (aa 91-100) of VP7 and regions 8-1 (aa146-151) and 8-3 (aa113-118 and 125-135) of VP4 (VP8*). PMID:26321159

  17. 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.

  18. Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors

    SciTech Connect

    Chang, C.; Kokontis, J.; Liao, S. )

    1988-10-01

    Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural Ars. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens.

  19. Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification

    PubMed Central

    Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

    2015-01-01

    We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique. PMID:26154567

  20. Snake venoms. The amino acid sequences of two proteinase inhibitor homologues from Dendroaspis angusticeps venom.

    PubMed

    Joubert, F J; Taljaard, N

    1980-05-01

    Toxins C13S1C3 and C13S2C3 from D. angusticeps venom were purified by gel filtration and ion exchange chromatography. Whereas C13S1C3 contains 57 amino acids, C13S2C3 contains 59 but each include six half-cystine residues. The complete primary structure of the low toxicity proteins have been elucidated. The sequences and the invariant residues of toxins C13S1C3 and C13S2C3 from D. angusticeps venom resemble, respectively, those of the proteinase inhibitor homologues K and I from D. polylepis polylepis venom and they are also homologous to the active proteinase inhibitors from various sources. In C13S1C3 and K the active site lysyl residue of active bovine pancreatic proteinase inhibitor is conserved but the site residue alanine, is replaced by lysine. In C13S2C3 and I the active site residue is replaced by tyrosine. PMID:7429422

  1. Irreversible and reversible topoisomerase II DNA cleavage stimulated by clerocidin: sequence specificity and structural drug determinants.

    PubMed

    Binaschi, M; Zagotto, G; Palumbo, M; Zunino, F; Farinosi, R; Capranico, G

    1997-05-01

    In contrast to other topoisomerase II poisons, the microbial terpenoid clerocidin was shown to stimulate irreversible topoisomerase II-mediated DNA cleavage. To establish the structural determinants for drug activity, in this study we have investigated intensity patterns and sequence specificity of clerocidin-stimulated DNA cleavage using 5'-end 32P-labeled DNA fragments. At a majority of the sites, clerocidin-stimulated cleavage did not revert upon NaCl addition; nevertheless, at some sites, cleavage completely reverted. Statistical analyses showed that drug-preferred bases were different in the two cases: guanine and cytosine were highly preferred at position -1 at irreversible and reversible sites, respectively. These results demonstrated that cleavage irreversibility was site selective and required a guanine at the 3' end of the cut. Further experiments revealed that some irreversible sites showed an abnormal electrophoretic mobility in sequencing gels with respect to cleaved bands generated by 4-(9-acridinylamino)methanesulfon-m-anisidide, suggesting a chemical alteration of the DNA strand. Interestingly, the ability to stimulate irreversible cleavage progressively decreased over time when clerocidin was stored in ethanol. Under these conditions, nuclear magnetic resonance measurements demonstrated that the drug underwent structural modifications that involved the C-12-C-15 side chain. Thus, the results indicate that a specific moiety of clerocidin may react with the DNA (guanine at -1) in the ternary complex, resulting in cleavage irreversibility and in altered DNA mobility in sequencing gels. PMID:9135013

  2. Multiwavelength spectrophotometric determination of acidity constants of some azo dyes.

    PubMed

    Shamsipur, Mojtaba; Maddah, Bozorgmehr; Hemmateenejad, Bahram; Rouhani, Shohreh; Haghbeen, Kamaladin; Alizadeh, Kamal

    2008-06-01

    A multiwavelength spectrophotometric titration method was applied to study the acidity constants of some azo dyes in water. The UV-vis absorption spectra of azo dye solutions were recorded in the course of their pH-metric titration with a standard base solution. The protolytic equilibrium constants, spectral profiles, concentration diagrams and also the number of components have been calculated. The quantitative effects of the substituents on the acidity of the studied azo dyes were investigated by the linear free energy relationship (LFER) using Hammet sigma constant (sigma) and field and resonance effects of Kamlet and Taft (f and Re, respectively). PMID:17719268

  3. Multiwavelength spectrophotometric determination of acidity constants of some azo dyes

    NASA Astrophysics Data System (ADS)

    Shamsipur, Mojtaba; Maddah, Bozorgmehr; Hemmateenejad, Bahram; Rouhani, Shohreh; Haghbeen, Kamaladin; Alizadeh, Kamal

    2008-06-01

    A multiwavelength spectrophotometric titration method was applied to study the acidity constants of some azo dyes in water. The UV-vis absorption spectra of azo dye solutions were recorded in the course of their pH-metric titration with a standard base solution. The protolytic equilibrium constants, spectral profiles, concentration diagrams and also the number of components have been calculated. The quantitative effects of the substituents on the acidity of the studied azo dyes were investigated by the linear free energy relationship (LFER) using Hammet sigma constant ( σ) and field and resonance effects of Kamlet and Taft ( f and ℜ, respectively).

  4. Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus Populus

    SciTech Connect

    Abraham, Paul E; Adams, Rachel M; Tuskan, Gerald A; Hettich, Robert {Bob} L

    2013-01-01

    The genetic diversity across natural populations of the model organism, Populus, is extensive, containing a single nucleotide polymorphism roughly every 200 base pairs. When deviations from the reference genome occur in coding regions, they can impact protein sequences. Rather than relying on a static reference database to profile protein expression, we employed a peptide sequence tagging (PST) approach capable of decoding the plasticity of the Populus proteome. Using shotgun proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6,653 unexpected sequence variants. Through manual validation, our study investigated how the most abundant chemical modification (methionine oxidation) could masquerade as a sequence variant (AlaSer) when few site-determining ions existed. In fact, precise localization of an oxidation site for peptides with more than one potential placement was indeterminate for 70% of the MS/MS spectra. We demonstrate that additional fragment ions made available by high energy collisional dissociation enhances the robustness of the peptide sequence tagging approach (81% of oxidation events could be exclusively localized to a methionine). We are confident that augmenting fragmentation processes for a PST approach will further improve the identification of single amino acid polymorphism in Populus and potentially other species as well.

  5. Distributions of amino acids suggest that certain residue types more effectively determine protein secondary structure.

    PubMed

    Saraswathi, S; Fernández-Martínez, J L; Koliński, A; Jernigan, R L; Kloczkowski, A

    2013-10-01

    Exponential growth in the number of available protein sequences is unmatched by the slower growth in the number of structures. As a result, the development of efficient and fast protein secondary structure prediction methods is essential for the broad comprehension of protein structures. Computational methods that can efficiently determine secondary structure can in turn facilitate protein tertiary structure prediction, since most methods rely initially on secondary structure predictions. Recently, we have developed a fast learning optimized prediction methodology (FLOPRED) for predicting protein secondary structure (Saraswathi et al. in JMM 18:4275, 2012). Data are generated by using knowledge-based potentials combined with structure information from the CATH database. A neural network-based extreme learning machine (ELM) and advanced particle swarm optimization (PSO) are used with this data to obtain better and faster convergence to more accurate secondary structure predicted results. A five-fold cross-validated testing accuracy of 83.8 % and a segment overlap (SOV) score of 78.3 % are obtained in this study. Secondary structure predictions and their accuracy are usually presented for three secondary structure elements: α-helix, β-strand and coil but rarely have the results been analyzed with respect to their constituent amino acids. In this paper, we use the results obtained with FLOPRED to provide detailed behaviors for different amino acid types in the secondary structure prediction. We investigate the influence of the composition, physico-chemical properties and position specific occurrence preferences of amino acids within secondary structure elements. In addition, we identify the correlation between these properties and prediction accuracy. The present detailed results suggest several important ways that secondary structure predictions can be improved in the future that might lead to improved protein design and engineering. PMID:23907551

  6. The delta EEG (sleep)-inducing peptide (DSIP). XI. Amino-acid analysis, sequence, synthesis and activity of the nonapeptide.

    PubMed

    Schoenenberger, G A; Maier, P F; Tobler, H J; Wilson, K; Monnier, M

    1978-09-01

    A peptide which induces slow-wave EEG (sleep) after intraventricular infusion into the brain has been isolated from the extracorporeal dialysate of cerebral venous blood in rabbits submitted to hypnogenic electrical stimulation of the intralaminar thalamic area. It was shown by amino-acid analysis and sequence determination to be Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu and named "Delta Sleep-Inducing Peptide" (DSIP). This compound was synthesized as well as 5 possible metabolic products (1--8, 2--9, 2--8, 1--4 and 5--9), 2 nonapeptide analogues (with one and two amino-acids exchanged) and a related tripeptide (Trp-Ser-Glu). All 9 synthetic peptides were infused intraventricularly in rabbits (6 nmol/kg in 0.05 ml of CSF-like solution over 3.5 min) and tested under double-blind conditions. A total of 61 rabbits including controls were used. The EEG from the frontal neocortex and the limbic archicortex were subjected to direct fast-Fourier transformation and analyzed by an 1108 computer system. A highly specific delta and spindle EEG-enhancing effect of the synthetic DSIP could be demonstrated. The mean increase of EEG delta activity reached 35% in the neocortex and limbic cortex as compared to control animals receiving CSF-like solution or any of the other 8 peptides. The final chemical characterization of the synthetic DSIP revealed that only the pure alpha-aspartyl peptide is highly active in contrast to its beta-Asp isomer. A neurohumoral modulating and programming activity was suggested. PMID:568769

  7. C-terminal peptides of rhodopsin. Determination of the optimum sequence for recognition of retinal transducin.

    PubMed Central

    Takemoto, D J; Morrison, D; Davis, L C; Takemoto, L J

    1986-01-01

    In vertebrate retinal rod outer segments, transducin, a guanine-nucleotide-binding protein, mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase. Whereas the T alpha subunit (39 kDa) of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunits (35 and 10 kDa) may function to physically link T alpha with photolysed rhodopsin. We have previously reported that a site of binding of transducin is on the C-terminus of bovine rhodopsin. By using competition with synthetic peptides, the recognition region was localized to bovine opsin amino acid residues 317-339. Further studies are detailed which determine the boundaries of this binding site on rhodopsin, as well as some of the critical amino acids needed for transducin binding. These results suggest that the serine and threonine residues in the rhodopsin C-terminal peptides Rhod-1 and Rhod-3 are critical for reconstitution of transducin GTPase activity. PMID:3461782

  8. Nucleotide and predicted amino acid sequence of a cDNA clone encoding part of human transketolase.

    PubMed

    Abedinia, M; Layfield, R; Jones, S M; Nixon, P F; Mattick, J S

    1992-03-31

    Transketolase is a key enzyme in the pentose-phosphate pathway which has been implicated in the latent human genetic disease, Wernicke-Korsakoff syndrome. Here we report the cloning and partial characterisation of the coding sequences encoding human transketolase from a human brain cDNA library. The library was screened with oligonucleotide probes based on the amino acid sequence of proteolytic fragments of the purified protein. Northern blots showed that the transketolase mRNA is approximately 2.2 kb, close to the minimum expected, of which approximately 60% was represented in the largest cDNA clone. Sequence analysis of the transketolase coding sequences reveals a number of homologies with related enzymes from other species. PMID:1567394

  9. Comparison of the Legionella pneumophila population structure as determined by sequence-based typing and whole genome sequencing

    PubMed Central

    2013-01-01

    Background Legionella pneumophila is an opportunistic pathogen of humans where the source of infection is usually from contaminated man-made water systems. When an outbreak of Legionnaires’ disease caused by L. pneumophila occurs, it is necessary to discover the source of infection. A seven allele sequence-based typing scheme (SBT) has been very successful in providing the means to attribute outbreaks of L. pneumophila to a particular source or sources. Particular sequence types described by this scheme are known to exhibit specific phenotypes. For instance some types are seen often in clinical cases but are rarely isolated from the environment and vice versa. Of those causing human disease some types are thought to be more likely to cause more severe disease. It is possible that the genetic basis for these differences are vertically inherited and associated with particular genetic lineages within the population. In order to provide a framework within which to test this hypothesis and others relating to the population biology of L. pneumophila, a set of genomes covering the known diversity of the organism is required. Results Firstly, this study describes a means to group L. pneumophila strains into pragmatic clusters, using a methodology that takes into consideration the genetic forces operating on the population. These clusters can be used as a standardised nomenclature, so those wishing to describe a group of strains can do so. Secondly, the clusters generated from the first part of the study were used to select strains rationally for whole genome sequencing (WGS). The data generated was used to compare phylogenies derived from SBT and WGS. In general the SBT sequence type (ST) accurately reflects the whole genome-based genotype. Where there are exceptions and recombination has resulted in the ST no longer reflecting the genetic lineage described by the whole genome sequence, the clustering technique employed detects these sequence types as being admixed

  10. Sequencing around 5-Hydroxyconiferyl Alcohol-Derived Units in Caffeic Acid O-Methyltransferase-Deficient Poplar Lignins1[OA

    PubMed Central

    Lu, Fachuang; Marita, Jane M.; Lapierre, Catherine; Jouanin, Lise; Morreel, Kris; Boerjan, Wout; Ralph, John

    2010-01-01

    Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its β-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure. PMID:20427467

  11. Sequencing around 5-hydroxyconiferyl alcohol-derived units in caffeic acid O-methyltransferase-deficient poplar lignins.

    PubMed

    Lu, Fachuang; Marita, Jane M; Lapierre, Catherine; Jouanin, Lise; Morreel, Kris; Boerjan, Wout; Ralph, John

    2010-06-01

    Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its beta-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure. PMID:20427467

  12. Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion.

    PubMed Central

    Rubens, C; Heffron, F; Falkow, S

    1976-01-01

    Insertion of the transposable deoxyribonucleic acid sequence that specifies the TEM beta-lactamase (TnA) occurred in at least 19 sites on the 5.5 x 10(6)-dalton plasmid RSF1010. There was no significant difference in the frequency of transposition or in the distribution of TnA insertion sites for recombinant plasmids isolated from recombination-proficient (rec+) or recombination-deficient (rec-) bacterial host cells. The site and orientation of TnA insertions were determined by both heteroduplex analysis and enzymatic digestion with restriction endonucleases. Insertion in the gene encoding for sulfonamide resistance occurred without circular permutation in one or the other of two distinct orientations. Insertions in orientation P were strongly polar on distal gene expression, whereas insertions in orientation M were mutagenic but not polar. In addition, we have observed that TnA elements from different R plasmids show fine structural heterogeneity, and that TnA insertion at a site adjacent to the origin of replication causes an increase in plasmid copy number. Images PMID:789346

  13. The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes.

    PubMed

    Fushitani, K; Higashiyama, K; Moriyama, E N; Imai, K; Hosokawa, K

    1996-09-01

    To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds. PMID:8752011

  14. Determination of volatile fatty acids in landfill leachates by ion-exclusion chromatography.

    PubMed

    Yamamoto, Atsushi; Yasuhara, Akio; Kodama, Shuji; Matsunaga, Akinobu; Suzuki, Shigeru; Mohri, Shino; Yamada, Masato

    2004-03-01

    An ion-exclusion chromatographic method with on-line desalinization for the determination of volatile fatty acids in landfill leachates is described. Highly sensitive conductivity detection of the organic acids was achieved by using dilute p-hydroxybenzoic acid solution as an eluent. Interference with mineral acids was reduced by treatment with barium chloride solution prior to desalinization. A silver-loaded cation-exchange guard column for the desalinization was installed in series with the analytical column to avoid the contamination of organic acids. This method features detection limits of 0.01 mg L(-1) formic acid, 0.02 mg L(-1) acetic acid, 0.05 mg L(-1) propionic acid, and 0.1 mg L(-1) butyric acid, respectively, with an injection of 20 microL sample. Application of the on-line desalinization LC method is illustrated for leachate samples from a Japanese sanitary landfill. PMID:15334921

  15. Determination of proton affinities and acidity constants of sugars.

    PubMed

    Feng, Shuting; Bagia, Christina; Mpourmpakis, Giannis

    2013-06-20

    Proton transfer reactions play a key role in the conversion of biomass derived sugars to chemicals. In this study, we employ high level ab initio theoretical methods, in tandem with solvation effects to calculate the proton affinities (PA) and acidity constants (pKa) of various d-glucose and d-fructose tautomers (protonation-deprotonation processes). In addition, we compare the theoretically derived pH values of sugar solutions against experimentally measured pH values in our lab. Our results demonstrate that the protonation of any of the O atoms of the sugars is thermodynamically preferred without any significant variation in the PA values. Intramolecular hydrogen transfers, dehydration reactions, and ring-opening processes were observed, resulting from the protonation of specific hydroxyl groups on the sugars. Regarding the deprotonation processes (pKa), we found that the sugars' anomeric hydroxyls exhibit the highest acidity. The theoretically calculated pH values of sugar solutions are in excellent agreement with experimental pH measurements at low sugar concentrations. At higher sugar concentrations the calculations predict less acidic solutions than the experiments. In this case, we expect the sugars to act as solvents increasing the proton solvation energy and the acidity of the solutions. We demonstrated through linear relationships that the pKa values are correlated with the relative stability of the conjugate bases. The latter is related to hydrogen bonding and polarization of the C-O(-) bond. A plausible explanation for the good performance of the direct method in calculating the pKa values of sugars can be the presence of intramolecular hydrogen bonds on the conjugate base. Both theory and experiments manifest that fructose is a stronger acid than glucose, which is of significant importance in self-catalyzed biomass-relevant dehydration reactions. PMID:23706015

  16. 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

  17. 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

  18. Overlapping activator sequences determined for two oppositely oriented promoters in halophilic Archaea

    PubMed Central

    Bauer, Martina; Marschaus, Larissa; Reuff, Muriel; Besche, Verena; Sartorius-Neef, Simone; Pfeifer, Felicitas

    2008-01-01

    Transcription of the genomic region involved in gas vesicle formation in Halobacterium salinarum (p-vac) and Haloferax mediterranei (mc-vac) is driven by two divergent promoters, PA and PD, separated by only 35 nt. Both promoters are activated by the transcription activator GvpE which in the case of PmcA requires a 20-nt sequence (UAS) consisting of two conserved 8-nt sequence portions located upstream of BRE. Here, we determined the two UAS elements in the promoter region of p-vac by scanning mutageneses using constructs containing PpD (without PpA) fused to the bgaH reporter gene encoding an enzyme with β-galactosidase activity, or the dual reporter construct pApD with PpD fused to bgaH and PpA to an altered version of gvpA. The two UAS elements found exhibited a similar extension and distance to BRE as previously determined for the UAS in PmcA. Their distal 8-nt portions almost completely overlapped in the centre of PpD–PpA, and mutations in this region negatively affected the GvpE-mediated activation of both promoters. Any alteration of the distance between BRE and UAS resulted in the loss of the GvpE activation, as did a complete substitution of the proximal 8-nt portion, underlining that a close location of UAS and BRE was very important. PMID:18056077

  19. Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

    PubMed Central

    Foster, Patricia L.; Lee, Heewook; Popodi, Ellen; Townes, Jesse P.; Tang, Haixu

    2015-01-01

    A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10−3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold. PMID:26460006

  20. Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system.

    PubMed Central

    Hoshino, T; Kose, K

    1990-01-01

    A DNA fragment of Pseudomonas aeruginosa PAO containing genes specifying the high-affinity branched-chain amino acid transport system (LIV-I) was isolated. The fragment contained the braC gene, encoding the binding protein for branched-chain amino acids, and the 4-kilobase DNA segment adjacent to 3' of braC. The nucleotide sequence of the 4-kilobase DNA fragment was determined and found to contain four open reading frames, designated braD, braE, braF, and braG. The braD and braE genes specify very hydrophobic proteins of 307 and 417 amino acid residues, respectively. The braD gene product showed extensive homology (67% identical) to the livH gene product, a component required for the Escherichia coli high-affinity branched-chain amino acid transport systems. The braF and braG genes encode proteins of 255 and 233 amino acids, respectively, both containing amino acid sequences typical of proteins with ATP-binding sites. By using a T7 RNA polymerase/promoter system together with plasmids having various deletions in the braDEFG region, the braD, braE, braF, and braG gene products were identified as proteins with apparent Mrs of 25,500, 34,000, 30,000, and 27,000, respectively. These proteins were found among cell membrane proteins on a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie blue. Images PMID:2120183

  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. Molecular cloning and expression of partial cDNAs and deduced amino acid sequence of a carboxyl-terminal fragment of human apolipoprotein B-100.

    PubMed Central

    Wei, C F; Chen, S H; Yang, C Y; Marcel, Y L; Milne, R W; Li, W H; Sparrow, J T; Gotto, A M; Chan, L

    1985-01-01

    Apolipoprotein (apo) B-100 cDNAs were identified in a human liver cDNA library cloned in the expression vector lambda gt11. The beta-galactosidase-apoB-100 fusion protein was detected by two independently produced low density lipoprotein polyclonal antisera and by three apoB-100 monoclonal antibodies that crossreact with apoB-74. It was not recognized by two apoB-100 monoclonal antibodies that crossreact with apoB-26. The longest clone, lambda B8, was completely sequenced. It contains a 2.8-kilobase DNA fragment containing the codons for the carboxyl-terminal 836 amino acid residues of apo-B-100, as well as the 3' untranslated region of apoB-100 mRNA. We have thus mapped apoB-74 to the carboxyl-terminal portion of apoB-100. The deduced amino acid sequence of the cloned DNA matches the sequences of 14 apoB-100 peptides determined in our laboratory. Minor differences in amino acid sequence were noted in three of the peptides, suggesting polymorphism of apoB-100 at the protein and DNA levels. Secondary structure predictions reveal an unusual pattern for apolipoproteins, consisting of beta-structure (24%), alpha-helical content (33%), and random structure (30%). Ten amphipathic helical regions of 10-24 residues were identified. This carboxyl-terminal fragment of apoB-100 is considerably more hydrophobic than other apolipoproteins with known structure. Its lipid binding regions might include stretches of highly hydrophobic beta-sheets as well as amphipathic helices. Our findings on apoB structure might be important for understanding the role of apoB-100-containing lipoproteins in atherosclerosis. PMID:2932736

  3. DETERMINATION OF CARBOXYLIC ACIDS BY ION-EXCLUSION CHROMATOGRAPHY WITH NON-SUPPRESSED CONDUCTIVITY AND OPTICAL DETECTORS

    EPA Science Inventory

    Determination of carboxylic acids using non-suppressed conductivity and UV detections is described. The background conductance of 1-octanesulfonic acid, hexane sulfonic acid and sulfuric acid at varying concentrations was determined. Using 0.2 mM 1-octanesulfonic acid as a mobile...

  4. DETERMINATION OF CARBOXYLIC ACIDS BY ION-EXCLUSION CHROMATOGRAPHY WITH NON-SUPPRESSED CONDUCTIVITY AND OPTICAL DETECTORS

    EPA Science Inventory

    Determination of carboxylic acids using non-suppressed conductivity and UV detections is described. he background conductance of I-octanesulfonic acid, hexane sulfonic acid and sulfuric acid at varying concentrations was determined. sing 0.2 MM I-octanesulfonic acid as a mobile p...

  5. Probing the Specificity Determinants of Amino Acid Recognition by Arginase

    SciTech Connect

    Shishova, E.; Di Costanzo, L; Emig, F; Ash, D; Christianson, D

    2009-01-01

    Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. In order to better understand the molecular basis of inhibitor affinity, we have employed site-directed mutagenesis, enzyme kinetics, and X-ray crystallography to probe the molecular recognition of the amino acid moiety (i.e., the ?-amino and ?-carboxylate groups) of substrate l-arginine and inhibitors in the active site of arginase I. Specifically, we focus on (1) a water-mediated hydrogen bond between the substrate ?-carboxylate and T135, (2) a direct hydrogen bond between the substrate ?-carboxylate and N130, and (3) a direct charged hydrogen bond between the substrate ?-amino group and D183. Amino acid substitutions for T135, N130, and D183 generally compromise substrate affinity as reflected by increased KM values but have less pronounced effects on catalytic function as reflected by minimal variations of kcat. As with substrate KM values, inhibitor Kd values increase for binding to enzyme mutants and suggest that the relative contribution of intermolecular interactions to amino acid affinity in the arginase active site is water-mediated hydrogen bond < direct hydrogen bond < direct charged hydrogen bond. Structural comparisons of arginase with the related binuclear manganese metalloenzymes agmatinase and proclavaminic acid amidinohydrolase suggest that the evolution of substrate recognition in the arginase fold occurs by mutation of residues contained in specificity loops flanking the mouth of the active site (especially loops 4 and 5), thereby allowing diverse guanidinium substrates to be accommodated for catalysis.

  6. Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

    PubMed Central

    Mann, K; Deutzmann, R; Aumailley, M; Timpl, R; Raimondi, L; Yamada, Y; Pan, T C; Conway, D; Chu, M L

    1989-01-01

    The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28-residue signal peptide. The data support a previously proposed dumb-bell model of nidogen by demonstrating a large N-terminal globular domain (641 residues), five EGF-like repeats constituting the rod-like domain (248 residues) and a smaller C-terminal globule (328 residues). Two more EGF-like repeats interrupt the N-terminal and terminate the C-terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta-hydroxylation, two N-linked carbohydrate acceptor sites and, within one of the EGF-like repeats an Arg-Gly-Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C-terminal globule but not yet precisely localized. Images PMID:2496973

  7. Jack bean α-mannosidase: amino acid sequencing and N-glycosylation analysis of a valuable glycomics tool.

    PubMed

    Gnanesh Kumar, B S; Pohlentz, Gottfried; Schulte, Mona; Mormann, Michael; Siva Kumar, Nadimpalli

    2014-03-01

    Jack bean (Canavalia ensiformis) seeds contain several biologically important proteins among which α-mannosidase (EC 3.2.1.24) has been purified, its biochemical properties studied and widely used in glycan analysis. In the present study, we have used the purified enzyme and derived its amino acid sequence covering both the known subunits (molecular mass of ∼66,000 and ∼44,000 Da) hitherto not known in its entirety. Peptide de novo sequencing and structural elucidation of N-glycopeptides obtained either directly from proteolytic digestion or after zwitterionic hydrophilic interaction liquid chromatography solid phase extraction-based separation were performed by use of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry and low-energy collision-induced dissociation experiments. De novo sequencing provided new insights into the disulfide linkage organization, intersection of subunits and complete N-glycan structures along with site specificities. The primary sequence suggests that the enzyme belongs to glycosyl hydrolase family 38 and the N-glycan sequence analysis revealed high-mannose oligosaccharides, which were found to be heterogeneous with varying number of hexoses viz, Man8-9GlcNAc2 and Glc1Man9GlcNAc2 in an evolutionarily conserved N-glycosylation site. This site with two proximal cysteines is present in all the acidic α-mannosidases reported so far in eukaryotes. Further, a truncated paucimannose type was identified to be lacking terminal two mannose, Man1(Xyl)GlcNAc2 (Fuc). PMID:24295789

  8. A Short Sequence within Two Purine-Rich Enhancers Determines 5′ Splice Site Specificity

    PubMed Central

    Elrick, Leslie L.; Humphrey, Mary Beth; Cooper, Thomas A.; Berget, Susan M.

    1998-01-01

    Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3′ splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5′ splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5′ splice site choice when placed between competing 5′ splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5′ splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5′ splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5′ splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3′ end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5′ splice site selectivity was different than that shown to be maximally important for activation of a 3′ splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in

  9. Determination of perfluoroalkyl carboxylic, sulfonic, and phosphonic acids in food.

    PubMed

    Ullah, Shahid; Alsberg, Tomas; Vestergren, Robin; Berger, Urs

    2012-11-01

    A sensitive and accurate method was developed and validated for simultaneous analysis of perfluoroalkyl carboxylic acids, sulfonic acids, and phosphonic acids (PFPAs) at low picograms per gram concentrations in a variety of food matrices. The method employed extraction with acetonitrile/water and cleanup on a mixed-mode co-polymeric sorbent (C8 + quaternary amine) using solid-phase extraction. High-performance liquid chromatographic separation was achieved on a C18 column using a mobile phase gradient containing 5 mM 1-methyl piperidine for optimal chromatographic resolution of PFPAs. A quadrupole time-of-flight high-resolution mass spectrometer operating in negative ion mode was used as detector. Method detection limits were in the range of 0.002 to 0.02 ng g(-1) for all analytes. Sample preparation (extraction and cleanup) recoveries at a spiking level of 0.1 ng g(-1) to a baby food composite were in the range of 59 to 98 %. A strong matrix effect was observed in the analysis of PFPAs in food extracts, which was tentatively assigned to sorption of PFPAs to the injection vial in the solvent-based calibration standard. The method was successfully applied to a range of different food matrices including duplicate diet samples, vegetables, meat, and fish samples. PMID:22955674

  10. Complete amino acid sequence of the lentil trypsin-chymotrypsin inhibitor LCI-1.7 and a discussion of atypical binding sites of Bowman-Birk inhibitors.

    PubMed

    Weder, Jürgen K P; Hinkers, Sabine C

    2004-06-30

    The complete primary structure of the lentil (Lens culinaris) trypsin-chymotrypsin inhibitor LCI-1.7 was determined by conventional methods in order to find relationships between partial sequences and the difference in action against human and bovine chymotrypsin. As other Bowman-Birk type inhibitors, LCI-1.7 contained 68 amino acid residues, seven disulfide bridges, and two reactive sites, Arg16-Ser17 for trypsin and Tyr42-Ser43 for chymotrypsin. Evaluation of sequence homologies showed that it belonged to the group III Bowman-Birk inhibitors. The atypical additional binding site of LCI-1.7 for human chymotrypsin was discussed and compared with such binding sites of two other Bowman-Birk inhibitors, the Bowman-Birk soybean proteinase inhibitor BBI, and the lima bean proteinase inhibitor LBI I, for human and bovine trypsin and chymotrypsin. A concept to reduce the action of these inhibitors against human enzymes by genetic engineering was proposed. PMID:15212472

  11. Analysis of the complete sequences of two biologically distinct Zucchini yellow mosaic virus isolates further evidences the involvement of a single amino acid in the virus pathogenicity.

    PubMed

    Nováková, S; Svoboda, J; Glasa, M

    2014-01-01

    The complete genome sequences of two Slovak Zucchini yellow mosaic virus isolates (ZYMV-H and ZYMV-SE04T) were determined. These isolates differ significantly in their pathogenicity, producing either severe or very mild symptoms on susceptible cucurbit hosts. The viral genome of both isolates consisted of 9593 nucleotides in size, and contained an open reading frame encoding a single polyprotein of 3080 amino acids. Despite their different biological properties, an extremely high nucleotide identity could be noted (99.8%), resulting in differences of only 5 aa, located in the HC-Pro, P3, and NIb, respectively. In silico analysis including 5 additional fully-sequenced and phylogenetically closely-related isolates known to induce different symptoms in cucurbits was performed. This suggested that the key single mutation responsible for virus pathogenicity is likely located in the N-terminal part of P3, adjacent to the PIPO. PMID:25518719

  12. Combining sequence-based prediction methods and circular dichroism and infrared spectroscopic data to improve protein secondary structure determinations

    PubMed Central

    Lees, Jonathan G; Janes, Robert W

    2008-01-01

    Background A number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available. Results In this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements. Conclusion Combining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained. PMID:18197968

  13. The amino-acid sequence of the glucose/mannose-specific lectin isolated from Parkia platycephala seeds reveals three tandemly arranged jacalin-related domains.

    PubMed

    Mann, K; Farias, C M; Del Sol, F G; Santos, C F; Grangeiro, T B; Nagano, C S; Cavada, B S; Calvete, J J

    2001-08-01

    A mannose/glucose-specific lectin was isolated from seeds of Parkia platycephala, the most primitive subfamily of Leguminosae plants. The molecular mass of the purified lectin determined by mass spectrometry was 47 946 +/- 6 Da (by electrospray ionization) and 47 951 +/- 9 Da (by matrix-assisted laser-desoption ionization). The apparent molecular mass of the lectin in solutions of pH in the range 4.5-8.5 determined by analytical ultracentrifugation equilibrium sedimentation was 94 +/- 3 kDa, showing that the protein behaved as a non-pH-dependent dimer. The amino-acid sequence of the Parkia lectin was determined by Edman degradation of overlapping peptides. This is the first report of the primary structure of a Mimosoideae lectin. The protein contained a blocked N-terminus and a single, nonglycosylated polypeptide chain composed of three tandemly arranged homologous domains. Each of these domains shares sequence similarity with jacalin-related lectin monomers from Asteraceae, Convolvulaceae, Moraceae, Musaceae, Gramineae, and Fagaceae plant families. Based on this homology, we predict that each Parkia lectin repeat may display a beta prism fold similar to that observed in the crystal structure of the lectin from Helianthus tuberosus. The P. platycephala lectin also shows sequence similarity with stress- and pathogen-upregulated defence genes of a number of different plants, suggesting a common ancestry for jacalin-related lectins and inducible defence proteins. PMID:11502201

  14. Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-IIIB4

    PubMed Central

    Swart, L. S.; Haylett, T.

    1971-01-01

    The complete amino acid sequence of protein SCMKB-IIIB4 is presented. It is closely related to the sequence of protein SCMKB-IIIB3 (Haylett, Swart & Parris, 1971) differing in only four positions. The peptic and thermolysin peptides of protein SCMKB-IIIB4 were analysed by the dansyl–Edman method (Gray, 1967) and by tritium-labelling of C-terminal residues (Matsuo, Fujimoto & Tatsuno, 1966). This protein is the third member of a group of high-sulphur wool proteins with molecular weight of about 11400. It consists of 98 residues and has acetylalanine and carboxymethylcysteine as N- and C-terminal residues respectively. PMID:4942536

  15. Creation and structure determination of an artificial protein with three complete sequence repeats.

    PubMed

    Adachi, Motoyasu; Shimizu, Rumi; Kuroki, Ryota; Blaber, Michael

    2013-11-01

    Symfoil-4P is a de novo protein exhibiting the threefold symmetrical β-trefoil fold designed based on the human acidic fibroblast growth factor. First three asparagine-glycine sequences of Symfoil-4P are replaced with glutamine-glycine (Symfoil-QG) or serine-glycine (Symfoil-SG) sequences protecting from deamidation, and His-Symfoil-II was prepared by introducing a protease digestion site into Symfoil-QG so that Symfoil-II has three complete repeats after removal of the N-terminal histidine tag. The Symfoil-QG and SG and His-Symfoil-II proteins were expressed in Eschericha coli as soluble protein, and purified by nickel affinity chromatography. Symfoil-II was further purified by anion-exchange chromatography after removing the HisTag by proteolysis. Both Symfoil-QG and Symfoil-II were crystallized in 0.1 M Tris-HCl buffer (pH 7.0) containing 1.8 M ammonium sulfate as precipitant at 293 K; several crystal forms were observed for Symfoil-QG and II. The maximum diffraction of Symfoil-QG and II crystals were 1.5 and 1.1 Å resolution, respectively. The Symfoil-II without histidine tag diffracted better than Symfoil-QG with N-terminal histidine tag. Although the crystal packing of Symfoil-II is slightly different from Symfoil-QG and other crystals of Symfoil derivatives having the N-terminal histidine tag, the refined crystal structure of Symfoil-II showed pseudo-threefold symmetry as expected from other Symfoils. Since the removal of the unstructured N-terminal histidine tag did not affect the threefold structure of Symfoil, the improvement of diffraction quality of Symfoil-II may be caused by molecular characteristics of Symfoil-II such as molecular stability. PMID:24121347

  16. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

    PubMed Central

    Patel, Chandra N.; Koh, David W.; Jacobson, Myron K.; Oliveira, Marcos A.

    2005-01-01

    PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG. PMID:15658938

  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. Clofibrate-induced cytochrome P450-lauric acid omega hydroxylase(P450LA omega):purification, cDNA cloning, sequence and regulation

    SciTech Connect

    Hardwick, J.P.; Song, B.J.; Gonzalez, F.J.

    1986-05-01

    A cytochrome P450 that hydroxylates lauric acid at the 12 position (P450LA omega) was isolated from liver microsomes of clofibrate treated rats. P450LA omega was immunologically distinct from P450s a,b,c,d,e,f,g,h,j,PB1, and PCN1. Polyclonal antibody against P450LA omega was utilized to screen a gt11 cDNA library. A clone (pP450LA omega), was isolated and its sequence determined. The P450LA omega mRNA is a minimum 2387 nts in length and codes for a P450 of Mr.58,222 daltons. This protein shares less than 35% amino acid similarity with P450s b,c,d,e,f,PB1, and PCN1; however, it does contain a hydrophobic amino terminal peptide and a conserved sequence surrounding the Cys residue at position 456, which is similar to other microsomal P450s. P450LA omega is present at high levels in untreated rat kidney and is induced by clofibrate in both kidney and liver. This induction is the result of an accumulation of mRNA through a rapid transcriptional activation of the P450LA gene. Southern blotting data suggest the presence of 2 or 3 genes in the P450LA omega family. This P450 gene family may be associated with arachidonic acid and prostraglandin metabolism in kidney and other tissues.

  19. Unifying bacteria from decaying wood with various ubiquitous Gibbsiella species as G. acetica sp. nov. based on nucleotide sequence similarities and their acetic acid secretion.

    PubMed

    Geider, Klaus; Gernold, Marina; Jock, Susanne; Wensing, Annette; Völksch, Beate; Gross, Jürgen; Spiteller, Dieter

    2015-12-01

    Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37 °C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov. PMID:26071988

  20. Sequence determination of the heavy-chain constant region in four immunoglobulin classes of Mongolian gerbils (Meriones unguiculatus).

    PubMed

    Ukaji, Takao; Sumiyama, Daisuke; Kai, Osamu

    2012-01-01

    We determined partial cDNA sequences of four immunoglobulin (Ig) classes-IgM, IgG1, IgE, and IgA-of Mongolian gerbil (Meriones unguiculatus). Each deduced Ig heavy-chain constant (IGHC) region-Cµ, Cγ1, Cε, and Cα-is structurally similar to its counterparts in the mouse and rat, and phylogenetic analysis suggests that the gerbil Igs are evolutionarily close to their counterparts. In spite of the high sequence homology to the other rodent Cγ sequences, the gerbil Cγ1 sequence differs from our previously reported Cγ2. This result indicates that the gerbil has at least two IgG subclasses. These four gerbil IGHC cDNA sequences will be useful for determining gerbil Ig isotypes and examining the expression of gerbil Ig mRNAs in response to parasitic and bacterial infections. PMID:22531724

  1. Quality control of herbs: determination of amino acids in Althaea officinalis, Matricaria chamomilla and Taraxacum officinale.

    PubMed

    Qureshi, Muhammad Nasimullah; Stecher, Guenther; Bonn, Guenther Karl

    2014-05-01

    Analysis of raw materials and final products need reliable methods for the standardization of natural product drugs. Legal guideline also emphasizes on the qualitative and quantitative analyses of the plant constituents in an herbal product. In this study, thin layer chromatography (TLC) and amino acid analyzer was used for the determination of amino acids in plant extracts. Samples for this study were standards and aqueous extracts from Althaea officinalis, Matricaria chamomilla and Taraxacum officinale. Different amino acids in the extracts were detected through TLC. An automatic amino acid analyzer was used for the quantification of amino acids in the plant extracts under study. PMID:24811801

  2. The sequence of sequencers: The history of sequencing DNA

    PubMed Central

    Heather, James M.; Chain, Benjamin

    2016-01-01

    Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way. PMID:26554401

  3. Whole-Exome Sequencing Enables Rapid Determination of Xeroderma Pigmentosum Molecular Etiology

    PubMed Central

    Ortega-Recalde, Oscar; Vergara, Jéssica Inés; Fonseca, Dora Janeth; Ríos, Xiomara; Mosquera, Hernando; Bermúdez, Olga María; Medina, Claudia Liliana; Vargas, Clara Inés; Pallares, Argemiro Enrique; Restrepo, Carlos Martín; Laissue, Paul

    2013-01-01

    Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by extreme sensitivity to actinic pigmentation changes in the skin and increased incidence of skin cancer. In some cases, patients are affected by neurological alterations. XP is caused by mutations in 8 distinct genes (XPA through XPG and XPV). The XP-V (variant) subtype of the disease results from mutations in a gene (XPV, also named POLH) which encodes for Polη, a member of the Y-DNA polymerase family. Although the presence and severity of skin and neurological dysfunctions differ between XP subtypes, there are overlapping clinical features among subtypes such that the sub-type cannot be deduced from the clinical features. In this study, in order to overcome this drawback, we undertook whole-exome sequencing in two XP sibs and their father. We identified a novel homozygous nonsense mutation (c.897T>G, p.Y299X) in POLH which causes the disease. Our results demonstrate that next generation sequencing is a powerful approach to rapid determination of XP genetic etiology. PMID:23755135

  4. [Determination of 16S rRNA gene sequence for a new ANAMMOX bacterial species].

    PubMed

    Zu, Bo; Zhang, Dai-jun; Yan, Qing

    2008-02-01

    The anaerobic ammonium oxidation (ANAMMOX) activity of the sludge was about 9.84 x 10(-4) mg x (mg x h)(-1) by measuring the simultaneous consumption of ammonium and nitrite under anoxic conditions in the batch tests. The consumption of NO2(-) -N and NH4+ -N was 1.311 for ANAMMOX bacteria. The partial 16S rDNA sequence was obtained by using molecule biology methods. Crude DNA of the total bacteria in granular sludge from EGSB reactor was extracted and purified. Then, PCR amplification by using specific primer, clone and sequence determination was performed. ANAMMOX bacterial species(anaerobic ammonium-oxidizing Planctomycete cquenviron-1) which was enrichment cultivated from EGSB reactor were the same genera with Candidatus "Anammoxoglobus propionicus" and Candidatus "Jettenia asiatica" by analyzing phylogenetic tree. The maximum identities of anaerobic ammonium-oxidizing Planctomycete cquenviron-1 with other ANAMMOX bacterial species was about 93%. The results showed that a new ANAMMOX bacterial species which was enrichment cultivated from EGSB reactor was found and anaerobic ammonium-oxidizing Planctomycete cquenviron-1 was denominated. PMID:18613522

  5. Identifying a base in a nucleic acid

    DOEpatents

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

    2005-02-08

    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.

  6. Determination of phenolic acids in olive oil by capillary electrophoresis.

    PubMed

    Buiarelli, Francesca; Di Berardino, Sonia; Coccioli, Franco; Jasionowska, Renata; Russo, Mario Vincenzo

    2004-01-01

    A CZE method for the separation and quantitation of phenolic acids (cinnamic, syringic, p-coumaric, vanillic, caffeic, 3,4-dihydroxyphenylacetic, protocatechuic), extracted from extra virgin olive oil, was developed. The sample preparation involved the LLE and SPE extraction methods. CE separation was performed in a fused silica capillary of I.D.= 50microm using as a BGE 40 mM borate buffer at pH=9.2. The separation voltage was 18kV with corresponding current of 27-28 microA. Detection was accomplished with UV-detector at lambda=200nm. The proposed method was fully validated. A good repeatability of migration time (RSD% ranged from 0.81 to 1.63) and of corrected peak area (RSD% from 2.89 to 5.77) was obtained. The linearity of detector response in the range from 5 to 50 ppm was checked, obtaining the correlation coefficient R2 values in the range: 0.9919-0.9997. Some phenolic acids in real oil samples were detected and quantified with the proposed method. PMID:15506620

  7. Determining the Δ47 acid fractionation in dolomites

    NASA Astrophysics Data System (ADS)

    Murray, Sean T.; Arienzo, Monica M.; Swart, Peter K.

    2016-02-01

    This paper reports the fractionation of Δ47 during the digestion of dolomite in phosphoric acid between 25 °C and 90 °C using five different samples, including three of Pliocene age from the Bahamas, one from the Jurassic in the Middle East, and one obtained from the National Institute of Standards (NIST 88b). The composition of the dolomites analyzed varied from Ca0.56Mg0.44CO3 to Ca0.50Mg0.50CO3. Fractionation values were also compared between the common acid bath and sealed vessel techniques at various temperatures. No statistically significant differences were observed either between these two methods or as a function of the dolomite's stoichiometry. These data produce a difference in fractionation of 0.153 ± 0.011‰ for dolomite samples digested at 90 °C compared to those reacted at 25 °C, a value higher and statistically different to previously published values. Utilization of this value in a study of dolomites from a core drilled on the island of San Salvador in the Bahamas yielded temperatures and δ18Ofluid values which agree with previous interpretations on the formation of these dolomites. Application of this value to other published studies produces lower estimates of temperature and δ18Ofluid values for the dolomitization process that are more consistent with the geologic models suggested for these studies.

  8. High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element.

    PubMed

    Roy, Subhadeep; Tanious, Farial A; Wilson, W David; Ly, Danith H; Armitage, Bruce A

    2007-09-18

    Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed. PMID:17718513

  9. 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

  10. Ferredoxin:NADP oxidoreductase of Cyanophora paradoxa: purification, partial characterization, and N-terminal amino acid sequence.

    PubMed

    Gebhart, U B; Maier, T L; Stevanović, S; Bayer, M G; Schenk, H E

    1992-06-01

    The ferredoxin:NADP+ oxidoreductase of the protist Cyanophora paradoxa, as a descendant of a former symbiotic consortium, an important model organism in view of the Endosymbiosis Theory, is the first enzyme purified from a formerly original endocytobiont (cyanelle) that is found to be encoded in the nucleus of the host. This cyanoplast enzyme was isolated by FPLC (19% yield) and characterized with respect to the uv-vis spectrum, pH optimum (pH 9), molecular mass of 34 kDa, and an N-terminal amino acid sequence (24 residues). The enzyme shows, as known from other organisms, molecular heterogeneity. The N-terminus of a further ferredoxin:NADP+ oxidoreductase polypeptide represents a shorter sequence missing the first four amino acids of the mature enzyme. PMID:1392619

  11. Purification, characterization, and amino acid sequencing of a. delta. /sup 5/-3-oxosteroid isomerase from Pseudomonas putida biotype B

    SciTech Connect

    Linden, K.G.

    1986-01-01

    Studies were performed on the ..delta../sup 5/-3-oxosteroid isomerase from Pseudomonas putida biotype B. The studies have involved three broad areas: improvement in the purification of the enzyme, further characterization of the purified enzyme, and completion of the amino acid sequence of the enzyme. For the purification of the enzyme, techniques for removing the isomerase from whole cells were studied, the effects of ionic strength on the binding of the isomerase to steroidal affinity resins was explored, and a new affinity resin was developed. Absorption spectra and the proton NMR spectra of the isomerase were obtained. Amino acid sequencing of the oxosteroid isomerase indicates that the enzyme is a dimeric protein consisting of two identical subunits each consisting of a polypeptide chain of 131 residues and a M/sub r/ = 14,536.

  12. 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 ...

  13. Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing.

    PubMed Central

    Patarca, R; Dorta, B; Ramirez, J L

    1982-01-01

    As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed. PMID:6278402

  14. Snake venoms. The amino-acid sequence of trypsin inhibitor E of Dendroaspis polylepis polylepis (Black Mamba) venom.

    PubMed

    Joubert, F J; Strydom, D J

    1978-06-01

    Trypsin inhibitor E from black mamba venom comprises 59 amino acid residues in a single polypeptide chain, cross-linked by three intrachain disulphide bridges. The complete primary structure of inhibitor E was elucidated. The sequence is homologous with trypsin inhibitors from different sources. Unique among this homologous series of proteinase inhibitors, inhibitor E has an affinity for transition metal ions, exemplified here by Cu2 and Co2+. PMID:668688

  15. Draft Genome Sequence of Escherichia coli Strain VKPM B-10182, Producing the Enzyme for Synthesis of Cephalosporin Acids

    PubMed Central

    Mardanov, Andrey V.; Eldarov, Mikhail A.; Sklyarenko, Anna V.; Dumina, Maria V.; Beletsky, Alexey V.; Yarotsky, Sergey V.

    2014-01-01

    Escherichia coli strain VKPM B-10182, obtained by chemical mutagenesis from E. coli strain ATCC 9637, produces cephalosporin acid synthetase employed in the synthesis of β-lactam antibiotics, such as cefazolin. The draft genome sequence of strain VKPM B-10182 revealed 32 indels and 1,780 point mutations that might account for the improvement in antibiotic synthesis that we observed. PMID:25414512

  16. Determination of Critical Experiment Correlations Using the Sampler Sequence Within SCALE 6.2

    SciTech Connect

    Marshall, William BJ J; Rearden, Bradley T

    2015-01-01

    The validation of neutron transport methods used in nuclear criticality safety analyses is required by consensus American National Standards Institute/American Nuclear Society (ANSI/ANS) standards. In the last decade, there has been an increased interest in correlations among critical experiments used in validation that have shared physical attributes and which impact the independence of each measurement. The statistical methods included in many of the frequently cited guidance documents on performing validation calculations incorporate the assumption that all individual measurements are independent, so little guidance is available to practitioners on the topic. Typical guidance includes recommendations to select experiments from multiple facilities and experiment series in an attempt to minimize the impact of correlations or common-cause errors in experiments. Recent efforts have been made both to determine the magnitude of such correlations between experiments and to develop and apply methods for adjusting the bias and bias uncertainty to account for the correlations. This paper describes recent work performed at Oak Ridge National Laboratory using the Sampler sequence from the SCALE code system to develop experimental correlations using a Monte Carlo sampling technique. Sampler will be available for the first time with the release of SCALE 6.2, and a brief introduction to the methods used to calculate experiment correlations within this new sequence is presented in this paper. Techniques to utilize these correlations in the establishment of upper subcritical limits are the subject of a companion paper and will not be discussed here. Example experimental uncertainties and correlation coefficients are presented for a variety of low-enriched uranium water-moderated lattice experiments selected for use in a benchmark exercise by the Working Party on Nuclear Criticality Safety Subgroup on Uncertainty Analysis in Criticality Safety Analyses. The results include

  17. The determination of ascorbic acid and uric acid in human seminal plasma using an HPLC with UV detection.

    PubMed

    Kanďár, Roman; Drábková, Petra; Hampl, Radek

    2011-09-15

    Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p>0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma. PMID:21871848

  18. Determination of free and total phenolic acids in plant-derived foods by HPLC with diode-array detection.

    PubMed

    Mattila, Pirjo; Kumpulainen, Jorma

    2002-06-19

    A high-performance liquid chromatographic (HPLC) method with diode-array detection (DAD) was used to identify and quantify free and total phenolic acids (m-hydroxybenzoic acid, p-hydroxybenzoic acid, protocatechuic acid, gallic acid, vanillic acid, syringic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, chlorogenic acid, and ellagic acid) in plant foods. Free phenolic acids were extracted with a mixture of methanol and 10% acetic acid. Bound phenolic acids were liberated using first alkaline and then acid hydrolysis followed by extraction with diethyl ether/ethyl acetate (1:1). All fractions were quantified separately by HPLC. After HPLC quantification, results of alkali and acid hydrolysates were calculated to represent total phenolic acids. Ellagic acid was quantified separately after long (20 h) acid hydrolysis. The methods developed were effective for the determination of phenolic acids in plant foods. DAD response was linear for all phenolic acids within the ranges evaluated, with correlation coefficients exceeding 0.999. Coefficients of variation for 4-8 sample replicates were consistently below 10%. Recovery tests of phenolic acids were performed for every hydrolysis condition using several samples. Recoveries were generally good (mean >90%) with the exceptions of gallic acid and, in some cases, caffeic acid samples. PMID:12059140

  19. Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

    PubMed

    Yohda, Masafumi; Yagi, Osami; Takechi, Ayane; Kitajima, Mizuki; Matsuda, Hisashi; Miyamura, Naoaki; Aizawa, Tomoko; Nakajima, Mutsuyasu; Sunairi, Michio; Daiba, Akito; Miyajima, Takashi; Teruya, Morimi; Teruya, Kuniko; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Juan, Ayaka; Nakano, Kazuma; Aoyama, Misako; Terabayashi, Yasunobu; Satou, Kazuhito; Hirano, Takashi

    2015-07-01

    A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium. PMID:25579666

  20. 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

  1. A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

    PubMed Central

    Childs-Disney, Jessica L.; Disney, Matthew D.

    2008-01-01

    Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone. PMID:18065718

  2. Electrochemical determination of folic acid: A short review.

    PubMed

    Akbar, S; Anwar, A; Kanwal, Q

    2016-10-01

    Folic acid (FA) is an electroactive compound of biological origin. It helps our body to produce and maintain healthy cells. It can significantly reduce the occurrence of neural tube defects and also prevents change in DNA structure. FA deficiency can lead to various health risks. Therefore, a sensitive, specific, and reproducible way of FA detection is essential. A number of analytical methods are in practice for the quantification of FA. However, electroanalytical methods are attracting much attention because of their advantage over conventional methods, as they are fast, simple, sensitive, and cost effective. Moreover, modification of electrodes offers control over size and morphology which allows miniaturization for applicability in portable electrochemical devices. PMID:27449133

  3. A novel T-cell-defined HLA-DR polymorphism not predicted from the linear amino acid sequence.

    PubMed

    Termijtelen, A; van den Elsen, P; Koning, F; de Koster, S; Schroeijers, W; Vanderkerckhove, B

    1989-09-01

    Recent investigations have shown that alloreactive T cells are capable of responding to structures defined by specific linear amino acid sequences on class II molecules. In the present study we show that also a polymorphism can be recognized that is not defined by such linear amino acid sequences. Two human T-cell clones, sensitized to DRw13 haplotypes, are described. The description of clone c50 serves to exemplify the first model. This DRB1-specific clone responds to stimulator cells that carry DR molecules, different in their DRB1 first and second hypervariable regions (HV1 and HV2) but identical in their HV3 regions (i.e., DRw13,Dw18; DRw13,Dw19; DR4,Dw10; and DRw11,LDVII). The second clone, c1443, behaves nonconventionally. It responds to DRw13,Dw18; DRw13,Dw19; and DR4,Dw4 stimulator cells, although no specific amino acid sequence is shared between these specificities. The latter pattern of reactivity suggests the existence of a novel polymorphism recognized by alloreactive T cells. This particular polymorphism may also be biologically significant. PMID:2476425

  4. Structural Determinants for Transport Across the Intestinal Bile Acid Transporter Using C-24 Bile Acid Conjugates

    PubMed Central

    Rais, Rana; Acharya, Chayan; MacKerell, Alexander D.; Polli, James E.

    2010-01-01

    The human apical sodium dependent bile acid transporter (hASBT) re-absorbs gram quantities of bile acid daily and is a potential prodrug target to increase oral drug absorption. In the absence of a high resolution hASBT crystal structure, 3D-QSAR modeling may prove beneficial in designing prodrug targets to hASBT. The objective was to derive a conformationally sampled pharmacophore 3D–QSAR (CSP-SAR) model for the uptake of bile acid conjugates by hASBT. A series of bile acid conjugates of glutamyl chenodeoxycholate were evaluated in terms of Km and normalized Vmax(normVmax) using hASBT-MDCK cells. All mono-anionic conjugates were potent substrates. Dianions, cations and zwitterions, which bound with a high affinity, were not substrates. CSP-SAR models were derived using structural and physicochemical descriptors, and evaluated via cross-validation. The best CSP-SAR model for Km included two structural and two physiochemical descriptors, where substrate hydrophobicity enhanced affinity. A best CSP-SAR model for Km/normVmax employed one structural and three physicochemical descriptors, also indicating hydrophobicity enhanced efficiency. Overall, the bile acid C-24 region accommodated a range of substituted anilines, provided a single negative charge was present near C-24. In comparing uptake findings to prior inhibition results, increased hydrophobicity enhanced activity, with dianions and zwitterions hindering activity. PMID:20939504

  5. 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

  6. 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

  7. 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....

  8. Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identifies sequences that determine recognitional specificity in Cf-4 and Cf-9.

    PubMed Central

    Thomas, C M; Jones, D A; Parniske, M; Harrison, K; Balint-Kurti, P J; Hatzixanthis, K; Jones, J D

    1997-01-01

    In many interactions between plants and their pathogens, resistance to infection is specified by plant resistance (R) genes and corresponding pathogen avirulence (Avr) genes. In tomato, the Cf-4 and Cf-9 resistance genes map to the same location but confer resistance to Cladosporium fulvum through recognition of different avirulence determinants (AVR4 and AVR9) by a molecular mechanism that has yet to be determined. Here, we describe the cloning and characterization of Cf-4, which also encodes a membrane-anchored extracellular glycoprotein. Cf-4 contains 25 leucine-rich repeats, which is two fewer than Cf-9. The proteins have > 91% identical amino acids. DNA sequence comparison suggests that Cf-4 and Cf-9 are derived from a common progenitor sequence. Amino acid differences distinguishing Cf-4 and Cf-9 are confined to their N termini, delimiting a region that determines the recognitional specificity of ligand binding. The majority of these differences are in residues interstitial to those of the leucine-rich repeat consensus motif. Many of these residues are predicted to form a solvent-exposed surface that can interact with the cognate ligand. Both Cf-4 and Cf-9 are located within a 36-kb region comprising five tandemly duplicated homologous genes. These results provide further insight into the molecular basis of pathogen perception by plants and the organization of complex R gene loci. PMID:9437864

  9. [Gas chromatographic determination of formic acid in urine as carbon monoxide (author's transl)].

    PubMed

    Angerer, J

    1976-02-01

    A gas chromatographic method for determining formic acid in human urine is described. The analytical reliability of this method fullfills the criteria of statistical quality control. The rate of recovery is 101.2 to 105.7% the variability coefficients lie between 2.9 and 7.2%. The selectivity of this method is demonstrated by analysing a group of components normally occuring in urine which did not interfere with the determination of formic acid. The detection limit of about 4.3 mumol/1 formic acid in urine permits the determination of the concentration of formic acid in the urine of normal persons. The concentrations of formic acid in the urine of a group of normal persons lies between 0 and 2.79 mmol/1. The average concentration was 0.39 +/- 0.60 mmol/1. PMID:1249528

  10. [Special qualification of a photometric procedure for determination of salicylic acid in therapeutic drug monitoring].

    PubMed

    Martens, J; Meyer, F P

    1995-01-01

    A procedure for the determination of salicylic acid from human serum is presented. It is based on an acidic extraction, a basic reextraction and the detection of salicylic acid as its iron-III-complex by photometry. The procedure is quantitative over a wide range of linearity, easy to carry out and is especially suitable for therapeutic drug monitoring in the treatment of juvenile rheumatoid arthritis. PMID:7886124

  11. Uronic acid determination by high performance liquid chromatography with postcolumn fluorescence derivatization.

    PubMed

    Kakita, Hirotaka; Kamishima, Hiroshi; Inouye, Kuniyo

    2006-10-01

    To develop a fluorimetric high performance liquid chromatography (HPLC) technique for uronic acid microanalysis, a saline mobile phase and the postcolumn fluorimetric determination were combined. The detection limits of D-glucuronic, D-galacturonic and D-mannuronic acids were 7.19, 23.88 and 7.08 pmol, respectively. The proposed method was successfully applied to uronic acid microanalysis in a polysaccharide hydrolysate and a drink. PMID:16956616

  12. 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

  13. Sequence-independent and reversible photocontrol of transcription/expression systems using a photosensitive nucleic acid binder

    PubMed Central

    Estévez-Torres, André; Crozatier, Cécile; Diguet, Antoine; Hara, Tomoaki; Saito, Hirohide; Yoshikawa, Kenichi; Baigl, Damien

    2009-01-01

    To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression (a set of reactions including both transcription and translation) were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures. PMID:19617550

  14. Determination of trans fatty acid levels by FTIR in processed foods in Australia.

    PubMed

    McCarthy, Justine; Barr, Daniel; Sinclair, Andrew

    2008-01-01

    Health authorities around the world advise 'limiting consumption of trans fatty acid', however in Australia the trans fatty acid (TFA) content is not required to be listed in the nutrition information panel unless a declaration or nutrient claim is made about fatty acids or cholesterol. Since there is limited knowledge about trans fatty acid levels in processed foods available in Australia, this study aimed to determine the levels of TFA in selected food items known to be sources of TFA from previously published studies. Food items (n=92) that contain vegetable oil and a total fat content greater than 5% were included. This criterion was used in conjunction with a review of similar studies where food items were found to contain high levels of trans fatty acids. Lipids were extracted using solvents. Gravimetric methods were used to determine total fat content and trans fatty acid levels were quantified by Attenuated Total Reflectance Fourier Transform Infrared spectroscopy. High levels of trans fatty acids were found in certain items in the Australian food supply, with a high degree of variability. Of the samples analysed, 13 contained greater than 1 g of trans fatty acids per serving size, the highest value was 8.1 g/serving. Apart from when the nutrition information panel states that the content is less than a designated low level, food labels sold in Australia do not indicate trans fatty acid levels. We suggested that health authorities seek ways to assist consumers to limit their intakes of trans fatty acids. PMID:18818158

  15. Mapping the sex determination locus in the Atlantic halibut (Hippoglossus hippoglossus) using RAD sequencing

    PubMed Central

    2013-01-01

    Background Atlantic halibut (Hippoglossus hippoglossus) is a high-value, niche market species for cold-water marine aquaculture. Production of monosex female stocks is desirable in commercial production since females grow faster and mature later than males. Understanding the sex determination mechanism and developing sex-associated markers will shorten the time for the development of monosex female production, thus decreasing the costs of farming. Results Halibut juveniles were masculinised with 17 α-methyldihydrotestosterone (MDHT) and grown to maturity. Progeny groups from four treated males were reared and sexed. Two of these groups (n = 26 and 70) consisted of only females, while the other two (n = 30 and 71) contained balanced sex ratios (50% and 48% females respectively). DNA from parents and offspring from the two mixed-sex families were used as a template for Restriction-site Associated DNA (RAD) sequencing. The 648 million raw reads produced 90,105 unique RAD-tags. A linkage map was constructed based on 5703 Single Nucleotide Polymorphism (SNP) markers and 7 microsatellites consisting of 24 linkage groups, which corresponds to the number of chromosome pairs in this species. A major sex determining locus was mapped to linkage group 13 in both families. Assays for 10 SNPs with significant association with phenotypic sex were tested in both population data and in 3 additional families. Using a variety of machine-learning algorithms 97% correct classification could be obtained with the 3% of errors being phenotypic males predicted to be females. Conclusion Altogether our findings support the hypothesis that the Atlantic halibut has an XX/XY sex determination system. Assays are described for sex-associated DNA markers developed from the RAD sequencing analysis to fast track progeny testing and implement monosex female halibut production for an immediate improvement in productivity. These should also help to speed up the inclusion of neomales derived

  16. Whole genome sequencing provides insights into the genetic determinants of invasiveness in Salmonella Dublin.

    PubMed

    Mohammed, M; Cormican, M

    2016-08-01

    Salmonella enterica subsp. enterica serovar Dublin (S. Dublin) is one of the non-typhoidal Salmonella (NTS); however, a relatively high proportion of human infections are associated with invasive disease. We applied whole genome sequencing to representative invasive and non-invasive clinical isolates of S. Dublin to determine the genomic variations among them and to investigate the underlying genetic determinants associated with invasiveness in S. Dublin. Although no particular genomic variation was found to differentiate in invasive and non-invasive isolates four virulence factors were detected within the genome of all isolates including two different type VI secretion systems (T6SS) encoded on two Salmonella pathogenicity islands (SPI), including SPI-6 (T6SSSPI-6) and SPI-19 (T6SSSPI-19), an intact lambdoid prophage (Gifsy-2-like prophage) that contributes significantly to the virulence and pathogenesis of Salmonella serotypes in addition to a virulence plasmid. These four virulence factors may all contribute to the potential of S. Dublin to cause invasive disease in humans. PMID:26996313

  17. Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP

    PubMed Central

    Tariq, Muhammad Akram; Pourmand, Nader

    2010-01-01

    Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing-based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to “tailor” DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated “dyad” pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost-effective DNA length measurement for STRs and resequencing of small DNA fragments. PMID:20408144

  18. Fast skeletal muscle transcriptome of the Gilthead sea bream (Sparus aurata) determined by next generation sequencing

    PubMed Central

    2012-01-01

    Background The gilthead sea bream (Sparus aurata L.) occurs around the Mediterranean and along Eastern Atlantic coasts from Great Britain to Senegal. It is tolerant of a wide range of temperatures and salinities and is often found in brackish coastal lagoons and estuarine areas, particularly early in its life cycle. Gilthead sea bream are extensively cultivated in the Mediterranean with an annual production of 125,000 metric tonnes. Here we present a de novo assembly of the fast skeletal muscle transcriptome of gilthead sea bream using 454 reads and identify gene paralogues, splice variants and microsatellite repeats. An annotated transcriptome of the skeletal muscle will facilitate understanding of the genetic and molecular basis of traits linked to production in this economically important species. Results Around 2.7 million reads of mRNA sequence data were generated from the fast myotomal of adult fish (~2 kg) and juvenile fish (~0.09 kg) that had been either fed to satiation, fasted for 3-5d or transferred to low (11°C) or high (33°C) temperatures for 3-5d. Newbler v2.5 assembly resulted in 43,461 isotigs >100 bp. The number of sequences annotated by searching protein and gene ontology databases was 10,465. The average coverage of the annotated isotigs was x40 containing 5655 unique gene IDs and 785 full-length cDNAs coding for proteins containing 58–1536 amino acids. The v2.5 assembly was found to be of good quality based on validation using 200 full-length cDNAs from GenBank. Annotated isotigs from the reference transcriptome were attributable to 344 KEGG pathway maps. We identified 26 gene paralogues (20 of them teleost-specific) and 43 splice variants, of which 12 had functional domains missing that were likely to affect their biological function. Many key transcription factors, signaling molecules and structural proteins necessary for myogenesis and muscle growth have been identified. Physiological status affected the number of reads that mapped

  19. An automatic system for acidity determination based on sequential injection titration and the monosegmented flow approach.

    PubMed

    Kozak, Joanna; Wójtowicz, Marzena; Gawenda, Nadzieja; Kościelniak, Paweł

    2011-06-15

    An automatic sequential injection system, combining monosegmented flow analysis, sequential injection analysis and sequential injection titration is proposed for acidity determination. The system enables controllable sample dilution and generation of standards of required concentration in a monosegmented sequential injection manner, sequential injection titration of the prepared solutions, data collecting, and handling. It has been tested on spectrophotometric determination of acetic, citric and phosphoric acids with sodium hydroxide used as a titrant and phenolphthalein or thymolphthalein (in the case of phosphoric acid determination) as indicators. Accuracy better than |4.4|% (RE) and repeatability better than 2.9% (RSD) have been obtained. It has been applied to the determination of total acidity in vinegars and various soft drinks. The system provides low sample (less than 0.3 mL) consumption. On average, analysis of a sample takes several minutes. PMID:21641455

  20. A Simple Spectrophotometric Method for the Determination of Thiobarbituric Acid Reactive Substances in Fried Fast Foods.

    PubMed

    Zeb, Alam; Ullah, Fareed

    2016-01-01

    A simple and highly sensitive spectrophotometric method was developed for the determination of thiobarbituric acid reactive substances (TBARS) as a marker for lipid peroxidation in fried fast foods. The method uses the reaction of malondialdehyde (MDA) and TBA in the glacial acetic acid medium. The method was precise, sensitive, and highly reproducible for quantitative determination of TBARS. The precision of extractions and analytical procedure was very high as compared to the reported methods. The method was used to determine the TBARS contents in the fried fast foods such as Shami kebab, samosa, fried bread, and potato chips. Shami kebab, samosa, and potato chips have higher amount of TBARS in glacial acetic acid-water extraction system than their corresponding pure glacial acetic acid and vice versa in fried bread samples. The method can successfully be used for the determination of TBARS in other food matrices, especially in quality control of food industries. PMID:27123360

  1. A Simple Spectrophotometric Method for the Determination of Thiobarbituric Acid Reactive Substances in Fried Fast Foods

    PubMed Central

    Zeb, Alam; Ullah, Fareed

    2016-01-01

    A simple and highly sensitive spectrophotometric method was developed for the determination of thiobarbituric acid reactive substances (TBARS) as a marker for lipid peroxidation in fried fast foods. The method uses the reaction of malondialdehyde (MDA) and TBA in the glacial acetic acid medium. The method was precise, sensitive, and highly reproducible for quantitative determination of TBARS. The precision of extractions and analytical procedure was very high as compared to the reported methods. The method was used to determine the TBARS contents in the fried fast foods such as Shami kebab, samosa, fried bread, and potato chips. Shami kebab, samosa, and potato chips have higher amount of TBARS in glacial acetic acid-water extraction system than their corresponding pure glacial acetic acid and vice versa in fried bread samples. The method can successfully be used for the determination of TBARS in other food matrices, especially in quality control of food industries. PMID:27123360

  2. Ascorbic Acid Determination in Natural Orange Juice: As a Teaching Tool of Coulometry and Polarography.

    ERIC Educational Resources Information Center

    Bertotti, Mauro; And Others

    1995-01-01

    Describes an experiment designed to determine ascorbic acid concentrations in natural orange juice. The experiment is used with undergraduate pharmacy students to allow understanding of the principles of operation of the coulometer and polarograph. (DDR)

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

    PubMed

    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

  5. Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases.

    PubMed

    Stephenson, F H; Ballard, B T; Boyer, H W; Rosenberg, J M; Greene, P J

    1989-12-21

    The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms. PMID:2695392

  6. The Implementation of "The n-term" Formula to Improve Student Ability in Determining the Rules of a Numeric Sequence

    ERIC Educational Resources Information Center

    In'am, Akhsanul; Hajar, Siti

    2013-01-01

    A good-quality teacher may determines a good-quality learning, thus good-quality students will be the results. In order to have a good-quality learning, a lot of strategies and methods can be adopted. The objective of this research is to improve students' ability in determining the rules of a numeric sequence and analysing the effectiveness of the…

  7. Determination of the Efficiency of Mixed-Acid Digestions of Sediments

    SciTech Connect

    Huerta Vazquez, Alejandra I.; Gill, Gary A.

    2007-01-01

    Mixed-acid digestion is a method often used for the determination of elemental analysis of sediment samples. It is crucial that efficiency details associated with the digestion method be well understood on an element by element basis. Battelle’s Marine Sciences Laboratory Standard Operating Procedure for Sediment Mixed-Acid Digestions was modified to identify conditions which produce optimal recovery of elements. The parameters that were adjusted for testing were mass of sediment, mixed-acid volume, mixed-acid composition and digestion time. Digestion involves treatment of the sediment sample with mixed-acid mixtures at 135º C ± 10º in a Teflon® digestion bomb. Typical analytical methods include Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and Inductively Coupled Plasma – Mass Spectrometry (ICP-MS). Initial experiments involved determining the optimal ratio of acid volume to mass of sediment. Experiments were designed to identify the point at which insufficient acid was used to effectively digest a given mass of sediment. When the mass of sediment was varied between 0.2 and 1.0 gram using a 4 mL aqua regia acid mixture (3 mL hydrochloric acid and 1 mL nitric acid), there was no effect on the recovery of the elements Al, Ba, Ca, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Sr, Ti, V, and Zn. The next experiments focused on a time study to resolve the shortest digestive time for optimal elemental recovery. Two masses of sediment were investigated, 0.25 and 0.7 g, again utilizing aqua regia digestion (4 mL). Maximum recovery was reached after 4 hours of digestion; additional digestion time released no or only minimal amounts of elements from the sediments. The final set of experiments was designed to identify optimal conditions for the total digestion of sediment using a mixture of hydrochloric acid, nitric acid, hydrofluoric acid, hydrogen peroxide, and boric acid. These experiments were designed to determine the optimal volume of hydrofluoric acid

  8. Cutaneous retinoic acid levels determine hair follicle development and downgrowth.

    PubMed

    Okano, Junko; Levy, Clara; Lichti, Ulrike; Sun, Hong-Wei; Yuspa, Stuart H; Sakai, Yasuo; Morasso, Maria I

    2012-11-16

    Retinoic acid (RA) is essential during embryogenesis and for tissue homeostasis, whereas excess RA is well known as a teratogen. In humans, excess RA is associated with hair loss. In the present study, we demonstrate that specific levels of RA, regulated by Cyp26b1, one of the RA-degrading enzymes, are required for hair follicle (hf) morphogenesis. Mice with embryonic ablation of Cyp26b1 (Cyp26b1(-/-)) have excessive endogenous RA, resulting in arrest of hf growth at the hair germ stage. The altered hf development is rescued by grafting the mutant skin on immunodeficient mice. Our results show that normalization of RA levels is associated with reinitiation of hf development. Conditional deficiency of Cyp26b1 in the dermis (En1Cre;Cyp26b1f/-) results in decreased hair follicle density and specific effect on hair type, indicating that RA levels also influence regulators of hair bending. Our results support the model of RA-dependent dermal signals regulating hf downgrowth and bending. To elucidate target gene pathways of RA, we performed microarray and RNA-Seq profiling of genes differentially expressed in Cyp26b1(-/-) skin and En1Cre;Cyp26b1f/- tissues. We show specific effects on the Wnt-catenin pathway and on members of the Runx, Fox, and Sox transcription factor families, indicating that RA modulates pathways and factors implicated in hf downgrowth and bending. Our results establish that proper RA distribution is essential for morphogenesis, development, and differentiation of hfs. PMID:23007396

  9. Cutaneous Retinoic Acid Levels Determine Hair Follicle Development and Downgrowth*

    PubMed Central

    Okano, Junko; Levy, Clara; Lichti, Ulrike; Sun, Hong-Wei; Yuspa, Stuart H.; Sakai, Yasuo; Morasso, Maria I.

    2012-01-01

    Retinoic acid (RA) is essential during embryogenesis and for tissue homeostasis, whereas excess RA is well known as a teratogen. In humans, excess RA is associated with hair loss. In the present study, we demonstrate that specific levels of RA, regulated by Cyp26b1, one of the RA-degrading enzymes, are required for hair follicle (hf) morphogenesis. Mice with embryonic ablation of Cyp26b1 (Cyp26b1−/−) have excessive endogenous RA, resulting in arrest of hf growth at the hair germ stage. The altered hf development is rescued by grafting the mutant skin on immunodeficient mice. Our results show that normalization of RA levels is associated with reinitiation of hf development. Conditional deficiency of Cyp26b1 in the dermis (En1Cre;Cyp26b1f/−) results in decreased hair follicle density and specific effect on hair type, indicating that RA levels also influence regulators of hair bending. Our results support the model of RA-dependent dermal signals regulating hf downgrowth and bending. To elucidate target gene pathways of RA, we performed microarray and RNA-Seq profiling of genes differentially expressed in Cyp26b1−/− skin and En1Cre;Cyp26b1f/− tissues. We show specific effects on the Wnt-catenin pathway and on members of the Runx, Fox, and Sox transcription factor families, indicating that RA modulates pathways and factors implicated in hf downgrowth and bending. Our results establish that proper RA distribution is essential for morphogenesis, development, and differentiation of hfs. PMID:23007396

  10. Fast online determination of surfactant inhibition in acidic phase bioreactors.

    PubMed

    Feitkenhauer, H

    2004-01-01

    Surfactants have been shown to inhibit the anaerobic digestion process severely, with the methanogenic microorganisms being the most affected. The diverse nature of surfactants used even in one (e.g. textile finishing) plant makes an online determination of surfactants sometimes very difficult and expensive. Therefore a fast online determination of inhibitory effects on the acidogenic microorganisms (first step of the degradation cascade) can help to give an early warning signal or to calculate a "pseudo"-surfactant concentration. In a two-phase system this information can be used to protect the methanogenic reactor against surfactant overloading and its long term negative effects. In this paper it is shown that the inhibition is a consequence of microbial inhibition and is not caused by an inactivation of extracellular hydrolytic enzymes (released by the cells for biopolymer cleavage). A titration technique was successfully employed to measure the surfactant inhibition in a laboratory-scale acidification reactor. Additional experiments demonstrate (using sodium dodecyl sulfate as the model substance) how inhibitory effects (and strategies to overcome inhibitory effects) can be investigated efficiently. PMID:14979534

  11. Sensitive and Selective Determination of Orotic Acid in Biological Specimens Using a Novel Fluorogenic Reaction.

    PubMed

    Yin, Sheng; Dragusha, Shpend; Ejupi, Valon; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki

    2015-07-01

    Orotic acid is an intermediate in the synthesis pathway of uridine-5'-monophosphate, and increases in body fluids of patients suffering from hereditary disorders such as orotic aciduria and hyperammonemia. In this study, we developed a spectrofluorometric method with or without high-performance liquid chromatography for the selective and sensitive quantification of orotic acid in human biological specimens, using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent. This reagent provided intensive fluorescence for only orotic acid amongst 62 compounds including structurally related bio-substances such as nucleic acid bases, nucleosides, nucleotides, amino acids, vitamins, bilirubin, uric acid, urea, creatine, creatinine and sugars. Under optimized reaction conditions, orotic acid was reacted with 4-TFMBAO, K3[Fe(CN)6] and K2CO3 in an aqueous solution. The fluorescence produced from the orotic acid derivative was measured at an excitation of 340 nm and an emission of 460 nm. A concentration of 1.2 μM orotic acid per 1.0 mM creatinine in normal urine and 0.64 nmol orotic acid per 5.0 × 10(5) HeLa cells were determined by this method. The present method permitted the facile quantification of orotic acid in healthy human urine and cultured HeLa cells by spectrofluorometry and/or high-performance liquid chromatography. PMID:26026930

  12. Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity

    PubMed Central

    Mahro, Martin; Brás, Natércia F.; Cerqueira, Nuno M. F. S. A.; Teutloff, Christian; Coelho, Catarina; Romão, Maria João; Leimkühler, Silke

    2013-01-01

    In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis. PMID:24358164

  13. Determination of bile acids in pharmaceutical formulations using micellar electrokinetic chromatography.

    PubMed

    Rodríguez, V G; Lucangioli, S E; Fernández Otero, G C; Carducci, C N

    2000-08-15

    A micellar electrokinetic chromatography method (MEKC) has been developed and validated for the determination of bile acids (BA) such as ursodeoxycholic acid (UDCA), dehydrocholic acid (DHCA) and deoxycholic acid (DCA) in pharmaceuticals for quality control purpose. The background electrolyte consisted of 20 mM borate-phosphate buffer containing 50 mM sodium dodecylsulfate (SDS), and acetonitrile as additive. UV detection was set at 185 nm. Selectivity, linearity, range, repeatability, intermediate precision and accuracy showed good results. Comparison of the values obtained by MEKC and HPLC methods were in close agreement. PMID:10933529

  14. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    DOEpatents

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  15. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    2001-06-05

    A computer system (1) for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area (814) and sample sequences in another area (816) on a display device (3).

  16. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    1999-10-26

    A computer system (1) for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area (814) and sample sequences in another area (816) on a display device (3).

  17. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, M.S.

    1998-08-18

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device. 27 figs.

  18. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    2003-08-19

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

  19. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.

    1998-08-18

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

  20. Computer-aided visualization and analysis system for sequence evaluation

    DOEpatents

    Chee, Mark S.; Wang, Chunwei; Jevons, Luis C.; Bernhart, Derek H.; Lipshutz, Robert J.

    2004-05-11

    A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.