The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

PubMed Central

Naccache, Samia N.; Greninger, Alexander L.; Lee, Deanna; Coffey, Lark L.; Phan, Tung; Rein-Weston, Annie; Aronsohn, Andrew; Hackett, John; Delwart, Eric L.

2013-01-01

Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing. PMID:24027301

Use of synthetic analogues in confirmation of structure of the peptide antibiotics Maltacines

NASA Astrophysics Data System (ADS)

Hagelin, Gunnar; Indrevoll, Bård; Hoeg-Jensen, Thomas

2007-12-01

Maltacines comprise a family of cyclic peptide lactone antibiotics produced by a strain of Bacillus subtilis. The previously proposed amino acid sequences of the linear ring-opened molecules show similarity to the lipopeptide antibiotic Fengycin IX that is also produced by a strain of B. subtilisE There were some discrepancies in the Maltacin data that could not be explained. To address this and gain more information into the structure of the linear ring-opened Maltacines, the two members D1c, E1b and Fengycin IX acid were synthesised and their MS2, MS3 and MS4 spectra compared. The similarity of the product ion spectra of Maltacin and Fengycin IX acid revealed that proline occupies an internal position in Maltacin. This finding led to revision of the interpretation of the amino acid sequences of the Maltacines. The proposed new structures of the Maltacines shows that the cyclic part of the molecules is the same as in Fengycin IX acid and Fengycin XII acid, but they have unique N-terminal sequences not found in Fengycins, and thus represent novel lipopeptide antibiotics.

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

PubMed Central

Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

2015-01-01

The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity.

PubMed

Nam, Bo-Hye; Seo, Jung-Kil; Lee, Min Jeong; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Park, Nam Gyu

2015-07-01

An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation, characterization, and primary structure of rubredoxin from the photosynthetic bacterium, Heliobacillus mobilis

NASA Technical Reports Server (NTRS)

Lee, W. Y.; Brune, D. C.; LoBrutto, R.; Blankenship, R. E.

1995-01-01

Rubredoxin is a small nonheme iron protein that serves as an electron carrier in bacterial systems. Rubredoxin has now been isolated and characterized from the strictly anaerobic phototroph, Heliobacillus mobilis. THe molecular mass (5671.3 Da from the amino acid sequence) was confirmed and partial formylation of the N-terminal methionyl residue was established by matrix-assisted laser desorption mass spectroscopy. The complete 52-amino-acid sequence was determined by a combination of N-terminal sequencing by Edman degradation and C-terminal sequencing by a novel method using carboxypeptidase treatment in conjunction with amino acid analysis and laser desorption time of flight mass spectrometry. The molar absorption coefficient of Hc. mobilis rubredoxin at 490 nm is 6.9 mM-1 cm-1 and the midpoint redox potential at pH 8.0 is -46 mV. The EPR spectrum of the oxidized form shows resonances at g = 9.66 and 4.30 due to a high-spin ferric iron. The amino acid sequence is homologous to those of rubredoxins from other species, in particular, the gram-positive bacteria, and the phototrophic green sulfur bacteria, and the evolutionary implications of this are discussed.

A Novel Phytase with Sequence Similarity to Purple Acid Phosphatases Is Expressed in Cotyledons of Germinating Soybean Seedlings 1

PubMed Central

Hegeman, Carla E.; Grabau, Elizabeth A.

2001-01-01

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases. PMID:11500558

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

PubMed

Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

2015-12-15

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicholas, R.A.; Suzuki, H.; Hirota, Y.

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed thatmore » the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.« less

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.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

PubMed

Camicia, Federico; Paredes, Rodolfo; Chalar, Cora; Galanti, Norbel; Kamenetzky, Laura; Gutierrez, Ariana; Rosenzvit, Mara C

2008-03-31

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.

PubMed

Wolffe, E J; Gause, W C; Pelfrey, C M; Holland, S M; Steinberg, A D; August, J T

1990-01-05

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.

A new variant of antimetabolic protein, arcelin from an Indian bean, Lablab purpureus (Linn.) and its effect on the stored product pest, Callosobruchus maculatus.

PubMed

Janarthanan, Sundaram; Sakthivelkumar, Shanmugavel; Veeramani, Velayutham; Radhika, Dixit; Muthukrishanan, Subbaratnam

2012-12-15

The anti-metabolic or insecticidal gene, arcelin (Arl) was isolated, cloned and sequenced using sequence specific degenerate primers from the seeds of Lablab purpureus collected from the Western Ghats, Tamil Nadu, India. The L. purpureus arcelin nucleotide sequence was homologous to Arl-3 and Arl-4 alleles from Phaseolus spp. The protein it encodes has 70% amino acid identity with the amino acid sequences of Arl-3I, Arl-3III, Arl-4 precursor, Arl-4 and Arl-4I. The partially purified arcelin from the seeds of L. purpureus using an artificial diet confirmed the complete retardation of development of the stored product pest Callosobruchus maculatus at 0.2% w/w arcelin-incorporated artificial seeds. Copyright © 2012 Elsevier Ltd. All rights reserved.

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus.

PubMed

Hammond, R W; Crosslin, J M

1995-04-01

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

PubMed Central

Lange, Allison; McLane, Laura M.; Mills, Ryan E.; Devine, Scott E.; Corbett, Anita H.

2010-01-01

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 amino acid linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 amino acid linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 amino acid linker. Our studies reveal that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition PMID:20028483

Relevance and Diversity of Nitrospira Populations in Biofilters of Brackish RAS

PubMed Central

Kruse, Myriam; Keuter, Sabine; Bakker, Evert; Spieck, Eva; Eggers, Till; Lipski, André

2013-01-01

Lithoautotrophic nitrite-oxidizing bacterial populations from moving-bed biofilters of brackish recirculation aquaculture systems (RAS; shrimp and barramundi) were tested for their metabolic activity and phylogenetic diversity. Samples from the biofilters were labeled with 13C-bicarbonate and supplemented with nitrite at concentrations of 0.3, 3 and 10 mM, and incubated at 17 and 28°C, respectively. The biofilm material was analyzed by fatty acid methyl ester - stable isotope probing (FAME-SIP). High portions of up to 45% of Nitrospira-related labeled lipid markers were found confirming that Nitrospira is the major autotrophic nitrite oxidizer in these brackish systems with high nitrogen loads. Other nitrite-oxidizing bacteria such as Nitrobacter or Nitrotoga were functionally not relevant in the investigated biofilters. Nitrospira-related 16S rRNA gene sequences were obtained from the samples with 10 mM nitrite and analyzed by a cloning approach. Sequence studies revealed four different phylogenetic clusters within the marine sublineage IV of Nitrospira, though most sequences clustered with the type strain of Nitrospira marina and with a strain isolated from a marine RAS. Three lipids dominated the whole fatty acid profiles of nitrite-oxidizing marine and brackish enrichments of Nitrospira sublineage IV organisms. The membranes included two marker lipids (16∶1 cis7 and 16∶1 cis11) combined with the non-specific acid 16∶0 as major compounds and confirmed these marker lipids as characteristic for sublineage IV species. The predominant labeling of these characteristic fatty acids and the phylogenetic sequence analyses of the marine Nitrospira sublineage IV identified organisms of this sublineage as main autotrophic nitrite-oxidizers in the investigated brackish biofilter systems. PMID:23705006

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12.

PubMed

Claverie-Martin, F; Diaz-Torres, M R; Kushner, S R

1987-01-01

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

PubMed Central

Aymerich, T; Holo, H; Håvarstein, L S; Hugas, M; Garriga, M; Nes, I F

1996-01-01

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin. PMID:8633865

Metabolic Acetate Therapy Improves Phenotype in the Tremor Rat Model of Canavan Disease

DTIC Science & Technology

2010-05-13

treated and untreated female tremor rats (pɘ.05). PA Phosphatidic acid , PC phosphatidylcholine, SM sphingomyelin, PI phosphatidylinositol, PE...was used to confirm the ASPA-deficient phenotype of homozygous tremor rats. The ASPA antibodies were generated against an 18 amino acid sequence from... acid ; 40:50:2:0.2 v/v) solvent front advanced 2/3rds plate height and dried. Solvent system 2 (diethyl ether: hexane; 6:94 v/v) was run the full plate

Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

PubMed

Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

2017-12-01

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

Sequence determination and analysis of the NSs genes of two tospoviruses.

PubMed

Hallwass, Mariana; Leastro, Mikhail O; Lima, Mirtes F; Inoue-Nagata, Alice K; Resende, Renato O

2012-03-01

The tospoviruses groundnut ringspot virus (GRSV) and zucchini lethal chlorosis virus (ZLCV) cause severe losses in many crops, especially in solanaceous and cucurbit species. In this study, the non-structural NSs gene and the 5'UTRs of these two biologically distinct tospoviruses were cloned and sequenced. The NSs sequence of GRSV and ZLCV were both 1,404 nucleotides long. Pairwise comparison showed that the NSs amino acid sequence of GRSV shared 69.6% identity with that of ZLCV and 75.9% identity with that of TSWV, while the NSs sequence of ZLCV and TSWV shared 67.9% identity. Phylogenetic analysis based on NSs sequences confirmed that these viruses cluster in the American clade.

Endotheliotropic elephant herpes virus (EEHV) infection. The first PCR-confirmed fatal case in Asia.

PubMed

Reid, C E; Hildebrandt, T B; Marx, N; Hunt, M; Thy, N; Reynes, J M; Schaftenaar, W; Fickel, J

2006-06-01

Since 1995, 4 suspected cases of Endotheliotropic Elephant Herpes Virus (EEHV) infection, i.e. based on clinical presentation, have occurred in Asia without resulting in epidemic outbreaks as expected. In order to confirm the presence of EEHV on the continent of Asia, viral DNA particles from liver samples of a wild-caught 3-year-old elephant found dead at a Cambodian elephant sanctuary and clinically diagnosed with EEHV, were PCR processed using known EEHV strain primers. The presence of EEHV viral nucleic acids was confirmed and the nucleic acids had a 99% sequence similarity to the U.S.A strain (gene bank locus: AF117265) and 97% sequence similarity to the European strain (gene bank locus: AF354746) assigning this case to the EEHV-1 cluster. More than the confirmation of EEHV on the continent of Asia, is the phylogenic relationship to the USA and European strains with no corresponding contact or transport of USA or European elephants to Asia. Thus, this brings many of the traditional theories into question. Although almost forgotten, this disease is still ramped in captive elephant populations worldwide and continues to devastate particularly the neonatal and weaning-age population. Special attention and continued research are needed specifically in the area of basic virology and epidemiology.

SENCA: A Multilayered Codon Model to Study the Origins and Dynamics of Codon Usage

PubMed Central

Pouyet, Fanny; Bailly-Bechet, Marc; Mouchiroud, Dominique; Guéguen, Laurent

2016-01-01

Gene sequences are the target of evolution operating at different levels, including the nucleotide, codon, and amino acid levels. Disentangling the impact of those different levels on gene sequences requires developing a probabilistic model with three layers. Here we present SENCA (site evolution of nucleotides, codons, and amino acids), a codon substitution model that separately describes 1) nucleotide processes which apply on all sites of a sequence such as the mutational bias, 2) preferences between synonymous codons, and 3) preferences among amino acids. We argue that most synonymous substitutions are not neutral and that SENCA provides more accurate estimates of selection compared with more classical codon sequence models. We study the forces that drive the genomic content evolution, intraspecifically in the core genome of 21 prokaryotes and interspecifically for five Enterobacteria. We retrieve the existence of a universal mutational bias toward AT, and that taking into account selection on synonymous codon usage has consequences on the measurement of selection on nonsynonymous substitutions. We also confirm that codon usage bias is mostly driven by selection on preferred codons. We propose new summary statistics to measure the relative importance of the different evolutionary processes acting on sequences. PMID:27401173

Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes.

PubMed

Stolterfoht, Holly; Schwendenwein, Daniel; Sensen, Christoph W; Rudroff, Florian; Winkler, Margit

2017-09-10

Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene.

PubMed

Trcek, Janja

2005-10-01

Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S-23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNAIle and tRNAAla, the sequences of 16S-23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. acetispecific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for revealing the misclassification of strain IFO 3283 into the species A. aceti.

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

PubMed

Okochi, Mina; Muto, Masaki; Yanai, Kentaro; Tanaka, Masayoshi; Onodera, Takeshi; Wang, Jin; Ueda, Hiroshi; Toko, Kiyoshi

2017-10-09

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 μM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Aqueous mineralogy and stratigraphy at and around the proposed Mawrth Vallis MSL Landing Site: New insights into the aqueous history of the region

USGS Publications Warehouse

Dobrea, Eldar Z. Noe; Michalski, Joseph; Swayze, Gregg

2011-01-01

In this work, we have confirmed the mineralogical stratigraphy previously inferred by other authors, but also demonstrate the presence of additional minerals, including a possible acid-leaching product near the top of the sequence, an Mh-OH bearing phyllosilicate at the to of the sequence, and potentially a Ca-sulfate at the bottom of the phyllosilicate sequence. The latter has important implications regarding the relative timing of sulfate vs clay formation on Mars.

The molecular mechanism for interaction of ceruloplasmin and myeloperoxidase

NASA Astrophysics Data System (ADS)

Bakhautdin, Bakytzhan; Bakhautdin, Esen Göksöy

2016-04-01

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change.

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

PubMed

Lerner, D R; Raikhel, N V

1992-06-05

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.

Cloning and characterization of the novel D-aspartyl endopeptidase, paenidase, from Paenibacillus sp. B38.

PubMed

Nirasawa, Satoru; Nakahara, Kazuhiko; Takahashi, Saori

2018-02-27

Paenidase is the first microorganism-derived D-aspartyl endopeptidase that specifically recognizes an internal D-Asp residue to cleave [D-Asp]-X peptide bonds. Using peptide sequences obtained from the protein, we performed PCR with degenerate primers to amplify the paenidase I-encoding gene. Nucleotide sequencing revealed that mature paenidase I consists of 322 amino acid residues and that the protein is encoded as a pro-protein with a 197-amino-acid N-terminal extension compared to the mature protein. Paenidase I exhibits amino acid sequence similarity to several penicillin-binding proteins. In addition, paenidase I was classified into peptidase family S12 based on a MEROPS database search. Family S12 contains serine-type D-Ala-D-Ala carboxypeptidases that have three active site residues (Ser, Lys, and Tyr) in the conserved motifs Ser-Xaa-Thr-Lys and Tyr-Xaa-Asn. These motifs were conserved in the primary structure of paenidase I, and the role of these residues was confirmed by site-directed mutagenesis.

Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, Mikael R.; Salazar, Margarita; Schaap, Peter

2011-06-01

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regionsmore » have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases and protein transporters.« less

Nucleotide and deduced amino acid sequence of the envelope gene of the Vasilchenko strain of TBE virus; comparison with other flaviviruses.

PubMed

Gritsun, T S; Frolova, T V; Pogodina, V V; Lashkevich, V A; Venugopal, K; Gould, E A

1993-02-01

A strain of tick-borne encephalitis virus known as Vasilchenko (Vs) exhibits relatively low virulence characteristics in monkeys, Syrian hamsters and humans. The gene encoding the envelope glycoprotein of this virus was cloned and sequenced. Alignment of the sequence with those of other known tick-borne flaviviruses and identification of the recognised amino acid genetic marker EHLPTA confirmed its identity as a member of the TBE complex. However, Vs virus was distinguishable from eastern and western tick-borne serotypes by the presence of the sequence AQQ at amino acid positions 232-234 and also by the presence of other specific amino acid substitutions which may be genetic markers for these viruses and could determine their pathogenetic characteristics. When compared with other tick-borne flaviviruses, Vs virus had 12 unique amino acid substitutions including an additional potential glycosylation site at position (315-317). The Vs virus strain shared closest nucleotide and amino acid homology (84.5% and 95.5% respectively) with western and far eastern strains of tick-borne encephalitis virus. Comparison with the far eastern serotype of tick-borne encephalitis virus, by cross-immunoelectrophoresis of Vs virions and PAGE analysis of the extracted virion proteins, revealed differences in surface charge and virus stability that may account for the different virulence characteristics of Vs virus. These results support and enlarge upon previous data obtained from molecular and serological analysis.

A novel chaotic based image encryption using a hybrid model of deoxyribonucleic acid and cellular automata

NASA Astrophysics Data System (ADS)

Enayatifar, Rasul; Sadaei, Hossein Javedani; Abdullah, Abdul Hanan; Lee, Malrey; Isnin, Ismail Fauzi

2015-08-01

Currently, there are many studies have conducted on developing security of the digital image in order to protect such data while they are sending on the internet. This work aims to propose a new approach based on a hybrid model of the Tinkerbell chaotic map, deoxyribonucleic acid (DNA) and cellular automata (CA). DNA rules, DNA sequence XOR operator and CA rules are used simultaneously to encrypt the plain-image pixels. To determine rule number in DNA sequence and also CA, a 2-dimension Tinkerbell chaotic map is employed. Experimental results and computer simulations, both confirm that the proposed scheme not only demonstrates outstanding encryption, but also resists various typical attacks.

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase.

PubMed

Kumar, Sumit; Khan, Rizwan Hasan; Khare, S K

2016-01-01

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. "FVHLFEW" was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.

Murine recessive hereditary spherocytosis, sph/sph, is caused by a mutation in the erythroid alpha-spectrin gene.

PubMed

Wandersee, N J; Birkenmeier, C S; Gifford, E J; Mohandas, N; Barker, J E

2000-01-01

Spectrin, a heterodimer of alpha- and beta-subunits, is the major protein component of the red blood cell membrane skeleton. The mouse mutation, sph, causes an alpha-spectrin-deficient hereditary spherocytosis with the severe phenotype typical of recessive hereditary spherocytosis in humans. The sph mutation maps to the erythroid alpha-spectrin locus, Spna1, on Chromosome 1. Scanning electron microscopy, osmotic gradient ektacytometry, cDNA cloning, RT-PCR, nucleic acid sequencing, and Northern blot analyses were used to characterize the wild type and sph alleles of the Spna1 locus. Our results confirm the spherocytic nature of sph/sph red blood cells and document a mild spherocytic transition in the +/sph heterozygotes. Sequencing of the full length coding region of the Spna1 wild type allele from the C57BL/6J strain of mice reveals a 2414 residue deduced amino acid sequence that shows the typical 106-amino-acid repeat structure previously described for other members of the spectrin protein family. Sequence analysis of RT-PCR clones from sph/sph alpha-spectrin mRNA identified a single base deletion in repeat 5 that would cause a frame shift and premature termination of the protein. This deletion was confirmed in sph/sph genomic DNA. Northern blot analyses of the distribution of Spna1 mRNA in non-erythroid tissues detects the expression of 8, 2.5 and 2.0 kb transcripts in adult heart. These results predict the heart as an additional site where alpha-spectrin mutations may produce a phenotype and raise the possibility that a novel functional class of small alpha-spectrin isoforms may exist.

The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function.

PubMed

Papadaki, Amalia; Politou, Anastasia S; Smirlis, Despina; Kotini, Maria P; Kourou, Konstadina; Papamarcaki, Thomais; Boleti, Haralabia

2015-05-01

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-11-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed Central

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-01-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM. Images PMID:1656067

Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase.

PubMed

Jackson, R G; Lim, E K; Li, Y; Kowalczyk, M; Sandberg, G; Hoggett, J; Ashford, D A; Bowles, D J

2001-02-09

Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDP-glucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDP-glucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis.

Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

PubMed Central

Andersen, Mikael R.; Salazar, Margarita P.; Schaap, Peter J.; van de Vondervoort, Peter J.I.; Culley, David; Thykaer, Jette; Frisvad, Jens C.; Nielsen, Kristian F.; Albang, Richard; Albermann, Kaj; Berka, Randy M.; Braus, Gerhard H.; Braus-Stromeyer, Susanna A.; Corrochano, Luis M.; Dai, Ziyu; van Dijck, Piet W.M.; Hofmann, Gerald; Lasure, Linda L.; Magnuson, Jon K.; Menke, Hildegard; Meijer, Martin; Meijer, Susan L.; Nielsen, Jakob B.; Nielsen, Michael L.; van Ooyen, Albert J.J.; Pel, Herman J.; Poulsen, Lars; Samson, Rob A.; Stam, Hein; Tsang, Adrian; van den Brink, Johannes M.; Atkins, Alex; Aerts, Andrea; Shapiro, Harris; Pangilinan, Jasmyn; Salamov, Asaf; Lou, Yigong; Lindquist, Erika; Lucas, Susan; Grimwood, Jane; Grigoriev, Igor V.; Kubicek, Christian P.; Martinez, Diego; van Peij, Noël N.M.E.; Roubos, Johannes A.; Nielsen, Jens; Baker, Scott E.

2011-01-01

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi. PMID:21543515

Phylogenetic analysis of mitochondrial protein coding genes confirms the reciprocal paraphyly of Hexapoda and Crustacea

PubMed Central

Carapelli, Antonio; Liò, Pietro; Nardi, Francesco; van der Wath, Elizabeth; Frati, Francesco

2007-01-01

Background The phylogeny of Arthropoda is still a matter of harsh debate among systematists, and significant disagreement exists between morphological and molecular studies. In particular, while the taxon joining hexapods and crustaceans (the Pancrustacea) is now widely accepted among zoologists, the relationships among its basal lineages, and particularly the supposed reciprocal paraphyly of Crustacea and Hexapoda, continues to represent a challenge. Several genes, as well as different molecular markers, have been used to tackle this problem in molecular phylogenetic studies, with the mitochondrial DNA being one of the molecules of choice. In this study, we have assembled the largest data set available so far for Pancrustacea, consisting of 100 complete (or almost complete) sequences of mitochondrial genomes. After removal of unalignable sequence regions and highly rearranged genomes, we used nucleotide and inferred amino acid sequences of the 13 protein coding genes to reconstruct the phylogenetic relationships among major lineages of Pancrustacea. The analysis was performed with Bayesian inference, and for the amino acid sequences a new, Pancrustacea-specific, matrix of amino acid replacement was developed and used in this study. Results Two largely congruent trees were obtained from the analysis of nucleotide and amino acid datasets. In particular, the best tree obtained based on the new matrix of amino acid replacement (MtPan) was preferred over those obtained using previously available matrices (MtArt and MtRev) because of its higher likelihood score. The most remarkable result is the reciprocal paraphyly of Hexapoda and Crustacea, with some lineages of crustaceans (namely the Malacostraca, Cephalocarida and, possibly, the Branchiopoda) being more closely related to the Insecta s.s. (Ectognatha) than two orders of basal hexapods, Collembola and Diplura. Our results confirm that the mitochondrial genome, unlike analyses based on morphological data or nuclear genes, consistently supports the non monophyly of Hexapoda. Conclusion The finding of the reciprocal paraphyly of Hexapoda and Crustacea suggests an evolutionary scenario in which the acquisition of the hexapod condition may have occurred several times independently in lineages descending from different crustacean-like ancestors, possibly as a consequence of the process of terrestrialization. If this hypothesis was confirmed, we should therefore re-think our interpretation of the evolution of the Arthropoda, where terrestrialization may have led to the acquisition of similar anatomical features by convergence. At the same time, the disagreement between reconstructions based on morphological, nuclear and mitochondrial data sets seems to remain, despite the use of larger data sets and more powerful analytical methods. PMID:17767736

Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

PubMed Central

Zhang, Shaofeng; Basile, Franco

2011-01-01

A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

PubMed

Fam, Rachel R S; Hiong, Kum C; Choo, Celine Y L; Wong, Wai P; Chew, Shit F; Ip, Yuen K

2018-05-20

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances. Copyright © 2018 Elsevier B.V. All rights reserved.

Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele.

PubMed

van der Leij, F R; Visser, R G; Ponstein, A S; Jacobsen, E; Feenstra, W J

1991-08-01

The genomic sequence of the potato gene for starch granule-bound starch synthase (GBSS; "waxy protein") has been determined for the wild-type allele of a monoploid genotype from which an amylose-free (amf) mutant was derived, and for the mutant part of the amf allele. Comparison of the wild-type sequence with a cDNA sequence from the literature and a newly isolated cDNA revealed the presence of 13 introns, the first of which is located in the untranslated leader. The promoter contains a G-box-like sequence. The deduced amino acid sequence of the precursor of GBSS shows a high degree of identity with monocot waxy protein sequences in the region corresponding to the mature form of the enzyme. The transit peptide of 77 amino acids, required for routing of the precursor to the plastids, shows much less identity with the transit peptides of the other waxy preproteins, but resembles the hydropathic distributions of these peptides. Alignment of the amino acid sequences of the four mature starch synthases with the Escherichia coli glgA gene product revealed the presence of at least three conserved boxes; there is no homology with previously proposed starch-binding domains of other enzymes involved in starch metabolism. We report the use of chimeric constructs with wild-type and amf sequences to localize, via complementation experiments, the region of the amf allele in which the mutation resides. Direct sequencing of polymerase chain reaction products confirmed that the amf mutation is a deletion of a single AT basepair in the region coding for the transit peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and molecular characterization of partial FSH and LH receptor genes in Arabian camels (Camelus dromedarius)

PubMed Central

Jelokhani-Niaraki, Saber; Tahmoorespur, Mojtaba; Bitaraf-Sani, Morteza

2015-01-01

Very little is known about LHR and FSHR genes of domestic dromedary camels. The main objective of this study was to determine and analyze partial genomic regions of FSHR and LHR genes in dromedary camels for the first time. To this end, a total of50 DNA samples belonging to dromedary camels raised in Iran were sent for sequencing (25 samples of each gene). We compared the nucleotide sequences of Camelus dromedarius with corresponding sequences of previously published FSHR and LHR genes in bactrian camels and other species. According to the data, the same nucleotide variation was identified in both regions of the two camel species. The alignment of deduced protein sequences of the two different species revealed an amino acid variation at the FSHR region. No evidence of amino acid variation was observed, however, in LHR sequences. Phylogenetic analysis indicated that both camel species had a close relationship and clustered together in a separate branch. This was further confirmed by genetic distance values illustrating significant sequence identity between Camelus dromedarius and Camelus bactrianus. Interestingly, sequence comparisons revealed heterozygote patterns in FSHR sequences isolated from dromedary camels of Iran. In comparison to other species, this camel contains three amino acid substitutions at 5, 67, and 105 positions in the FSHR coding region. These positions are found exclusively in camels and can be considered as species specific. The results of our study can be used for hormone functionality research (FSHR and LHR) as well as reproduction-linked polymorphisms and breeding programs. PMID:27844002

Isolation and molecular characterization of partial FSH and LH receptor genes in Arabian camels (Camelus dromedarius).

PubMed

Jelokhani-Niaraki, Saber; Tahmoorespur, Mojtaba; Bitaraf-Sani, Morteza

2015-06-01

Very little is known about LHR and FSHR genes of domestic dromedary camels. The main objective of this study was to determine and analyze partial genomic regions of FSHR and LHR genes in dromedary camels for the first time. To this end, a total of50 DNA samples belonging to dromedary camels raised in Iran were sent for sequencing (25 samples of each gene). We compared the nucleotide sequences of Camelus dromedarius with corresponding sequences of previously published FSHR and LHR genes in bactrian camels and other species. According to the data, the same nucleotide variation was identified in both regions of the two camel species. The alignment of deduced protein sequences of the two different species revealed an amino acid variation at the FSHR region. No evidence of amino acid variation was observed, however, in LHR sequences. Phylogenetic analysis indicated that both camel species had a close relationship and clustered together in a separate branch. This was further confirmed by genetic distance values illustrating significant sequence identity between Camelus dromedarius and Camelus bactrianus . Interestingly, sequence comparisons revealed heterozygote patterns in FSHR sequences isolated from dromedary camels of Iran. In comparison to other species, this camel contains three amino acid substitutions at 5, 67, and 105 positions in the FSHR coding region. These positions are found exclusively in camels and can be considered as species specific. The results of our study can be used for hormone functionality research ( FSHR and LHR ) as well as reproduction-linked polymorphisms and breeding programs.

Identification and profiling of conserved and novel microRNAs involved in oil and oleic acid production during embryogenesis in Carya cathayensis Sarg.

PubMed

Wang, Zhengjia; Huang, Ruiming; Sun, Zhichao; Zhang, Tong; Huang, Jianqin

2017-05-01

MicroRNAs (miRNAs) are important regulators of plant development and fruit formation. Mature embryos of hickory (Carya cathayensis Sarg.) nuts contain more than 70% oil (comprising 90% unsaturated fatty acids), along with a substantial amount of oleic acid. To understand the roles of miRNAs involved in oil and oleic acid production during hickory embryogenesis, three small RNA libraries from different stages of embryogenesis were constructed. Deep sequencing of these three libraries identified 95 conserved miRNAs with 19 miRNA*s, 7 novel miRNAs (as well as their corresponding miRNA*s), and 26 potentially novel miRNAs. The analysis identified 15 miRNAs involved in oil and oleic acid production that are differentially expressed during embryogenesis in hickory. Among them, nine miRNA sequences, including eight conserved and one novel, were confirmed by qRT-PCR. In addition, 145 target genes of the novel miRNAs were predicted using a bioinformatic approach. Our results provide a framework for better understanding the roles of miRNAs during embryogenesis in hickory.

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit.

PubMed

Dong, J G; Kim, W T; Yip, W K; Thompson, G A; Li, L; Bennett, A B; Yang, S F

1991-08-01

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)(+) RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3'-end was intact, it lacked a portion of sequence at the 5'-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5'-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.

Phylogeographic analysis of porcine reproductive and respiratory syndrome virus 1 in Guangdong province, Southern China.

PubMed

Zhai, Shao-Lun; Lin, Tao; Zhou, Xia; Pei, Zhang-Fu; Wei, Zu-Zhang; Zhang, He; Wen, Xiao-Hui; Chen, Qin-Ling; Lv, Dian-Hong; Wei, Wen-Kang

2018-05-10

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered an important economic pathogen for the international swine industry. At present, both PRRSV-1 and PRRSV-2 have been confirmed to be co-circulating in China. However, there is little available information about the prevalence or distribution of PRRSV-1 in Guangdong province, southern China. In this study, we performed molecular detection of PRRSV-1 in 750 samples collected from 50 farms in 15 major pig farming regions in this province. After RT-PCR testing, 64% (32/50) of farms were confirmed as PRRSV-1-positive. Surprisingly, PRRSV-1 was circulating on at least one pig farm in all 15 regions; of the 750 samples, 186 samples (24.8%) were positive for PRRSV-1. Furthermore, 15 representative PRRSV-1 ORF5 sequences (606 bp) (n = 1 per region) were obtained from those PRRSV-1-positive regions. Sequence alignment analysis indicated that they shared 81.8% ~ 100% nucleotide and 81.2% ~ 100% amino acid similarity with each other. Although all current PRRSV-1 sequences were divided into pandemic subtype 1, most of them had unique glycoprotein-5 amino acid sequences that are significantly different from other known PRRSV-1 isolates. To conclude, the present findings revealed wide geographical distribution of PRRSV-1 in Guangdong province, southern China. This study further extends the epidemiological significance of PRRSV-1 in China.

Toscana Virus Genome Stability: Data from a Meningoencephalitis Case in Mantua, Italy

PubMed Central

Baggieri, Melissa; Gattuso, Gianni; Fortuna, Claudia; Remoli, Maria Elena; Vaccari, Gabriele; Zaccaria, Guendalina; Marchi, Antonella; Bucci, Paola; Benedetti, Eleonora; Fiorentini, Cristiano; Nicoletti, Loredana

2014-01-01

Abstract In July of 2013, samples from a patient with a neurological syndrome were collected from Mantua hospital and sent to the National Reference Laboratory for Arboviruses (National Institute of Health, Rome). On the basis of the symptoms, serological and molecular assays were performed to diagnose either West Nile virus (WNV) or Toscana virus (TOSV) infection. Molecular and serological tests confirmed TOSV infection. Virus isolation was obtained from cerebrospinal fluid. A full genome sequence was determined from this TOSV strain with next-generation sequencing using Ion Torrent technology. Nucleotide and amino acidic sequences grouped phylogenetically with lineage TOSV A and showed a low genome variability. PMID:25514123

Isolation and sequence analysis of the Pseudomonas syringae pv. tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase.

PubMed

Morris, V L; Jackson, D P; Grattan, M; Ainsworth, T; Cuppels, D A

1995-04-01

Pseudomonas syringae pv. tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings. It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes. We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism. Uptake studies have shown that DC3481 is not deficient in transport. A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced. The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases. However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively). PGMs not requiring the cofactor DPG are usually found in plants and algae. Enzyme assays confirmed that P. syringae PGM activity required an intact ORF1. Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P. syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM. PGM activity appears essential for the growth and pathogenicity of P. syringae pv. tomato on its host plant.

Isolation and sequence analysis of the Pseudomonas syringae pv. tomato gene encoding a 2,3-diphosphoglycerate-independent phosphoglyceromutase.

PubMed Central

Morris, V L; Jackson, D P; Grattan, M; Ainsworth, T; Cuppels, D A

1995-01-01

Pseudomonas syringae pv. tomato DC3481, a Tn5-induced mutant of the tomato pathogen DC3000, cannot grow and elicit disease symptoms on tomato seedlings. It also cannot grow on minimal medium containing malate, citrate, or succinate, three of the major organic acids found in tomatoes. We report here that this mutant also cannot use, as a sole carbon and/or energy source, a wide variety of hexoses and intermediates of hexose catabolism. Uptake studies have shown that DC3481 is not deficient in transport. A 3.8-kb EcoRI fragment of DC3000 DNA, which complements the Tn5 mutation, has been cloned and sequenced. The deduced amino acid sequences of two of the three open reading frames (ORFs) present on this fragment, ORF2 and ORF3, had no significant homology with sequences in the GenBank databases. However, the 510-amino-acid sequence of ORF1, the site of the Tn5 insertion, strongly resembled the deduced amino acid sequences of the Bacillus subtilis and Zea mays genes encoding 2,3-diphosphoglycerate (DPG)-independent phosphoglyceromutase (PGM) (52% identity and 72% similarity and 37% identity and 57% similarity, respectively). PGMs not requiring the cofactor DPG are usually found in plants and algae. Enzyme assays confirmed that P. syringae PGM activity required an intact ORF1. Not only is DC3481 the first PGM-deficient pseudomonad mutant to be described, but the P. syringae pgm gene is the first gram-negative bacterial gene identified that appears to code for a DPG-independent PGM. PGM activity appears essential for the growth and pathogenicity of P. syringae pv. tomato on its host plant. PMID:7896694

High-Throughput rRNA Gene Sequencing Reveals High
and Complex Bacterial Diversity Associated with
Brazilian Coffee Bean Fermentation

PubMed Central

Vinícius de Melo, Gilberto

2018-01-01

Summary Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97% of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing.

Bacteremia Caused by Gordonia bronchialis in a Patient with Sequestrated Lung

PubMed Central

Sng, Li-Hwei; Koh, T. H.; Toney, S. R.; Floyd, M.; Butler, W. R.; Tan, B. H.

2004-01-01

Gordonia species have been recognized as pathogens in immunocompromised and immunocompetent patients. We report the first case of bacteremia due to Gordonia bronchialis in a diabetic patient with a sequestrated lung. Species identification was confirmed with mycolic acid analysis by high-performance liquid chromatography and sequencing of the 16S rRNA gene. PMID:15184495

Fine tangled pili expressed by Haemophilus ducreyi are a novel class of pili.

PubMed Central

Brentjens, R J; Ketterer, M; Apicella, M A; Spinola, S M

1996-01-01

Haemophilus ducreyi synthesizes fine, tangled pili composed predominantly of a protein whose apparent molecular weight is 24,000 (24K). A hybridoma, 2D8, produced a monoclonal antibody (MAb) that bound to a 24K protein in H. ducreyi strains isolated from diverse geographic locations. A lambda gt11 H. ducreyi library was screened with MAb 2D8. A 3.5-kb chromosomal insert from one reactive plaque was amplified and ligated into the pCRII vector. The recombinant plasmid, designated pHD24, expressed a 24K protein in Escherichia coli INV alpha F that bound MAb 2D8. The coding sequence of the 24K gene was localized by exonuclease III digestion. The insert contained a 570-bp open reading frame, designated ftpA (fine, tangled pili). Translation of ftpA predicted a polypeptide with a molecular weight of 21.1K. The predicted N-terminal amino acid sequence of the polypeptide encoded by ftpA was identical to the N-terminal amino acid sequence of purified pilin and lacked a cleavable signal sequence. Primer extension analysis of ftpA confirmed the lack of a leader peptide. The predicted amino acid sequence lacked homology to known pilin sequences but shared homology with the sequences of E. coli Dps and Treponema pallidum antigen TpF1 or 4D, proteins which associate to form ordered rings. An isogenic pilin mutant, H. ducreyi 35000ftpA::mTn3(Cm), was constructed by shuttle mutagenesis and did not contain pili when examined by electron microscopy. We conclude that H. ducreyi synthesizes fine, tangled pili that are composed of a unique major subunit, which may be exported by a signal sequence independent mechanism. PMID:8550517

Some properties and cDNA cloning of proteinaceous toxins from two species of lionfish (Pterois antennata and Pterois volitans).

PubMed

Kiriake, Aya; Shiomi, Kazuo

2011-11-01

Lionfish, members of the genera Pterois, Parapterois and Dendrochirus, are well known to be venomous, having venomous glandular tissues in dorsal, pelvic and anal spines. The lionfish toxins have been shown to cross-react with the stonefish toxins by neutralization tests using the commercial stonefish antivenom, although their chemical properties including structures have been little characterized. In this study, an antiserum against neoverrucotoxin, the stonefish Synanceia verrucosa toxin, was first raised in a guinea pig and used in immunoblotting and inhibition immunoblotting to confirm that two species of Pterois lionfish (P. antennata and P. volitans) contain a 75kDa protein (corresponding to the toxin subunit) cross-reacting with neoverrucotoxin. Then, the amino acid sequences of the P. antennata and P. volitans toxins were successfully determined by cDNA cloning using primers designed from the highly conserved sequences of the stonefish toxins. Notably, either α-subunits (699 amino acid residues) or β-subunits (698 amino acid residues) of the P. antennata and P. volitans toxins share as high as 99% sequence identity with each other. Furthermore, both α- and β-subunits of the lionfish toxins exhibit high sequence identity (70-80% identity) with each other and also with the β-subunits of the stonefish toxins. As reported for the stonefish toxins, the lionfish toxins also contain a B30.2/SPRY domain (comprising nearly 200 amino acid residues) in the C-terminal region of each subunit. Copyright © 2011 Elsevier Ltd. All rights reserved.

Sequence Based Structural Characterization and Genetic Diversity Analysis of Full Length TLR4 CDS in Crossbred and Indigenous Cattle.

PubMed

Mishra, Chinmoy; Kumar, Subodh; Sonwane, Arvind Asaram; Yathish, H M; Chaudhary, Rajni

2017-01-02

The exploration of candidate genes for immune response in cattle may be vital for improving our understanding regarding the species specific response to pathogens. Toll-like receptor 4 (TLR4) is mostly involved in protection against the deleterious effects of Gram negative pathogens. Approximately 2.6 kb long cDNA sequence of TLR4 gene covering the entire coding region was characterized in two Indian milk cattle (Vrindavani and Tharparkar). The phylogenetic analysis confirmed that the bovine TLR4 was apparently evolved from an ancestral form that predated the appearance of vertebrates, and it is grouped with buffalo, yak, and mithun TLR4s. Sequence analysis revealed a 2526-nucleotide long open reading frame (ORF) encoding 841 amino acids, similar to other cattle breeds. The calculated molecular weight of the translated ORF was 96144 and 96040.9 Da; the isoelectric point was 6.35 and 6.42 in Vrindavani and Tharparkar cattle, respectively. The Simple Modular Architecture Research Tool (SMART) analysis identified 14 leucine rich repeats (LRR) motifs in bovine TLR4 protein. The deduced TLR4 amino acid sequence of Tharparkar had 4 different substitutions as compared to Bos taurus, Sahiwal, and Vrindavani. The signal peptide cleavage site predicted to lie between 16th and 17th amino acid of mature peptide. The transmebrane helix was identified between 635-657 amino acids in the mature peptide.

Comparative analysis of barophily-related amino acid content in protein domains of Pyrococcus abyssi and Pyrococcus furiosus.

PubMed

Yafremava, Liudmila S; Di Giulio, Massimo; Caetano-Anollés, Gustavo

2013-01-01

Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.

Sarcocystis spp. Infection in two Red Panda Cubs (Ailurus fulgens).

PubMed

Zoll, W M; Needle, D B; French, S J; Lim, A; Bolin, S; Langohr, I; Agnew, D

2015-01-01

Two neonatal male red panda (Ailurus fulgens) littermates were submitted for necropsy examination. One animal was found dead with no prior signs of illness; the other had a brief history of laboured breathing. Post-mortem examination revealed disseminated protozoal infection. To further characterize the causative agent, transmission electron microscopy (TEM), immunohistochemistry (IHC), polymerase chain reaction (PCR) and amplification and nucleic acid sequencing were performed. IHC was negative for Toxoplasma gondii and Neospora caninum, but was positive for a Sarcocystis spp. TEM of cardiac muscle and lung revealed numerous intracellular apicomplexan protozoa within parasitophorous vacuoles. PCR and nucleic acid sequencing of partial 18S rRNA and the internal transcribed spacer (ITS)-1 region confirmed a Sarcocystis spp. that shared 99% sequence homology to Sarcocystis neurona and Sarcocystis dasypi. This represents the first report of sarcocystosis in red pandas. The histopathological, immunohistochemical, molecular and ultrastructural findings are supportive of vertical transmission resulting in fatal disseminated disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interferon-gamma of the giant panda (Ailuropoda melanoleuca): complementary DNA cloning, expression, and phylogenetic analysis.

PubMed

Tao, Yaqiong; Zeng, Bo; Xu, Liu; Yue, Bisong; Yang, Dong; Zou, Fangdong

2010-01-01

Interferon-gamma (IFN-gamma) is the only member of type II IFN and is vital in the regulation of immune and inflammatory responses. Herein we report the cloning, expression, and sequence analysis of IFN-gamma from the giant panda (Ailuropoda melanoleuca). The open reading frame of this gene is 501 base pair in length and encodes a polypeptide consisting of 166 amino acids. All conserved N-linked glycosylation sites and cysteine residues among carnivores were found in the predicted amino acid sequence of the giant panda. Recombinant giant panda IFN-gamma with a V5 epitope and polyhistidine tag was expressed in HEK293 host cells and confirmed by Western blotting. Phylogenetic analysis of mammalian IFN-gamma-coding sequences indicated that the giant panda IFN-gamma was closest to that of carnivores, then to ungulates and dolphin, and shared a distant relationship with mouse and human. These results represent a first step into the study of IFN-gamma in giant panda.

Peracetic acid-ionic liquid pretreatment to enhance enzymatic saccharification of lignocellulosic biomass.

PubMed

Uju; Abe, Kojiro; Uemura, Nobuyuki; Oshima, Toyoji; Goto, Masahiro; Kamiya, Noriho

2013-06-01

To enhance enzymatic saccharification of pine biomass, the pretreatment reagents peracetic acid (PAA) and ionic liquid (IL) were validated in single reagent pretreatments or combination pretreatments with different sequences. In a 1h saccharification, 5-25% cellulose conversion was obtained from the single pretreatment of PAA or IL. In contrast, a marked enhancement in conversion rates was achieved by PAA-IL combination pretreatments (45-70%). The PAA followed by IL (PAA+IL) pretreatment sequence was the most effective for preparing an enzymatic digestible regenerated biomass with 250-fold higher glucose formation rates than untreated biomass and 2- to 12-fold higher than single pretreatments with PAA or IL alone. Structural analysis confirmed that this pretreatment resulted in biomass with highly porous structural fibers associated with the reduction of lignin content and acetyl groups. Using the PAA+IL sequence, biomass loading in the pretreatment step can be increased from 5% to 15% without significant decrease in cellulose conversion. Copyright © 2013 Elsevier Ltd. All rights reserved.

Homology analysis and cross-immunogenicity of OmpA from pathogenic Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis.

PubMed

Chen, Yuhuang; Duan, Ran; Li, Xu; Li, Kewei; Liang, Junrong; Liu, Chang; Qiu, Haiyan; Xiao, Yuchun; Jing, Huaiqi; Wang, Xin

2015-12-01

The outer membrane protein A (OmpA) is one of the intra-species conserved proteins with immunogenicity widely found in the family of Enterobacteriaceae. Here we first confirmed OmpA is conserved in the three pathogenic Yersinia: Yersinia pestis, Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica, with high homology at the nucleotide level and at the amino acid sequence level. The identity of ompA sequences for 262 Y. pestis strains, 134 Y. pseudotuberculosis strains and 219 pathogenic Y. enterocolitica strains are 100%, 98.8% and 97.7% similar. The main pattern of OmpA of pathogenic Yersinia are 86.2% and 88.8% identical at the nucleotide and amino acid sequence levels, respectively. Immunological analysis showed the immunogenicity of each OmpA and cross-immunogenicity of OmpA for pathogenic Yersinia where OmpA may be a vaccine candidate for Y. pestis and other pathogenic Yersinia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus.

PubMed

Fiore, Nicola; Fajardo, Thor V M; Prodan, Simona; Herranz, María Carmen; Aparicio, Frederic; Montealegre, Jaime; Elena, Santiago F; Pallás, Vicente; Sánchez-Navarro, Jesús

2008-01-01

Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.

Conservation and variability of West Nile virus proteins.

PubMed

Koo, Qi Ying; Khan, Asif M; Jung, Keun-Ok; Ramdas, Shweta; Miotto, Olivo; Tan, Tin Wee; Brusic, Vladimir; Salmon, Jerome; August, J Thomas

2009-01-01

West Nile virus (WNV) has emerged globally as an increasingly important pathogen for humans and domestic animals. Studies of the evolutionary diversity of the virus over its known history will help to elucidate conserved sites, and characterize their correspondence to other pathogens and their relevance to the immune system. We describe a large-scale analysis of the entire WNV proteome, aimed at identifying and characterizing evolutionarily conserved amino acid sequences. This study, which used 2,746 WNV protein sequences collected from the NCBI GenPept database, focused on analysis of peptides of length 9 amino acids or more, which are immunologically relevant as potential T-cell epitopes. Entropy-based analysis of the diversity of WNV sequences, revealed the presence of numerous evolutionarily stable nonamer positions across the proteome (entropy value of < or = 1). The representation (frequency) of nonamers variant to the predominant peptide at these stable positions was, generally, low (< or = 10% of the WNV sequences analyzed). Eighty-eight fragments of length 9-29 amino acids, representing approximately 34% of the WNV polyprotein length, were identified to be identical and evolutionarily stable in all analyzed WNV sequences. Of the 88 completely conserved sequences, 67 are also present in other flaviviruses, and several have been associated with the functional and structural properties of viral proteins. Immunoinformatic analysis revealed that the majority (78/88) of conserved sequences are potentially immunogenic, while 44 contained experimentally confirmed human T-cell epitopes. This study identified a comprehensive catalogue of completely conserved WNV sequences, many of which are shared by other flaviviruses, and majority are potential epitopes. The complete conservation of these immunologically relevant sequences through the entire recorded WNV history suggests they will be valuable as components of peptide-specific vaccines or other therapeutic applications, for sequence-specific diagnosis of a wide-range of Flavivirus infections, and for studies of homologous sequences among other flaviviruses.

Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1

PubMed Central

2014-01-01

Background Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal. Methods V. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). Results All V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498–511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti. Conclusions In 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti. PMID:25022982

Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli.

PubMed

Salony; Garg, N; Baranwal, R; Chhabra, M; Mishra, S; Chaudhuri, T K; Bisaria, V S

2008-02-01

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% alpha-helical, 26% beta-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher beta-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.

Phylogenetic analysis and molecular methods for the detection of lymphocystis disease virus from yellow perch, Perca flavescens (Mitchell).

PubMed

Palmer, L J; Hogan, N S; van den Heuvel, M R

2012-09-01

Lymphocystis disease is a prevalent, non-fatal disease that affects many teleost fish and is caused by the DNA virus lymphocystis disease virus (LCDV). Lymphocystis-like lesions have been observed in yellow perch, Perca flavescens (Mitchell), in lakes in northern Alberta, Canada. In an effort to confirm the identity of the virus causing these lesions, DNA was extracted from these lesions and PCR with genotype generic LCDV primers specific to the major capsid protein (MCP) gene was performed. A 1357-base pair nucleotide sequence corresponding to a peptide length of 452 amino acids of the MCP gene was sequenced, confirming the lesions as being lymphocystis disease lesions. Phylogenetic analysis of the generated amino acid sequence revealed the perch LCDV isolate to be a distinct and novel genotype. From the obtained sequence, a real-time PCR identification method was developed using fluorgenic LUX primers. The identification method was used to detect the presence/absence of LCDV in yellow perch from two lakes, one where lymphocystis disease was observed to occur and the other where the disease had not been observed. All samples of fin, spleen and liver tested negative for LCDV in the lake where lymphocystis disease had not been observed. The second lake had a 2.6% incidence of LCD, and virus was detected in tissue samples from all individuals tested regardless of whether they were expressing the disease or not. However, estimated viral copy number in spleen and liver of symptomatic perch was four orders of magnitude higher than that in asymptomatic perch. © 2012 Blackwell Publishing Ltd.

Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa.

PubMed Central

Bryant, D A; de Lorimier, R; Lambert, D H; Dubbs, J M; Stirewalt, V L; Stevens, S E; Porter, R D; Tam, J; Jay, E

1985-01-01

The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome. PMID:2987916

Isolation and distribution of a novel iron-oxidizing crenarchaeon from acidic geothermal springs in Yellowstone National Park.

PubMed

Kozubal, M; Macur, R E; Korf, S; Taylor, W P; Ackerman, G G; Nagy, A; Inskeep, W P

2008-02-01

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75 degrees C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65 degrees C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80 degrees C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide.

PubMed

Pérez Sirkin, Daniela I; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M; Vissio, Paula G; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide

PubMed Central

Pérez Sirkin, Daniela I.; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M.; Vissio, Paula G.; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation. PMID:28878737

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting.

PubMed

Andrés-Barrao, Cristina; Benagli, Cinzia; Chappuis, Malou; Ortega Pérez, Ruben; Tonolla, Mauro; Barja, François

2013-03-01

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species. Copyright © 2012 Elsevier GmbH. All rights reserved.

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis.

PubMed

Bulau, Patrick; Okuno, Atsuro; Thome, Elke; Schmitz, Tina; Peter-Katalinic, Jasna; Keller, Rainer

2005-11-01

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.

Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles.

PubMed

McMeel, O M; Hoey, E M; Ferguson, A

2001-01-01

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lima, Cassia A.; Sasaki, Sergio D.; Tanaka, Aparecida S.

2006-08-18

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M{sub r} of 11kDa. Bmcystatin gene was cloned in pET 26b vector andmore » the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K{sub i} value of 0.1 and 0.6nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.« less

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus.

PubMed

Lima, Cassia A; Sasaki, Sergio D; Tanaka, Aparecida S

2006-08-18

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.

Burkholderia sacchari DSM 17165: A source of compositionally-tunable block-copolymeric short-chain poly(hydroxyalkanoates) from xylose and levulinic acid.

PubMed

Ashby, Richard D; Solaiman, Daniel K Y; Nuñez, Alberto; Strahan, Gary D; Johnston, David B

2018-04-01

Burkholderia sacchari was used to produce poly-3-hydroxybutyrate-co-3-hydroxyvalerate block copolymers from xylose and levulinic acid. Levulinic acid was the preferred substrate resulting in 3-hydroxyvalerate (3HV) contents as high as 95 mol% at 24 h. The 3HB:3HV ratios were controlled by the initial levulinic acid media concentration and fermentation length. Higher levulinic acid concentrations and longer durations, resulted in polymers with two glass transition temperatures, each approximating those associated with poly-3HB and poly-3HV. 13 C NMR confirmed the presence of high concentrations of 3HB-3HB and 3HV-3HV homopolymeric dyads, while mass spectrometry of the partial hydrolysis products did not conform to Bernoullian statistics for randomness, confirming block sequences. MS/MS analysis of specific oligomers showed the mass-loss of 86 amu (a 3HB unit) and 100 amu (a 3HV unit) attesting to some randomness within the polymers. This study verifies the potential for producing Poly-3HB-block-3HV copolymers from inexpensive biorenewable feedstocks without sequential addition of carbon sources. Published by Elsevier Ltd.

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

PubMed Central

Le Bourhis, Anne-Gaëlle; Saunier, Katiana; Doré, Joël; Carlier, Jean-Philippe; Chamba, Jean-François; Popoff, Michel-Robert; Tholozan, Jean-Luc

2005-01-01

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g. PMID:15640166

Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus L

PubMed Central

Comino, Cinzia; Lanteri, Sergio; Portis, Ezio; Acquadro, Alberto; Romani, Annalisa; Hehn, Alain; Larbat, Romain; Bourgaud, Frédéric

2007-01-01

Background Cynara cardunculus L. is an edible plant of pharmaceutical interest, in particular with respect to the polyphenolic content of its leaves. It includes three taxa: globe artichoke, cultivated cardoon, and wild cardoon. The dominating phenolics are the di-caffeoylquinic acids (such as cynarin), which are largely restricted to Cynara species, along with their precursor, chlorogenic acid (CGA). The scope of this study is to better understand CGA synthesis in this plant. Results A gene sequence encoding a hydroxycinnamoyltransferase (HCT) involved in the synthesis of CGA, was identified. Isolation of the gene sequence was achieved by using a PCR strategy with degenerated primers targeted to conserved regions of orthologous HCT sequences available. We have isolated a 717 bp cDNA which shares 84% aminoacid identity and 92% similarity with a tobacco gene responsible for the biosynthesis of CGA from p-coumaroyl-CoA and quinic acid. In silico studies revealed the globe artichoke HCT sequence clustering with one of the main acyltransferase groups (i.e. anthranilate N-hydroxycinnamoyl/benzoyltransferase). Heterologous expression of the full length HCT (GenBank accession DQ104740) cDNA in E. coli demonstrated that the recombinant enzyme efficiently synthesizes both chlorogenic acid and p-coumaroyl quinate from quinic acid and caffeoyl-CoA or p-coumaroyl-CoA, respectively, confirming its identity as a hydroxycinnamoyl-CoA: quinate HCT. Variable levels of HCT expression were shown among wild and cultivated forms of C. cardunculus subspecies. The level of expression was correlated with CGA content. Conclusion The data support the predicted involvement of the Cynara cardunculus HCT in the biosynthesis of CGA before and/or after the hydroxylation step of hydroxycinnamoyl esters. PMID:17374149

Application of small RNA sequencing to identify microRNAs in acute kidney injury and fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pellegrini, Kathryn L.

Establishing a microRNA (miRNA) expression profile in affected tissues provides an important foundation for the discovery of miRNAs involved in the development or progression of pathologic conditions. We conducted small RNA sequencing to generate a temporal profile of miRNA expression in the kidneys using a mouse model of folic acid-induced (250 mg/kg i.p.) kidney injury and fibrosis. From the 103 miRNAs that were differentially expressed over the time course (> 2-fold, p < 0.05), we chose to further investigate miR-18a-5p, which is expressed during the acute stage of the injury; miR-132-3p, which is upregulated during transition between acute and fibroticmore » injury; and miR-146b-5p, which is highly expressed at the peak of fibrosis. Using qRT-PCR, we confirmed the increased expression of these candidate miRNAs in the folic acid model as well as in other established mouse models of acute injury (ischemia/reperfusion injury) and fibrosis (unilateral ureteral obstruction). In situ hybridization confirmed high expression of miR-18a-5p, miR-132-3p and miR-146b-5p throughout the kidney cortex in mice and humans with severe kidney injury or fibrosis. When primary human proximal tubular epithelial cells were treated with model nephrotoxicants such as cadmium chloride (CdCl{sub 2}), arsenic trioxide, aristolochic acid (AA), potassium dichromate (K{sub 2}Cr{sub 2}O{sub 7}) and cisplatin, miRNA-132-3p was upregulated 4.3-fold after AA treatment and 1.5-fold after K{sub 2}Cr{sub 2}O{sub 7} and CdCl{sub 2} treatment. These results demonstrate the application of temporal small RNA sequencing to identify miR-18a, miR-132 and miR-146b as differentially expressed miRNAs during distinct phases of kidney injury and fibrosis progression. - Highlights: • We used small RNA sequencing to identify differentially expressed miRNAs in kidney. • Distinct patterns were found for acute injury and fibrotic stages in the kidney. • Upregulation of miR-18a, -132 and -146b was confirmed in mice and human kidneys.« less

Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae).

PubMed

Sousa, Juliana C; Berto, Raquel F; Gois, Elicélia A; Fontenele-Cardi, Nauíla C; Honório, José E R; Konno, Katsuhiro; Richardson, Michael; Rocha, Marcos F G; Camargo, Antônio A C M; Pimenta, Daniel C; Cardi, Bruno A; Carvalho, Krishnamurti M

2009-07-01

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C(18) column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 microM, 50 microM, and 75 microM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 microM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing.

PubMed

Huo, Heqiang; Henry, Isabelle M; Coppoolse, Eric R; Verhoef-Post, Miriam; Schut, Johan W; de Rooij, Han; Vogelaar, Aat; Joosen, Ronny V L; Woudenberg, Leo; Comai, Luca; Bradford, Kent J

2016-11-01

Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single-nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild-type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

PubMed

Klaassen, V A; Boeshore, M; Dolja, V V; Falk, B W

1994-07-01

Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.

Confirmation of a novel siadenovirus species detected in raptors: partial sequence and phylogenetic analysis.

PubMed

Kovács, Endre R; Benko, Mária

2009-03-01

Partial genome characterisation of a novel adenovirus, found recently in organ samples of multiple species of dead birds of prey, was carried out by sequence analysis of PCR-amplified DNA fragments. The virus, named as raptor adenovirus 1 (RAdV-1), has originally been detected by a nested PCR method with consensus primers targeting the adenoviral DNA polymerase gene. Phylogenetic analysis with the deduced amino acid sequence of the small PCR product has implied a new siadenovirus type present in the samples. Since virus isolation attempts remained unsuccessful, further characterisation of this putative novel siadenovirus was carried out with the use of PCR on the infected organ samples. The DNA sequence of the central genome part of RAdV-1, encompassing nine full (pTP, 52K, pIIIa, III, pVII, pX, pVI, hexon, protease) and two partial (DNA polymerase and DBP) genes and exceeding 12 kb pairs in size, was determined. Phylogenetic tree reconstructions, based on several genes, unambiguously confirmed the preliminary classification of RAdV-1 as a new species within the genus Siadenovirus. Further study of RAdV-1 is of interest since it represents a rare adenovirus genus of yet undetermined host origin.

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.

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.

NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics

NASA Astrophysics Data System (ADS)

Volpon, Laurent; Tsan, Pascale; Majer, Zsuzsa; Vass, Elemer; Hollósi, Miklós; Noguéra, Valérie; Lancelin, Jean-Marc; Besson, Françoise

2007-08-01

Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven α-amino acids of configuration LDDLLDL and one β-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common β-amino fatty acid 8- L-Asn1- D-Tyr2- D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo ( L-Asp1- D-Tyr2- D-Asn3- L-Ser4- L-Gln5- D-Ser6- L-Thr7-β-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.

Sequence diversity among badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and Nigeria.

PubMed

Eni, A O; Hughes, J d'A; Asiedu, R; Rey, M E C

2008-01-01

We analysed the sequence diversity in the reverse transcriptase (RT)/ribonuclease H (RNaseH) coding region of 19 badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin, and Nigeria. Phylogenetic analysis of the deduced amino acid sequences revealed that the isolates are broadly divided into two distinct species, each clustering with Dioscorea alata bacilliform virus (DaBV) and Dioscorea sansibarensis bacilliform virus (DsBV). Fourteen isolates had 90-96% amino acid identity with DaBV, while four isolates had 83-84% amino acid identity with DsBV. One isolate from Benin, BN4Dr, was distinct and had 77 and 75% amino acid identity with DaBV and DsBV, respectively, and may be a member of a new badnavirus species infecting yam in West Africa. Viruses of the two main species were present in Ghana, Togo and Benin and were observed to infect both D. alata and D. rotundata indiscriminately. This is the first confirmed report of DsBV infection in yam in Ghana and Togo. The results of this study demonstrate that members of two distinct species of badnaviruses infect yam in the West African yam zone and suggest a putative new species, BN4Dr. We also conclude that these species are not confined to limited geographic regions or specific for yam host species. However, the three badnavirus species are serologically related. The sequence information obtained from this study can be used to develop PCR-based diagnostics to detect members of the various species and/or strains of badnaviruses infecting yam in West Africa.

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.

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.

Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

PubMed

Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

2016-09-12

CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function.

The production of Multiple Small Peptaibol Families by Single 14-Module Peptide Synthetases in Trichoderma/Hypocrea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Degenkolb, Thomas; Aghchehb, Razieh Karimi; Dieckmann, Ralf

2012-03-01

The most common peptaibibiotic structures are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of 3 Trichoderma strains of the major clades reveal the presence of up to 3 types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid adding modules. We here provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina) and T. atroviride produces both 11- and 14- residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis ofmore » their amino acid activating domains and modules. The structures of these peptides may be predicted from the gene structures and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 new sequences), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.« less

Prevalence of Escherichia coli sequence type 131 and its H30 subclone among E. coli isolates in a French hospital.

PubMed

Lafolie, Jeremy; Nicolas-Chanoine, Marie-Hélène; Grenouillet, Frédéric; Hocquet, Didier; Bertrand, Xavier

2014-11-01

The prevalence of Escherichia coli sequence type 131 (ST131) and its subclone H30 was assessed among a collection of 490 E. coli isolated in 2013 in a French university hospital. The prevalence of ST131 was 4% among bloodstream isolates (regardless of antimicrobial resistance) and 17.2% among extended-spectrum β-lactamase (ESBL)-producing isolates. Although a much lower prevalence of ST131 was found among bloodstream E. coli isolates compared with other countries, a large predominance of H30 subclone within ST131 was confirmed. It was also confirmed that, among ESBL-producing E. coli, ST131 isolates were more frequently resistant to amoxicillin/clavulanic acid, ceftazidime, fluoroquinolones and aminoglycosides than non-ST131 isolates. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

PubMed

Vera-Cabrera, L; Johnson, W M; Welsh, O; Resendiz-Uresti, F L; Salinas-Carmona, M C

1999-06-01

An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.

Weissella fabaria sp. nov., from a Ghanaian cocoa fermentation.

PubMed

De Bruyne, Katrien; Camu, Nicholas; De Vuyst, Luc; Vandamme, Peter

2010-09-01

Two lactic acid bacteria, strains 257(T) and 252, were isolated from traditional heap fermentations of Ghanaian cocoa beans. 16S rRNA gene sequence analysis of these strains allocated them to the genus Weissella, showing 99.5 % 16S rRNA gene sequence similarity towards Weissella ghanensis LMG 24286(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and biochemical tests confirmed their unique taxonomic position. DNA-DNA hybridization experiments towards their nearest phylogenetic neighbour demonstrated that the two strains represent a novel species, for which we propose the name Weissella fabaria sp. nov., with strain 257(T) (=LMG 24289(T) =DSM 21416(T)) as the type strain. Additional sequence analysis using pheS gene sequences proved useful for identification of all Weissella-Leuconostoc-Oenococcus species and for the recognition of the novel species.

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates.

PubMed

Smail, D A; Bain, N; Bruno, D W; King, J A; Thompson, F; Pendrey, D J; Morrice, S; Cunningham, C O

2006-01-01

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).

Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park

PubMed Central

Hamamura, Natsuko; Olson, Sarah H.; Ward, David M.; Inskeep, William P.

2005-01-01

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the α-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils. PMID:16204508

Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park.

PubMed

Hamamura, Natsuko; Olson, Sarah H; Ward, David M; Inskeep, William P

2005-10-01

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the alpha-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.

The molecular epidemiological study of bovine leukemia virus infection in Myanmar cattle.

PubMed

Polat, Meripet; Moe, Hla Hla; Shimogiri, Takeshi; Moe, Kyaw Kyaw; Takeshima, Shin-Nosuke; Aida, Yoko

2017-02-01

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide and affects both health status and productivity. However, no studies have examined the distribution of BLV in Myanmar, and the genetic characteristics of Myanmar BLV strains are unknown. Therefore, the aim of this study was to detect BLV infection in Myanmar and examine genetic variability. Blood samples were obtained from 66 cattle from different farms in four townships of the Nay Pyi Taw Union Territory of central Myanmar. BLV provirus was detected by nested PCR and real-time PCR targeting BLV long terminal repeats. Results were confirmed by nested PCR targeting the BLV env-gp51 gene and real-time PCR targeting the BLV tax gene. Out of 66 samples, six (9.1 %) were positive for BLV provirus. A phylogenetic tree, constructed using five distinct partial and complete env-gp51 sequences from BLV strains isolated from three different townships, indicated that Myanmar strains were genotype-10. A phylogenetic tree constructed from whole genome sequences obtained by sequencing cloned, overlapping PCR products from two Myanmar strains confirmed the existence of genotype-10 in Myanmar. Comparative analysis of complete genome sequences identified genotype-10-specific amino acid substitutions in both structural and non-structural genes, thereby distinguishing genotype-10 strains from other known genotypes. This study provides information regarding BLV infection levels in Myanmar and confirms that genotype-10 is circulating in Myanmar.

Purification and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171.

PubMed

Kumari, Archana; Akkoç, Nefise; Akçelik, Mustafa

2012-04-01

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.

Replacement of all arginine residues with canavanine in MazF-bs mRNA interferase changes its specificity.

PubMed

Ishida, Yojiro; Park, Jung-Ho; Mao, Lili; Yamaguchi, Yoshihiro; Inouye, Masayori

2013-03-15

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.

RNA sequencing analysis of transcriptional change in the freshwater mussel Elliptio complanata after environmentally relevant sodium chloride exposure

USGS Publications Warehouse

Robertson, Laura S.; Galbraith, Heather S.; Iwanowicz, Deborah; Blakeslee, Carrie J.; Cornman, Robert S.

2017-01-01

To identify potential biomarkers of salt stress in a freshwater sentinel species, we examined transcriptional responses of the common mussel Elliptio complanata to controlled sodium chloride (NaCl) exposures. Ribonucleic acid sequencing (RNA-Seq) of mantle tissue identified 481 transcripts differentially expressed in adult mussels exposed to 2 ppt NaCl (1.2 ppt chloride) for 7 d, of which 290 had nonoverlapping intervals. Differentially expressed gene categories included ion and transmembrane transport, oxidoreductase activity, maintenance of protein folding, and amino acid metabolism. The rate-limiting enzyme for synthesis of taurine, an amino acid frequently linked to osmotic stress in aquatic species, was upregulated, as was the transmembrane ion pump sodium/potassium adenosine 5′-triphosphatase. These patterns confirm a primary transcriptional response to the experimental dose, albeit likely overlapping with nonspecific secondary stress responses. Substantial involvement of the heat shock protein 70 chaperone family and the water-transporting aquaporin family was not detected, however, in contrast to some studies in other bivalves. A subset of the most significantly regulated genes was confirmed by quantitative polymerase chain reaction in an independent sample. Cluster analysis showed separation of mussels exposed to 2 ppt NaCl from control mussels in multivariate space, but mussels exposed to 1 ppt NaCl were largely indistinguishable from controls. Transcriptome-scale analysis of salt exposure under laboratory conditions efficiently identified candidate biomarkers for further functional analysis and field validation

Characterization of capsaicin synthase and identification of its gene (csy1) for pungency factor capsaicin in pepper (Capsicum sp.)

PubMed Central

Prasad, B. C. Narasimha; Kumar, Vinod; Gururaj, H. B.; Parimalan, R.; Giridhar, P.; Ravishankar, G. A.

2006-01-01

Capsaicin is a unique alkaloid of the plant kingdom restricted to the genus Capsicum. Capsaicin is the pungency factor, a bioactive molecule of food and of medicinal importance. Capsaicin is useful as a counterirritant, antiarthritic, analgesic, antioxidant, and anticancer agent. Capsaicin biosynthesis involves condensation of vanillylamine and 8-methyl nonenoic acid, brought about by capsaicin synthase (CS). We found that CS activity correlated with genotype-specific capsaicin levels. We purified and characterized CS (≈35 kDa). Immunolocalization studies confirmed that CS is specifically localized to the placental tissues of Capsicum fruits. Western blot analysis revealed concomitant enhancement of CS levels and capsaicin accumulation during fruit development. We determined the N-terminal amino acid sequence of purified CS, cloned the CS gene (csy1) and sequenced full-length cDNA (981 bp). The deduced amino acid sequence of CS from full-length cDNA was 38 kDa. Functionality of csy1 through heterologous expression in recombinant Escherichia coli was also demonstrated. Here we report the gene responsible for capsaicin biosynthesis, which is unique to Capsicum spp. With this information on the CS gene, speculation on the gene for pungency is unequivocally resolved. Our findings have implications in the regulation of capsaicin levels in Capsicum genotypes. PMID:16938870

Bioaugmentation with Clostridium tyrobutyricum to improve butyric acid production through direct rice straw bioconversion.

PubMed

Chi, Xue; Li, Jianzheng; Wang, Xin; Zhang, Yafei; Leu, Shao-Yuan; Wang, Ying

2018-05-02

One-pot bioconversion is an economically attractive biorefinery strategy to reduce enzyme consumption. Direct conversion of lignocellulosic biomass for butyric acid production is still challenging because of competition among microorganisms. In a consolidated hydrolysis/fermentation bioprocessing (CBP) the microbial structure may eventually prefer the production of caproic acid rather than butyric acid production. This paper presents a new bioaugmentation approach for high butyric acid production from rice straw. By dosing 0.03 g/L of Clostridium tyrobutyricum ATCC 25755 in the CBP, an increase of 226% higher butyric acid was yielded. The selectivity and concentration also increased to 60.7% and 18.05 g/L, respectively. DNA-sequencing confirmed the shift of bacterial community in the augmented CBP. Butyric acid producer was enriched in the bioaugmented bacterial community and the bacteria related to long chain acids production was degenerated. The findings may be useful in future research and process design to enhance productivity of desired bio-products. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gene structure and evolution of transthyretin in the order Chiroptera.

PubMed

Khwanmunee, Jiraporn; Leelawatwattana, Ladda; Prapunpoj, Porntip

2016-02-01

Bats are mammals in the order Chiroptera. Although many extensive morphologic and molecular genetics analyses have been attempted, phylogenetic relationships of bats has not been completely resolved. The paraphyly of microbats is of particular controversy that needs to be confirmed. In this study, we attempted to use the nucleotide sequence of transthyretin (TTR) intron 1 to resolve the relationship among bats. To explore its utility, the complete sequences of TTR gene and intron 1 region of bats in Vespertilionidae: genus Eptesicus (Eptesicus fuscus) and genus Myotis (Myotis brandtii, Myotis davidii, and Myotis lucifugus), and Pteropodidae (Pteropus alecto and Pteropus vampyrus) were extracted from the retrieved sequences, whereas those of Rhinoluphus affinis and Scotophilus kuhlii were amplified and sequenced. The derived overall amino sequences of bat TTRs were found to be very similar to those in other eutherians but differed from those in other classes of vertebrates. However, missing of amino acids from N-terminal or C-terminal region was observed. The phylogenetic analysis of amino acid sequences suggested bat and other eutherian TTRs lineal descent from a single most recent common ancestor which differed from those of non-placental mammals and the other classes of vertebrates. The splicing of bat TTR precursor mRNAs was similar to those of other eutherian but different from those of marsupial, bird, reptile and amphibian. Based on TTR intron 1 sequence, the inferred evolutionary relationship within Chiroptera revealed more closely relatedness of R. affinis to megabats than to microbats. Accordingly, the paraphyly of microbats was suggested.

Transcriptional regulation of fatty acid biosynthesis in mycobacteria

PubMed Central

Mondino, S.; Gago, G.; Gramajo, H.

2013-01-01

SUMMARY The main purpose of our study is to understand how mycobacteria exert control over the biosynthesis of their membrane lipids and find out the key components of the regulatory network that control fatty acid biosynthesis at the transcriptional level. In this paper we describe the identification and purification of FasR, a transcriptional regulator from Mycobacterium sp. that controls the expression of the fatty acid synthase (fas) and the 4-phosphopantetheinyl transferase (acpS) encoding genes, whose products are involved in the fatty acid and mycolic acid biosynthesis pathways. In vitro studies demonstrated that fas and acpS genes are part of the same transcriptional unit and that FasR specifically binds to three conserved operator sequences present in the fas-acpS promoter region (Pfas). The construction and further characterization of a fasR conditional mutant confirmed that FasR is a transcriptional activator of the fas-acpS operon and that this protein is essential for mycobacteria viability. Furthermore, the combined used of Pfas-lacZ fusions in different fasR backgrounds and electrophoretic mobility shift assays experiments, strongly suggested that long-chain acyl-CoAs are the effector molecules that modulate the affinity of FasR for its DNA binding sequences and therefore the expression of the essential fas-acpS operon. PMID:23721164

Method for isolating chromosomal DNA in preparation for hybridization in suspension

DOEpatents

Lucas, Joe N.

2000-01-01

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. Chromosomal DNA in a sample containing cell debris is prepared for hybridization in suspension by treating the mixture with RNase. The treated DNA can also be fixed prior to hybridization.

Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semelr, B.L.; Anderson, C.W.; Hanecak, R.

1982-02-01

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNAmore » sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex.« less

Molecular characterization of infectious bursal disease viruses from Pakistan.

PubMed

Shabbir, Muhammad Zubair; Ali, Muhammad; Abbas, Muhammad; Chaudhry, Umer Naveed; Zia-Ur-Rehman; Munir, Muhammad

2016-07-01

Since the first report of infectious bursal disease in Pakistan in 1987, outbreaks have been common even in vaccinated flocks. Despite appropriate administration of vaccines, concerns arise if the circulating strains are different from the ones used in the vaccine. Here, we sequenced the hypervariable region (HVR) of the VP2 gene of circulating strains of infectious bursal disease virus (IBDV) originating from outbreaks (n = 4) in broiler flocks in Pakistan. Nucleotide sequencing followed by phylogeny and deduced amino acid sequence analysis showed the circulating strains to be very virulent (vv) and identified characteristic residues at position 222 (A), 242 (I), 256 (I), 294 (I) and 299 (S). In addition, a substitution at positions 221 (Q→H) was found to be exclusive to Pakistani strains in our analysis, although a larger dataset is required to confirm this finding. Compared to vaccine strains that are commonly used in Pakistan, substitution mutations were found at key amino acid positions in VP2 that may be responsible for potential changes in neutralization epitopes and vaccine failure.

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park▿ †

PubMed Central

Kozubal, M.; Macur, R. E.; Korf, S.; Taylor, W. P.; Ackerman, G. G.; Nagy, A.; Inskeep, W. P.

2008-01-01

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP. PMID:18083851

The regulatory network of cluster-root function and development in phosphate-deficient white lupin (Lupinus albus) identified by transcriptome sequencing.

PubMed

Wang, Zhengrui; Straub, Daniel; Yang, Huaiyu; Kania, Angelika; Shen, Jianbo; Ludewig, Uwe; Neumann, Günter

2014-07-01

Lupinus albus serves as model plant for root-induced mobilization of sparingly soluble soil phosphates via the formation of cluster-roots (CRs) that mediate secretion of protons, citrate, phenolics and acid phosphatases (APases). This study employed next-generation sequencing to investigate the molecular mechanisms behind these complex adaptive responses at the transcriptome level. We compared different stages of CR development, including pre-emergent (PE), juvenile (JU) and the mature (MA) stages. The results confirmed that the primary metabolism underwent significant modifications during CR maturation, promoting the biosynthesis of organic acids, as had been deduced from physiological studies. Citrate catabolism was downregulated, associated with citrate accumulation in MA clusters. Upregulation of the phenylpropanoid pathway reflected the accumulation of phenolics. Specific transcript expression of ALMT and MATE transporter genes correlated with the exudation of citrate and flavonoids. The expression of transcripts related to nucleotide degradation and APases in MA clusters coincided with the re-mobilization and hydrolysis of organic phosphate resources. Most interestingly, hormone-related gene expression suggested a central role of ethylene during CR maturation. This was associated with the upregulation of the iron (Fe)-deficiency regulated network that mediates ethylene-induced expression of Fe-deficiency responses in other species. Finally, transcripts related to abscisic acid and jasmonic acid were upregulated in MA clusters, while auxin- and brassinosteroid-related genes and cytokinin receptors were most strongly expressed during CR initiation. Key regulations proposed by the RNA-seq data were confirmed by quantitative real-time polymerase chain reaction (RT-qPCR) and some physiological analyses. A model for the gene network regulating CR development and function is presented. © 2014 Scandinavian Plant Physiology Society.

Marked Genomic Diversity of Norovirus Genogroup I Strains in a Waterborne Outbreak

PubMed Central

Hannoun, Charles; Larsson, Charlotte U.; Bergström, Tomas

2012-01-01

Marked norovirus (NoV) diversity was detected in patient samples from a large community outbreak of gastroenteritis with waterborne epidemiology affecting approximately 2,400 people. NoV was detected in 33 of 50 patient samples examined by group-specific real-time reverse transcription-PCR. NoV genotype I (GI) strains predominated in 31 patients, with mixed GI infections occurring in 5 of these patients. Sequence analysis of RNA-dependent polymerase-N/S capsid-coding regions (∼900 nucleotides in length) confirmed the dominance of the GI strains (n = 36). Strains of NoV GI.4 (n = 21) and GI.7 (n = 9) were identified, but six strains required full capsid amino acid analyses (530 to 550 amino acids) based on control sequencing of cloned amplicons before the virus genotype could be determined. Three strains were assigned to a new NoV GI genotype, proposed as GI.9, based on capsid amino acid analyses showing 26% dissimilarity from the established genotypes GI.1 to GI.8. Three other strains grouped in a sub-branch of GI.3 with 13 to 15% amino acid dissimilarity to GI.3 GenBank reference strains. Phylogenetic analysis (2.1 kb) of 10 representative strains confirmed these genotype clusters. Strains of NoV GII.4 (n = 1), NoV GII.6 (n = 2), sapovirus GII.2 (n = 1), rotavirus (n = 3), adenovirus (n = 1), and Campylobacter spp. (n = 2) were detected as single infections or as mixtures with NoV GI. Marked NoV GI diversity detected in patients was consistent with epidemiologic evidence of waterborne NoV infections, suggesting human fecal contamination of the water supply. Recognition of NoV diversity in a cluster of patients provided a useful warning marker of waterborne contamination in the Lilla Edet outbreak. PMID:22247153

DNA Sequence Polymorphism of the Lactate Dehydrogenase Genefrom Iranian Plasmodium vivax and Plasmodium falciparum Isolates.

PubMed

Getacher Feleke, Daniel; Nateghpour, Mehdi; Motevalli Haghi, Afsaneh; Hajjaran, Homa; Farivar, Leila; Mohebali, Mehdi; Raoofian, Reza

2015-01-01

Parasite lactate dehydrogenase (pLDH) is extensively employed as malaria rapid diagnostic tests (RDTs). Moreover, it is a well-known drug target candidate. However, the genetic diversity of this gene might influence performance of RDT kits and its drug target candidacy. This study aimed to determine polymorphism of pLDH gene from Iranian isolates of P. vivax and P. falciparum. Genomic DNA was extracted from whole blood of microscopically confirmed P. vivax and P. falciparum infected patients. pLDH gene of P. falciparum and P. vivax was amplified using conventional PCR from 43 symptomatic malaria patients from Sistan and Baluchistan Province, Southeast Iran from 2012 to 2013. Sequence analysis of 15 P. vivax LDH showed fourteen had 100% identity with P. vivax Sal-1 and Belem strains. Two nucleotide substitutions were detected with only one resulted in amino acid change. Analysis of P. falciparum LDH sequences showed six of the seven sequences had 100% homology with P. falciparum 3D7 and Mzr-1. Moreover, PfLDH displayed three nucleotide changes that resulted in changing only one amino acid. PvLDH and PfLDH showed 75%-76% nucleotide and 90.4%-90.76% amino acid homology. pLDH gene from Iranian P. falciparum and P. vivax isolates displayed 98.8-100% homology with 1-3 nucleotide substitutions. This indicated this gene was relatively conserved. Additional studies can be done weather this genetic variation can influence the performance of pLDH based RDTs or not.

Structure-based conformational preferences of amino acids

PubMed Central

Koehl, Patrice; Levitt, Michael

1999-01-01

Proteins can be very tolerant to amino acid substitution, even within their core. Understanding the factors responsible for this behavior is of critical importance for protein engineering and design. Mutations in proteins have been quantified in terms of the changes in stability they induce. For example, guest residues in specific secondary structures have been used as probes of conformational preferences of amino acids, yielding propensity scales. Predicting these amino acid propensities would be a good test of any new potential energy functions used to mimic protein stability. We have recently developed a protein design procedure that optimizes whole sequences for a given target conformation based on the knowledge of the template backbone and on a semiempirical potential energy function. This energy function is purely physical, including steric interactions based on a Lennard-Jones potential, electrostatics based on a Coulomb potential, and hydrophobicity in the form of an environment free energy based on accessible surface area and interatomic contact areas. Sequences designed by this procedure for 10 different proteins were analyzed to extract conformational preferences for amino acids. The resulting structure-based propensity scales show significant agreements with experimental propensity scale values, both for α-helices and β-sheets. These results indicate that amino acid conformational preferences are a natural consequence of the potential energy we use. This confirms the accuracy of our potential and indicates that such preferences should not be added as a design criterion. PMID:10535955

Characterization of papain-like isoenzymes from latex of Asclepias curassavica by molecular biology validated by proteomic approach.

PubMed

Obregón, Walter D; Liggieri, Constanza S; Trejo, Sebastian A; Avilés, Francesc X; Vairo-Cavalli, Sandra E; Priolo, Nora S

2009-01-01

Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders. These pharmacological actions have been attributed to the presence of cysteine proteases in these milky latices. Asclepias curassavica (Asclepiadaceae), "scarlet milkweed" is a perennial subshrub native to South America. In the current paper we report a new approach directed at the selective biochemical and molecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for the proteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases were digested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. No identification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceae latex cysteine proteinases available in databases. From total RNA extracted from latex samples, cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained by cloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequences of 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII, characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain the full-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment and phylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group of papain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMF could be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with very similar physicochemical and functional properties, with advantages over other conventional methods (for instance enzyme kinetics) that are time consuming and afford less reliable results.

Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Claffey, K.P.; Herrera, V.L.; Brecher, P.

1987-12-01

A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a lambda gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundantmore » mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source.« less

[Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

PubMed

Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

2003-02-01

Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

Burkholderia cordobensis sp. nov., from agricultural soils.

PubMed

Draghi, Walter O; Peeters, Charlotte; Cnockaert, Margo; Snauwaert, Cindy; Wall, Luis G; Zorreguieta, Angeles; Vandamme, Peter

2014-06-01

Two Gram-negative, rod-shaped bacteria were isolated from agricultural soils in Córdoba province in central Argentina. Their 16S rRNA gene sequences demonstrated that they belong to the genus Burkholderia, with Burkholderia zhejiangensis as most closely related formally named species; this relationship was confirmed through comparative gyrB sequence analysis. Whole-cell fatty acid analysis supported their assignment to the genus Burkholderia. Burkholderia sp. strain YI23, for which a whole-genome sequence is available, represents the same taxon, as demonstrated by its highly similar 16S rRNA (100% similarity) and gyrB (99.1-99.7%) gene sequences. The results of DNA-DNA hybridization experiments and physiological and biochemical characterization further substantiated the genotypic and phenotypic distinctiveness of the Argentinian soil isolates, for which the name Burkholderia cordobensis sp. nov. is proposed, with strain MMP81(T) ( = LMG 27620(T) = CCUG 64368(T)) as the type strain. © 2014 IUMS.

A novel ABCD1 mutation detected by next generation sequencing in presumed hereditary spastic paraplegia: A 30-year diagnostic delay caused by misleading biochemical findings.

PubMed

Koutsis, Georgios; Lynch, David S; Tucci, Arianna; Houlden, Henry; Karadima, Georgia; Panas, Marios

2015-08-15

To present a Greek family in which 5 male and 2 female members developed progressive spastic paraplegia. Plasma very long chain fatty acids (VLCFA) were reportedly normal at first testing in an affected male and for over 30 years the presumed diagnosis was hereditary spastic paraplegia (HSP). Targeted next generation sequencing (NGS) was used as a further diagnostic tool. Targeted exome sequencing in the proband, followed by Sanger sequencing confirmation; mutation segregation testing in multiple family members and plasma VLCFA measurement in the proband. NGS of the proband revealed a novel frameshift mutation in ABCD1 (c.1174_1178del, p.Leu392Serfs*7), bringing an end to diagnostic uncertainty by establishing the diagnosis of adrenomyeloneuropathy (AMN), the myelopathic phenotype of X-linked adrenoleukodystrophy (ALD). The mutation segregated in all family members and the diagnosis of AMN/ALD was confirmed by plasma VLCFA measurement. Confounding factors that delayed the diagnosis are presented. This report highlights the diagnostic utility of NGS in patients with undiagnosed spastic paraplegia, establishing a molecular diagnosis of AMN, allowing proper genetic counseling and management, and overcoming the diagnostic delay that can be rarely caused by false negative VLCFA analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Pantoea allii sp. nov., isolated from onion plants and seed.

PubMed

Brady, Carrie L; Goszczynska, Teresa; Venter, Stephanus N; Cleenwerck, Ilse; De Vos, Paul; Gitaitis, Ronald D; Coutinho, Teresa A

2011-04-01

Eight yellow-pigmented, Gram-negative, rod-shaped, oxidase-negative, motile, facultatively anaerobic bacteria were isolated from onion seed in South Africa and from an onion plant exhibiting centre rot symptoms in the USA. The isolates were assigned to the genus Pantoea on the basis of phenotypic and biochemical tests. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), based on gyrB, rpoB, infB and atpD sequences, confirmed the allocation of the isolates to the genus Pantoea. MLSA further indicated that the isolates represented a novel species, which was phylogenetically most closely related to Pantoea ananatis and Pantoea stewartii. Amplified fragment length polymorphism analysis also placed the isolates into a cluster separate from P. ananatis and P. stewartii. Compared with type strains of species of the genus Pantoea that showed >97 % 16S rRNA gene sequence similarity with strain BD 390(T), the isolates exhibited 11-55 % whole-genome DNA-DNA relatedness, which confirmed the classification of the isolates in a novel species. The most useful phenotypic characteristics for the differentiation of the isolates from their closest phylogenetic neighbours are production of acid from amygdalin and utilization of adonitol and sorbitol. A novel species, Pantoea allii sp. nov., is proposed, with type strain BD 390(T) ( = LMG 24248(T)).

C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus.

PubMed

Li, Zhong; Yalcin, Talat; Cassady, Carolyn J

2006-07-01

Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.

Structure characterization of lipocyclopeptide antibiotics, aspartocins A, B & C, by ESI-MSMS and ESI-nozzle-skimmer-MSMS.

PubMed

Siegel, Marshall M; Kong, Fangming; Feng, Xidong; Carter, Guy T

2009-12-01

Three lipocyclopeptide antibiotics, aspartocins A (1), B (2), and C (3), were obtained from the aspartocin complex by HPLC separation methodology. Their structures were elucidated using previously published chemical degradation results coupled with spectroscopic studies including ESI-MS, ESI-Nozzle Skimmer-MSMS and NMR. All three aspartocin compounds share the same cyclic decapeptide core of cyclo [Dab2 (Asp1-FA)-Pip3-MeAsp4-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11]. They differ only in the fatty acid side chain moiety (FA) corresponding to (Z)-13-methyltetradec-3-ene-carbonyl, (+,Z)-12-methyltetradec-3-ene-carbonyl and (Z)-12-methyltridec-3-ene-carbonyl for aspartocins A (1), B (2), and C (3), respectively. All of the sequence ions were observed by ESI-MSMS of the doubly charged parent ions. However, a number of the sequence ions observed were of low abundance. To fully sequence the lipocyclopeptide antibiotic structures, these low abundance sequence ions together with complementary sequence ions were confirmed by ESI-Nozzle-Skimmer-MSMS of the singly charged linear peptide parent fragment ions H-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11-Dab2(1+)-Asp1-FA. Cyclization of the aspartocins was demonstrated to occur via the beta-amino group of Dab2 from ions of moderate intensity in the ESI-MSMS spectra. As the fatty acid moieties do not undergo internal fragmentations under the experimental ESI mass spectral conditions used, the 14 Da mass difference between the fatty acid moieties of aspartocins A (1) and B (2) versus aspartocin C (3) was used as an internal mass tag to differentiate fragment ions containing fatty acid moieties and those not containing the fatty acid moieties. The most numerous and abundant fragment ions observed in the tandem mass spectra are due to the cleavage of the tertiary nitrogen amide of the pipecolic acid residue-3 (16 fragment ions) and the proline residue-11 (7 fragment ions). In addition, the neutral loss of ethanimine from alpha,beta-diaminobutyric acid residue 9 was observed for the parent molecular ion and for 7 fragment ions. Copyright 2009 John Wiley & Sons, Ltd.

Functional Genomics Analysis of Singapore Grouper Iridovirus: Complete Sequence Determination and Proteomic Analysis

PubMed Central

Song, Wen Jun; Qin, Qi Wei; Qiu, Jin; Huang, Can Hua; Wang, Fan; Hew, Choy Leong

2004-01-01

Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products. PMID:15507645

Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus (Digenea): Species Differentiation Based On mtDNA (Barcode) and Partial LSU–rDNA Sequences

USGS Publications Warehouse

Bergmame, Laura; Huffman, Jane; Cole, Rebecca; Dayanandan, Selvadurai; Tkach, Vasyl; McLaughlin, J. Daniel

2011-01-01

Flukes belonging to Sphaeridiotrema are important parasites of waterfowl, and 2 morphologically similar species Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus, have been implicated in waterfowl mortality in North America. Cytochrome oxidase I (barcode region) and partial LSU-rDNA sequences from specimens of S. globulus and S. pseudoglobulus, obtained from naturally and experimentally infected hosts from New Jersey and Quebec, respectively, confirmed that these species were distinct. Barcode sequences of the 2 species differed at 92 of 590 nucleotide positions (15.6%) and the translated sequences differed by 13 amino acid residues. Partial LSU-rDNA sequences differed at 29 of 1,208 nucleotide positions (2.4%). Additional barcode sequences from specimens collected from waterfowl in Wisconsin and Minnesota and morphometric data obtained from specimens acquired along the north shore of Lake Superior revealed the presence of S. pseudoglobulus in these areas. Although morphometric data suggested the presence of S. globulus in the Lake Superior sample, it was not found among the specimens sequenced from Wisconsin or Minnesota.

Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus (Digenea): Species Differentiation Based on mtDNA (Barcode) and Partial LSUrDNA Sequences

USGS Publications Warehouse

Bergmame, L.; Huffman, J.; Cole, R.; Dayanandan, S.; Tkach, V.; McLaughlin, J.D.

2011-01-01

Flukes belonging to Sphaeridiotrema are important parasites of waterfowl, and 2 morphologically similar species Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus, have been implicated in waterfowl mortality in North America. Cytochrome oxidase I (barcode region) and partial LSU-rDNA sequences from specimens of S. globulus and S. pseudoglobulus, obtained from naturally and experimentally infected hosts from New Jersey and Quebec, respectively, confirmed that these species were distinct. Barcode sequences of the 2 species differed at 92 of 590 nucleotide positions (15.6%) and the translated sequences differed by 13 amino acid residues. Partial LSU-rDNA sequences differed at 29 of 1,208 nucleotide positions (2.4%). Additional barcode sequences from specimens collected from waterfowl in Wisconsin and Minnesota and morphometric data obtained from specimens acquired along the north shore of Lake Superior revealed the presence of S. pseudoglobulus in these areas. Although morphometric data suggested the presence of S. globulus in the Lake Superior sample, it was not found among the specimens sequenced from Wisconsin or Minnesota. ?? 2011 American Society of Parasitologists.

A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364

DOE Office of Scientific and Technical Information (OSTI.GOV)

Littrell, BobbiJo R

The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm.more » Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364 is phosphorylated by a PKC-dependent mechanism.« less

[Molecular epidemiological analysis of rubella virus isolates from 2001 to 2011 in Shanghai, China].

PubMed

Li, Chong-Shan; Yang, Yu-Ying; Wang, Jian-Guo; Zhu, Zhen; Tang, Wei; Li, Zhi; Sun, Xiao-Dong; Xu, Wen-Bo

2012-03-01

Throat swabs collected from patients whose serum was measles IgM negative and rubella IgM positive during 2001-2011 were used to conduct cell culture for rubella virus. After identification of cell culture with RT-PCR, nucleotide of gene E1 of rubella virus was amplified and sequenced, followed by molecular epidemiological analysis. A total of 31 rubella viruses were isolated from 60 throat swabs. Compared 27 isolates with the WHO reference strains of all genotypes, phylogenetic tree was constructed based on the amplified 739 nucleotide fragment. These isolates belonged to two different genotypes respectively. Isolates 11009, 11052 and 11106 in 2011 belonged to genotype 2B, and others belonged to genotype 1E. Most of mutations were nonsense mutation, and sequence of amino acid was highly conserved. Amino acid sequence of most isolates of genotype 1E was identical, which suggested rubella viruses from same transmission chain might be transmitted continually since 2001. Rubella virus genotype 2B was found to be popular for the first time in Shanghai in 2011. The nucleotide sequences of these genotype 2B isolates showed 99% identity compared with that of isolates recently from Vietnam, Japan and Argentina. The resources of these strains were not confirmed due to the absence of rubella virus surveillance before.

Nucleic acid arrays and methods of synthesis

DOEpatents

Sabanayagam, Chandran R.; Sano, Takeshi; Misasi, John; Hatch, Anson; Cantor, Charles

2001-01-01

The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.

Assessment of Epstein-Barr virus nucleic acids in gastric but not in breast cancer by next-generation sequencing of pooled Mexican samples

PubMed Central

Fuentes-Pananá, Ezequiel M; Larios-Serrato, Violeta; Méndez-Tenorio, Alfonso; Morales-Sánchez, Abigail; Arias, Carlos F; Torres, Javier

2016-01-01

Gastric (GC) and breast (BrC) cancer are two of the most common and deadly tumours. Different lines of evidence suggest a possible causative role of viral infections for both GC and BrC. Wide genome sequencing (WGS) technologies allow searching for viral agents in tissues of patients with cancer. These technologies have already contributed to establish virus-cancer associations as well as to discovery new tumour viruses. The objective of this study was to document possible associations of viral infection with GC and BrC in Mexican patients. In order to gain idea about cost effective conditions of experimental sequencing, we first carried out an in silico simulation of WGS. The next-generation-platform IlluminaGallx was then used to sequence GC and BrC tumour samples. While we did not find viral sequences in tissues from BrC patients, multiple reads matching Epstein-Barr virus (EBV) sequences were found in GC tissues. An end-point polymerase chain reaction confirmed an enrichment of EBV sequences in one of the GC samples sequenced, validating the next-generation sequencing-bioinformatics pipeline. PMID:26910355

Assessment of Epstein-Barr virus nucleic acids in gastric but not in breast cancer by next-generation sequencing of pooled Mexican samples.

PubMed

Fuentes-Pananá, Ezequiel M; Larios-Serrato, Violeta; Méndez-Tenorio, Alfonso; Morales-Sánchez, Abigail; Arias, Carlos F; Torres, Javier

2016-03-01

Gastric (GC) and breast (BrC) cancer are two of the most common and deadly tumours. Different lines of evidence suggest a possible causative role of viral infections for both GC and BrC. Wide genome sequencing (WGS) technologies allow searching for viral agents in tissues of patients with cancer. These technologies have already contributed to establish virus-cancer associations as well as to discovery new tumour viruses. The objective of this study was to document possible associations of viral infection with GC and BrC in Mexican patients. In order to gain idea about cost effective conditions of experimental sequencing, we first carried out an in silico simulation of WGS. The next-generation-platform IlluminaGallx was then used to sequence GC and BrC tumour samples. While we did not find viral sequences in tissues from BrC patients, multiple reads matching Epstein-Barr virus (EBV) sequences were found in GC tissues. An end-point polymerase chain reaction confirmed an enrichment of EBV sequences in one of the GC samples sequenced, validating the next-generation sequencing-bioinformatics pipeline.

Retention of nucleic acids in ion-pair reversed-phase high-performance liquid chromatography depends not only on base composition but also on base sequence.

PubMed

Qiao, Jun-Qin; Liang, Chao; Wei, Lan-Chun; Cao, Zhao-Ming; Lian, Hong-Zhen

2016-12-01

The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C 18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insights into the diversity of eukaryotes in acid mine drainage biofilm communities.

PubMed

Baker, Brett J; Tyson, Gene W; Goosherst, Lindsey; Banfield, Jillian F

2009-04-01

Microscopic eukaryotes are known to have important ecosystem functions, but their diversity in most environments remains vastly unexplored. Here we analyzed an 18S rRNA gene library from a subsurface iron- and sulfur-oxidizing microbial community growing in highly acidic (pH < 0.9) runoff within the Richmond Mine at Iron Mountain (northern California). Phylogenetic analysis revealed that the majority (68%) of the sequences belonged to fungi. Protists falling into the deeply branching lineage named the acidophilic protist clade (APC) and the class Heterolobosea were also present. The APC group represents kingdom-level novelty, with <76% sequence similarity to 18S rRNA gene sequences of organisms from other environments. Fluorescently labeled oligonucleotide rRNA probes were designed to target each of these groups in biofilm samples, enabling abundance and morphological characterization. Results revealed that the populations vary significantly with the habitat and no group is ubiquitous. Surprisingly, many of the eukaryotic lineages (with the exception of the APC) are closely related to neutrophiles, suggesting that they recently adapted to this extreme environment. Molecular analyses presented here confirm that the number of eukaryotic species associated with the acid mine drainage (AMD) communities is low. This finding is consistent with previous results showing a limited diversity of archaea, bacteria, and viruses in AMD environments and suggests that the environmental pressures and interplay between the members of these communities limit species diversity at all trophic levels.

Amino Acid Substitution in Trichophyton rubrum Squalene Epoxidase Associated with Resistance to Terbinafine

PubMed Central

Osborne, Colin S.; Leitner, Ingrid; Favre, Bertrand; Ryder, Neil S.

2005-01-01

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE. PMID:15980358

Identification of Group B Streptococcal Sip Protein, Which Elicits Cross-Protective Immunity

PubMed Central

Brodeur, Bernard R.; Boyer, Martine; Charlebois, Isabelle; Hamel, Josée; Couture, France; Rioux, Clément R.; Martin, Denis

2000-01-01

A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the sip gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6.84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the sip gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate. PMID:10992461

Mercury remediation potential of a mercury resistant strain Sphingopyxis sp. SE2 isolated from contaminated soil.

PubMed

Mahbub, Khandaker Rayhan; Krishnan, Kannan; Naidu, Ravi; Megharaj, Mallavarapu

2017-01-01

A mercury resistant bacterial strain SE2 was isolated from contaminated soil. The 16s rRNA gene sequencing confirms the strain as Sphingopyxis belongs to the Sphingomonadaceae family of the α-Proteobacteria group. The isolate showed high resistance to mercury with estimated concentrations of Hg that caused 50% reduction in growth (EC 50 ) of 5.97 and 6.22mg/L and minimum inhibitory concentrations (MICs) of 32.19 and 34.95mg/L in minimal and rich media, respectively. The qualitative detection of volatilized mercury and the presence of mercuric reductase enzyme proved that the strain SE2 can potentially remediate mercury. ICP-QQQ-MS analysis of the remaining mercury in experimental broths indicated that a maximum of 44% mercury was volatilized within 6hr by live SE2 culture. Furthermore a small quantity (23%) of mercury was accumulated in live cell pellets. While no volatilization was caused by dead cells, sorption of mercury was confirmed. The mercuric reductase gene merA was amplified and sequenced. Homology was observed among the amino acid sequences of mercuric reductase enzyme of different organisms from α-Proteobacteria and ascomycota groups. Copyright © 2016. Published by Elsevier B.V.

Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes.

PubMed

Diallinas, G; Gorfinkiel, L; Arst, H N; Cecchetto, G; Scazzocchio, C

1995-04-14

In Aspergillus nidulans, loss-of-function mutations in the uapA and azgA genes, encoding the major uric acid-xanthine and hypoxanthine-adenine-guanine permeases, respectively, result in impaired utilization of these purines as sole nitrogen sources. The residual growth of the mutant strains is due to the activity of a broad specificity purine permease. We have identified uapC, the gene coding for this third permease through the isolation of both gain-of-function and loss-of-function mutations. Uptake studies with wild-type and mutant strains confirmed the genetic analysis and showed that the UapC protein contributes 30% and 8-10% to uric acid and hypoxanthine transport rates, respectively. The uapC gene was cloned, its expression studied, its sequence and transcript map established, and the sequence of its putative product analyzed. uapC message accumulation is: (i) weakly induced by 2-thiouric acid; (ii) repressed by ammonium; (iii) dependent on functional uaY and areA regulatory gene products (mediating uric acid induction and nitrogen metabolite repression, respectively); (iv) increased by uapC gain-of-function mutations which specifically, but partially, suppress a leucine to valine mutation in the zinc finger of the protein coded by the areA gene. The putative uapC gene product is a highly hydrophobic protein of 580 amino acids (M(r) = 61,251) including 12-14 putative transmembrane segments. The UapC protein is highly similar (58% identity) to the UapA permease and significantly similar (23-34% identity) to a number of bacterial transporters. Comparisons of the sequences and hydropathy profiles of members of this novel family of transporters yield insights into their structure, functionally important residues, and possible evolutionary relationships.

Molecular dynamics simulations of the auxin-binding protein 1 in complex with indole-3-acetic acid and naphthalen-1-acetic acid.

PubMed

Grandits, Melanie; Oostenbrink, Chris

2014-10-01

Auxin-binding protein 1 (ABP1) is suggested to be an auxin receptor which plays an important role in several processes in green plants. Maize ABP1 was simulated with the natural auxin indole-3-acetic acid (IAA) and the synthetic analog naphthalen-1-acetic acid (NAA), to elucidate the role of the KDEL sequence and the helix at the C-terminus. The KDEL sequence weakens the intermolecular interactions between the monomers but stabilizes the C-terminal helix. Conformational changes at the C-terminus occur within the KDEL sequence and are influenced by the binding of the simulated ligands. This observation helps to explain experimental findings on ABP1 interactions with antibodies that are modulated by the presence of auxin, and supports the hypothesis that ABP1 acts as an auxin receptor. Stable hydrogen bonds between the monomers are formed between Glu40 and Glu62, Arg10 and Thr97, Lys39, and Glu62 in all simulations. The amino acids Ile22, Leu25, Trp44, Pro55, Ile130, and Phe149 are located in the binding pocket and are involved in hydrophobic interactions with the ring system of the ligand. Trp151 is stably involved in a face to end interaction with the ligand. The calculated free energy of binding using the linear interaction energy approach showed a higher binding affinity for NAA as compared to IAA. Our simulations confirm the asymmetric behavior of the two monomers, the stronger interaction of NAA than IAA and offers insight into the possible mechanism of ABP1 as an auxin receptor. © 2014 Wiley Periodicals, Inc.

The emergence and evolution of life in a "fatty acid world" based on quantum mechanics.

PubMed

Tamulis, Arvydas; Grigalavicius, Mantas

2011-02-01

Quantum mechanical based electron correlation interactions among molecules are the source of the weak hydrogen and Van der Waals bonds that are critical to the self-assembly of artificial fatty acid micelles. Life on Earth or elsewhere could have emerged in the form of self-reproducing photoactive fatty acid micelles, which gradually evolved into nucleotide-containing micelles due to the enhanced ability of nucleotide-coupled sensitizer molecules to absorb visible light. Comparison of the calculated absorption spectra of micelles with and without nucleotides confirmed this idea and supports the idea of the emergence and evolution of nucleotides in minimal cells of a so-called Fatty Acid World. Furthermore, the nucleotide-caused wavelength shift and broadening of the absorption pattern potentially gives these molecules an additional valuable role, other than a purely genetic one in the early stages of the development of life. From the information theory point of view, the nucleotide sequences in such micelles carry positional information providing better electron transport along the nucleotide-sensitizer chain and, in addition, providing complimentary copies of that information for the next generation. Nucleotide sequences, which in the first period of evolution of fatty acid molecules were useful just for better absorbance of the light in the longer wavelength region, later in the PNA or RNA World, took on the role of genetic information storage.

A natural mutation-led truncation in one of the two aluminum-activated malate transporter-like genes at the Ma locus is associated with low fruit acidity in apple.

PubMed

Bai, Yang; Dougherty, Laura; Li, Mingjun; Fazio, Gennaro; Cheng, Lailiang; Xu, Kenong

2012-08-01

Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150 kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65-82 kb containing 12-19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of genotype Mama) that cover the two distinct haplotypes at the Ma locus. Gene expression profiling in 18 apple germplasm accessions suggested that Ma1 is the major determinant at the Ma locus controlling fruit acidity as Ma1 is expressed at a much higher level than Ma2 and the Ma1 expression is significantly correlated with fruit titratable acidity (R (2) = 0.4543, P = 0.0021). In the coding sequences of low acidity alleles of Ma1 and Ma2, sequence variations at the amino acid level between Golden Delicious and G.41 were not detected. But the alleles for high acidity vary considerably between the two genotypes. The low acidity allele of Ma1, Ma1-1455A, is mainly characterized by a mutation at base 1455 in the open reading frame. The mutation leads to a premature stop codon that truncates the carboxyl terminus of Ma1-1455A by 84 amino acids compared with Ma1-1455G. A survey of 29 apple germplasm accessions using marker CAPS(1455) that targets the SNP(1455) in Ma1 showed that the CAPS(1455A) allele was associated completely with high pH and highly with low titratable acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma for low pH or high acidity in apple.

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

... for nucleotide and/or amino acid sequence data. 1.822 Section 1.822 Patents, Trademarks, and... Amino Acid Sequences § 1.822 Symbols and format to be used for nucleotide and/or amino acid sequence data. (a) The symbols and format to be used for nucleotide and/or amino acid sequence data shall...

Sequencing of T-superfamily conotoxins from Conus virgo: pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry.

PubMed

Mandal, Amit Kumar; Ramasamy, Mani Ramakrishnan Santhana; Sabareesh, Varatharajan; Openshaw, Matthew E; Krishnan, Kozhalmannom S; Balaram, Padmanabhan

2007-08-01

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH(2), and Vi1361, ZCCPTMPECCRI-NH(2), which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation of w(n)- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

A new missense mutation in the BCKDHB gene causes the classic form of maple syrup urine disease (MSUD).

PubMed

Miryounesi, Mohammad; Ghafouri-Fard, Soudeh; Goodarzi, Hamedreza; Fardaei, Majid

2015-05-01

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disease caused by mutations in the BCKDHA, BCKDHB, DBT and DLD genes, which encode the E1α, E1β, E2 and E3 subunits of the branched chain α ketoacid dehydrogenase (BCKD) complex, respectively. This complex is involved in the metabolism of branched-chain amino acids. In this study, we analyzed the DNA sequences of BCKDHA and BCKDHB genes in an infant who suffered from MSUD and died at the age of 6 months. We found a new missense mutation in exon 5 of BCKDHB gene (c.508C>T). The heterozygosity of the parents for the mentioned nucleotide change was confirmed by direct sequence analysis of the corresponding segment. Another missense mutation has been found in the same codon previously and shown by in silico analyses to be deleterious. This report provides further evidence that this amino acid change can cause classic MSUD.

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. Copyright © 2015. Published by Elsevier GmbH.

Single substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin.

PubMed

Rasoolizadeh, Asieh; Goulet, Marie-Claire; Sainsbury, Frank; Cloutier, Conrad; Michaud, Dominique

2016-04-01

A causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection. © 2016 Federation of European Biochemical Societies.

Identification and Analysis of Jasmonate Pathway Genes in Coffea canephora (Robusta Coffee) by In Silico Approach.

PubMed

Bharathi, Kosaraju; Sreenath, H L

2017-07-01

Coffea canephora is the commonly cultivated coffee species in the world along with Coffea arabica . Different pests and pathogens affect the production and quality of the coffee. Jasmonic acid (JA) is a plant hormone which plays an important role in plants growth, development, and defense mechanisms, particularly against insect pests. The key enzymes involved in the production of JA are lipoxygenase, allene oxide synthase, allene oxide cyclase, and 12-oxo-phytodienoic reductase. There is no report on the genes involved in JA pathway in coffee plants. We made an attempt to identify and analyze the genes coding for these enzymes in C. canephora . First, protein sequences of jasmonate pathway genes from model plant Arabidopsis thaliana were identified in the National Center for Biotechnology Information (NCBI) database. These protein sequences were used to search the web-based database Coffee Genome Hub to identify homologous protein sequences in C. canephora genome using Basic Local Alignment Search Tool (BLAST). Homologous protein sequences for key genes were identified in the C. canephora genome database. Protein sequences of the top matches were in turn used to search in NCBI database using BLAST tool to confirm the identity of the selected proteins and to identify closely related genes in species. The protein sequences from C. canephora database and the top matches in NCBI were aligned, and phylogenetic trees were constructed using MEGA6 software and identified the genetic distance of the respective genes. The study identified the four key genes of JA pathway in C. canephora , confirming the conserved nature of the pathway in coffee. The study expected to be useful to further explore the defense mechanisms of coffee plants. JA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC, and OPR are identified in C. canephora (robusta coffee) by bioinformatic approaches confirming the conserved nature of the pathway in coffee. The findings are useful to understand the defense mechanisms of C. canephora and coffee breeding in the long run. JA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC and OPR were identified and analyzed in C. canephora (robusta coffee) by in silico approach. The study has confirmed the conserved nature of JA pathway in coffee; the findings are useful to further explore the defense mechanisms of coffee plants. Abbreviations used: C. canephora : Coffea canephora ; C. arabica : Coffea arabica ; JA: Jasmonic acid; CGH: Coffee Genome Hub; NCBI: National Centre for Biotechnology Information; BLAST: Basic Local Alignment Search Tool; A. thaliana : Arabidopsis thaliana ; LOX: Lipoxygenase, AOS: Allene oxide synthase; AOC: Allene oxide cyclase; OPR: 12 oxo phytodienoic reductase.

Verification of Ribosomal Proteins of Aspergillus fumigatus for Use as Biomarkers in MALDI-TOF MS Identification.

PubMed

Nakamura, Sayaka; Sato, Hiroaki; Tanaka, Reiko; Yaguchi, Takashi

2016-01-01

We have previously proposed a rapid identification method for bacterial strains based on the profiles of their ribosomal subunit proteins (RSPs), observed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method can perform phylogenetic characterization based on the mass of housekeeping RSP biomarkers, ideally calculated from amino acid sequence information registered in public protein databases. With the aim of extending its field of application to medical mycology, this study investigates the actual state of information of RSPs of eukaryotic fungi registered in public protein databases through the characterization of ribosomal protein fractions extracted from genome-sequenced Aspergillus fumigatus strains Af293 and A1163 as a model. In this process, we have found that the public protein databases harbor problems. The RSP names are in confusion, so we have provisionally unified them using the yeast naming system. The most serious problem is that many incorrect sequences are registered in the public protein databases. Surprisingly, more than half of the sequences are incorrect, due chiefly to mis-annotation of exon/intron structures. These errors could be corrected by a combination of in silico inspection by sequence homology analysis and MALDI-TOF MS measurements. We were also able to confirm conserved post-translational modifications in eleven RSPs. After these verifications, the masses of 31 expressed RSPs under 20,000 Da could be accurately confirmed. These RSPs have a potential to be useful biomarkers for identifying clinical isolates of A. fumigatus .

New insights into plant glycoside hydrolase family 32 in Agave species

PubMed Central

Avila de Dios, Emmanuel; Gomez Vargas, Alan D.; Damián Santos, Maura L.; Simpson, June

2015-01-01

In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana. PMID:26300895

New insights into plant glycoside hydrolase family 32 in Agave species.

PubMed

Avila de Dios, Emmanuel; Gomez Vargas, Alan D; Damián Santos, Maura L; Simpson, June

2015-01-01

In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

Probiotic Potential of Lactobacillus Strains with Antifungal Activity Isolated from Animal Manure.

PubMed

Ilavenil, Soundharrajan; Park, Hyung Soo; Vijayakumar, Mayakrishnan; Arasu, Mariadhas Valan; Kim, Da Hye; Ravikumar, Sivanesan; Choi, Ki Choon

2015-01-01

The aim of the study was to isolate and characterize the lactic acid bacteria (LAB) from animal manure. Among the thirty LAB strains, four strains, namely, KCC-25, KCC-26, KCC-27, and KCC-28, showed good cell growth and antifungal activity and were selected for further characterization. Biochemical and physiology properties of strains confirmed that the strains are related to the Lactobacillus sp.; further, the 16S rRNA sequencing confirmed 99.99% sequence similarity towards Lactobacillus plantarum. The strains exhibited susceptibility against commonly used antibiotics with negative hemolytic property. Strains KCC-25, KCC-26, KCC-27, and KCC-28 showed strong antifungal activity against Aspergillus fumigatus, Penicillium chrysogenum, Penicillium roqueforti, Botrytis elliptica, and Fusarium oxysporum, respectively. Fermentation studies noted that the strains were able to produce significant amount of lactic, acetic, and succinic acids. Further, the production of extracellular proteolytic and glycolytic enzymes, survival under low pH, bile salts, and gastric juice together with positive bile salt hydrolase (Bsh) activity, cholesterol lowering, cell surface hydrophobicity, and aggregation properties were the strains advantages. Thus, KCC-25, KCC-26, KCC-27, and KCC-28 could have the survival ability in the harsh condition of the digestive system in the gastrointestinal tract. In conclusion, novel L. plantarum KCC-25, KCC-26, KCC-27, and KCC-28 could be considered as potential antimicrobial probiotic strains.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-06-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed Central

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-01-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. Finally, we expect the design rules that we uncovered and the thioesterase variants that we identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain-length oleochemicals.« less

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE PAGES

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.; ...

2017-04-20

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. Finally, we expect the design rules that we uncovered and the thioesterase variants that we identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain-length oleochemicals.« less

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.

Novel Group of Leaderless Multipeptide Bacteriocins from Gram-Positive Bacteria.

PubMed

Ovchinnikov, Kirill V; Chi, Hai; Mehmeti, Ibrahim; Holo, Helge; Nes, Ingolf F; Diep, Dzung B

2016-09-01

From raw milk we found 10 Lactococcus garvieae isolates that produce a new broad-spectrum bacteriocin. Though the isolates were obtained from different farms, they turned out to possess identical inhibitory spectra, fermentation profiles of sugars, and repetitive sequence-based PCR (rep-PCR) DNA patterns, indicating that they produce the same bacteriocin. One of the isolates (L. garvieae KS1546) was chosen for further assessment. Purification and peptide sequencing combined with genome sequencing revealed that the antimicrobial activity was due to a bacteriocin unit composed of three similar peptides of 32 to 34 amino acids. The three peptides are produced without leader sequences, and their genes are located next to each other in an operon-like structure, adjacent to the genes normally involved in bacteriocin transport (ABC transporter) and self-immunity. The bacteriocin, termed garvicin KS (GarKS), showed sequence homology to four multipeptide bacteriocins in databases: the known staphylococcal aureocin A70, consisting of four peptides, and three unannotated putative multipeptide bacteriocins produced by Bacillus cereus All these multipeptide bacteriocin loci show conserved genetic organization, including being located adjacent to conserved genetic determinants (Cro/cI and integrase) which are normally associated with mobile genetic elements or genome rearrangements. The antimicrobial activity of all multipeptide bacteriocins was confirmed with synthetic peptides, and all were shown to have broad antimicrobial spectra, with GarKS being the most active of them. The inhibitory spectrum of GarKS includes important pathogens belonging to the genera Staphylococcus, Bacillus, Listeria, and Enterococcus Bacterial resistance to antibiotics is a very serious global problem. There are no new antibiotics with novel antimicrobial mechanisms in clinical trials. Bacteriocins use antimicrobial mechanisms different from those of antibiotics and can kill antibiotic-resistant bacteria, but the number of bacteriocins with very broad antimicrobial spectra is very small. In this study, we have found and purified a novel three-peptide bacteriocin, garvicin KS. By homology search, we were able to find one known and three novel sequence-related bacteriocins consisting of 3 or 4 peptides. None of the peptides has modified amino acids in its sequence. Thus, the activity of all bacteriocins was confirmed with chemically synthesized peptides. All of them, especially garvicin KS, have very broad antibacterial spectra, thus representing a great potential in antimicrobial applications in the food industry and medicine. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Novel Group of Leaderless Multipeptide Bacteriocins from Gram-Positive Bacteria

PubMed Central

Chi, Hai; Mehmeti, Ibrahim; Holo, Helge; Nes, Ingolf F.

2016-01-01

ABSTRACT From raw milk we found 10 Lactococcus garvieae isolates that produce a new broad-spectrum bacteriocin. Though the isolates were obtained from different farms, they turned out to possess identical inhibitory spectra, fermentation profiles of sugars, and repetitive sequence-based PCR (rep-PCR) DNA patterns, indicating that they produce the same bacteriocin. One of the isolates (L. garvieae KS1546) was chosen for further assessment. Purification and peptide sequencing combined with genome sequencing revealed that the antimicrobial activity was due to a bacteriocin unit composed of three similar peptides of 32 to 34 amino acids. The three peptides are produced without leader sequences, and their genes are located next to each other in an operon-like structure, adjacent to the genes normally involved in bacteriocin transport (ABC transporter) and self-immunity. The bacteriocin, termed garvicin KS (GarKS), showed sequence homology to four multipeptide bacteriocins in databases: the known staphylococcal aureocin A70, consisting of four peptides, and three unannotated putative multipeptide bacteriocins produced by Bacillus cereus. All these multipeptide bacteriocin loci show conserved genetic organization, including being located adjacent to conserved genetic determinants (Cro/cI and integrase) which are normally associated with mobile genetic elements or genome rearrangements. The antimicrobial activity of all multipeptide bacteriocins was confirmed with synthetic peptides, and all were shown to have broad antimicrobial spectra, with GarKS being the most active of them. The inhibitory spectrum of GarKS includes important pathogens belonging to the genera Staphylococcus, Bacillus, Listeria, and Enterococcus. IMPORTANCE Bacterial resistance to antibiotics is a very serious global problem. There are no new antibiotics with novel antimicrobial mechanisms in clinical trials. Bacteriocins use antimicrobial mechanisms different from those of antibiotics and can kill antibiotic-resistant bacteria, but the number of bacteriocins with very broad antimicrobial spectra is very small. In this study, we have found and purified a novel three-peptide bacteriocin, garvicin KS. By homology search, we were able to find one known and three novel sequence-related bacteriocins consisting of 3 or 4 peptides. None of the peptides has modified amino acids in its sequence. Thus, the activity of all bacteriocins was confirmed with chemically synthesized peptides. All of them, especially garvicin KS, have very broad antibacterial spectra, thus representing a great potential in antimicrobial applications in the food industry and medicine. PMID:27316965

A novel, highly divergent ssDNA virus identified in Brazil infecting apple, pear and grapevine.

PubMed

Basso, Marcos Fernando; da Silva, José Cleydson Ferreira; Fajardo, Thor Vinícius Martins; Fontes, Elizabeth Pacheco Batista; Zerbini, Francisco Murilo

2015-12-02

Fruit trees of temperate and tropical climates are of great economical importance worldwide and several viruses have been reported affecting their productivity and longevity. Fruit trees of different Brazilian regions displaying virus-like symptoms were evaluated for infection by circular DNA viruses. Seventy-four fruit trees were sampled and a novel, highly divergent, monopartite circular ssDNA virus was cloned from apple, pear and grapevine trees. Forty-five complete viral genomes were sequenced, with a size of approx. 3.4 kb and organized into five ORFs. Deduced amino acid sequences showed identities in the range of 38% with unclassified circular ssDNA viruses, nanoviruses and alphasatellites (putative Replication-associated protein, Rep), and begomo-, curto- and mastreviruses (putative coat protein, CP, and movement protein, MP). A large intergenic region contains a short palindromic sequence capable of forming a hairpin-like structure with the loop sequence TAGTATTAC, identical to the conserved nonanucleotide of circoviruses, nanoviruses and alphasatellites. Recombination events were not detected and phylogenetic analysis showed a relationship with circo-, nano- and geminiviruses. PCR confirmed the presence of this novel ssDNA virus in field plants. Infectivity tests using the cloned viral genome confirmed its ability to infect apple and pear tree seedlings, but not Nicotiana benthamiana. The name "Temperate fruit decay-associated virus" (TFDaV) is proposed for this novel virus. Copyright © 2015 Elsevier B.V. All rights reserved.

Site-directed mutagenesis of Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin: effect on polyhedron structure.

PubMed

Bravo-Patiño, A; Ibarra, J E

2000-01-01

Amino acids Lys34, His36, and Phe37 were substituted by PCR-mediated, site-directed mutagenesis for three Trp's in the AcNPV polyhedrin sequence. Phase contrast microscopy revealed refringent, amorphous polyhedra in the nuclei of infected cells. Electron microscopy confirmed a great variation in form and size of the mutated polyhedra. Although crystallization of the mutated polyhedrin occurred, it was irregular within each polyhedron. Virion occlusion was also severely affected. Virions were partially occluded, or only one virion was occluded per polyhedron. Results suggest that the substitution of these three amino acids affected the morphology of polyhedra, the uniformity of crystallization within each polyhedron, and the virion occlusion.

Influence of physicochemical treatments on iron-based spent catalyst for catalytic oxidation of toluene.

PubMed

Kim, Sang Chai; Shim, Wang Geun

2008-06-15

The catalytic oxidation of toluene was studied over an iron-based spent and regenerated catalysts. Air, hydrogen, or four different acid solutions (oxalic acid (C2H2O4), citric acid (C6H8O7), acetic acid (CH3COOH), and nitric acid (HNO3)) were employed to regenerate the spent catalyst. The properties of pretreated spent catalyst were characterized by the Brunauer Emmett Teller (BET), inductively coupled plasma (ICP), temperature programmed reduction (TPR), and X-ray diffraction (XRD) analyses. The air pretreatment significantly enhanced the catalytic activity of the spent catalyst in the pretreatment temperature range of 200-400 degrees C, but its catalytic activity diminished at the pretreatment temperature of 600 degrees C. The catalytic activity sequence with respect to the air pretreatment temperatures was 400 degrees C>200 degrees C>parent>600 degrees C. The TPR results indicated that the catalytic activity was correlated with both the oxygen mobility and the amount of available oxygen on the catalyst. In contrast, the hydrogen pretreatment had a negative effect on the catalytic activity, and toluene conversion decreased with increasing pretreatment temperatures (200-600 degrees C). The XRD and TPR results confirmed the formation of metallic iron which had a negative effect on the catalytic activity with increasing pretreatment temperature. The acid pretreatment improved the catalytic activity of the spent catalyst. The catalytic activity sequence with respect to different acids pretreatment was found to be oxalic acid>citric acid>acetic acid>or=nitric acid>parent. The TPR results of acid pretreated samples showed an increased amount of available oxygen which gave a positive effect on the catalytic activity. Accordingly, air or acid pretreatments were more promising methods of regenerating the iron-based spent catalyst. In particular, the oxalic acid pretreatment was found to be most effective in the formation of FeC2O4 species which contributed highly to the catalytic combustion of toluene.

Moraxella catarrhalis synthesizes an autotransporter that is an acid phosphatase.

PubMed

Hoopman, Todd C; Wang, Wei; Brautigam, Chad A; Sedillo, Jennifer L; Reilly, Thomas J; Hansen, Eric J

2008-02-01

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

Moraxella catarrhalis Synthesizes an Autotransporter That Is an Acid Phosphatase▿

PubMed Central

Hoopman, Todd C.; Wang, Wei; Brautigam, Chad A.; Sedillo, Jennifer L.; Reilly, Thomas J.; Hansen, Eric J.

2008-01-01

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10−10) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity. PMID:18065547

Prospecting Russula senecis: a delicacy among the tribes of West Bengal

PubMed Central

Khatua, Somanjana; Dutta, Arun Kumar

2015-01-01

Russula senecis, a worldwide distributed mushroom, is exclusively popular among the tribal communities of West Bengal for food purposes. The present study focuses on its reliable taxonomic identification through macro- and micro-morphological features, DNA barcoding, confirmation of its systematic placement by phylogenetic analyses, myco-chemicals and functional activities. For the first time, the complete Internal Transcribed Spacer region of R. senecis has been sequenced and its taxonomic position within subsection Foetentinae under series Ingratae of the subgen. Ingratula is confirmed through phylogenetic analysis. For exploration of its medicinal properties, dried basidiocarps were subjected for preparation of a heat stable phenol rich extract (RusePre) using water and ethanol as solvent system. The antioxidant activity was evaluated through hydroxyl radical scavenging (EC50 5 µg/ml), chelating ability of ferrous ion (EC50 0.158 mg/ml), DPPH radical scavenging (EC50 1.34 mg/ml), reducing power (EC50 2.495 mg/ml) and total antioxidant activity methods (13.44 µg ascorbic acid equivalent/mg of extract). RusePre exhibited antimicrobial potentiality against Listeria monocytogenes, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Furthermore, different parameters were tested to investigate its chemical composition, which revealed the presence of appreciable quantity of phenolic compounds, along with carotenoids and ascorbic acid. HPLC-UV fingerprint indicated the probable existence of at least 13 phenolics, of which 10 were identified (pyrogallol > kaempferol > quercetin > chlorogenic acid > ferulic acid, cinnamic acid > vanillic acid > salicylic acid > p-coumaric acid > gallic acid). Result from the present work suggests that the fraction, RusePre, may open novel prospect as a functional ingredient in antioxidant supplements and in drugs to treat infectious disease. PMID:25780764

Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex.

PubMed

Abdelkafi, Slim; Ogata, Hiroyuki; Barouh, Nathalie; Fouquet, Benjamin; Lebrun, Régine; Pina, Michel; Scheirlinckx, Frantz; Villeneuve, Pierre; Carrière, Frédéric

2009-11-01

An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser(35)-Asp(307)-His(310)) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.

[Identification of lactic acid bacteria in commercial yogurt and their antibiotic resistance].

PubMed

Qin, Yuxuan; Li, Jing; Wang, Qiuya; Gao, Kexin; Zhu, Baoli; Lv, Na

2013-08-04

To identify lactic acid bacteria (LAB) in commercial yogurts and investigate their antibiotic resistance. LABs were cultured from 5 yogurt brands and the isolates were identified at the species level by 16S rRNA sequence. Genotyping was performed by repetitive extragenic palindromic PCR (rep-PCR). The sensitivity to 7 antibiotics was tested for all LAB isolates by Kirby-Bauer paper diffusion (K-B method). Meanwhile, 9 antibiotic resistance genes (ARGs), including erythromycin resistance genes (ermA and ermB) and tetracycline resistance genes (tetM, tetK, tetS, tetQ, tetO, tetL and tetW), were detected by PCR amplification in the identified LAB isolates. The PCR products were confirmed by sequencing. Total 100 LABs were isolated, including 23 Lactobacillus delbrueckii ssp. bulgaricus, 26 Lactobacillus casei, 30 Streptococcus thermophilus, 5 Lactobacillus acidophilus, 6 Lactobacillus plantarum, and 10 Lactobacillus paracasei. The drug susceptibility test shows that all 100 isolates were resistant to gentamicin and streptomycin, 42 isolates were resistant to vancomycin, and on the contrary all were sensitive to cefalexin, erythromycin, tetracycline and oxytetracycline. Moreover, 5 ARGs were found in the 28 sequencing confirmed isolates, ermB gene was detected in 8 isolates, tet K in 4 isolates, tetL in 2 isolates, tetM in 4 isolates, tetO in 2 isolates. erm A, tet S, tet Q and tet W genes were not detected in the isolates. Antibiotic resistance genes were found in 53.57% (15/28) sequenced isolates, 2 -3 antibiotic resistance genes were detected in 4 isolates of L. delbrueckii ssp. bulgaricus. Some LABs were not labeled in commercial yogurt products. Antibiotic resistance genes tend to be found in the starter culture of L. delbrueckii ssp. Bulgaricus and S. thermophilus. All the LAB isolates were sensitive to erythromycin and tetracycline, even though some carried erythromycin and/or tetracycline resistance genes. We proved again that LAB could carry antibiotic resistance gene(s) though it is sensitive to antibiotics.

Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne

PubMed Central

Welker, Frido; Hajdinjak, Mateja; Talamo, Sahra; Jaouen, Klervia; Dannemann, Michael; David, Francine; Julien, Michèle; Meyer, Matthias; Barnes, Ian; Brace, Selina; Kamminga, Pepijn; Fischer, Roman; Kessler, Benedikt M.; Stewart, John R.; Pääbo, Svante; Collins, Matthew J.; Hublin, Jean-Jacques

2016-01-01

In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this “transitional” assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal–Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne. PMID:27638212

Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne.

PubMed

Welker, Frido; Hajdinjak, Mateja; Talamo, Sahra; Jaouen, Klervia; Dannemann, Michael; David, Francine; Julien, Michèle; Meyer, Matthias; Kelso, Janet; Barnes, Ian; Brace, Selina; Kamminga, Pepijn; Fischer, Roman; Kessler, Benedikt M; Stewart, John R; Pääbo, Svante; Collins, Matthew J; Hublin, Jean-Jacques

2016-10-04

In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this "transitional" assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal-Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne.

Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus

PubMed Central

2010-01-01

Background Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. Results Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. Conclusions The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes. PMID:20534175

Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus.

PubMed

Chulakasian, Songkhla; Lee, Min-Shiuh; Wang, Chi-Young; Chiou, Shyan-Song; Lin, Kuan-Hsun; Lin, Fong-Yuan; Hsu, Tien-Huan; Wong, Min-Liang; Chang, Tien-Jye; Hsu, Wei-Li

2010-06-10

Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

PubMed Central

Kumar, Rajinder; Adams, Brian; Oldenburg, Anja; Musiyenko, Alla; Barik, Sailen

2002-01-01

Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA) led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes. PMID:12057017

The gastrin/cholecystokinin-B receptor on prostate cells--a novel target for bifunctional prostate cancer imaging.

PubMed

Sturzu, Alexander; Klose, Uwe; Sheikh, Sumbla; Echner, Hartmut; Kalbacher, Hubert; Deeg, Martin; Nägele, Thomas; Schwentner, Christian; Ernemann, Ulrike; Heckl, Stefan

2014-02-14

The means of identifying prostate carcinoma and its metastases are limited. The contrast agents used in magnetic resonance imaging clinical diagnostics are not taken up into the tumor cells, but only accumulate in the interstitial space of the highly vasculated tumor. We examined the gastrin/cholecystokinin-B receptor as a possible target for prostate-specific detection using the C-terminal seven amino acid sequence of the gastrin peptide hormone. The correct sequence and a scrambled control sequence were coupled to the fluorescent dye rhodamine and the magnetic resonance imaging contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Expression analysis of the gastrin receptor mRNA was performed by reverse transcriptase polymerase chain reaction on PC3 prostate carcinoma cells, U373 glioma, U2OS osteosarcoma and Colo205 colon carcinoma cells. After having confirmed elevated expression of gastrin receptor in PC3 cells and very low expression of the receptor in Colo205 cells, these two cell lines were used to create tumor xenografts on nude mice for in vivo experiments. Confocal lasers scanning microscopy and magnetic resonance imaging showed a high specificity of the correct conjugate for the PC3 xenografts. Staining of the PC3 xenografts was much weaker with the scrambled conjugate while the Colo205 xenografts showed no marked staining with any of the conjugates. In vitro experiments comparing the correct and scrambled conjugates on PC3 cells by magnetic resonance relaxometry and fluorescence-activated cell sorting confirmed markedly higher specificity of the correct conjugate. The investigations show that the gastrin receptor is a promising tumor cell surface target for future prostate-cancer-specific imaging applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus).

PubMed

Johne, Reimar; Dremsek, Paul; Kindler, Eveline; Schielke, Anika; Plenge-Bönig, Anita; Gregersen, Henrike; Wessels, Ute; Schmidt, Katja; Rietschel, Wolfram; Groschup, Martin H; Guenther, Sebastian; Heckel, Gerald; Ulrich, Rainer G

2012-07-01

Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals. Copyright © 2012 Elsevier B.V. All rights reserved.

Vitellogenin Receptor Mutation Leads to the Oogenesis Mutant Phenotype “scanty vitellin” of the Silkworm, Bombyx mori*

PubMed Central

Lin, Ying; Meng, Yan; Wang, Yan-Xia; Luo, Juan; Katsuma, Susumu; Yang, Cong-Wen; Banno, Yutaka; Kusakabe, Takahiro; Shimada, Toru; Xia, Qing-You

2013-01-01

In insects, the vitellogenin receptor (VgR) mediates the uptake of vitellogenin (Vg) from the hemolymph by developing oocytes. The oogenesis mutant scanty vitellin (vit) of Bombyx mori (Bm) lacks vitellin and 30-kDa proteins, but B. mori egg-specific protein and BmVg are normal. The vit eggs are white and smaller compared with the pale yellow eggs of the wild type and are embryonic lethal. This study found that a mutation in the B. mori VgR gene (BmVgR) is responsible for the vit phenotype. We cloned the cDNA sequences encoding WT and vit BmVgR. The functional domains of BmVgR are similar to those of other low-density lipoprotein receptors. When compared with the wild type, a 235-bp genomic sequence in vit BmVgR is substituted for a 7-bp sequence. This mutation has resulted in a 50-amino acid deletion in the third Class B region of the first epidermal growth factor (EGF1) domain. BmVgR is expressed specifically in oocytes, and the transcriptional level is changed dramatically and consistently with maturation of oocytes during the previtellogenic periods. Linkage analysis confirmed that BmVgR is mutated in the vit mutant. The coimmunoprecipitation assay confirmed that mutated BmVgR is able to bind BmVg but that BmVg cannot be dissociated under acidic conditions. The WT phenotype determined by RNA interference was similar to that of the vit phenotype for nutritional deficiency, such as BmVg and 30-kDa proteins. These results showed that BmVgR has an important role in transporting proteins for egg formation and embryonic development in B. mori. PMID:23515308

The twilight zone of cis element alignments.

PubMed

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-02-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein-DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein-DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments.

The twilight zone of cis element alignments

PubMed Central

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-01-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein–DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein–DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments. PMID:23268451

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.

Helicobacter pylori Heat Shock Protein A: Serologic Responses and Genetic Diversity

PubMed Central

Ng, Enders K. W.; Thompson, Stuart A.; Pérez-Pérez, Guillermo I.; Kansau, Imad; van der Ende, Arie; Labigne, Agnès; Sung, Joseph J. Y.; Chung, S. C. Sydney; Blaser, Martin J.

1999-01-01

Helicobacter pylori synthesizes an unusual GroES homolog, heat shock protein A (HspA). The present study was aimed at an assessment of the serological response to HspA in a group of Chinese patients with defined gastroduodenal pathologies and determination of whether diversity is present in the nucleotide sequences encoding HspA in isolates from these patients. Serum samples collected from 154 patients who had an upper gastrointestinal pathology and the presence of H. pylori defined by biopsy were tested for an immunoglobulin G (IgG) serologic response to H. pylori HspA by an enzyme linked immunosorbant assay. HspA-encoding nucleotide sequences in H. pylori isolates from 14 patients (7 seropositive and 7 seronegative for HspA) were analyzed by PCR and direct sequencing of the PCR products. The sequencing results were compared to those of 48 isolates from other parts of the world. Of the 154 known H. pylori-positive patients, 54 (35.1%) were seropositive for HspA. The A domain (GroES homology) of HspA was highly conserved in the 14 isolates tested. Although the B domain (metal-binding site unique to H. pylori) resembled that in the known major variant, particular amino acid substitutions allowed definition of an HspA variant associated with isolates from East Asia. There were no associations between patient characteristics and HspA seropositivity or amino acid sequences. We confirmed in this study that the clinical outcomes of H. pylori infection are not related to HspA antigenicity or to sequence variation. However, B-domain sequence variation may be a marker for the study of the genetic diversity of H. pylori strains of different geographic origins. PMID:10225839

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

Purification and characterization of Campylobacter rectus surface layer proteins.

PubMed Central

Nitta, H; Holt, S C; Ebersole, J L

1997-01-01

Campylobacter rectus is a putative periodontopathogen which expresses a proteinaceous surface layer (S-layer) external to the outer membrane. S-layers are considered to play a protective role for the microorganism in hostile environments. The S-layer proteins from six different C. rectus strains (five human isolates and a nonhuman primate [NHP] isolate) were isolated, purified, and characterized. The S-layer proteins of these strains varied in molecular mass (ca. 150 to 166 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They all reacted with monospecific rabbit antiserum to the purified S-layer of C. rectus 314, but a quantitative enzyme-linked immunosorbent assay demonstrated a strong antigenic relationship among the five human strains, while the NHP strain, 6250, showed weaker reactivity. Amino acid composition analysis showed that the S-layers of four C. rectus strains contained large proportions of acidic amino acids (13 to 27%) and that >34% of the amino acid residues were hydrophobic. Amino acid sequence analysis of six S-layer proteins revealed that the first 15 amino-terminal amino acids were identical and showed seven residues of identity with the amino-terminal sequence of the Campylobacter fetus S-layer protein SapA1. CNBr peptide profiles of the S-layer proteins from C. rectus 314, ATCC 33238, and 6250 confirmed that the S-layer proteins from the human strains were similar to each other and somewhat different from that of the NHP isolate (strain 6250). However, the S-layer proteins from the two human isolates do show some structural heterogeneity. For example, there was a 17-kDa fragment unique to the C. rectus 314 S-layer. The amino-terminal sequence of this peptide had homology with the C. rectus 51-kDa porin and was composed of nearly 50% hydrophobic residues. Thus, the S-layer protein from C. rectus has structural heterogeneity among different human strains and immunoheterogeneity with the NHP strain. PMID:9009300

Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.

PubMed

do Nascimento, Adriana Mendes; Cuvillier-Hot, Virginie; Barchuk, Angel Roberto; Simões, Zilá Luz Paulino; Hartfelder, Klaus

2004-05-01

Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species.

Characterization of strains of Weissella fabalis sp. nov. and Fructobacillus tropaeoli from spontaneous cocoa bean fermentations.

PubMed

Snauwaert, Isabel; Papalexandratou, Zoi; De Vuyst, Luc; Vandamme, Peter

2013-05-01

Six facultatively anaerobic, non-motile lactic acid bacteria were isolated from spontaneous cocoa bean fermentations carried out in Brazil, Ecuador and Malaysia. Phylogenetic analysis revealed that one of these strains, designated M75(T), isolated from a Brazilian cocoa bean fermentation, had the highest 16S rRNA gene sequence similarity towards Weissella fabaria LMG 24289(T) (97.7%), W. ghanensis LMG 24286(T) (93.3%) and W. beninensis LMG 25373(T) (93.4%). The remaining lactic acid bacteria isolates, represented by strain M622, showed the highest 16S rRNA gene sequence similarity towards the type strain of Fructobacillus tropaeoli (99.9%), a recently described species isolated from a flower in South Africa. pheS gene sequence analysis indicated that the former strain represented a novel species, whereas pheS, rpoA and atpA gene sequence analysis indicated that the remaining five strains belonged to F. tropaeoli; these results were confirmed by DNA-DNA hybridization experiments towards their respective nearest phylogenetic neighbours. Additionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved successful for the identification of species of the genera Weissella and Fructobacillus and for the recognition of the novel species. We propose to classify strain M75(T) ( = LMG 26217(T)  = CCUG 61472(T)) as the type strain of the novel species Weissella fabalis sp. nov.

Complete genome sequences of cowpea polerovirus 1 and cowpea polerovirus 2 infecting cowpea plants in Burkina Faso.

PubMed

Palanga, Essowè; Martin, Darren P; Galzi, Serge; Zabré, Jean; Bouda, Zakaria; Neya, James Bouma; Sawadogo, Mahamadou; Traore, Oumar; Peterschmitt, Michel; Roumagnac, Philippe; Filloux, Denis

2017-07-01

The full-length genome sequences of two novel poleroviruses found infecting cowpea plants, cowpea polerovirus 1 (CPPV1) and cowpea polerovirus 2 (CPPV2), were determined using overlapping RT-PCR and RACE-PCR. Whereas the 5845-nt CPPV1 genome was most similar to chickpea chlorotic stunt virus (73% identity), the 5945-nt CPPV2 genome was most similar to phasey bean mild yellow virus (86% identity). The CPPV1 and CPPV2 genomes both have a typical polerovirus genome organization. Phylogenetic analysis of the inferred P1-P2 and P3 amino acid sequences confirmed that CPPV1 and CPPV2 are indeed poleroviruses. Four apparently unique recombination events were detected within a dataset of 12 full polerovirus genome sequences, including two events in the CPPV2 genome. Based on the current species demarcation criteria for the family Luteoviridae, we tentatively propose that CPPV1 and CPPV2 should be considered members of novel polerovirus species.

Isolation and cloning of a metalloproteinase from king cobra snake venom.

PubMed

Guo, Xiao-Xi; Zeng, Lin; Lee, Wen-Hui; Zhang, Yun; Jin, Yang

2007-06-01

A 50 kDa fibrinogenolytic protease, ohagin, from the venom of Ophiophagus hannah was isolated by a combination of gel filtration, ion-exchange and heparin affinity chromatography. Ohagin specifically degraded the alpha-chain of human fibrinogen and the proteolytic activity was completely abolished by EDTA, but not by PMSF, suggesting it is a metalloproteinase. It dose-dependently inhibited platelet aggregation induced by ADP, TMVA and stejnulxin. The full sequence of ohagin was deduced by cDNA cloning and confirmed by protein sequencing and peptide mass fingerprinting. The full-length cDNA sequence of ohagin encodes an open reading frame of 611 amino acids that includes signal peptide, proprotein and mature protein comprising metalloproteinase, disintegrin-like and cysteine-rich domains, suggesting it belongs to P-III class metalloproteinase. In addition, P-III class metalloproteinases from the venom glands of Naja atra, Bungarus multicinctus and Bungarus fasciatus were also cloned in this study. Sequence analysis and phylogenetic analysis indicated that metalloproteinases from elapid snake venoms form a new subgroup of P-III SVMPs.

Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production.

PubMed

Mukai, Nobuhiko; Masaki, Kazuo; Fujii, Tsutomu; Iefuji, Haruyuki

2014-07-01

Among industrial yeasts used for alcoholic beverage production, most wine and weizen beer yeasts decarboxylate ferulic acid to 4-vinylguaiacol, which has a smoke-like flavor, whereas sake, shochu, top-fermenting, and bottom-fermenting yeast strains lack this ability. However, the factors underlying this difference among industrial yeasts are not clear. We previously confirmed that both PAD1 (phenylacrylic acid decarboxylase gene, YDR538W) and FDC1 (ferulic acid decarboxylase gene, YDR539W) are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. In the present study, single nucleotide polymorphisms (SNPs) of PAD1 and FDC1 in sake, shochu, wine, weizen, top-fermenting, bottom-fermenting, and laboratory yeast strains were examined to clarify the differences in ferulic acid decarboxylation ability between these types of yeast. For PAD1, a nonsense mutation was observed in the gene sequence of standard top-fermenting yeast. Gene sequence analysis of FDC1 revealed that sake, shochu, and standard top-fermenting yeasts contained a nonsense mutation, whereas a frameshift mutation was identified in the FDC1 gene of bottom-fermenting yeast. No nonsense or frameshift mutations were detected in laboratory, wine, or weizen beer yeast strains. When FDC1 was introduced into sake and shochu yeast strains, the transformants exhibited ferulic acid decarboxylation activity. Our findings indicate that a positive relationship exists between SNPs in PAD1 and FDC1 genes and the ferulic acid decarboxylation ability of industrial yeast strains. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

RNA sequencing analysis of transcriptional change in the freshwater mussel Elliptio complanata after environmentally relevant sodium chloride exposure.

PubMed

Robertson, Laura S; Galbraith, Heather S; Iwanowicz, Deborah; Blakeslee, Carrie J; Cornman, R Scott

2017-09-01

To identify potential biomarkers of salt stress in a freshwater sentinel species, we examined transcriptional responses of the common mussel Elliptio complanata to controlled sodium chloride (NaCl) exposures. Ribonucleic acid sequencing (RNA-Seq) of mantle tissue identified 481 transcripts differentially expressed in adult mussels exposed to 2 ppt NaCl (1.2 ppt chloride) for 7 d, of which 290 had nonoverlapping intervals. Differentially expressed gene categories included ion and transmembrane transport, oxidoreductase activity, maintenance of protein folding, and amino acid metabolism. The rate-limiting enzyme for synthesis of taurine, an amino acid frequently linked to osmotic stress in aquatic species, was upregulated, as was the transmembrane ion pump sodium/potassium adenosine 5'-triphosphatase. These patterns confirm a primary transcriptional response to the experimental dose, albeit likely overlapping with nonspecific secondary stress responses. Substantial involvement of the heat shock protein 70 chaperone family and the water-transporting aquaporin family was not detected, however, in contrast to some studies in other bivalves. A subset of the most significantly regulated genes was confirmed by quantitative polymerase chain reaction in an independent sample. Cluster analysis showed separation of mussels exposed to 2 ppt NaCl from control mussels in multivariate space, but mussels exposed to 1 ppt NaCl were largely indistinguishable from controls. Transcriptome-scale analysis of salt exposure under laboratory conditions efficiently identified candidate biomarkers for further functional analysis and field validation. Environ Toxicol Chem 2017;36:2352-2366. © Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America. © 2017 SETAC.

Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

PubMed Central

Ehrenshaft, M; Daub, M E

1994-01-01

We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures. Images PMID:8085820

Gene expression profiling in human precision cut liver slices in response to the FXR agonist obeticholic acid.

PubMed

Ijssennagger, Noortje; Janssen, Aafke W F; Milona, Alexandra; Ramos Pittol, José M; Hollman, Danielle A A; Mokry, Michal; Betzel, Bark; Berends, Frits J; Janssen, Ignace M; van Mil, Saskia W C; Kersten, Sander

2016-05-01

The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA. hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days. Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTβ), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6. Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Structure-Specific Ribonucleases for MS-Based Elucidation of Higher-Order RNA Structure

NASA Astrophysics Data System (ADS)

Scalabrin, Matteo; Siu, Yik; Asare-Okai, Papa Nii; Fabris, Daniele

2014-07-01

Supported by high-throughput sequencing technologies, structure-specific nucleases are experiencing a renaissance as biochemical probes for genome-wide mapping of nucleic acid structure. This report explores the benefits and pitfalls of the application of Mung bean (Mb) and V1 nuclease, which attack specifically single- and double-stranded regions of nucleic acids, as possible structural probes to be employed in combination with MS detection. Both enzymes were found capable of operating in ammonium-based solutions that are preferred for high-resolution analysis by direct infusion electrospray ionization (ESI). Sequence analysis by tandem mass spectrometry (MS/MS) was performed to confirm mapping assignments and to resolve possible ambiguities arising from the concomitant formation of isobaric products with identical base composition and different sequences. The observed products grouped together into ladder-type series that facilitated their assignment to unique regions of the substrate, but revealed also a certain level of uncertainty in identifying the boundaries between paired and unpaired regions. Various experimental factors that are known to stabilize nucleic acid structure, such as higher ionic strength, presence of Mg(II), etc., increased the accuracy of cleavage information, but did not completely eliminate deviations from expected results. These observations suggest extreme caution in interpreting the results afforded by these types of reagents. Regardless of the analytical platform of choice, the results highlighted the need to repeat probing experiments under the most diverse possible conditions to recognize potential artifacts and to increase the level of confidence in the observed structural information.

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.

Characterization and cloning of laccase gene from Hericium coralloides NBRC 7716 suitable for production of epitheaflagallin 3-O-gallate.

PubMed

Itoh, Nobuya; Takagi, Shinya; Miki, Asami; Kurokawa, Junji

2016-01-01

Epitheaflagallin 3-O-gallate (ETFGg) is a minor polyphenol found in black tea extract, which has good physiological functions. It is synthesized from epigallocatechin gallate (EGCg) with gallic acid via laccase oxidation. Various basidiomycetes and fungi were screened to find a suitable laccase for the production of ETFGg. A basidiomycete, Hericium coralloides NBRC 7716, produced an appropriate extracellular laccase. The purified laccase produced twice the level of ETFGg compared with commercially available laccase from Trametes sp. The enzyme, termed Lcc2, is a monomeric protein with an apparent molecular mass of 67.2 kDa. The N-terminal amino acid sequence of Lcc2 is quite different from laccase isolated from the fruiting bodies of Hericium. Lcc2 showed similar substrate specificity to known laccases and could oxidize various phenolic substrates, including pyrogallol, gallic acid, and 2,6-dimethoxyphenol. The full-length lcc2 gene was obtained by PCR using degenerate primers, which were designed based on the N-terminal amino acid sequence of Lcc2 and conserved copper-binding sites of laccases, and 5'-, and 3'-RACE PCR with mRNA. The Lcc2 gene showed homology with Lentinula edodes laccase (sharing 77% amino acid identity with Lcc6). We successfully produced extracellular Lcc2 using a heterologous expression system with Saccharomyces cerevisiae. Moreover, it was confirmed that the recombinant laccase generates similar levels of ETFGg as the native enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2011 CFR

2011-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2013 CFR

2013-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2012 CFR

2012-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2010 CFR

2010-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2014 CFR

2014-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties.

PubMed

Upadhyay, Atul K; Chacko, Anita R; Gandhimathi, A; Ghosh, Pritha; Harini, K; Joseph, Agnel P; Joshi, Adwait G; Karpe, Snehal D; Kaushik, Swati; Kuravadi, Nagesh; Lingu, Chandana S; Mahita, J; Malarini, Ramya; Malhotra, Sony; Malini, Manoharan; Mathew, Oommen K; Mutt, Eshita; Naika, Mahantesha; Nitish, Sathyanarayanan; Pasha, Shaik Naseer; Raghavender, Upadhyayula S; Rajamani, Anantharamanan; Shilpa, S; Shingate, Prashant N; Singh, Heikham Russiachand; Sukhwal, Anshul; Sunitha, Margaret S; Sumathi, Manojkumar; Ramaswamy, S; Gowda, Malali; Sowdhamini, Ramanathan

2015-08-28

Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is 'Tulsi' (or 'Tulasi' or 'Thulasi') and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were generated for the whole genome sequenced with the Illumina Hiseq 1000, resulting in an assembled genome of 374 Mb, with a genome coverage of 61 % (612 Mb estimated genome size). We have also studied transcriptomes (RNA-Seq) of two subtypes of O. tenuiflorum, Krishna and Rama Tulsi and report the relative expression of genes in both the varieties. The pathways leading to the production of medicinally-important specialized metabolites have been studied in detail, in relation to similar pathways in Arabidopsis thaliana and other plants. Expression levels of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi were observed to be relatively high, explaining the purple colouration of Krishna Tulsi leaves. The expression of six important genes identified from genome data were validated by performing q-RT-PCR in different tissues of five different species, which shows the high extent of urosolic acid-producing genes in young leaves of the Rama subtype. In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases including cancer was confirmed using mass spectrometry. The availability of the whole genome of O.tenuiflorum and our sequence analysis suggests that small amino acid changes at the functional sites of genes involved in metabolite synthesis pathways confer special medicinal properties to this herb.

Molecular characterization of KGH, the first human isolate of rabies virus in Korea.

PubMed

Park, Jun-Sun; Kim, Chi-Kyeong; Kim, Su Yeon; Ju, Young Ran

2013-04-01

The complete genome sequence of the KGH strain of the first human rabies virus, which was isolated from a skin biopsy of a patient with rabies, whose symptoms developed due to bites from a raccoon dog in 2001. The size of the KGH strain genome was determined to be 11,928 nucleotides (nt) with a leader sequence of 58 nt, nucleoprotein gene of 1,353 nt, phosphoprotein gene of 894 nt, matrix protein gene of 609 nt, glycoprotein gene of 1,575 nt, RNA-dependent RNA polymerase gene of 6,384 nt, and trailer region of 69 nt. Sequence similarity was compared with 39 fully sequenced rabies virus genomes currently available, and the result showed 70.6-91.6 % at the nucleotide level, and 82.8-97.9 % at the amino acid level. The deduced amino acids in the viral protein were compared with those of other rabies viruses, and various functional regions were investigated. As a result, we found that the KGH strain only had a unique amino acid substitution that was identified to be associated either with host immune response and pathogenicity in the N protein, or with a related region regulating STAT1 in the P protein, and related to pathogenicity in G protein. Based on phylogenetic analyses using the complete genome of 39 rabies viruses, the KGH strain was determined to be closely related with the NNV-RAB-H strain and transplant rabies virus serotype 1, which are Indian isolates, and was confirmed to belong to the Arctic-like 2 clade. The KGH strain was most closely related to the SKRRD0204HC and SKRRD0205HC strain when compared with Korean animal isolates, which was separated around the same time and place, and belonged to the Gangwon III subgroup.

Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem.

PubMed

Illeghems, Koen; De Vuyst, Luc; Weckx, Stefan

2013-08-01

Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process. The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition. Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.

Differential gene expression profiles in the venom gland/sac of Eumenes pomiformis (Hymenoptera: Eumenidae).

PubMed

Baek, Ji Hyeong; Lee, Si Hyeock

2010-06-01

To search for novel transcripts encoding biologically active venom components, a subtractive cDNA library specific to the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (ESTs) were clustered and assembled into 102 contigs (31 multiple sequences and 71 singletons). In total, 37 cDNAs were found in the library via BLASTx searching and manual annotation. Eight contigs (337 ESTs) encoding short venom peptides (10 to 16 amino acids) occupied 62% of the library. The deduced amino acid sequence (78 amino acids) of a novel venom peptide transcript shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known to be K(+) channel blockers, implying that this novel peptide may play a role in the paralysis of prey. In addition to phospholipase A2 and hyaluronidase, which are known to be the main components of wasp venoms, several transcripts encoding enzymes, including three metallopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, amines, and neurotransmitters, were also isolated from the library. The presence of a transcript encoding a putative insulin/insulin-like peptide binding protein suggests that solitary hunting wasps use their venom to control their prey, leading to larval growth cessation. The abundance of these venom components in the venom gland/sac and in the alimentary canal was confirmed by quantitative real-time PCR. Discovery of venom gland/sac-specific transcripts should promote further studies on biologically active components in the venom of solitary hunting wasps. Copyright 2010 Elsevier Ltd. All rights reserved.

Cloning and expression of a CYP720B orthologue involved in the biosynthesis of diterpene resin acids in Pinus brutia.

PubMed

Semiz, Asli; Sen, Alaattin

2015-03-01

Cytochrome P450 monooxygenases mediate a broad range of oxidative reactions involved in the biosynthesis of both primary and secondary metabolites in plants. Until now, only two P450 genes, CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, have been functionally characterised and described in the literature. The purpose of this study was to describe the cloning and expression of CYP720B from Pinus brutia due to its suggested role in the synthesis of bioactive compounds used for chemical defence against insects. A PCR product of the P. brutia CYP720B gene was cloned into the pCR8/GW/TOPO cloning vector. After optimising the sequence for codon usage in yeast, it was transferred into the inducible expression vector pYES-DEST52 and transfected into the S. cerevisiae INVSc1 strain. Sequence analysis showed that the P. brutia CYP720B gene contains an open reading frame of 1,464 nucleotides, which encodes a 53,570 Da putative protein of 487 amino acid residues. The putative protein contains the classic heme-binding sequence motif that is conserved in all P450 enzymes. It shares 99 and 61% identity with the deduced amino acid sequences of CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, respectively. Recombinant CYP720B protein expression was confirmed using western blot analysis. Furthermore, recombinant CYP720B was functionally active, showing a Soret peak at approximately 448 nm in the reduced CO difference spectra. These data suggest that the cloned gene is an orthologue of CYP720B in P. brutia and might be involved in DRA biosynthesis.

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

PubMed

Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

2013-10-15

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus

NASA Astrophysics Data System (ADS)

Knolhoff, Ann M.; Zheng, Jie; McFarland, Melinda A.; Luo, Yan; Callahan, John H.; Brown, Eric W.; Croley, Timothy R.

2015-08-01

The rise of antimicrobial resistance necessitates the discovery and/or production of novel antibiotics. Isolated strains of Paenibacillus alvei were previously shown to exhibit antimicrobial activity against a number of pathogens, such as E. coli, Salmonella, and methicillin-resistant Staphylococcus aureus (MRSA). The responsible antimicrobial compounds were isolated from these Paenibacillus strains and a combination of low and high resolution mass spectrometry with multiple-stage tandem mass spectrometry was used for identification. A group of closely related cyclic lipopeptides was identified, differing primarily by fatty acid chain length and one of two possible amino acid substitutions. Variation in the fatty acid length resulted in mass differences of 14 Da and yielded groups of related MSn spectra. Despite the inherent complexity of MS/MS spectra of cyclic compounds, straightforward analysis of these spectra was accomplished by determining differences in complementary product ion series between compounds that differ in molecular weight by 14 Da. The primary peptide sequence assignment was confirmed through genome mining; the combination of these analytical tools represents a workflow that can be used for the identification of complex antibiotics. The compounds also share amino acid sequence similarity to a previously identified broad-spectrum antibiotic isolated from Paenibacillus. The presence of such a wide distribution of related compounds produced by the same organism represents a novel class of broad-spectrum antibiotic compounds.

Pyrin gene and mutants thereof, which cause familial Mediterranean fever

DOEpatents

Kastner, Daniel L [Bethesda, MD; Aksentijevichh, Ivona [Bethesda, MD; Centola, Michael [Tacoma Park, MD; Deng, Zuoming [Gaithersburg, MD; Sood, Ramen [Rockville, MD; Collins, Francis S [Rockville, MD; Blake, Trevor [Laytonsville, MD; Liu, P Paul [Ellicott City, MD; Fischel-Ghodsian, Nathan [Los Angeles, CA; Gumucio, Deborah L [Ann Arbor, MI; Richards, Robert I [North Adelaide, AU; Ricke, Darrell O [San Diego, CA; Doggett, Norman A [Santa Cruz, NM; Pras, Mordechai [Tel-Hashomer, IL

2003-09-30

The invention provides the nucleic acid sequence encoding the protein associated with familial Mediterranean fever (FMF). The cDNA sequence is designated as MEFV. The invention is also directed towards fragments of the DNA sequence, as well as the corresponding sequence for the RNA transcript and fragments thereof. Another aspect of the invention provides the amino acid sequence for a protein (pyrin) associated with FMF. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. The invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain at the C-terminal of the protein. These mutants include M6801, M694V, K695R, and V726A. Additionally, the invention includes methods for diagnosing a patient at risk for having FMF and kits therefor.

Comparison of the performance of laboratory tests in the diagnosis of feline infectious peritonitis.

PubMed

Stranieri, Angelica; Giordano, Alessia; Paltrinieri, Saverio; Giudice, Chiara; Cannito, Valentina; Lauzi, Stefania

2018-05-01

We compared the performance of clinicopathologic and molecular tests used in the antemortem diagnosis of feline infectious peritonitis (FIP). From 16 FIP and 14 non-FIP cats, we evaluated retrospectively the sensitivity, specificity, and likelihood ratios (LRs) of serum protein electrophoresis, α 1 -acid glycoprotein (AGP) on peripheral blood, screening reverse-transcription nested PCR (RT-nPCR) on the 3'-untranslated region (3'-UTR), and spike (S) gene sequencing on peripheral blood, body cavity effusions, and tissue, as well as body cavity cytology and delta total nucleated cell count (ΔTNC). Any of these tests on blood, and especially the molecular tests, may support or confirm a clinical diagnosis of FIP. A negative result does not exclude the disease except for AGP. Cytology, 3'-UTR PCR, and ΔTNC may confirm a clinical diagnosis on effusions; cytology or 3'-UTR PCR may exclude FIP. Conversely, S gene sequencing is not recommended based on the LRs. On tissues, S gene sequencing is preferable when histology is highly consistent with FIP, and 3'-UTR PCR when FIP is unlikely. Combining one test with high LR+ with one with low LR- (e.g., molecular tests and AGP on blood, ΔTNC and cytology in effusions) may improve the diagnostic power of the most used laboratory tests.

Transcriptomic Analysis Identifies Candidate Genes Related to Intramuscular Fat Deposition and Fatty Acid Composition in the Breast Muscle of Squabs (Columba)

PubMed Central

Ye, Manhong; Zhou, Bin; Wei, Shanshan; Ding, MengMeng; Lu, Xinghui; Shi, Xuehao; Ding, Jiatong; Yang, Shengmei; Wei, Wanhong

2016-01-01

Despite the fact that squab is consumed throughout the world because of its high nutritional value and appreciated sensory attributes, aspects related to its characterization, and in particular genetic issues, have rarely been studied. In this study, meat traits in terms of pH, water-holding capacity, intramuscular fat content, and fatty acid profile of the breast muscle of squabs from two meat pigeon breeds were determined. Breed-specific differences were detected in fat-related traits of intramuscular fat content and fatty acid composition. RNA-Sequencing was applied to compare the transcriptomes of muscle and liver tissues between squabs of two breeds to identify candidate genes associated with the differences in the capacity of fat deposition. A total of 27 differentially expressed genes assigned to pathways of lipid metabolism were identified, of which, six genes belonged to the peroxisome proliferator-activated receptor signaling pathway along with four other genes. Our results confirmed in part previous reports in livestock and provided also a number of genes which had not been related to fat deposition so far. These genes can serve as a basis for further investigations to screen markers closely associated with intramuscular fat content and fatty acid composition in squabs. The data from this study were deposited in the National Center for Biotechnology Information (NCBI)’s Sequence Read Archive under the accession numbers SRX1680021 and SRX1680022. This is the first transcriptome analysis of the muscle and liver tissue in Columba using next generation sequencing technology. Data provided here are of potential value to dissect functional genes influencing fat deposition in squabs. PMID:27175015

Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

NASA Astrophysics Data System (ADS)

Anderson, Lissa C.; Håkansson, Maria; Walse, Björn; Nilsson, Carol L.

2017-09-01

Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a 45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. [Figure not available: see fulltext.

Connective tissue activation. XXXII. Structural and biologic characteristics of mesenchymal cell-derived connective tissue activating peptide-V.

PubMed

Cabral, A R; Cole, L A; Walz, D A; Castor, C W

1987-12-01

Connective tissue activating peptide-V (CTAP-V) is a single-chain, mesenchymal cell-derived anionic protein with large and small molecular forms (Mr of 28,000 and 16,000, respectively), as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins have similar specific activities with respect to stimulation of hyaluronic acid and DNA formation in human synovial fibroblast cultures. S-carboxymethylation or removal of sialic acid residues did not modify CTAP-V biologic activity. Rabbit antibodies raised separately against each of the purified CTAP-V proteins reacted, on immunodiffusion and on Western blot, with each antigen and neutralized mitogenic activity. The amino-terminal amino acid sequence of the CTAP-V proteins, determined by 2 laboratories, confirmed their structural similarities. The amino-terminal sequence through 37 residues was demonstrated for the smaller protein. The first 10 residues of CTAP-V (28 kd) were identical to the N-terminal decapeptide of CTAP-V (16 kd). The C-terminal sequence, determined by carboxypeptidase Y digestion, was the same for both CTAP-V molecular species. The 2 CTAP-V peptides had similar amino acid compositions, whether residues were expressed as a percent of the total or were normalized to mannose. Reduction of native CTAP-V protein released sulfhydryl groups in a protein:disulfide ratio of 1:2; this suggests that CTAP-V contains 2 intramolecular disulfide bonds. Clearly, CTAP-V is a glycoprotein. The carbohydrate content of CTAP-V (16 kd) and CTAP-V (28 kd) is 27% and 25%, respectively. CTAP-V may have significance in relation to autocrine mechanisms for growth regulation of connective tissue cells and other cell types.

Criteria for confirming sequence periodicity identified by Fourier transform analysis: application to GCR2, a candidate plant GPCR?

PubMed

Illingworth, Christopher J R; Parkes, Kevin E; Snell, Christopher R; Mullineaux, Philip M; Reynolds, Christopher A

2008-03-01

Methods to determine periodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the alpha-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.

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

... DEPARTMENT OF COMMERCE Patent and Trademark Office Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence Disclosures ACTION: Proposed collection; comment request... Patent applications that contain nucleotide and/or amino acid sequence disclosures must include a copy of...

Verification of Ribosomal Proteins of Aspergillus fumigatus for Use as Biomarkers in MALDI-TOF MS Identification

PubMed Central

Nakamura, Sayaka; Sato, Hiroaki; Tanaka, Reiko; Yaguchi, Takashi

2016-01-01

We have previously proposed a rapid identification method for bacterial strains based on the profiles of their ribosomal subunit proteins (RSPs), observed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method can perform phylogenetic characterization based on the mass of housekeeping RSP biomarkers, ideally calculated from amino acid sequence information registered in public protein databases. With the aim of extending its field of application to medical mycology, this study investigates the actual state of information of RSPs of eukaryotic fungi registered in public protein databases through the characterization of ribosomal protein fractions extracted from genome-sequenced Aspergillus fumigatus strains Af293 and A1163 as a model. In this process, we have found that the public protein databases harbor problems. The RSP names are in confusion, so we have provisionally unified them using the yeast naming system. The most serious problem is that many incorrect sequences are registered in the public protein databases. Surprisingly, more than half of the sequences are incorrect, due chiefly to mis-annotation of exon/intron structures. These errors could be corrected by a combination of in silico inspection by sequence homology analysis and MALDI-TOF MS measurements. We were also able to confirm conserved post-translational modifications in eleven RSPs. After these verifications, the masses of 31 expressed RSPs under 20,000 Da could be accurately confirmed. These RSPs have a potential to be useful biomarkers for identifying clinical isolates of A. fumigatus. PMID:27843740

Amino acid sequence analysis of the annexin super-gene family of proteins.

PubMed

Barton, G J; Newman, R H; Freemont, P S; Crumpton, M J

1991-06-15

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.

Sequence of the amino-terminal region of rat liver ribosomal proteins S4, S6, S8, L6, L7a, L18, L27, L30, L37, L37a, and L39.

PubMed

Wittmann-Liebold, B; Geissler, A W; Lin, A; Wool, I G

1979-01-01

The sequence of the amino-terminal region of eleven rat liver ribosomal proteins--S4, S6, S8, L6, L7a, L18, L27, L30, L37a, and L39--was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N-terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino-terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry.

Cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor L.; Brow, Mary Ann D.; Dahlberg, James E.

2007-12-11

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 structure-specific 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.

Invasive cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

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 structure-specific 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.

Invasive cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

2002-01-01

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 structure-specific 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.

Cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow; Mary Ann D.; Dahlberg, James E.

2010-11-09

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 structure-specific 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.

Cleavage of nucleic acids

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

2000-01-01

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 structure-specific 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.

Nucleic acid detection assays

DOEpatents

Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

2005-04-05

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 structure-specific 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 Oral Microbiota in Health and Disease: An Overview of Molecular Findings.

PubMed

Siqueira, José F; Rôças, Isabela N

2017-01-01

Culture-independent nucleic acid technologies have been extensively applied to the analysis of oral bacterial communities associated with healthy and diseased conditions. These methods have confirmed and substantially expanded the findings from culture studies to reveal the oral microbial inhabitants and candidate pathogens associated with the major oral diseases. Over 1000 bacterial distinct species-level taxa have been identified in the oral cavity and studies using next-generation DNA sequencing approaches indicate that the breadth of bacterial diversity may be even much larger. Nucleic acid technologies have also been helpful in profiling bacterial communities and identifying disease-related patterns. This chapter provides an overview of the diversity and taxonomy of oral bacteria associated with health and disease.

Identification of single-nucleotide polymorphisms of the prion protein gene in sika deer (Cervus nippon laiouanus)

PubMed Central

Jeong, Hyun-Jeong; Lee, Joong-Bok; Park, Seung-Yong; Song, Chang-Seon; Kim, Bo-Sook; Rho, Jung-Rae; Yoo, Mi-Hyun; Jeong, Byung-Hoon; Kim, Yong-Sun

2007-01-01

Polymorphisms of the prion protein gene (PRNP) have been detected in several cervid species. In order to confirm the genetic variations, this study examined the DNA sequences of the PRNP obtained from 33 captive sika deer (Cervus nippon laiouanus) in Korea. A total of three single-nucleotide polymorphisms (SNPs) at codons 100, 136 and 226 in the PRNP of the sika deer were identified. The polymorphic site located at codon 100 has not been reported. The SNPs detected at codons 100 and 226 induced amino acid substitutions. The SNP at codon 136 was a silent mutation that does not induce any amino acid change. The genotype and allele frequencies were determined for each of the SNPs. PMID:17679779

Micromonospora halotolerans sp. nov., isolated from the rhizosphere of a Pisum sativum plant.

PubMed

Carro, Lorena; Pukall, Rüdiger; Spröer, Cathrin; Kroppenstedt, Reiner M; Trujillo, Martha E

2013-06-01

A filamentous actinomycete strain designated CR18(T) was isolated on humic acid agar from the rhizosphere of a Pisum sativum plant collected in Spain. This isolate was observed to grow optimally at 28 °C, pH 7.0 and in the presence of 5 % NaCl. Phylogenetic analyses based on the 16S rRNA gene sequence indicated a close relationship with the type strains of Micromonospora chersina and Micromonospora endolithica. A further analysis based on a concatenated DNA sequence stretch of 4,523 bp that included partial sequences of the atpD, gyrB, recA, rpoB and 16S rRNA genes clearly differentiated the new strain from recognized Micromonospora species compared. DNA-DNA hybridization studies further supported the taxonomic position of strain CR18(T) as a novel genomic species. Chemotaxonomic analyses which included whole cell sugars, polar lipids, fatty acid profiles and menaquinone composition confirmed the affiliation of the new strain to the genus Micromonospora and also highlighted differences at the species level. These studies were finally complemented with an array of physiological tests to help differentiate between the new strain and its phylogenetic neighbours. Consequently, strain CR18(T) (= CECT 7890(T) = DSM 45598(T)) is proposed as the type strain of a novel species, Micromonospora halotolerans sp. nov.

Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites.

PubMed

Grijseels, Sietske; Nielsen, Jens Christian; Randelovic, Milica; Nielsen, Jens; Nielsen, Kristian Fog; Workman, Mhairi; Frisvad, Jens Christian

2016-10-14

A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311 T  = IBT 12289 T ). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted in the identification of 62 putative biosynthetic gene clusters. Extracts of P. arizonense were analysed for secondary metabolites and austalides, pyripyropenes, tryptoquivalines, fumagillin, pseurotin A, curvulinic acid and xanthoepocin were detected. A comparative analysis against known pathways enabled the proposal of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential industrial applications for the new species P. arizonense. The description and availability of the genome sequence of P. arizonense, further provides the basis for biotechnological exploitation of this species.

Penicillium arizonense, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites

PubMed Central

Grijseels, Sietske; Nielsen, Jens Christian; Randelovic, Milica; Nielsen, Jens; Nielsen, Kristian Fog; Workman, Mhairi; Frisvad, Jens Christian

2016-01-01

A new soil-borne species belonging to the Penicillium section Canescentia is described, Penicillium arizonense sp. nov. (type strain CBS 141311T = IBT 12289T). The genome was sequenced and assembled into 33.7 Mb containing 12,502 predicted genes. A phylogenetic assessment based on marker genes confirmed the grouping of P. arizonense within section Canescentia. Compared to related species, P. arizonense proved to encode a high number of proteins involved in carbohydrate metabolism, in particular hemicellulases. Mining the genome for genes involved in secondary metabolite biosynthesis resulted in the identification of 62 putative biosynthetic gene clusters. Extracts of P. arizonense were analysed for secondary metabolites and austalides, pyripyropenes, tryptoquivalines, fumagillin, pseurotin A, curvulinic acid and xanthoepocin were detected. A comparative analysis against known pathways enabled the proposal of biosynthetic gene clusters in P. arizonense responsible for the synthesis of all detected compounds except curvulinic acid. The capacity to produce biomass degrading enzymes and the identification of a high chemical diversity in secreted bioactive secondary metabolites, offers a broad range of potential industrial applications for the new species P. arizonense. The description and availability of the genome sequence of P. arizonense, further provides the basis for biotechnological exploitation of this species. PMID:27739446

Structure of CARB-4 and AER-1 CarbenicillinHydrolyzing β-Lactamases

PubMed Central

Sanschagrin, François; Bejaoui, Noureddine; Levesque, Roger C.

1998-01-01

We determined the nucleotide sequences of blaCARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing β-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these β-lactamases varied from 98.7 to 99.9%, while that between these genes and blaCARB-4 encoding CARB-4 was 86.3%. The blaCARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that blaAER-1 shared 60.8% DNA identity with blaPSE-3 encoding PSE-3. The deduced AER-1 β-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A β-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking blaCARB-4 and blaAER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution. PMID:9687391

Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome.

PubMed

Villate, Olatz; Ibarluzea, Nekane; Fraile-Bethencourt, Eugenia; Valenzuela, Alberto; Velasco, Eladio A; Grozeva, Detelina; Raymond, F L; Botella, María P; Tejada, María-Isabel

2018-01-01

Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T) was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD ® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG) 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.

Method for nucleic acid hybridization using single-stranded DNA binding protein

DOEpatents

Tabor, Stanley; Richardson, Charles C.

1996-01-01

Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

PubMed

Reddy, M K; Nair, S; Singh, B N; Mudgil, Y; Tewari, K K; Sopory, S K

2001-01-24

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

PubMed

Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

2012-08-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

Structural characterization and comparative modeling of PD-Ls 1-3, type 1 ribosome-inactivating proteins from summer leaves of Phytolacca dioica L.

PubMed

Di Maro, Antimo; Chambery, Angela; Carafa, Vincenzo; Costantini, Susan; Colonna, Giovanni; Parente, Augusto

2009-03-01

The amino acid sequence and glycan structure of PD-L1, PD-L2 and PD-L3, type 1 ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves, were determined using a combined approach based on peptide mapping, Edman degradation and ESI-Q-TOF MS in precursor ion discovery mode. The comparative analysis of the 261 amino acid residue sequences showed that PD-L1 and PD-L2 have identical primary structure, as it is the case of PD-L3 and PD-L4. Furthermore, the primary structure of PD-Ls 1-2 and PD-Ls 3-4 have 81.6% identity (85.1% similarity). The ESI-Q-TOF MS analysis confirmed that PD-Ls 1-3 were glycosylated at different sites. In particular, PD-L1 contained three glycidic chains with the well known paucidomannosidic structure (Man)(3) (GlcNAc)(2) (Fuc)(1) (Xyl)(1) linked to Asn10, Asn43 and Asn255. PD-L2 was glycosylated at Asn10 and Asn43, and PD-L3 was glycosylated only at Asn10. PD-L4 was confirmed to be not glycosylated. Despite an overall high structural similarity, the comparative modeling of PD-L1, PD-L2, PD-L3 and PD-L4 has shown potential influences of the glycidic chains on their adenine polynucleotide glycosylase activity on different substrates.

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

PubMed

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

The primary structures of two yeast enolase genes. Homology between the 5' noncoding flanking regions of yeast enolase and glyceraldehyde-3-phosphate dehydrogenase genes.

PubMed

Holland, M J; Holland, J P; Thill, G P; Jackson, K A

1981-02-10

Segments of yeast genomic DNA containing two enolase structural genes have been isolated by subculture cloning procedures using a cDNA hybridization probe synthesized from purified yeast enolase mRNA. Based on restriction endonuclease and transcriptional maps of these two segments of yeast DNA, each hybrid plasmid contains a region of extensive nucleotide sequence homology which forms hybrids with the cDNA probe. The DNA sequences which flank this homologous region in the two hybrid plasmids are nonhomologous indicating that these sequences are nontandemly repeated in the yeast genome. The complete nucleotide sequence of the coding as well as the flanking noncoding regions of these genes has been determined. The amino acid sequence predicted from one reading frame of both structural genes is extremely similar to that determined for yeast enolase (Chin, C. C. Q., Brewer, J. M., Eckard, E., and Wold, F. (1981) J. Biol. Chem. 256, 1370-1376), confirming that these isolated structural genes encode yeast enolase. The nucleotide sequences of the coding regions of the genes are approximately 95% homologous, and neither gene contains an intervening sequence. Codon utilization in the enolase genes follows the same biased pattern previously described for two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). DNA blotting analysis confirmed that the isolated segments of yeast DNA are colinear with yeast genomic DNA and that there are two nontandemly repeated enolase genes per haploid yeast genome. The noncoding portions of the two enolase genes adjacent to the initiation and termination codons are approximately 70% homologous and contain sequences thought to be involved in the synthesis and processing messenger RNA. Finally there are regions of extensive homology between the two enolase structural genes and two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes within the 5- noncoding portions of these glycolytic genes.

Lelliottia aquatilis sp. nov., isolated from drinking water.

PubMed

Kämpfer, Peter; Glaeser, Stefanie P; Packroff, Gabriele; Behringer, Katja; Exner, Martin; Chakraborty, Trinad; Schmithausen, Ricarda M; Doijad, Swapnil

2018-06-22

Five beige-pigmented, oxidase-negative bacterial isolates, 6331-17 T , 6332-17, 6333-17, 6334-17 and 9827-07, isolated either from a drinking water storage reservoir or drinking water in 2006 and 2017 in Germany, were examined in detail applying by a polyphasic taxonomic approach. Cells of the isolates were rod-shaped and Gram-stain-negative. Comparison of the 16S rRNA gene sequences of these five isolates showed highest sequence similarities to Lelliottia amnigena (99.98 %) and Lelliottia nimipressuralis (99.99 %). Multilocus sequence analyses based on concatenated partial rpoB, gyrB, infB and atpD sequences confirmed the clustering of these isolates with Lelliottia species, but also revealed a clear distinction to the closest related type strains. Analysis of the genome sequences of these isolates indicated >70 % in silico DNA-DNA hybridization and high average nucleotide identities between strains. Nevertheless, they showed only <70 and <95 % similarity to the type strains of these two Lelliottia species. The fatty acid profiles of these isolates were very similar and consisted of the major fatty acids C16:0, C17 : 0cyclo, C15 : 0iso 2-OH/C16 : 1ω7c and C18 : 1ω7c. In addition, physiological/biochemical tests revealed high phenotypic similarity to each other. These cumulative data indicate that these isolates represent a novel Lelliottia species, for which the name Lelliottia aquatilis sp. nov. is proposed, with strain 6331-17 T (=CCM 8846 T =CIP 111609 T =LMG 30560 T ) as the type strain.

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

PubMed

Vivante, Asaf; Mann, Nina; Yonath, Hagith; Weiss, Anna-Carina; Getwan, Maike; Kaminski, Michael M; Bohnenpoll, Tobias; Teyssier, Catherine; Chen, Jing; Shril, Shirlee; van der Ven, Amelie T; Ityel, Hadas; Schmidt, Johanna Magdalena; Widmeier, Eugen; Bauer, Stuart B; Sanna-Cherchi, Simone; Gharavi, Ali G; Lu, Weining; Magen, Daniella; Shukrun, Rachel; Lifton, Richard P; Tasic, Velibor; Stanescu, Horia C; Cavaillès, Vincent; Kleta, Robert; Anikster, Yair; Dekel, Benjamin; Kispert, Andreas; Lienkamp, Soeren S; Hildebrandt, Friedhelm

2017-08-01

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene ( NRIP1 ) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RAR α , and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RAR α RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease. Copyright © 2017 by the American Society of Nephrology.

Pseudomonas kribbensis sp. nov., isolated from garden soils in Daejeon, Korea.

PubMed

Chang, Dong-Ho; Rhee, Moon-Soo; Kim, Ji-Sun; Lee, Yookyung; Park, Mi Young; Kim, Haseong; Lee, Seung-Goo; Kim, Byoung-Chan

2016-11-01

Two bacterial strains, 46-1 and 46-2 T , were isolated from garden soil. These strains were observed to be aerobic, Gram-stain negative, rod-shaped, non-spore-forming, motile and catalase and oxidase positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two strains shared 100 % sequence similarity with each other and belong to the genus Pseudomonas in the class Gammaproteobacteria. The concatenated 16S rRNA, gyrB, rpoB and rpoD gene sequences further confirmed that the isolates belong to the Pseudomonas koreensis subgroup (SG), with P. koreensis Ps 9-14 T , Pseudomonas moraviensis 1B4 T and Pseudomonas granadensis F-278,770 T as their close relatives (>96 % pairwise similarity). DNA-DNA hybridization with the closely related type strain P. koreensis SG revealed a low level of relatedness (<50 %). A cladogram constructed using whole-cell matrix-assisted laser desorption/ionization time-of-flight (WC-MALDI-TOF) MS analysis showed the isolates formed a completely separate monophyletic group. The isolates were negative for utilization of glycogen, D-psicose, α-keto butyric acid, α-keto valeric acid, succinamic acid and D, L-α-glycerol phosphate. In contrast, all these reactions were positive in P. koreensis JCM 14769 T and P. moraviensis DSM 16007 T . The fatty acid C 17:0 cyclo was detected as one of the major cellular fatty acids (>15 %) in the isolates but it was a minor component (<4 %) in both reference type strains. In contrast, the fatty acid, C 12:0 was not observed in the isolates but was present in both reference strains. Based on differences such as phylogenetic position, low-level DNA-DNA hybridization, WC-MALDI-TOF MS analysis, fluorescence pigmentation, fatty acid profiles, and substrate utilization, we propose that the isolates 46-1 and 46-2 T represent a novel species of the genus Pseudomonas, for which the name Pseudomonas kribbensis sp. nov. is proposed; the type strain is 46-2 T (=KCTC 32541 T  = DSM 100278 T ).

Identification of a novel nidovirus in an outbreak of fatal respiratory disease in ball pythons (Python regius).

PubMed

Uccellini, Lorenzo; Ossiboff, Robert J; de Matos, Ricardo E C; Morrisey, James K; Petrosov, Alexandra; Navarrete-Macias, Isamara; Jain, Komal; Hicks, Allison L; Buckles, Elizabeth L; Tokarz, Rafal; McAloose, Denise; Lipkin, Walter Ian

2014-08-08

Respiratory infections are important causes of morbidity and mortality in reptiles; however, the causative agents are only infrequently identified. Pneumonia, tracheitis and esophagitis were reported in a collection of ball pythons (Python regius). Eight of 12 snakes had evidence of bacterial pneumonia. High-throughput sequencing of total extracted nucleic acids from lung, esophagus and spleen revealed a novel nidovirus. PCR indicated the presence of viral RNA in lung, trachea, esophagus, liver, and spleen. In situ hybridization confirmed the presence of intracellular, intracytoplasmic viral nucleic acids in the lungs of infected snakes. Phylogenetic analysis based on a 1,136 amino acid segment of the polyprotein suggests that this virus may represent a new species in the subfamily Torovirinae. This report of a novel nidovirus in ball pythons may provide insight into the pathogenesis of respiratory disease in this species and enhances our knowledge of the diversity of nidoviruses.

MYCOBACTERIUM ABSCESSUS PNEUMONIA IN AN ATLANTIC BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS)

PubMed Central

Clayton, Leigh Ann; Stamper, M. Andrew; Whitaker, Brent R.; Hadfield, Catherine A.; Simons, Brian; Mankowski, Joseph L.

2013-01-01

Mycobacterium abscessus pneumonia was diagnosed antemortem in a 23-yr-old male Atlantic bottlenose dolphin (Tursiops truncatus). Clinical signs included lethargy, hyporexia, coughing, and bloody respiratory discharge. Diagnostic findings included neutrophilic leukocytosis, anemia, elevated erythrocyte sedimentation rate, and repeated forceful exhaled breath (sputum) cytology, with acute inflammatory cells and acid-fast positive beaded rods. The bacteria were initially identified free in the sputum sample and subsequently were seen within neutrophils. A culture was positive for a rapidly growing, white, colony-forming organism confirmed as M. abscessus by polymerase chain reaction and DNA sequencing. Clinical signs initially resolved with multidrug therapy. Concurrent Pseudomonas aeruginosa infection complicated clinical management and contributed to terminal decline. The dolphin was euthanized 5 mo after initial diagnosis. Necropsy results demonstrated acid-fast positive bacteria in lung tissue and supported the diagnosis of M. abscessus pneumonia. Acid-fast stains and mycobacteria cultures should be considered when evaluating ill dolphins. PMID:23272373

Characterization of a novel hepadnavirus in the white sucker (Catostomus commersonii) from the Great Lakes Region of the USA

USGS Publications Warehouse

Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Conway, Carla M.; Winton, James R.; Blazer, Vicki S.

2015-01-01

The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the USA. Dideoxysequencing confirmed the white sucker hepatitis B virus (WSHBV) has a circular genome (3542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase and surface proteins was low and ranged from 16-27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively non-coding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV other hepadnaviruses, and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, of which only 2.4% were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker.

The Initial Evaluation of Patients After Positive Newborn Screening: Recommended Algorithms Leading to a Confirmed Diagnosis of Pompe Disease.

PubMed

Burton, Barbara K; Kronn, David F; Hwu, Wuh-Liang; Kishnani, Priya S

2017-07-01

Newborn screening (NBS) for Pompe disease is done through analysis of acid α-glucosidase (GAA) activity in dried blood spots. When GAA levels are below established cutoff values, then second-tier testing is required to confirm or refute a diagnosis of Pompe disease. This article in the "Newborn Screening, Diagnosis, and Treatment for Pompe Disease" guidance supplement provides recommendations for confirmatory testing after a positive NBS result indicative of Pompe disease is obtained. Two algorithms were developed by the Pompe Disease Newborn Screening Working Group, a group of international experts on both NBS and Pompe disease, based on whether DNA sequencing is performed as part of the screening method. Using the recommendations in either algorithm will lead to 1 of 3 diagnoses: classic infantile-onset Pompe disease, late-onset Pompe disease, or no disease/not affected/carrier. Mutation analysis of the GAA gene is essential for confirming the biochemical diagnosis of Pompe disease. For NBS laboratories that do not have DNA sequencing capabilities, the responsibility of obtaining sequencing of the GAA gene will fall on the referral center. The recommendations for confirmatory testing and the initial evaluation are intended for a broad global audience. However, the Working Group recognizes that clinical practices, standards of care, and resource capabilities vary not only regionally, but also by testing centers. Individual patient needs and health status as well as local/regional insurance reimbursement programs and regulations also must be considered. Copyright © 2017 by the American Academy of Pediatrics.

Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

PubMed

Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

2015-03-01

The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Two distinct CXC chemokine receptors (CXCR3 and CXCR4) from the big-belly seahorse Hippocampus abdominalis: Molecular perspectives and immune defensive role upon pathogenic stress.

PubMed

Priyathilaka, Thanthrige Thiunuwan; Oh, Minyoung; Bathige, S D N K; De Zoysa, Mahanama; Lee, Jehee

2017-06-01

CXC chemokine receptor 3 (CXCR3) and 4 (CXCR4) are members of the seven transmembrane G protein coupled receptor family, involved in pivotal physiological functions. In this study, seahorse CXCR3 and CXCR4 (designated as HaCXCR3 and HaCXCR4) cDNA sequences were identified from the transcriptome library and subsequently molecularly characterized. HaCXCR3 and HaCXCR4 encoded 363 and 373 amino acid long polypeptides, respectively. The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors, including seven transmembrane domains and a G protein coupled receptors family 1 profile with characteristic DRY motifs. Amino acid sequence comparison and phylogenetic analysis of these two CXC chemokine receptors revealed a close relationship to their corresponding teleost counterparts. Quantitative real time PCR analysis revealed that HaCXCR3 and HaCXCR4 were ubiquitously expressed in all the tested tissues, with highest expression levels in blood cells. The seahorse blood cells and kidney HaCXCR3 and HaCXCR4 mRNA expressions were differently modulated when challenged with Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid, confirming their involvement in post immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Expression and analysis of the nucleoprotein of paramyxovirus Tianjin strain].

PubMed

Wang, Qing; Li, Mei; Shi, Li-Ying; Yuan, Li-Jun; Wang, Wen-Xiu

2008-05-01

Paramyxovirus Tianjin strain is a novel strain of virus causing common cotton-eared marmoset fatal infection. To investigate the relationship between the gene structure and function of nucleoprotein (NP) of Tianjin strain, NP gene of paramyxovirus Tianjin strain was cloned and three domains of NP were expressed. The homologous and phylogenetic analysis of NP sequences among the paramyxovirus Tianjin strain and eight strains of Sendai viruses from GenBank were performed. The results indicated the recombinant proteins NP1, NP2 and NP3 showed the native antigenicity to the polyclonal antiserum of paramyxovirus Tianjin strain, ranking as NP3>NP1>NP2 (precedence order). The homology of NP nucleotide and the deduced amino acid sequences between paramyxovirus Tianjin strain and Sendai virus BB1 strain were 94.5%, 96.2%, respectively, whereas the identity were 85.1% - 88.7% and 92.4% - 94.7% among Tianjin strain and the 7 strains of Sendai viruses from GenBank respectively. There were 15 unique amino acid substitutions in Tianjin strain NP protein and 11 common amino acid substitutions same with BB1 strain. This research confirmed that paramyxovirus Tianjin strain might be a new genotype of Sendai virus and can be helpful in the establishment of detection assay applying recombinant NP as antigen instead of the whole virions.

Molecular characterization and gene expression patterns of retinoid receptors, in normal and regenerating tissues of the sea cucumber, Holothuria glaberrima.

PubMed

Viera-Vera, Jorge; García-Arrarás, José E

2018-05-15

Retinoic acid receptors (RAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors that synchronize intricate signaling networks in metazoans. Dimer formation between these two nuclear receptors mediates the recruitment of co-regulatory complexes coordinating the progression of signaling cascades during developmental and regenerative events. In the present study we identified and characterized the receptors for retinoic acid in the sea cucumber Holothuria glaberrima; a model system capable of regenerative organogenesis during adulthood. Molecular characterizations revealed the presence of three isoforms of RAR and two of RXR as a consequence of alternative splicing events. Various analyses including: primary structure sequencing, phylogenetic analysis, protein domain prediction, and multiple sequence alignment further confirmed their identity. Semiquantitative reverse transcription PCR analysis of each receptor isoform herein identified showed that the retinoid receptors are expressed in all tissues sampled: the mesenteries, respiratory trees, muscles, gonads, and the digestive tract. During regenerative organogenesis two of the receptors (RAR-L and RXR-T) showed differential expression in the posterior segment while RAR-S is differentially expressed in the anterior segment of the intestine. This work presents the first description of the components relaying the signaling for retinoic acid within this model system. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the Zebrafish larvae assay.

PubMed

Fitzgerald, Ciarán; Gallagher, Eimear; O'Connor, Paula; Prieto, José; Mora-Soler, Leticia; Grealy, Maura; Hayes, Maria

2013-12-01

The vascular inflammatory role of platelet activating factor acetylhydrolase (PAF-AH) is thought to be due to the formation of lysophosphatidyl choline and oxidized non-esterified fatty acids. This enzyme is considered a promising therapeutic target for the prevention of atherosclerosis and there is a need to expand the available chemical templates of PAF-AH inhibitors. This study demonstrated how natural PAF-AH inhibitory peptides were isolated and characterized from the red macroalga Palmaria palmata. The dried powdered alga was hydrolyzed using the food grade enzyme papain, and the resultant peptide containing fraction generated using RP-HPLC. Several oligopeptides were identified as potential PAF-AH inhibitors following bio-guided fractionation, and the amino acid sequences of these oligopeptides were confirmed by Q-TOF-MS and microwave-assisted solid phase de novo synthesis. The most promising PAF-AH inhibitory peptide had the amino acid sequence NIGK and a PAF-AH IC50 value of 2.32 mM. This peptide may constitute a valid drug template for PAF-AH inhibitors. Furthermore the P. palmata hydrolysate was nontoxic when assayed using the Zebrafish toxicity model at a concentration of 1mg/ml. Copyright © 2013 Elsevier Inc. All rights reserved.

Biochemistry of terminal deoxynucleotidyltransferase. Identification and unity of ribo- and deoxyribonucleoside triphosphate binding site in terminal deoxynucleotidyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, V.N.; Modak, M.J.

Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with (/sup 32/P)ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of (/sup 32/P)ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identicalmore » with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide. The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.« less

Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (Oryza sativa L.)

PubMed Central

Zhao, Jie

2010-01-01

Arabinogalactan proteins (AGPs) comprise a family of hydroxyproline-rich glycoproteins that are implicated in plant growth and development. In this study, 69 AGPs are identified from the rice genome, including 13 classical AGPs, 15 arabinogalactan (AG) peptides, three non-classical AGPs, three early nodulin-like AGPs (eNod-like AGPs), eight non-specific lipid transfer protein-like AGPs (nsLTP-like AGPs), and 27 fasciclin-like AGPs (FLAs). The results from expressed sequence tags, microarrays, and massively parallel signature sequencing tags are used to analyse the expression of AGP-encoding genes, which is confirmed by real-time PCR. The results reveal that several rice AGP-encoding genes are predominantly expressed in anthers and display differential expression patterns in response to abscisic acid, gibberellic acid, and abiotic stresses. Based on the results obtained from this analysis, an attempt has been made to link the protein structures and expression patterns of rice AGP-encoding genes to their functions. Taken together, the genome-wide identification and expression analysis of the rice AGP gene family might facilitate further functional studies of rice AGPs. PMID:20423940

Composition for nucleic acid sequencing

DOEpatents

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

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.

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.

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.

Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC

NASA Astrophysics Data System (ADS)

Barton, Janice S.; Tang, Chung-Fei; Reed, Steven S.

2000-02-01

Amino acid composition and sequence determination, important techniques for characterizing peptides and proteins, are essential for predicting conformation and studying sequence alignment. This experiment presents improved, fundamental methods of sequence analysis for an upper-division biochemistry laboratory. Working in pairs, students use the Edman reagent to prepare phenylthiohydantoin derivatives of amino acids for determination of the sequence of an unknown dipeptide. With a single HPLC technique, students identify both the N-terminal amino acid and the composition of the dipeptide. This method yields good precision of retention times and allows use of a broad range of amino acids as components of the dipeptide. Students learn fundamental principles and techniques of sequence analysis and HPLC.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2014-02-25

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-05-16

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-04-01

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2010-10-12

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVIII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-05-23

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl8, and the corresponding EGVIII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVIII, recombinant EGVIII proteins and methods for producing the same.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2010-10-05

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-06-06

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2009-05-05

The present invention provides an endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2013-07-16

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2012-02-14

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2015-04-14

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

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 target sequence.

Fibrinogen Lincoln: a new truncated alpha chain variant with delayed clotting.

PubMed

Ridgway, H J; Brennan, S O; Gibbons, S; George, P M

1996-04-01

A patient referred for preoperative investigation of prolonged bleeding and easy bruising was found to have increased thrombin and reptilase times; however, the thrombin catalysed release of fibrinopeptides A and B was normal. Analysis of five other family members, spanning three generations, indicated that three had a similar defect and suggested autosomal dominant inheritance. Non-reducing SDS-PAGE of purified fibrinogen from affected individuals showed that the 340 kD form of their fibrinogen ran as a doublet. SSCP (single-stranded conformational polymorphism) analysis of exon 5 of the A alpha gene, which encodes the C-terminal half of the chain, confirmed the presence of a mutation. Cycle sequencing of PCR amplified DNA revealed a 13 base pair deletion (nt 4758-4770), resulting in a frame-shift at Ala 475, which translates as four new amino acids before terminating at a new stop codon (-476His-Cys-Leu-Ala-Stop). The presence of a circulating truncated A alpha chain was confirmed when SDS-PAGE gels were probed with an alpha chain specific antisera; which showed that the variant A alpha chain comigrated with gamma chains. The truncation results in a variant A alpha chain with a deletion of 131 amino acids (480-610), and four new amino acids at the C-terminal.

Capillary electrophoresis, a method for the determination of nucleic acid ligands covalently attached to quantum dots representing a donor of Förster resonance energy transfer.

PubMed

Datinská, Vladimíra; Klepárník, Karel; Belšánová, Barbora; Minárik, Marek; Foret, František

2018-05-09

The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single-stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a non-complementary strands. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Identification and Functional Analysis of the Nocardithiocin Gene Cluster in Nocardia pseudobrasiliensis

PubMed Central

Sakai, Kanae; Komaki, Hisayuki; Gonoi, Tohru

2015-01-01

Nocardithiocin is a thiopeptide compound isolated from the opportunistic pathogen Nocardia pseudobrasiliensis. It shows a strong activity against acid-fast bacteria and is also active against rifampicin-resistant Mycobacterium tuberculosis. Here, we report the identification of the nocardithiocin gene cluster in N. pseudobrasiliensis IFM 0761 based on conserved thiopeptide biosynthesis gene sequence and the whole genome sequence. The predicted gene cluster was confirmed by gene disruption and complementation. As expected, strains containing the disrupted gene did not produce nocardithiocin while gene complementation restored nocardithiocin production in these strains. The predicted cluster was further analyzed using RNA-seq which showed that the nocardithiocin gene cluster contains 12 genes within a 15.2-kb region. This finding will promote the improvement of nocardithiocin productivity and its derivatives production. PMID:26588225

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.

Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

PubMed Central

Thomsen, Martin Christen Frølund; Nielsen, Morten

2012-01-01

Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed). PMID:22638583

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

PubMed Central

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

2010-01-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reiser, Steven E.; Somerville, Chris R.

The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

Geodermatophilus tzadiensis sp. nov., a UV radiation-resistant bacterium isolated from sand of the Saharan desert.

PubMed

Montero-Calasanz, Maria del Carmen; Göker, Markus; Broughton, William J; Cattaneo, Arlette; Favet, Jocelyne; Pötter, Gabriele; Rohde, Manfred; Spröer, Cathrin; Schumann, Peter; Klenk, Hans-Peter; Gorbushina, Anna A

2013-05-01

Three novel Gram-positive, aerobic, actinobacterial strains, CF5/2(T), CF5/1 and CF7/1, were isolated in 2007 during environmental screening of arid desert soil in the Sahara desert, Chad. Results from riboprinting, MALDI-TOF protein spectra and 16S rRNA sequence analysis confirmed that all three strains belonged to the same species. Phylogenetic analysis of 16S rRNA sequences with the strains' closest relatives indicated that they represented a distinct species. The three novel strains also shared a number of physiological and biochemical characteristics distinct from previously named Geodermatophilus species. The novel strains' peptidoglycan contained meso-diaminopimelic acid; their main phospholipids were phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and a small amount of phosphatidylglycerol; MK-9(H4) was the dominant menaquinone. The major cellular fatty acids were the branched-chain saturated acids iso-C16:0 and iso-C15:0. Galactose was detected as diagnostic sugar. Based on these chemotaxonomic results, 16S rRNA gene sequence analysis and DNA-DNA hybridization between strain CF5/2(T) and the type strains of Geodermatophilus saharensis, Geodermatophilus arenarius, Geodermatophilus nigrescens, Geodermatophilus telluris and Geodermatophilus siccatus, the isolates CF5/2(T), CF5/1 and CF7/1 are proposed to represent a novel species, Geodermatophilus tzadiensis, with type strain CF5/2(T)=DSM 45416=MTCC 11411 and two reference strains, CF5/1 (DSM 45415) and CF7/1 (DSM 45420). Copyright © 2013 Elsevier GmbH. All rights reserved.

Lactobacillus kefiri shows inter-strain variations in the amino acid sequence of the S-layer proteins.

PubMed

Malamud, Mariano; Carasi, Paula; Bronsoms, Sílvia; Trejo, Sebastián A; Serradell, María de Los Angeles

2017-04-01

The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea, and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri. Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L. kefiri, and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L. kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2013-01-29

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

BGL6 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2012-10-02

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL5 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-02-28

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

BGL5 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-03-18

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunn-Coleman, Nigel; Ward, Michael

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2014-03-04

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2015-04-14

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2014-03-25

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2015-08-11

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2007-09-25

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-04-01

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2011-12-06

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

BGL4 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-05-16

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2011-06-14

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Ward, Michael [San Francisco, CA

2009-09-01

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2012-10-30

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-01-22

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

Positive selection of digestive Cys proteases in herbivorous Coleoptera.

PubMed

Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

2015-10-01

Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grigoriev, Igor V.; Baker, Scott E.; Andersen, Mikael R.

2011-04-28

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regionsmore » have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up-regulation of genes relevant to glucoamylase A production, such as tRNA-synthases and protein transporters. Our results and datasets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.[Supplemental materials (10 figures, three text documents and 16 tables) have been made available. The whole genome sequence for A. niger ATCC 1015 is available from NBCI under acc. no ACJE00000000. The up-dated sequence for A. niger CBS 513.88 is available from EMBL under acc. no AM269948-AM270415. The sequence data from the phylogeny study has been submitted to NCBI (GU296686-296739). Microarray data from this study is submitted to GEO as series GSE10983. Accession for reviewers is possible through: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi token GSE10983] The dsmM_ANIGERa_coll511030F library and platform information is deposited at GEO under number GPL6758« less

Optical spectra, electronic structure and aromaticity of benzannulated N-heterocyclic carbene and its analogues of the type C6H4(NR)2E: (E = Si, Ge, Sn, Pb).

PubMed

Aysin, Rinat R; Bukalov, Sergey S; Leites, Larissa A; Zabula, Alexander V

2017-07-11

A series of benzannulated N-heterocyclic compounds containing divalent 14 group atoms, C 6 H 4 (NR) 2 E II , E = C, Si, Ge, Sn, Pb, have been studied by various experimental (vibrational and UV-vis spectroscopy) and theoretical (NICS, ISE, ACID) techniques. The methods used confirm 10 π-electron delocalization (aromaticity) in these heterocycles, however, the aromaticity sequences estimated by the criteria based on different physical properties do not coincide.

The nucleotide sequence of Watermelon mosaic virus (WMV, Potyvirus) reveals interspecific recombination between two related potyviruses in the 5' part of the genome.

PubMed

Desbiez, C; Lecoq, H

2004-08-01

Watermelon mosaic virus (WMV, Potyvirus) is a potyvirus with a worldwide distribution, mostly in temperate and mediterranean regions. According to the partial sequences that were available, WMV appeared to share high sequence similarity with Soybean mosaic virus (SMV), and it was almost considered as a strain of SMV in spite of its different and much broader host range. Like SMV, it was also related to legume-infecting potyviruses belonging to the " Bean common mosaic virus (BCMV) subgroup". In this paper we obtained the full-length sequence of WMV, and we confirmed that this virus is very closely related to SMV in most of its genome; however, there is evidence for an interspecific recombination in the P1 protein, as the P1 of WMV was 135 amino-acids longer than that of SMV, and the N-terminal half of the P1 showed no relation to SMV but was 85% identical to BCMV. This suggests that WMV has emerged through an ancestral recombination event, and supports the distinction of WMV and SMV as separate taxonomic units.

Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences.

PubMed Central

Lim, P O; Sears, B B

1992-01-01

The families within the class Mollicutes are distinguished by their morphologies, nutritional requirements, and abilities to metabolize certain compounds. Biosystematic classification of the plant-pathogenic mycoplasmalike organisms (MLOs) has been difficult because these organisms have not been cultured in vitro, and hence their nutritional requirements have not been determined nor have physiological characterizations been possible. To investigate the evolutionary relationship of the MLOs to other members of the class Mollicutes, a segment of a ribosomal protein operon was cloned and sequenced from an aster yellows-type MLO which is pathogenic for members of the genus Oenothera and from Acholeplasma laidlawii. The deduced amino acid sequence data from the rpl22 and rps3 genes indicate that the MLOs are more closely related to A. laidlawii than to animal mycoplasmas, confirming previous results from 16S rRNA sequence comparisons. This conclusion is also supported by the finding that the UGA codon is not read as a tryptophan codon in the MLO and A. laidlawii, in contrast to its usage in Mycoplasma capricolum. PMID:1556079

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.

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.

Novel pathogenic variant (c.3178G>A) in the SMC1A gene in a family with Cornelia de Lange syndrome identified by exome sequencing.

PubMed

Jang, Mi Ae; Lee, Chang Woo; Kim, Jin Kyung; Ki, Chang Seok

2015-11-01

Cornelia de Lange syndrome (CdLS) is a clinically and genetically heterogeneous congenital anomaly. Mutations in the NIPBL gene account for a half of the affected individuals. We describe a family with CdLS carrying a novel pathogenic variant of the SMC1A gene identified by exome sequencing. The proband was a 3-yr-old boy presenting with a developmental delay. He had distinctive facial features without major structural anomalies and tested negative for the NIPBL gene. His younger sister, mother, and maternal grandmother presented with mild mental retardation. By exome sequencing of the proband, a novel SMC1A variant, c.3178G>A, was identified, which was expected to cause an amino acid substitution (p.Glu1060Lys) in the highly conserved coiled-coil domain of the SMC1A protein. Sanger sequencing confirmed that the three female relatives with mental retardation also carry this variant. Our results reveal that SMC1A gene defects are associated with milder phenotypes of CdLS. Furthermore, we showed that exome sequencing could be a useful tool to identify pathogenic variants in patients with CdLS.

Genetic characterization of avian metapneumovirus subtype C isolated from pheasants in a live bird market.

PubMed

Lee, Eun ho; Song, Min-Suk; Shin, Jin-Young; Lee, Young-Min; Kim, Chul-Joong; Lee, Young Sik; Kim, Hyunggee; Choi, Young Ki

2007-09-01

Complete nucleotide sequences of two avian metapneumoviruses (aMPV), designated PL-1 and PL-2, were isolated from pheasants, revealing novel sequences of the first aMPV to be fully sequenced in Korea. The complete genome of both PL-1 and PL-2 was composed of 13,170 nucleotides. Phylogenetic analysis revealed that PL-1 belonged to aMPV subtype C, sharing higher homology in deduced amino acid sequence identities with hMPV, rather than with aMPV subtypes A and B. Replication of PL-1 in experimentally re-infected pheasants was confirmed by reverse transcription (RT)-polymerase chain reaction (PCR). Chickens and mice were experimentally inoculated with PL-1 to test the replication potential of PL-1 in other species. Although one specimen from the nasal turbinates of an inoculated chicken showed a slight trace of viral replication at 3 days post-infection (dpi), all of the infected mice were negative for aMPV by RT-PCR throughout the experiment, suggesting that PL-1 does not readily infect mammals. This is the first report of the isolation and complete genomic sequence of aMPV subtype C originating from pheasants.

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device.

PubMed

Nishiyama, Kazusa; Takakusagi, Yoichi; Kusayanagi, Tomoe; Matsumoto, Yuki; Habu, Shiori; Kuramochi, Kouji; Sugawara, Fumio; Sakaguchi, Kengo; Takahashi, Hideyo; Natsugari, Hideaki; Kobayashi, Susumu

2009-01-01

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.

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.

Human jagged polypeptide, encoding nucleic acids and methods of use

DOEpatents

Li, Linheng; Hood, Leroy

2000-01-01

The present invention provides an isolated polypeptide exhibiting substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the polypeptide does not have the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the nucleotide sequence does not encode the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Also provided herein is a method of inhibiting differentiation of hematopoietic progenitor cells by contacting the progenitor cells with an isolated JAGGED polypeptide, or active fragment thereof. The invention additionally provides a method of diagnosing Alagille Syndrome in an individual. The method consists of detecting an Alagille Syndrome disease-associated mutation linked to a JAGGED locus.

Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.

PubMed

Oberg, Craig J; Oberg, Taylor S; Culumber, Michele D; Ortakci, Fatih; Broadbent, Jeffery R; McMahon, Donald J

2016-01-01

A Gram-stain positive, rod-shaped, non-spore-forming strain (WDC04T), which may be associated with late gas production in cheese, was isolated from aged Cheddar cheese following incubation on MRS agar (pH 5.2) at 6 °C for 35 days. Strain WDC04T had 97 % 16S rRNA gene sequence similarity with Lactobacillus hokkaidonensis DSM 26202T, Lactobacillus oligofermentans 533, 'Lactobacillus danicus' 9M3, Lactobacillus suebicus CCUG 32233T and Lactobacillus vaccinostercus DSM 20634T. API 50 CH carbohydrate fermentation panels indicated strain WDC04T could only utilize one of the 50 substrates tested, ribose, although it does slowly utilize galactose. In the API ZYM system, strain WDC04T was positive for leucine arylamidase, valine arylamidase, cysteine arylamidase (weakly), naphthol-AS-BI-phosphohydrolase and β-galactosidase activities. Total genomic DNA was sequenced from strain WDC04T using a whole-genome shotgun strategy on a 454 GS Titanium pyrosequencer. The sequence was assembled into a 1.90 Mbp draft genome consisting of 105 contigs with preliminary genome annotation performed using the RAST algorithm (rast.nmpdr.org). Genome analysis confirmed the pentose phosphate pathway for ribose metabolism as well as galactose, N-acetylglucosamine, and glycerol fermentation pathways. Genomic analysis places strain WDC04T in the obligately heterofermentative group of lactobacilli and metabolic results confirm this conclusion. The result of genome sequencing, along with 16S rRNA gene sequence analysis, indicates WDC04T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus wasatchensis sp. nov. is proposed. The type strain is WDC04T ( = DSM 29958T = LMG 28678T).

Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom.

PubMed

Romero-Vargas, Frey Francisco; Ponce-Soto, Luis Alberto; Martins-de-Souza, Daniel; Marangoni, Sergio

2010-01-01

This work reports the purification, biological characterization and amino acid sequence of two new basic PLA(2) isoforms, Cdc-9 and Cdc-10, purified from the Crotalus durissus cumanensis venom by one step analytical chromatography reverse phase HPLC. The molecular masses of the PLA(2) were 14,175+/-2.7 Da for Cdc-9 and 14,228+/-3.5 Da for Cdc-10 both deduced by primary structure and confirmed by MALDI-TOF. The isoforms presented an amino acid sequence of 122 amino acid residues, being Cdc-9: SLVQFNKMIK FETRKSGLPF YAAYGCYCGW GGQRPKDATD RCCFVHDCCY GKVAKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLS TYKNEYMFYP DSRCREPPEY TC with pI value of 8.25 and Cdc-10: SLLQFNKMIK FETRKSGVPF YAAYGCYCGW GGRRPKDPTD RCCFVHDCCY GKLTKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLN TYKNEYMFYP DSRCRGPPEY TC with a pI value of 8.46, showing highly conserved Ca(2+)-binding and catalytic sites. The PLA(2) activity decreased when the isoforms Cdc-9 and Cdc-10 were incubated with 4-bromophenacyl bromide (p-BPB), anhydrous acetic acid and p-nitrobenzene sulfonyl fluoride (NBSF) when compared with the activity of both native isoforms. In mice, the PLA(2) isoforms Cdc-9 and Cdc-10 induced myonecrosis and edema. Myotoxic and edema activities were reduced after treatment of the isoforms with p-BPB; acetylation of the lysine residues and the treatment of PLA(2) with NBSF have also induced edema reduction. However, p-BPB strongly diminishes the local and systemic myotoxic effects.

Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2016-02-16

The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well asmore » the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.« less

Polypeptide having swollenin activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elizabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica D; Damveld, Robbertus Antonius

2015-11-04

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

2015-09-01

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having cellobiohydrolase activity and uses thereof

DOEpatents

Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

2015-09-15

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having acetyl xylan esterase activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2015-10-20

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having carbohydrate degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica Diana; Damveld, Robbertus Antonius

2015-08-18

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Complementary DNA sequencing and identification of mRNAs from the venomous gland of Agkistrodon piscivorus leucostoma.

PubMed

Jia, Ying; Cantu, Bruno A; Sánchez, Elda E; Pérez, John C

2008-06-15

To advance our knowledge on the snake venom composition and transcripts expressed in venom gland at the molecular level, we constructed a cDNA library from the venom gland of Agkistrodon piscivorus leucostoma for the generation of expressed sequence tags (ESTs) database. From the randomly sequenced 2112 independent clones, we have obtained ESTs for 1309 (62%) cDNAs, which showed significant deduced amino acid sequence similarity (scores >80) to previously characterized proteins in National Center for Biotechnology Information (NCBI) database. Ribosomal proteins make up 47 clones (2%) and the remaining 756 (36%) cDNAs represent either unknown identity or show BLASTX sequence identity scores of <80 with known GenBank accessions. The most highly expressed gene encoding phospholipase A(2) (PLA(2)) accounting for 35% of A. p. leucostoma venom gland cDNAs was identified and further confirmed by crude venom applied to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and protein sequencing. A total of 180 representative genes were obtained from the sequence assemblies and deposited to EST database. Clones showing sequence identity to disintegrins, thrombin-like enzymes, hemorrhagic toxins, fibrinogen clotting inhibitors and plasminogen activators were also identified in our EST database. These data can be used to develop a research program that will help us identify genes encoding proteins that are of medical importance or proteins involved in the mechanisms of the toxin venom.

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

Code of Federal Regulations, 2010 CFR

2010-07-01

... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide and...

37 CFR 5.31-5.33 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-07-01

... from abandonment 1.135 Amino Acid Sequences. (See Nucleotide and/or Amino Acid Sequences) Appeal to... Appeals and Interference 41.47 Of rejection of an application 1.104(a) Nucleotide and/or Amino Acid...) Symbols for nucleotide and/or amino acid sequence data 1.822 T Tables in patent applications 1.58 Terminal...

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Nucleotide and/or amino acid... Biotechnology Invention Disclosures Application Disclosures Containing Nucleotide And/or Amino Acid Sequences § 1.821 Nucleotide and/or amino acid sequence disclosures in patent applications. (a) Nucleotide and...

Sequence-based identification of inositol monophosphatase-like histidinol-phosphate phosphatases (HisN) in Corynebacterium glutamicum, Actinobacteria, and beyond.

PubMed

Kulis-Horn, Robert Kasimir; Rückert, Christian; Kalinowski, Jörn; Persicke, Marcus

2017-07-18

The eighth step of L-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). Three unrelated HolPase families are known so far. Two of them are well studied: HAD-type HolPases known from Gammaproteobacteria like Escherichia coli or Salmonella enterica and PHP-type HolPases known from yeast and Firmicutes like Bacillus subtilis. However, the third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. Moreover, there exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in L-histidine biosynthesis. Deletion of hisN, the gene encoding the IMPase-like HolPase in C. glutamicum, does not result in complete L-histidine auxotrophy. Out of four hisN homologs present in the genome of C. glutamicum (impA, suhB, cysQ, and cg0911), only cg0911 encodes an enzyme with HolPase activity. The enzymatic properties of HisN and Cg0911 were determined, delivering the first available kinetic data for IMPase-like HolPases. Additionally, we analyzed the amino acid sequences of potential HisN, ImpA, SuhB, CysQ and Cg0911 orthologs from bacteria and identified six conserved sequence motifs for each group of orthologs. Mutational studies confirmed the importance of a highly conserved aspartate residue accompanied by several aromatic amino acid residues present in motif 5 for HolPase activity. Several bacterial proteins containing all identified HolPase motifs, but showing only moderate sequence similarity to HisN from C. glutamicum, were experimentally confirmed as IMPase-like HolPases, demonstrating the value of the identified motifs. Based on the confirmed IMPase-like HolPases two profile Hidden Markov Models (HMMs) were build using an iterative approach. These HMMs allow the fast, reliable detection and differentiation of the two paralog groups from each other and other IMPases. The kinetic data obtained for HisN from C. glutamicum, as an example for an IMPase-like HolPases, shows remarkable differences in enzyme properties as compared to HAD- or PHP-type HolPases. The six sequence motifs and the HMMs presented in this study can be used to reliably differentiate between IMPase-like HolPases and IMPase-like proteins with no such activity, with the potential to enhance current and future genome annotations. A phylogenetic analysis reveals that IMPase-like HolPases are not only present in Actinobacteria and plant but can be found in further bacterial phyla, including, among others, Proteobacteria, Chlorobi and Planctomycetes.

Molecular characterization and phylogenetic study of African swine fever virus isolates from recent outbreaks in Uganda (2010–2013)

PubMed Central

2013-01-01

Background African swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in Eastern Africa particularly in Uganda where outbreaks regularly occur. Sequence analysis of variable genome regions have been extensively used for molecular epidemiological studies of African swine fever virus (ASFV) isolates. By combining p72, P54 and pB602L (CVR), a high level resolution approach is achieved for viral discrimination. The major aim of this study therefore, was to investigate the genetic relatedness of ASF outbreaks that occurred between 2010 and 2013 in Uganda to contribute to the clarification of the epidemiological situation over a four year period. Methods Tissue samples from infected domestic pigs associated with an ASF outbreak from 15 districts in Uganda were confirmed as being infected with ASFV using a p72 gene-based polymerase chain reaction amplification (PCR) assay recommended by OIE. The analysis was conducted by genotyping based on sequence data from three single copy ASFV genes. The E183L gene encoding the structural protein P54 and part of the gene encoding the p72 protein was used to delineate genotypes. Intra-genotypic resolution of viral relationships was achieved by analysis of tetramer amino acid repeats within the hypervariable CVR of the B602L gene. Results Twenty one (21) ASF outbreaks were confirmed by the p72 ASF diagnostic PCR, however; only 17 isolates were successfully aligned after sequencing. Our entire isolates cluster with previous ASF viruses in genotype IX isolated in Uganda and Kenya using p72 and P54 genes. Analysis of the CVR gene generated three sub-groups one with 23 tetrameric amino acid repeats (TRS) with an additional CAST sequence, the second with 22 TRS while one isolate Ug13. Kampala1 had 13 TRS. Conclusion We identified two new CVR subgroups different from previous studies. This study constitutes the first detailed assessment of the molecular epidemiology of ASFV in domestic pigs in the different regions of Uganda. PMID:23914918

Unraveling the sequence and structure of the protein osteocalcin from a 42 ka fossil horse

NASA Astrophysics Data System (ADS)

Ostrom, Peggy H.; Gandhi, Hasand; Strahler, John R.; Walker, Angela K.; Andrews, Philip C.; Leykam, Joseph; Stafford, Thomas W.; Kelly, Robert L.; Walker, Danny N.; Buckley, Mike; Humpula, James

2006-04-01

We report the first complete amino acid sequence and evidence of secondary structure for osteocalcin from a temperate fossil. The osteocalcin derives from a 42 ka equid bone excavated from Juniper Cave, Wyoming. Results were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS) and Edman sequencing with independent confirmation of the sequence in two laboratories. The ancient sequence was compared to that of three modern taxa: horse ( Equus caballus), zebra ( Equus grevyi), and donkey ( Equus asinus). Although there was no difference in sequence among modern taxa, MALDI-MS and Edman sequencing show that residues 48 and 49 of our modern horse are Thr, Ala rather than Pro, Val as previously reported (Carstanjen B., Wattiez, R., Armory, H., Lepage, O.M., Remy, B., 2002. Isolation and characterization of equine osteocalcin. Ann. Med. Vet.146(1), 31-38). MALDI-MS and Edman sequencing data indicate that the osteocalcin sequence of the 42 ka fossil is similar to that of modern horse. Previously inaccessible structural attributes for ancient osteocalcin were observed. Glu 39 rather than Gln 39 is consistent with deamidation, a process known to occur during fossilization and aging. Two post-translational modifications were documented: Hyp 9 and a disulfide bridge. The latter suggests at least partial retention of secondary structure. As has been done for ancient DNA research, we recommend standards for preparation and criteria for authenticating results of ancient protein sequencing.

Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

PubMed Central

Sloma, A; Rudolph, C F; Rufo, G A; Sullivan, B J; Theriault, K A; Ally, D; Pero, J

1990-01-01

The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation. Images FIG. 2 FIG. 7 PMID:2105291

Phylogenetic and Structural Analysis of the Pluripotency Factor Sex-Determining Region Y box2 Gene of Camelus dromedarius (cSox2).

PubMed

Alawad, Abdullah; Alharbi, Sultan; Alhazzaa, Othman; Alagrafi, Faisal; Alkhrayef, Mohammed; Alhamdan, Ziyad; Alenazi, Abdullah; Al-Johi, Hasan; Alanazi, Ibrahim O; Hammad, Mohamed

2016-01-01

Although the sequencing information of Sox2 cDNA for many mammalian is available, the Sox2 cDNA of Camelus dromedaries has not yet been characterized. The objective of this study was to sequence and characterize Sox2 cDNA from the brain of C. dromedarius (also known as Arabian camel). A full coding sequence of the Sox2 gene from the brain of C. dromedarius was amplified by reverse transcription PCRjmc and then sequenced using the 3730XL series platform Sequencer (Applied Biosystem) for the first time. The cDNA sequence displayed an open reading frame of 822 nucleotides, encoding a protein of 273 amino acids. The molecular weight and the isoelectric point of the translated protein were calculated as 29.825 kDa and 10.11, respectively, using bioinformatics analysis. The predicted cSox2 protein sequence exhibited high identity: 99% for Homo sapiens, Mus musculus, Bos taurus, and Vicugna pacos; 98% for Sus scrofa and 93% for Camelus ferus. A 3D structure was built based on the available crystal structure of the HMG-box domain of human stem cell transcription factor Sox2 (PDB: 2 LE4) with 81 residues and predicting bioinformatics software for 273 amino acid residues. The comparison confirms the presence of the HMG-box domain in the cSox2 protein. The orthologous phylogenetic analysis showed that the Sox2 isoform from C. dromedarius was grouped with humans, alpacas, cattle, and pigs. We believe that this genetic and structural information will be a helpful source for the annotation. Furthermore, Sox2 is one of the transcription factors that contributes to the generation-induced pluripotent stem cells (iPSCs), which in turn will probably help generate camel induced pluripotent stem cells (CiPSCs).

Novel Single-Base Deletional Mutation in Major Intrinsic Protein (MIP) in Autosomal Dominant Cataract

PubMed Central

Geyer, David D.; Spence, M. Anne; Johannes, Meriam; Flodman, Pamela; Clancy, Kevin P.; Berry, Rebecca; Sparkes, Robert S.; Jonsen, Matthew D.; Isenberg, Sherwin J.; Bateman, J. Bronwyn

2006-01-01

PURPOSE To further elucidate the cataract phenotype, and identify the gene and mutation for autosomal dominant cataract (ADC) in an American family of European descent (ADC2) by sequencing the major intrinsic protein gene (MIP), a candidate based on linkage to chromosome 12q13. DESIGN Observational case series and laboratory experimental study. METHODS We examined two at-risk individuals in ADC2. We PCR-amplified and sequenced all four exons and all intron-exon boundaries of the MIP gene from genomic and cloned DNA in affected members to confirm one variant as the putative mutation. RESULTS We found a novel single deletion of nucleotide (nt) 3223 (within codon 235) in exon four, causing a frameshift that alters 41 of 45 subsequent amino acids and creates a premature stop codon. CONCLUSIONS We identified a novel single base pair deletion in the MIP gene and conclude that it is a pathogenic sequence alteration. PMID:16564824

Epidemiological and molecular analysis of a waterborne outbreak of norovirus GII.4.

PubMed

Zhou, X; Li, H; Sun, L; Mo, Y; Chen, S; Wu, X; Liang, J; Zheng, H; Ke, C; Varma, J K; Klena, J D; Chen, Q; Zou, L; Yang, X

2012-12-01

Contaminated water is one of the main sources of norovirus (NoV) gastroenteritis outbreaks globally. Waterborne NoV outbreaks are infrequently attributed to GII.4 NoV. In September 2009, a NoV outbreak affected a small school in Guangdong Province, China. Epidemiological investigations indicated that household use water, supplied by a well, was the probable source (relative risk 1·9). NoV nucleic acid material in concentrated well-water samples was detected using real-time RT-PCR. Nucleotide sequences of NoV extracted from diarrhoea and well-water specimens were identical and had the greatest sequence identity to corresponding sequences from the epidemic strain GII.4-2006b. Our report documents the first laboratory-confirmed waterborne outbreak caused by GII.4 NoV genotype in China. Our investigations indicate that well water, intended exclusively for household use but not for consumption, caused this outbreak. The results of this report serve as a reminder that private well water intended for household use should be tested for NoV.

Weissella ghanensis sp. nov., isolated from a Ghanaian cocoa fermentation.

PubMed

De Bruyne, Katrien; Camu, Nicholas; Lefebvre, Karen; De Vuyst, Luc; Vandamme, Peter

2008-12-01

During a study on lactic acid bacteria (and their species diversity) in spontaneous heap fermentations of Ghanaian cocoa beans, two strains, designated 215(T) and 194B, were isolated. A phylogenetic analysis based on 16S rRNA gene sequences demonstrated that these strains represented a distinct lineage close to the genus Weissella and showing only 92.1 % 16S rRNA gene sequence similarity with respect to their closest neighbour, Weissella soli LMG 20113(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and physiological and biochemical tests confirmed the unique taxonomic position of the two novel isolates. On the basis of the results of the morphological and biochemical tests and 16S rRNA gene sequence analysis, strains 215(T) and 194B represent the most peripheral lineage of the genus Weissella, for which we propose the name Weissella ghanensis sp. nov. The type strain is 215(T) (=LMG 24286(T)=DSM 19935(T)).

[Application of the polymerase chain reaction (PCR) in the diagnosis of Hb S-beta(+)-thalassemia].

PubMed

Harano, K; Harano, T; Kushida, Y; Ueda, S

1991-08-01

Isoelectric focusing of the hemolysate prepared from a two-year-old American black boy with microcytic hypochromia showed the presence of a high percentage (63.3%) of such Hb variant as Hb S, while the levels of Hb A, Hb F and Hb A2 were 20.0%, 12.7%, and 4.0%, respectively. The ratio of the non-alpha-chain to the alpha-chain of the biosynthesized globin chains was 0.49. The variant was identified as Hb S by amino acid analysis of the abnormal peptide (beta T-1) and digestion of DNA amplified by the polymerase chain reaction with enzyme Eco 81 I. This was further confirmed by DNA sequencing. DNA sequencing of a beta-gene without the beta s-mutation revealed a nucleotide change of T to C in the polyadenylation signal sequence AATAAA 3' to the beta-gene, resulting in beta(+)-thalassemia. These results are consistent with the existence of a beta s-gene and a beta(+)-thalassemia gene in trans.

An amino acid sequence coded by the exon 2 of the BoLA DRB3 gene associated with a BoLA class I specificity constitutes a likely genetic marker of resistance to dermatophilosis in Brahman zebu cattle of Martinique (FWI).

PubMed

Maillard, J C; Martinez, D; Bensaid, A

1996-07-23

One hundred and twenty-seven Brahman cattle from several locations in Martinique (FWI), reared under different environmental conditions, were followed over three years and checked for clinical signs of dermatophilosis. To confirm that these animals had been in contact with the pathogen Dermatophilus congolensis, their sera were tested by ELISA. On the basis of this epidemiological study, 12 animals were classified as resistant (seropositive without clinical signs), belonging to herds in which the prevalence of the disease ranged from 25 to nearly 98%. Eighteen animals classified as highly susceptible displayed severe characteristic skin lesions. These 30 selected animals were typed for class I antigens of the major histocompatibility complex (MHC). MHC class II genes were analyzed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques, on the exon 2 of the bovine leucocyte antigen (BoLA) DRB3 gene. Several alleles were found, according to patterns provided by the restriction enzymes used: Fnu 4HI, Dpn II, Hae III, and Rsa I. A particular sequence "EIAY" at amino acid positions 66/67/74/78 located in the antigen recognition sites (ARS) was found in the 12 animals classified as resistant, and 10 of them displayed also class I BoLA-A8 specificity. On the other hand, only 3 out of the 18 susceptible animals showed simultaneously the BoLA-DRB3 "EIAY" sequence and BoLA-A8 specificity. Interestingly, a serine residue at position 30 of the ARS was found in 8 of the susceptible animals and was completely absent from all resistant animals. Furthermore, in a same animal, the serine at position 30 and the EIAY sequence were never found simultaneously on the same haplotype. These results show a strong correlation between the resistant character to dermatophilosis and the association of MHC haplotypes: the BoLA-A8 specificity and the BoLA-DRB3 "EIAY" sequence at ARS positions 66/67/74/78 with the lack of serine in position 30. To confirm these results, family segregation studies are in progress and some interesting observations have been obtained.

β-Globin gene sequencing of hemoglobin Austin revises the historically reported electrophoretic migration pattern.

PubMed

Racsa, Lori D; Luu, Hung S; Park, Jason Y; Mitui, Midori; Timmons, Charles F

2014-06-01

Hemoglobin (Hb) Austin was defined in 1977, using amino acid sequencing of samples from 3 unrelated Mexican-Americans, as a substitution of serine for arginine at position 40 of the β-globin chain (Arg40Ser). Its electrophoretic migration on both cellulose acetate (pH 8.4) and citrate agar (pH 6.2) was reported between Hb F and Hb A, and this description persists in reference literature. OBJECTIVES.-To review the clinical features and redefine the diagnostic characteristics of Hb Austin. Eight samples from 6 unrelated individuals and 2 siblings, all with Hispanic surnames, were submitted for abnormal Hb identification between June 2010 and September 2011. High-performance liquid chromatography, isoelectric focusing (IEF), citrate agar electrophoresis, and bidirectional DNA sequencing of the entire β-globin gene were performed. DNA sequencing confirmed all 8 individuals to be heterozygous for Hb Austin (Arg40Ser). Retention time on high-performance liquid chromatography and migration on citrate agar electrophoresis were consistent with that identification. Migration on IEF, however, was not between Hb F and Hb A, as predicted from the report of cellulose acetate electrophoresis. By IEF, Hb Austin migrated anodal to ("faster than") Hb A. Hemoglobin Austin (Arg40Ser) appears on IEF as a "fast," anodally migrating, Hb variant, just as would be expected from its amino acid substitution. The cited historic report is, at best, not applicable to IEF and is probably erroneous. Our observation of 8 cases in 16 months suggests that this variant may be relatively common in some Hispanic populations, making its recognition important. Furthermore, gene sequencing is proving itself a powerful and reliable tool for definitive identification of Hb variants.

Thermophilic cellobiohydrolase

DOEpatents

Sapra, Rajat; Park, Joshua I.; Datta, Supratim; Simmons, Blake A.

2017-04-18

The present invention provides for a composition comprising a polypeptide comprising a first amino acid sequence having at least 70% identity with the amino acid sequence of Csac GH5 wherein said first amino acid sequence has a thermostable or thermophilic cellobiohydrolase (CBH) or exoglucanase activity.

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.

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.

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.

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.

Cell culture compositions

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

2014-03-18

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

Identification of angiotensin I in several vertebrate species: its structural and functional evolution.

PubMed

Takei, Yoshio; Joss, Jean M P; Kloas, Werner; Rankin, J Cliff

2004-02-01

In order to delineate further the molecular evolution of the renin-angiotensin system in vertebrates, angiotensin I (ANG I) has been isolated after incubation of plasma and kidney extracts of emu (Dromiceus novaehollandiae), axolotl (Ambystoma mexicanum), and sea lamprey (Petromyzon marinus). The identified sequences were [Asp1, Val5, Asn9] ANG I in emu, [Asp1, Val5, His9] ANG I in axolotl, and [Asn1, Val5, Thr9] ANG I in sea lamprey. These results confirmed the previous findings that tetrapods have Asp and fishes including cyclostomes have Asn at the N-terminus, and that the amino acid residue at position 9 of ANG I was highly variable but, those at other positions were well conserved among different species. Since Asp and Asn are convertible during incubation, angiotensinogen sequences were searched in the genome and/or EST database to determine the N-terminal amino acid residue from the gene. The screening detected 12 tetrapod (10 mammalian, one avian, and one amphibian) and seven teleostean angiotensinogen sequences. Among them, all tetrapods have [Asp1] ANG except for Xenopus, and all teleosts have [Asn1] ANG, thereby confirming the above rule. Comparison of the vasopressor activity in the eel revealed that [Asn1] ANG I and II were more potent than [Asp1] peptides, which was opposite to the previous results in mammals and birds, in which [Asp1] ANG I and II were more potent. Collectively, the present results support the general rule that tetrapods have [Asp1] ANG and fishes including cyclostomes have [Asn1] ANG. However, an aquatic anuran (Xenopus) has [Asn1] ANG in its gene despite another aquatic urodele (axolotl) has [Asp1] ANG. From the functional viewpoint, homologous [Asn1] ANG was more potent in fish as is homologous [Asp1] ANG in tetrapods, suggesting that ANG II molecule has undergone co-evolution with its receptor during vertebrate phylogeny.

Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris

PubMed Central

Jiménez, Juan J.; Gútiez, Loreto; Cintas, Luis M.; Herranz, Carmen; Hernández, Pablo E.

2015-01-01

We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins. PMID:25821820

Nucleotide sequence of the 3' terminal region of lettuce mosaic potyvirus RNA shows a Gln/Val dipeptide at the cleavage site between the polymerase and the coat protein.

PubMed

Dinant, S; Lot, H; Albouy, J; Kuziak, C; Meyer, M; Astier-Manifacier, S

1991-01-01

DNA complementary to the 3' terminal 1651 nucleotides of the genome of the common strain of lettuce mosaic virus (LMV-O) has been cloned and sequenced. Microsequencing of the N-terminus enabled localization of the coat protein gene in this sequence. It showed also that the LMV coat protein coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein by cleavage at an unusual Q/V dipeptide between the polymerase and the coat protein. This is the first report of such a site for cleavage of a potyvirus polyprotein, where only Q/A, Q/S, and Q/G cleavage sites have been reported. The LMV coat protein gene encodes a 278 amino acid polypeptide with a calculated Mr of 31,171 and is flanked by a region which has a high degree of homology with the putative polymerase and a 3' untranslated region of 211 nucleotides in length. Percentage of homology with the coat protein of other potyviruses confirms that LMV is a distinct member of this group. Moreover, amino acid homologies noticed with the coat protein of potexvirus, bymovirus, and carlavirus elongated plant viruses suggest a functional significance for the conserved domains.

Recombinant lactoferrin (Lf) of Vechur cow, the critical breed of Bos indicus and the Lf gene variants.

PubMed

Anisha, Shashidharan; Bhasker, Salini; Mohankumar, Chinnamma

2012-03-01

Vechur cow, categorized as a critically maintained breed by the FAO, is a unique breed of Bos indicus due to its extremely small size, less fodder intake, adaptability, easy domestication and traditional medicinal property of the milk. Lactoferrin (Lf) is an iron-binding glycoprotein that is found predominantly in the milk of mammals. The full coding region of Lf gene of Vechur cow was cloned, sequenced and expressed in a prokaryotic system. Antibacterial activity of the recombinant Lf showed suppression of bacterial growth. To the best of our knowledge this is the first time that the full coding region of Lf gene of B. indicus Vechur breed is sequenced, successfully expressed in a prokaryotic system and characterized. Comparative analysis of Lf gene sequence of five Vechur cows with B. taurus revealed 15 SNPs in the exon region associated with 11 amino acid substitutions. The amino acid arginine was noticed as a pronounced substitution and the tertiary structure analysis of the BLfV protein confirmed the positions of arginine in the β sheet region, random coil and helix region 1. Based on the recent reports on the nutritional therapies of arginine supplementation for wound healing and for cardiovascular diseases, the higher level of arginine in the lactoferrin protein of Vechur cow milk provides enormous scope for further therapeutic studies. Copyright © 2011 Elsevier B.V. All rights reserved.

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

PubMed Central

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

Labeled nucleotide phosphate (NP) probes

DOEpatents

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

2009-02-03

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.

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase1

PubMed Central

Yasuno, Rie; Wada, Hajime

1998-01-01

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide. PMID:9808738

Towards proteomic analysis of milk proteins in historical building materials

NASA Astrophysics Data System (ADS)

Kuckova, S.; Crhova, M.; Vankova, L.; Hnizda, A.; Hynek, R.; Kodicek, M.

2009-07-01

The addition of proteinaceous binders to mortars and plasters has a long tradition. The protein additions were identified in many sacral and secular historical buildings. For this method of peptide mass mapping, three model mortar samples with protein additives were prepared. These samples were analysed fresh (1-2 weeks old) and after 9 months of natural ageing. The optimal duration of tryptic cleavage (2 h) and the lowest amount of material needed for relevant analysis of fresh and weathered samples were found; the sufficient amounts of weathered and fresh mortars were set to 0.05 and 0.005 g. The list of main tryptic peptides coming from milk additives (bovine milk, curd, and whey), their relative intensities and theoretical amino acid sequences assignment is presented. Several sequences have been "de novo" confirmed by mass spectrometry.

Identification of a type-D feruloyl esterase from Neurospora crassa.

PubMed

Crepin, V F; Faulds, C B; Connerton, I F

2004-02-01

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.

Trichoderma .beta.-glucosidase

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-01-03

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

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).

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).

Carbohydrate degrading polypeptide and uses thereof

DOEpatents

Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

2015-10-20

The invention relates to a polypeptide having carbohydrate material degrading activity which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Nucleic Acid Amplification Testing and Sequencing Combined with Acid-Fast Staining in Needle Biopsy Lung Tissues for the Diagnosis of Smear-Negative Pulmonary Tuberculosis.

PubMed

Jiang, Faming; Huang, Weiwei; Wang, Ye; Tian, Panwen; Chen, Xuerong; Liang, Zongan

2016-01-01

Smear-negative pulmonary tuberculosis (PTB) is common and difficult to diagnose. In this study, we investigated the diagnostic value of nucleic acid amplification testing and sequencing combined with acid-fast bacteria (AFB) staining of needle biopsy lung tissues for patients with suspected smear-negative PTB. Patients with suspected smear-negative PTB who underwent percutaneous transthoracic needle biopsy between May 1, 2012, and June 30, 2015, were enrolled in this retrospective study. Patients with AFB in sputum smears were excluded. All lung biopsy specimens were fixed in formalin, embedded in paraffin, and subjected to acid-fast staining and tuberculous polymerase chain reaction (TB-PCR). For patients with positive AFB and negative TB-PCR results in lung tissues, probe assays and 16S rRNA sequencing were used for identification of nontuberculous mycobacteria (NTM). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of PCR and AFB staining were calculated separately and in combination. Among the 220 eligible patients, 133 were diagnosed with TB (men/women: 76/57; age range: 17-80 years, confirmed TB: 9, probable TB: 124). Forty-eight patients who were diagnosed with other specific diseases were assigned as negative controls, and 39 patients with indeterminate final diagnosis were excluded from statistical analysis. The sensitivity, specificity, PPV, NPV, and accuracy of histological AFB (HAFB) for the diagnosis of smear-negative were 61.7% (82/133), 100% (48/48), 100% (82/82), 48.5% (48/181), and 71.8% (130/181), respectively. The sensitivity, specificity, PPV, and NPV of histological PCR were 89.5% (119/133), 95.8% (46/48), 98.3% (119/121), and 76.7% (46/60), respectively, demonstrating that histological PCR had significantly higher accuracy (91.2% [165/181]) than histological acid-fast staining (71.8% [130/181]), P < 0.001. Parallel testing of histological AFB staining and PCR showed the sensitivity, specificity, PPV, NPV, and accuracy to be 94.0% (125/133), 95.8% (46/48), 98.4% (125/127), 85.2% (46/54), and 94.5% (171/181), respectively. Among patients with positive AFB and negative PCR results in lung tissue specimens, two were diagnosed with NTM infections (Mycobacterium avium-intracellulare complex and Mycobacterium kansasii). Nucleic acid amplification testing combined with acid-fast staining in lung biopsy tissues can lead to early and accurate diagnosis in patients with smear-negative pulmonary tuberculosis. For patients with positive histological AFB and negative tuberculous PCR results in lung tissue, NTM infection should be suspected and could be identified by specific probe assays or 16S rRNA sequencing.

Automated Sanger Analysis Pipeline (ASAP): A Tool for Rapidly Analyzing Sanger Sequencing Data with Minimum User Interference.

PubMed

Singh, Aditya; Bhatia, Prateek

2016-12-01

Sanger sequencing platforms, such as applied biosystems instruments, generate chromatogram files. Generally, for 1 region of a sequence, we use both forward and reverse primers to sequence that area, in that way, we have 2 sequences that need to be aligned and a consensus generated before mutation detection studies. This work is cumbersome and takes time, especially if the gene is large with many exons. Hence, we devised a rapid automated command system to filter, build, and align consensus sequences and also optionally extract exonic regions, translate them in all frames, and perform an amino acid alignment starting from raw sequence data within a very short time. In full capabilities of Automated Mutation Analysis Pipeline (ASAP), it is able to read "*.ab1" chromatogram files through command line interface, convert it to the FASTQ format, trim the low-quality regions, reverse-complement the reverse sequence, create a consensus sequence, extract the exonic regions using a reference exonic sequence, translate the sequence in all frames, and align the nucleic acid and amino acid sequences to reference nucleic acid and amino acid sequences, respectively. All files are created and can be used for further analysis. ASAP is available as Python 3.x executable at https://github.com/aditya-88/ASAP. The version described in this paper is 0.28.

Nucleic acid analysis using terminal-phosphate-labeled nucleotides

DOEpatents

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

2008-04-22

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.

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: cloning and expression of gntP and gntK in Escherichia coli.

PubMed

Porco, Antonietta; Gamero, Elida E; Mylonás, Elena; Istúriz, Tomás

2008-01-01

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral medium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constitutively expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli M1-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

Evaluation of commercial soy sauce koji strains of Aspergillus oryzae for γ-aminobutyric acid (GABA) production.

PubMed

Ab Kadir, Safuan; Wan-Mohtar, Wan Abd Al Qadr Imad; Mohammad, Rosfarizan; Abdul Halim Lim, Sarina; Sabo Mohammed, Abdulkarim; Saari, Nazamid

2016-10-01

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P < 0.05) among themselves. Based on the A. oryzae strains, highest GABA concentration was obtained from NSK (194 mg/L) followed by NSZ (63 mg/L), NSJ (51.53 mg/L) and NST (31.66 mg/L). Therefore, A. oryzae NSK was characterized and the sequence was found to be similar to A. oryzae and A. flavus with 99 % similarity. The evolutionary distance (K nuc) between sequences of identical fungal species was calculated and a phylogenetic tree prepared from the K nuc data showed that the isolate belonged to the A. oryzae species. This finding may allow the development of GABA-rich ingredients using A. oryzae NSK as a starter culture for soy sauce production.

Gracilibacillus aidingensis sp. nov., a novel moderately halophilic bacterium isolated from Aiding salt lake.

PubMed

Guan, Tong-Wei; Tian, Lei; Li, En-Yuan; Tang, Shu-Kun; Zhang, Xiao-Ping

2017-11-01

A novel Gram-positive, aerobe, moderately halophilic bacterium was isolated from saline soil of Aiding lake in Xinjiang, north-west of China, designated strain YIM 98001 T . Cells were rod-shaped, motile and grew at 5-20% (w/v) NaCl (optimum 10%), pH 6-10 (optimum pH 7.0) and 4-45 °C (optimum 37 °C). The major cellular fatty acids were anteiso C 15:0 , anteiso C 17:0 , iso C 15:0 . The predominant respiratory quinone was MK-7. Diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid were the major polar lipids. Meso-diaminopimelic acid was the diagnostic diamino acid of the cell-wall peptidoglycan. The G+C content was 36.46 mol%. 16S rRNA gene sequence analysis showed that the strain belongs to the family Bacillaceae, with the highest sequence similarity to the type strain Gracilibacillus thailandensis TP2-8 T (96.84%), followed by Gracilibacillus saliphilus YIM 91119 T (96.78%) and Gracilibacillus ureilyticus MF38 T (96.57%), thus confirming the affiliation of strain YIM 98001 T to the genus Gracilibacillus. The polyphasic approach indicates that strain YIM 98001 T represents a novel species of the genus Gracilibacillus, for which the name Gracilibacillus aidingensis is proposed. The type strain is YIM 98001 T (=KCTC 42683 T  = DSMZ 104330 T ).

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

PubMed Central

Lange, T; Hedden, P; Graebe, J E

1994-01-01

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases. Images PMID:8078921

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

PubMed

Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

2016-11-01

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analysis of the recE locus of Escherichia coli K-12 by use of polyclonal antibodies to exonuclease VIII.

PubMed Central

Luisi-DeLuca, C; Clark, A J; Kolodner, R D

1988-01-01

Exonuclease VIII (exoVIII) of Escherichia coli has been purified from a strain carrying a plasmid-encoded recE gene by using a new procedure. This procedure yielded 30 times more protein per gram of cells, and the protein had a twofold higher specific activity than the enzyme purified by the previously published procedure (J. W. Joseph and R. Kolodner, J. Biol. Chem. 258:10411-10417, 1983). The sequence of the 12 N-terminal amino acids was also obtained and found to correspond to one of the open reading frames predicted from the nucleic acid sequence of the recE region of Rac (C. Chu, A. Templin, and A. J. Clark, manuscript in preparation). Polyclonal antibodies directed against purified exoVIII were also prepared. Cell-free extracts prepared from strains containing a wide range of chromosomal- or plasmid-encoded point, insertion, and deletion mutations which result in expression of exoVIII were examined by Western blot (immunoblot) analysis. This analysis showed that two point sbcA mutations (sbcA5 and sbcA23) and the sbc insertion mutations led to the synthesis of the 140-kilodalton (kDa) polypeptide of wild-type exoVIII. Plasmid-encoded partial deletion mutations of recE reduced the size of the cross-reacting protein(s) in direct proportion to the size of the deletion, even though exonuclease activity was still present. The analysis suggests that 39 kDa of the 140-kDa exoVIII subunit is all that is essential for exonuclease activity. One of the truncated but functional exonucleases (the pRAC3 exonuclease) has been purified and confirmed to be a 41-kDa polypeptide. The first 18 amino acids from the N terminus of the 41-kDa pRAC3 exonuclease were sequenced and fond to correspond to one of the translational start signals predicted from the nucleotide sequence of radC (Chu et al., in preparation). Images PMID:3056915

Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, beta-1,3-Endoglucanase, in Soybean.

PubMed

Takeuchi, Y; Yoshikawa, M; Takeba, G; Tanaka, K; Shibata, D; Horino, O

1990-06-01

Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.

Streptomyces jeddahensis sp. nov., an oleaginous bacterium isolated from desert soil.

PubMed

Röttig, Annika; Atasayar, Ewelina; Meier-Kolthoff, Jan Philipp; Spröer, Cathrin; Schumann, Peter; Schauer, Jennifer; Steinbüchel, Alexander

2017-06-01

A novel strain, G25T, was isolated from desert soil collected near Jeddah in Saudi Arabia. The strain could accumulate nearly 65 % of its cell dry weight as fatty acids, grow on a broad range of carbon sources and tolerate temperatures of up to 50 °C. With respect to to its 16S rRNA gene sequence, G25T is most closely related to Streptomyces massasporeus DSM 40035T, Streptomyces hawaiiensis DSM 40042T, Streptomyces indiaensis DSM 43803T, Streptomyces luteogriseus DSM 40483T and Streptomyces purpurascens DSM 40310T. Conventional DNA-DNA hybridization (DDH) values ranged from 18.7 to 46.9 % when G25T was compared with these reference strains. Furthermore, digital DDH values between the draft genome sequence of G25T and the genome sequences of other species of the genus Streptomyces were also significantly below the threshold of 70 %. The DNA G+C content of the draft genome sequence, consisting of 8.46 Mbp, was 70.3 %. The prevalent cellular fatty acids of G25T comprised anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and iso-C16 : 0. The predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The polar lipids profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannosides as well as unidentified phospholipids and phosphoaminolipids. The cell wall contained ll-diaminopimelic acid. Whole-cell sugars were predominantly glucose with small traces of ribose and mannose. The results of the polyphasic approach confirmed that this isolate represents a novel species of the genus Streptomyces, for which the name Streptomyces jeddahensis sp. nov. is proposed. The type strain of this species is G25T (=DSM 101878T =LMG 29545T =NCCB 100603T).

Proteogenomic Analysis of Polymorphisms and Gene Annotation Divergences in Prokaryotes using a Clustered Mass Spectrometry-Friendly Database*

PubMed Central

de Souza, Gustavo A.; Arntzen, Magnus Ø.; Fortuin, Suereta; Schürch, Anita C.; Målen, Hiwa; McEvoy, Christopher R. E.; van Soolingen, Dick; Thiede, Bernd; Warren, Robin M.; Wiker, Harald G.

2011-01-01

Precise annotation of genes or open reading frames is still a difficult task that results in divergence even for data generated from the same genomic sequence. This has an impact in further proteomic studies, and also compromises the characterization of clinical isolates with many specific genetic variations that may not be represented in the selected database. We recently developed software called multistrain mass spectrometry prokaryotic database builder (MSMSpdbb) that can merge protein databases from several sources and be applied on any prokaryotic organism, in a proteomic-friendly approach. We generated a database for the Mycobacterium tuberculosis complex (using three strains of Mycobacterium bovis and five of M. tuberculosis), and analyzed data collected from two laboratory strains and two clinical isolates of M. tuberculosis. We identified 2561 proteins, of which 24 were present in M. tuberculosis H37Rv samples, but not annotated in the M. tuberculosis H37Rv genome. We were also able to identify 280 nonsynonymous single amino acid polymorphisms and confirm 367 translational start sites. As a proof of concept we applied the database to whole-genome DNA sequencing data of one of the clinical isolates, which allowed the validation of 116 predicted single amino acid polymorphisms and the annotation of 131 N-terminal start sites. Moreover we identified regions not present in the original M. tuberculosis H37Rv sequence, indicating strain divergence or errors in the reference sequence. In conclusion, we demonstrated the potential of using a merged database to better characterize laboratory or clinical bacterial strains. PMID:21030493

Comparative genome and evolutionary analysis of naturally occurring Beilong virus in brown and black rats.

PubMed

Woo, Patrick C Y; Wong, Annette Y P; Wong, Beatrice H L; Lam, Carol S F; Fan, Rachel Y Y; Lau, Susanna K P; Yuen, Kwok-Yung

2016-11-01

Recently, we reported the presence of Beilong virus in spleen and kidney samples of brown rats and black rats, suggesting that these rodents could be natural reservoirs of Beilong virus. In this study, four genomes of Beilong virus from brown rats and black rats were sequenced. Similar to the Beilong virus genome sequenced from kidney mesangial cell line culture, those of J-virus from house mouse and Tailam virus from Sikkim rats, these four genomes from naturally occurring Beilong virus also contain the eight genes (3'-N-P/V/C-M-F-SH-TM-G-L-5'). In these four genomes, the attachment glycoprotein encoded by the G gene consists of 1046 amino acids; but for the original Beilong virus genome sequenced from kidney mesangial cell line, the G CDS was predicted to be prematurely terminated at position 2205 (TGG→TAG), resulting in a 734-amino-acid truncated G protein. This phenomenon of a lack of nonsense mutation in naturally occurring Beilong viruses was confirmed by sequencing this region of 15 additional rodent samples. Phylogenetic analyses showed that the cell line and naturally occurring Beilong viruses were closely clustered, without separation into subgroups. In addition, these viruses were further clustered with J-virus and Tailam virus, with high bootstrap supports of >90%, forming a distinct group in Paramyxoviridae. Brown rats and black rats are natural reservoirs of Beilong virus. Our results also supports that the recently proposed genus, Jeilongvirus, should encompass Beilong virus, J-virus and Tailam virus as members. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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.

Variability of the protein sequences of lcrV between epidemic and atypical rhamnose-positive strains of Yersinia pestis.

PubMed

Anisimov, Andrey P; Panfertsev, Evgeniy A; Svetoch, Tat'yana E; Dentovskaya, Svetlana V

2007-01-01

Sequencing of lcrV genes and comparison of the deduced amino acid sequences from ten Y. pestis strains belonging mostly to the group of atypical rhamnose-positive isolates (non-pestis subspecies or pestoides group) showed that the LcrV proteins analyzed could be classified into five sequence types. This classification was based on major amino acid polymorphisms among LcrV proteins in the four "hot points" of the protein sequences. Some additional minor polymorphisms were found throughout these sequence types. The "hot points" corresponded to amino acids 18 (Lys --> Asn), 72 (Lys --> Arg), 273 (Cys --> Ser), and 324-326 (Ser-Gly-Lys --> Arg) in the LcrV sequence of the reference Y. pestis strain CO92. One possible explanation for polymorphism in amino acid sequences of LcrV among different strains is that strain-specific variation resulted from adaptation of the plague pathogen to different rodent and lagomorph hosts.

A cluster of two human cases of tick-borne encephalitis (TBE) transmitted by unpasteurised goat milk and cheese in Germany, May 2016.

PubMed

Brockmann, S O; Oehme, R; Buckenmaier, T; Beer, M; Jeffery-Smith, A; Spannenkrebs, M; Haag-Milz, S; Wagner-Wiening, C; Schlegel, C; Fritz, J; Zange, S; Bestehorn, M; Lindau, A; Hoffmann, D; Tiberi, S; Mackenstedt, U; Dobler, G

2018-04-01

In May 2016, two cases of tick-borne encephalitis (TBE) were confirmed by serology (positive IgM and IgG antibodies against TBE virus (TBEV) in serum), with a possible link to raw milk and cheese from a goat farm in a region in Baden-Württemberg, Germany not previously known as TBE-endemic. The outbreak investigation identified 32 consumers of goat dairy products (29 consumers, one farm employee, two owners) of whom none had IgM antibodies against TBEV 3-8 weeks after consumption. Of the 27 notified TBE cases in the State, none reported consumption of raw goat milk or cheese from the suspected farm. Five of 22 cheese samples from 18 different batches were RT-qPCR-positive for TBEV -genome, and two of the five samples were confirmed by virus isolation, indicating viability of TBEV in the cheese. Nine of the 45 goats had neutralising TBEV antibodies, two of them with a high titre indicating recent infection. One of 412 Ixodes ricinus was RT-qPCR-positive, and sequencing of the E gene from nucleic acid extracted from the tick confirmed TBEV. Phylogenetic analyses of tick and cheese isolates showed 100% amino acid homology in the E gene and a close relation to TBEV strains from Switzerland and Austria.

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses

USDA-ARS?s Scientific Manuscript database

Background: Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected ...

.beta.-glucosidase 5 (BGL5) compositions

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2010-06-01

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

Evidence of Divergent Amino Acid Usage in Comparative Analyses of R5- and X4-Associated HIV-1 Vpr Sequences

PubMed Central

Antell, Gregory C.; Zhong, Wen; Kercher, Katherine; Passic, Shendra; Williams, Jean; Liu, Yucheng; James, Tony; Jacobson, Jeffrey M.; Szep, Zsofia

2017-01-01

Vpr is an HIV-1 accessory protein that plays numerous roles during viral replication, and some of which are cell type dependent. To test the hypothesis that HIV-1 tropism extends beyond the envelope into the vpr gene, studies were performed to identify the associations between coreceptor usage and Vpr variation in HIV-1-infected patients. Colinear HIV-1 Env-V3 and Vpr amino acid sequences were obtained from the LANL HIV-1 sequence database and from well-suppressed patients in the Drexel/Temple Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. Genotypic classification of Env-V3 sequences as X4 (CXCR4-utilizing) or R5 (CCR5-utilizing) was used to group colinear Vpr sequences. To reveal the sequences associated with a specific coreceptor usage genotype, Vpr amino acid sequences were assessed for amino acid diversity and Jensen-Shannon divergence between the two groups. Five amino acid alphabets were used to comprehensively examine the impact of amino acid substitutions involving side chains with similar physiochemical properties. Positions 36, 37, 41, 89, and 96 of Vpr were characterized by statistically significant divergence across multiple alphabets when X4 and R5 sequence groups were compared. In addition, consensus amino acid switches were found at positions 37 and 41 in comparisons of the R5 and X4 sequence populations. These results suggest an evolutionary link between Vpr and gp120 in HIV-1-infected patients. PMID:28620613

A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone

PubMed Central

Serra, Montserrat; Piña, Benjamin; Abad, José Luis; Camps, Francisco; Fabriàs, Gemma

2007-01-01

The sex pheromone of the female processionary moth, Thaumetopoea pityocampa, is a unique C16 enyne acetate that is biosynthesized from palmitic acid. Three consecutive desaturation reactions transform this saturated precursor into the triunsaturated fatty acyl intermediate: formation of (Z)-11-hexadecenoic acid, acetylenation to 11-hexadecynoic acid, and final Δ13 desaturation to (Z)-13-hexadecen-11-ynoic acid. By using degenerate primers common to all reported insect desaturases, a single cDNA sequence was isolated from total RNA of T. pityocampa female pheromone glands. The full-length transcript of this putative desaturase was expressed in elo1Δ/ole1Δ yeast mutants (both elongase 1 and Δ9 desaturase-deficient) for functional assays. The construct fully rescued the Δole1 yeast phenotype, confirming its desaturase activity. Analysis of the unsaturated products from transformed yeast extracts demonstrated that the cloned enzyme showed Δ11 desaturase, Δ11 acetylenase, and Δ13 desaturase activities. Therefore, this single desaturase may account for the three desaturation steps involved in the sex pheromone biosynthetic pathway of the processionary moth. PMID:17921252

A Unique Case of Classic Kaposi's sarcoma restricted to the toes.

PubMed

Renteria, Anne S; Marshall, Vickie A; Sun, Yanyu; Chockalingam, Porselvi; Cooper, Jay S; Huang, Yiwu; Whitby, Denise

2013-01-01

Kaposi's sarcoma associated-herpesvirus causes all forms of Kaposi's sarcoma, and six major subtypes have been described based on the amino acid sequences of the open reading frame K1. A 71-year-old man from China, HIV negative, presented with nodules on the dorsal aspect of his toes. Biopsy confirmed the diagnosis of Kaposi's sarcoma and virology studies of his blood and saliva confirmed the presence of Kaposi's sarcoma associated-herpesvirus infection. Viral genotyping was consistent with subtype C3. Intervention has been deferred as our patient has remained clinically asymptomatic and without evident growth of his lesions over a 2-year follow up. We herein report the first known case of Kaposi's sarcoma restricted to the toes caused by the viral subtype C3 in an HIV-negative patient from Harbin, China.

Methods of diagnosing alagille syndrome

DOEpatents

Li, Linheng; Hood, Leroy; Krantz, Ian D.; Spinner, Nancy B.

2004-03-09

The present invention provides an isolated polypeptide exhibiting substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the polypeptide does not have the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the nucleotide sequence does not encode the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Also provided herein is a method of inhibiting differentiation of hematopoietic progenitor cells by contacting the progenitor cells with an isolated JAGGED polypeptide, or active fragment thereof. The invention additionally provides a method of diagnosing Alagille Syndrome in an individual. The method consists of detecting an Alagille Syndrome disease-associated mutation linked to a JAGGED locus.

Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor.

PubMed

Hirado, M; Tsunasawa, S; Sakiyama, F; Niinobe, M; Fujii, S

1985-07-01

The complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S-carboxymethylated protein. Achromobacter peptidase digestion was successfully used to isolate two disulfide-containing peptides. The inhibitor consists of 112 amino acids with an Mr of 12787. Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110. A high degree of homology in the sequence was found between the colostrum inhibitor and human gamma-trace, human salivary acidic protein and chicken egg-white cystatin.

[Clinical features and ETFDH mutations of children with late-onset glutaric aciduria type II: a report of two cases].

PubMed

Cheng, Yan-Yang; Tang, Yue; Liu, Ao-Jie; Wei, Li; Lin, Lan; Zhang, Jing; Zhi, Liang

2017-09-01

To investigate the clinical and genetic features of two families with late-onset glutaric aciduria type II caused by ETFDH mutations. Target gene sequence capture and next generation sequencing were used for sequencing of suspected patients and their family members. The patients' clinical features were retrospectively analyzed and literature review was performed. The probands of the two families had a clinical onset at the ages of 10 years and 5.5 years respectively, with the clinical manifestations of muscle weakness and muscle pain. Laboratory examinations revealed significant increases in the serum levels of creatine kinase, creatine kinase-MB, and lactate dehydrogenase. Tandem mass spectrometry showed increases in various types of acylcarnitines. The analysis of urine organic acids showed an increase in glutaric acid. Electromyography showed myogenic damage in both patients. Gene detection showed two novel mutations in the ETFDH gene (c.1331T>C from the mother and c.824C>T from the father) in patient 1, and the patient's younger brother carried the c.1331T>C mutation but had a normal phenotype. In patient 2, there was a novel mutation (c.177insT from the father) and a known mutation (c.1474T>C from the mother) in the ETFDH gene. Several family members carried such mutations. Both patients were diagnosed with glutaric aciduria type II. Their symptoms were improved after high-dose vitamin B2 treatment. For patients with unexplained muscle weakness and pain, serum creatine kinase, acylcarnitines, and urinary organic acids should be measured, and the possibility of glutaric aciduria type II should be considered. Genetic detection is helpful to make a confirmed diagnosis.

In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function.

PubMed

Follin, Elna; Karlsson, Maria; Lundegaard, Claus; Nielsen, Morten; Wallin, Stefan; Paulsson, Kajsa; Westerdahl, Helena

2013-04-01

The major histocompatibility complex (MHC) genes are the most polymorphic genes found in the vertebrate genome, and they encode proteins that play an essential role in the adaptive immune response. Many songbirds (passerines) have been shown to have a large number of transcribed MHC class I genes compared to most mammals. To elucidate the reason for this large number of genes, we compared 14 MHC class I alleles (α1-α3 domains), from great reed warbler, house sparrow and tree sparrow, via phylogenetic analysis, homology modelling and in silico peptide-binding predictions to investigate their functional and genetic relationships. We found more pronounced clustering of the MHC class I allomorphs (allele specific proteins) in regards to their function (peptide-binding specificities) compared to their genetic relationships (amino acid sequences), indicating that the high number of alleles is of functional significance. The MHC class I allomorphs from house sparrow and tree sparrow, species that diverged 10 million years ago (MYA), had overlapping peptide-binding specificities, and these similarities across species were also confirmed in phylogenetic analyses based on amino acid sequences. Notably, there were also overlapping peptide-binding specificities in the allomorphs from house sparrow and great reed warbler, although these species diverged 30 MYA. This overlap was not found in a tree based on amino acid sequences. Our interpretation is that convergent evolution on the level of the protein function, possibly driven by selection from shared pathogens, has resulted in allomorphs with similar peptide-binding repertoires, although trans-species evolution in combination with gene conversion cannot be ruled out.

GENETIC CHARACTERIZATION OF CANINE PARVOVIRUS IN SYMPATRIC FREE-RANGING WILD CARNIVORES IN PORTUGAL.

PubMed

Miranda, Carla; Santos, Nuno; Parrish, Colin; Thompson, Gertrude

2017-10-01

Since its emergence in the 1970s, canine parvovirus (CPV) has been reported in domestic and nondomestic carnivores worldwide with severe implications on their health and survival. Here, we aim to better understand CPV circulation in multihost-pathogens systems by characterizing CPV DNA or viruses in 227 free-ranging wild carnivores of 12 species from Portugal. Collected samples during 1995-2011 were analyzed by PCR and sequence analysis. The canine parvovirus DNA was detected in 4 (2%) animals of two species, namely in wolves (Canis lupus; 3/63, 5%, 95% confidence interval=1.6-3.15) and in a stone marten (Martes foina; 1/36, 3%, 95% confidence interval=0.5-14.2). Viruses in two wolves had VP2 residue 426 as aspartic acid (so-called CPV-2b) and the third had VP2 residue 426 as asparagine (CPV-2a), while the virus in the stone marten uniquely had VP2 residue 426 as glutamic acid (CPV-2c). The comparative analysis of the full-length VP2 gene of our isolates showed other nonsynonymous mutations. The phylogenetic analysis demonstrated that the sequences from wolves clustered together, showing a close relationship with European domestic dogs (Canis lupus familiaris) and wolf strains while the viral sequence from the stone marten grouped with other viruses contained the glutamic acid VP2 426 along with raccoon (Procyon lotor), bobcat (Lynx rufus), and domestic dog strains. This study confirmed that wild carnivores in Portugal are infected by CPV variants, strongly suggesting viral transmission between the wild and domestic populations and suggesting a need for a better understanding of the epidemiology of the disease and its management in wild populations.

Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in Lactobacillus johnsonii 100-100

PubMed Central

Elkins, Christopher A.; Savage, Dwayne C.

1998-01-01

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [14C]taurocholic acid that were increased as much as threefold over control levels. [14C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes. PMID:9721268

Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS.

PubMed

Prince, J T; Alberti, L; Healy, P A; Nauman, S J; Stallcup, W B

1991-11-01

The NILE glycoprotein is a rat neuronal cell adhesion molecule which has been reported to be very similar in structure, function, and distribution to the mouse L1 glycoprotein. Here we report the complete nucleotide sequence of the NILE message (5,208 nucleotides) and the deduced amino acid sequence of the NILE polypeptide (1,257 amino acids). The predicted NILE protein is 96% identical to L1 at the amino acid level, confirming that the two molecules are homologues. The sequence information shows that NILE is a transmembrane molecule with an extensive ectodomain and a much smaller cytoplasmic domain. The extracellular portion of the molecule contains six immunoglobulin C-2 type domains followed by five fibronectin type III repeats. These two structural motifs are characteristic of several other cell adhesion molecules. The cytoplasmic tails of NILE and L1 are identical to each other and distinct from the cytoplasmic regions of all other cell adhesion molecules except Ng-CAM and neuroglian. Several possible sites for phosphorylation are present in the cytoplasmic tail of NILE. Antisera were produced against two NILE-beta-galactosidase fusion proteins containing distinct segments of the NILE polypeptide: the cytoplasmic domain and the segment containing fibronectin type III repeats. Immunoblots with these antisera and Northern blots with a NILE cDNA probe indicate that NILE continues to be expressed in most areas of the mature rat brain. This contradicts previous immunofluorescence data, which suggested that NILE was substantially down-regulated in maturing nerve fiber tracts. This raises the possibility that NILE could be masked in situ by interactions with other cell surface molecules.

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.

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.

Tomato ASR1 abrogates the response to abscisic acid and glucose in Arabidopsis by competing with ABI4 for DNA binding.

PubMed

Shkolnik, Doron; Bar-Zvi, Dudy

2008-05-01

The manipulation of transacting factors is commonly used to achieve a wide change in the expression of a large number of genes in transgenic plants as a result of a change in the expression of a single gene product. This is mostly achieved by the overexpression of transactivator or repressor proteins. In this study, it is demonstrated that the overexpression of an exogenous DNA-binding protein can be used to compete with the expression of an endogenous transcription factor sharing the same DNA-binding sequence. Arabidopsis was transformed with cDNA encoding tomato abscisic acid stress ripening 1 (ASR1), a sequence-specific DNA protein that has no orthologues in the Arabidopsis genome. ASR1-overexpressing (ASR1-OE) plants display an abscisic acid-insensitive 4 (abi4) phenotype: seed germination is not sensitive to inhibition by abscisic acid (ABA), glucose, NaCl and paclobutrazol. ASR1 binds coupling element 1 (CE1), a cis-acting element bound by the ABI4 transcription factor, located in the ABI4-regulated promoters, including that of the ABI4 gene. Chromatin immunoprecipitation demonstrates that ASR1 is bound in vivo to the promoter of the ABI4 gene in ASR1-OE plants, but not to promoters of genes known to be regulated by the transcription factors ABI3 or ABI5. Real-time polymerase chain reaction (PCR) analysis confirmed that the expression of ABI4 and ABI4-regulated genes is markedly reduced in ASR1-OE plants. Therefore, it is concluded that the abi4 phenotype of ASR1-OE plants is the result of competition between the foreign ASR1 and the endogenous ABI4 on specific promoter DNA sequences. The biotechnological advantage of using this approach in crop plants from the Brassicaceae family to reduce the transactivation activity of ABI4 is discussed.

Genetic characterization of the hemagglutinin genes of wild-type measles virus circulating in china, 1993-2009.

PubMed

Xu, Songtao; Zhang, Yan; Zhu, Zhen; Liu, Chunyu; Mao, Naiying; Ji, Yixin; Wang, Huiling; Jiang, Xiaohong; Li, Chongshan; Tang, Wei; Feng, Daxing; Wang, Changyin; Zheng, Lei; Lei, Yue; Ling, Hua; Zhao, Chunfang; Ma, Yan; He, Jilan; Wang, Yan; Li, Ping; Guan, Ronghui; Zhou, Shujie; Zhou, Jianhui; Wang, Shuang; Zhang, Hong; Zheng, Huanying; Liu, Leng; Ma, Hemuti; Guan, Jing; Lu, Peishan; Feng, Yan; Zhang, Yanjun; Zhou, Shunde; Xiong, Ying; Ba, Zhuoma; Chen, Hui; Yang, Xiuhui; Bo, Fang; Ma, Yujie; Liang, Yong; Lei, Yake; Gu, Suyi; Liu, Wei; Chen, Meng; Featherstone, David; Jee, Youngmee; Bellini, William J; Rota, Paul A; Xu, Wenbo

2013-01-01

China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993-2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10(-3) substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure. Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.

Acidovorax valerianellae sp. nov., a novel pathogen of lamb's lettuce [Valerianella locusta (L.) Laterr].

PubMed

Gardan, Louis; Stead, David E; Dauga, Catherine; Gillis, Moniek

2003-05-01

Bacterial spot disease of lamb's lettuce [Valerianella locusta (L.) Laterr.] was first observed in fields in 1991. This new bacterial disease is localized in western France in high-technology field production of lamb's lettuce for the preparation of ready-to-use salad. Nineteen strains isolated in 1992 and 1993 from typical black leaf spots of naturally infected lamb's lettuce were characterized and compared with reference strains of Acidovorax and Delftia. The pathogenicity of the 19 strains was confirmed by artificial inoculation. Biochemical and physiological tests, fatty acid profiles, DNA-DNA hybridization and other nucleic acid-based tests were performed. A numerical taxonomic analysis of the 19 lamb's lettuce strains showed a single homogeneous phenon closely related to previously described phytopathogenic taxa of the genus Acidovorax. DNA-DNA hybridization studies showed that the lamb's lettuce strains were 91-100% related to a representative strain, strain CFBP 4730(T), and constituted a discrete DNA hybridization group, indicating that they belong to the same novel species. Results from DNA-rRNA hybridization, 16S rRNA sequence analysis and fatty acid analysis studies confirmed that this novel species belongs to the beta-subclass of the Proteobacteria and, more specifically, to the family Comamonadaceae and the genus Acidovorax. The name Acidovorax valerianellae sp. nov. is proposed for this novel taxon of phytopathogenic bacteria. The type strain is strain CFBP 4730(T) (= NCPPB 4283(T)).

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region

PubMed Central

Ghasemi, Faezeh; Ghayour-Mobarhan, Majid; Pasdar, Alireza; Pourianfar, Hamid; Reza Aghasadeghi, Mohammad; Gouklani, Hamed; Meshkat, Zahra

2016-01-01

Background The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions. Objectives The aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it. Methods Specific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequenced Results Quantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture. Conclusions Real-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines. PMID:27882063

Characterization of a novel flavivirus isolated from Culex (Melanoconion) ocossa mosquitoes from Iquitos, Peru.

PubMed

Evangelista, Julio; Cruz, Cristhopher; Guevara, Carolina; Astete, Helvio; Carey, Cristiam; Kochel, Tadeusz J; Morrison, Amy C; Williams, Maya; Halsey, Eric S; Forshey, Brett M

2013-06-01

We describe the isolation and characterization of a novel flavivirus, isolated from a pool of Culex (Melanoconion) ocossa Dyar and Knab mosquitoes collected in 2009 in an urban area of the Amazon basin city of Iquitos, Peru. Flavivirus infection was detected by indirect immunofluorescent assay of inoculated C6/36 cells using polyclonal flavivirus antibodies (St. Louis encephalitis virus, yellow fever virus and dengue virus type 1) and confirmed by RT-PCR. Based on partial sequencing of the E and NS5 gene regions, the virus isolate was most closely related to the mosquito-borne flaviviruses but divergent from known species, with less than 45 and 71 % pairwise amino acid identity in the E and NS5 gene products, respectively. Phylogenetic analysis of E and NS5 amino acid sequences demonstrated that this flavivirus grouped with mosquito-borne flaviviruses, forming a clade with Nounané virus (NOUV). Like NOUV, no replication was detected in a variety of mammalian cells (Vero-76, Vero-E6, BHK, LLCMK, MDCK, A549 and RD) or in intracerebrally inoculated newborn mice. We tentatively designate this genetically distinct flavivirus as representing a novel species, Nanay virus, after the river near where it was first detected.

Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples

PubMed Central

Passaretti, Teresa V.; Jose, Reashma; Cole, Jocelyn; Coorevits, An; Carpenter, Andrea N.; Jose, Sherly; Van Landschoot, Anita; Izard, Jacques; Kohlerschmidt, Donna J.; Vandamme, Peter; Dewhirst, Floyd E.; Fisher, Mark A.; Musser, Kimberlee A.

2013-01-01

A polyphasic analysis was undertaken of seven independent isolates of Gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7–100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA–DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica. Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria. The name Neisseria oralis sp. nov. (type strain 6332T  = DSM 25276T  = LMG 26725T) is proposed. PMID:22798652

Lactobacillus durianis sp. nov., isolated from an acid-fermented condiment (tempoyak) in Malaysia.

PubMed

Leisner, J J; Vancanneyt, M; Lefebvre, K; Vandemeulebroecke, K; Hoste, B; Vilalta, N Euras; Rusul, G; Swings, J

2002-05-01

Lactic acid bacteria (LAB) are the predominant micro-organisms in tempoyak, a Malaysian acid-fermented condiment. In a study on the diversity of LAB in this product, three isolates could not be identified using SDS-PAGE of whole-cell proteins or API 50 CH. The taxonomic position of the three isolates was clarified in the present study. 16S rDNA sequencing classified a representative strain in the genus Lactobacillus, clearly separated from all known species, and most closely related to the Lactobacillus reuteri phylogenetic group. DNA-DNA hybridization experiments and an extensive phenotypic description confirm that the strains represent a single and separate novel species among the obligately heterofermentative lactobacilli. The three isolates are distinguished at the intra-species level by plasmid profiling, pulsed-field gel electrophoresis of macro-restriction fragments and biochemical features. The name Lactobacillus durianis sp. nov. is proposed for the novel taxon and the type strain is LMG 19193T (= CCUG 45405T).

Isolation and identification of lactic acid bacteria from fermented red dragon fruit juices.

PubMed

Ong, Yien Yien; Tan, Wen Siang; Rosfarizan, Mohamad; Chan, Eng Seng; Tey, Beng Ti

2012-10-01

Red dragon fruit or red pitaya is rich in potassium, fiber, and antioxidants. Its nutritional properties and unique flesh color have made it an attractive raw material of various types of food products and beverages including fermented beverages or enzyme drinks. In this study, phenotypic and genotypic methods were used to confirm the identity of lactic acid bacteria (LAB) appeared in fermented red dragon fruit (Hylocereus polyrhizus) beverages. A total of 21 isolates of LAB were isolated and characterized. They belonged to the genus of Enterococcus based on their biochemical characteristics. The isolates can be clustered into two groups by using the randomly amplified polymorphic DNA method. Nucleotide sequencing and restriction fragment length polymorphism of the 16S rRNA region suggested that they were either Enterococcus faecalis or Enterococcus durans. Current research revealed the use of biochemical analyses and molecular approaches to identify the microbial population particularly lactic acid bacteria from fermented red dragon fruit juices. © 2012 Institute of Food Technologists®

Biocompatible and biodegradable fibrinogen microspheres for tumor-targeted doxorubicin delivery

PubMed Central

Joo, Jae Yeon; Park, Gil Yong; An, Seong Soo A

2015-01-01

In the development of effective drug delivery carriers, many researchers have focused on the usage of nontoxic and biocompatible materials and surface modification with targeting molecules for tumor-specific drug delivery. Fibrinogen (Fbg), an abundant glycoprotein in plasma, could be a potential candidate for developing drug carriers because of its biocompatibility and tumor-targeting property via arginine–glycine–aspartate (RGD) peptide sequences. Doxorubicin (DOX), a chemotherapeutic agent, was covalently conjugated to Fbg, and the microspheres were prepared. Acid-labile and non-cleavable linkers were used for the conjugation of DOX to Fbg, resulting in an acid-triggered drug release under a mild acidic condition and a slow-controlled drug release, respectively. In vitro cytotoxicity tests confirmed low cytotoxicity in normal cells and high antitumor effect toward cancer cells. In addition, it was discovered that a longer linker could make the binding of cells to Fbg drug carriers easier. Therefore, DOX–linker–Fbg microspheres could be a suitable drug carrier for safer and effective drug delivery. PMID:26366073

Mutation analysis of GLDC, AMT and GCSH in cataract captive-bred vervet monkeys (Chlorocebus aethiops).

PubMed

Chauke, Chesa G; Magwebu, Zandisiwe E; Sharma, Jyoti R; Arieff, Zainunisha; Seier, Jürgen V

2016-08-01

Non-ketotic hyperglycinaemia (NKH) is an autosomal recessive inborn error of glycine metabolism characterized by accumulation of glycine in body fluids and various neurological symptoms. This study describes the first screening of NKH in cataract captive-bred vervet monkeys (Chlorocebus aethiops). Glycine dehydrogenase (GLDC), aminomethyltransferase (AMT) and glycine cleavage system H protein (GCSH) were prioritized. Mutation analysis of the complete coding sequence of GLDC and AMT revealed six novel single-base substitutions, of which three were non-synonymous missense and three were silent nucleotide changes. Although deleterious effects of the three amino acid substitutions were not evaluated, one substitution of GLDC gene (S44R) could be disease-causing because of its drastic amino acid change, affecting amino acids conserved in different primate species. This study confirms the diagnosis of NKH for the first time in vervet monkeys with cataracts. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

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 structure-specific 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 on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Nucleic acid detection kits

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

2005-03-29

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 structure-specific 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 on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

Detection of nucleic acids by multiple sequential invasive cleavages 02

DOEpatents

Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann D.

2002-01-01

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 structure-specific 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 on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Detection of nucleic acids by multiple sequential invasive cleavages

DOEpatents

Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

2012-10-16

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 structure-specific 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 on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Sequence and expression of the genes for HPr (ptsH) and enzyme I (ptsI) of the phosphoenolpyruvate-dependent phosphotransferase transport system from Streptococcus mutans.

PubMed Central

Boyd, D A; Cvitkovitch, D G; Hamilton, I R

1994-01-01

We report the sequencing of a 2,242-bp region of the Streptococcus mutants NG5 genome containing the genes for ptsH and ptsI, which encode HPr and enzyme I (EI), respectively, of the phosphoenolpyruvate-dependent phosphotransferase transport system. The sequence was obtained from two cloned overlapping genomic fragments; one expresses HPr and a truncated EI, while the other expresses a full-length EI in Escherichia coli, as determined by Western immunoblotting. The ptsI gene appeared to be expressed from a region located in the ptsH gene. The S. mutans NG5 pts operon does not appear to be linked to other phosphotransferase transport system proteins as has been found in other bacteria. A positive fermentation pattern on MacConkey-glucose plates by an E. coli ptsI mutant harboring the S. mutans NG5 ptsI gene on a plasmid indicated that the S. mutans NG5 EI can complement a defect in the E. coli gene. This was confirmed by protein phosphorylation experiments with 32P-labeled phosphoenolpyruvate indicating phosphotransfer from the S. mutans NG5 EI to the E. coli HPr. Two forms of the cloned EI, both truncated to varying degrees in the C-terminal region, were inefficiently phosphorylated and unable to complement fully the ptsI defect in the E. coli mutant. The deduced amino acid sequence of HPr shows a high degree of homology, particularly around the active site, to the same protein from other gram-positive bacteria, notably, S. salivarius, and to a lesser extent with those of gram-negative bacteria. The deduced amino acid sequence of S. mutans NG5 EI also shares several regions of homology with other sequenced EIs, notably, with the region around the active site, a region that contains the only conserved cystidyl residue among the various proteins and which may be involved in substrate binding. Images PMID:8132321

Variants of the D{sub 5} dopamine receptor gene found in patients with schizophrenia: Identification of a nonsense mutation and multiple missense changes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sobell, J.L.; Lind, T.J.; Sommer, S.S.

To determine whether mutations in the D{sub 5} dopamine receptor (D{sub 5}DR) gene are associated with schizophrenia, the gene was examined in 78 unrelated schizophrenic individuals. After amplification by the polymerase chain reaction, products were examined by dideoxy fingerprinting (ddF), a highly sensitive screening method related to single strand conformational polymorphism analysis. All samples with unusual ddF patterns were sequenced to precisely identify the sequence change. In the 156 D{sub 5}DR alleles examined, nine sequence changes were identified. Four of the nine did not affect protein structure; of these, three were silent changes and one was a transition in themore » 3{prime} untranslated region. The remaining five sequence changes result in protein alterations: of these, one is a missense change in a non-conserved amino acid, 3 are missense changes in amino acids that are conserved in some dopamine D{sub 5} receptors and the last is a nonsense mutation. To investigate whether the nonsense mutation was associated with schizophrenia, 400 additional schizophrenic cases of western European descent and 1914 ethnically-similar controls were screened for the change. One additional schizophrenic carrier was identified and verified by direct genomic sequencing (allele frequency: .0013), but eight carriers also were found and confirmed among the non-schizophrenics (allele frequency: .0021)(p>.25). The gene was re-examined in all newly identified carriers of the nonsense mutation by direct sequencing and/or ddF in search of additional mutations. None were identified. Family studies also were conducted to investigate possible cosegregation of the mutation with other neuropsychiatric diseases, but this was not demonstrated. Thus, the mutation does not appear to be associated with an increased risk of schizophrenia nor does an initial analysis suggest cosegregation with other neuropsychiatric disorders or symptom complexes.« less

Gene encoding the group B streptococcal protein R4, its presence in clinical reference laboratory isolates & R4 protein pepsin sensitivity.

PubMed

Smith, B L; Flores, A; Dechaine, J; Krepela, J; Bergdall, A; Ferrieri, P

2004-05-01

R proteins were first identified by Lancefield in group B Streptococcus (GBS) as resistant to trypsin at pH8 and sensitive to pepsin at pH2. The R4 protein found predominantly in type III and some type II and V invasive isolates conforms to these criteria. The Rib protein, although structurally and epidemiologically similar to R4, was reported as resistant to both proteases. We report here the gene encoding the R4 protein from a type III group B streptococcal isolate (76-043) well characterized in our laboratory. Trypsin extracted GBS proteins were assayed for protease sensitivities by double-diffusion Ouchterlony using varying conditions for the enzyme pepsin. Standard haemoglobin assay was used to examine pepsin enzymatic activity. Thirty clinical isolates of varying protein profiles identified by double-diffusion from our reference strain laboratory were screened by PCR and Southern technique. SDS-PAGE gel purified R4 amino acid sequences were determined and used to design oligonucleotide primers for screening a 76-043 genomic library. R4 was sensitive to pepsin at pH2 but appeared resistant at pH4, the reported pH used for Rib. By standard haemoglobin assay and trypsin extract studies of R4 protein, pepsin was shown to be active at pH2, yet easily inactivated; assays of GBS surface proteins are critical at pH2. Of the amino acids initially sequenced from R4, 88 per cent (61/69) showed identity to Rib; the r4 nucleotide sequence was identical to that of rib. All isolates with strong positive protein reactions for R4 were positive in both PCR and Southern technique, whereas isolates expressing alpha, beta, R1/R4, and R5 (BPS) protein profiles were not. Sequenced PCR products aligned with identity to the R4 and Rib nucleotide sequences and confirmed the identity of these proteins and their molecular sequences.

Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis

PubMed Central

2014-01-01

Background The raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains. Results The obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum. Conclusions Raw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action. PMID:24521354

Sequence, distribution and chromosomal context of class I and class II pilin genes of Neisseria meningitidis identified in whole genome sequences

PubMed Central

2014-01-01

Background Neisseria meningitidis expresses type four pili (Tfp) which are important for colonisation and virulence. Tfp have been considered as one of the most variable structures on the bacterial surface due to high frequency gene conversion, resulting in amino acid sequence variation of the major pilin subunit (PilE). Meningococci express either a class I or a class II pilE gene and recent work has indicated that class II pilins do not undergo antigenic variation, as class II pilE genes encode conserved pilin subunits. The purpose of this work was to use whole genome sequences to further investigate the frequency and variability of the class II pilE genes in meningococcal isolate collections. Results We analysed over 600 publically available whole genome sequences of N. meningitidis isolates to determine the sequence and genomic organization of pilE. We confirmed that meningococcal strains belonging to a limited number of clonal complexes (ccs, namely cc1, cc5, cc8, cc11 and cc174) harbour a class II pilE gene which is conserved in terms of sequence and chromosomal context. We also identified pilS cassettes in all isolates with class II pilE, however, our analysis indicates that these do not serve as donor sequences for pilE/pilS recombination. Furthermore, our work reveals that the class II pilE locus lacks the DNA sequence motifs that enable (G4) or enhance (Sma/Cla repeat) pilin antigenic variation. Finally, through analysis of pilin genes in commensal Neisseria species we found that meningococcal class II pilE genes are closely related to pilE from Neisseria lactamica and Neisseria polysaccharea, suggesting horizontal transfer among these species. Conclusions Class II pilins can be defined by their amino acid sequence and genomic context and are present in meningococcal isolates which have persisted and spread globally. The absence of G4 and Sma/Cla sequences adjacent to the class II pilE genes is consistent with the lack of pilin subunit variation in these isolates, although horizontal transfer may generate class II pilin diversity. This study supports the suggestion that high frequency antigenic variation of pilin is not universal in pathogenic Neisseria. PMID:24690385

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

PubMed Central

Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

1986-01-01

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

Method of increasing conversion of a fatty acid to its corresponding dicarboxylic acid

DOEpatents

Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

2004-09-14

A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

Cross-over clinical trial for evaluating the safety of camel's milk intake in patients who are allergic to cow's milk protein.

PubMed

Navarrete-Rodríguez, E M; Ríos-Villalobos, L A; Alcocer-Arreguín, C R; Del-Rio-Navarro, B E; Del Rio-Chivardi, J M; Saucedo-Ramírez, O J; Sienra-Monge, J J L; Frias, R V

Cow's milk protein allergy (CMPA) affects between 0.6 and 0.9% of the general population, and its treatment implies the total elimination of the intake of this protein. Camel's milk has been suggested as an alternative for patients over one year of age who suffer from CMPA due to the difference in the amino acid sequence from that of cow's milk. The objective of this study was to evaluate the safety and tolerability of camel's milk in children with CMPA. Crossed clinical trial for the use of camel's milk vs. amino acid formula, carried out at the Dr. Federico Gómez Children's Hospital of Mexico (HIMFG) on patients between one and 18 years of age with diagnosed CMPA confirmed through double-blind, placebo-controlled food challenges (DBPCFCs). Only those whose allergies were confirmed were randomly placed into two groups: those to be administered camel's milk and those to be administered the amino-acid formula for two weeks, followed by a six-week wash-out period, and then a group crossing for a further two weeks. 49 patients with suspected CMPA were included in the study; the diagnosis was confirmed through DBPCFCs in 15 patients, who were those who participated in the study. After having been administered camel's milk, none of the patients presented adverse effects. Camel's milk is safe and tolerable in patients above one year of age with CMPA and can be considered as a good alternative given the benefit of its taste compared to other formulas. Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

PubMed

Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

1995-04-01

The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

Bioremediation potential of a highly mercury resistant bacterial strain Sphingobium SA2 isolated from contaminated soil.

PubMed

Mahbub, Khandaker Rayhan; Krishnan, Kannan; Megharaj, Mallavarapu; Naidu, Ravi

2016-02-01

A mercury resistant bacterial strain, SA2, was isolated from soil contaminated with mercury. The 16S rRNA gene sequence of this isolate showed 99% sequence similarity to the genera Sphingobium and Sphingomonas of α-proteobacteria group. However, the isolate formed a distinct phyletic line with the genus Sphingobium suggesting the strain belongs to Sphingobium sp. Toxicity studies indicated resistance to high levels of mercury with estimated EC50 values 4.5 mg L(-1) and 44.15 mg L(-1) and MIC values 5.1 mg L(-1) and 48.48 mg L(-1) in minimal and rich media, respectively. The strain SA2 was able to volatilize mercury by producing mercuric reductase enzyme which makes it potential candidate for remediating mercury. ICP-QQQ-MS analysis of Hg supplemented culture solutions confirmed that almost 79% mercury in the culture suspension was volatilized in 6 h. A very small amount of mercury was observed to accumulate in cell pellets which was also evident according to ESEM-EDX analysis. The mercuric reductase gene merA was amplified and sequenced. The deduced amino acid sequence demonstrated sequence homology with α-proteobacteria and Ascomycota group. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) in Serbia.

PubMed

Savic, Bozidar; Milicevic, Vesna; Jakic-Dimic, Dobrila; Bojkovski, Jovan; Prodanovic, Radisa; Kureljusic, Branislav; Potkonjak, Aleksandar; Savic, Borivoje

2012-01-01

Porcine circovirus type 2 (PCV2) is the main causative agent of postweaning multisystemic wasting syndrome (PMWS). To characterize and determine the genetic diversity of PCV2 in the porcine population of Serbia, nucleotide and deduced amino acid sequences of the open reading frame 2 (ORF2) of PCV2 collected from the tissues of pigs that either had died as a result of PMWS or did not exhibit disease symptoms were analyzed. Sequencing and phylogenetic analysis showed considerable diversity among PCV2 ORF2 sequences and the existence of two main PCV2 genotypes, PCV2b and PCV2a, with at least three clusters, 1A/B, 1C and 2D. In order to provide further proof that the 1C strain is circulating in the porcine population, the whole viral genome of one PCV2 isolate was sequenced. Genotyping and phylogenetic analysis using the entire viral genome sequences confirmed that there was a PMWS-associated 1C strain emerging in Serbia. Our analysis also showed that PCV2b is dominant in the porcine population, and that it is exclusively associated with PMWS occurrences in the country. These data constitute a useful basis for further epidemiological studies regarding the heterogeneity of PCV2 strains on the European continent.

Gluconacetobacter medellinensis sp. nov., cellulose- and non-cellulose-producing acetic acid bacteria isolated from vinegar.

PubMed

Castro, Cristina; Cleenwerck, Ilse; Trcek, Janja; Zuluaga, Robin; De Vos, Paul; Caro, Gloria; Aguirre, Ricardo; Putaux, Jean-Luc; Gañán, Piedad

2013-03-01

The phylogenetic position of a cellulose-producing acetic acid bacterium, strain ID13488, isolated from commercially available Colombian homemade fruit vinegar, was investigated. Analyses using nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rRNA gene internal transcribed spacer (ITS) sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated the micro-organism to the genus Gluconacetobacter, and more precisely to the Gluconacetobacter xylinus group. Moreover, the data suggested that the micro-organism belongs to a novel species in this genus, together with LMG 1693(T), a non-cellulose-producing strain isolated from vinegar by Kondo and previously classified as a strain of Gluconacetobacter xylinus. DNA-DNA hybridizations confirmed this finding, revealing a DNA-DNA relatedness value of 81 % between strains ID13488 and LMG 1693(T), and values <70 % between strain LMG 1693(T) and the type strains of the closest phylogenetic neighbours. Additionally, the classification of strains ID13488 and LMG 1693(T) into a single novel species was supported by amplified fragment length polymorphism (AFLP) and (GTG)5-PCR DNA fingerprinting data, as well as by phenotypic data. Strains ID13488 and LMG 1693(T) could be differentiated from closely related species of the genus Gluconacetobacter by their ability to produce 2- and 5-keto-d-gluconic acid from d-glucose, their ability to produce acid from sucrose, but not from 1-propanol, and their ability to grow on 3 % ethanol in the absence of acetic acid and on ethanol, d-ribose, d-xylose, sucrose, sorbitol, d-mannitol and d-gluconate as carbon sources. The DNA G+C content of strains ID13488 and LMG 1693(T) was 58.0 and 60.7 mol%, respectively. The major ubiquinone of LMG 1693(T) was Q-10. Taken together these data indicate that strains ID13488 and LMG 1693(T) represent a novel species of the genus Gluconacetobacter for which the name Gluconacetobacter medellinensis sp. nov. is proposed. The type strain is LMG 1693(T) ( = NBRC 3288(T) = Kondo 51(T)).

WEB-server for search of a periodicity in amino acid and nucleotide sequences

NASA Astrophysics Data System (ADS)

E Frenkel, F.; Skryabin, K. G.; Korotkov, E. V.

2017-12-01

A new web server (http://victoria.biengi.ac.ru/splinter/login.php) was designed and developed to search for periodicity in nucleotide and amino acid sequences. The web server operation is based upon a new mathematical method of searching for multiple alignments, which is founded on the position weight matrices optimization, as well as on implementation of the two-dimensional dynamic programming. This approach allows the construction of multiple alignments of the indistinctly similar amino acid and nucleotide sequences that accumulated more than 1.5 substitutions per a single amino acid or a nucleotide without performing the sequences paired comparisons. The article examines the principles of the web server operation and two examples of studying amino acid and nucleotide sequences, as well as information that could be obtained using the web server.

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis

PubMed Central

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections. PMID:21687530

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis.

PubMed

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Dreiseikelmann, Brigitte

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections.

Management of adult-onset methylmalonic acidemia with hypotonia and acute respiratory failure: A case report.

PubMed

Zhao, Zhanqi; Chu, Chan-Ching; Chang, Mei-Yun; Chang, Hao-Tai; Hsu, Yeong-Long

2018-06-01

Methylmalonic acidemia (MMA) is an autosomal recessive disease of organic acidemia. We report a 26-year-old male who presented with metabolic acidosis, acute renal failure required hemodialysis and acute respiratory failure required mechanical ventilation support. Progressive hypotonia of muscles made weaning from mechanical ventilator difficult. High level of serum methylmalonic acid and the mut genotype sequences confirmed the diagnosis of this adult-onset MMA. Two mut genotype sequences were found by analyzing all coding exons and exon-intron junctions. One genotype was well documented (Exon 6 Mutation, c. 1280G>A. p. G427D, heterozygous). The other mut genotype sequence had never been reported elsewhere (Intron 6 Novel, c. 1333-13_c. 1333-8delTTTTTC, heterozygous). Diet modification, medication, regular hemodialysis and physical rehabilitation. Weaning strategy adjusted with help of electrical impedance tomography. The muscle power of the patient gradually recovered. Extubation of the patient was successful and he was discharged without oxygen required. This case gives us the lesson that MMA can be newly diagnosed in adult patient. A new mut genotype sequence was discovered. The use of electrical impedance tomography to select a suitable method for inspiratory muscle training was possible and useful.

Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence.

PubMed Central

DeWitt, D L; Smith, W L

1988-01-01

Prostaglandin G/H synthase (8,11,14-icosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) catalyzes the first step in the formation of prostaglandins and thromboxanes, the conversion of arachidonic acid to prostaglandin endoperoxides G and H. This enzyme is the site of action of nonsteroidal anti-inflammatory drugs. We have isolated a 2.7-kilobase complementary DNA (cDNA) encompassing the entire coding region of prostaglandin G/H synthase from sheep vesicular glands. This cDNA, cloned from a lambda gt 10 library prepared from poly(A)+ RNA of vesicular glands, hybridizes with a single 2.75-kilobase mRNA species. The cDNA clone was selected using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the purified enzyme. The full-length cDNA encodes a protein of 600 amino acids, including a signal sequence of 24 amino acids. Identification of the cDNA as coding for prostaglandin G/H synthase is based on comparison of amino acid sequences of seven peptides comprising 103 amino acids with the amino acid sequence deduced from the nucleotide sequence of the cDNA. The molecular weight of the unglycosylated enzyme lacking the signal peptide is 65,621. The synthase is a glycoprotein, and there are three potential sites for N-glycosylation, two of them in the amino-terminal half of the molecule. The serine reported to be acetylated by aspirin is at position 530, near the carboxyl terminus. There is no significant similarity between the sequence of the synthase and that of any other protein in amino acid or nucleotide sequence libraries, and a heme binding site(s) is not apparent from the amino acid sequence. The availability of a full-length cDNA clone coding for prostaglandin G/H synthase should facilitate studies of the regulation of expression of this enzyme and the structural features important for catalysis and for interaction with anti-inflammatory drugs. Images PMID:3125548

PubDNA Finder: a web database linking full-text articles to sequences of nucleic acids.

PubMed

García-Remesal, Miguel; Cuevas, Alejandro; Pérez-Rey, David; Martín, Luis; Anguita, Alberto; de la Iglesia, Diana; de la Calle, Guillermo; Crespo, José; Maojo, Víctor

2010-11-01

PubDNA Finder is an online repository that we have created to link PubMed Central manuscripts to the sequences of nucleic acids appearing in them. It extends the search capabilities provided by PubMed Central by enabling researchers to perform advanced searches involving sequences of nucleic acids. This includes, among other features (i) searching for papers mentioning one or more specific sequences of nucleic acids and (ii) retrieving the genetic sequences appearing in different articles. These additional query capabilities are provided by a searchable index that we created by using the full text of the 176 672 papers available at PubMed Central at the time of writing and the sequences of nucleic acids appearing in them. To automatically extract the genetic sequences occurring in each paper, we used an original method we have developed. The database is updated monthly by automatically connecting to the PubMed Central FTP site to retrieve and index new manuscripts. Users can query the database via the web interface provided. PubDNA Finder can be freely accessed at http://servet.dia.fi.upm.es:8080/pubdnafinder

Intellectual disability and bleeding diathesis due to deficient CMP--sialic acid transport.

PubMed

Mohamed, Miski; Ashikov, Angel; Guillard, Mailys; Robben, Joris H; Schmidt, Samuel; van den Heuvel, B; de Brouwer, Arjan P M; Gerardy-Schahn, Rita; Deen, Peter M T; Wevers, Ron A; Lefeber, Dirk J; Morava, Eva

2013-08-13

To identify the underlying genetic defect in a patient with intellectual disability, seizures, ataxia, macrothrombocytopenia, renal and cardiac involvement, and abnormal protein glycosylation. Genetic studies involved homozygosity mapping by 250K single nucleotide polymorphism array and SLC35A1 sequencing. Functional studies included biochemical assays for N-glycosylation and mucin-type O-glycosylation and SLC35A1-encoded cytidine 5'-monophosphosialic acid (CMP-sialic acid) transport after heterologous expression in yeast. We performed biochemical analysis and found combined N- and O-glycosylation abnormalities and specific reduction in sialylation in this patient. Homozygosity mapping revealed homozygosity for the CMP-sialic acid transporter SLC35A1. Mutation analysis identified a homozygous c.303G > C (p.Gln101His) missense mutation that was heterozygous in both parents. Functional analysis of mutant SLC35A1 showed normal Golgi localization but 50% reduction in transport activity of CMP-sialic acid in vitro. We confirm an autosomal recessive, generalized sialylation defect due to mutations in SLC35A1. The primary neurologic presentation consisting of ataxia, intellectual disability, and seizures, in combination with bleeding diathesis and proteinuria, is discriminative from a previous case described with deficient sialic acid transporter. Our study underlines the importance of sialylation for normal CNS development and regular organ function.

Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes.

PubMed

He, Dandan; Chiou, Jiachi; Zeng, Zhenling; Chan, Edward Wai-Chi; Liu, Jian-Hua; Chen, Sheng

2016-10-01

Clinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between the blaCTX-M-15 and blaCTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

PubMed Central

He, Dandan; Chiou, Jiachi; Zeng, Zhenling; Chan, Edward Wai-Chi

2016-01-01

Clinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between the blaCTX-M-15 and blaCTX-M-14 elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity. PMID:27480856

Antimycobacterial Activity: A New Pharmacological Target for Conotoxins Found in the First Reported Conotoxin from Conasprella ximenes.

PubMed

Figueroa-Montiel, Andrea; Bernáldez, Johanna; Jiménez, Samanta; Ueberhide, Beatrix; González, Luis Javier; Licea-Navarro, Alexei

2018-01-23

Mycobacterium tuberculosis is the etiological agent of tuberculosis, an airborne infectious disease that is a leading cause of human morbidity and mortality worldwide. We report here the first conotoxin that is able to inhibit the growth of M. tuberculosis at a concentration similar to that of two other drugs that are currently used in clinics. Furthermore, it is also the first conopeptide that has been isolated from the venom of Conasprella ximenes. The venom gland transcriptome of C. ximenes was sequenced to construct a database with 24,284 non-redundant transcripts. The conopeptide was purified from the venom using reverse phase high performance liquid chromatography (RP-HPLC) and was analyzed using electrospray ionization-mass spectrometry (ESI-MS/MS). No automatic identification above the identity threshold with 1% of the false discovery rate was obtained; however, a 10-amino-acid sequence tag, manually extracted from the MS/MS spectra, allowed for the identification of a conotoxin in the transcriptome database. Electron transfer higher energy collision dissociation (EThcD) fragmentation of the native conotoxin confirmed the N-terminal sequence (1-14), while LC-MS/MS analysis of the tryptic digest of the reduced and S-alkylated conotoxin confirmed the C-terminal region (15-36). The expected and experimental molecular masses corresponded, within sub-ppm mass error. The 37-mer peptide (MW 4109.69 Da), containing eight cysteine residues, was named I1_xm11a, according to the current nomenclature for this type of molecule.

Integrative analysis workflow for the structural and functional classification of C-type lectins

PubMed Central

2011-01-01

Background It is important to understand the roles of C-type lectins in the immune system due to their ubiquity and diverse range of functions in animal cells. It has been observed that currently confirmed C-type lectins share a highly conserved domain known as the C-type carbohydrate recognition domain (CRD). Using the sequence profile of the CRD, an increasing number of putative C-type lectins have been identified. Hence, it is highly needed to develop a systematic framework that enables us to elucidate their carbohydrate (glycan) recognition function, and discover their physiological and pathological roles. Results Presented herein is an integrated workflow for characterizing the sequence and structural features of novel C-type lectins. Our workflow utilizes web-based queries and available software suites to annotate features that can be found on the C-type lectin, given its amino acid sequence. At the same time, it incorporates modeling and analysis of glycans - a major class of ligands that interact with C-type lectins. Thereafter, the results are analyzed together with context-specific knowledge to filter off unlikely predictions. This allows researchers to design their subsequent experiments to confirm the functions of the C-type lectins in a systematic manner. Conclusions The efficacy and usefulness of our proposed immunoinformatics workflow was demonstrated by applying our integrated workflow to a novel C-type lectin -CLEC17A - and we report some of its possible functions that warrants further validation through wet-lab experiments. PMID:22372988

Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older than 55 ka

NASA Astrophysics Data System (ADS)

Nielsen-Marsh, Christina M.; Ostrom, Peggy H.; Gandhi, Hasand; Shapiro, Beth; Cooper, Alan; Hauschka, Peter V.; Collins, Matthew J.

2002-12-01

We report the first complete sequences of the protein osteocalcin from small amounts (20 mg) of two bison bone (Bison priscus) dated to older than 55.6 ka and older than 58.9 ka. Osteocalcin was purified using new gravity columns (never exposed to protein) followed by microbore reversed-phase high-performance liquid chromatography. Sequencing of osteocalcin employed two methods of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS): peptide mass mapping (PMM) and post-source decay (PSD). The PMM shows that ancient and modern bison osteocalcin have the same mass to charge (m/z) distribution, indicating an identical protein sequence and absence of diagenetic products. This was confirmed by PSD of the m/z 2066 tryptic peptide (residues 1 19); the mass spectra from ancient and modern peptides were identical. The 129 mass unit difference in the molecular ion between cow (Bos taurus) and bison is caused by a single amino-acid substitution between the taxa (Trp in cow is replaced by Gly in bison at residue 5). Bison mitochondrial control region DNA sequences were obtained from the older than 55.6 ka fossil. These results suggest that DNA and protein sequences can be used to directly investigate molecular phylogenies over a considerable time period, the absolute limit of which is yet to be determined.

Rondonin an antifungal peptide from spider (Acanthoscurria rondoniae) haemolymph

PubMed Central

Riciluca, K.C.T.; Sayegh, R.S.R.; Melo, R.L.; Silva, P.I.

2012-01-01

Antimicrobial activities were detected in the haemolymph of the spider Acanthoscurrria rondoniae. A novel antifungal peptide, rondonin, was purified by reverse phase high performance liquid chromatography (RP-HPLC). Rondonin has an amino acid sequence of IIIQYEGHKH and a molecular mass of 1236.776 Da. This peptide has identity to a C-terminal fragment of the “d” subunit of haemocyanin from the spiders Eurypelma californicum and Acanthoscurria gomesiana. A synthetic peptide mimicking rondonin had identical characteristics to those of the isolated material, confirming its sequence. The synthetic peptide was active only against fungus. These data led us to conclude that the antifungal activity detected in the plasma of these spiders is the result of enzymatic processing of a protein that delivers oxygen in the haemolymph of many chelicerate. Several studies have suggested that haemocyanins are involved in the arthropod immune system, and the activity of this haemocyanin fragment reinforces this idea. PMID:24371568

Gold nanoparticle capture within protein crystal scaffolds.

PubMed

Kowalski, Ann E; Huber, Thaddaus R; Ni, Thomas W; Hartje, Luke F; Appel, Karina L; Yost, Jarad W; Ackerson, Christopher J; Snow, Christopher D

2016-07-07

DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)∼17 (nitrilotriacetic acid)∼1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography.

New species of Bordetella, Bordetella ansorpii sp. nov., isolated from the purulent exudate of an epidermal cyst.

PubMed

Ko, Kwan Soo; Peck, Kyong Ran; Oh, Won Sup; Lee, Nam Yong; Lee, Jang Ho; Song, Jae-Hoon

2005-05-01

A gram-negative bacillus, SMC-8986(T), which was isolated from the purulent exudate of an epidermal cyst but could not be identified by a conventional microbiologic method, was characterized by a variety of phenotypic and genotypic analyses. Sequences of the 16S rRNA gene revealed that this bacterium belongs to the genus Bordetella but diverged distinctly from previously described Bordetella species. Analyses of cellular fatty acid composition and performance of biochemical tests confirmed that this bacterium is distinct from other Bordetella species. Furthermore, the results of comparative sequence analyses of two protein-coding genes (risA and ompA) also showed that this strain represents a new species within the genus Bordetella. Based on the evaluated phenotypic and genotypic characteristics, it is proposed that SMC-8986(T) should be classified as a new species, namely Bordetella ansorpii sp. nov.

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peters, J.; Peters, M.; Lottspeich, F.

1987-11-01

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate (HPI))-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and M/sub r/ estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%)more » of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.« less

Artificial mismatch hybridization

DOEpatents

Guo, Zhen; Smith, Lloyd M.

1998-01-01

An improved nucleic acid hybridization process is provided which employs a modified oligonucleotide and improves the ability to discriminate a control nucleic acid target from a variant nucleic acid target containing a sequence variation. The modified probe contains at least one artificial mismatch relative to the control nucleic acid target in addition to any mismatch(es) arising from the sequence variation. The invention has direct and advantageous application to numerous existing hybridization methods, including, applications that employ, for example, the Polymerase Chain Reaction, allele-specific nucleic acid sequencing methods, and diagnostic hybridization methods.

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.

Preparation and evaluation of a 68Ga-labeled RGD-containing octapeptide for noninvasive imaging of angiogenesis: biodistribution in non-human primate

PubMed Central

Velikyan, Irina; Lindhe, Örjan

2018-01-01

Monitoring general disease marker such as angiogenesis may contribute to the development of personalized medicine and improve therapy outcome. Readily availability of positron emitter based imaging agents providing quantification would expand clinical positron emission tomography (PET) applications. Generator produced 68Ga provides PET images of high resolution and the half-life time frame is compatible with the pharmacokinetics of small peptides comprising arginine-glycine-aspartic acid (RGD) sequence specific to αvβ3 integrin receptors. The main objective of this study was to develop a method for 68Ga-labeling of RGD containing bicyclic octapeptide ([68Ga]Ga-DOTA-RGD) with high specific radioactivity and preclinically assess its imaging potential. DOTA-RGD was labeled using generator eluate preconcentration technique and microwave heating. The binding and organ distribution properties of [68Ga]Ga-DOTA-RGD were tested in vitro by autoradiography of frozen tumor sections, and in vivo in mice carrying a Lewis Lung carcinoma graft (LL2), and in non-human primate (NHP). Another peptide with aspartic acid-glycine-phenylalanine sequence was used as a negative control. The full 68Ga radioactivity eluted from two generators was quantitatively incorporated into 3-8 nanomoles of the peptide conjugates. The target binding specificity was confirmed by blocking experiments. The specific uptake in the LL2 mice model was observed in vivo and confirmed in the corresponding ex vivo biodistribution experiments. Increased accumulation of the radioactivity was detected in the wall of the uterus of the female NHP probably indicating neovascularization. [68Ga]Ga-DOTA-RGD demonstrated potential for the imaging of angiogenesis. PMID:29531858

Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis and identification of the phenylthiohydantoin-D-amino acid residue of [D-Ala2]-methionine enkephalin.

PubMed

Kurosu, Y; Murayama, K; Shindo, N; Shisa, Y; Ishioka, N

1996-11-01

This is an initial report to propose a protein sequence analysis system with DL differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE system. After fractionation of phenyl-thiohydantoin (PTH)-amino acids using a protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, beta-escin and others. As a model peptide, [D-Ala2]-methionine enkephalin (L-Tyr-D-Ala-Gly-L-Phe-L-Met), was used and the sequence with DL differentiation was determined, with the exception of the fourth amino acid, L-Phe, using our proposed system.

Micromonospora luteifusca sp. nov. isolated from cultivated Pisum sativum.

PubMed

Carro, Lorena; Riesco, Raúl; Spröer, Cathrin; Trujillo, Martha E

2016-06-01

Three novel actinobacterial strains, GUI2(T), GUI42 and CR21 isolated from nodular tissues and the rhizosphere of a sweet pea plant collected in Cañizal, Spain were identified according to their 16S rRNA gene sequences as new members of the genus Micromonospora. The closest phylogenetic members were found to be Micromonospora saelicesensis (99.2%) "Micromonospora zeae" (99.1%), "Micromonospora jinlongensis" (99%), Micromonospora lupini (98.9%) and Micromonospora zamorensis (98.8%). To resolve their full taxonomic position, four additional genes (atpD, gyrB, recA, rpoB) were partially sequenced and compared to available Micromonospora type strain sequences. DNA-DNA hybridization, BOX-PCR and ARDRA profiles confirmed that these strains represent a novel genomic species. All strains contained meso-diaminopimelic and hydroxy-diaminopimelic acids in their cell wall. Their fatty acid profiles comprised iso-C15:0, iso-C16:0 and anteiso-C15:0 as major components. The polar lipids diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol were found in the type strain GUI2(T) which also contained MK-10(H4) as the major menaquinone. Physiological and biochemical characteristics also differentiated the new isolates. Based on the integration of the above studies, strains GUI2(T), GUI42 and CR21 represent a novel Micromonospora species and we propose the name Micromonospora luteifusca sp. nov. The type strain is GUI2(T) (=CECT 8846(T); =DSM 100204(T)). Copyright © 2016 Elsevier GmbH. All rights reserved.

Genetic basis for mycophenolic acid production and strain-dependent production variability in Penicillium roqueforti.

PubMed

Gillot, Guillaume; Jany, Jean-Luc; Dominguez-Santos, Rebeca; Poirier, Elisabeth; Debaets, Stella; Hidalgo, Pedro I; Ullán, Ricardo V; Coton, Emmanuel; Coton, Monika

2017-04-01

Mycophenolic acid (MPA) is a secondary metabolite produced by various Penicillium species including Penicillium roqueforti. The MPA biosynthetic pathway was recently described in Penicillium brevicompactum. In this study, an in silico analysis of the P. roqueforti FM164 genome sequence localized a 23.5-kb putative MPA gene cluster. The cluster contains seven genes putatively coding seven proteins (MpaA, MpaB, MpaC, MpaDE, MpaF, MpaG, MpaH) and is highly similar (i.e. gene synteny, sequence homology) to the P. brevicompactum cluster. To confirm the involvement of this gene cluster in MPA biosynthesis, gene silencing using RNA interference targeting mpaC, encoding a putative polyketide synthase, was performed in a high MPA-producing P. roqueforti strain (F43-1). In the obtained transformants, decreased MPA production (measured by LC-Q-TOF/MS) was correlated to reduced mpaC gene expression by Q-RT-PCR. In parallel, mycotoxin quantification on multiple P. roqueforti strains suggested strain-dependent MPA-production. Thus, the entire MPA cluster was sequenced for P. roqueforti strains with contrasted MPA production and a 174bp deletion in mpaC was observed in low MPA-producers. PCRs directed towards the deleted region among 55 strains showed an excellent correlation with MPA quantification. Our results indicated the clear involvement of mpaC gene as well as surrounding cluster in P. roqueforti MPA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Spectrum of mutations in Glutaryl-CoA dehydrogenase gene in glutaric aciduria type I--Study from South India.

PubMed

Radha Rama Devi, A; Ramesh, Vakkalagadda A; Nagarajaram, H A; Satish, S P S; Jayanthi, U; Lingappa, Lokesh

2016-01-01

Glutaric aciduria type I is an autosomal recessive organic acid disorder. The primary defect is the deficiency of Glutaryl-CoA dehydrogenase (EC number 1.3.99.7) enzyme that is involved in the catabolic pathways of the amino acids l-lysine, l-hydroxylysine, and l-tryptophan. It is a treatable neuro-metabolic disorder. Early diagnosis and treatment helps in preventing brain damage. The Glutaryl-CoA dehydrogenase gene (GCDH) gene was sequenced to identify disease causing mutations by direct sequencing of all the exons in twelve patients who were biochemically confirmed with GA I. We identified eleven mutations of which nine are homozygous mutations, one heterozygous and two synonymous mutations. Among the eleven mutations, four mutations p.Q162R, p.P286S, p.W225X in two families and p.V410M are novel. A milder clinical presentation is observed in those families who are either heterozygous or with a benign synonymous SNP. Multiple sequence alignment (MSA) of GCDH with its homologues revealed that the observed novel mutations are not tolerated by protein structure and function. The present study indicates genetic heterogeneity in GCDH gene mutations among South Indian population. Genetic analysis is useful in prenatal diagnosis and prevention. Mutation analysis is a useful tool in the absence of non-availability of enzyme assay in GA I. Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Identification of equine influenza virus infection in Asian wild horses (Equus przewalskii).

PubMed

Yin, Xin; Lu, Gang; Guo, Wei; Qi, Ting; Ma, Jian; Zhu, Chao; Zhao, Shihua; Pan, Jialiang; Xiang, Wenhua

2014-05-01

An outbreak of equine influenza was observed in the Asian wild horse population in Xinjiang Province, China, in 2007. Nasal swabs were collected from wild horses and inoculated into 9-10-day SPF embryonated eggs. The complete genome of the isolate was sequenced. A comparison of the amino acid sequence revealed that the isolate was an equine influenza virus strain, which we named A/equine/Xinjiang/4/2007. Each gene of the virus was found to have greater than 99 % homology to equine influenza virus strains of the Florida-2 sublineage, which were circulating simultaneously in China, and a lesser amount of homology was found to the strain A/equine/Qinghai/1/1994 (European lineage), which was isolated during the last outbreak in China. These observations were confirmed by phylogenetic analysis. In addition, the deduced amino acid sequence of the neuraminidase of the A/equine/Xinjiang/4/2007 strain was identical to that of A/equine/California/8560/2002, an American isolate, and was found to be similar to those of Florida-2 strains found in other countries by comparing them with nine other field strains that were isolated in China from 2007 to 2008. It is suggested that the neuraminidase segment of A/equine/Xinjiang/4/2007 may have been obtained from equine influenza virus strains from other countries. We report for the first time an outbreak of equine influenza in the Asian wild horse population, and the complete genome of the virus is provided and analyzed.

Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnell, W.O.; Kyung Cheol Yi; Conway, T.

1990-12-01

The Zymomonas mobilis genes that encode glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) were cloned independently by genetic complementation of specific defects in Escherichia coli metabolism. The identify of these cloned genes was confirmed by various biochemical means. Nucleotide sequence analysis established that these three genes are clustered on the genome and revealed an additional open reading frame in this region that has significant amino acid identity to the E.coli xylose-proton symporter and the human glucose transporter. On the basis of this evidence and structural analysis of the deduced primary amino acid sequence, this gene is believed tomore » encode the Z. mobilis glucose-facilitated diffusion protein, glf. The four genes in the 6-kb cluster are organized in the order glf, zwf, edd, glk. The glf and zwf genes are separated by 146 bp. The zwf and edd genes overlap by 8 bp, and their expression may be translationally coupled. The edd and glk genes are separated by 203 bp. The glk gene is followed by tandem transcriptional terminators. The four genes appear to be organized in an operon. Such an arrangement of the genes that govern glucose uptake and the first three steps of the Entner-Doudoroff glycolytic pathway provides the organism with a mechanism for carefully regulating the levels of the enzymes that control carbon flux into the pathway.« less

Improved nucleic acid descriptors for siRNA efficacy prediction.

PubMed

Sciabola, Simone; Cao, Qing; Orozco, Modesto; Faustino, Ignacio; Stanton, Robert V

2013-02-01

Although considerable progress has been made recently in understanding how gene silencing is mediated by the RNAi pathway, the rational design of effective sequences is still a challenging task. In this article, we demonstrate that including three-dimensional descriptors improved the discrimination between active and inactive small interfering RNAs (siRNAs) in a statistical model. Five descriptor types were used: (i) nucleotide position along the siRNA sequence, (ii) nucleotide composition in terms of presence/absence of specific combinations of di- and trinucleotides, (iii) nucleotide interactions by means of a modified auto- and cross-covariance function, (iv) nucleotide thermodynamic stability derived by the nearest neighbor model representation and (v) nucleic acid structure flexibility. The duplex flexibility descriptors are derived from extended molecular dynamics simulations, which are able to describe the sequence-dependent elastic properties of RNA duplexes, even for non-standard oligonucleotides. The matrix of descriptors was analysed using three statistical packages in R (partial least squares, random forest, and support vector machine), and the most predictive model was implemented in a modeling tool we have made publicly available through SourceForge. Our implementation of new RNA descriptors coupled with appropriate statistical algorithms resulted in improved model performance for the selection of siRNA candidates when compared with publicly available siRNA prediction tools and previously published test sets. Additional validation studies based on in-house RNA interference projects confirmed the robustness of the scoring procedure in prospective studies.

Aspartic cathepsin D endopeptidase contributes to extracellular digestion in clawed lobsters Homarus americanus and Homarus gammarus.

PubMed

Rojo, Liliana; Muhlia-Almazan, Adriana; Saborowski, Reinhard; García-Carreño, Fernando

2010-11-01

Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding.

Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria.

PubMed

Miller, Kennon; Harrington, Susan M; Procop, Gary W

2015-08-01

New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum . Testing of AFB smear-negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

[Analysis of FABP4 expression pattern in rump fat deposition and metabolism of Altay sheep].

PubMed

Ruixia, Xu; Lei, Gao; Weili, Zhao; Wei, Zhang; Guangchao, Song; Shangquan, Gan; Guoqing, Shi

2015-02-01

FABP4 (Fatty acid binding protein 4) is a hot candidate gene in fat deposition and lipid metabolism and participates in the transport and metabolism of intracellular free fatty acids. We aim to study the role of FABP4 in fat deposition and metabolism of the rump fat in Altay sheep. In this study, bioinformatics method was used to analyze the protein sequence homology among 10 species, and RT-PCR was employed to detect FABP4 tissue profiling of Altay sheep. An animal model simulating the rump fat deposition and metabolism of Altay sheep was established by continuous starvation, and qPCR and iTRAQ (isobaric tags for relative and absolute quantitation) were used to detecte FABP4 mRNA and protein expression changes in the control and continuous starvation groups, respectively. Sequence analysis showed that FABP4 protein sequence is highly conserved among species, suggesting an important biological function during evolution for FABP4. The RT-PCR result confirmed that FABP4 mRNA was highly expressed in intestinal and rump fat, suggesting that FABP4 plays an important physiological role in fat tissues. We did not find significant differences in FABP4 mRNA and protein between control and continuous starvation groups (P>0.05), which indicates that FABP4 may not be the key gene in fat deposition and metabolism in Altay sheep.The results above lay a foundation for further studies of FABP4 in rump or tail fat.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

PubMed

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Complete genome sequence of Acidihalobacter prosperus strain F5, an extremely acidophilic, iron- and sulfur-oxidizing halophile with potential industrial applicability in saline water bioleaching of chalcopyrite.

PubMed

Khaleque, Himel N; Corbett, Melissa K; Ramsay, Joshua P; Kaksonen, Anna H; Boxall, Naomi J; Watkin, Elizabeth L J

2017-11-20

Successful process development for the bioleaching of mineral ores, particularly the refractory copper sulfide ore chalcopyrite, remains a challenge in regions where freshwater is scarce and source water contains high concentrations of chloride ion. In this study, a pure isolate of Acidihalobacter prosperus strain F5 was characterized for its ability to leach base metals from sulfide ores (pyrite, chalcopyrite and pentlandite) at increasing chloride ion concentrations. F5 successfully released base metals from ores including pyrite and pentlandite at up to 30gL -1 chloride ion and chalcopyrite up to 18gL -1 chloride ion. In order to understand the genetic mechanisms of tolerance to high acid, saline and heavy metal stress the genome of F5 was sequenced and analysed. As well as being the first strain of Ac. prosperus to be isolated from Australia it is also the first complete genome of the Ac. prosperus species to be sequenced. The F5 genome contains genes involved in the biosynthesis of compatible solutes and genes encoding monovalent cation/proton antiporters and heavy metal transporters which could explain its abilities to tolerate high salinity, acidity and heavy metal stress. Genome analysis also confirmed the presence of genes involved in copper tolerance. The study demonstrates the potential biotechnological applicability of Ac. prosperus strain F5 for saline water bioleaching of mineral ores. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Acα2-6Galβ1-4GlcNAc human-type influenza receptor

PubMed Central

Kadirvelraj, Renuka; Grant, Oliver C; Goldstein, Irwin J; Winter, Harry C; Tateno, Hiroaki; Fadda, Elisa; Woods, Robert J

2011-01-01

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7 Å) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding. PMID:21436237

Isolation, identification and screening of antimicrobial thermophilic Streptomyces sp. Al-Dhabi-1 isolated from Tharban hot spring, Saudi Arabia.

PubMed

Al-Dhabi, Naif Abdullah; Esmail, Galal Ali; Duraipandiyan, Veeramuthu; Valan Arasu, Mariadhas; Salem-Bekhit, Mounir M

2016-01-01

The strain Streptomyces sp. Al-Dhabi-1 was isolated from soil sediments collected from Tharban hot spring in the southern west of Saudi Arabia using actinomycetes isolation agar and starch casein agar at 55 °C. Identification of the isolate was done according to morphological, physiological and biochemical characteristics and 16S rRNA sequence similarity as well. 16S rRNA sequence and blast analyses confirmed that the isolate belonging to the genus Streptomyces. The sequence was submitted to GenBank with accession number (KF815080). Ethyl acetate extract of Streptomyces sp. Al-Dhabi-1 showed good antimicrobial activities against tested pathogenic microbes. Minimum inhibitory concentration results showed that the best values were observed against S. agalactiae (<0.039 mg/ml) and Klebsiella pneumonia (0.125 mg/ml). Minimum inhibitory concentration of Al-Dhabi-1 against fungi; Cryptococcus neoformans (0.078 mg/ml), C. albicans (0.156 mg/ml), A. niger (0.625 mg/ml), and T. mentagrophytes (0.156 mg/ml). GC-MS analysis was used for the chemical profile of ethyl acetate extract. Benzeneacetic acid (16.02 %) and acetic acid 2-phenylethyl ester (10.35 %) were the major compounds among 31 substances found the ethyl acetate extract. According to the results of antimicrobial activity against pathogenic microbes, it is clear that the actinomycetes from hot springs with extreme environments are promising source for antimicrobial compounds.

Identification and Analysis of Novel Amino-Acid Sequence Repeats in Bacillus anthracis str. Ames Proteome Using Computational Tools

PubMed Central

Hemalatha, G. R.; Rao, D. Satyanarayana; Guruprasad, L.

2007-01-01

We have identified four repeats and ten domains that are novel in proteins encoded by the Bacillus anthracis str. Ames proteome using automated in silico methods. A “repeat” corresponds to a region comprising less than 55-amino-acid residues that occur more than once in the protein sequence and sometimes present in tandem. A “domain” corresponds to a conserved region with greater than 55-amino-acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 57-amino-acid-residue PxV domain, (2) 122-amino-acid-residue FxF domain, (3) 111-amino-acid-residue YEFF domain, (4) 109-amino-acid-residue IMxxH domain, (5) 103-amino-acid-residue VxxT domain, (6) 84-amino-acid-residue ExW domain, (7) 104-amino-acid-residue NTGFIG domain, (8) 36-amino-acid-residue NxGK repeat, (9) 95-amino-acid-residue VYV domain, (10) 75-amino-acid-residue KEWE domain, (11) 59-amino-acid-residue AFL domain, (12) 53-amino-acid-residue RIDVK repeat, (13) (a) 41-amino-acid-residue AGQF repeat and (b) 42-amino-acid-residue GSAL repeat. A repeat or domain type is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure. PMID:17538688

Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing.

PubMed

Lawton, Samantha J; Weis, Allison M; Byrne, Barbara A; Fritz, Heather; Taff, Conor C; Townsend, Andrea K; Weimer, Bart C; Mete, Aslı; Wheeler, Sarah; Boyce, Walter M

2018-05-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

Genome sequence analysis of dengue virus 1 isolated in Key West, Florida.

PubMed

Shin, Dongyoung; Richards, Stephanie L; Alto, Barry W; Bettinardi, David J; Smartt, Chelsea T

2013-01-01

Dengue virus (DENV) is transmitted to humans through the bite of mosquitoes. In November 2010, a dengue outbreak was reported in Monroe County in southern Florida (FL), including greater than 20 confirmed human cases. The virus collected from the human cases was verified as DENV serotype 1 (DENV-1) and one isolate was provided for sequence analysis. RNA was extracted from the DENV-1 isolate and was used in reverse transcription polymerase chain reaction (RT-PCR) to amplify PCR fragments to sequence. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the entire genome. The DENV-1 isolate found in Key West (KW), FL was sequenced for whole genome characterization. Sequence assembly, Genbank searches, and recombination analyses were performed to verify the identity of the genome sequences and to determine percent similarity to known DENV-1 sequences. We show that the KW DENV-1 strain is 99% identical to Nicaraguan and Mexican DENV-1 strains. Phylogenetic and recombination analyses suggest that the DENV-1 isolated in KW originated from Nicaragua (NI) and the KW strain may circulate in KW. Also, recombination analysis results detected recombination events in the KW strain compared to DENV-1 strains from Puerto Rico. We evaluate the relative growth of KW strain of DENV-1 compared to other dengue viruses to determine whether the underlying genetics of the strain is associated with a replicative advantage, an important consideration since local transmission of DENV may result because domestic tourism can spread DENVs.

In Vivo Emergence of Resistance to Novel Cephalosporin-β-Lactamase Inhibitor Combinations through the Duplication of Amino Acid D149 from OXA-2 β-Lactamase (OXA-539) in Sequence Type 235 Pseudomonas aeruginosa.

PubMed

Fraile-Ribot, Pablo A; Mulet, Xavier; Cabot, Gabriel; Del Barrio-Tofiño, Ester; Juan, Carlos; Pérez, José L; Oliver, Antonio

2017-09-01

Resistance development to novel cephalosporin-β-lactamase inhibitor combinations during ceftazidime treatment of a surgical infection by Pseudomonas aeruginosa was investigated. Both initial (97C2) and final (98G1) isolates belonged to the high-risk clone sequence type (ST) 235 and were resistant to carbapenems ( oprD ), fluoroquinolones (GyrA-T83I, ParC-S87L), and aminoglycosides ( aacA7/aacA8/aadA6 ). 98G1 also showed resistance to ceftazidime, ceftazidime-avibactam, and ceftolozane-tazobactam. Sequencing identified bla OXA-2 in 97C2, but 98G1 contained a 3-bp insertion leading to the duplication of the key residue D149 (designated OXA-539). Evaluation of PAO1 transformants producing cloned OXA-2 or OXA-539 confirmed that D149 duplication was the cause of resistance. Active surveillance of the emergence of resistance to these new valuable agents is warranted. Copyright © 2017 American Society for Microbiology.

In Vivo Emergence of Resistance to Novel Cephalosporin–β-Lactamase Inhibitor Combinations through the Duplication of Amino Acid D149 from OXA-2 β-Lactamase (OXA-539) in Sequence Type 235 Pseudomonas aeruginosa

PubMed Central

Fraile-Ribot, Pablo A.; Mulet, Xavier; del Barrio-Tofiño, Ester; Juan, Carlos; Pérez, José L.

2017-01-01

ABSTRACT Resistance development to novel cephalosporin–β-lactamase inhibitor combinations during ceftazidime treatment of a surgical infection by Pseudomonas aeruginosa was investigated. Both initial (97C2) and final (98G1) isolates belonged to the high-risk clone sequence type (ST) 235 and were resistant to carbapenems (oprD), fluoroquinolones (GyrA-T83I, ParC-S87L), and aminoglycosides (aacA7/aacA8/aadA6). 98G1 also showed resistance to ceftazidime, ceftazidime-avibactam, and ceftolozane-tazobactam. Sequencing identified blaOXA-2 in 97C2, but 98G1 contained a 3-bp insertion leading to the duplication of the key residue D149 (designated OXA-539). Evaluation of PAO1 transformants producing cloned OXA-2 or OXA-539 confirmed that D149 duplication was the cause of resistance. Active surveillance of the emergence of resistance to these new valuable agents is warranted. PMID:28674059

Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus.

PubMed

Livingston, B T; Shaw, R; Bailey, A; Wilt, F

1991-12-01

In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus pictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide, a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to proteins that form a unique beta-spiral supersecondary structure.

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

Code of Federal Regulations, 2010 CFR

2010-07-01

... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Form and format for... And/or Amino Acid Sequences § 1.824 Form and format for nucleotide and/or amino acid sequence... Code for Information Interchange (ASCII) text. No other formats shall be allowed. (3) The computer...

SLC6A1 Mutation and Ketogenic Diet in Epilepsy With Myoclonic-Atonic Seizures.

PubMed

Palmer, Samantha; Towne, Meghan C; Pearl, Phillip L; Pelletier, Renee C; Genetti, Casie A; Shi, Jiahai; Beggs, Alan H; Agrawal, Pankaj B; Brownstein, Catherine A

2016-11-01

Epilepsy with myoclonic-atonic seizures, also known as myoclonic-astatic epilepsy or Doose syndrome, has been recently linked to variants in the SLC6A1 gene. Epilepsy with myoclonic-atonic seizures is often refractory to antiepileptic drugs, and the ketogenic diet is known for treating medically intractable seizures, although the mechanism of action is largely unknown. We report a novel SLC6A1 variant in a patient with epilepsy with myoclonic-atonic seizures, analyze its effects, and suggest a mechanism of action for the ketogenic diet. We describe a ten-year-old girl with epilepsy with myoclonic-atonic seizures and a de novo SLC6A1 mutation who responded well to the ketogenic diet. She carried a c.491G>A mutation predicted to cause p.Cys164Tyr amino acid change, which was identified using whole exome sequencing and confirmed by Sanger sequencing. High-resolution structural modeling was used to analyze the likely effects of the mutation. The SLC6A1 gene encodes a transporter that removes gamma-aminobutyric acid from the synaptic cleft. Mutations in SLC6A1 are known to disrupt the gamma-aminobutyric acid transporter protein 1, affecting gamma-aminobutyric acid levels and causing seizures. The p.Cys164Tyr variant found in our study has not been previously reported, expanding on the variants linked to epilepsy with myoclonic-atonic seizures. A 10-year-old girl with a novel SLC6A1 mutation and epilepsy with myoclonic-atonic seizures had an excellent clinical response to the ketogenic diet. An effect of the diet on gamma-aminobutyric acid reuptake mediated by gamma-aminobutyric acid transporter protein 1 is suggested. A personalized approach to epilepsy with myoclonic-atonic seizures patients carrying SLC6A1 mutation and a relationship between epilepsy with myoclonic-atonic seizures due to SLC6A1 mutations, GABAergic drugs, and the ketogenic diet warrants further exploration. Copyright © 2016 Elsevier Inc. All rights reserved.

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids.

PubMed

Nickling, Jessica H; Baumann, Tobias; Schmitt, Franz-Josef; Bartholomae, Maike; Kuipers, Oscar P; Friedrich, Thomas; Budisa, Nediljko

2018-05-04

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pimkin, Maxim; Miller, C. Glenn; Blakesley, Lauryn

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the proteinmore » identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.« less

The hemocyanin from a living fossil, the cephalopod Nautilus pompilius: protein structure, gene organization, and evolution.

PubMed

Bergmann, Sandra; Lieb, Bernhard; Ruth, Peter; Markl, Jürgen

2006-03-01

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M(r) = approximately 3.5 million), assembled from 10 identical copies of an approximately 350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of approximately 50 kDa each (FUs a-g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5' UTR of 58 bp, a 3' UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M(r) = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin "OdH" from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 +/- 24 million years ago, in close agreement with fossil records from the early Devonian.

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction*

PubMed Central

Rahman, Kh. Shamsur; Chowdhury, Erfan Ullah; Sachse, Konrad; Kaltenboeck, Bernhard

2016-01-01

X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences. PMID:27189949

Phylogenetic relationships and taxonomic position of Chlorella-like isolates from low pH environments (pH < 3.0)

PubMed Central

Huss, Volker AR; Ciniglia, Claudia; Cennamo, Paola; Cozzolino, Salvatore; Pinto, Gabriele; Pollio, Antonino

2002-01-01

Background Little is known about phytoplankton communities inhabiting low pH environments such as volcanic and geothermal sites or acidic waters. Only specialised organisms are able to tolerate such extreme conditions. There is, thus, low species diversity. We have characterised the previously isolated acid tolerant Chlorella-like microalgae Viridiella fridericiana and Chlorella protothecoides var. acidicola by microscopical and biomolecular methods in order to assess their phylogenetic relationships. Results Both isolates belong to the trebouxiophycean lineage of chlorophytes. 18S and ITS1 sequence data clearly confirm that Viridiella fridericiana constitutes a new genus apart from the morphologically similar and likewise acid tolerant microalga Chlorella saccharophila. Chlorella protothecoides var. acidicola on the other hand is not a variety of Chlorella protothecoides but falls within a heterogeneous cluster consisting of Nannochloris, "Chlorella" spec. Yanaqocha, and Koliella, and is most closely related to algae which were also isolated from extreme environments. Conclusions The distribution of acid tolerant strains in the 18S rRNA tree shows that acquisition of acid tolerance was unlikely a monophyletic event in green microalgae. We propose that different strains have independently adapted to extreme environments. Some of them have spread worldwide and were able to colonise other extreme habitats. Considering the problems of successfully isolating acid tolerant strains, acidic soils could represent an unsuspected source of biological diversity with high potential for biotechnological utilisations. PMID:12194702

Application of 2D graphic representation of protein sequence based on Huffman tree method.

PubMed

Qi, Zhao-Hui; Feng, Jun; Qi, Xiao-Qin; Li, Ling

2012-05-01

Based on Huffman tree method, we propose a new 2D graphic representation of protein sequence. This representation can completely avoid loss of information in the transfer of data from a protein sequence to its graphic representation. The method consists of two parts. One is about the 0-1 codes of 20 amino acids by Huffman tree with amino acid frequency. The amino acid frequency is defined as the statistical number of an amino acid in the analyzed protein sequences. The other is about the 2D graphic representation of protein sequence based on the 0-1 codes. Then the applications of the method on ten ND5 genes and seven Escherichia coli strains are presented in detail. The results show that the proposed model may provide us with some new sights to understand the evolution patterns determined from protein sequences and complete genomes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Opsin cDNA sequences of a UV and green rhodopsin of the satyrine butterfly Bicyclus anynana.

PubMed

Vanhoutte, K J A; Eggen, B J L; Janssen, J J M; Stavenga, D G

2002-11-01

The cDNAs of an ultraviolet (UV) and long-wavelength (LW) (green) absorbing rhodopsin of the bush brown Bicyclus anynana were partially identified. The UV sequence, encoding 377 amino acids, is 76-79% identical to the UV sequences of the papilionids Papilio glaucus and Papilio xuthus and the moth Manduca sexta. A dendrogram derived from aligning the amino acid sequences reveals an equidistant position of Bicyclus between Papilio and Manduca. The sequence of the green opsin cDNA fragment, which encodes 242 amino acids, represents six of the seven transmembrane regions. At the amino acid level, this fragment is more than 80% identical to the corresponding LW opsin sequences of Dryas, Heliconius, Papilio (rhodopsin 2) and Manduca. Whereas three LW absorbing rhodopsins were identified in the papilionid butterflies, only one green opsin was found in B. anynana.

Complete amino acid sequence of ananain and a comparison with stem bromelain and other plant cysteine proteases.

PubMed Central

Lee, K L; Albee, K L; Bernasconi, R J; Edmunds, T

1997-01-01

The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain. PMID:9355753

Genotypic diversity of stress response in Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus.

PubMed

Ricciardi, Annamaria; Parente, Eugenio; Guidone, Angela; Ianniello, Rocco Gerardo; Zotta, Teresa; Abu Sayem, S M; Varcamonti, Mario

2012-07-02

Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum are three closely related species which are widespread in food and non-food environments, and are important as starter bacteria or probiotics. In order to evaluate the phenotypic diversity of stress tolerance in the L. plantarum group and the ability to mount an adaptive heat shock response, the survival of exponential and stationary phase and of heat adapted exponential phase cells of six L. plantarum subsp. plantarum, one L. plantarum subsp. argentoratensis, one L. pentosus and two L. paraplantarum strains selected in a previous work upon exposure to oxidative, heat, detergent, starvation and acid stresses was compared to that of the L. plantarum WCFS1 strain. Furthermore, to evaluate the genotypic diversity in stress response genes, ten genes (encoding for chaperones DnaK, GroES and GroEL, regulators CtsR, HrcA and CcpA, ATPases/proteases ClpL, ClpP, ClpX and protease FtsH) were amplified using primers derived from the WCFS1 genome sequence and submitted to restriction with one or two endonucleases. The results were compared by univariate and multivariate statistical methods. In addition, the amplicons for hrcA and ctsR were sequenced and compared by multiple sequence alignment and polymorphism analysis. Although there was evidence of a generalized stress response in the stationary phase, with increase of oxidative, heat, and, to a lesser extent, starvation stress tolerance, and for adaptive heat stress response, with increased tolerance to heat, acid and detergent, different growth phases and adaptation patterns were found. Principal component analysis showed that while heat, acid and detergent stresses respond similarly to growth phase and adaptation, tolerance to oxidative and starvation stresses implies completely unrelated mechanisms. A dendrogram obtained using the data from multilocus restriction typing (MLRT) of stress response genes clearly separated two groups of L. plantarum strains from the other species but there was no correlation between genotypic grouping and grouping obtained on the basis of the stress response pattern, nor with the phylograms obtained from hrcA and ctsR sequences. Differences in sequence in L. plantarum strains were mostly due to single nucleotide polymorphisms with a high frequency of synonymous nucleotide changes and, while hrcA was characterized by an excess of low frequency polymorphism, very low diversity was found in ctsR sequences. Sequence alignment of hrcA allowed a correct discrimination of the strains at the species level, thus confirming the relevance of stress response genes for taxonomy. Copyright © 2012 Elsevier B.V. All rights reserved.

WebLogo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crooks, Gavin E.

WebLogo is a web based application designed to make the generation of sequence logos as easy and painless as possible. Sequesnce logos are a graphical representation of an amino acid or nucleic acid multiple sequence alignment developed by Tom Schneider and Mike Stephens. Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. In general, a sequence logo provides a richermore » and more precise description of, for example, a binding site, than would a consensus sequence.« less

Contribution of Tryptophan Residues to the Combining Site of a Monoclonal Anti Dinitrophenyl Spin-Label Antibody

DTIC Science & Technology

1987-01-01

identified in the difference spectra, implying that: there are five to seven tryptophans within 17 A of the spin-label hapten. Amino acid sequences...of the heavy, and light chains were obtained by a combination of amino acid and DNA sequencing. A molecular model’ was constructed from the sequence...Clore & acids yields detailed information about the amino acid com- Gronenborn, 1982, 1983). This technique should also identify position of the combining

Characterization of metabolic network of oxalic acid biosynthesis through RNA seq data analysis of developing spikes of finger millet (Eleusine coracana): Deciphering the role of key genes involved in oxalate formation in relation to grain calcium accumulation.

PubMed

Akbar, Naved; Gupta, Supriya; Tiwari, Apoorv; Singh, K P; Kumar, Anil

2018-04-05

In the present study, we identified seven major genes of oxalic acid biosynthesis pathway (SGAT, GGAT, ICL, GLO, MHAR, APO and OXO) from developing spike transcriptome of finger millet using rice as a reference. Sequence alignment of identified genes showed high similarity with their respective homolog in rice except for OXO and GLO. Transcript abundance (FPKM) reflects the higher accumulation of identified genes in GP-1 (low calcium genotype) as compared to GP-45 (high calcium genotype) which was further confirmed by qRT-PCR analysis, indicating differential oxalate formation in both genotypes. Determination of oxalic acid and tartaric acid content in developing spikes explain that higher oxalic acid content in GP-1 however, tartaric acid content was more in GP-45. Higher calcium content in GP-45 and lower oxalate accumulation may be due to the diversion of more ascorbic acid into tartaric acid and may correspond to less formation of calcium oxalate. Our results suggest that more than one pathway for oxalic acid biosynthesis might be present in finger millet with probable predominance of ascorbate-tartarate pathway rather than glyoxalate-oxalate conversion. Thus, finger millet can be use as an excellent model system for understanding more specific role of nutrients-antinutrients interactions, as evident from the present study. Copyright © 2018 Elsevier B.V. All rights reserved.

Prediction of selenoprotein T structure and its response to selenium deficiency in chicken immune organs.

PubMed

You, Lu; Liu, Ci; Yang, Zi-Jiang; Li, Ming; Li, Shu

2014-08-01

Selenoprotein T (SelT) is associated with the regulation of calcium homeostasis and neuroendocrine secretion. SelT can also change cell adhesion and is involved in redox regulation and cell fixation. However, the structure and function of chicken SelT and its response to selenium (Se) remains unclear. In the present study, 150 1-day-old chickens were randomly divided into a low Se group (L group, fed a Se-deficient diet containing 0.020 mg/kg Se) and a control group (C group, fed a diet containing sodium selenite at 0.2 mg/kg Se). The immune organs (spleen, thymus, and bursa of Fabricius) were collected at 15, 25, 35, 45, and 55 days of age. We performed a sequence analysis and predicted the structure and function of SelT. We also investigated the effects of Se deficiency on the expression of SelT, selenophosphate synthetase-1 (SPS1), and selenocysteine synthase (SecS) using RT-PCR and the oxidative stress in the chicken immune organs. The data showed that the coding sequence (CDS) and deduced amino acid sequence of SelT were highly similar to those of 17 other animals. Se deficiency induced lower (P < 0.05) levels of SelT, SPS1, and SecS, reduced the catalase (CAT) activity, and increased the levels of hydrogen peroxide (H2O2) and hydroxyl radical (-OH) in immune organs. In conclusion, the CDS and deduced amino acid sequence of chicken SelT are highly homologous to those of various mammals. The redox function and response to the Se deficiency of chicken SelT may be conserved. A Se-deficient diet led to a decrease in SelT, SecS, and SPS1 and induced oxidative stress in the chicken immune organs. To our knowledge, this is the first report of predictions of chicken SelT structure and function. The present study demonstrated the relationship between the selenoprotein synthases (SPS1, SecS) and SelT expression in the chicken immune organs and further confirmed oxidative stress caused by Se deficiency. Thus, the information presented in this study is helpful to understand chicken SelT structure and function. Meanwhile, the present research also confirmed the negative effects of Se deficiency on chicken immune organs.

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

Role of chicken astrovirus as a causative agent of gout in commercial broilers in India.

PubMed

Bulbule, N R; Mandakhalikar, K D; Kapgate, S S; Deshmukh, V V; Schat, K A; Chawak, M M

2013-01-01

Several outbreaks of gout were reported in commercial broilers in India during 2011 and 2012, causing up to 40% mortality in the birds. Gross and histopathological observations confirmed gout. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis from kidney samples of gout-affected birds indicated the presence of chicken astrovirus (CAstV) in 41.7% of cases and a mixed infection of CAstV and avian nephritis virus (ANV) in 36.4% of cases. CAstV isolated from gout-affected kidneys by inoculating embryonated specific pathogen free (SPF) eggs showed dwarfing in embryos and a cytopathic effect in chicken embryo kidney cells. Inoculation of 1-day-old SPF and broiler chicks with CAstVs caused gout and mortality between 4 and 10 days post inoculation. Virus isolation and qRT-PCR analysis showed the presence of only CAstV in inoculated chicks. Sequence analysis of capsid genes indicated a major group of Indian CAstVs that displayed 92.0 to 99.2% intergroup amino acid identity and 83.9 to 90.4% identity with subgroup Bi CAstVs of UK origin. We designated this group Indian Bi. Analysis of the partial polymerase amino acid sequences of our isolates indicated two groups of CAstVs (Indian 1 and 2) that displayed 90.2 to 95.5% amino acid identity between them. We thus report for the first time that, in addition to infectious bronchitis virus and ANV, CAstVs are a causative agent of gout.

Different HIV-1 env frames: gp120 and ASP (antisense protein) biosynthesis, and theirs co-variation tropic amino acid signatures in X4- and R5-viruses.

PubMed

Dimonte, Salvatore

2017-01-01

Antisense protein (ASP) is the new actor of viral life of Human Immunodeficiency Virus type 1 (HIV-1) although proposed above 20 years ago. The asp ORF is into complementary strand of the gp120/gp41 junction of env gene. The ASP biological role remains little known. Knowing the Env markers of viral tropism, a dataset of sequences (660 strains) was used to analyze the hypothetical ASP involvement in CCR5 (R5) and/or CXCR4 (X4) co-receptor interaction. Preliminarily, prevalence of ASP and gp120 V3 mutations was performed; following association among mutations were elaborate. The classical V3 tropic-signatures were confirmed, and 36 R5- and 22 X4-tropic ASP mutations were found. Moreover, by analyzing the ASP sequences, 36 out of 179 amino acid positions significantly associated with different co-receptor usage were found. Several statistically significant associations between gp120 V3 and ASP mutations were observed. The dendrogram showed the existence of a cluster associated with R5-usage and a large cluster associated with X4-usage. These results show that gp120 V3 and specific amino acid changes in ASP are associated together with CXCR4 and/or CCR5-usage. These findings implement previous observations on unclear ASP functions. J. Med. Virol. 89:112-122, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Acidithiobacillus ferrooxidans metabolism: from genome sequence to industrial applications.

PubMed

Valdés, Jorge; Pedroso, Inti; Quatrini, Raquel; Dodson, Robert J; Tettelin, Herve; Blake, Robert; Eisen, Jonathan A; Holmes, David S

2008-12-11

Acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for the industrial recovery of copper (bioleaching or biomining). It is a chemolithoautrophic, gamma-proteobacterium using energy from the oxidation of iron- and sulfur-containing minerals for growth. It thrives at extremely low pH (pH 1-2) and fixes both carbon and nitrogen from the atmosphere. It solubilizes copper and other metals from rocks and plays an important role in nutrient and metal biogeochemical cycling in acid environments. The lack of a well-developed system for genetic manipulation has prevented thorough exploration of its physiology. Also, confusion has been caused by prior metabolic models constructed based upon the examination of multiple, and sometimes distantly related, strains of the microorganism. The genome of the type strain A. ferrooxidans ATCC 23270 was sequenced and annotated to identify general features and provide a framework for in silico metabolic reconstruction. Earlier models of iron and sulfur oxidation, biofilm formation, quorum sensing, inorganic ion uptake, and amino acid metabolism are confirmed and extended. Initial models are presented for central carbon metabolism, anaerobic metabolism (including sulfur reduction, hydrogen metabolism and nitrogen fixation), stress responses, DNA repair, and metal and toxic compound fluxes. Bioinformatics analysis provides a valuable platform for gene discovery and functional prediction that helps explain the activity of A. ferrooxidans in industrial bioleaching and its role as a primary producer in acidic environments. An analysis of the genome of the type strain provides a coherent view of its gene content and metabolic potential.

Participation of cysteine-rich secretory proteins (CRISP) in mammalian sperm-egg interaction.

PubMed

Cohen, Débora J; Busso, Dolores; Da Ros, Vanina; Ellerman, Diego A; Maldera, Julieta A; Goldweic, Nadia; Cuasnicu, Patricia S

2008-01-01

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. CRISP1 (cysteine-rich secretory protein 1) is an epididymal protein thought to participate in gamete fusion through its binding to egg-complementary sites. Structure-function studies using recombinant fragments of CRISP1 as well as synthetic peptides reveal that its egg-binding ability resides in a 12 amino acid region corresponding to an evolutionary conserved motif of the CRISP family, named Signature 2 (S2). Further experiments analyzing both the ability of other CRISP proteins to bind to the rat egg and the amino acid sequence of their S2 regions show that the amino acid sequence of the S2 is needed for CRISP1 to interact with the egg. CRISP1 appears to be involved in the first step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. The observation that sperm testicular CRISP2 is also able to bind to the egg surface suggests a role for this protein in gamete fusion. Subsequent experiments confirmed the participation of CRISP2 in this step of fertilization and revealed that CRISP1 and CRISP2 interact with common egg surface binding sites. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization. These observations contribute to a better understanding of the molecular mechanisms underlying mammalian fertilization.

Role of CadC and CadD in the 2,4-dichlorophenoxyacetic acid oxygenase system of Sphingomonas agrestis 58-1.

PubMed

Kijima, Kumiko; Mita, Hajime; Kawakami, Mitsuyasu; Amada, Kei

2018-02-02

In the present study, we confirm that 2,4-dichlorophenoxyacetic acid (2,4-D) oxygenase from Sphingomonas agrestis 58-1 belongs to the family of Rieske non-heme iron aromatic ring-hydroxylating oxygenases, which comprise a core enzyme (oxygenase), ferredoxin, and oxidoreductase. It has previously been shown that cadAB genes are necessary for the conversion of 2,4-D to 2,4-dichlorophenol; however, the respective roles of ferredoxin and oxidoreductase in the 2,4-D oxygenase system from S. agrestis 58-1 remain unknown. Using nucleotide sequence analysis of the plasmid pCADAB1 from Sphingomonas sp. ERG5, which degrades 4-chloro-2-methylphenoxyacetic acid and 2,4-D, Nielsen et al. identified orf95, upstream of cadA, and orf98, downstream of cadB, which were predicted and designated as cadD (oxidoreductase) and cadC (ferredoxin), respectively (Nielsen et al., PLoS One, 8, 1-9, 2013). These designations were the result of sequence analysis; therefore, we constructed an expression system of CadABC and CadABCD in Escherichia coli and assayed their enzyme activities. Our findings indicate that CadC is essential for the activity of 2,4-D oxygenase and CadD promotes CadABC activity in recombinant E. coli cells. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Sulfate-reducing bacteria mediate thionation of diphenylarsinic acid under anaerobic conditions.

PubMed

Guan, Ling; Shiiya, Ayaka; Hisatomi, Shihoko; Fujii, Kunihiko; Nonaka, Masanori; Harada, Naoki

2015-02-01

Diphenylarsinic acid (DPAA) is often found as a toxic intermediate metabolite of diphenylchloroarsine or diphenylcyanoarsine that were produced as chemical warfare agents and were buried in soil after the World Wars. In our previous study Guan et al. (J Hazard Mater 241-242:355-362, 2012), after application of sulfate and carbon sources, anaerobic transformation of DPAA in soil was enhanced with the production of diphenylthioarsinic acid (DPTAA) as a main metabolite. This study aimed to isolate and characterize anaerobic soil microorganisms responsible for the metabolism of DPAA. First, we obtained four microbial consortia capable of transforming DPAA to DPTAA at a high transformation rate of more than 80% after 4 weeks of incubation. Sequencing for the bacterial 16S rRNA gene clone libraries constructed from the consortia revealed that all the positive consortia contained Desulfotomaculum acetoxidans species. In contrast, the absence of dissimilatory sulfite reductase gene (dsrAB) which is unique to sulfate-reducing bacteria was confirmed in the negative consortia showing no DPAA reduction. Finally, strain DEA14 showing transformation of DPAA to DPTAA was isolated from one of the positive consortia. The isolate was assigned to D. acetoxidans based on the partial 16S rDNA sequence analysis. Thionation of DPAA was also carried out in a pure culture of a known sulfate-reducing bacterial strain, Desulfovibrio aerotolerans JCM 12613(T). These facts indicate that sulfate-reducing bacteria are microorganisms responsible for the transformation of DPAA to DPTAA under anaerobic conditions.

New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanase, Toshihiko; Takayanagi, Ryoichi; Oba, Koichi

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for themore » mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients` genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene. 31 refs., 4 figs., 2 tabs.« less

Sequence of a cDNA encoding pancreatic preprosomatostatin-22.

PubMed Central

Magazin, M; Minth, C D; Funckes, C L; Deschenes, R; Tavianini, M A; Dixon, J E

1982-01-01

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels. Images PMID:6127673

Characterization of Bacillus megaterium, Bacillus pumilus, and Paenibacillus polymyxa isolated from a Pinot noir wine from Western Washington State.

PubMed

von Cosmos, Nicolas H; Watson, Bruce A; Fellman, J K; Mattinson, D S; Edwards, Charles G

2017-10-01

This report provides the first confirmed evidence of Bacillus-like bacteria present in a wine from Washington State. These bacteria were isolated from a 2013 Pinot noir wine whose aroma was sensorially described as being 'dirty' or 'pond scum.' Based on physiological traits and genetic sequencing, three bacterial isolates were identified as Bacillus megaterium (strain NHO-1), Bacillus pumilus (strain NHO-2), and Paenibacillus polymyxa (strain NHO-3). These bacteria grew in synthetic media of low pH (pH 3.5) while some survived ethanol concentrations up to 15% v/v. However, none tolerated molecular SO 2 concentrations ≥0.4 mg/l. Growth of strains NHO-1 and NHO-3 in a Merlot grape juice resulted in increases of titratable and volatile acidities while decreases in titratable acidity were noted for NHO-2. Copyright © 2017. Published by Elsevier Ltd.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

PubMed

Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

1996-07-15

Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

Identification and characterization of Theileria ovis surface protein (ToSp) resembled TaSp in Theileria annulata.

PubMed

Shayan, P; Jafari, S; Fattahi, R; Ebrahimzade, E; Amininia, N; Changizi, E

2016-05-01

Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions which caused high economic loses in the livestock industry. Theileria annulata surface protein (TaSp) was used previously as a tool for serological analysis in livestock. Since the amino acid sequences of TaSp is, at least, in part very conserved in T. annulata, Theileria lestoquardi and Theileria china I and II, it is very important to determine the amino acid sequence of this protein in Theileria ovis as well, to avoid false interpretation of serological data based on this protein in small animal. In the present study, the nucleotide sequence and amino acid sequence of T. ovis surface protein (ToSp) were determined. The comparison of the nucleotide sequence of ToSp showed 96, 96, 99, and 86 % homology to the corresponding nucleotide sequence of TaSp genes by T. annulata, T. China I, T. China II and T. lestoquardi, previously registered in GenBank under accession nos. AJ316260.1, AY274329.1, DQ120058.1, and EF092924.1 respectively. The amino acid sequence analysis showed 95, 81, 98 and 70 % homology to the corresponding amino acid sequence of T. annulata, T chinaI, T china II and T. lestoquardi, registered in GenBank under accession nos. CAC87478.1, AAP36993.1, AAZ30365.1 and AAP36999.11, respectively. Interestingly, in contrast to the C terminus, a significant difference in amino acid sequence in the N teminus of the ToSp protein could be determined compared to the other known corresponding TaSp sequences, which make this region attractive for designing of a suitable tool for serological diagnosis.

Brain cDNA clone for human cholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

McTiernan, C.; Adkins, S.; Chatonnet, A.

1987-10-01

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum.more » The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase.« less

Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

PubMed Central

Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

2014-01-01

ABSTRACT Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3′ consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCE Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. PMID:24599996

Virulence-associated genome mutations of murine rotavirus identified by alternating serial passages in mice and cell cultures.

PubMed

Tsugawa, Takeshi; Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

2014-05-01

Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3' consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.

Cloning and expression of cDNA coding for bouganin.

PubMed

den Hartog, Marcel T; Lubelli, Chiara; Boon, Louis; Heerkens, Sijmie; Ortiz Buijsse, Antonio P; de Boer, Mark; Stirpe, Fiorenzo

2002-03-01

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.

Method for altering antibody light chain interactions

DOEpatents

Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

2002-01-01

A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones✰

PubMed Central

Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Venermo, Maria S Söderlund; Young, Neal S.; Brown, Kevin E.

2008-01-01

Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus. PMID:18252260

VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones.

PubMed

Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Söderlund-Venermo, Maria; Young, Neal S; Brown, Kevin E

2008-05-10

Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

Molecular Microbial Analysis of Lactobacillus Strains Isolated from the Gut of Calves for Potential Probiotic Use

PubMed Central

Soto, Lorena P.; Frizzo, Laureano S.; Bertozzi, Ezequiel; Avataneo, Elizabeth; Sequeira, Gabriel J.; Rosmini, Marcelo R.

2010-01-01

The intestinal microbiota has an influence on the growth and health status of the hosts. This is of particular interest in animals reared using intensive farming practices. Hence, it is necessary to know more about complexity of the beneficial intestinal microbiota. The use of molecular methods has revolutionized microbial identification by improving its quality and effectiveness. The specific aim of the study was to analyze predominant species of Lactobacillus in intestinal microbial ecosystem of young calves. Forty-two lactic acid bacteria (LAB) isolated from intestinal tract of young calves were characterized by: Amplified Ribosomal DNA Restriction Analysis (ARDRA), by using Hae III, Msp I, and Hinf I restriction enzymes, and 16S rDNA gene sequencing. ARDRA screening revealed nine unique patterns among 42 isolates, with the same pattern for 29 of the isolates. Gene fragments of 16S rDNA of 19 strains representing different patterns were sequenced to confirm the identification of these species. These results confirmed that ARDRA is a good tool for identification and discrimination of bacterial species isolated from complex ecosystem and between closely related groups. This paper provides information about the LAB species predominant in intestinal tract of young calves that could provide beneficial effects when administered as probiotic. PMID:20445780

A survey of wild marine fish identifies a potential origin of an outbreak of viral haemorrhagic septicaemia in wrasse, Labridae, used as cleaner fish on marine Atlantic salmon, Salmo salar L., farms.

PubMed

Wallace, I S; Donald, K; Munro, L A; Murray, W; Pert, C C; Stagg, H; Hall, M; Bain, N

2015-06-01

Viral haemorrhagic septicaemia virus (VHSV) was isolated from five species of wrasse (Labridae) used as biological controls for parasitic sea lice predominantly, Lepeophtheirus salmonis (Krøyer, 1837), on marine Atlantic salmon, Salmo salar L., farms in Shetland. As part of the epidemiological investigation, 1400 wild marine fish were caught and screened in pools of 10 for VHSV using virus isolation. Eleven pools (8%) were confirmed VHSV positive from: grey gurnard, Eutrigla gurnardus L.; Atlantic herring, Clupea harengus L.; Norway pout, Trisopterus esmarkii (Nilsson); plaice, Pleuronectes platessa L.; sprat, Sprattus sprattus L. and whiting, Merlangius merlangus L. The isolation of VHSV from grey gurnard is the first documented report in this species. Nucleic acid sequencing of the partial nucleocapsid (N) and glycoprotein (G) genes was carried out for viral characterization. Sequence analysis confirmed that all wild isolates were genotype III the same as the wrasse and there was a close genetic similarity between the isolates from wild fish and wrasse on the farms. Infection from these local wild marine fish is the most likely source of VHSV isolated from wrasse on the fish farms. © 2014 Crown Copyright. Journal of Fish Diseases © 2014 John Wiley & Sons Ltd.

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 WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. This incorporation by reference was... ST.25 (1998), Appendix 2, Tables 1 and 3, shall be listed in a given sequence as “n” or “Xaa... acids. (1) The amino acids in a protein or peptide sequence shall be listed using the three-letter...

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

... in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. This incorporation by reference was... ST.25 (1998), Appendix 2, Tables 1 and 3, shall be listed in a given sequence as “n” or “Xaa... acids. (1) The amino acids in a protein or peptide sequence shall be listed using the three-letter...

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 WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. This incorporation by reference was... ST.25 (1998), Appendix 2, Tables 1 and 3, shall be listed in a given sequence as “n” or “Xaa... acids. (1) The amino acids in a protein or peptide sequence shall be listed using the three-letter...

Use of CYP52A2A promoter to increase gene expression in yeast

DOEpatents

Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

2004-01-06

A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

Method of Identifying a Base in a Nucleic Acid

DOEpatents

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

1999-01-01

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.

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.

Role of Alicyclobacillus acidoterrestris in the development of a disinfectant taint in shelf-stable fruit juice.

PubMed

Jensen, N; Whitfield, F B

2003-01-01

This study was undertaken to identify the bacterium and metabolic products contributing to a disinfectant taint in shelf-stable fruit juice and to determine some of the growth conditions for the organism. Microbiological examination of tainted and untainted fruit juice drinks detected low numbers of acid-dependent, thermotolerant, spore-forming bacteria in the tainted juices only. The presence of omega-cyclohexyl fatty acids was confirmed in two of the isolates by cell membrane fatty acid analysis. The isolates were subsequently identified as Alicyclobacillus acidoterrestris by partial 16S rDNA sequencing. Studies on the isolates showed growth at pH 2.5-6.0 and 19.5-58 degrees C. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 2,6-dibromophenol (2,6-DBP) and 2,6-dichlorophenol (2,6-DCP) in the tainted juice. Challenge studies in a mixed fruit drink inoculated with the two isolates and the type strain of A. acidoterrestris, incubated at 44-46 degrees C for 4 d, showed the production of both metabolites, which were confirmed and quantified by GC/MS. The results show that A. acidoterrestris can produce 2,6-DBP and 2,6-DCP in shelf-stable juices. This is the first report detailing experimental methodology showing that A. acidoterrestris can produce 2,6-DCP in foods. Control of storage temperatures (to < 20 degrees C) immediately after processing may provide an effective control measure for the fruit juice industry to prevent spoilage by A. acidoterrestris.

Poly(ornithine-co-arginine-co-glycine-co-aspartic Acid): Preparation via NCA Polymerization and its Potential as a Polymeric Tumor-Penetrating Agent.

PubMed

Yu, Haiyang; Tang, Zhaohui; Zhang, Dawei; Song, Wantong; Duan, Taicheng; Gu, Jingkai; Chen, Xuesi

2015-06-01

A novel random copolypeptide of ornithine, arginine, glycine, and aspartic acid [Poly(ornithine-co-arginine-co-glycine-co-aspartic acid), Poly(O,R,G,D)] has been prepared through ring-opening polymerization of N-δ-carbobenzoxy-l-ornithine N-carboxyanhydride [Orn(Cbz)-NCA)], l-glycine N-carboxyanhydride (Gly-NCA) and β-benzyl l-aspartate N-carboxyanhydride [Asp(Bn)-NCA], following by subsequent deprotection and guanidization. The structure of Poly(O,R,G,D) was confirmed by nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). Low cytotoxicity of Poly(O,R,G,D) was confirmed from MTT assay. The Poly(O,R,G,D) contain some internal sequences of RXXR (X = O, R, G, or D) that could be proteolytically cleaved to expose the cryptic CendR element and bind to Neuropilin-1. This would lead to vascular and tissue permeabilization. Therefore trypsin-cleaved Poly(O,R,G,D) increase the vascular leakage of Evans blue from dermal microvessels at the injection site in vivo skin permeability assay. The intratumoral injection of the Poly(O,R,G,D) significantly enhanced the concentration of cisplatin-loaded nanoparticles in MCF-7 solid tumors. These results show that Poly(O,R,G,D) could increase the vascular leakage and tissue penetration of nanoparticles in a solid tumor and can be used as a potential polymeric tumor-penetrating agent. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.