[Study on the genetic difference of SEO type Hantaviruses].

PubMed

Zhang, X; Zhou, S; Wang, H; Hu, J; Guan, Z; Liu, H

2000-10-01

To understand the genetic type of Hantaviruses and the difference between them caused by rodents in Beijing and to furhter explore the source of the infectious factors. Hantavirus RNA, isolated from lungs of rodents captured in Beijing and positive with Hantavirus antigens with frozen sectioning and Immunofluorescent assay, were reverse-transcribed and amplified with PCR with Hantavirus-specific primers. Five of the PCR amplifications were discovered and sequenced with 300 bp sequence data of M segments (from 2003 - 2302nt according cDNA of seoul 8039 strain). Nucleotide sequence homology showed that they were sequences of SEO-type Hantavirus. Compared with SEO type Hantavirus, the nucleotide sequence homology of these samples was more than 94% while the homology of amonia acid sequence was more than 98%. When compared with HNT type Hantavirus, the homology of nucleotide sequence became less than 72% with the homology of amonia acid sequence less than 81%. Similar to other Hantavirus of SEO type, their nucleotide sequences and deduced amino acid sequences were highly preserved. Phylogenetic tree analysis showed that the five viruses could be divided into at least 4 branches. It was quite likely that there were at least two sub-type SEO viruses with 4 branches that were circulating in Beijing.

Cloning and sequence analysis of the invertase gene INV 1 from the yeast Pichia anomala.

PubMed

Pérez, J A; Rodríguez, J; Rodríguez, L; Ruiz, T

1996-02-01

A genomic library from the yeast Pichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant of Saccharomyces cerevisiae. The cloned gene, INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also with Kluyveromyces marxianus inulinase, a yeast beta-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5' and 3' non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification.

PubMed

Sinclair, Robert M; Ravantti, Janne J; Bamford, Dennis H

2017-04-15

Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. Copyright © 2017 Sinclair et al.

Nucleic and Amino Acid Sequences Support Structure-Based Viral Classification

PubMed Central

Sinclair, Robert M.; Ravantti, Janne J.

2017-01-01

ABSTRACT Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds. IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids. PMID:28122979

SNBRFinder: A Sequence-Based Hybrid Algorithm for Enhanced Prediction of Nucleic Acid-Binding Residues.

PubMed

Yang, Xiaoxia; Wang, Jia; Sun, Jun; Liu, Rong

2015-01-01

Protein-nucleic acid interactions are central to various fundamental biological processes. Automated methods capable of reliably identifying DNA- and RNA-binding residues in protein sequence are assuming ever-increasing importance. The majority of current algorithms rely on feature-based prediction, but their accuracy remains to be further improved. Here we propose a sequence-based hybrid algorithm SNBRFinder (Sequence-based Nucleic acid-Binding Residue Finder) by merging a feature predictor SNBRFinderF and a template predictor SNBRFinderT. SNBRFinderF was established using the support vector machine whose inputs include sequence profile and other complementary sequence descriptors, while SNBRFinderT was implemented with the sequence alignment algorithm based on profile hidden Markov models to capture the weakly homologous template of query sequence. Experimental results show that SNBRFinderF was clearly superior to the commonly used sequence profile-based predictor and SNBRFinderT can achieve comparable performance to the structure-based template methods. Leveraging the complementary relationship between these two predictors, SNBRFinder reasonably improved the performance of both DNA- and RNA-binding residue predictions. More importantly, the sequence-based hybrid prediction reached competitive performance relative to our previous structure-based counterpart. Our extensive and stringent comparisons show that SNBRFinder has obvious advantages over the existing sequence-based prediction algorithms. The value of our algorithm is highlighted by establishing an easy-to-use web server that is freely accessible at http://ibi.hzau.edu.cn/SNBRFinder.

Identification of the likely translational start of Mycobacterium tuberculosis GyrB.

PubMed

Karkare, Shantanu; Brown, Amanda C; Parish, Tanya; Maxwell, Anthony

2013-07-15

Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A₂B₂ complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus. There are discrepancies between the M. tuberculosis GyrB sequences retrieved from different databases, including sequences annotated with or without the additional 40 amino acids. This has resulted in differences in the GyrB sequence numbering that has led to the reporting of previously known fluoroquinolone-resistance mutations as novel mutations. We have expressed M. tuberculosis GyrB with and without the extra 40 amino acids in Escherichia coli and shown that both can be produced as soluble, active proteins. Supercoiling and other assays of the two proteins show no differences, suggesting that the additional 40 amino acids have no effect on the enzyme in vitro. RT-PCR analysis of M. tuberculosis mRNA shows that transcripts that could yield both the longer and shorter protein are present. However, promoter analysis showed that only the promoter elements leading to the shorter GyrB (lacking the additional 40 amino acids) had significant activity. We conclude that the most probable translational start codon for M. tuberculosis GyrB is GTG (Val) which results in translation of a protein of 674 amino acids (74 kDa).

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.

Amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui.

PubMed

Hatakeyama, T; Hatakeyama, T

1990-07-06

The complete amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui were determined. Protein HL30 was found to be acetylated at its N-terminal amino acid and shows homology to the eukaryotic ribosomal proteins YL34 from yeast and RL31 from rat. Protein HmaL5 was homologous to the protein L5 from Escherichia coli and Bacillus stearothermophilus as well as to YL16 from yeast. HmaL5 shows more similarities to its eukaryotic counterpart than to eubacterial ones.

[Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix].

PubMed

Li, Jun; Long, Deng-Kai; Zhou, Tao; Ding, Ling; Zheng, Wei; Jiang, Wei-Ke

2016-07-01

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress. Copyright© by the Chinese Pharmaceutical Association.

The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase.

PubMed Central

Haggarty, N W; Dunbar, B; Fothergill, L A

1983-01-01

The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase, comprising 239 residues, was determined. The sequence was deduced from the four cyanogen bromide fragments, and from the peptides derived from these fragments after digestion with a number of proteolytic enzymes. Comparison of this sequence with that of the yeast glycolytic enzyme, phosphoglycerate mutase, shows that these enzymes are 47% identical. Most, but not all, of the residues implicated as being important for the activity of the glycolytic mutase are conserved in the erythrocyte diphosphoglycerate mutase. PMID:6313356

Isolation and in silico analysis of a novel H+-pyrophosphatase gene orthologue from the halophytic grass Leptochloa fusca

NASA Astrophysics Data System (ADS)

Rauf, Muhammad; Saeed, Nasir A.; Habib, Imran; Ahmed, Moddassir; Shahzad, Khurram; Mansoor, Shahid; Ali, Rashid

2017-02-01

Structure prediction can provide information about function and active sites of protein which helps to design new functional proteins. H+-pyrophosphatase is transmembrane protein involved in establishing proton motive force for active transport of Na+ across membrane by Na+/H+ antiporters. A full length novel H+-pyrophosphatase gene was isolated from halophytic grass Leptochloa fusca using RT-PCR and RACE method. Full length LfVP1 gene sequence of 2292 nucleotides encodes protein of 764 amino acids. DNA and protein sequences were used for characterization using bioinformatics tools. Various important potential sites were predicted by PROSITE webserver. Primary structural analysis showed LfVP1 as stable protein and Grand average hydropathy (GRAVY) indicated that LfVP1 protein has good hydrosolubility. Secondary structure analysis showed that LfVP1 protein sequence contains significant proportion of alpha helix and random coil. Protein membrane topology suggested the presence of 14 transmembrane domains and presence of catalytic domain in TM3. Three dimensional structure from LfVP1 protein sequence also indicated the presence of 14 transmembrane domains and hydrophobicity surface model showed amino acid hydrophobicity. Ramachandran plot showed that 98% amino acid residues were predicted in the favored region.

Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.)*

PubMed Central

Liu, Chang-ying; Lü, Rui-hua; Li, Jun; Zhao, Ai-chun; Wang, Xi-ling; Diane, Umuhoza; Wang, Xiao-hong; Wang, Chuan-hong; Yu, Ya-sheng; Han, Shu-mei; Lu, Cheng; Yu, Mao-de

2014-01-01

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) are encoded by multigene families and are involved in fruit ripening by catalyzing the production of ethylene throughout the development of fruit. However, there are no reports on ACS or ACO genes in mulberry, partly because of the limited molecular research background. In this study, we have obtained five ACS gene sequences and two ACO gene sequences from Morus Genome Database. Sequence alignment and phylogenetic analysis of MaACO1 and MaACO2 showed that their amino acids are conserved compared with ACO proteins from other species. MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III, with different C-terminal sequences. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) expression analysis showed that the transcripts of MaACS genes were strongly expressed in fruit, and more weakly in other tissues. The expression of MaACO1 and MaACO2 showed different patterns in various mulberry tissues. MaACS and MaACO genes demonstrated two patterns throughout the development of mulberry fruit, and both of them were strongly up-regulated by abscisic acid (ABA) and ethephon. PMID:25001221

Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences.

PubMed

Arend, J; Warzecha, H; Stöckigt, J

2000-01-01

Plant cell suspension cultures of Rauvolfia are able to produce a high amount of arbutin by glucosylation of exogenously added hydroquinone. A four step purification procedure using anion exchange, hydrophobic interaction, hydroxyapatite-chromatography and chromatofocusing delivered in a yield of 0.5%, an approximately 390 fold enrichment of the involved glucosyltransferase. SDS-PAGE showed a M(r) for the enzyme of 52 kDa. Proteolysis of the pure enzyme with endoproteinase LysC revealed six peptide fragments with 9-23 amino acids which were sequenced. Sequence alignment of the six peptides showed high homologies to glycosyltransferases from other higher plants.

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui. Comparison of the ribosomal protein S11 family.

PubMed

Kimura, M; Kimura, J; Hatakeyama, T

1988-11-21

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45-49%) than to the eubacterial counterparts (35%).

Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST.

PubMed

Goonesekere, Nalin Cw

2009-01-01

The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP) database. We show that when incorporated into the homology search algorithms BLAST and PSI-blast, the structure-based substitution matrices enhance the efficacy of detecting remote homologs.

Sequencing proteins with transverse ionic transport in nanochannels.

PubMed

Boynton, Paul; Di Ventra, Massimiliano

2016-05-03

De novo protein sequencing is essential for understanding cellular processes that govern the function of living organisms and all sequence modifications that occur after a protein has been constructed from its corresponding DNA code. By obtaining the order of the amino acids that compose a given protein one can then determine both its secondary and tertiary structures through structure prediction, which is used to create models for protein aggregation diseases such as Alzheimer's Disease. Here, we propose a new technique for de novo protein sequencing that involves translocating a polypeptide through a synthetic nanochannel and measuring the ionic current of each amino acid through an intersecting perpendicular nanochannel. We find that the distribution of ionic currents for each of the 20 proteinogenic amino acids encoded by eukaryotic genes is statistically distinct, showing this technique's potential for de novo protein sequencing.

Microbial Diversity of Acidic Hot Spring (Kawah Hujan B) in Geothermal Field of Kamojang Area, West Java-Indonesia

PubMed Central

Aditiawati, Pingkan; Yohandini, Heni; Madayanti, Fida; Akhmaloka

2009-01-01

Microbial communities in an acidic hot spring, namely Kawah Hujan B, at Kamojang geothermal field, West Java-Indonesia was examined using culture dependent and culture independent strategies. Chemical analysis of the hot spring water showed a characteristic of acidic-sulfate geothermal activity that contained high sulfate concentrations and low pH values (pH 1.8 to 1.9). Microbial community present in the spring was characterized by 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) analysis. The majority of the sequences recovered from culture-independent method were closely related to Crenarchaeota and Proteobacteria phyla. However, detail comparison among the member of Crenarchaeota showing some sequences variation compared to that the published data especially on the hypervariable and variable regions. In addition, the sequences did not belong to certain genus. Meanwhile, the 16S Rdna sequences from culture-dependent samples revealed mostly close to Firmicute and gamma Proteobacteria. PMID:19440252

Microbial diversity of acidic hot spring (kawah hujan B) in geothermal field of kamojang area, west java-indonesia.

PubMed

Aditiawati, Pingkan; Yohandini, Heni; Madayanti, Fida; Akhmaloka

2009-01-01

Microbial communities in an acidic hot spring, namely Kawah Hujan B, at Kamojang geothermal field, West Java-Indonesia was examined using culture dependent and culture independent strategies. Chemical analysis of the hot spring water showed a characteristic of acidic-sulfate geothermal activity that contained high sulfate concentrations and low pH values (pH 1.8 to 1.9). Microbial community present in the spring was characterized by 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) analysis. The majority of the sequences recovered from culture-independent method were closely related to Crenarchaeota and Proteobacteria phyla. However, detail comparison among the member of Crenarchaeota showing some sequences variation compared to that the published data especially on the hypervariable and variable regions. In addition, the sequences did not belong to certain genus. Meanwhile, the 16S Rdna sequences from culture-dependent samples revealed mostly close to Firmicute and gamma Proteobacteria.

TaALMT1 promoter sequence compositions, acid tolerance, and Al tolerance in wheat cultivars and landraces from Sichuan in China.

PubMed

Han, C; Dai, S F; Liu, D C; Pu, Z J; Wei, Y M; Zheng, Y L; Wen, D J; Zhao, L; Yan, Z H

2013-11-18

Previous genetic studies on wheat from various sources have indicated that aluminum (Al) tolerance may have originated independently in USA, Brazil, and China. Here, TaALMT1 promoter sequences of 92 landraces and cultivars from Sichuan, China, were sequenced. Five promoter types (I', II, III, IV, and V) were observed in 39 cultivars, and only three promoter types (I, II, and III) were observed in 53 landraces. Among the wheat collections worldwide, only the Chinese Spring (CS) landrace native to Sichuan, China, carried the TaALMT1 promoter type III. Besides CS, two other Sichuan-bred landraces and six cultivars with TaALMT1 promoter type III were identified in this study. In the phylogenetic tree constructed based on the TaALMT1 promoter sequences, type III formed a separate branch, which was supported by a high bootstrap value. It is likely that TaALMT1 promoter type III originated from Sichuan-bred wheat landraces of China. In addition, the landraces with promoter type I showed the lowest Al tolerance among all landraces and cultivars. Furthermore, the cultivars with promoter type IV showed better Al tolerance than landraces with promoter type II. A comparison of acid tolerance and Al tolerance between cultivars and landraces showed that the landraces had better acid tolerance than the cultivars, whereas the cultivars showed better Al tolerance than the landraces. Moreover, significant difference in Al tolerance was also observed between the cultivars raised by the National Ministry of Agriculture and by Sichuan Province. Among the landraces from different regions, those from the East showed better acid tolerance and Al tolerance than those from the South and West of Sichuan. Additional Al-tolerant and acid-tolerant wheat lines were also identified.

Complementary DNA cloning and molecular evolution of opine dehydrogenases in some marine invertebrates.

PubMed

Kimura, Tomohiro; Nakano, Toshiki; Yamaguchi, Toshiyasu; Sato, Minoru; Ogawa, Tomohisa; Muramoto, Koji; Yokoyama, Takehiko; Kan-No, Nobuhiro; Nagahisa, Eizou; Janssen, Frank; Grieshaber, Manfred K

2004-01-01

The complete complementary DNA sequences of genes presumably coding for opine dehydrogenases from Arabella iricolor (sandworm), Haliotis discus hannai (abalone), and Patinopecten yessoensis (scallop) were determined, and partial cDNA sequences were derived for Meretrix lusoria (Japanese hard clam) and Spisula sachalinensis (Sakhalin surf clam). The primers ODH-9F and ODH-11R proved useful for amplifying the sequences for opine dehydrogenases from the 4 mollusk species investigated in this study. The sequence of the sandworm was obtained using primers constructed from the amino acid sequence of tauropine dehydrogenase, the main opine dehydrogenase in A. iricolor. The complete cDNA sequence of A. iricolor, H. discus hannai, and P. yessoensis encode 397, 400, and 405 amino acids, respectively. All sequences were aligned and compared with published databank sequences of Loligo opalescens, Loligo vulgaris (squid), Sepia officinalis (cuttlefish), and Pecten maximus (scallop). As expected, a high level of homology was observed for the cDNA from closely related species, such as for cephalopods or scallops, whereas cDNA from the other species showed lower-level homologies. A similar trend was observed when the deduced amino acid sequences were compared. Furthermore, alignment of these sequences revealed some structural motifs that are possibly related to the binding sites of the substrates. The phylogenetic trees derived from the nucleotide and amino acid sequences were consistent with the classification of species resulting from classical taxonomic analyses.

The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli.

PubMed

Martin, Audrey; Daniel, Jaiyanth

2018-02-05

Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function. Copyright © 2018 Elsevier Inc. All rights reserved.

Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

PubMed Central

Chan, Jasper Fuk-Woo; Choi, Garnet Kwan-Yue; Tsang, Alan Ka-Lun; Tee, Kah-Meng; Lam, Ho-Yin; Yip, Cyril Chik-Yan; To, Kelvin Kai-Wang; Cheng, Vincent Chi-Chung; Yeung, Man-Lung; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Chan, Kwok-Hung; Tang, Bone Siu-Fai

2015-01-01

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses. PMID:26019210

Cloning and functional characterization of SAD genes in potato.

PubMed

Li, Fei; Bian, Chun Song; Xu, Jian Fei; Pang, Wan Fu; Liu, Jie; Duan, Shao Guang; Lei, Zun-Guo; Jiwan, Palta; Jin, Li-Ping

2015-01-01

Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

Rhodotorula svalbardensis sp. nov., a novel yeast species isolated from cryoconite holes of Ny-Ålesund, Arctic.

PubMed

Singh, Purnima; Singh, Shiv M; Tsuji, Masaharu; Prasad, Gandham S; Hoshino, Tamotsu

2014-02-01

A psychrophilic yeast species was isolated from glacier cryoconite holes of Svalbard. Nucleotide sequences of the strains were studied using D1/D2 domain, ITS region and partial sequences of mitochondrial cytochrome b gene. The strains belonged to a clade of psychrophilic yeasts, but showed marked differences from related species in the D1/D2 domain and biochemical characters. Effects of temperature, salt and media on growth of the cultures were also studied. Screening of the cultures for amylase, cellulase, protease, lipase, urease and catalase activities was carried out. The strains expressed high amylase and lipase activities. Freeze tolerance ability of the isolates indicated the formation of unique hexagonal ice crystal structures due to presence of 'antifreeze proteins' (AFPs). FAME analysis of cultures showed a unique trend of increase in unsaturated fatty acids with decrease in temperature. The major fatty acids recorded were oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, myristic acid and pentadecanoic acid. Based on sequence data and, physiological and morphological properties of the strains, we propose a novel species, Rhodotorula svalbardensis and designate strains MLB-I (CCP-II) and CRY-YB-1 (CBS 12863, JCM 19699, JCM 19700, MTCC 10952) as its type strains (Etymology: sval.bar.den'sis. N.L. fem. adj. svalbardensis pertaining to Svalbard). Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Purification, characterization, and cDNA cloning of a novel acidic endoglycoceramidase from the jellyfish, Cyanea nozakii.

PubMed

Horibata, Y; Okino, N; Ichinose, S; Omori, A; Ito, M

2000-10-06

Endoglycoceramidase (EC ) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here the purification, characterization, and cDNA cloning of a novel endoglycoceramidase from the jellyfish, Cyanea nozakii. The purified enzyme showed a single protein band estimated to be 51 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed a pH optimum of 3.0 and was activated by Triton X-100 and Lubrol PX but not by sodium taurodeoxycholate. This enzyme preferentially hydrolyzed gangliosides, especially GT1b and GQ1b, whereas neutral glycosphingolipids were somewhat resistant to hydrolysis by the enzyme. A full-length cDNA encoding the enzyme was cloned by 5'- and 3'-rapid amplification of cDNA ends using a partial amino acid sequence of the purified enzyme. The open reading frame of 1509 nucleotides encoded a polypeptide of 503 amino acids including a signal sequence of 25 residues and six potential N-glycosylation sites. Interestingly, the Asn-Glu-Pro sequence, which is the putative active site of Rhodococcus endoglycoceramidase, was conserved in the deduced amino acid sequences. This is the first report of the cloning of an endoglycoceramidase from a eukaryote.

[Molecular cloning and characterization of an acetylcholinesterase gene Dd-ace-2 from sweet potato stem nematode Ditylenchus destructor].

PubMed

Ding, Zhong; Peng, Deliang; Huang, Wenkun; He, Wenting; Gao, Bida

2008-02-01

A cDNA, named Dd-ace-2, encoding an acetylcholinesterase (AChE, EC3.1.1.7), was isolated from sweet-potato-stem nematode, Ditylenchus destructor. The nucleotide and amino acid sequences among different nematode species were compared and analyzed with DNAMAN5.0, MEGA3.0 softwares. The results showed that the complete nucleotide sequence of Dd-ace-2 gene of Ditylenchus destructor contains 2425 base pairs from which deduced 734 amino acids (GenBank accession No. EF583058). The homology rates of amino acid sequences of Dd-ace-2 gene between Ditylenchus destructor and Meloidogyne incognita, Caenorhabditis elegans, Dictyocaulus viviparous were 48.0%, 42.7%, 42.1% respectively. The mature acetylcholinesterase sequences of Ditylenchus destructor may encode by the first 701 residues of deduced 734 amino acids.The conserved motifs involved in the catalytic triad, the choline binding site and 10 aromatic residues lining the catalytic gorge were present in the Dd-ace-2 deduced protein. Phylogenetic analysis based on AChEs of other nematodes and species showed that the deduced AChE formed the same cluster with ACE-2s.

The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain

PubMed Central

Husain, S. S.; Lowe, G.

1970-01-01

Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-14C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin. PMID:5420046

Sequence-dependent DNA deformability studied using molecular dynamics simulations.

PubMed

Fujii, Satoshi; Kono, Hidetoshi; Takenaka, Shigeori; Go, Nobuhiro; Sarai, Akinori

2007-01-01

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein-DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein-DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.

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.

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

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

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

Rattlesnake Neurotoxin Structure, Mechanism of Action, Immunology and Molecular Biology

DTIC Science & Technology

1992-09-10

and Kaiser, 1990). Sequencing of the three peptides present in the acidic subunit, two of which are blocked by pyroglutamate , represents a significant...deblock with pyroglutamate aminopeptidase were unsuccessful. The B-chain contained 35 amino acids and showed 91% amino acid identity witn the...similarities of all rattlesnake neurotoxins, showed that the acidic subunit plays more than a chaperone role for the basic subunit and is clearly

Comparative analysis of the prion protein gene sequences in African lion.

PubMed

Wu, Chang-De; Pang, Wan-Yong; Zhao, De-Ming

2006-10-01

The prion protein gene of African lion (Panthera Leo) was first cloned and polymorphisms screened. The results suggest that the prion protein gene of eight African lions is highly homogenous. The amino acid sequences of the prion protein (PrP) of all samples tested were identical. Four single nucleotide polymorphisms (C42T, C81A, C420T, T600C) in the prion protein gene (Prnp) of African lion were found, but no amino acid substitutions. Sequence analysis showed that the higher homology is observed to felis catus AF003087 (96.7%) and to sheep number M31313.1 (96.2%) Genbank accessed. With respect to all the mammalian prion protein sequences compared, the African lion prion protein sequence has three amino acid substitutions. The homology might in turn affect the potential intermolecular interactions critical for cross species transmission of prion disease.

Canine Lat1: molecular structure, distribution and its expression in cancer samples.

PubMed

Ochiai, Hideharu; Morishita, Taiki; Onda, Ken; Sugiyama, Hiroki; Maruo, Takuya

2012-07-01

A full-length cDNA sequence of canine L-type amino acid transporter 1 (Lat1) was determined from a canine brain. The sequence was 1828 bp long and was predicted to encode 485 amino acid polypeptides. The deduced amino acid sequence of canine Lat1 showed 93.2% and 91.1% similarities to those of humans and rats, respectively. Northern blot analysis detected Lat1 expression in the cerebellum at 4 kb, and Western blot analysis showed a single band at 40 kDa. RT-PCR analysis revealed a distinct expression of Lat1 in the pancreas and testis in addition to the cerebrum and cerebellum. Notably, Lat1 expression was observed in the tissues of thyroid cancer, melanoma and hemangiopericytoma. Although the cancer samples examined were not enough, Lat1 may serve as a useful biomarker of cancer cells in veterinary clinic.

Sequence signatures of allosteric proteins towards rational design.

PubMed

Namboodiri, Saritha; Verma, Chandra; Dhar, Pawan K; Giuliani, Alessandro; Nair, Achuthsankar S

2010-12-01

Allostery is the phenomenon of changes in the structure and activity of proteins that appear as a consequence of ligand binding at sites other than the active site. Studying mechanistic basis of allostery leading to protein design with predetermined functional endpoints is an important unmet need of synthetic biology. Here, we screened the amino acid sequence landscape in search of sequence-signatures of allostery using Recurrence Quantitative Analysis (RQA) method. A characteristic vector, comprised of 10 features extracted from RQA was defined for amino acid sequences. Using Principal Component Analysis, four factors were found to be important determinants of allosteric behavior. Our sequence-based predictor method shows 82.6% accuracy, 85.7% sensitivity and 77.9% specificity with the current dataset. Further, we show that Laminarity-Mean-hydrophobicity representing repeated hydrophobic patches is the most crucial indicator of allostery. To our best knowledge this is the first report that describes sequence determinants of allostery based on hydrophobicity. As an outcome of these findings, we plan to explore possibility of inducing allostery in proteins.

Codes in the codons: construction of a codon/amino acid periodic table and a study of the nature of specific nucleic acid-protein interactions.

PubMed

Benyo, B; Biro, J C; Benyo, Z

2004-01-01

The theory of "codon-amino acid coevolution" was first proposed by Woese in 1967. It suggests that there is a stereochemical matching - that is, affinity - between amino acids and certain of the base triplet sequences that code for those amino acids. We have constructed a common periodic table of codons and amino acids, where the nucleic acid table showed perfect axial symmetry for codons and the corresponding amino acid table also displayed periodicity regarding the biochemical properties (charge and hydrophobicity) of the 20 amino acids and the position of the stop signals. The table indicates that the middle (2/sup nd/) amino acid in the codon has a prominent role in determining some of the structural features of the amino acids. The possibility that physical contact between codons and amino acids might exist was tested on restriction enzymes. Many recognition site-like sequences were found in the coding sequences of these enzymes and as many as 73 examples of codon-amino acid co-location were observed in the 7 known 3D structures (December 2003) of endonuclease-nucleic acid complexes. These results indicate that the smallest possible units of specific nucleic acid-protein interaction are indeed the stereochemically compatible codons and amino acids.

Cloning and sequence analysis of Hemonchus contortus HC58cDNA.

PubMed

Muleke, Charles I; Ruofeng, Yan; Lixin, Xu; Xinwen, Bo; Xiangrui, Li

2007-06-01

The complete coding sequence of Hemonchus contortus HC58cDNA was generated by rapid amplification of cDNA ends and polymerase chain reaction using primers based on the 5' and 3' ends of the parasite mRNA, accession no. AF305964. The HC58cDNA gene was 851 bp long, with open reading frame of 717 bp, precursors to 239 amino acids coding for approximately 27 kDa protein. Analysis of amino acid sequence revealed conserved residues of cysteine, histidine, asparagine, occluding loop pattern, hemoglobinase motif and glutamine of the oxyanion hole characteristic of cathepsin B like proteases (CBL). Comparison of the predicted amino acid sequences showed the protein shared 33.5-58.7% identity to cathepsin B homologues in the papain clan CA family (family C1). Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the CBL, suggesting that HC58cDNA was a member of the papain family.

Functional identification and regulatory analysis of Δ6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10.

PubMed

Jiang, Xianzhang; Liu, Hongjiao; Niu, Yongchao; Qi, Feng; Zhang, Mingliang; Huang, Jianzhong

2017-03-01

To enlarge the diversity of the desaturases associated with PUFA biosynthesis and to better understand the transcriptional regulation of desaturases, a Δ 6 -desaturase gene (Md6) from Mucor sp. and its 5'-upstream sequence was functionally identified in Saccharomyces cerevisiae. Expression of the Δ 6 -fatty acid desaturase (Md6) in S. cerevisiae showed that Md6 could convert linolenic acid to γ-linolenic acid. Computational analysis of the promoter of Md6 suggested it contains several eukaryotic fundamental transcription regulatory elements. In vivo functional analysis of the promoter showed the 5'-upstream sequence of Md6 could initiate expression of GFP and Md6 itself in S. cerevisiae. A series deletion analysis of the promoter suggested that sequence between -919 to -784 bp (relative to start site) named as eMd6 is the key factor for high activity of Δ 6 -desaturase. The activity of Δ 6 -desaturase was increased by 2.8-fold and 2.5-fold when the eMd6 sequence was placed upstream of -434 with forward or reverse orientations respectively. To our best knowledge, the native promoter of Md6 from Mucor is the strongest promoter for Δ 6 -desaturase reported so far and the sequence between -919 to -784 bp is an enhancer for Δ 6 -desaturase activity.

Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library.

PubMed

Kim, B S; Kim, S Y; Park, J; Park, W; Hwang, K Y; Yoon, Y J; Oh, W K; Kim, B Y; Ahn, J S

2007-05-01

Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome. We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, syk181, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids. Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of syk181 shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds. SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.

Molecular cloning and sequence analysis of full-length growth hormone cDNAs from six important economic fishes.

PubMed

Zhang, Jing-Nan; Song, Ping; Hu, Jia-Rui; Mo, Sai-Jun; Peng, Mao-Yu; Zhou, Wei; Zou, Ji-Xing; Hu, Yin-Chang

2005-01-01

In this study,the full-length cDNAs of GH (Growth Hormone) gene was isolated from six important economic fishes, Siniperca kneri, Epinephelus coioides, Monopterus albus, Silurus asotus, Misgurnus anguillicaudatus and Carassius auratus gibelio Bloch. It is the first time to clone these GH sequences except E. coioides GH. The lengths of the above cDNAs are as follows: 953 bp, 1 023 bp, 825 bp, 1 082 bp, 1 154 bp and 1 180 bp. Each sequence includes an ORF of about 600 bp which encodes a protein of about 200 amino acid: S. kneri, E. coioides and M. albus GHs of 204 amino acid, S. asotus GH of 200 amino acid, M. anguillicaudatus and C. auratus gibelio GHs of 210 amino acid. Then detailed sequence analysis of the six GHs with many other fish sequences was performed. The six sequences all showed high homology to other sequences, especially to sequences within the same order, and many conserved residues were identified, most localized in five domains. The phylogenetic trees (MP and NJ) of many fish GH ORF sequences (including the new six) with Amia calva as outgroup were generally resolved and largely congruent with the morphology-based tree though some incongruities were observed, suggesting GH ORF should be paid more attention to in teleostean phylogeny.

Characterization and mapping of cDNA encoding aspartate aminotransferase in rice, Oryza sativa L.

PubMed

Song, J; Yamamoto, K; Shomura, A; Yano, M; Minobe, Y; Sasaki, T

1996-10-31

Fifteen cDNA clones, putatively identified as encoding aspartate aminotransferase (AST, EC 2.6.1.1.), were isolated and partially sequenced. Together with six previously isolated clones putatively identified to encode ASTs (Sasaki, et al. 1994, Plant Journal 6, 615-624), their sequences were characterized and classified into 4 cDNA species. Two of the isolated clones, C60213 and C2079, were full-length cDNAs, and their complete nucleotide sequences were determined. C60213 was 1612 bp long and its deduced amino acid sequence showed 88% homology with that of Panicum miliaceum L. mitochondrial AST. The C60213-encoded protein had an N-terminal amino acid sequence that was characteristic of a mitochondrial transit peptide. On the other hand, C2079 was 1546 bp long and had 91% amino acid sequence homology with P. miliaceum L. cytosolic AST but lacked in the transit peptide sequence. The homologies of nucleotide sequences and deduced amino acid sequences of C2079 and C60213 were 54% and 52%, respectively. C2079 and C60213 were mapped on chromosomes 1 and 6, respectively, by restriction fragment length polymorphism linkage analysis. Northern blot analysis using C2079 as a probe revealed much higher transcript levels in callus and root than in green and etiolated shoots, suggesting tissue-specific variations of AST gene expression.

Detection and characterization of hepatitis A virus circulating in Egypt.

PubMed

Hamza, Hazem; Abd-Elshafy, Dina Nadeem; Fayed, Sayed A; Bahgat, Mahmoud Mohamed; El-Esnawy, Nagwa Abass; Abdel-Mobdy, Emam

2017-07-01

Hepatitis A virus (HAV) still poses a considerable problem worldwide. In the current study, hepatitis A virus was recovered from wastewater samples collected from three wastewater treatment plants over one year. Using RT-PCR, HAV was detected in 43 out of 68 samples (63.2%) representing both inlet and outlet. Eleven positive samples were subjected to sequencing targeting the VP1-2A junction region. Phylogenetic analysis revealed that all samples belonged to subgenotype IB with few substitutions at the amino acid level. The complete sequence of one isolate (HAV/Egy/BI-11/2015) showed that the similarity at the amino acid level was not reflected at the nucleotide level. However, the deduced amino acid sequence derived from the complete nucleotide sequence showed distinct substitutions in the 2B, 2C, and 3A regions. Recombination analysis revealed a recombination event between X75215 (subgenotype IA) and AF268396 (subgenotype IB) involving a portion of the 2B nonstructural protein coding region (nucleotides 3757-3868) assuming the herein characterized sequence an actual recombinant. Despite the role of recombination in picornaviruses evolution, its involvement in HAV evolution has rarely been reported, and this may be due to the limited available complete HAV sequences. To our knowledge, this represents the first characterized complete sequence of an Egyptian isolate and the described recombination event provides an important update on the circulating HAV strains in Egypt.

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.

Molecular cloning of the pheromone biosynthesis-activating neuropeptide in Helicoverpa zea.

PubMed Central

Davis, M T; Vakharia, V N; Henry, J; Kempe, T G; Raina, A K

1992-01-01

Pheromone biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone biosynthesis in female Helicoverpa (Heliothis) zea. Two oligonucleotide probes representing two overlapping amino acid regions of PBAN were used to screen 2.5 x 10(5) recombinant plaques, and a positive recombinant clone was isolated. Sequence analysis of the isolated clone showed that the PBAN gene is interrupted after the codon encoding amino acid 14 by a 0.63-kilobase (kb) intron. Preceding the PBAN amino acid sequence is a 10-amino acid sequence containing a pentapeptide Phe-Thr-Pro-Arg-Leu, which is followed by a Gly-Arg-Arg processing site. Immediately after the PBAN amino acid sequence is a Gly-Arg processing site and a short stretch of 10 amino acids. This 10-amino acid sequence contains a repeat of the PBAN C-terminal pentapeptide Phe-Ser-Pro-Arg-Leu and is terminated by another Gly-Arg processing site. It is suggested that the PBAN gene in H. zea might carry, besides PBAN, a 7- and an 8-residue amidated peptide, which share with PBAN the core C-terminal pentapeptide Phe-(Ser or Thr)-Pro-Arg-Leu-NH2. The C-terminal pentapeptide sequence of PBAN represents the minimum sequence required for pheromonotropic activity in H. zea and also bears a high degree of homology to the pyrokinin family of insect peptides with myotropic activity. It is possible that the putative heptapeptide and octapeptide might be new members of the pyrokinin family, with pheromonotropic and/or myotropic activities. Thus, the PBAN gene products, besides affecting sexual behavior, might have broad influence on many biological processes in H. zea. Images PMID:1729680

Structural study of Bombyx mori silk fibroin during processing for regeneration

NASA Astrophysics Data System (ADS)

Ha, Sung-Won

Bombyx mori silk fibroin has excellent mechanical properties combined with flexibility, tissue compatibility, and high oxygen permeability in the wet condition. This important material should be dissolved and regenerated to be utilized as useful forms such as gel, film, fiber, powder, or non-woven. However, it has long been a problem that the regenerated fibroin materials show poor mechanical properties and brittleness. These problems were technically solved by improving a fiber processing method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the original silk fibers. This improved technique for the fiber processing of Bombyx mori silk fibroin may be used as a model system for other semi-crystalline fiber forming proteins, becoming available through biotechnology. The physical and chemical properties of the regenerated fibers were characterized by SinTechRTM tensile testing, X-ray diffraction, solid state 13C NMR spectroscopy, and SEM. Unlike synthetic polymers, the molecular weight distribution of Bombyx mori silk fibroin is mono-disperse because silk fibroin is synthesized from DNA template. Genetic studies have revealed the entire amino acid sequence of Bombyx mori silk fibroin. It is known that the crystalline silk II structure is composed of hexa-amino acid sequences, GAGAGS. However, in the amino acid sequence of Bombyx mori silk fibroin heavy chain, there are present 11 chemically irregular but evolutionarily conserved sequences with about 31 amino acid residues (irregular GT˜GT sequences). The structure and role of these irregular sequences have remained unknown. One of the most frequently appearing irregular sequences was synthesized by a peptide synthesizer. The three-dimensional structure of this irregular silk peptide was studied by the high resolution two-dimensional NMR technique. The three-dimensional structure of this peptide shows that it makes a turn or loop structure (distorted O shape), which means the proceeding backbone direction is changed 180° by this sequence. This may facilitate the beta-sheet formation of the crystal forming building blocks, GAGAGS/GY˜GY sequences, in fibroin heavy chain. It may also facilitate the solubilization of the fibroin heavy chain within the silk gland.

Aggregation of peptides in the tube model with correlated sidechain orientations

NASA Astrophysics Data System (ADS)

Hung, Nguyen Ba; Hoang, Trinh Xuan

2015-06-01

The ability of proteins and peptides to aggregate and form toxic amyloid fibrils is associated with a range of diseases including BSE (or mad cow), Alzheimer's and Parkinson's Diseases. In this study, we investigate the the role of amino acid sequence in the aggregation propensity by using a modified tube model with a new procedure for hydrophobic interaction. In this model, the amino acid sidechains are not considered explicitly, but their orientations are taken into account in the formation of hydrophobic contact. Extensive Monte Carlo simulations for systems of short peptides are carried out with the use of parallel tempering technique. Our results show that the propensity to form and the structures of the aggregates strongly depend on the amino acid sequence and the number of peptides. Some sequences may not aggregate at all at a presumable physiological temperature while other can easily form fibril-like, β-sheet struture. Our study provides an insight into the principles of how the formation of amyloid can be governed by amino acid sequence.

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

NASA Technical Reports Server (NTRS)

Funderburgh, J. L.; Funderburgh, M. L.; Brown, S. J.; Vergnes, J. P.; Hassell, J. R.; Mann, M. M.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1993-01-01

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

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

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

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

1989-11-01

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

Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

NASA Astrophysics Data System (ADS)

Zhao, Chunling; Ju, Jiyu

2015-06-01

The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

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.

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)

Molecular cloning and sequencing of the cDNA and gene for a novel elastinolytic metalloproteinase from Aspergillus fumigatus and its expression in Escherichia coli.

PubMed Central

Sirakova, T D; Markaryan, A; Kolattukudy, P E

1994-01-01

An extracellular elastinolytic metalloproteinase, purified from Aspergillus fumigatus isolated from an aspergillosis and patient/and an internal peptide derived from it were subjected to N-terminal sequencing. Oligonucleotide primers based on these sequences were used to PCR amplify a segment of the metalloproteinase cDNA, which was used as a probe to isolate the cDNA and gene for this enzyme. The gene sequence matched exactly with the cDNA sequence except for the four introns that interrupted the open reading frame. According to the deduced amino acid sequence, the metalloproteinase has a signal sequence and 227 additional amino acids preceding the sequence for the mature protein of 389 amino acids with a calculated molecular mass of 42 kDa, which is close to the size of the purified mature fungal proteinase. This sequence contains segments that matched both the N terminus of the mature protein and the internal peptide. A. fumigatus metalloproteinase contains some of the conserved zinc-binding and active-site motifs characteristic of metalloproteinases but shows no overall homology with known metalloproteinases. The cDNA of the mature protein when introduced into Escherichia coli directed the expression of a protein with a size, N-terminal sequence, and immunological cross-reactivity identical to those of the native fungal enzyme. Although the enzyme in the inclusion bodies could not be renatured, expression at 30 degrees C yielded soluble enzyme that showed chromatographic behavior identical to that of the native fungal enzyme and catalyzed hydrolysis of elastin. The metalloproteinase gene described here was not found in Aspergillus flavus. Images PMID:7927676

Characterization of durum wheat high molecular weight glutenin subunits Bx20 and By20 sequences by a molecular and proteomic approach.

PubMed

Santagati, Vito Davide; Sestili, Francesco; Lafiandra, Domenico; D'Ovidio, Renato; Rogniaux, Helene; Masci, Stefania

2016-07-01

Wheat high molecular weight glutenin subunit variation is important because of its great influence on glutenin polymer structure, that is related to dough technological properties. Among the different subunits, the pair Bx20 and By20 is known to have a negative effect on quality, but the reasons are not clear: Bx20 has two cysteines, which theoretically make this subunit a chain extender of the glutenin polymer, just like the other Bx subunits, showing four cysteines, two of which should be involved in intra-molecular disulfide bonds. By20 has never been characterized so far at molecular level. Here we report the nucleotide sequences of Bx20 and By20 genes isolated from the durum wheat cultivar 'Lira 45' and the validation of the corresponding deduced amino acid sequences by using MALDI-TOF and LC-MS/MS. Four nucleotide differences were identified in the Bx20 gene with respect to the deduced sequence present in NCBI, causing two amino acid substitutions. For the By20 subunit, nucleotide and amino acid sequences revealed a great similarity to By15, both at gene and protein levels, showing five nucleotide changes generating two amino acid differences. No evidence of post-translational modifications has been found. Hypotheses are formulated in regard to relationships with technological quality. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of a Non-Conservative Sequence on the Properties of β-glucuronidase from Aspergillus terreus Li-20

PubMed Central

Liu, Yanli; Huangfu, Jie; Qi, Feng; Kaleem, Imdad; E, Wenwen; Li, Chun

2012-01-01

We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1–592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co2+, Ca2+ and Ni2+ showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s−1), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme. PMID:22347419

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%.

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

PubMed

He, Ying-Ying; Lee, Wei-Hui; Zhang, Yun

2004-09-01

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.

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.

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.

Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India.

PubMed

Pudupakam, Raghavendra Sumanth; Raghunath, Shobana; Pudupakam, Meghanath; Daggupati, Sreenivasulu

2017-03-01

Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India

PubMed Central

Pudupakam, Raghavendra Sumanth; Raghunath, Shobana; Pudupakam, Meghanath; Daggupati, Sreenivasulu

2017-01-01

Aim: Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. Materials and Methods: The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. Results: The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Conclusion: Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability. PMID:28435199

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349.

PubMed

Imabayashi, Yuki; Suzuki, Shun'ichi; Kawasaki, Hisashi; Nakamatsu, Tsuyoshi

2016-01-01

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.

[Comparative genomics and evolutionary analysis of CRISPR loci in acetic acid bacteria].

PubMed

Xia, Kai; Liang, Xin-le; Li, Yu-dong

2015-12-01

The clustered regularly interspaced short palindromic repeat (CRISPR) is a widespread adaptive immunity system that exists in most archaea and many bacteria against foreign DNA, such as phages, viruses and plasmids. In general, CRISPR system consists of direct repeat, leader, spacer and CRISPR-associated sequences. Acetic acid bacteria (AAB) play an important role in industrial fermentation of vinegar and bioelectrochemistry. To investigate the polymorphism and evolution pattern of CRISPR loci in acetic acid bacteria, bioinformatic analyses were performed on 48 species from three main genera (Acetobacter, Gluconacetobacter and Gluconobacter) with whole genome sequences available from the NCBI database. The results showed that the CRISPR system existed in 32 species of the 48 strains studied. Most of the CRISPR-Cas system in AAB belonged to type I CRISPR-Cas system (subtype E and C), but type II CRISPR-Cas system which contain cas9 gene was only found in the genus Acetobacter and Gluconacetobacter. The repeat sequences of some CRISPR were highly conserved among species from different genera, and the leader sequences of some CRISPR possessed conservative motif, which was associated with regulated promoters. Moreover, phylogenetic analysis of cas1 demonstrated that they were suitable for classification of species. The conservation of cas1 genes was associated with that of repeat sequences among different strains, suggesting they were subjected to similar functional constraints. Moreover, the number of spacer was positively correlated with the number of prophages and insertion sequences, indicating the acetic acid bacteria were continually invaded by new foreign DNA. The comparative analysis of CRISR loci in acetic acid bacteria provided the basis for investigating the molecular mechanism of different acetic acid tolerance and genome stability in acetic acid bacteria.

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.

The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids. Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts.

PubMed

Jiang, W; Gupta, D; Gallagher, D; Davis, S; Bhavanandan, V P

2000-04-01

We previously elucidated five distinct protein domains (I-V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of approximately 55 tandem repeats of 329 amino acids and that domains III-V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2. 2-q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92-94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68-94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.

Amino- and carboxyl-terminal amino acid sequences of proteins coded by gag gene of murine leukemia virus

PubMed Central

Oroszlan, Stephen; Henderson, Louis E.; Stephenson, John R.; Copeland, Terry D.; Long, Cedric W.; Ihle, James N.; Gilden, Raymond V.

1978-01-01

The amino- and carboxyl-terminal amino acid sequences of proteins (p10, p12, p15, and p30) coded by the gag gene of Rauscher and AKR murine leukemia viruses were determined. Among these proteins, p15 from both viruses appears to have a blocked amino end. Proline was found to be the common NH2 terminus of both p30s and both p12s, and alanine of both p10s. The amino-terminal sequences of p30s are identical, as are those of p10s, while the p12 sequences are clearly distinctive but also show substantial homology. The carboxyl-terminal amino acids of both viral p30s and p12s are leucine and phenylalanine, respectively. Rauscher leukemia virus p15 has tyrosine as the carboxyl terminus while AKR virus p15 has phenylalanine in this position. The compositional and sequence data provide definite chemical criteria for the identification of analogous gag gene products and for the comparison of viral proteins isolated in different laboratories. On the basis of amino acid sequences and the previously proposed H-p15-p12-p30-p10-COOH peptide sequence in the precursor polyprotein, a model for cleavage sites involved in the post-translational processing of the precursor coded for by the gag gene is proposed. PMID:206897

[Molecular cloning and characterization in silico of phospholipase A(2) transcript isolated from Lachesis muta peruvian snake venom].

PubMed

Jimenez, Karim L; Zavaleta, Amparo I; Izaguirre, Victor; Yarleque, Armando; Inga, Rosio R

2010-01-01

Isolate and characterize in silico gene phospholipase A(2) (PLA(2)) isolated from Lachesis muta venom of the Peruvian Amazon. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13,976 kDa and 5.66 respectively. The aminoacid sequence was called Lm-PLA(2)-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA(2) from Lachesis stenophrys (93%) and other PLA(2) snake venoms and over 80% of other sPLA(2) family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA(2)-Peru grouped with other acidic [Asp(49)] sPLA(2) previously isolated from Bothriechis schlegelii venom showing 89 % nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA(2) group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. The nucleotide sequence corresponding to the first transcript of gene from PLA(2) cloned of Lachesis muta venom, snake from the Peruvian rainforest.

Complete genome analysis of jasmine virus T from Jasminum sambac in China.

PubMed

Tang, Yajun; Gao, Fangluan; Yang, Zhen; Wu, Zujian; Yang, Liang

2016-07-01

The genome of a potyvirus (isolate JaVT_FZ) recovered from jasmine (Jasminum sambac L.) showing yellow ringspot symptoms in Fuzhou, China, was sequenced. JaVT_FZ is closely related to seven other potyviruses with completely sequenced genomes, with which it shares 66-70 % nucleotide and 52-56 % amino acid sequence identity. However, the coat protein (CP) gene shares 82-92 % nucleotide and 90-97 % amino acid sequence identity with those of two partially sequenced potyviruses, named jasmine potyvirus T (JaVT-jasmine) and jasmine yellow mosaic potyvirus (JaYMV-India), respectively. This suggests that JaVT_FZ, JaVT-jasmine and JaYMV-India should be regarded as members of a single potyvirus species, for which the name "Jasmine virus T" has priority.

Terminal sequence importance of de novo proteins from binary-patterned library: stable artificial proteins with 11- or 12-amino acid alphabet.

PubMed

Okura, Hiromichi; Takahashi, Tsuyoshi; Mihara, Hisakazu

2012-06-01

Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

Novel rod-shaped viruses isolated from garlic, Allium sativum, possessing a unique genome organization.

PubMed

Sumi, S; Tsuneyoshi, T; Furutani, H

1993-09-01

Rod-shaped flexuous viruses were partially purified from garlic plants (Allium sativum) showing typical mosaic symptoms. The genome was shown to be composed of RNA with a poly(A) tail of an estimated size of 10 kb as shown by denaturing agarose gel electrophoresis. We constructed cDNA libraries and screened four independent clones, which were designated GV-A, GV-B, GV-C and GV-D, using Northern and Southern blot hybridization. Nucleotide sequence determination of the cDNAs, two of which correspond to nearly one-third of the virus genomic RNA, shows that all of these viruses possess an identical genomic structure and that also at least four proteins are encoded in the viral cDNA, their M(r)s being estimated to be 15K, 27K, 40K and 11K. The 15K open reading frame (ORF) encodes the core-like sequence of a zinc finger protein preceded by a cluster of basic amino acid residues. The 27K ORF probably encodes the viral coat protein (CP), based on both the existence of some conserved sequences observed in many other rod-shaped or flexuous virus CPs and an overall amino acid sequence similarity to potexvirus and carlavirus CPs. The 11K ORF shows significant amino acid sequence similarities to the corresponding 12K proteins of the potexviruses and carlaviruses. On the other hand, the 40K ORF product does not resemble any other plant virus gene products reported so far. The genomic organization in the 3' region of the garlic viruses resembles, but clearly differs from, that of carlaviruses. Phylogenetic analysis based upon the amino acid sequence of the viral capsid protein also indicates that the garlic viruses have a unique and distinct domain different from those of the potexvirus and carlavirus groups. The results suggest that the garlic viruses described here belong to an unclassified and new virus group closely related to the carlaviruses.

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

PubMed Central

Eberhardt, S; Korn, S; Lottspeich, F; Bacher, A

1997-01-01

Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria. PMID:9139911

[Complete genome sequencing of polymalic acid-producing strain Aureobasidium pullulans CCTCC M2012223].

PubMed

Wang, Yongkang; Song, Xiaodan; Li, Xiaorong; Yang, Sang-tian; Zou, Xiang

2017-01-04

To explore the genome sequence of Aureobasidium pullulans CCTCC M2012223, analyze the key genes related to the biosynthesis of important metabolites, and provide genetic background for metabolic engineering. Complete genome of A. pullulans CCTCC M2012223 was sequenced by Illumina HiSeq high throughput sequencing platform. Then, fragment assembly, gene prediction, functional annotation, and GO/COG cluster were analyzed in comparison with those of other five A. pullulans varieties. The complete genome sequence of A. pullulans CCTCC M2012223 was 30756831 bp with an average GC content of 47.49%, and 9452 genes were successfully predicted. Genome-wide analysis showed that A. pullulans CCTCC M2012223 had the biggest genome assembly size. Protein sequences involved in the pullulan and polymalic acid pathway were highly conservative in all of six A. pullulans varieties. Although both A. pullulans CCTCC M2012223 and A. pullulans var. melanogenum have a close affinity, some point mutation and inserts were occurred in protein sequences involved in melanin biosynthesis. Genome information of A. pullulans CCTCC M2012223 was annotated and genes involved in melanin, pullulan and polymalic acid pathway were compared, which would provide a theoretical basis for genetic modification of metabolic pathway in A. pullulans.

Perlwapin, an Abalone Nacre Protein with Three Four-Disulfide Core (Whey Acidic Protein) Domains, Inhibits the Growth of Calcium Carbonate Crystals

PubMed Central

Treccani, Laura; Mann, Karlheinz; Heinemann, Fabian; Fritz, Monika

2006-01-01

We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of ∼40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre. PMID:16861275

Identification and characterization of a NBS–LRR class resistance gene analog in Pistacia atlantica subsp. Kurdica

PubMed Central

Bahramnejad, Bahman

2014-01-01

P. atlantica subsp. Kurdica, with the local name of Baneh, is a wild medicinal plant which grows in Kurdistan, Iran. The identification of resistance gene analogs holds great promise for the development of resistant cultivars. A PCR approach with degenerate primers designed according to conserved NBS-LRR (nucleotide binding site-leucine rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from P. atlantica subsp. Kurdica. A DNA fragment of the expected 500-bp size was amplified. The nucleotide sequence of this amplicon was obtained through sequencing and the predicted amino acid sequence compared to the amino acid sequences of known R-genes revealed significant sequence similarity. Alignment of the deduced amino acid sequence of P. atlantica subsp. Kurdica resistance gene analog (RGA) showed strong identity, ranging from 68% to 77%, to the non-toll interleukin receptor (non-TIR) R-gene subfamily from other plants. A P-loop motif (GMMGGEGKTT), a conserved and hydrophobic motif GLPLAL, a kinase-2a motif (LLVLDDV), when replaced by IAVFDDI in PAKRGA1 and a kinase-3a (FGPGSRIII) were presented in all RGA. A phylogenetic tree, based on the deduced amino-acid sequences of PAKRGA1 and RGAs from different species indicated that they were separated in two clusters, PAKRGA1 being on cluster II. The isolated NBS analogs can be eventually used as guidelines to isolate numerous R-genes in Pistachio. PMID:27843981

Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition.

PubMed

Corominas, Jordi; Ramayo-Caldas, Yuliaxis; Puig-Oliveras, Anna; Estellé, Jordi; Castelló, Anna; Alves, Estefania; Pena, Ramona N; Ballester, Maria; Folch, Josep M

2013-12-01

In pigs, adipose tissue is one of the principal organs involved in the regulation of lipid metabolism. It is particularly involved in the overall fatty acid synthesis with consequences in other lipid-target organs such as muscles and the liver. With this in mind, we have used massive, parallel high-throughput sequencing technologies to characterize the porcine adipose tissue transcriptome architecture in six Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition (three per group). High-throughput RNA sequencing was used to generate a whole characterization of adipose tissue (backfat) transcriptome. A total of 4,130 putative unannotated protein-coding sequences were identified in the 20% of reads which mapped in intergenic regions. Furthermore, 36% of the unmapped reads were represented by interspersed repeats, SINEs being the most abundant elements. Differential expression analyses identified 396 candidate genes among divergent animals for intramuscular fatty acid composition. Sixty-two percent of these genes (247/396) presented higher expression in the group of pigs with higher content of intramuscular SFA and MUFA, while the remaining 149 showed higher expression in the group with higher content of PUFA. Pathway analysis related these genes to biological functions and canonical pathways controlling lipid and fatty acid metabolisms. In concordance with the phenotypic classification of animals, the major metabolic pathway differentially modulated between groups was de novo lipogenesis, the group with more PUFA being the one that showed lower expression of lipogenic genes. These results will help in the identification of genetic variants at loci that affect fatty acid composition traits. The implications of these results range from the improvement of porcine meat quality traits to the application of the pig as an animal model of human metabolic diseases.

Molecular Recognition and Structural Influences on Function in Bio-nanosystems of Nucleic Acids and Proteins

NASA Astrophysics Data System (ADS)

Sethaphong, Latsavongsakda

This work examines smart material properties of rational self-assembly and molecular recognition found in nano-biosystems. Exploiting the sequence and structural information encoded within nucleic acids and proteins will permit programmed synthesis of nanomaterials and help create molecular machines that may carry out new roles involving chemical catalysis and bioenergy. Responsive to different ionic environments thru self-reorgnization, nucleic acids (NA) are nature's signature smart material; organisms such as viruses and bacteria use features of NAs to react to their environment and orchestrate their lifecycle. Furthermore, nucleic acid systems (both RNA and DNA) are currently exploited as scaffolds; recent applications have been showcased to build bioelectronics and biotemplated nanostructures via directed assembly of multidimensional nanoelectronic devices 1. Since the most stable and rudimentary structure of nucleic acids is the helical duplex, these were modeled in order to examine the influence of the microenvironment, sequence, and cation-dependent perturbations of their canonical forms. Due to their negatively charged phosphate backbone, NA's rely on counterions to overcome the inherent repulsive forces that arise from the assembly of two complementary strands. As a realistic model system, we chose the HIV-TAR helix (PDB ID: 397D) to study specific sequence motifs on cation sequestration. At physiologically relevant concentrations of sodium and potassium ions, we observed sequence based effects where purine stretches were adept in retaining high residency cations. The transitional space between adenine and guanosine nucleotides (ApG step) in a sequence proved the most favorable. This work was the first to directly show these subtle interactions of sequence based cationic sequestration and may be useful for controlling metallization of nucleic acids in conductive nanowires. Extending the study further, we explored the degree to which the structure of NA duplexes alone interacted with cations distinct from a specific sequence. Under physiologically relevant conditions, a duplex of RNA polyguanine-polycitidine was highly responsive and able to sequester cations to the middle of the purine stretches. The least responsive structure was a DNA polyadenine-polythymine duplex. A random sequence DNA duplex contorted into an RNA-like helix resulted in cationic dynamics similar to RNA systems. These studies showed that cation diffusive binding events in nucleic acid duplex structures are sequence specific and heavily influenced by structural aspects helical forms to account for much of the differences observed. Although structural information in nucleic acids is encoded within their sequence, linking amino acid sequence to protein structure is murkier; the structural information within proteins is encoded by the folding process itself: a complex phenomenon driven toward the equilibrium state of the active conformation. Upwards of two thirds of a protein's sequence can be substituted with similar amino acids without significantly perturbing its function; conserved residues of about 10% seem to be vital; since evolutionary selection pressure in proteins operates 3-dimenionally, a linear sequence is partially informative. We explored this problem by folding de-novo the cytosolic portion of the membrane protein, cellulose synthase, CESA1 from upland cotton, Gossypium hirsutum (Ghcesa1). The cytoplasmic region was generated by homology modeling and refined with molecular dynamics. These mutations impair local structural flexibility which likely results in cellulose that is produced at a lower rate and is less crystalline. Additional modeling of fragments of cellulose synthases from the model plant, Arabidopsis thaliana, offered novel insights into the function of conserved cytosolic domains within plant cellulose synthases. Transport mechanisms related to the transmembrane region revealed significant differences between plants and a bacterial complex. These studies generated possible mutations that may allow for the creation of new synthases and identified other avenues of research in order to develop technologies that may alter the crystallinity and other useful properties of cellulose. 1. Karplus, K., SAM-T08, HMM-based protein structure prediction. Nucleic Acids Research, 2009. 37: p. W492-W497.

The cDNA sequence of a neutral horseradish peroxidase.

PubMed

Bartonek-Roxå, E; Eriksson, H; Mattiasson, B

1991-02-16

A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.

Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from Prevotella intermedia

PubMed Central

Chen, Xiaochi; Ansai, Toshihiro; Awano, Shuji; Iida, Toshiya; Barik, Sailen; Takehara, Tadamichi

1999-01-01

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an Mr of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined. PMID:10559178

Cloning and molecular characterization of the betaine aldehyde dehydrogenase involved in the biosynthesis of glycine betaine in white shrimp (Litopenaeus vannamei).

PubMed

Delgado-Gaytán, María F; Rosas-Rodríguez, Jesús A; Yepiz-Plascencia, Gloria; Figueroa-Soto, Ciria G; Valenzuela-Soto, Elisa M

2017-10-01

The enzyme betaine aldehyde dehydrogenase (BADH) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB), a very efficient osmolyte accumulated during osmotic stress. In this study, we determined the nucleotide sequence of the cDNA for the BADH from the white shrimp Litopenaeus vannamei (LvBADH). The cDNA was 1882 bp long, with a complete open reading frame of 1524 bp, encoding 507 amino acids with a predicted molecular mass of 54.15 kDa and a pI of 5.4. The predicted LvBADH amino acid sequence shares a high degree of identity with marine invertebrate BADHs. Catalytic residues (C-298, E-264 and N-167) and the decapeptide VTLELGGKSP involved in nucleotide binding and highly conserved in BADHs were identified in the amino acid sequence. Phylogenetic analyses classified LvBADH in a clade that includes ALDH9 sequences from marine invertebrates. Molecular modeling of LvBADH revealed that the protein has amino acid residues and sequence motifs essential for the function of the ALDH9 family of enzymes. LvBADH modeling showed three potential monovalent cation binding sites, one site is located in an intra-subunit cavity; other in an inter-subunit cavity and a third in a central-cavity of the protein. The results show that LvBADH shares a high degree of identity with BADH sequences from marine invertebrates and enzymes that belong to the ALDH9 family. Our findings suggest that the LvBADH has molecular mechanisms of regulation similar to those of other BADHs belonging to the ALDH9 family, and that BADH might be playing a role in the osmoregulation capacity of L. vannamei. Copyright © 2017 Elsevier B.V. All rights reserved.

The isolation, purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius (daddy sculpin).

PubMed

Cutfield, J F; Cutfield, S M; Carne, A; Emdin, S O; Falkmer, S

1986-07-01

Insulin from the principal islets of the teleost fish, Cottus scorpius (daddy sculpin), has been isolated and sequenced. Purification involved acid/alcohol extraction, gel filtration, and reverse-phase high-performance liquid chromatography to yield nearly 1 mg pure insulin/g wet weight islet tissue. Biological potency was estimated as 40% compared to porcine insulin. The sculpin insulin crystallised in the absence of zinc ions although zinc is known to be present in the islets in significant amounts. Two other hormones, glucagon and pancreatic polypeptide, were copurified with the insulin, and an N-terminal sequence for pancreatic polypeptide was determined. The primary structure of sculpin insulin shows a number of sequence changes unique so far amongst teleost fish. These changes occur at A14 (Arg), A15 (Val), and B2 (Asp). The B chain contains 29 amino acids and there is no N-terminal extension as seen with several other fish. Presumably as a result of the amino acid substitutions, sculpin insulin does not readily form crystals containing zinc-insulin hexamers, despite the presence of the coordinating B10 His.

Comparative analysis of ribosomal protein L5 sequences from bacteria of the genus Thermus.

PubMed

Jahn, O; Hartmann, R K; Boeckh, T; Erdmann, V A

1991-06-01

The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted). Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons. Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene. Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence. The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%). From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine. Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25. The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.

Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

PubMed

Yarimizu, Tohru; Nakamura, Mikiko; Hoshida, Hisashi; Akada, Rinji

2015-02-14

Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences. Mutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield. We present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters

PubMed Central

Bao, Weichen; Mi, Zhihui; Xu, Haiyan; Zheng, Yi; Kwok, Lai Yu; Zhang, Heping; Zhang, Wenyi

2016-01-01

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, γ-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the organic acids content. By SMRT analysis of the microbial composition in eight silage samples, we found that the bacterial species number and relative abundances shifted apparently after fermentation. Such changes were specific to the LAB species in the additives. Particularly, Bacillus megaterium was the initial dominant species in the raw materials; and after the fermentation process, Pediococcus acidilactici and Lactobacillus plantarum became the most prevalent species, both of which were intrinsically present in the LAB additives. Our data have demonstrated that the SMRT sequencing platform is applicable in assessing the quality of silage. PMID:27340760

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters.

PubMed

Bao, Weichen; Mi, Zhihui; Xu, Haiyan; Zheng, Yi; Kwok, Lai Yu; Zhang, Heping; Zhang, Wenyi

2016-06-24

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, γ-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the organic acids content. By SMRT analysis of the microbial composition in eight silage samples, we found that the bacterial species number and relative abundances shifted apparently after fermentation. Such changes were specific to the LAB species in the additives. Particularly, Bacillus megaterium was the initial dominant species in the raw materials; and after the fermentation process, Pediococcus acidilactici and Lactobacillus plantarum became the most prevalent species, both of which were intrinsically present in the LAB additives. Our data have demonstrated that the SMRT sequencing platform is applicable in assessing the quality of silage.

Quantum-Sequencing: Biophysics of quantum tunneling through nucleic acids

NASA Astrophysics Data System (ADS)

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

2014-03-01

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

Terminal region sequence variations in variola virus DNA.

PubMed

Massung, R F; Loparev, V N; Knight, J C; Totmenin, A V; Chizhikov, V E; Parsons, J M; Safronov, P F; Gutorov, V V; Shchelkunov, S N; Esposito, J J

1996-07-15

Genome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of > or = 98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of > or = 65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted.

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...

Acid lipase inhibitor in chicken plasma identified as apolipoprotein A-I.

PubMed

Fujii, M; Higuchi, T; Mukai, S; Yonekura, M; Yano, T; Kawaguchi, H; Nonaka, K; Fukunaga, T; Sugimoto, Y; Yamada, S

1996-10-01

We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895-898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken. The purified inhibitor migrated with the same mobility on SDS-PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hev ein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

2000-07-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

CDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

Preferential amino acid sequences in alumina-catalyzed peptide bond formation.

PubMed

Bujdák, J; Rode, B M

2002-05-21

The catalytic effect of activated alumina on amino acid condensation was investigated. The readiness of amino acids to form peptide sequences was estimated on the basis of the yield of dipeptides and was found to decrease in the order glycine (Gly), alanine (Ala), leucine (Leu), valine (Val), proline (Pro). For example, approximately 15% Gly was converted to the dipeptide (Gly(2)), 5% to cyclic anhydride (cyc(Gly(2))) and small amounts of tri- (Gly(3)) and tetrapeptide (Gly(4)) were formed after 28 days. On the other hand, only trace amounts of Pro(2) were formed from proline under the same conditions. Preferential formation of certain sequences was observed in the mixed reaction systems containing two amino acids. For example, almost ten times more Gly-Val than Val-Gly was formed in the Gly+Val reaction system. The preferred sequences can be explained on the basis of an inductive effect that side groups have on the nucleophilicity and electrophilicity, respectively, of the amino and carboxyl groups. A comparison with published data of amino acid reactions in other reaction systems revealed that the main trends of preferential sequence formation were the same as those described for the salt-induced peptide formation (SIPF) reaction. The results of this work and other previously published papers show that alumina and related mineral surfaces might have played a crucial role in the prebiotic formation of the first peptides on the primitive earth.

A Score of the Ability of a Three-Dimensional Protein Model to Retrieve Its Own Sequence as a Quantitative Measure of Its Quality and Appropriateness

PubMed Central

Martínez-Castilla, León P.; Rodríguez-Sotres, Rogelio

2010-01-01

Background Despite the remarkable progress of bioinformatics, how the primary structure of a protein leads to a three-dimensional fold, and in turn determines its function remains an elusive question. Alignments of sequences with known function can be used to identify proteins with the same or similar function with high success. However, identification of function-related and structure-related amino acid positions is only possible after a detailed study of every protein. Folding pattern diversity seems to be much narrower than sequence diversity, and the amino acid sequences of natural proteins have evolved under a selective pressure comprising structural and functional requirements acting in parallel. Principal Findings The approach described in this work begins by generating a large number of amino acid sequences using ROSETTA [Dantas G et al. (2003) J Mol Biol 332:449–460], a program with notable robustness in the assignment of amino acids to a known three-dimensional structure. The resulting sequence-sets showed no conservation of amino acids at active sites, or protein-protein interfaces. Hidden Markov models built from the resulting sequence sets were used to search sequence databases. Surprisingly, the models retrieved from the database sequences belonged to proteins with the same or a very similar function. Given an appropriate cutoff, the rate of false positives was zero. According to our results, this protocol, here referred to as Rd.HMM, detects fine structural details on the folding patterns, that seem to be tightly linked to the fitness of a structural framework for a specific biological function. Conclusion Because the sequence of the native protein used to create the Rd.HMM model was always amongst the top hits, the procedure is a reliable tool to score, very accurately, the quality and appropriateness of computer-modeled 3D-structures, without the need for spectroscopy data. However, Rd.HMM is very sensitive to the conformational features of the models' backbone. PMID:20830209

Contribution of silent mutations to thermal adaptation of RNA bacteriophage Qβ.

PubMed

Kashiwagi, Akiko; Sugawara, Ryu; Sano Tsushima, Fumie; Kumagai, Tomofumi; Yomo, Tetsuya

2014-10-01

Changes in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qβ in which the culture temperature was increased from 37.2°C to 41.2°C and finally to an inhibitory temperature of 43.6°C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qβ strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2°C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature. Recent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qβ based on experimental evolution during which Qβ showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and one mutation in the untranslated region that spread widely in the Qβ population during the adaptation process at moderately high temperature provided a suitable genetic background to alter the fitness contribution of subsequent mutations from deleterious to beneficial at a higher temperature. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular Cloning and Characterization of cDNA Encoding a Putative Stress-Induced Heat-Shock Protein from Camelus dromedarius

PubMed Central

Elrobh, Mohamed S.; Alanazi, Mohammad S.; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Bazzi, Mohammad D.

2011-01-01

Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B′) from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77–91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80–94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species. PMID:21845074

The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

USGS Publications Warehouse

Bjorklund, H.V.; Higman, K.H.; Kurath, G.

1996-01-01

The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2–33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

USGS Publications Warehouse

Bjorklund, H.V.; Higman, K.H.; Kurath, G.

1996-01-01

The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2-33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

Investigation of the protein osteocalcin of Camelops hesternus: Sequence, structure and phylogenetic implications

NASA Astrophysics Data System (ADS)

Humpula, James F.; Ostrom, Peggy H.; Gandhi, Hasand; Strahler, John R.; Walker, Angela K.; Stafford, Thomas W.; Smith, James J.; Voorhies, Michael R.; George Corner, R.; Andrews, Phillip C.

2007-12-01

Ancient DNA sequences offer an extraordinary opportunity to unravel the evolutionary history of ancient organisms. Protein sequences offer another reservoir of genetic information that has recently become tractable through the application of mass spectrometric techniques. The extent to which ancient protein sequences resolve phylogenetic relationships, however, has not been explored. We determined the osteocalcin amino acid sequence from the bone of an extinct Camelid (21 ka, Camelops hesternus) excavated from Isleta Cave, New Mexico and three bones of extant camelids: bactrian camel ( Camelus bactrianus); dromedary camel ( Camelus dromedarius) and guanaco ( Llama guanacoe) for a diagenetic and phylogenetic assessment. There was no difference in sequence among the four taxa. Structural attributes observed in both modern and ancient osteocalcin include a post-translation modification, Hyp 9, deamidation of Gln 35 and Gln 39, and oxidation of Met 36. Carbamylation of the N-terminus in ancient osteocalcin may result in blockage and explain previous difficulties in sequencing ancient proteins via Edman degradation. A phylogenetic analysis using osteocalcin sequences of 25 vertebrate taxa was conducted to explore osteocalcin protein evolution and the utility of osteocalcin sequences for delineating phylogenetic relationships. The maximum likelihood tree closely reflected generally recognized taxonomic relationships. For example, maximum likelihood analysis recovered rodents, birds and, within hominins, the Homo-Pan-Gorilla trichotomy. Within Artiodactyla, character state analysis showed that a substitution of Pro 4 for His 4 defines the Capra-Ovis clade within Artiodactyla. Homoplasy in our analysis indicated that osteocalcin evolution is not a perfect indicator of species evolution. Limited sequence availability prevented assigning functional significance to sequence changes. Our preliminary analysis of osteocalcin evolution represents an initial step towards a complete character analysis aimed at determining the evolutionary history of this functionally significant protein. We emphasize that ancient protein sequencing and phylogenetic analyses using amino acid sequences must pay close attention to post-translational modifications, amino acid substitutions due to diagenetic alteration and the impacts of isobaric amino acids on mass shifts and sequence alignments.

Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

PubMed

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

Purification, characterization, gene cloning and nucleotide sequencing of D: -stereospecific amino acid amidase from soil bacterium: Delftia acidovorans.

PubMed

Hongpattarakere, Tipparat; Komeda, Hidenobu; Asano, Yasuhisa

2005-12-01

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.

PubMed

Gallage, Nethaji J; Hansen, Esben H; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

2014-06-19

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme

PubMed Central

Gallage, Nethaji J.; Hansen, Esben H.; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

2014-01-01

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco. PMID:24941968

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

PubMed Central

Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

1993-01-01

A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

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.

Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation

PubMed Central

Li, Ming; Wang, Rui; Xiang, Hua

2014-01-01

The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring ‘self’ DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition. PMID:24803673

Characterization of rat calcitonin mRNA.

PubMed Central

Amara, S G; David, D N; Rosenfeld, M G; Roos, B A; Evans, R M

1980-01-01

A chimeric plasmic containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. The cloned calcitonin DNA was used to characterize the expression of calcitonin mRNA. Cytoplasmic mRNAs from calcitonin-producing rat medullary thyroid carcinoma lines and from normal rat thyroid glands contain a single species, 1050 nucleotides long, whch hybridizes to the cloned calcitonin cDNA. The concentration of calcitonin mRNA sequences is greater in those tumors that produce larger amounts of immunoreactive calcitonin. RNAs from other endocrine tissues, including anterior and neurointermediate lobes of rat pituitary, contain no detectable calcitonin mRNA. Images PMID:6933496

Effect of amino acid sequence and pH on nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV.

PubMed

Hong, Yooseong; Legge, Raymond L; Zhang, S; Chen, P

2003-01-01

Atomic force microscopy (AFM) and axisymmetric drop shape analysis-profile (ASDA-P) were used to investigate the mechanism of self-assembly of peptides. The peptides chosen consisted of 16 alternating hydrophobic and hydrophilic amino acids, where the hydrophilic residues possess alternating negative and positive charges. Two types of peptides, AEAEAKAKAEAEAKAK (EAK16-II) and AEAEAEAEAKAKAKAK (EAK16-IV), were investigated in terms of nanostructure formation through self-assembly. The experimental results, which focused on the effects of the amino acid sequence and pH, show that the nanostructures formed by the peptides are dependent on the amino acid sequence and the pH of the solution. For pH conditions around neutrality, one of the peptides used in this study, EAK16-IV, forms globular assemblies and has lower surface tension at air-water interfaces than another peptide, EAK16-II, which forms fibrillar assemblies at the same pH. When the pH is lowered below 6.5 or raised above 7.5, there is a transition from globular to fibrillar structures for EAK16-IV, but EAK16-II does not show any structural transition. Surface tension measurements using ADSA-P showed different surface activities of peptides at air-water interfaces. EAK16-II does not show a significant difference in surface tension for the pH range between 4 and 9. However, EAK16-IV shows a noticeable decrease in surface tension at pH around neutrality, indicating that the formation of globular assemblies is related to the molecular hydrophobicity.

Deep Sequencing Reveals the Complete Genome and Evidence for Transcriptional Activity of the First Virus-Like Sequences Identified in Aristotelia chilensis (Maqui Berry)

PubMed Central

Villacreses, Javier; Rojas-Herrera, Marcelo; Sánchez, Carolina; Hewstone, Nicole; Undurraga, Soledad F.; Alzate, Juan F.; Manque, Patricio; Maracaja-Coutinho, Vinicius; Polanco, Victor

2015-01-01

Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant. PMID:25855242

Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

PubMed

Graham, L A; Bendena, W G; Walker, V K

1996-02-01

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

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

PubMed Central

2013-01-01

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

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.

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

Su, Li; Xu, Xun; Zhao, Hui

A synthetic deca-peptide corresponding to the amino acid sequence Arg{sup 54}-Trp{sup 63} of human tissue-type plasminogen activator (t-PA) kringle 2 domain, named TKII-10, is produced and tested for its ability to inhibit endothelial cell proliferation, migration, tube formation in vitro, and angiogenesis in vivo. At the same time, another peptide TKII-10S composed of the same 10 amino acids as TKII-10, but in a different sequence, is also produced and tested. The results show that TKII-10 potently inhibits VEGF-stimulated endothelial cell migration and tube formation in a dose-dependent, as well as sequence-dependent, manner in vitro while it is inactive in inhibitingmore » endothelial cell proliferation. Furthermore, TKII-10 potently inhibits angiogenesis in chick chorioallantoic membrane and mouse cornea. The middle four amino acids DGDA in their sequence play an important role in TKII-10 angiogenesis inhibition{sub .} These results suggest that TKII-10 is a novel angiogenesis inhibitor that may serve as a prototype for antiangiogenic drug development.« less

Gordonia caeni sp. nov., isolated from sludge of a sewage disposal plant.

PubMed

Srinivasan, Sathiyaraj; Park, Giho; Yang, Hyejin; Hwang, Supyong; Bae, Yoonjung; Jung, Yong-An; Kim, Myung Kyum; Lee, Myungjin

2012-11-01

A Gram-stain-positive, strictly aerobic, short-rod-shaped, non-motile strain (designated MJ32(T)) was isolated from a sludge sample of the Daejeon sewage disposal plant in South Korea. A polyphasic approach was applied to study the taxonomic position of strain MJ32(T). Strain MJ32(T) showed highest 16S rRNA gene sequence similarity to Gordonia hirsuta DSM 44140(T) (98.1%) and Gordonia hydrophobica DSM 44015(T) (97.0%); levels of sequence similarity to the type strains of other recognized Gordonia species were less than 97.0%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJ32(T) belonged to the clade formed by members of the genus Gordonia in the family Gordoniaceae. The G+C content of the genomic DNA of strain MJ32(T) was 69.2 mol%. Chemotaxonomically, strain MJ32(T) showed features typical of the genus Gordonia. The predominant respiratory quinone was MK-9(H(2)), the mycolic acids present had C(56)-C(60) carbon atoms, and the major fatty acids were C(16:0) (34.6%), tuberculostearic acid (21.8%), C(16:1)ω7c (19.5%) and C(18:1)ω9c (12.7%). The peptidoglycan type was based on meso-2,6-diaminopimelic acid as the diagnostic diamino acid with glycolated sugars. On the basis of phylogenetic inference, fatty acid profile and other phenotypic properties, strain MJ32(T) is considered to represent a novel species of the genus Gordonia, for which the name Gordonia caeni sp. nov. is proposed. The type strain is MJ32(T) (=KCTC 19771(T)=JCM 16923(T)).

Complete Amino Acid Sequence of a Copper/Zinc-Superoxide Dismutase from Ginger Rhizome.

PubMed

Nishiyama, Yuki; Fukamizo, Tamo; Yoneda, Kazunari; Araki, Tomohiro

2017-04-01

Superoxide dismutase (SOD) is an antioxidant enzyme protecting cells from oxidative stress. Ginger (Zingiber officinale) is known for its antioxidant properties, however, there are no data on SODs from ginger rhizomes. In this study, we purified SOD from the rhizome of Z. officinale (Zo-SOD) and determined its complete amino acid sequence using N terminal sequencing, amino acid analysis, and de novo sequencing by tandem mass spectrometry. Zo-SOD consists of 151 amino acids with two signature Cu/Zn-SOD motifs and has high similarity to other plant Cu/Zn-SODs. Multiple sequence alignment showed that Cu/Zn-binding residues and cysteines forming a disulfide bond, which are highly conserved in Cu/Zn-SODs, are also present in Zo-SOD. Phylogenetic analysis revealed that plant Cu/Zn-SODs clustered into distinct chloroplastic, cytoplasmic, and intermediate groups. Among them, only chloroplastic enzymes carried amino acid substitutions in the region functionally important for enzymatic activity, suggesting that chloroplastic SODs may have a function distinct from those of SODs localized in other subcellular compartments. The nucleotide sequence of the Zo-SOD coding region was obtained by reverse-translation, and the gene was synthesized, cloned, and expressed. The recombinant Zo-SOD demonstrated pH stability in the range of 5-10, which is similar to other reported Cu/Zn-SODs, and thermal stability in the range of 10-60 °C, which is higher than that for most plant Cu/Zn-SODs but lower compared to the enzyme from a Z. officinale relative Curcuma aromatica.

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors.

PubMed

Torhan, April Smith; Cheewatrakoolpong, Boonlert; Kwee, Lia; Greenfeder, Scott

2007-09-01

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species. [(3)H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.

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.

Genetic variation assessment of acid lime accessions collected from south of Iran using SSR and ISSR molecular markers.

PubMed

Sharafi, Ata Allah; Abkenar, Asad Asadi; Sharafi, Ali; Masaeli, Mohammad

2016-01-01

Iran has a long history of acid lime cultivation and propagation. In this study, genetic variation in 28 acid lime accessions from five regions of south of Iran, and their relatedness with other 19 citrus cultivars were analyzed using Simple Sequence Repeat (SSR) and Inter-Simple Sequence Repeat (ISSR) molecular markers. Nine primers for SSR and nine ISSR primers were used for allele scoring. In total, 49 SSR and 131 ISSR polymorphic alleles were detected. Cluster analysis of SSR and ISSR data showed that most of the acid lime accessions (19 genotypes) have hybrid origin and genetically distance with nucellar of Mexican lime (9 genotypes). As nucellar of Mexican lime are susceptible to phytoplasma, these acid lime genotypes can be used to evaluate their tolerance against biotic constricts like lime "witches' broom disease".

Identification of an Amino Acid Domain Encoded by the Capsid Protein Gene of Porcine Circovirus Type 2 that Modulates Viral Protein Distribution During Replication

USDA-ARS?s Scientific Manuscript database

Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in ...

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

PubMed

Nimata, Masaomi; Okada, Hideki; Kurihara, Kei; Sugimoto, Tsukasa; Honjoh, Tsutomu; Kuroda, Kazuhiko; Yano, Takeo; Tachibana, Hirofumi; Shoji, Masahiro

2018-01-01

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

Identification and Characterization of Novel Surface Proteins in Lactobacillus johnsonii and Lactobacillus gasseri

PubMed Central

Ventura, Marco; Jankovic, Ivana; Walker, D. Carey; Pridmore, R. David; Zink, Ralf

2002-01-01

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family. PMID:12450842

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.

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.

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

PubMed Central

D'Souza, T M; Boominathan, K; Reddy, C A

1996-01-01

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. PMID:8837429

Cloning and sequence analysis of Galleria mellonella juvenile hormone binding protein--a search for ancestors and relatives.

PubMed

Rodriguez Parkitna, Jan M; Ozyhar, Andrzej; Wiśniewski, Jacek R; Kochman, Marian

2002-09-01

Juvenile hormone binding proteins (JHBPs) serve as specific carriers of juvenile hormone (JH) in insect hemolymph. As shown in this report, Galleria mellonella JHBP is encoded by a cDNA of 1063 nucleotides. The pre-protein consists of 245 amino acids with a 20 amino acid leader sequence. The concentration of the JHBP mRNA reaches a maximum on the third day of the last larval instar, and decreases five-fold towards pupation. Comparison of amino acid sequences of JHBPs from Bombyx mori, Heliothis virescens, Manduca sexta and G. mellonella shows that 57 positions out of 226 are occupied by identical amino acids. A phylogeny tree was constructed from 32 proteins, which function could be associated to JH. It has three major branches: (i) ligand binding domains of nuclear receptors, (ii) JHBPs and JH esterases (JHEs), and (iii) hypothetical proteins found in Drosophila melanogaster genome. Despite the close positioning of JHEs and JHBPs on the tree, which probably arises from the presence of a common JH binding motif, these proteins are unlikely to belong to the same family. Detailed analysis of the secondary structure modeling shows that JHBPs may contain a beta-barrel motif flanked by alpha-helices and thus be evolutionary related to the same superfamily as calycins.

A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate.

PubMed

Goldman, G H; Temmerman, W; Jacobs, D; Contreras, R; Van Montagu, M; Herrera-Estrella, A

1993-07-01

We characterized a Trichoderma viride strain that is resistant to the antimitotic drug methyl benzimidazole-2-yl-carbamate (MBC). This species has two beta-tubulin genes (tub1 and tub2) and by reverse genetics we showed that a mutation in the tub2 gene confers MBC resistance in this strain. Comparison of the tub2 sequence of the mutant strain with that of the wild type revealed that a single amino acid substitution of tyrosine for histidine at a position 6 is responsible for the MBC tolerance. Furthermore, we showed that this gene can be used as a homologous dominant selectable marker in T. viride transformation. Both tubulin genes were completely sequenced. They differ by 48 residues and the degree of identity between their deduced amino acid sequences is 86.3%.

Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae.

PubMed

Roosendaal, E; Jacobs, A A; Rathman, P; Sondermeyer, C; Stegehuis, F; Oudega, B; de Graaf, F K

1987-09-01

Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.

Predicting residue-wise contact orders in proteins by support vector regression.

PubMed

Song, Jiangning; Burrage, Kevin

2006-10-03

The residue-wise contact order (RWCO) describes the sequence separations between the residues of interest and its contacting residues in a protein sequence. It is a new kind of one-dimensional protein structure that represents the extent of long-range contacts and is considered as a generalization of contact order. Together with secondary structure, accessible surface area, the B factor, and contact number, RWCO provides comprehensive and indispensable important information to reconstructing the protein three-dimensional structure from a set of one-dimensional structural properties. Accurately predicting RWCO values could have many important applications in protein three-dimensional structure prediction and protein folding rate prediction, and give deep insights into protein sequence-structure relationships. We developed a novel approach to predict residue-wise contact order values in proteins based on support vector regression (SVR), starting from primary amino acid sequences. We explored seven different sequence encoding schemes to examine their effects on the prediction performance, including local sequence in the form of PSI-BLAST profiles, local sequence plus amino acid composition, local sequence plus molecular weight, local sequence plus secondary structure predicted by PSIPRED, local sequence plus molecular weight and amino acid composition, local sequence plus molecular weight and predicted secondary structure, and local sequence plus molecular weight, amino acid composition and predicted secondary structure. When using local sequences with multiple sequence alignments in the form of PSI-BLAST profiles, we could predict the RWCO distribution with a Pearson correlation coefficient (CC) between the predicted and observed RWCO values of 0.55, and root mean square error (RMSE) of 0.82, based on a well-defined dataset with 680 protein sequences. Moreover, by incorporating global features such as molecular weight and amino acid composition we could further improve the prediction performance with the CC to 0.57 and an RMSE of 0.79. In addition, combining the predicted secondary structure by PSIPRED was found to significantly improve the prediction performance and could yield the best prediction accuracy with a CC of 0.60 and RMSE of 0.78, which provided at least comparable performance compared with the other existing methods. The SVR method shows a prediction performance competitive with or at least comparable to the previously developed linear regression-based methods for predicting RWCO values. In contrast to support vector classification (SVC), SVR is very good at estimating the raw value profiles of the samples. The successful application of the SVR approach in this study reinforces the fact that support vector regression is a powerful tool in extracting the protein sequence-structure relationship and in estimating the protein structural profiles from amino acid sequences.

Molecular Characterization of Geographically Different Banana bunchy top virus Isolates in India.

PubMed

Selvarajan, R; Mary Sheeba, M; Balasubramanian, V; Rajmohan, R; Dhevi, N Lakshmi; Sasireka, T

2010-10-01

Banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBTV) is one of the most devastating diseases of banana and poses a serious threat for cultivars like Hill Banana (Syn: Virupakshi) and Grand Naine in India. In this study, we have cloned and sequenced the complete genome comprised of six DNA components of BBTV infecting Hill Banana grown in lower Pulney hills, Tamil Nadu State, India. The complete genome sequence of this hill banana isolate showed high degree of similarity with the corresponding sequences of BBTV isolates originating from Lucknow, Uttar Pradesh State, India, and from Fiji, Egypt, Pakistan, and Australia. In addition, sixteen coat protein (CP) and thirteen replicase genes (Rep) sequences of BBTV isolates collected from different banana growing states of India were cloned and sequenced. The replicase sequences of 13 isolates showed high degree of similarity with that of South Pacific group of BBTV isolates. However, the CP gene of BBTV isolates from Shervroy and Kodaikanal hills of Tamil Nadu showed higher amino acid sequence variability compared to other isolates. Another hill banana isolate from Meghalaya state had 23 nucleotide substitutions in the CP gene but the amino acid sequence was conserved. This is the first report of the characterization of a complete genome of BBTV occurring in the high altitudes of India. Our study revealed that the Indian BBTV isolates with distinct geographical origins belongs to the South Pacific group, except Shervroy and Kodaikanal hill isolates which neither belong to the South Pacific nor the Asian group.

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.

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.

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.

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

PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino-Acid Sequences.

PubMed

Mirarab, Siavash; Nguyen, Nam; Guo, Sheng; Wang, Li-San; Kim, Junhyong; Warnow, Tandy

2015-05-01

We introduce PASTA, a new multiple sequence alignment algorithm. PASTA uses a new technique to produce an alignment given a guide tree that enables it to be both highly scalable and very accurate. We present a study on biological and simulated data with up to 200,000 sequences, showing that PASTA produces highly accurate alignments, improving on the accuracy and scalability of the leading alignment methods (including SATé). We also show that trees estimated on PASTA alignments are highly accurate--slightly better than SATé trees, but with substantial improvements relative to other methods. Finally, PASTA is faster than SATé, highly parallelizable, and requires relatively little memory.

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...

Epitope mapping of the variable repetitive region with the MB antigen of Ureaplasma urealyticum.

PubMed Central

Zheng, X; Lau, K; Frazier, M; Cassell, G H; Watson, H L

1996-01-01

One of the major surface structures of Ureaplasma urealyticum recognized by antibodies of patients during infection is the MB antigen. Previously, we showed by Western blot (immunoblot) analysis that any one of the anti-MB monoclonal antibodies (MAbs) 3B1.5, 5B1.1, and 10C6.6 could block the binding of patient antibodies to MB. Subsequent DNA sequencing revealed that a unique six-amino-acid direct tandem repeat region composed the carboxy two-thirds of this antigen. In the present study, using antibody-reactive peptide scanning of this repeat region, we demonstrated that the amino acids defining the epitopes for MAbs 3B1.5 5B1.1 and 10C6.6 are EQP, GK, and KEQPA, respectively. Peptide scanning analysis of an infected patient's serum antibody response showed that the dominant epitope was defined by the sequence PAGK. Mapping of these continuous epitopes revealed overlap between all MAb and patient polyclonal antibody binding sites, thus explaining the ability of a single MAb to apparently block all polyclonal antibody binding sites. We also show that a single amino acid difference in the sequence of the repeats of serovars 3 and 14 accounts for the lack of reactivity with serovar 14 of two of the serovar 3-specific MAbs. Finally, the data demonstrate the need to obtain the sequences of the mba genes of all serovars before an effective serovar-specific antibody detection method can be developed. PMID:8914774

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.

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

PubMed Central

Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo

2012-01-01

Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba. PMID:23284650

Gene Cloning and Characterization of the Very Large NAD-Dependent l-Glutamate Dehydrogenase from the Psychrophile Janthinobacterium lividum, Isolated from Cold Soil▿

PubMed Central

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-01-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. PMID:17526698

Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-08-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

Protein Tertiary Structure Prediction Based on Main Chain Angle Using a Hybrid Bees Colony Optimization Algorithm

NASA Astrophysics Data System (ADS)

Mahmood, Zakaria N.; Mahmuddin, Massudi; Mahmood, Mohammed Nooraldeen

Encoding proteins of amino acid sequence to predict classified into their respective families and subfamilies is important research area. However for a given protein, knowing the exact action whether hormonal, enzymatic, transmembranal or nuclear receptors does not depend solely on amino acid sequence but on the way the amino acid thread folds as well. This study provides a prototype system that able to predict a protein tertiary structure. Several methods are used to develop and evaluate the system to produce better accuracy in protein 3D structure prediction. The Bees Optimization algorithm which inspired from the honey bees food foraging method, is used in the searching phase. In this study, the experiment is conducted on short sequence proteins that have been used by the previous researches using well-known tools. The proposed approach shows a promising result.

Amino acid sequence of human cholinesterase. Annual report, 30 September 1984-30 September 1985

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

Lockridge, O.

1985-10-01

The active-site serine residue is located 198 amino acids from the N-terminal. The active-site peptide was isolated from three different genetic types of human serum cholinesterase: from usual, atypical, and atypical-silent genotypes. It was found that the amino acid sequence of the active-site peptide was identical in all three genotypes. Comparison of the complete sequences of cholinesterase from human serum and acetylcholinesterase from the electric organ of Torpedo californica shows an identity of 53%. Cholinesterase is of interest to the Department of Defense because cholinesterase protects against organophosphate poisons of the type used in chemical warfare. The structural results presentedmore » here will serve as the basis for cloning the gene for cholinesterase. The potential uses of large amounts of cholinesterase would be for cleaning up spills of organophosphates and possibly for detoxifying exposed personnel.« less

Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20.

PubMed

Tork, Sanaa E; Aly, Magda M; Alakilli, Saleha Y; Al-Seeni, Madeha N

2015-03-01

γ-poly glutamic acid (γ-PGA) has received considerable attention for pharmaceutical and biomedical applications. γ-PGA from the newly isolate Bacillus licheniformis NRC20 was purified and characterized using diffusion distance agar plate, mass spectrometry and thin layer chromatography. All analysis indicated that γ-PGA is a homopolymer composed of glutamic acid. Its molecular weight was determined to be 1266 kDa. It was composed of L- and D-glutamic acid residues. An amplicon of 3050 represents the γ-PGA-coding genes was obtained, sequenced and submitted in genbank database. Its amino acid sequence showed high similarity with that obtained from B. licheniformis strains. The bacterium NRC 20 was independent of L-glutamic acid but the polymer production enhanced when cultivated in medium containing L-glutamic acid as the sole nitrogen source. Finally we can conclude that γ-PGA production from B. licheniformis NRC20 has many promised applications in medicine, industry and nanotechnology. Copyright © 2014 Elsevier B.V. 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)

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

Structure and Sequence Search on Aptamer-Protein Docking

NASA Astrophysics Data System (ADS)

Xiao, Jiajie; Bonin, Keith; Guthold, Martin; Salsbury, Freddie

2015-03-01

Interactions between proteins and deoxyribonucleic acid (DNA) play a significant role in the living systems, especially through gene regulation. However, short nucleic acids sequences (aptamers) with specific binding affinity to specific proteins exhibit clinical potential as therapeutics. Our capillary and gel electrophoresis selection experiments show that specific sequences of aptamers can be selected that bind specific proteins. Computationally, given the experimentally-determined structure and sequence of a thrombin-binding aptamer, we can successfully dock the aptamer onto thrombin in agreement with experimental structures of the complex. In order to further study the conformational flexibility of this thrombin-binding aptamer and to potentially develop a predictive computational model of aptamer-binding, we use GPU-enabled molecular dynamics simulations to both examine the conformational flexibility of the aptamer in the absence of binding to thrombin, and to determine our ability to fold an aptamer. This study should help further de-novo predictions of aptamer sequences by enabling the study of structural and sequence-dependent effects on aptamer-protein docking specificity.

Cloning and characterization of two novel DNases from Streptococcus pyogenes.

PubMed

Hasegawa, Tadao; Torii, Keizo; Hashikawa, Shinnosuke; Iinuma, Yoshitsugu; Ohta, Michio

2002-06-01

The proteins in the culture supernatant (exoproteins) from Streptococcus pyogenes serotype M1 were separated by two-dimensional gel electrophoresis, and their N-terminal amino acid sequences were determined. The amino acid sequences were compared to sequences in the S. pyogenes genome database. The coding sequence showed similarity to sequences of two genes, mf2-v ( mf2 variant) and mf3, which had sequence similarity to genes encoding mitogenic factor (MF); MF has DNase activity. The recombinant genes were expressed in Escherichia coli and the proteins were synthesized. Mf2-v and Mf3 had DNase activity. The activity of Mf2-v was localized to the C-terminal half of the protein. The mf3 gene was shown to be present in most clinically isolated strains of S. pyogenes tested, and the mf2gene was detected in 20% of the isolates. The products of the mf2 and mf3 genes in clinically isolated S. pyogenes strains were thus shown to be DNases.

Molecular Signatures of Microbial Metabolism in an Actively Growing, Silicified, Microbial Structure from Yellowstone National Park

NASA Astrophysics Data System (ADS)

Ferreira, M.; Creveling, J.; Hilburn, I.; Karlsson, E.; Pepe-Ranney, C.; Spear, J.; Dawson, S.; Geobio2008, I.

2008-12-01

Silicified structures that exhibit a putative biologic component in their formation permeate the rock record as stromatolites. We have studied a silicified microbial structure from a hot spring in Yellowstone National Park using phenotypic, phylogenetic, and metagenomic analyses to determine microbial carbon metabolic pathways and the phylogenetic affiliations of microbes present in this unique structure. In this multi-faceted approach, dominant physiologies, specifically with regards to anaerobic and aerobic metabolisms, were inferred from 16S rRNA gene sequences and 454 sequencing data from bulk DNA samples of the structure. Carbon utilization as indicated by ECO Biolog plates showed abundant heterotrophy and heterotrophic diversity throughout the microbial structure. Microbes within the structure are able to utilize all tested sources of carbohydrates, lipids/fatty acids, and protein/amino acids as carbon sources. ECO plate testing of the hot spring water yielded considerable less carbohydrate consumption (only 4 out of 13 tested carbohydrates) and similar lipids/fatty acids and protein/amino acids consumption (2 out of 3 and 5 out of 5 tested sources respectively). Full length 16S rRNA gene sequences and metagenomic 454 pyrosequencing of community DNA showed limited diversity among primary producers. From the 16S data, the majority of the autotrophs are inferred to utilize the Calvin cycle for CO2 fixation, followed by 3-hydroxypropionate/4- hydroxybutyrate CO2 fixation. However, an analysis of the metagenomic data compared to the KEGG database does not show genes directly involved with Calvin cycle carbon fixation. Further BLAST searches of our data failed to find significant matches within our 6514 metagenomic sequences to known RuBisCo sequences taken from the NCBI database. This is likely due to a far under-sampled dataset of metagenomic sequences, and the low number (958) that had matches to the KEGG pathways database. Anaerobic versus aerobic physiology also can be estimated from the 16S clone libraries. Phylogenetic analysis of recovered 16S sequences suggests that 15% of the 16S sequences can be attributed to anaerobic microbes while 42% likely come from aerobes. The remaining 43% of 16S rRNA gene sequences belong to metabolically unassigned phyla both known and novel. This preliminary study demonstrates that the small spatially stratified silicified microbial structure present on the margins of a hot spring contains a rich and complex microbial community with different trophic levels and enzymatic pathways.

A novel isoform of vertebrate ancient opsin in a smelt fish, Plecoglossus altivelis.

PubMed

Minamoto, Toshifumi; Shimizu, Isamu

2002-01-11

Vertebrate ancient (VA) opsin of nonvisual pigment in fishes was reported to exist in two isoforms, i.e., short and long variants with an unusual predicted amino acid sequence length compared to vertebrate visual opsins. Here we cloned an isoform (Pal-VAM) of VA opsin showing the usual opsin length in addition to the long type isoform (Pal-VAL) from a smelt fish, Plecoglossus altivelis. Pal-VAM and Pal-VAL were composed of 346 and 387 amino acids, respectively. The deduced amino acid sequences of these variants were identical to each other within the first 342 residues, but they showed divergence in the carboxyl-terminal sequence. Pal-VAL corresponded to the long isoform found in zebrafish and carp, and Pal-VAM was identified as a new type of VA opsin variant. Southern blotting experiments indicated that the VA opsin gene of the smelt is present as a single copy, and RT-PCR analysis revealed that Pal-VAM and Pal-VAL mRNA were expressed in both the eyes and brain. In situ hybridization showed that Pal-VAM and Pal-VAL mRNA are expressed in amacrine cells in the retina. Pal-VAM is a new probably functional nonvisual photoreceptive molecule in fish. (c)2002 Elsevier Science.

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.

Sequence dependent aggregation of peptides and fibril formation

NASA Astrophysics Data System (ADS)

Hung, Nguyen Ba; Le, Duy-Manh; Hoang, Trinh X.

2017-09-01

Deciphering the links between amino acid sequence and amyloid fibril formation is key for understanding protein misfolding diseases. Here we use Monte Carlo simulations to study the aggregation of short peptides in a coarse-grained model with hydrophobic-polar (HP) amino acid sequences and correlated side chain orientations for hydrophobic contacts. A significant heterogeneity is observed in the aggregate structures and in the thermodynamics of aggregation for systems of different HP sequences and different numbers of peptides. Fibril-like ordered aggregates are found for several sequences that contain the common HPH pattern, while other sequences may form helix bundles or disordered aggregates. A wide variation of the aggregation transition temperatures among sequences, even among those of the same hydrophobic fraction, indicates that not all sequences undergo aggregation at a presumable physiological temperature. The transition is found to be the most cooperative for sequences forming fibril-like structures. For a fibril-prone sequence, it is shown that fibril formation follows the nucleation and growth mechanism. Interestingly, a binary mixture of peptides of an aggregation-prone and a non-aggregation-prone sequence shows the association and conversion of the latter to the fibrillar structure. Our study highlights the role of a sequence in selecting fibril-like aggregates and also the impact of a structural template on fibril formation by peptides of unrelated sequences.

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

PubMed

Kachhap, Sangita; Singh, Balvinder

2015-01-01

In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

Pseudoclavibacter caeni sp. nov., isolated from sludge of a sewage disposal plant.

PubMed

Srinivasan, Sathiyaraj; Kim, Hyun Sook; Kim, Myung Kyum; Lee, Myungjin

2012-04-01

A Gram-positive, strictly aerobic, rod-shaped, non-motile bacterial strain, designated MJ28T, was isolated from a sludge sample from the Daejeon sewage disposal plant in South Korea. A polyphasic approach was applied to study the taxonomic position of strain MJ28T. Strain MJ28T showed highest 16S rRNA gene sequence similarity to Pseudoclavibacter soli KP02T (95.2 %). Levels of 16S rRNA gene sequence similarity to the type strains of other Pseudoclavibacter species were less than 94.0 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJ28T belonged to the clade formed by members of the genus Pseudoclavibacter in the family Microbacteriaceae. The G+C content of the genomic DNA of strain MJ28T was 65.8 mol%. The chemotaxonomic characteristics of strain MJ28T showed features typical of the genus Pseudoclavibacter, with MK-9 as the predominant respiratory quinone, 2,4-diaminobutryic acid as the diamino acid in the peptidoglycan, and anteiso-C17:0 (44.6 %), anteiso-C15:0 (35.7 %) and C16:0 (9.5 %) as the major fatty acids. On the basis of phylogenetic inference, fatty acid profile and other phenotypic properties, strain MJ28T is considered to represent a novel species of the genus Pseudoclavibacter, for which the name Pseudoclavibacter caeni sp. nov. is proposed. The type strain is MJ28T (=KCTC 19773T=JCM 16921T).

A newly constructed primer pair for the PCR amplification, cloning and sequencing of the flagellin (flaA) gene from isolatesof urease-negative Campylobacter lari.

PubMed

Sekizuka, Tsuyoshi; Yokoi, Taeko; Murayama, Ohoshi; Millar, B Cherie; Moore, Johne; Matsuda, Motoo

2005-08-01

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564-572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70-75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.

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...

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.

Characterization of acid-tolerant H/CO-utilizing methanogenic enrichment cultures from an acidic peat bog in New York State.

PubMed

Bräuer, Suzanna L; Yashiro, Erika; Ueno, Norikiyo G; Yavitt, Joseph B; Zinder, Stephen H

2006-08-01

Two methanogenic cultures were enriched from acidic peat soil using a growth medium buffered to c. pH 5. One culture, 6A, was obtained from peat after incubation with H(2)/CO(2), whereas culture NTA was derived from a 10(-4) dilution of untreated peat into a modified medium. 16S rRNA gene clone libraries from each culture contained one methanogen and two bacterial sequences. The methanogen 16S rRNA gene sequences were 99% identical with each other and belonged to the novel "R-10/Fen cluster" family of the Methanomicrobiales, whereas their mcrA sequences were 96% identical. One bacterial 16S rRNA gene sequence from culture 6A belonged to the Bacteroidetes and showed 99% identity with sequences from methanogenic enrichments from German and Russian bogs. The other sequence belonged to the Firmicutes and was identical to a thick rod-shaped citrate-utilizing organism isolated from culture 6A, the numbers of which decreased when the Ti (III) chelator was switched from citrate to nitrilotriacetate. Bacterial clones from the NTA culture clustered in the Delta- and Betaproteobacteria. Both cultures contained thin rods, presumably the methanogens, as the predominant morphotype, and represent a significant advance in characterization of the novel acidiphilic R-10 family methanogens.

Cloning and sequence determination of the gene coding for the pyruvate phosphate dikinase of Entamoeba histolytica.

PubMed

Saavedra-Lira, E; Pérez-Montfort, R

1994-05-16

We isolated three overlapping clones from a DNA genomic library of Entamoeba histolytica strain HM1:IMSS, whose translated nucleotide (nt) sequence shows similarities of 51, 48 and 47% with the amino acid (aa) sequences reported for the pyruvate phosphate dikinases from Bacteroides symbiosus, maize and Flaveria trinervia, respectively. The reading frame determined codes for a protein of 886 aa.

From a marine neuropeptide to antimicrobial pseudopeptides containing aza-β(3)-amino acids: structure and activity

PubMed Central

Laurencin, Mathieu; Legrand, Baptiste; Duval, Emilie; Henry, Joël; Baudy-Floc'H, Michèle; Zatylny-Gaudin, Céline; Bondon, Arnaud

2012-01-01

Incorporation of aza-β3-amino acids into endogenous neuropeptide from mollusks (ALSGDAFLRF-NH2) with weak antimicrobial activities allows us to design new AMPs sequences. We find that, depending on the nature of the substitution, these could result either in inactive pseudopeptides or in a drastic enhancement of the antimicrobial activity without high cytotoxicity resulted. Structural studies perform by NMR and circular dichroism on the pseudopeptides show the impact of aza-β3-amino acids on the peptide structures. We obtain the first three-dimensional structures of pseudopeptides containing aza-β3-amino acids in aqueous micellar SDS and demonstrate that hydrazino turn can be formed in aqueous solution. Overall, these results demonstrate the ability to modulate AMPs activities through structural modifications induced by the nature and the position of these amino acid analogs in the peptide sequences. PMID:22320306

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes

PubMed Central

Jia, Baolei; Hao, Lujiang; Xuan, Yuan Hu; Jeon, Che Ok

2018-01-01

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas. PMID:29872447

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes.

PubMed

Jia, Baolei; Hao, Lujiang; Xuan, Yuan Hu; Jeon, Che Ok

2018-01-01

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas.

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

PubMed

Sailaja, B; Anjum, Najreen; Patil, Yogesh K; Agarwal, Surekha; Malathi, P; Krishnaveni, D; Balachandran, S M; Viraktamath, B C; Mangrauthia, Satendra K

2013-12-01

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.

Novel poly-uridine insertion in the 3'UTR and E2 amino acid substitutions in a low virulent classical swine fever virus.

PubMed

Coronado, Liani; Liniger, Matthias; Muñoz-González, Sara; Postel, Alexander; Pérez, Lester Josue; Pérez-Simó, Marta; Perera, Carmen Laura; Frías-Lepoureau, Maria Teresa; Rosell, Rosa; Grundhoff, Adam; Indenbirken, Daniela; Alawi, Malik; Fischer, Nicole; Becher, Paul; Ruggli, Nicolas; Ganges, Llilianne

2017-03-01

In this study, we compared the virulence in weaner pigs of the Pinar del Rio isolate and the virulent Margarita strain. The latter caused the Cuban classical swine fever (CSF) outbreak of 1993. Our results showed that the Pinar del Rio virus isolated during an endemic phase is clearly of low virulence. We analysed the complete nucleotide sequence of the Pinar del Rio virus isolated after persistence in newborn piglets, as well as the genome sequence of the inoculum. The consensus genome sequence of the Pinar del Rio virus remained completely unchanged after 28days of persistent infection in swine. More importantly, a unique poly-uridine tract was discovered in the 3'UTR of the Pinar del Rio virus, which was not found in the Margarita virus or any other known CSFV sequences. Based on RNA secondary structure prediction, the poly-uridine tract results in a long single-stranded intervening sequence (SS) between the stem-loops I and II of the 3'UTR, without major changes in the stem- loop structures when compared to the Margarita virus. The possible implications of this novel insertion on persistence and attenuation remain to be investigated. In addition, comparison of the amino acid sequence of the viral proteins E rns , E1, E2 and p7 of the Margarita and Pinar del Rio viruses showed that all non-conservative amino acid substitutions acquired by the Pinar del Rio isolate clustered in E2, with two of them being located within the B/C domain. Immunisation and cross-neutralisation experiments in pigs and rabbits suggest differences between these two viruses, which may be attributable to the amino acid differences observed in E2. Altogether, these data provide fresh insights into viral molecular features which might be associated with the attenuation and adaptation of CSFV for persistence in the field. Copyright © 2017 Elsevier B.V. All rights reserved.

cDNA cloning of Brassica napus malonyl-CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant.

PubMed

Simon, J W; Slabas, A R

1998-09-18

The GenBank database was searched using the E. coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated. A 272-bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E. coli MCAT. A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library. This resulted in the isolation of a 1200-bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids. The protein shows 47% homology to the E. coli MCAT amino acid sequence in the coding region for the mature protein. Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E. coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA. This is the first functional evidence supporting the identification of a plant cDNA for MCAT.

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.

Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli.

PubMed Central

Saeed, A M; Magnuson, N S; Sriranganathan, N; Burger, D; Cosand, W

1984-01-01

Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata. Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E. coli. Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition. This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine. The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E. coli. In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E. coli and to the sequence predicted from the last 18 codons of the transposon Tn1681. There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E. coli, which may indicate the existence of identical bioactive configuration among ST peptides of E. coli strains of various host origins. These data support the hypothesis that STs produced by human, bovine, and porcine E. coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon. Images PMID:6376355

In silico analysis of subtilisin from Glaciozyma antarctica PI12

NASA Astrophysics Data System (ADS)

Mustafha, Siti Mardhiah; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad; Kamaruddin, Shazilah; Bakar, Farah Diba Abu

2015-09-01

Subtilisin constitute as a major player in industrial enzymes that has a wide range of application especially in the detergent industry. In this study, a cDNA encoding for subtilisin (GaSUBT) was extracted from the psychrophilic yeast, Glaciozyma antarctica PI12, PCR amplified and sequenced. Various bioinformatics tools were used to characterize the GaSUBT. GaSUBT contains 1587 bp nucleotides encoding for 529 amino acids. The predicted molecular weight of the deduced protein is 55.34 kDa with an isoelectric point of 6.25. GaSUBT was predicted to possess a signal peptide and pro-peptide consisting of a peptidase inhibitor I9 sequence. From the sequence alignment analysis of deduced amino acids with other subtilisins in the NCBI database showed that the sequences surrounding the catalytic triad that forms the catalytic domain are well conserved.

Characterization of gene encoding amylopullulanase from plant-originated lactic acid bacterium, Lactobacillus plantarum L137.

PubMed

Kim, Jong-Hyun; Sunako, Michihiro; Ono, Hisayo; Murooka, Yoshikatsu; Fukusaki, Eiichiro; Yamashita, Mitsuo

2008-11-01

A starch-hydrolyzing lactic acid bacterium, Lactobacillus plantarum L137, was isolated from traditional fermented food made from fish and rice in the Philippines. A gene (apuA) encoding an amylolytic enzyme from Lactobacillus plantarum L137 was cloned, and its nucleotide sequence was determined. The apuA gene consisted of an open reading frame of 6171 bp encoding a protein of 2056 amino acids, the molecular mass of which was calculated to be 215,625 Da. The catalytic domains of amylase and pullulanase were located in the same region within the middle of the N-terminal region. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the N-terminal region, a six-amino-acid sequence (Asp-Ala/Thr-Ala-Asn-Ser-Thr) is repeated 39 times, and a three-amino-acid sequence (Gln-Pro-Thr) is repeated 50 times in the C-terminal region. The apuA gene was subcloned in L. plantarum NCL21, which is a plasmid-cured derivative of the wild-type L137 strain and has no amylopullulanase activity, and the gene was overexpressed under the control of its own promoter. The ApuA enzyme from this recombinant L. plantarum NCL21 harboring apuA gene was purified. The enzyme has both alpha-amylase and pullulanase activities. The N-terminal sequence of the purified enzyme showed that the signal peptide was cleaved at Ala(36) and the molecular mass of the mature extracellular enzyme is 211,537 Da. The major reaction products from soluble starch were maltotriose (G3) and maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. From these results, we concluded that ApuA is an amylolytic enzyme belonging to the amylopullulanase family.

Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

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

Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M{sub r} of its subunit was 77,000. The cells converted ({sup 14}C)-L-phenylalanine into ({sup 14}C)-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading framemore » capable of coding for a polypeptide with 707 amino acids (M{sub r} 77,137), a 22-bp 5{prime}-noncoding region and a 207-bp 3{prime}-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.« less

A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum.

PubMed Central

de la Cruz, J; Pintor-Toro, J A; Benítez, T; Llobell, A; Romero, L C

1995-01-01

The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls. PMID:7592488

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.

Venom characterization of the Amazonian scorpion Tityus metuendus.

PubMed

Batista, C V F; Martins, J G; Restano-Cassulini, R; Coronas, F I V; Zamudio, F Z; Procópio, R; Possani, L D

2018-03-01

The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and β-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Taxonomic status of the Tyulek virus (TLKV) (Orthomyxoviridae, Quaranjavirus, Quaranfil group) isolated from the ticks Argas vulgaris Filippova, 1961 (Argasidae) from the birds burrow nest biotopes in the Kyrgyzstan].

PubMed

L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Shchetinin, A M; Deriabin, P G; Aristova, V A; Gitel'man, A K; Samokhvalov, E I; Botikov, A G

2014-01-01

The Tyulek virus (TLKV) was isolated from the ticks Argas vulgaris Filippova, 1961 (Argasidae), collected from the burrow biotopes in multispecies birds colony in the Aksu river floodplain near Tyulek village (northern part of Chu Valley, Kyrgyzstan). Recently, the TLKV was assigned to the Quaranfil group (including the Quaranfil virus (QRFV), Johnston Atoll virus (JAV), Lake Chad virus) that is a novel genus of the Quaranjavirus in the Orthomyxoviridae family. In his work, the complete genome (ID GenBank KJ438647-8) sequence of the TLKV was determined using next-generation sequencing (Illumina platform). Comparison of deduced amino acid sequences shows closed relationship of the TLKV with QRFV and JAV (86% and 84% identity for PB1 and about 70% for PB2 and PA, respectively). The identity level of the TLKV and QRFV in outer glycoprotein GP is 72% and 80% for nucleotide and amino acid sequences, respectively. The phylogenetic analysis showed that the TLKV belongs to the genus of the Quaranjavirus in the family Orthomyxoviridae.

Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum.

PubMed Central

Witter, K; Cahill, D J; Werner, T; Ziegler, I; Rödl, W; Bacher, A; Gütlich, M

1996-01-01

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM. PMID:8870645

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.

Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp.

PubMed Central

Yoshida, Kenji; Tanaka, Kosei; Yoshida, Ken-ichi

2014-01-01

N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and Vmax values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1). PMID:24375143

Location of a major antigenic site involved in Ross River virus neutralization.

PubMed

Vrati, S; Fernon, C A; Dalgarno, L; Weir, R C

1988-02-01

The location of a major antigenic domain involved in the neutralization of an alphavirus, Ross River virus, has been defined in terms of its position in the amino acid sequence of the E2 glycoprotein. The domain encompasses three topographically close epitopes which were identified using three E2-specific neutralizing monoclonal antibodies in competitive binding assays. Nucleotide sequencing of the structural protein genes of monoclonal antibody-selected antigenic variants showed that for each variant there was a single nucleotide change in the E2 gene leading to a nonconservative amino acid substitution in E2. Changes were at positions 216, 234, and 246-251 in the amino acid sequence. The epitopes are in a region of E2 which, though not strongly conserved as to sequence among Ross River virus, Semliki Forest virus, and Sindbis virus, is conserved in its hydropathy profile among the three alphaviruses. The epitopes lie between two asparagine-linked glycosylation sites (residues 200 and 262) in E2. They are conserved as to position between the mouse virulent T48 strain and the mouse avirulent NB5092 strain.

Molecular cloning of pepsinogens A and C from adult newt (Cynops pyrrhogaster) stomach.

PubMed

Inokuchi, Tomofumi; Ikuzawa, Masayuki; Yamazaki, Shin; Watanabe, Yukari; Shiota, Koushiro; Katoh, Takuma; Kobayashi, Ken-Ichiro

2013-08-01

The full-length cDNAs of three pepsinogens (Pgs) were cloned from the stomach of newt, Cynops pyrrhogaster, and nucleotide sequences of the full-length cDNAs were determined. Molecular phylogenetic analysis showed that two Pgs, named PgC1 and PgC2, belong to the pepsinogen C group, and one Pg, named PgA, belongs to the pepsinogen A group. The sequences contain an open reading frame (ORF) encoding 385 amino acid residues for PgC1, 383 amino acid residues for PgC2 and 377 amino acid residues for PgA. In addition, all of the three amino acid sequences conserve some unique characteristics such as six cysteine residues and putative active site two aspartic acid residues. All of the pepsinogen mRNAs were detected in the stomach by RT-PCR but not in other organs. Although a slight difference at the time of the start of expression was seen among the three pepsinogen genes, all of them were expressed in the larval stage after hatching. This is the first report on cloning of pepsinogens from urodele stomach. Copyright © 2013 Elsevier Inc. All rights reserved.

Catalytic behavior of perchloric acid on silica mesoporous SBA-15 as a green heterogeneous Bronsted acid in heterocyclic multicomponent reactions

NASA Astrophysics Data System (ADS)

Baghban, Ali; Doustkhah, Esmail; Rostamnia, Sadegh

2018-04-01

Catalytic behavior of perchloric acid when supported to mesoporous silica SBA-15 (SBA-15/HClO4) was investigated as a heterogeneous Bronsted acid. Its reactivity and leaching possibility were studied in cascade ring opening-cyclocondensation sequence of diketene and alcohol with aldehyde in the presence of either of urea or ammonium acetate. Results showed that this catalyst can be highly recyclable for several cycles.

Diacylglycerol acyltransferase 2 of Mortierella alpina with specificity on long-chain polyunsaturated fatty acids: A potential tool for reconstituting lipids with nutritional value.

PubMed

Jeennor, Sukanya; Veerana, Mayura; Anantayanon, Jutamas; Panchanawaporn, Sarocha; Chutrakul, Chanikul; Laoteng, Kobkul

2017-12-10

Based on available genome sequences and bioinformatics tools, we searched for an uncharacterized open reading frame of Mortierella alpina (MaDGAT2) using diacylglycerol acyltransferase sequence (fungal DGAT type 2B) as a query. Functional characterization of the identified native and codon-optimized M. alpina genes were then performed by heterologous expression in Saccharomyces cerevisiae strain defective in synthesis of neutral lipid (NL). Lipid analysis of the yeast tranformant carrying MaDGAT2 showed that the NL biosynthesis and lipid particle formation were restored by the gene complementation. Substrate specificity study of the fungal enzyme by fatty acid supplementation in the transformant cultures showed that it had a broad specificity on saturated and unsaturated fatty acid substrates for esterification into triacylglycerol (TAG). The n-6 polyunsaturated fatty acids (PUFAs) with 18 and 20 carbon atoms, including linoleic acid, γ-linolenic acid, dihomo γ-linolenic and arachidonic acid could be incorporated into TAG fraction in the yeast cells. Interestingly, among n-3 PUFAs tested, the MaDGAT2 enzyme preferred eicosapentaenoic acid (EPA) substrate as its highly proportional constituent found in TAG fraction. This study provides a potential genetic tool for reconstituting oils rich in long-chain PUFAs with nutritional value. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and expression analysis of a novel R-type lectin from the coleopteran beetle, Tenebrio molitor.

PubMed

Kim, Dong Hyun; Patnaik, Bharat Bhusan; Seo, Gi Won; Kang, Seong Min; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

2013-11-01

We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, Tenebrio molitor. The cDNA sequence is comprised of 495 bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type beta-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram (-) bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, Listeria monocytogenes suggesting putative function in innate immunity. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744.

PubMed Central

Zenno, S; Saigo, K; Kanoh, H; Inouye, S

1994-01-01

The gene encoding the major NAD(P)H-flavin oxidoreductase (flavin reductase) of the luminous bacterium Vibrio fischeri ATCC 7744 was isolated by using synthetic oligonucleotide probes corresponding to the N-terminal amino acid sequence of the enzyme. Nucleotide sequence analysis suggested that the major flavin reductase of V. fischeri consisted of 218 amino acids and had a calculated molecular weight of 24,562. Cloned flavin reductase expressed in Escherichia coli was purified virtually to homogeneity, and its basic biochemical properties were examined. As in the major flavin reductase in crude extracts of V. fischeri, cloned flavin reductase showed broad substrate specificity and served well as a catalyst to supply reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. The major flavin reductase of V. fischeri not only showed significant similarity in amino acid sequence to oxygen-insensitive NAD(P)H nitroreductases of Salmonella typhimurium, Enterobacter cloacae, and E. coli but also was associated with a low level of nitroreductase activity. The major flavin reductase of V. fischeri and the nitroreductases of members of the family Enterobacteriaceae would thus appear closely related in evolution and form a novel protein family. Images PMID:8206830

Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

PubMed Central

Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi

2003-01-01

We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic. PMID:12614195

Error catastrophe and phase transition in the empirical fitness landscape of HIV

NASA Astrophysics Data System (ADS)

Hart, Gregory R.; Ferguson, Andrew L.

2015-03-01

We have translated clinical sequence databases of the p6 HIV protein into an empirical fitness landscape quantifying viral replicative capacity as a function of the amino acid sequence. We show that the viral population resides close to a phase transition in sequence space corresponding to an "error catastrophe" beyond which there is lethal accumulation of mutations. Our model predicts that the phase transition may be induced by drug therapies that elevate the mutation rate, or by forcing mutations at particular amino acids. Applying immune pressure to any combination of killer T-cell targets cannot induce the transition, providing a rationale for why the viral protein can exist close to the error catastrophe without sustaining fatal fitness penalties due to adaptive immunity.

Distribution and diversity of Verrucomicrobia methanotrophs in geothermal and acidic environments.

PubMed

Sharp, Christine E; Smirnova, Angela V; Graham, Jaime M; Stott, Matthew B; Khadka, Roshan; Moore, Tim R; Grasby, Stephen E; Strack, Maria; Dunfield, Peter F

2014-06-01

Recently, methanotrophic members of the phylum Verrucomicrobia have been described, but little is known about their distribution in nature. We surveyed methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in samples covering a broad temperature range (22.5-81.6°C), but only in acidic conditions (pH 1.8-5.0) and only in geothermal environments, not in acidic bogs or fens. Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic Verrucomicrobia were observed. Those detected in high-temperature geothermal samples (44.1-81.6°C) grouped with known thermoacidiphilic 'Methylacidiphilum' isolates. A second group dominated in moderate-temperature geothermal samples (22.5-40.1°C) and a representative mesophilic methanotroph from this group was isolated (strain LP2A). Genome sequencing verified that strain LP2A possessed particulate methane monooxygenase, but its 16S rRNA gene sequence identity to 'Methylacidiphilum infernorum' strain V4 was only 90.6%. A third group clustered distantly with known methanotrophic Verrucomicrobia. Using pmoA-gene targeted quantitative polymerase chain reaction, two geothermal soil profiles showed a dominance of LP2A-like pmoA sequences in the cooler surface layers and 'Methylacidiphilum'-like pmoA sequences in deeper, hotter layers. Based on these results, there appears to be a thermophilic group and a mesophilic group of methanotrophic Verrucomicrobia. However, both were detected only in acidic geothermal environments. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Identification of Delta5-fatty acid desaturase from the cellular slime mold dictyostelium discoideum.

PubMed

Saito, T; Ochiai, H

1999-10-01

cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Fragoso, Rodrigo R; Silva, Rodrigo O; Gomes, Eliane A; Franco, Octávio L; Dias, Simoni C; Cordeiro, Célia M T; Monnerat, Rose G; Grossi-De-Sá, Maria F

2004-09-01

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated. Copyright 2004 Elsevier Ltd.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing.

PubMed

Margaglione, M; Santacroce, R; Colaizzo, D; Seripa, D; Vecchione, G; Lupone, M R; De Lucia, D; Fortina, P; Grandone, E; Perricone, C; Di Minno, G

2000-10-01

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the gamma-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated gamma-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

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.

PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino-Acid Sequences

PubMed Central

Mirarab, Siavash; Nguyen, Nam; Guo, Sheng; Wang, Li-San; Kim, Junhyong

2015-01-01

Abstract We introduce PASTA, a new multiple sequence alignment algorithm. PASTA uses a new technique to produce an alignment given a guide tree that enables it to be both highly scalable and very accurate. We present a study on biological and simulated data with up to 200,000 sequences, showing that PASTA produces highly accurate alignments, improving on the accuracy and scalability of the leading alignment methods (including SATé). We also show that trees estimated on PASTA alignments are highly accurate—slightly better than SATé trees, but with substantial improvements relative to other methods. Finally, PASTA is faster than SATé, highly parallelizable, and requires relatively little memory. PMID:25549288

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.

The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.

PubMed Central

Cladaras, C; Hadzopoulou-Cladaras, M; Nolte, R T; Atkinson, D; Zannis, V I

1986-01-01

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B-100 (apoB-100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5' and 3' untranslated regions which are 128 and 301 nucleotides respectively. The DNA-derived protein sequence shows that apoB-100 is 513,000 daltons and contains 4560 amino acids including a 24-amino-acid-long signal peptide. The mol. wt of apoB-100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non-amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid-binding domains of apoB-100. The protein contains 25 cysteines and 20 potential N-glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor-binding domain of apoB-100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15-kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB-100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript. Images Fig. 4. Fig. 6. PMID:3030729

Sequence-based prediction of protein-binding sites in DNA: comparative study of two SVM models.

PubMed

Park, Byungkyu; Im, Jinyong; Tuvshinjargal, Narankhuu; Lee, Wook; Han, Kyungsook

2014-11-01

As many structures of protein-DNA complexes have been known in the past years, several computational methods have been developed to predict DNA-binding sites in proteins. However, its inverse problem (i.e., predicting protein-binding sites in DNA) has received much less attention. One of the reasons is that the differences between the interaction propensities of nucleotides are much smaller than those between amino acids. Another reason is that DNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding DNA nucleotides is much harder than predicting DNA-binding amino acids. We computed the interaction propensity (IP) of nucleotide triplets with amino acids using an extensive dataset of protein-DNA complexes, and developed two support vector machine (SVM) models that predict protein-binding nucleotides from sequence data alone. One SVM model predicts protein-binding nucleotides using DNA sequence data alone, and the other SVM model predicts protein-binding nucleotides using both DNA and protein sequences. In a 10-fold cross-validation with 1519 DNA sequences, the SVM model that uses DNA sequence data only predicted protein-binding nucleotides with an accuracy of 67.0%, an F-measure of 67.1%, and a Matthews correlation coefficient (MCC) of 0.340. With an independent dataset of 181 DNAs that were not used in training, it achieved an accuracy of 66.2%, an F-measure 66.3% and a MCC of 0.324. Another SVM model that uses both DNA and protein sequences achieved an accuracy of 69.6%, an F-measure of 69.6%, and a MCC of 0.383 in a 10-fold cross-validation with 1519 DNA sequences and 859 protein sequences. With an independent dataset of 181 DNAs and 143 proteins, it showed an accuracy of 67.3%, an F-measure of 66.5% and a MCC of 0.329. Both in cross-validation and independent testing, the second SVM model that used both DNA and protein sequence data showed better performance than the first model that used DNA sequence data. To the best of our knowledge, this is the first attempt to predict protein-binding nucleotides in a given DNA sequence from the sequence data alone. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria.

PubMed

Wuyts, Sander; Van Beeck, Wannes; Oerlemans, Eline F M; Wittouck, Stijn; Claes, Ingmar J J; De Boeck, Ilke; Weckx, Stefan; Lievens, Bart; De Vuyst, Luc; Lebeer, Sarah

2018-06-15

Spontaneous vegetable fermentations, with their rich flavors and postulated health benefits, are regaining popularity. However, their microbiology is still poorly understood, therefore raising concerns about food safety. In addition, such spontaneous fermentations form interesting cases of man-made microbial ecosystems. Here, samples from 38 carrot juice fermentations were collected through a citizen science initiative, in addition to three laboratory fermentations. Culturing showed that Enterobacteriaceae were outcompeted by lactic acid bacteria (LAB) between 3 and 13 days of fermentation. Metabolite-target analysis showed that lactic acid and mannitol were highly produced, as well as the biogenic amine cadaverine. High-throughput 16S rRNA gene sequencing revealed that mainly species of Leuconostoc and Lactobacillus (as identified by 8 and 20 amplicon sequence variants [ASVs], respectively) mediated the fermentations in subsequent order. The analyses at the DNA level still detected a high number of Enterobacteriaceae , but their relative abundance was low when RNA-based sequencing was performed to detect presumptive metabolically active bacterial cells. In addition, this method greatly reduced host read contamination. Phylogenetic placement indicated a high LAB diversity, with ASVs from nine different phylogenetic groups of the Lactobacillus genus complex. However, fermentation experiments with isolates showed that only strains belonging to the most prevalent phylogenetic groups preserved the fermentation dynamics. The carrot juice fermentation thus forms a robust man-made microbial ecosystem suitable for studies on LAB diversity and niche specificity. IMPORTANCE The usage of fermented food products by professional chefs is steadily growing worldwide. Meanwhile, this interest has also increased at the household level. However, many of these artisanal food products remain understudied. Here, an extensive microbial analysis was performed of spontaneous fermented carrot juices which are used as nonalcoholic alternatives for wine in a Belgian Michelin star restaurant. Samples were collected through an active citizen science approach with 38 participants, in addition to three laboratory fermentations. Identification of the main microbial players revealed that mainly species of Leuconostoc and Lactobacillus mediated the fermentations in subsequent order. In addition, a high diversity of lactic acid bacteria was found; however, fermentation experiments with isolates showed that only strains belonging to the most prevalent lactic acid bacteria preserved the fermentation dynamics. Finally, this study showed that the usage of RNA-based 16S rRNA amplicon sequencing greatly reduces host read contamination. Copyright © 2018 American Society for Microbiology.

Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form.

PubMed Central

Sikorav, J L; Duval, N; Anselmet, A; Bon, S; Krejci, E; Legay, C; Osterlund, M; Reimund, B; Massoulié, J

1988-01-01

In this paper, we show the existence of alternative splicing in the 3' region of the coding sequence of Torpedo acetylcholinesterase (AChE). We describe two cDNA structures which both diverge from the previously described coding sequence of the catalytic subunit of asymmetric (A) forms (Schumacher et al., 1986; Sikorav et al., 1987). They both contain a coding sequence followed by a non-coding sequence and a poly(A) stretch. Both of these structures were shown to exist in poly(A)+ RNAs, by S1 mapping experiments. The divergent region encoded by the first sequence corresponds to the precursor of the globular dimeric form (G2a), since it contains the expected C-terminal amino acids, Ala-Cys. These amino acids are followed by a 29 amino acid extension which contains a hydrophobic segment and must be replaced by a glycolipid in the mature protein. Analyses of intact G2a AChE showed that the common domain of the protein contains intersubunit disulphide bonds. The divergent region of the second type of cDNA consists of an adjacent genomic sequence, which is removed as an intron in A and Ga mRNAs, but may encode a distinct, less abundant catalytic subunit. The structures of the cDNA clones indicate that they are derived from minor mRNAs, shorter than the three major transcripts which have been described previously (14.5, 10.5 and 5.5 kb). Oligonucleotide probes specific for the asymmetric and globular terminal regions hybridize with the three major transcripts, indicating that their size is determined by 3'-untranslated regions which are not related to the differential splicing leading to A and Ga forms. Images PMID:3181125

Electron-Transfer Ion/Ion Reactions of Doubly Protonated Peptides: Effect of Elevated Bath Gas Temperature

PubMed Central

Pitteri, Sharon J.; Chrisman, Paul A.; McLuckey, Scott A.

2005-01-01

In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (~100%) was observed for small peptides (≤7 amino acids). For medium-sized peptides composed of 8–11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum. PMID:16131079

Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds.

PubMed

Schütt, Burkhardt Siegfried; Abbadi, Amine; Loddenkötter, Brigitte; Brummel, Monika; Spener, Friedrich

2002-09-01

With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.

Performance of 1,2-indanedione and the need for sequential treatment of fingerprints.

PubMed

Mangle, Milery Figuera; Xu, Xioama; de Puit, M

2015-09-01

The use of 1,2-indanedione-ZnCl2 (IND-Zn) for the visualisation of fingermarks on porous materials has been widely accepted. The use of the reagent in comparison with others has been well described. To what extent IND or IND-Zn reacts with amino acids, in comparison to ninhydrin, has not been described to date. In this technical note we describe the analysis of amino acids with LCMS with the purpose of understanding the reactivity of ninhydrin, IND-Zn and the sequence thereof. The consumption of amino acids by these visualisation reagents is a feature we propose to use for calculations on the reactivity of these reagents. By using recently developed methods for the quantification of amino acids, we determined the consumption of these entities by visualisation reagents. We show that the differences in reactivity between IND and ninhydrin are not as big as the differences between 1,8-diazafluoren-9-one (DFO) and ninhydrin. We also show that it is of great importance to use IND-Zn and ninhydrin in sequence, in order to fully consume the amino acids present in fingermarks. Copyright © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.

Molecular cloning of a cDNA encoding the glycoprotein of hen oviduct microsomal signal peptidase.

PubMed Central

Newsome, A L; McLean, J W; Lively, M O

1992-01-01

Detergent-solubilized hen oviduct signal peptidase has been characterized previously as an apparent complex of a 19 kDa protein and a 23 kDa glycoprotein (GP23) [Baker & Lively (1987) Biochemistry 26, 8561-8567]. A cDNA clone encoding GP23 from a chicken oviduct lambda gt11 cDNA library has now been characterized. The cDNA encodes a protein of 180 amino acid residues with a single site for asparagine-linked glycosylation that has been directly identified by amino acid sequence analysis of a tryptic-digest peptide containing the glycosylated site. Immunoblot analysis reveals cross-reactivity with a dog pancreas protein. Comparison of the deduced amino acid sequence of GP23 with the 22/23 kDa glycoprotein of dog microsomal signal peptidase [Shelness, Kanwar & Blobel (1988) J. Biol. Chem. 263, 17063-17070], one of five proteins associated with this enzyme, reveals that the amino acid sequences are 90% identical. Thus the signal peptidase glycoprotein is as highly conserved as the sequences of cytochromes c and b from these same species and is likely to be found in a similar form in many, if not all, vertebrate species. The data also show conclusively that the dog and avian signal peptidases have at least one protein subunit in common. Images Fig. 1. PMID:1546959

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.

Cloning and characterization of acid invertase genes in the roots of the metallophyte Kummerowia stipulacea (Maxim.) Makino from two populations: Differential expression under copper stress.

PubMed

Zhang, Luan; Xiong, Zhi-ting; Xu, Zhong-rui; Liu, Chen; Cai, Shen-wen

2014-06-01

The roots of metallophytes serve as the key interface between plants and heavy metal-contaminated underground environments. It is known that the roots of metallicolous plants show a higher activity of acid invertase enzymes than those of non-metallicolous plants when under copper stress. To test whether the higher activity of acid invertases is the result of increased expression of acid invertase genes or variations in the amino acid sequences between the two population types, we isolated full cDNAs for acid invertases from two populations of Kummerowia stipulacea (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, expressed them in Pichia pastoris, and conducted real-time PCR to determine differences in transcript levels during Cu stress. Heterologous expression of acid invertase cDNAs in P. pastoris indicated that variations in the amino acid sequences of acid invertases between the two populations played no significant role in determining enzyme characteristics. Seedlings of K. stipulacea were exposed to 0.3µM Cu(2+) (control) and 10µM Cu(2+) for 7 days under hydroponics׳ conditions. The transcript levels of acid invertases in metallicolous plants were significantly higher than in non-metallicolous plants when under copper stress. The results suggest that the expression of acid invertase genes in metallicolous plants of K. stipulacea differed from those in non-metallicolous plants under such conditions. In addition, the sugars may play an important role in regulating the transcript level of acid invertase genes and acid invertase genes may also be involved in root/shoot biomass allocation. Copyright © 2014 Elsevier Inc. All rights reserved.

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

PubMed Central

Beccari, T; Hoade, J; Orlacchio, A; Stirling, J L

1992-01-01

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse. Images Fig. 1. PMID:1379046

Culturable Facultative Methylotrophic Bacteria from the Cactus Neobuxbaumia macrocephala Possess the Locus xoxF and Consume Methanol in the Presence of Ce3+ and Ca2+

PubMed Central

del Rocío Bustillos-Cristales, María; Corona-Gutierrez, Ivan; Castañeda-Lucio, Miguel; Águila-Zempoaltécatl, Carolina; Seynos-García, Eduardo; Hernández-Lucas, Ismael; Muñoz-Rojas, Jesús; Medina-Aparicio, Liliana; Fuentes-Ramírez, Luis Ernesto

2017-01-01

Methanol-consuming culturable bacteria were isolated from the plant surface, rhizosphere, and inside the stem of Neobuxbaumia macrocephala. All 38 isolates were facultative methylotrophic microorganisms. Their classification included the Classes Actinobacteria, Sphingobacteriia, Alpha-, Beta-, and Gammaproteobacteria. The deduced amino acid sequences of methanol dehydrogenase obtained by PCR belonging to Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria showed high similarity to rare-earth element (REE)-dependent XoxF methanol dehydrogenases, particularly the group XoxF5. The sequences included Asp301, the REE-coordinating amino acid, present in all known XoxF dehydrogenases and absent in MxaF methanol dehydrogenases. The quantity of the isolates showed positive hybridization with a xoxF probe, but not with a mxaF probe. Isolates of all taxonomic groups showed methylotrophic growth in the presence of Ce3+ or Ca2+. The presence of xoxF-like sequences in methylotrophic bacteria from N. macrocephala and its potential relationship with their adaptability to xerophytic plants are discussed. PMID:28855445

High-Molecular-Mass Multi-c-Heme Cytochromes from Methylococcus capsulatus Bath†

PubMed Central

Bergmann, David J.; Zahn, James A.; DiSpirito, Alan A.

1999-01-01

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme c concentration was estimated to be 8.2 ± 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94,000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome. PMID:9922265

Gordonia westfalica sp. nov., a novel rubber-degrading actinomycete.

PubMed

Linos, Alexandros; Berekaa, Mahmoud M; Steinbüchel, Alexander; Kim, Kwang Kyu; Sproer, Cathrin; Kroppenstedt, Reiner M

2002-07-01

A cis-1,4-polyisoprene-degrading bacterium (strain Kb2T) was isolated from foul water taken from the inside of a deteriorated automobile tyre found on a farmer's field in Westfalia, Germany. The strain was aerobic, Gram-positive, exhibited orange smooth and rough colonies on complex nutrient agar, produced elementary branching hyphae that fragmented into rod/coccus-like elements and showed chemotaxonomic markers which were consistent with its classification within the genus Gordonia, i.e. the presence of mesodiaminopimelic acid, arabinose and galactose in whole-cell hydrolysates (cell-wall chemotype IV), N-glycolylmuramic acid in the peptidoglycan wall, a fatty-acid pattern composed of unbranched saturated and monounsaturated fatty acids plus tuberculostearic acid, mycolic acids comprising 56-60 carbon atoms and MK-9(H2) as the only menaquinone. The 16S rDNA sequence of strain Kb2T was found to be most similar to the 16S rDNA sequences of the type strains of Gordonia alkanivorans (DSM 44369T) and Gordonia nitida (KCTC 0605BPT). However, DNA-DNA relatedness data showed that strain Kb2T ( =DSM 44215T NRRL B-24152T) could be distinguished from these two species and represented a new species within the genus Gordonia, for which the name Gordonia westfalica is proposed.

Biochemical and Genetic Evidence that Enterococcus faecium L50 Produces Enterocins L50A and L50B, the sec-Dependent Enterocin P, and a Novel Bacteriocin Secreted without an N-Terminal Extension Termed Enterocin Q

PubMed Central

Cintas, Luis M.; Casaus, Pilar; Herranz, Carmen; Håvarstein, Leiv Sigve; Holo, Helge; Hernández, Pablo E.; Nes, Ingolf F.

2000-01-01

Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321–4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47°C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25°C and are not detected at 37°C or above. PMID:11073927

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.

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps.

PubMed

Rosa, Rafael Diego; Bandeira, Paula Terra; Barracco, Margherita Anna

2007-09-01

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.

fCCAC: functional canonical correlation analysis to evaluate covariance between nucleic acid sequencing datasets.

PubMed

Madrigal, Pedro

2017-03-01

Computational evaluation of variability across DNA or RNA sequencing datasets is a crucial step in genomic science, as it allows both to evaluate reproducibility of biological or technical replicates, and to compare different datasets to identify their potential correlations. Here we present fCCAC, an application of functional canonical correlation analysis to assess covariance of nucleic acid sequencing datasets such as chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). We show how this method differs from other measures of correlation, and exemplify how it can reveal shared covariance between histone modifications and DNA binding proteins, such as the relationship between the H3K4me3 chromatin mark and its epigenetic writers and readers. An R/Bioconductor package is available at http://bioconductor.org/packages/fCCAC/ . pmb59@cam.ac.uk. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

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

Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp.

PubMed

Deng, Peng; Tan, Xiaoqing; Wu, Ying; Bai, Qunhua; Jia, Yan; Xiao, Hong

2015-03-01

The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica , which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function.

Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp

PubMed Central

DENG, PENG; TAN, XIAOQING; WU, YING; BAI, QUNHUA; JIA, YAN; XIAO, HONG

2015-01-01

The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica, which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function. PMID:25667630

A novel alignment-free method to classify protein folding types by combining spectral graph clustering with Chou's pseudo amino acid composition.

PubMed

Tripathi, Pooja; Pandey, Paras N

2017-07-07

The present work employs pseudo amino acid composition (PseAAC) for encoding the protein sequences in their numeric form. Later this will be arranged in the similarity matrix, which serves as input for spectral graph clustering method. Spectral methods are used previously also for clustering of protein sequences, but they uses pair wise alignment scores of protein sequences, in similarity matrix. The alignment score depends on the length of sequences, so clustering short and long sequences together may not good idea. Therefore the idea of introducing PseAAC with spectral clustering algorithm came into scene. We extensively tested our method and compared its performance with other existing machine learning methods. It is consistently observed that, the number of clusters that we obtained for a given set of proteins is close to the number of superfamilies in that set and PseAAC combined with spectral graph clustering shows the best classification results. Copyright © 2017 Elsevier Ltd. All rights reserved.

Algal Species and Light Microenvironment in a Low-pH, Geothermal Microbial Mat Community

PubMed Central

Ferris, M. J.; Sheehan, K. B.; Kühl, M.; Cooksey, K.; Wigglesworth-Cooksey, B.; Harvey, R.; Henson, J. M.

2005-01-01

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to <1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of ≥49°C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of ≤39°C. PMID:16269755

Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing.

PubMed Central

Patarca, R; Dorta, B; Ramirez, J L

1982-01-01

As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed. PMID:6278402

Algal species and light microenvironment in a low-pH, geothermal microbial mat community.

PubMed

Ferris, M J; Sheehan, K B; Kühl, M; Cooksey, K; Wigglesworth-Cooksey, B; Harvey, R; Henson, J M

2005-11-01

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to < 1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of > or = 49 degrees C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of < or = 39 degrees C.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

PubMed

Martini, Laura; Meyer, Adam J; Ellefson, Jared W; Milligan, John N; Forlin, Michele; Ellington, Andrew D; Mansy, Sheref S

2015-10-16

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR

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

D`Souza, T.M.; Boominathan, K.; Reddy, C.A.

1996-10-01

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequences of each of the PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum,more » Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. 36 refs., 6 figs., 2 tabs.« less

Geothermal Resource Evaluation at Naval Air Station Fallon, Nevada

DTIC Science & Technology

1987-08-01

20 4. Mainside Topographic Map Showing Warm Wells and Thermal Gradient H oles... Oligocene and early Miocene periods. These troughs were sites of intense hyohtic to andesitic volcanism and coeval faulting. The orientation and age...volcanic sequence, (2) intervolcanic sediments in the volcanic sequence. (3) a fractured reservoir within uwderlying Mio- Oligocene acid tuffs and

Microbial ecological succession during municipal solid waste decomposition.

PubMed

Staley, Bryan F; de Los Reyes, Francis L; Wang, Ling; Barlaz, Morton A

2018-04-28

The decomposition of landfilled refuse proceeds through distinct phases, each defined by varying environmental factors such as volatile fatty acid concentration, pH, and substrate quality. The succession of microbial communities in response to these changing conditions was monitored in a laboratory-scale simulated landfill to minimize measurement difficulties experienced at field scale. 16S rRNA gene sequences retrieved at separate stages of decomposition showed significant succession in both Bacteria and methanogenic Archaea. A majority of Bacteria sequences in landfilled refuse belong to members of the phylum Firmicutes, while Proteobacteria levels fluctuated and Bacteroidetes levels increased as decomposition proceeded. Roughly 44% of archaeal sequences retrieved under conditions of low pH and high acetate were strictly hydrogenotrophic (Methanomicrobiales, Methanobacteriales). Methanosarcina was present at all stages of decomposition. Correspondence analysis showed bacterial population shifts were attributed to carboxylic acid concentration and solids hydrolysis, while archaeal populations were affected to a higher degree by pH. T-RFLP analysis showed specific taxonomic groups responded differently and exhibited unique responses during decomposition, suggesting that species composition and abundance within Bacteria and Archaea are highly dynamic. This study shows landfill microbial demographics are highly variable across both spatial and temporal transects.

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

Sharma, Manisha; Jamieson, Cara; Lui, Christina

β-catenin is a key mediator of Wnt signaling and its deregulated nuclear accumulation can drive cancer progression. While the central armadillo (Arm) repeats of β-catenin stimulate nuclear entry, the N- and C-terminal “tail” sequences are thought to regulate turnover and transactivation. We show here that the N- and C-tails are also potent transport sequences. The unstructured tails of β-catenin, when individually fused to a GFP-reporter, could enter and exit the nucleus rapidly in live cells. Proximity ligation assays and pull-down assays identified a weak interaction between the tail sequences and the FG-repeats of nucleoporins, consistent with a possible direct translocationmore » of β-catenin through the nuclear pore complex. Extensive alanine mutagenesis of the tail sequences revealed that nuclear translocation of β-catenin was dependent on specific uniformly distributed patches of hydrophobic residues, whereas the mutagenesis of acidic amino acids had no effect. Moreover, the mutation of hydrophobic patches within the N-tail and C-tail of full length β-catenin reduced nuclear transport rate and diminished its ability to activate transcription. We propose that the tail sequences can contribute to β-catenin transport and suggest a possible similar role for hydrophobic unstructured regions in other proteins. - Highlights: • We show that the N- and C-tails of beta-catenin possess nuclear transport activity. • Nuclear transport of the N- or C-tails requires specific hydrophobic amino acids. • Mutagenesis of the N-terminus diminished nuclear entry of full-length beta-catenin. • We propose the N-tail contributes to beta-catenin nuclear entry and transactivation.« less

Isolation, expression, and chromosomal localization of the human mitochondrial capsule selenoprotein gene (MCSP)

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

Aho, Hanne; Schwemmer, M.; Tessmann, D.

1996-03-01

The mitochondrial capsule selenoprotein (MCS) (HGMW-approved symbol MCSP) is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. We describe here the isolation of a cDNA, the exon-intron organization, the expression, and the chromosomal localization of the human MCS gene. Nucleotide sequence analysis of the human and mouse MCS cDNAs reveals that the 5{prime}- and 3{prime}-untranslated sequences are more conserved (71%) than the coding sequences (59%). The open reading frame encodes a 116-amino-acid protein and lacks the UGA codons, which have been reported to encode the selenocysteines in themore » N-terminal of the deduced mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein (39%). The most striking homology lies in the dicysteine motifs. Northern and Southern zooblot analyses reveal that the MCS gene in human, baboon, and bovine is more conserved than its counterparts in mouse and rat. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5{prime}-untranslated region at a position equivalent to that in the mouse and rat genes. Northern blot and in situ hybridization experiments demonstrate that the expression of the human MCS gene is restricted to haploid spermatids. The human gene was assigned to q21 of chromosome 1. 30 refs., 9 figs.« less

[Apply fourier transform infrared spectra coupled with two-dimensional correlation analysis to study the evolution of humic acids during composting].

PubMed

Bu, Gui-jun; Yu, Jing; Di, Hui-hui; Luo, Shi-jia; Zhou, Da-zhai; Xiao, Qiang

2015-02-01

The composition and structure of humic acids formed during composting play an important influence on the quality and mature of compost. In order to explore the composition and evolution mechanism, municipal solid wastes were collected to compost and humic and fulvic acids were obtained from these composted municipal solid wastes. Furthermore, fourier transform infrared spectra and two-dimensional correlation analysis were applied to study the composition and transformation of humic and fulvic acids during composting. The results from fourier transform infrared spectra showed that, the composition of humic acids was complex, and several absorbance peaks were observed at 2917-2924, 2844-2852, 2549, 1662, 1622, 1566, 1454, 1398, 1351, 990-1063, 839 and 711 cm(-1). Compared to humic acids, the composition of fulvci acids was simple, and only three peaks were detected at 1725, 1637 and 990 cm(-1). The appearance of these peaks showed that both humic and fulvic acids comprised the benzene originated from lignin and the polysaccharide. In addition, humic acids comprised a large number of aliphatic and protein which were hardly detected in fulvic acids. Aliphatic, polysaccharide, protein and lignin all were degraded during composting, however, the order of degradation was different between humic and fulvci acids. The result from two-dimensional correlation analysis showed that, organic compounds in humic acids were degraded in the following sequence: aliphatic> protein> polysaccharide and lignin, while that in fulvic acids was as following: protein> polysaccharide and aliphatic. A large number of carboxyl, alcohols and ethers were formed during the degradation process, and the carboxyl was transformed into carbonates. It can be concluded that, fourier transform infrared spectra coupled with two-dimensional correlation analysis not only can analyze the function group composition of humic substances, but also can characterize effectively the degradation sequence of these groups and identified the formation mechanism and dynamics of humic substances during composting.

Combinatorial interactions of two amino acids with a single base pair define target site specificity in plant dimeric homeodomain proteins

PubMed Central

Tron, Adriana E.; Bertoncini, Carlos W.; Palena, Claudia M.; Chan, Raquel L.; Gonzalez, Daniel H.

2001-01-01

Four groups of plant homeodomain proteins contain a dimerization motif closely linked to the homeodomain. We here show that two sunflower homeodomain proteins, Hahb-4 and HAHR1, which belong to the Hd-Zip I and GL2/Hd-Zip IV groups, respectively, show different binding preferences at a defined position of a pseudopalindromic DNA-binding site used as a target. HAHR1 shows a preference for the sequence 5′-CATT(A/T)AATG-3′, rather than 5′-CAAT(A/T)ATTG-3′, recognized by Hahb-4. To analyze the molecular basis of this behavior, we have constructed a set of mutants with exchanged residues (Phe→Ile and Ile→Phe) at position 47 of the homeodomain, together with chimeric proteins between HAHR1 and Hahb-4. The results obtained indicate that Phe47, but not Ile47, allows binding to 5′-CATT(A/T)AATG-3′. However, the preference for this sequence is determined, in addition, by amino acids located C-terminal to residue 53 of the HAHR1 homeodomain. A double mutant of Hahb-4 (Ile47→Phe/Ala54→Thr) shows the same binding behavior as HAHR1, suggesting that combinatorial interactions of amino acid residues at positions 47 and 54 of the homeodomain are involved in establishing the affinity and selectivity of plant dimeric homeodomain proteins with different DNA target sequences. PMID:11726696

Accurate prediction of protein–protein interactions from sequence alignments using a Bayesian method

PubMed Central

Burger, Lukas; van Nimwegen, Erik

2008-01-01

Accurate and large-scale prediction of protein–protein interactions directly from amino-acid sequences is one of the great challenges in computational biology. Here we present a new Bayesian network method that predicts interaction partners using only multiple alignments of amino-acid sequences of interacting protein domains, without tunable parameters, and without the need for any training examples. We first apply the method to bacterial two-component systems and comprehensively reconstruct two-component signaling networks across all sequenced bacteria. Comparisons of our predictions with known interactions show that our method infers interaction partners genome-wide with high accuracy. To demonstrate the general applicability of our method we show that it also accurately predicts interaction partners in a recent dataset of polyketide synthases. Analysis of the predicted genome-wide two-component signaling networks shows that cognates (interacting kinase/regulator pairs, which lie adjacent on the genome) and orphans (which lie isolated) form two relatively independent components of the signaling network in each genome. In addition, while most genes are predicted to have only a small number of interaction partners, we find that 10% of orphans form a separate class of ‘hub' nodes that distribute and integrate signals to and from up to tens of different interaction partners. PMID:18277381

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.

Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein.

PubMed

Tsuchiaka, Shinobu; Rahpaya, Sayed Samim; Otomaru, Konosuke; Aoki, Hiroshi; Kishimoto, Mai; Naoi, Yuki; Omatsu, Tsutomu; Sano, Kaori; Okazaki-Terashima, Sachiko; Katayama, Yukie; Oba, Mami; Nagai, Makoto; Mizutani, Tetsuya

2017-01-17

Bovine enterovirus (BEV) belongs to the species Enterovirus E or F, genus Enterovirus and family Picornaviridae. Although numerous studies have identified BEVs in the feces of cattle with diarrhea, the pathogenicity of BEVs remains unclear. Previously, we reported the detection of novel kobu-like virus in calf feces, by metagenomics analysis. In the present study, we identified a novel BEV in diarrheal feces collected for that survey. Complete genome sequences were determined by deep sequencing in feces. Secondary RNA structure analysis of the 5' untranslated region (UTR), phylogenetic tree construction and pairwise identity analysis were conducted. The complete genome sequences of BEV were genetically distant from other EVs and the VP1 coding region contained novel and unique amino acid sequences. We named this strain as BEV AN12/Bos taurus/JPN/2014 (referred to as BEV-AN12). According to genome analysis, the genome length of this virus is 7414 nucleotides excluding the poly (A) tail and its genome consists of a 5'UTR, open reading frame encoding a single polyprotein, and 3'UTR. The results of secondary RNA structure analysis showed that in the 5'UTR, BEV-AN12 had an additional clover leaf structure and small stem loop structure, similarly to other BEVs. In pairwise identity analysis, BEV-AN12 showed high amino acid (aa) identities to Enterovirus F in the polyprotein, P2 and P3 regions (aa identity ≥82.4%). Therefore, BEV-AN12 is closely related to Enterovirus F. However, aa sequences in the capsid protein regions, particularly the VP1 encoding region, showed significantly low aa identity to other viruses in genus Enterovirus (VP1 aa identity ≤58.6%). In addition, BEV-AN12 branched separately from Enterovirus E and F in phylogenetic trees based on the aa sequences of P1 and VP1, although it clustered with Enterovirus F in trees based on sequences in the P2 and P3 genome region. We identified novel BEV possessing highly divergent aa sequences in the VP1 coding region in Japan. According to species definition, we proposed naming this strain as "Enterovirus K", which is a novel species within genus Enterovirus. Further genomic studies are needed to understand the pathogenicity of BEVs.

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.

Centrocins: isolation and characterization of novel dimeric antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis.

PubMed

Li, Chun; Haug, Tor; Moe, Morten K; Styrvold, Olaf B; Stensvåg, Klara

2010-09-01

As immune effector molecules, antimicrobial peptides (AMPs) play an important role in the invertebrate immune system. Here, we present two novel AMPs, named centrocins 1 (4.5kDa) and 2 (4.4kDa), purified from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. The native peptides are cationic and show potent activities against Gram-positive and Gram-negative bacteria. The centrocins have an intramolecular heterodimeric structure, containing a heavy chain (30 amino acids) and a light chain (12 amino acids). The cDNA encoding the peptides and genomic sequences were cloned and sequenced. One putative isoform (centrocin 1b) was identified and one intron was found in the genes coding for the centrocins. The full length protein sequence of centrocin 1 consists of 119 amino acids, whereas centrocin 2 consists of 118 amino acids which both include a preprosequence of 51 or 50 amino acids for centrocins 1 and 2, respectively, and an interchain of 24 amino acids between the heavy and light chain. The difference of molecular mass between the native centrocins and the deduced sequences from cDNA indicates that the native centrocins contain a post-translational brominated tryptophan. In addition, two amino acids at the C-terminal, Gly-Arg, were removed from the light chains during the post-translational processing. The separate peptide chains of centrocin 1 were synthesized and the heavy chain alone was shown to be sufficient for antimicrobial activity. The genome of the closely related species, the purple sea urchin (S. purpuratus), was shown to contain two putative proteins with high similarity to the centrocins. Copyright 2010 Elsevier Ltd. All rights reserved.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed

Sakoda, H; Imanaka, T

1992-02-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed Central

Sakoda, H; Imanaka, T

1992-01-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH. Images PMID:1735726

Regional spread of HIV-1 M subtype B in middle-aged patients by random env-C2V4 region sequencing

PubMed Central

Stürmer, Martin; Zimmermann, Katrin; Fritzsche, Carlos; Reisinger, Emil; Doelken, Gottfried; Berger, Annemarie; Doerr, Hans W.; Eberle, Josef

2010-01-01

A transmission cluster of HIV-1 M:B was identified in 11 patients with a median age of 52 (range 26–65) in North-East Germany by C2V4 region sequencing of the env gene of HIV-1, who—except of one—were not aware of any risky behaviour. The 10 male and 1 female patients deteriorated immunologically, according to their information made available, within 4 years after a putative HIV acquisition. Nucleic acid sequence analysis showed a R5 virus in all patients and in 7 of 11 a crown motif of the V3 loop, GPGSALFTT, which is found rarely. Analysis of formation of this cluster showed that there is still a huge discrepancy between awareness and behaviour regarding HIV transmission in middle-aged patients, and that a local outbreak can be detected by nucleic acid analysis of the hypervariable env region. PMID:20217125

Physical Model of the Genotype-to-Phenotype Map of Proteins

NASA Astrophysics Data System (ADS)

Tlusty, Tsvi; Libchaber, Albert; Eckmann, Jean-Pierre

2017-04-01

How DNA is mapped to functional proteins is a basic question of living matter. We introduce and study a physical model of protein evolution which suggests a mechanical basis for this map. Many proteins rely on large-scale motion to function. We therefore treat protein as learning amorphous matter that evolves towards such a mechanical function: Genes are binary sequences that encode the connectivity of the amino acid network that makes a protein. The gene is evolved until the network forms a shear band across the protein, which allows for long-range, soft modes required for protein function. The evolution reduces the high-dimensional sequence space to a low-dimensional space of mechanical modes, in accord with the observed dimensional reduction between genotype and phenotype of proteins. Spectral analysis of the space of 1 06 solutions shows a strong correspondence between localization around the shear band of both mechanical modes and the sequence structure. Specifically, our model shows how mutations are correlated among amino acids whose interactions determine the functional mode.

Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

PubMed

Ferreira, A B; Oliveira, M N V de; Freitas, F S; Paiva, A D; Alfenas-Zerbini, P; Silva, D F da; Queiroz, M V de; Borges, A C; Moraes, C A de

2015-01-01

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-02-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed Central

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators. Images PMID:3257578

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases. PMID:21247434

Development of a sugar-binding residue prediction system from protein sequences using support vector machine.

PubMed

Banno, Masaki; Komiyama, Yusuke; Cao, Wei; Oku, Yuya; Ueki, Kokoro; Sumikoshi, Kazuya; Nakamura, Shugo; Terada, Tohru; Shimizu, Kentaro

2017-02-01

Several methods have been proposed for protein-sugar binding site prediction using machine learning algorithms. However, they are not effective to learn various properties of binding site residues caused by various interactions between proteins and sugars. In this study, we classified sugars into acidic and nonacidic sugars and showed that their binding sites have different amino acid occurrence frequencies. By using this result, we developed sugar-binding residue predictors dedicated to the two classes of sugars: an acid sugar binding predictor and a nonacidic sugar binding predictor. We also developed a combination predictor which combines the results of the two predictors. We showed that when a sugar is known to be an acidic sugar, the acidic sugar binding predictor achieves the best performance, and showed that when a sugar is known to be a nonacidic sugar or is not known to be either of the two classes, the combination predictor achieves the best performance. Our method uses only amino acid sequences for prediction. Support vector machine was used as a machine learning algorithm and the position-specific scoring matrix created by the position-specific iterative basic local alignment search tool was used as the feature vector. We evaluated the performance of the predictors using five-fold cross-validation. We have launched our system, as an open source freeware tool on the GitHub repository (https://doi.org/10.5281/zenodo.61513). Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The delta-subunit of murine guanine nucleotide exchange factor eIF-2B. Characterization of cDNAs predicts isoforms differing at the amino-terminal end.

PubMed

Henderson, R A; Krissansen, G W; Yong, R Y; Leung, E; Watson, J D; Dholakia, J N

1994-12-02

Protein synthesis in mammalian cells is regulated at the level of the guanine nucleotide exchange factor, eIF-2B, which catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. We have isolated and sequenced cDNA clones encoding the delta-subunit of murine eIF-2B. The cDNA sequence encodes a polypeptide of 544 amino acids with molecular mass of 60 kDa. Antibodies against a synthetic polypeptide of 30 amino acids deduced from the cDNA sequence specifically react with the delta-subunit of mammalian eIF-2B. The cDNA-derived amino acid sequence shows significant homology with the yeast translational regulator Gcd2, supporting the hypothesis that Gcd2 may be the yeast homolog of the delta-subunit of mammalian eIF-2B. Primer extension studies and anchor polymerase chain reaction analysis were performed to determine the 5'-end of the transcript for the delta-subunit of eIF-2B. Results of these experiments demonstrate two different mRNAs for the delta-subunit of eIF-2B in murine cells. The isolation and characterization of two different full-length cDNAs also predicts the presence of two alternate forms of the delta-subunit of eIF-2B in murine cells. These differ at their amino-terminal end but have identical nucleotide sequences coding for amino acids 31-544.

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.

cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus.

PubMed

Wu, Fang; Yan, Ming; Li, Yikun; Chang, Shaojie; Song, Xiaomin; Zhou, Zhaocai; Gong, Weimin

2003-12-19

SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized "hydrophobic probe" for proteins. This binding activity indicated another biological function of SPE-16.

Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans.

PubMed Central

Chernin, L S; De la Fuente, L; Sobolev, V; Haran, S; Vorgias, C E; Oppenheim, A B; Chet, I

1997-01-01

The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions. PMID:9055404

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

PubMed

Oba, Mami; Tsuchiaka, Shinobu; Omatsu, Tsutomu; Katayama, Yukie; Otomaru, Konosuke; Hirata, Teppei; Aoki, Hiroshi; Murata, Yoshiteru; Makino, Shinji; Nagai, Makoto; Mizutani, Tetsuya

2018-01-08

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae.

PubMed

Kim, Sunhwa; Matsuo, Ichiro; Ajisaka, Katsumi; Nakajima, Harushi; Kitamoto, Katsuhiko

2002-10-01

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.

Enterocin TW21, a novel bacteriocin from dochi-isolated Enterococcus faecium D081821.

PubMed

Chang, S-Y; Chen, Y-S; Pan, S-F; Lee, Y-S; Chang, C-H; Chang, C-H; Yu, B; Wu, H-C

2013-09-01

Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300·6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2 -ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative. © 2013 The Society for Applied Microbiology.

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.

The cel3 gene of Agaricus bisporus codes for a modular cellulase and is transcriptionally regulated by the carbon source.

PubMed Central

Chow, C M; Yagüe, E; Raguz, S; Wood, D A; Thurston, C F

1994-01-01

A 52-kDa protein, CEL3, has been separated from the culture filtrate of Agaricus bisporus during growth on cellulose. A PCR-derived probe was made, with a degenerate oligodeoxynucleotide derived from the amino acid sequence of a CEL3 CNBr cleavage product and was used to select cel3 cDNA clones from an A. bisporus cDNA library. Two allelic cDNAs were isolated. They showed 98.8% identity of their nucleotide sequences. The deduced amino acid sequence and domain architecture of CEL3 showed a high degree of similarity to those of cellobiohydrolase II of Trichoderma reesei. Functional expression of cel3 cDNA in Saccharomyces cerevisiae was achieved by placing it under the control of a constitutive promoter and fusing it to the yeast invertase signal sequence. Recombinant CEL3 secreted by yeast showed enzymatic activity towards crystalline cellulose. At long reaction times, CEL3 was also able to degrade carboxymethyl cellulose. Northern (RNA) analysis showed that cel3 gene expression was induced by cellulose and repressed by glucose, fructose, 2-deoxyglucose, and lactose. Glycerol, mannitol, sorbitol, and maltose were neutral carbon sources. Nuclear run-on analysis showed that the rate of synthesis of cel3 mRNA in cellulose-grown cultures was 13 times higher than that in glucose-grown cultures. A low basal rate of cel3 mRNA synthesis was observed in the nuclei isolated from glucose-grown mycelia. Images PMID:8085821

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

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

Takegami, T.; Semler, B.L.; Anderson, C.W.

1983-01-01

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have alsomore » revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.« less

Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue

PubMed Central

Jiang, Ming Feng; Hu, Ming Jun; Ren, Hong Hui; Wang, Li

2015-01-01

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity. PMID:26580446

Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I4

PubMed Central

Le Marrec, Claire; Hyronimus, Bertrand; Bressollier, Philippe; Verneuil, Bernard; Urdaci, Maria C.

2000-01-01

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I4 has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I4. Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI4 DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI4 when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus. PMID:11097892

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

PubMed

He, Amanda; Penix, Stephanie R; Basting, Preston J; Griffith, Jessie M; Creamer, Kaitlin E; Camperchioli, Dominic; Clark, Michelle W; Gonzales, Alexandra S; Chávez Erazo, Jorge Sebastian; George, Nadja S; Bhagwat, Arvind A; Slonczewski, Joan L

2017-06-15

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS 5 insertion or IS-mediated deletion in cadC , while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR , yhiM , and Gad). Other strains showed downregulation of H 2 consumption mediated by hydrogenases ( hya and hyb ) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes ( dmsABC , frdABCD , hybABO , nikABCDE , and nrfAC ). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism. IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain. Copyright © 2017 American Society for Microbiology.

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.

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.

Hidden Markov model-derived structural alphabet for proteins: the learning of protein local shapes captures sequence specificity.

PubMed

Camproux, A C; Tufféry, P

2005-08-05

Understanding and predicting protein structures depend on the complexity and the accuracy of the models used to represent them. We have recently set up a Hidden Markov Model to optimally compress protein three-dimensional conformations into a one-dimensional series of letters of a structural alphabet. Such a model learns simultaneously the shape of representative structural letters describing the local conformation and the logic of their connections, i.e. the transition matrix between the letters. Here, we move one step further and report some evidence that such a model of protein local architecture also captures some accurate amino acid features. All the letters have specific and distinct amino acid distributions. Moreover, we show that words of amino acids can have significant propensities for some letters. Perspectives point towards the prediction of the series of letters describing the structure of a protein from its amino acid sequence.

Scarabaecin, a novel cysteine-containing antifungal peptide from the rhinoceros beetle, Oryctes rhinoceros.

PubMed

Tomie, Tetsuya; Ishibashi, Jun; Furukawa, Seiichi; Kobayashi, Satoe; Sawahata, Ryoko; Asaoka, Ai; Tagawa, Michito; Yamakawa, Minoru

2003-07-25

A novel antifungal peptide, scarabaecin (4080Da), was isolated from the coconut rhinoceros beetle, Oryctes rhinoceros. Scarabaecin cDNA was cloned by reverse transcriptase-polymerase chain reactions (RT-PCR) using a primer based on the N-terminal amino acid sequence. The amino acid sequence deduced from scarabaecin cDNA showed no significant similarity to those of reported proteins. Chemically synthesized scarabaecin indicated antifungal activity against phytopathogenic fungi such as Pyricularia oryzae, Rhizoctonia solani, and Botrytis cinerea, but not against phytopathogenic bacteria. It showed weak activity against Bauberia bassiana, an insect pathogenic fungus, and Staphylococcus aureus, a pathogenic bacterium. Scarabaecin showed chitin binding property and its K(d) was 1.315 microM. A comparison of putative chitin-binding domains among scarabaecin, invertebrate, and plant chitin-binding proteins suggests that scarabaecin is a new member of chitin-binding antimicrobial proteins.

High molecular weight glutenin subunits in some durum wheat cultivars investigated by means of mass spectrometric techniques.

PubMed

Muccilli, Vera; Lo Bianco, Marisol; Cunsolo, Vincenzo; Saletti, Rosaria; Gallo, Giulia; Foti, Salvatore

2011-11-23

The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.

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...

Analysis of Protein Thermostability Enhancing Factors in Industrially Important Thermus Bacteria Species

PubMed Central

Kumwenda, Benjamin; Litthauer, Derek; Bishop, Özlem Tastan; Reva, Oleg

2013-01-01

Elucidation of evolutionary factors that enhance protein thermostability is a critical problem and was the focus of this work on Thermus species. Pairs of orthologous sequences of T. scotoductus SA-01 and T. thermophilus HB27, with the largest negative minimum folding energy (MFE) as predicted by the UNAFold algorithm, were statistically analyzed. Favored substitutions of amino acids residues and their properties were determined. Substitutions were analyzed in modeled protein structures to determine their locations and contribution to energy differences using PyMOL and FoldX programs respectively. Dominant trends in amino acid substitutions consistent with differences in thermostability between orthologous sequences were observed. T. thermophilus thermophilic proteins showed an increase in non-polar, tiny, and charged amino acids. An abundance of alanine substituted by serine and threonine, as well as arginine substituted by glutamine and lysine was observed in T. thermophilus HB27. Structural comparison showed that stabilizing mutations occurred on surfaces and loops in protein structures. PMID:24023508

Sequence space and the ongoing expansion of the protein universe.

PubMed

Povolotskaya, Inna S; Kondrashov, Fyodor A

2010-06-17

The need to maintain the structural and functional integrity of an evolving protein severely restricts the repertoire of acceptable amino-acid substitutions. However, it is not known whether these restrictions impose a global limit on how far homologous protein sequences can diverge from each other. Here we explore the limits of protein evolution using sequence divergence data. We formulate a computational approach to study the rate of divergence of distant protein sequences and measure this rate for ancient proteins, those that were present in the last universal common ancestor. We show that ancient proteins are still diverging from each other, indicating an ongoing expansion of the protein sequence universe. The slow rate of this divergence is imposed by the sparseness of functional protein sequences in sequence space and the ruggedness of the protein fitness landscape: approximately 98 per cent of sites cannot accept an amino-acid substitution at any given moment but a vast majority of all sites may eventually be permitted to evolve when other, compensatory, changes occur. Thus, approximately 3.5 x 10(9) yr has not been enough to reach the limit of divergent evolution of proteins, and for most proteins the limit of sequence similarity imposed by common function may not exceed that of random sequences.

The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize

PubMed Central

Bonen, Linda; Boer, Poppo H.; Gray, Michael W.

1984-01-01

We have determined the sequence of the wheat mitochondrial gene for cytochrome oxidase subunit II (COII) and find that its derived protein sequence differs from that of maize at only three amino acid positions. Unexpectedly, all three replacements are non-conservative ones. The wheat COII gene has a highly-conserved intron at the same position as in maize, but the wheat intron is 1.5 times longer because of an insert relative to its maize counterpart. Hybridization analysis of mitochondrial DNA from rye, pea, broad bean and cucumber indicates strong sequence conservation of COII coding sequences among all these higher plants. However, only rye and maize mitochondrial DNA show homology with wheat COII intron sequences and rye alone with intron-insert sequences. We find that a sequence identical to the region of the 5' exon corresponding to the transmembrane domain of the COII protein is present at a second genomic location in wheat mitochondria. These variations in COII gene structure and size, as well as the presence of repeated COII sequences, illustrate at the DNA sequence level, factors which contribute to higher plant mitochondrial DNA diversity and complexity. ImagesFig. 3.Fig. 4.Fig. 5. PMID:16453565

Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

PubMed

Heilmann, L J; Trewitt, P M; Kumaran, A K

1993-01-01

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

Shark complement: an assessment.

PubMed

Smith, S L

1998-12-01

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

Sequence diversity of hepatitis C virus 6a within the extended interferon sensitivity-determining region correlates with interferon-alpha/ribavirin treatment outcomes.

PubMed

Zhou, Daniel X M; Chan, Paul K S; Zhang, Tiejun; Tully, Damien C; Tam, John S

2010-10-01

Studies on the association between sequence variability of the interferon sensitivity-determining region (ISDR) of hepatitis C virus and the outcome of treatment have reached conflicting results. In this study, 25 patients infected with HCV 6a who had received interferon-alpha/ribavirin combination treatment were analyzed for the sequence variations. 14 of them had the full genome sequences obtained from a previous study, whereas the other 11 samples were sequenced for the extended ISDR (eISDR). This eISDR fragment covers 192 bp (64 amino acids) upstream and 201 bp (67 amino acids) downstream from the ISDR previously defined for HCV 1b. The comparison between interferon-alpha resistance and response groups for the amino acid mutations located in the full genome (6 and 8 patients respectively) as well as the mutations located in the eISDR (10 and 15 patients respectively) showed that the mutations I2160V, I2256V, V2292I (P<0.05) within eISDR were significantly associated with resistance to treatment. However, the extent of amino acid variations within previously defined ISDR was not associated with resistance to treatment as previously reported. Four amino acid variations I248V (P=0.03-0.06) within E1, R445K (P=0.02-0.05) and S747T (P=0.03) within E2, I861V (P=0.01) within NS2 which located outside the eISDR may also associate with treatment outcome as identified by a prescreening of variations within 14 HCV 6a full genomes. (c) 2010 Elsevier B.V. All rights reserved.

Cloning and Sequence Analysis of Vibrio halioticoli Genes Encoding Three Types of Polyguluronate Lyase.

PubMed

Sugimura; Sawabe; Ezura

2000-01-01

The alginate lyase-coding genes of Vibrio halioticoli IAM 14596(T), which was isolated from the gut of the abalone Haliotis discus hannai, were cloned using plasmid vector pUC 18, and expressed in Escherichia coli. Three alginate lyase-positive clones, pVHB, pVHC, and pVHE, were obtained, and all clones expressed the enzyme activity specific for polyguluronate. Three genes, alyVG1, alyVG2, and alyVG3, encoding polyguluronate lyase were sequenced: alyVG1 from pVHB was composed of a 1056-bp open reading frame (ORF) encoding 352 amino acid residues; alyVG2 gene from pVHC was composed of a 993-bp ORF encoding 331 amino acid residues; and alyVG3 gene from pVHE was composed of a 705-bp ORF encoding 235 amino acid residues. Comparison of nucleotide and deduced amino acid sequences among AlyVG1, AlyVG2, and AlyVG3 revealed low homologies. The identity value between AlyVG1 and AlyVG2 was 18.7%, and that between AlyVG2 and AlyVG3 was 17.0%. A higher identity value (26.0%) was observed between AlyVG1 and AlyVG3. Sequence comparison among known polyguluronate lyases including AlyVG1, AlyVG2, and AlyVG3 also did not reveal an identical region in these sequences. However, AlyVG1 showed the highest identity value (36.2%) and the highest similarity (73.3%) to AlyA from Klebsiella pneumoniae. A consensus region comprising nine amino acid (YFKAGXYXQ) in the carboxy-terminal region previously reported by Mallisard and colleagues was observed only in AlyVG1 and AlyVG2.

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.

ChAy/Bx, a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp. dicoccoides.

PubMed

Guo, Xiao-Hui; Bi, Zhe-Guang; Wu, Bi-Hua; Wang, Zhen-Zhen; Hu, Ji-Liang; Zheng, You-Liang; Liu, Deng-Cai

2013-12-01

High-molecular-weight glutenin subunits (HMW-GSs) are of considerable interest, because they play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. In this paper, ChAy/Bx, a novel chimeric HMW-GS gene from Triticum turgidum ssp. dicoccoides (AABB, 2n=4x=28) accession D129, was isolated and characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1,671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 nt sequence was highly homologous with the corresponding sequence of 1Ay type genes, while its final 366 nt sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein consisted of the N-terminus of 1Ay type subunit (the first 414 amino acid residues) and the C-terminus of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structure of this HMW glutenin chimera ChAy/Bx subunit might have unique effects on the end-use quality of wheat flour. Here we propose that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs. © 2013.

The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

PubMed Central

Uemura, K; Roelcke, D; Nagai, Y; Feizi, T

1984-01-01

The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue. The other three antibodies differ from anti-Pr2 in their lack of reaction with glycolipids of the ganglio series. They react with the NeuAc(alpha 2-3)Gal(beta 1-4)Glc/GlcNAc sequence as found in GM3 and in glycolipids of the neolacto series, but show a preference for the latter, longer sequences. Thus all four antibodies react with sialylated oligosaccharides containing i type (linear) and I type (branched) neolacto backbones. Fl antibody differs from the other three in its stronger reaction with branched neolacto sequences in accordance with its stronger agglutination of erythrocytes of I rather than i type. The four antibodies show a specificity for N-acetyl- rather than N-glycolyl-neuraminic acid. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6204642

Reclassification of Bacillus beijingensis Qiu et al. 2009 and Bacillus ginsengi Qiu et al. 2009 as Bhargavaea beijingensis comb. nov. and Bhargavaea ginsengi comb. nov. and emended description of the genus Bhargavaea.

PubMed

Verma, Pankaj; Pandey, Prashant Kumar; Gupta, Arvind Kumar; Seong, Chi Nam; Park, Seong Chan; Choe, Han Na; Baik, Keun Sik; Patole, Milind Shivaji; Shouche, Yogesh Shreepad

2012-10-01

We have carried out a polyphasic taxonomic characterization of Bacillus beijingensis DSM 19037(T) and Bacillus ginsengi DSM 19038(T), which are closely related phylogenetically to Bhargavaea cecembensis LMG 24411(T). All three strains are Gram-stain-positive, non-motile, moderately halotolerant and non-spore-forming. 16S rRNA gene sequence analyses showed that the strains constituted a coherent cluster, with sequence similarities between 99.7 and 98.7 %. The percentage similarity on the basis of amino acid sequences deduced from partial gyrB gene nucleotide sequences of these three type strains was 96.1-92.7 %. Phylogenetic trees based on the 16S rRNA gene and GyrB amino acid sequences, obtained by using three different algorithms, were consistent and showed that these three species constituted a deeply rooted cluster separated from the clades represented by the genera Bacillus, Planococcus, Planomicrobium, Sporosarcina, Lysinibacillus, Viridibacillus, Kurthia and Geobacillus, supporting their placement in the genus Bhargavaea. All three type strains have menaquinone MK-8 as the major respiratory quinone and showed similar fatty acid profiles. The main polar lipids present in the three type strains were diphosphatidylglycerol and phosphatidylglycerol, and the three strains showed peptidoglycan type A4α with L-lysine as the diagnostic diamino acid. The DNA G+C contents of Bacillus beijingensis DSM 19037(T), Bacillus ginsengi DSM 19038(T) and Bhargavaea cecembensis LMG 24411(T) were 53.1, 50.2 and 53.7 mol%, respectively. The level of DNA-DNA hybridization among the three strains was 57-39 %, indicating that they are members of different species of the genus Bhargavaea. The phenotypic data are consistent with the placement of these three species in a single genus and support their differentiation at the species level. On the basis of these data, we have emended the description of the genus Bhargavaea and propose the reclassification of Bacillus beijingensis and Bacillus ginsengi to the genus Bhargavaea, as Bhargavaea beijingensis comb. nov. (type strain ge10(T)  = DSM 19037(T)  = CGMCC 1.6762(T)) and Bhargavaea ginsengi comb. nov. (type strain ge14(T)  = DSM 19038(T)  = CGMCC 1.6763(T)).

Comparative analysis of microbial community of novel lactic acid fermentation inoculated with different undefined mixed cultures.

PubMed

Liang, Shaobo; Gliniewicz, Karol; Mendes-Soares, Helena; Settles, Matthew L; Forney, Larry J; Coats, Erik R; McDonald, Armando G

2015-03-01

Three undefined mixed cultures (activated sludge) from different municipal wastewater treatment plants were used as seeds in a novel lactic acid fermentation process fed with potato peel waste (PPW). Anaerobic sequencing batch fermenters were run under identical conditions to produce predominantly lactic acid. Illumina sequencing was used to examine the 16S rRNA genes of bacteria in the three seeds and fermenters. Results showed that the structure of microbial communities of three seeds were different. All three fermentation products had unique community structures that were dominated (>96%) by species of the genus Lactobacillus, while members of this genus constituted <0.1% in seeds. The species of Lactobacillus sp. differed among the three fermentations. Results of this study suggest the structure of microbial communities in lactic acid fermentation of PPW with undefined mixed cultures were robust and resilient, which provided engineering prospects for the microbial utilization of carbohydrate wastes to produce lactic acid. Copyright © 2014 Elsevier Ltd. All rights reserved.

Isolation and preliminary characterization of a Cd-binding protein from Tenebrio molitor (Coleoptera).

PubMed

Pedersen, S A; Kristiansen, E; Andersen, R A; Zachariassen, K E

2007-04-01

The effect of cadmium (Cd) exposure on Cd-binding ligands was investigated for the first time in a beetle (Coleoptera), using the mealworm Tenebrio molitor (L) as a model species. Exposure to Cd resulted in an approximate doubling of the Cd-binding capacity of the protein extracts from whole animals. Analysis showed that the increase was mainly explained by the induction of a Cd-binding protein of 7134.5 Da, with non-metallothionein characteristics. Amino acid analysis and de novo sequencing revealed that the protein has an unusually high content of the acidic amino acids aspartic and glutamic acid that may explain how this protein can bind Cd even without cysteine residues. Similarities in the amino acid composition suggest it to belong to a group of little studied proteins often referred to as "Cd-binding proteins without high cysteine content". This is the first report on isolation and peptide sequence determination of such a protein from a coleopteran.

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

Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library.

PubMed

Krumpe, Lauren R H; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2007-10-05

Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

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.

Neisseria arctica sp. nov. isolated from nonviable eggs of greater white-fronted geese (Anser albifrons) in Arctic Alaska

USGS Publications Warehouse

Hansen, Cristina M.; Himschoot, Elizabeth; Hare, Rebekah F.; Meixell, Brandt W.; Van Hemert, Caroline R.; Hueffer, Karsten

2017-01-01

During the summers of 2013 and 2014, isolates of a novel Gram-negative coccus in the Neisseria genus were obtained from the contents of nonviable greater white-fronted goose (Anser albifrons) eggs on the Arctic Coastal Plain of Alaska. We used a polyphasic approach to determine whether these isolates represent a novel species. 16S rRNA gene sequences, 23S rRNA gene sequences, and chaperonin 60 gene sequences suggested that these Alaskan isolates are members of a distinct species that is most closely related to Neisseria canis, N. animaloris, and N. shayeganii. Analysis of the rplF gene additionally showed that our isolates are unique and most closely related to N. weaveri. Average nucleotide identity of the whole genome sequence of our type strain was between 71.5% and 74.6% compared to close relatives, further supporting designation as a novel species. Fatty acid methyl ester analysis showed a predominance of C14:0, C16:0, and C16:1ω7c fatty acids. Finally, biochemical characteristics distinguished our isolates from other Neisseria species. The name Neisseria arctica (type strain KH1503T = ATCC TSD-57T = DSM 103136T) is proposed.

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

PubMed

Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Aspergillus bertholletius sp. nov. from Brazil Nuts

PubMed Central

Taniwaki, Marta H.; Pitt, John I.; Iamanaka, Beatriz T.; Sartori, Daniele; Copetti, Marina V.; Balajee, Arun; Fungaro, Maria Helena P.; Frisvad, Jens C.

2012-01-01

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial β-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial β-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228). PMID:22952594

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.

Precision Aliphatic Polyesters with Alternating Microstructures via Cross-Metathesis Polymerization: An Event of Sequence Control.

PubMed

Li, Zi-Long; Zeng, Fu-Rong; Ma, Ji-Mei; Sun, Lin-Hao; Zeng, Zhen; Jiang, Hong

2017-06-01

Sequence-regulated polymerization is realized upon sequential cross-metathesis polymerization (CMP) and exhaustive hydrogenation to afford precision aliphatic polyesters with alternating sequences. This strategy is particularly suitable for the arrangement of well-known monomer units including glycolic acid, lactic acid, and caprolactic acid on polymer chain in a predetermined sequence. First of all, structurally asymmetric monomers bearing acrylate and α-olefin terminuses are generated in an efficient and straightforward fashion. Subsequently, cross-metathesis (co)polymerization of M1 and M2 using the Hoveyda-Grubbs second-generation catalyst (HG-II) furnishes P1-P3, respectively. Finally, hydrogenation yields the desired saturated polyesters HP1-HP3. It is noteworthy that the ε-caprolactone-derived unit is generated in situ rather than introduced to tailor-made monomers prior to CMP. NMR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) results verify the microstructural periodicity of these precision polyesters. Differential scanning calorimetry (DSC) results reflect that polyesters without methyl side groups exhibit crystallinity, and unsaturated polyester samples show higher glass transition temperatures than their hydrogenated counterparts owing to structural rigidity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Predicting DNA binding proteins using support vector machine with hybrid fractal features.

PubMed

Niu, Xiao-Hui; Hu, Xue-Hai; Shi, Feng; Xia, Jing-Bo

2014-02-21

DNA-binding proteins play a vitally important role in many biological processes. Prediction of DNA-binding proteins from amino acid sequence is a significant but not fairly resolved scientific problem. Chaos game representation (CGR) investigates the patterns hidden in protein sequences, and visually reveals previously unknown structure. Fractal dimensions (FD) are good tools to measure sizes of complex, highly irregular geometric objects. In order to extract the intrinsic correlation with DNA-binding property from protein sequences, CGR algorithm, fractal dimension and amino acid composition are applied to formulate the numerical features of protein samples in this paper. Seven groups of features are extracted, which can be computed directly from the primary sequence, and each group is evaluated by the 10-fold cross-validation test and Jackknife test. Comparing the results of numerical experiments, the group of amino acid composition and fractal dimension (21-dimension vector) gets the best result, the average accuracy is 81.82% and average Matthew's correlation coefficient (MCC) is 0.6017. This resulting predictor is also compared with existing method DNA-Prot and shows better performances. © 2013 The Authors. Published by Elsevier Ltd All rights reserved.

Characterization of the cDNA coding for rat brain cysteine sulfinate decarboxylase: brain and liver enzymes are identical proteins encoded by two distinct mRNAs.

PubMed

Tappaz, M; Bitoun, M; Reymond, I; Sergeant, A

1999-09-01

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

PubMed Central

Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

1998-01-01

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

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.

Saccharomyces cerevisiae sigma 1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance.

PubMed

Takagi, H; Shichiri, M; Takemura, M; Mohri, M; Nakamori, S

2000-08-01

We discovered on the chromosome of Saccharomyces cerevisiae Sigma 1278b novel genes involved in L-proline analogue L-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the L-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Sigma 1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for L-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for L-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Sigma 1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for L-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to L-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.

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.

Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

NASA Astrophysics Data System (ADS)

Li, Jiakai; Wu, Xiangwei; Tan, Jing; Zhao, Ruixiang; Deng, Lingwei; Liu, Xiande

2015-07-01

P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 cDNA designated PtHSP20 was cloned from P. textile. The full-length cDNA of PtHSP20 is 1 090 bp long and contains a 5' untranslated region (UTR) of 93 bp, a 3' UTR of 475 bp, and an open reading frame (ORF) of 522 bp. The PtHSP20 cDNA encodes 173 amino acid residues and has a molecular mass of 20.22 kDa and an isoelectric point of 6.2. Its predicted amino acid sequence shows that PtHSP20 contains a typical α-crystallin domain (residues 77-171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, PtHSP20 shows moderate homology to other mollusk sHSPs. PtHSP20 mRNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of PtHSP20 mRNA was down-regulated in all of the tissues except the adductor muscle and gonad.

Isolation and Characterization of the PKAr Gene From a Plant Pathogen, Curvularia lunata.

PubMed

Liu, T; Ma, B C; Hou, J M; Zuo, Y H

2014-09-01

By using EST database from a full-length cDNA library of Curvularia lunata, we have isolated a 2.9 kb cDNA, termed PKAr. An ORF of 1,383 bp encoding a polypeptide of 460 amino acids with molecular weight 50.1 kDa, (GeneBank Acc. No. KF675744) was cloned. The deduced amino acid sequence of the PKAr shows 90 and 88 % identity with cAMP-dependent protein kinase A regulatory subunit from Alternaria alternate and Pyrenophora tritici-repentis Pt-1C-BFP, respectively. Database analysis revealed that the deduced amino acid sequence of PKAr shares considerable similarity with that of PKA regulatory subunits in other organisms, particularly in the conserved regions. No introns were identified within the 1,383 bp of ORF compared with PKAr genomic DNA sequence. Southern blot indicated that PKAr existed as a single copy per genome. The mRNA expression level of PKAr in different development stages were demonstrated using real-time quantitative PCR. The results showed that the level of PKAr expression was highest in vegetative growth mycelium, which indicated it might play an important role in the vegetative growth of C. lunata. These results provided a fundamental supporting research on the function of PKAr in plant pathogen, C. lunata.

Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases.

PubMed Central

Arimitsu, E; Aoki, S; Ishikura, S; Nakanishi, K; Matsuura, K; Hara, A

1999-01-01

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins. PMID:10477285

The CD8α gene in duck (Anatidae): cloning, characterization, and expression during viral infection.

PubMed

Xu, Qi; Chen, Yang; Zhao, Wen Ming; Huang, Zheng Yang; Duan, Xiu Jun; Tong, Yi Yu; Zhang, Yang; Li, Xiu; Chang, Guo Bin; Chen, Guo Hong

2015-02-01

Cluster of differentiation 8 alpha (CD8α) is critical for cell-mediated immune defense and T-cell development. Although CD8α sequences have been reported for several species, very little is known about CD8α in ducks. To elucidate the mechanisms involved in the innate and adaptive immune responses of ducks, we cloned CD8α coding sequences from domestic, Muscovy, Mallard, and Spotbill ducks using reverse transcription polymerase chain reaction (RT-PCR). Each sequence consisted of 714 nucleotides and encoded a signal peptide, an IgV-like domain, a stalk region, a transmembrane region, and a cytoplasmic tail. We identified 58 nucleotide differences and 37 amino acid differences among the four types of duck; of these, 53 nucleotide and 33 amino acid differences were between Muscovy ducks and the other duck species. The CD8α cDNA sequence from domestic duck consisted of a 61-nucleotide 5' untranslated region (UTR), a 714-nucleotide open reading frame, and an 849-nucleotide 3' UTR. Multiple sequence alignments showed that the amino acid sequence of CD8α is conserved in vertebrates. RT-PCR revealed that expression of CD8α mRNA of domestic ducks was highest in the thymus and very low in the kidney, cerebrum, cerebellum, and muscle. Immunohistochemical analyses detected CD8α on the splenic corpuscle and periarterial lymphatic sheath of the spleen. CD8α mRNA in domestic ducklings was initially up-regulated, and then down-regulated, in the thymus, spleen, and liver after treatment with duck hepatitis virus type I (DHV-1) or the immunostimulant polyriboinosinic polyribocytidylic acid (poly I:C).

Replacement of C305 in heart/muscle-type isozyme of human carnitine palmitoyltransferase I with aspartic acid and other amino acids.

PubMed

Matsuo, Taisuke; Yamamoto, Atsushi; Yamamoto, Takenori; Otsuki, Kaoru; Yamazaki, Naoshi; Kataoka, Masatoshi; Terada, Hiroshi; Shinohara, Yasuo

2010-04-01

Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.

Veillonella infantium sp. nov., an anaerobic, Gram-stain-negative coccus isolated from tongue biofilm of a Thai child.

PubMed

Mashima, Izumi; Liao, Yu-Chieh; Miyakawa, Hiroshi; Theodorea, Citra F; Thawboon, Boonyanit; Thaweboon, Sroisiri; Scannapieco, Frank A; Nakazawa, Futoshi

2018-04-01

A strain of a novel anaerobic, Gram-stain-negative coccus was isolated from the tongue biofilm of a Thai child. This strain was shown, at the phenotypic level and based on 16S rRNA gene sequencing, to be a member of the genus Veillonella. Comparative analysis of the 16S rRNA, dnaK and rpoB gene sequences indicated that phylogenetically the strain comprised a distinct novel branch within the genus Veillonella. The novel strain showed 99.8, 95.1 and 95.9 % similarity to partial 16S rRNA, dnaK and rpoB gene sequences, respectively, to the type strains of the two most closely related species, Veillonelladispar ATCC 17748 T and Veillonellatobetsuensis ATCC BAA-2400 T . The novel strain could be discriminated from previously reported species of the genus Veillonella based on partial dnaK and rpoB gene sequencing and average nucleotide identity values. The major acid end-product produced by this strain was acetic acid under anaerobic conditions in trypticase-yeast extract-haemin with 1 % (w/v) glucose or fructose medium. Lactate was fermented to acetic acid and propionic acid. Based on these observations, this strain represents a novel species, for which the name Veillonella infantium sp. nov. is proposed. The type strain is T11011-4 T (=JCM 31738 T =TSD-88 T ).

Identification of interleukin-8 converting enzyme as cathepsin L.

PubMed

Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

2003-06-26

IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

A comprehensive bioinformatic analysis of hepatitis D virus full-length genomes.

PubMed

Delfino, C M; Cerrudo, C S; Biglione, M; Oubiña, J R; Ghiringhelli, P D; Mathet, V L

2018-02-06

In association with hepatitis B virus (HBV), hepatitis delta virus (HDV) is a subviral agent that may promote severe acute and chronic forms of liver disease. Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes. However, since 2006, the original classification has been further expanded into eight clades/genotypes. The intergenotype divergence may be as high as 35%-40% over the entire RNA genome, whereas sequence heterogeneity among the isolates of a given genotype is <20%; furthermore, HDV recombinants have been clearly demonstrated. The genetic diversity of HDV is related to the geographic origin of the isolates. This study shows the first comprehensive bioinformatic analysis of the complete available set of HDV sequences, using both nucleotide and protein phylogenies (based on an evolutionary model selection, gamma distribution estimation, tree inference and phylogenetic distance estimation), protein composition analysis and comparison (based on the presence of invariant residues, molecular signatures, amino acid frequencies and mono- and di-amino acid compositional distances), as well as amino acid changes in sequence evolution. Taking into account the congruent and consistent results of both nucleotide and amino acid analyses of GenBank available sequences (recorded as of January, 2017), we propose that the eight hepatitis D virus genotypes may be grouped into three large genogroups fully supported by their shared characteristics. © 2018 John Wiley & Sons Ltd.

Functional dissection of the alphavirus capsid protease: sequence requirements for activity.

PubMed

Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M; Schaefer, Stephan

2010-11-18

The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

Identification of a hydratase and a class II aldolase involved in biodegradation of the organic solvent tetralin.

PubMed

Hernáez, M J; Floriano, B; Ríos, J J; Santero, E

2002-10-01

Two new genes whose products are involved in biodegradation of the organic solvent tetralin were identified. These genes, designated thnE and thnF, are located downstream of the previously identified thnD gene and code for a hydratase and an aldolase, respectively. A sequence comparison of enzymes similar to ThnE showed the significant similarity of hydratases involved in biodegradation pathways to 4-oxalocrotonate decarboxylases and established four separate groups of related enzymes. Consistent with the sequence information, characterization of the reaction catalyzed by ThnE showed that it hydrated a 10-carbon dicarboxylic acid. The only reaction product detected was the enol tautomer, 2,4-dihydroxydec-2-ene-1,10-dioic acid. The aldolase ThnF showed significant similarity to aldolases involved in different catabolic pathways whose substrates are dihydroxylated dicarboxylic acids and which yield pyruvate and a semialdehyde. The reaction products of the aldol cleavage reaction catalyzed by ThnF were identified as pyruvate and the seven-carbon acid pimelic semialdehyde. ThnF and similar aldolases showed conservation of the active site residues identified by the crystal structure of 2-dehydro-3-deoxy-galactarate aldolase, a class II aldolase with a novel reaction mechanism, suggesting that these similar enzymes are class II aldolases. In contrast, ThnF did not show similarity to 4-hydroxy-2-oxovalerate aldolases of other biodegradation pathways, which are significantly larger and apparently are class I aldolases.

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.

[Genetic characteristics of hemagglutinin in measles viruses isolated in Henan Province, China].

PubMed

Feng, Da-Xing; Seng, Ming-Hua; Liu, Qian; Zhang, Zhen-Ying

2014-03-01

This study aims to investigate the genetic characteristics of hemagglutinin in wild-type measles viruses in Henan Province, China and to provide a basis for measles control and elimination. Specimens were collected from suspected measles cases in Henan during 2008-2012. Cell culture was performed for virus isolation, and RT-PCR was used to amplify hemagglutinin gene. The PCR products were sequenced and analyzed, including construction of phylogenetic tree and analysis of the distance between the isolated virus and the reference virus; then, the variations in predicted amino acids were analyzed. The results showed that 12 measles viruses were isolated in Henan Province and identified as H1a genotype; the nucleotide and amino acid homologies were 98.0%-100% and 97.2%-99.8%, respectively. One glycosylation site changed in all the 12 sequences because of the amino acid mutation from serine to asparagine at the 240th site, as compared with Edmonston-wt. USA/54/A. Overall, the wild-type measles virus genotype circulating in Henan Province from 2008 to 2012 was H1a, with high homology between strains; there were some variations in amino acid sequences, resulting in glycosylation site deletion.

Nucleotide sequence of the gene for the Mr 32,000 thylakoid membrane protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of Mr 38,950

PubMed Central

Zurawski, Gerard; Bohnert, Hans J.; Whitfeld, Paul R.; Bottomley, Warwick

1982-01-01

The gene for the so-called Mr 32,000 rapidly labeled photosystem II thylakoid membrane protein (here designated psbA) of spinach (Spinacia oleracea) chloroplasts is located on the chloroplast DNA in the large single-copy region immediately adjacent to one of the inverted repeat sequences. In this paper we show that the size of the mRNA for this protein is ≈ 1.25 kilobases and that the direction of transcription is towards the inverted repeat unit. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of Mr 38,950. The nucleotide sequence of psbA from Nicotiana debneyi also has been determined, and comparison of the sequences from the two species shows them to be highly conserved (>95% homology) throughout the entire reading frame. Conservation of the amino acid sequence is absolute, there being no changes in a total of 353 residues. This leads us to conclude that the primary translation product of psbA must be a protein of Mr 38,950. The protein is characterized by the complete absence of lysine residues and is relatively rich in hydrophobic amino acids, which tend to be clustered. Transcription of spinach psbA starts about 86 base pairs before the first ATG codon. Immediately upstream from this point there is a sequence typical of that found in E. coli promoters. An almost identical sequence occurs in the equivalent region of N. debneyi DNA. Images PMID:16593262

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

Complementary DNA cloning and constitutive expression of cytochrome P450 1C1 in the gills of carp (Cyprinus carpio).

PubMed

Itakura, Takao; El-Kady, Mohamed; Mitsuo, Ryoichi; Kaminishi, Yoshio

2005-01-01

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from carp liver after intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 244 bp, an open reading frame of 1572 bp coding for 524 amino acids, a stop codon, and a 3' noncoding region of 965 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The deduced amino acid sequence of this cDNA was 82.1% and 80.2% similar to Japanese eel and scup CYP1C1 sequences, respectively, while it exhibited a similarity of 74.9% with the scup CYP1C2 sequence. The deduced amino acid sequence of carp CYP1C1 showed similarities with those of the reported CYP1B1s of teleosts and mammals of 48.4, 48.8, 48.2, 48.6, 45.3, and 45.5% for carp CYP1B1, carp CYP1B2, plaice CYP1B1, and human, rat, and mouse CYP1B1, respectively. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of the CYP1C subfamily to CYP1B than to CYP1A. The tree showed the possibility of the existence of CYP1C subfamily genes in mammalian species. Northern blot analysis for the liver, intestine, gills, and kidney showed no detectable induced expression but constitutive expression in the gill organs.

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

PubMed Central

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

2005-01-01

We have developed an algorithm for designing multiple sequences of nucleic acids that have a uniform melting temperature between the sequence and its complement and that do not hybridize non-specifically with each other based on the minimum free energy (ΔGmin). Sequences that satisfy these constraints can be utilized in computations, various engineering applications such as microarrays, and nano-fabrications. Our algorithm is a random generate-and-test algorithm: it generates a candidate sequence randomly and tests whether the sequence satisfies the constraints. The novelty of our algorithm is that the filtering method uses a greedy search to calculate ΔGmin. This effectively excludes inappropriate sequences before ΔGmin is calculated, thereby reducing computation time drastically when compared with an algorithm without the filtering. Experimental results in silico showed the superiority of the greedy search over the traditional approach based on the hamming distance. In addition, experimental results in vitro demonstrated that the experimental free energy (ΔGexp) of 126 sequences correlated well with ΔGmin (|R| = 0.90) than with the hamming distance (|R| = 0.80). These results validate the rationality of a thermodynamic approach. We implemented our algorithm in a graphic user interface-based program written in Java. PMID:15701762

Acid-base chemical reaction model for nucleation rates in the polluted atmospheric boundary layer.

PubMed

Chen, Modi; Titcombe, Mari; Jiang, Jingkun; Jen, Coty; Kuang, Chongai; Fischer, Marc L; Eisele, Fred L; Siepmann, J Ilja; Hanson, David R; Zhao, Jun; McMurry, Peter H

2012-11-13

Climate models show that particles formed by nucleation can affect cloud cover and, therefore, the earth's radiation budget. Measurements worldwide show that nucleation rates in the atmospheric boundary layer are positively correlated with concentrations of sulfuric acid vapor. However, current nucleation theories do not correctly predict either the observed nucleation rates or their functional dependence on sulfuric acid concentrations. This paper develops an alternative approach for modeling nucleation rates, based on a sequence of acid-base reactions. The model uses empirical estimates of sulfuric acid evaporation rates obtained from new measurements of neutral molecular clusters. The model predicts that nucleation rates equal the sulfuric acid vapor collision rate times a prefactor that is less than unity and that depends on the concentrations of basic gaseous compounds and preexisting particles. Predicted nucleation rates and their dependence on sulfuric acid vapor concentrations are in reasonable agreement with measurements from Mexico City and Atlanta.

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.

Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli.

PubMed

Perelle, S; Gibert, M; Boquet, P; Popoff, M R

1993-12-01

The iota toxin which is produced by Clostridium perfringens type E, is a binary toxin consisting of two independent polypeptides: Ia, which is an ADP-ribosyltransferase, and Ib, which is involved in the binding and internalization of the toxin into the cell. Two degenerate oligonucleotide probes deduced from partial amino acid sequence of each component of C. spiroforme toxin, which is closely related to the iota toxin, were used to clone three overlapping DNA fragments containing the iota-toxin genes from C. perfringens type E plasmid DNA. Two genes, in the same orientation, coding for Ia (387 amino acids) and Ib (875 amino acids) and separated by 243 noncoding nucleotides were identified. A predicted signal peptide was found for each component, and the secreted Ib displays two domains, the propeptide (172 amino acids) and the mature protein (664 amino acids). The Ia gene has been expressed in Escherichia coli and C. perfringens, under the control of its own promoter. The recombinant polypeptide obtained was recognized by Ia antibodies and ADP-ribosylated actin. The expression of the Ib gene was obtained in E. coli harboring a recombinant plasmid encompassing the putative promoter upstream of the Ia gene and the Ia and Ib genes. Two residues which have been found to be involved in the NAD+ binding site of diphtheria and pseudomonas toxins are conserved in the predicted Ia sequence (Glu-14 and Trp-19). The predicted amino acid Ib sequence shows 33.9% identity with and 54.4% similarity to the protective antigen of the anthrax toxin complex. In particular, the central region of Ib, which contains a predicted transmembrane segment (Leu-292 to Ser-308), presents 45% identity with the corresponding protective antigen sequence which is involved in the translocation of the toxin across the cell membrane.

Differences in acid tolerance between Bifidobacterium breve BB8 and its acid-resistant derivative B. breve BB8dpH, revealed by RNA-sequencing and physiological analysis.

PubMed

Yang, Xu; Hang, Xiaomin; Tan, Jing; Yang, Hong

2015-06-01

Bifidobacteria are common inhabitants of the human gastrointestinal tract, and their application has increased dramatically in recent years due to their health-promoting effects. The ability of bifidobacteria to tolerate acidic environments is particularly important for their function as probiotics because they encounter such environments in food products and during passage through the gastrointestinal tract. In this study, we generated a derivative, Bifidobacterium breve BB8dpH, which displayed a stable, acid-resistant phenotype. To investigate the possible reasons for the higher acid tolerance of B. breve BB8dpH, as compared with its parental strain B. breve BB8, a combined transcriptome and physiological approach was used to characterize differences between the two strains. An analysis of the transcriptome by RNA-sequencing indicated that the expression of 121 genes was increased by more than 2-fold, while the expression of 146 genes was reduced more than 2-fold, in B. breve BB8dpH. Validation of the RNA-sequencing data using real-time quantitative PCR analysis demonstrated that the RNA-sequencing results were highly reliable. The comparison analysis, based on differentially expressed genes, suggested that the acid tolerance of B. breve BB8dpH was enhanced by regulating the expression of genes involved in carbohydrate transport and metabolism, energy production, synthesis of cell envelope components (peptidoglycan and exopolysaccharide), synthesis and transport of glutamate and glutamine, and histidine synthesis. Furthermore, an analysis of physiological data showed that B. breve BB8dpH displayed higher production of exopolysaccharide and lower H(+)-ATPase activity than B. breve BB8. The results presented here will improve our understanding of acid tolerance in bifidobacteria, and they will lead to the development of new strategies to enhance the acid tolerance of bifidobacterial strains. Copyright © 2015 Elsevier Ltd. All rights reserved.

Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

PubMed Central

Kitagawa, Wataru; Takami, Sachiko; Miyauchi, Keisuke; Masai, Eiji; Kamagata, Yoichi; Tiedje, James M.; Fukuda, Masao

2002-01-01

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC. PMID:11751829

Effect of lysine to arginine mutagenesis in the V3 loop of HIV-1 gp120 on viral entry efficiency and neutralization.

PubMed

Schwalbe, Birco; Schreiber, Michael

2015-01-01

HIV-1 infection is characterized by an ongoing replication leading to T-lymphocyte decline which is paralleled by the switch from CCR5 to CXCR4 coreceptor usage. To predict coreceptor usage, several computer algorithms using gp120 V3 loop sequence data have been developed. In these algorithms an occupation of the V3 positions 11 and 25, by one of the amino acids lysine (K) or arginine (R), is an indicator for CXCR4 usage. Amino acids R and K dominate at these two positions, but can also be identified at positions 9 and 10. Generally, CXCR4-viruses possess V3 sequences, with an overall positive charge higher than the V3 sequences of R5-viruses. The net charge is calculated by subtracting the number of negatively charged amino acids (D, aspartic acid and E, glutamic acid) from the number of positively charged ones (K and R). In contrast to D and E, which are very similar in their polar and acidic properties, the characteristics of the R guanidinium group differ significantly from the K ammonium group. However, in coreceptor predictive computer algorithms R and K are both equally rated. The study was conducted to analyze differences in infectivity and coreceptor usage because of R-to-K mutations at the V3 positions 9, 10 and 11. V3 loop mutants with all possible RRR-to-KKK triplets were constructed and analyzed for coreceptor usage, infectivity and neutralization by SDF-1α and RANTES. Virus mutants R9R10R11 showed the highest infectivity rates, and were inhibited more efficiently in contrast to the K9K10K11 viruses. They also showed higher efficiency in a virus-gp120 paired infection assay. Especially V3 loop position 9 was relevant for a switch to higher infectivity when occupied by R. Thus, K-to-R exchanges play a role for enhanced viral entry efficiency and should therefore be considered when the viral phenotype is predicted based on V3 sequence data.

Effect of Lysine to Arginine Mutagenesis in the V3 Loop of HIV-1 gp120 on Viral Entry Efficiency and Neutralization

PubMed Central

Schwalbe, Birco; Schreiber, Michael

2015-01-01

HIV-1 infection is characterized by an ongoing replication leading to T-lymphocyte decline which is paralleled by the switch from CCR5 to CXCR4 coreceptor usage. To predict coreceptor usage, several computer algorithms using gp120 V3 loop sequence data have been developed. In these algorithms an occupation of the V3 positions 11 and 25, by one of the amino acids lysine (K) or arginine (R), is an indicator for CXCR4 usage. Amino acids R and K dominate at these two positions, but can also be identified at positions 9 and 10. Generally, CXCR4-viruses possess V3 sequences, with an overall positive charge higher than the V3 sequences of R5-viruses. The net charge is calculated by subtracting the number of negatively charged amino acids (D, aspartic acid and E, glutamic acid) from the number of positively charged ones (K and R). In contrast to D and E, which are very similar in their polar and acidic properties, the characteristics of the R guanidinium group differ significantly from the K ammonium group. However, in coreceptor predictive computer algorithms R and K are both equally rated. The study was conducted to analyze differences in infectivity and coreceptor usage because of R-to-K mutations at the V3 positions 9, 10 and 11. V3 loop mutants with all possible RRR-to-KKK triplets were constructed and analyzed for coreceptor usage, infectivity and neutralization by SDF-1α and RANTES. Virus mutants R9R10R11 showed the highest infectivity rates, and were inhibited more efficiently in contrast to the K9K10K11 viruses. They also showed higher efficiency in a virus-gp120 paired infection assay. Especially V3 loop position 9 was relevant for a switch to higher infectivity when occupied by R. Thus, K-to-R exchanges play a role for enhanced viral entry efficiency and should therefore be considered when the viral phenotype is predicted based on V3 sequence data. PMID:25785610

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.

Detection of Rickettsia helvetica and Candidatus R. tarasevichiae DNA in Ixodes persulcatus ticks collected in Northeastern European Russia (Komi Republic).

PubMed

Kartashov, Mikhail Yu; Glushkova, Ludmila I; Mikryukova, Tamara P; Korabelnikov, Igor V; Egorova, Yulia I; Tupota, Natalia L; Protopopova, Elena V; Konovalova, Svetlana N; Ternovoi, Vladimir A; Loktev, Valery B

2017-06-01

The number of tick-borne infections in the northern European regions of Russia has increased considerably in the last years. In the present study, 676 unfed adult Ixodes persulcatus ticks were collected in the Komi Republic from 2011 to 2013 to study tick-borne rickettsioses. Rickettsia spp. DNA was detected by PCR in 51 (7.6%) ticks. The nucleotide sequence analysis of gltA fragments (765bp) from 51 ticks indicated that 60.8% and 39.2% of the ticks were infected with Rickettsia helvetica and Candidatus R. tarasevichiae, respectively. The gltA fragments showed 100% identity with those of Candidatus R. tarasevichiae previously discovered in Siberia and China, whereas R. helvetica showed 99.9% sequence identity with European isolates. The ompB had 8 nucleotide substitutions, 6 of which resulted in amino acid substitutions. In the sca9 gene, 3 nucleotide substitutions were detected, and only one resulted in amino acid substitution. The smpA, ompW, and β-lactamase genes of R. helvetica also showed a high level of sequence identity. Copyright © 2017 Elsevier GmbH. All rights reserved.

BnNHL18A shows a localization change by stress-inducing chemical treatments

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

Lee, Suk-Bae; Ham, Byung-Kook; Park, Jeong Mee

2006-01-06

The two genes, named BnNHL18A and BnNHL18B, showing sequence homology with Arabidopsis NDR1/HIN1-like (NHL) genes, were isolated from cDNA library prepared with oilseed rape (Brassica napus) seedlings treated with NaCl. The transcript level of BnNHL18A was increased by sodium chloride, ethephon, hydrogen peroxide, methyl jasmonate, or salicylic acid treatment. The coding regions of BnNHL18A and BnNHL18B contain a sarcolipin (SLN)-like sequence. Analysis of the localization of smGFP fusion proteins showed that BnNHL18A is mainly localized to endoplasmic reticulum (ER). This result suggests that the SLN-like sequence plays a role in retaining proteins in ER membrane in plants. In response tomore » NaCl, hydrogen peroxide, ethephon, and salicylic acid treatments, the protein localization of BnNHL18A was changed. Our findings suggest a common function of BnNHL18A in biotic and abiotic stresses, and demonstrate the presence of the shared mechanism of protein translocalization between the responses to plant pathogen and to osmotic stress.« less

Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere.

PubMed

Cid, Fernanda P; Maruyama, Fumito; Murase, Kazunori; Graether, Steffen P; Larama, Giovanni; Bravo, Leon A; Jorquera, Milko A

2018-05-01

Genome analyses are being used to characterize plant growth-promoting (PGP) bacteria living in different plant compartiments. In this context, we have recently isolated bacteria from the phyllosphere of an Antarctic plant (Deschampsia antarctica) showing ice recrystallization inhibition (IRI), an activity related to the presence of antifreeze proteins (AFPs). In this study, the draft genomes of six phyllospheric bacteria showing IRI activity were sequenced and annotated according to their functional gene categories. Genome sizes ranged from 5.6 to 6.3 Mbp, and based on sequence analysis of the 16S rRNA genes, five strains were identified as Pseudomonas and one as Janthinobacterium. Interestingly, most strains showed genes associated with PGP traits, such as nutrient uptake (ammonia assimilation, nitrogen fixing, phosphatases, and organic acid production), bioactive metabolites (indole acetic acid and 1-aminocyclopropane-1-carboxylate deaminase), and antimicrobial compounds (hydrogen cyanide and pyoverdine). In relation with IRI activity, a search of putative AFPs using current bioinformatic tools was also carried out. Despite that genes associated with reported AFPs were not found in these genomes, genes connected to ice-nucleation proteins (InaA) were found in all Pseudomonas strains, but not in the Janthinobacterium strain.

Cloning and expression of a small heat and salt tolerant protein (Hsp22) from Chaetomium globosum.

PubMed

Aggarwal, Rashmi; Gupta, Sangeeta; Sharma, Sapna; Banerjee, Sagar; Singh, Priyanka

2012-11-01

The present study reports molecular characterization of small heat shock protein gene in Indian isolates of Chaetomium globosum, C. perlucidum, C. reflexum, C. cochlioides and C. cupreum. Six isolates of C. globosum and other species showed a band of 630bp using specific primers. Amplified cDNA product of C. globosum (Cg 1) cloned and sequenced showed 603bp open reading frame encoding 200 amino-acids. The protein sequence had a molecular mass of 22 kDa and was therefore, named Hsp22. BlastX analysis revealed that the gene codes for a protein homologous to previously characterized Hsp22.4 gene from C. globosum (AAR36902.1, XP 001229241.1) and shared 95% identity in amino acid sequence. It also showed varying degree of similarities with small Hsp protein from Neurospora spp. (60%), Myceliophthora sp. (59%), Glomerella sp. (50%), Hypocrea sp. (52%), and Fusarium spp. (51%). This gene was further cloned into pET28a (+) and transformed E. coli BL21 cells were induced by IPTG, and the expressed protein of 30 kDa was analyzed by SDS-PAGE. The IPTG induced transformants displayed significantly greater resistance to NaCl and Na2CO3 stresses.

Major proteins of yam bean tubers.

PubMed

Gomes, A V; Sirju-Charran, G; Barnes, J A

1997-09-01

The tuberous roots of the Mexican yam bean, jicama, (Pachyrhizus erosus L. Urban) contained large quantities of two acidic glycoproteins which accounted for more than 70% of the total soluble proteins (about 3 g per 100 g of tuber on a dry weight basis). The two major proteins, tentatively named YBG1 and YBG2, had apparent M(r)s of 28,000 and 26,000, respectively, by SDS-PAGE. A third protein named YBP22 which accounted for 2-5% of the total soluble proteins had an M(r) of 22,000. YBG1 and YBG2 exhibited great similarity on the basis of their amino acid composition and had identical N-terminal amino acid sequences. The first 23 amino acids in the N-terminal region of YBG2 were DDLPDYVDWRDYGAVTRIKNQGQ which showed strong homology with the papain class of cysteine proteases. YBG1 and YBG2 were found to bind to a Concanavalin A-Sepharose column and were also stained positively by a sensitive glycoprotein stain. Both glycoproteins exhibited cysteine proteolytic activity. In contrast, YBP22 showed sequence homology with several known protease inhibitors, and a polyclonal antibody raised against this protein cross reacted with soybean trypsin inhibitor.

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.

A Novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors.

PubMed

van Verk, Marcel C; Pappaioannou, Dimitri; Neeleman, Lyda; Bol, John F; Linthorst, Huub J M

2008-04-01

PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions -564 (box WK(1)) and -859 (box WK(2)). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC[C/T]) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK(1) box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a (as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoterbeta-glucuronidase (GUS) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35SNtWRKY12 and PR-1aGUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter.

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

Molecular cloning, characterization, and immunolocalization of two lactate dehydrogenase homologous genes from Taenia solium.

PubMed

Du, Wuying; Hu, Fengyu; Yang, Yabo; Hu, Dong; Hu, Xuchu; Yu, Xinbing; Xu, Jin; Dai, Jialin; Liao, Xinjiang; Huang, Jiang

2011-09-01

Two novel genes encoding lactate dehydrogenase A (LDHA) and B (LDHB) homologues, respectively, were identified from the cDNA libraries of adult Taenia solium (T. solium). The two deduced amino acid sequences both show more than 50% identity to the homologues for Danio rerio, Xenopus laevis, Schistosoma japonicum, Sus scrofa, Homo sapiens, et al. The identity of the amino acid sequence between TsLDHA and TsLDHB is 57.4%, and that of the nucleotide sequence is 61.5%. Recombinant TsLDHA homologue (rTsLDHA) and TsLDHB homologue (rTsLDHB) were expressed in Escherichia coli BL21/DE3 and purified. Though there were some differences in the sequence, the two LDH isozyme homologues show similarity in the conserved LDH domain, topological structure, primary immunological traits, localization on the tegument of T. solium adult, and partial physicochemical properties. The linear B-cell epitope analysis of TsLDHA and TsLDHB discovered a TsLDHA specific epitope. The purified rTsLDHA and rTsLDHB could be recognized by rat immuno-sera, serum from swine, or a patient infected with T. solium, respectively, but Western blot analysis showed cross-reactions, not only between these two LDH members but also with other common human tapeworms or helminths. The results suggested that the two LDH homologues are similar in the characteristics of LDH family, and they are not specific antigens for immunodiagnosis.

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.

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

PubMed

Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

2013-01-01

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in Thailand.

PubMed

Pohuang, Tawatchai; Chansiripornchai, Niwat; Tawatsin, Achara; Sasipreeyajan, Jiroj

2009-09-01

Thirteen field isolates of infectious bronchitis virus (IBV) were isolated from broiler flocks in Thailand between January and June 2008. The 878-bp of the S1 gene covering a hypervariable region was amplified and sequenced. Phylogenetic analysis based on that region revealed that these viruses were separated into two groups (I and II). IBV isolates in group I were not related to other IBV strains published in the GenBank database. Group 1 nucleotide sequence identities were less than 85% and amino acid sequence identities less than 84% in common with IBVs published in the GenBank database. This group likely represents the strains indigenous to Thailand. The isolates in group II showed a close relationship with Chinese IBVs. They had nucleotide sequence identities of 97-98% and amino acid sequence identities 96-98% in common with Chinese IBVs (strain A2, SH and QXIBV). This finding indicated that the recent Thai IBVs evolved separately and at least two groups of viruses are circulating in Thailand.

Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools.

PubMed

Kim, Sung Bae; Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

2017-09-11

The fabrication of artificial luciferases (ALucs) with unique optical properties has a fundamental impact on bioassays and molecular imaging. In this study, we developed a new lineage of ALucs with unique substrate preferences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools. The primary sequence was first created with a sequence logo generator resulting in a total of 11 sibling sequences. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases, and from a prior series of ALucs produced by our laboratory using a smaller basis set. The new lineage of ALucs were strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. A single-residue-level comparison of the C-terminal sequences of new ALucs reveals that some amino acids in the C-terminal ends are greatly influential on the optical intensities but limited in the color variance. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays.

Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry.

PubMed

Setner, Bartosz; Rudowska, Magdalena; Klem, Ewelina; Cebrat, Marek; Szewczuk, Zbigniew

2014-10-01

Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1-azabicyclo[2.2.2]octane (ABCO) or 1,4-diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI-MS) and longer retention times on the reverse-phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision-induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a- and b-type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision-induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI-MS/MS sequencing of peptides. Copyright © 2014 John Wiley & Sons, Ltd.

Complete Genome Sequences of Two Plant-Associated Pseudomonas putida Isolates with Increased Heavy-Metal Tolerance

PubMed Central

Cania, Barbara; Zadel, Urška; Schöler, Anne; Płaza, Grażyna A.; Schloter, Michael

2017-01-01

ABSTRACT We report here the complete genome sequences of two Pseudomonas putida isolates recovered from surface-sterilized roots of Sida hermaphrodita. The two isolates were characterized by an increased tolerance to zinc, cadmium, and lead. Furthermore, the strains showed typical plant growth-promoting properties, such as the production of indole acetic acid, cellulolytic enzymes, and siderophores. PMID:29167255

A homologue of the defender against the apoptotic death gene (dad1 )in UV-exposed Chlamydomonas cells is downregulated with the onset of programmed cell death.

PubMed

Moharikar, Swati; D'Souza, Jacinta S; Rao, Basuthkar J

2007-03-01

We report here the isolation of a homologue of the potential anti-apoptotic gene, defender against apoptotic death (dad1 )from Chlamydomonas reinhardtii cells.Using polymerase chain reaction (PCR),we investigated its expression in the execution process of programmed cell death (PCD)in UV-C exposed dying C.reinhardtii cells.Reverse- transcriptase (RT)-PCR showed that C.reinhardtii dad1 amplification was drastically reduced in UV-C exposed dying C.reinhardtii cells.We connect the downregulation of dad1 with the upregulation of apoptosis protease activating factor-1 (APAF-1)and the physiological changes that occur in C.reinhardtii cells upon exposure to 12 J/m 2 UV-C in order to show a reciprocal relationship between proapoptotic and inhibitor of apoptosis factors.The temporal changes indicate a correlation between the onset of cell death and dad1 downregulation.The sequence of the PCR product of the cDNA encoding the dad1 homologue was aligned with the annotated dad1 (C_20215)from the Chlamydomonas database (http://genome.jgi-psf.org:8080/annotator/servlet/jgi.annotation.Annotation?pDb=chlre2); Annotation?pDb=chlre2 );this sequence was found to show 100% identity,both at the nucleotide and amino acid level. The 327 bp transcript showed an open reading frame of 87 amino acid residues.The deduced amino acid sequence of the putative C.reinhardtii DAD1 homologue showed 54% identity with Oryza sativa, 56 identity with Drosophila melanogaster, 66% identity with Xenopus laevis, and 64% identity with Homo sapiens,Sus scrofa,Gallus gallus,Rattus norvegicus and Mus musculus.

An Amino Acid Code for β-sheet Packing Structure

PubMed Central

Joo, Hyun; Tsai, Jerry

2014-01-01

To understand the relationship between protein sequence and structure, this work extends the knob-socket model in an investigation of β-sheet packing. Over a comprehensive set of β-sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the 2 types of 4 residue packing cliques necessary to describe β-sheet packing were characterized. Both occur between 2 adjacent hydrogen bonded β-strands. First, defining the secondary structure packing within β-sheets, the combined socket or XY:HG pocket consists of 4 residues i,i+2 on one strand and j,j+2 on the other. Second, characterizing the tertiary packing between β-sheets, the knob-socket XY:H+B consists of a 3 residue XY:H socket (i,i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, 2 types of knob-sockets are found: side-chain and main-chain sockets. The amino acid composition of the pockets and knob-sockets reveal the sequence specificity of β-sheet packing. For β-sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side-chain and main-chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β-sheet structure and provide an intuitive topological mapping of β-sheet packing. PMID:24668690

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

PubMed

Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

1998-06-01

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Phylogenetic Characterizations of Highly Mutated EV-B106 Recombinants Showing Extensive Genetic Exchanges with Other EV-B in Xinjiang, China.

PubMed

Song, Yang; Zhang, Yong; Fan, Qin; Cui, Hui; Yan, Dongmei; Zhu, Shuangli; Tang, Haishu; Sun, Qiang; Wang, Dongyan; Xu, Wenbo

2017-02-23

Human enterovirus B106 (EV-B106) is a new member of the enterovirus B species. To date, only three nucleotide sequences of EV-B106 have been published, and only one full-length genome sequence (the Yunnan strain 148/YN/CHN/12) is available in the GenBank database. In this study, we conducted phylogenetic characterisation of four EV-B106 strains isolated in Xinjiang, China. Pairwise comparisons of the nucleotide sequences and the deduced amino acid sequences revealed that the four Xinjiang EV-B106 strains had only 80.5-80.8% nucleotide identity and 95.4-97.3% amino acid identity with the Yunnan EV-B106 strain, indicating high mutagenicity. Similarity plots and bootscanning analyses revealed that frequent intertypic recombination occurred in all four Xinjiang EV-B106 strains in the non-structural region. These four strains may share a donor sequence with the EV-B85 strain, which circulated in Xinjiang in 2011, indicating extensive genetic exchanges between these strains. All Xinjiang EV-B106 strains were temperature-sensitive. An antibody seroprevalence study against EV-B106 in two Xinjiang prefectures also showed low titres of neutralizing antibodies, suggesting limited exposure and transmission in the population. This study contributes the whole genome sequences of EV-B106 to the GenBank database and provides valuable information regarding the molecular epidemiology of EV-B106 in China.

Phylogenetic Characterizations of Highly Mutated EV-B106 Recombinants Showing Extensive Genetic Exchanges with Other EV-B in Xinjiang, China

PubMed Central

Song, Yang; Zhang, Yong; Fan, Qin; Cui, Hui; Yan, Dongmei; Zhu, Shuangli; Tang, Haishu; Sun, Qiang; Wang, Dongyan; Xu, Wenbo

2017-01-01

Human enterovirus B106 (EV-B106) is a new member of the enterovirus B species. To date, only three nucleotide sequences of EV-B106 have been published, and only one full-length genome sequence (the Yunnan strain 148/YN/CHN/12) is available in the GenBank database. In this study, we conducted phylogenetic characterisation of four EV-B106 strains isolated in Xinjiang, China. Pairwise comparisons of the nucleotide sequences and the deduced amino acid sequences revealed that the four Xinjiang EV-B106 strains had only 80.5–80.8% nucleotide identity and 95.4–97.3% amino acid identity with the Yunnan EV-B106 strain, indicating high mutagenicity. Similarity plots and bootscanning analyses revealed that frequent intertypic recombination occurred in all four Xinjiang EV-B106 strains in the non-structural region. These four strains may share a donor sequence with the EV-B85 strain, which circulated in Xinjiang in 2011, indicating extensive genetic exchanges between these strains. All Xinjiang EV-B106 strains were temperature-sensitive. An antibody seroprevalence study against EV-B106 in two Xinjiang prefectures also showed low titres of neutralizing antibodies, suggesting limited exposure and transmission in the population. This study contributes the whole genome sequences of EV-B106 to the GenBank database and provides valuable information regarding the molecular epidemiology of EV-B106 in China. PMID:28230168

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.

Adhesive Proteins of Stalked and Acorn Barnacles Display Homology with Low Sequence Similarities

PubMed Central

Jonker, Jaimie-Leigh; Abram, Florence; Pires, Elisabete; Varela Coelho, Ana; Grunwald, Ingo; Power, Anne Marie

2014-01-01

Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins ‘sticky’ has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7–16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18–26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa). PMID:25295513

Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities.

PubMed

Jonker, Jaimie-Leigh; Abram, Florence; Pires, Elisabete; Varela Coelho, Ana; Grunwald, Ingo; Power, Anne Marie

2014-01-01

Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins 'sticky' has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7-16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18-26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa).

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.

A chondroitin sulfate chain attached to the bone dentin matrix protein 1 NH2-terminal fragment.

PubMed

Qin, Chunlin; Huang, Bingzhen; Wygant, James N; McIntyre, Bradley W; McDonald, Charles H; Cook, Richard G; Butler, William T

2006-03-24

Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein shown by gene ablations to be critical for the proper mineralization of bone and dentin. In the extracellular matrix of these tissues DMP1 is present as fragments representing the NH2-terminal (37 kDa) and COOH-terminal (57 kDa) portions of the cDNA-deduced amino acid sequence. During our separation of bone noncollagenous proteins, we observed a high molecular weight, DMP1-related component (designated DMP1-PG). We purified DMP1-PG with a monoclonal anti-DMP1 antibody affinity column. Amino acid analysis and Edman degradation of tryptic peptides proved that the core protein for DMP1-PG is the 37-kDa fragment of DMP1. Chondroitinase treatments demonstrated that the slower migration rate of DMP1-PG is due to the presence of glycosaminoglycan. Quantitative disaccharide analysis indicated that the glycosaminoglycan is made predominantly of chondroitin 4-sulfate. Further analysis on tryptic peptides led us to conclude that a single glycosaminoglycan chain is linked to the core protein via Ser74, located in the Ser74-Gly75 dipeptide, an amino acid sequence specific for the attachment of glycosaminoglycans. Our findings show that in addition to its existence as a phosphoprotein, the NH2-terminal fragment from DMP1 occurs as a proteoglycan. Amino acid sequence alignment analysis showed that the Ser74-Gly75 dipeptide and its flanking regions are highly conserved among a wide range of species from caiman to the Homo sapiens, indicating that this glycosaminoglycan attachment domain has survived an extremely long period of evolution pressure, suggesting that the glycosaminoglycan may be critical for the basic biological functions of DMP1.

Nuclear localization and transactivation by Vitis CBF transcription factors are regulated by combinations of conserved amino acid domains.

PubMed

Carlow, Chevonne E; Faultless, J Trent; Lee, Christine; Siddiqua, Mahbuba; Edge, Alison; Nassuth, Annette

2017-09-01

The highly conserved CBF pathway is crucial in the regulation of plant responses to low temperatures. Extensive analysis of Arabidopsis CBF proteins revealed that their functions rely on several conserved amino acid domains although the exact function of each domain is disputed. The question was what functions similar domains have in CBFs from other, overwintering woody plants such as Vitis, which likely have a more involved regulation than the model plant Arabidopsis. A total of seven CBF genes were cloned and sequenced from V. riparia and the less frost tolerant V. vinifera. The deduced species-specific amino acid sequences differ in only a few amino acids, mostly in non-conserved regions. Amino acid sequence comparison and phylogenetic analysis showed two distinct groups of Vitis CBFs. One group contains CBF1, CBF2, CBF3 and CBF8 and the other group contains CBF4, CBF5 and CBF6. Transient transactivation assays showed that all Vitis CBFs except CBF5 activate via a CRT or DRE promoter element, whereby Vitis CBF3 and 4 prefer a CRT element. The hydrophobic domains in the C-terminal end of VrCBF6 were shown to be important for how well it activates. The putative nuclear localization domain of Vitis CBF1 was shown to be sufficient for nuclear localization, in contrast to previous reports for AtCBF1, and also important for transactivation. The latter highlights the value of careful analysis of domain functions instead of reliance on computer predictions and published data for other related proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

PubMed

Arndt, E; Scholzen, T; Krömer, W; Hatakeyama, T; Kimura, M

1991-06-01

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.

RNA Editing in Plant Mitochondria

NASA Astrophysics Data System (ADS)

Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

1989-12-01

Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

NASA Technical Reports Server (NTRS)

Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

1992-01-01

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

Computational analysis of sequence selection mechanisms.

PubMed

Meyerguz, Leonid; Grasso, Catherine; Kleinberg, Jon; Elber, Ron

2004-04-01

Mechanisms leading to gene variations are responsible for the diversity of species and are important components of the theory of evolution. One constraint on gene evolution is that of protein foldability; the three-dimensional shapes of proteins must be thermodynamically stable. We explore the impact of this constraint and calculate properties of foldable sequences using 3660 structures from the Protein Data Bank. We seek a selection function that receives sequences as input, and outputs survival probability based on sequence fitness to structure. We compute the number of sequences that match a particular protein structure with energy lower than the native sequence, the density of the number of sequences, the entropy, and the "selection" temperature. The mechanism of structure selection for sequences longer than 200 amino acids is approximately universal. For shorter sequences, it is not. We speculate on concrete evolutionary mechanisms that show this behavior.

A modular DNA signal translator for the controlled release of a protein by an aptamer.

PubMed

Beyer, Stefan; Simmel, Friedrich C

2006-01-01

Owing to the intimate linkage of sequence and structure in nucleic acids, DNA is an extremely attractive molecule for the development of molecular devices, in particular when a combination of information processing and chemomechanical tasks is desired. Many of the previously demonstrated devices are driven by hybridization between DNA 'effector' strands and specific recognition sequences on the device. For applications it is of great interest to link several of such molecular devices together within artificial reaction cascades. Often it will not be possible to choose DNA sequences freely, e.g. when functional nucleic acids such as aptamers are used. In such cases translation of an arbitrary 'input' sequence into a desired effector sequence may be required. Here we demonstrate a molecular 'translator' for information encoded in DNA and show how it can be used to control the release of a protein by an aptamer using an arbitrarily chosen DNA input strand. The function of the translator is based on branch migration and the action of the endonuclease FokI. The modular design of the translator facilitates the adaptation of the device to various input or output sequences.

A modular DNA signal translator for the controlled release of a protein by an aptamer

PubMed Central

Beyer, Stefan; Simmel, Friedrich C.

2006-01-01

Owing to the intimate linkage of sequence and structure in nucleic acids, DNA is an extremely attractive molecule for the development of molecular devices, in particular when a combination of information processing and chemomechanical tasks is desired. Many of the previously demonstrated devices are driven by hybridization between DNA ‘effector’ strands and specific recognition sequences on the device. For applications it is of great interest to link several of such molecular devices together within artificial reaction cascades. Often it will not be possible to choose DNA sequences freely, e.g. when functional nucleic acids such as aptamers are used. In such cases translation of an arbitrary ‘input’ sequence into a desired effector sequence may be required. Here we demonstrate a molecular ‘translator’ for information encoded in DNA and show how it can be used to control the release of a protein by an aptamer using an arbitrarily chosen DNA input strand. The function of the translator is based on branch migration and the action of the endonuclease FokI. The modular design of the translator facilitates the adaptation of the device to various input or output sequences. PMID:16547201

Statistical theory for protein combinatorial libraries. Packing interactions, backbone flexibility, and the sequence variability of a main-chain structure.

PubMed

Kono, H; Saven, J G

2001-02-23

Combinatorial experiments provide new ways to probe the determinants of protein folding and to identify novel folding amino acid sequences. These types of experiments, however, are complicated both by enormous conformational complexity and by large numbers of possible sequences. Therefore, a quantitative computational theory would be helpful in designing and interpreting these types of experiment. Here, we present and apply a statistically based, computational approach for identifying the properties of sequences compatible with a given main-chain structure. Protein side-chain conformations are included in an atom-based fashion. Calculations are performed for a variety of similar backbone structures to identify sequence properties that are robust with respect to minor changes in main-chain structure. Rather than specific sequences, the method yields the likelihood of each of the amino acids at preselected positions in a given protein structure. The theory may be used to quantify the characteristics of sequence space for a chosen structure without explicitly tabulating sequences. To account for hydrophobic effects, we introduce an environmental energy that it is consistent with other simple hydrophobicity scales and show that it is effective for side-chain modeling. We apply the method to calculate the identity probabilities of selected positions of the immunoglobulin light chain-binding domain of protein L, for which many variant folding sequences are available. The calculations compare favorably with the experimentally observed identity probabilities.

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism.

PubMed

Rae, Anne L; Casu, Rosanne E; Perroux, Jai M; Jackson, Mark A; Grof, Christopher P L

2011-06-15

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form. Copyright © 2010 Elsevier GmbH. All rights reserved.

Determination and analysis of the complete genome sequence of Paralichthys olivaceus rhabdovirus (PORV).

PubMed

Zhu, Ruo-Lin; Zhang, Qi-Ya

2014-04-01

Paralichthys olivaceus rhabdovirus (PORV), which is associated with high mortality rates in flounder, was isolated in China in 2005. Here, we provide an annotated sequence record of PORV, the genome of which comprises 11,182 nucleotides and contains six genes in the order 3'-N-P-M-G-NV-L-5'. Phylogenetic analysis based on glycoprotein sequences of PORV and other rhabdoviruses showed that PORV clusters with viral haemorrhagic septicemia virus (VHSV), genus Novirhabdovirus, family Rhabdoviridae. Further phylogenetic analysis of the combined amino acid sequences of six proteins of PORV and VHSV strains showed that PORV clusters with Korean strains and is closely related to Asian strains, all of which were isolated from flounder. In a comparison in which the sequences of the six proteins were combined, PORV shared the highest identity (98.3 %) with VHSV strain KJ2008 from Korea.

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.

Interaction of Mimetic Analogs of Insect Kinin Neuropeptides with Arthropod Receptors

DTIC Science & Technology

2010-01-01

stagnalis.20 The amino acid sequence of the B. microplus receptor showed most similarity to the CG10626 Drosophila melanogaster gene product and to the...25 The A. aegypti cDNA encodes a 584 amino acid residue protein of predicted molecular mass of 65.2 kDa. The mosquito kinin receptor cDNA was...charged acid moiety.36,37 Within the core pentapeptide, the aromatic residues Phe1 and Trp4 are the most important for activity whereas a wide range

Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

PubMed

Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

2015-12-04

In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

Molecular analysis of partial VP-2 gene amplified from rectal swab samples of diarrheic dogs in Pakistan confirms the circulation of canine parvovirus genetic variant CPV-2a and detects sequences of feline panleukopenia virus (FPV).

PubMed

Ahmed, Nisar; Riaz, Adeel; Zubair, Zahra; Saqib, Muhammad; Ijaz, Sehrish; Nawaz-Ul-Rehman, Muhammad Shah; Al-Qahtani, Ahmed; Mubin, Muhammad

2018-03-15

The infection in dogs due to canine parvovirus (CPV), is a highly contagious one with high mortality rate. The present study was undertaken for a detailed genetic analysis of partial VP2 gene i.e., 630 bp isolated from rectal swab samples of infected domestic and stray dogs from all areas of district Faisalabad. Monitoring of viruses is important, as continuous prevalence of viral infection might be associated with emergence of new virulent strains. In the present study, 40 rectal swab samples were collected from diarrheic dogs from different areas of district Faisalabad, Pakistan, in 2014-15 and screened for the presence of CPV by immunochromatography. Most of these dogs were stray dogs showing symptoms of diarrhea. Viral DNA was isolated and partial VP2 gene was amplified using gene specific primer pair Hfor/Hrev through PCR. Amplified fragments were cloned in pTZ57R/T (Fermentas) and completely sequenced. Sequences were analyzed and assembled by the Lasergene DNA analysis package (v8; DNAStar Inc., Madison, WI, USA). The results with immunochromatography showed that 33/40 (82%) of dogs were positive for CPV. We were able to amplify a fragment of 630 bp from 25 samples. In 25 samples the sequences of CPV-2a were detected showing the amino acid substitution Ser297Ala and presence of amino acid (426-Asn) in partial VP2 protein. Interestingly the BLAST analysis showed the of feline panleukopenia virus (FPV) sequences in 3 samples which were already positive for new CPV-2a, with 99% sequence homology to other FPV sequences present in GenBank. Phylogenetic analysis showed clustering of partial CPV-VP-2 gene with viruses from China, India, Japan and Uruguay identifying a new variant, whereas the 3 FPV sequences showed immediate ancestral relationship with viruses from Portugal, South Africa and USA. Interesting observation was that CPV are clustering away from the commercial vaccine strains. In this work we provide a better understanding of CPV prevailing in Pakistan at molecular level. The detection of FPV could be a case of real co-infection or a case of dual presence, due to ingestion of contaminated food.

Cloning and Characterization of an Outer Membrane Protein of Vibrio vulnificus Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence

PubMed Central

Litwin, Christine M.; Byrne, Burke L.

1998-01-01

Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis. PMID:9632577

Dynamic changes of yak (Bos grunniens) gut microbiota during growth revealed by polymerase chain reaction-denaturing gradient gel electrophoresis and metagenomics

PubMed Central

Nie, Yuanyang; Zhou, Zhiwei; Guan, Jiuqiang; Xia, Baixue; Luo, Xiaolin; Yang, Yang; Fu, Yu; Sun, Qun

2017-01-01

Objective To understand the dynamic structure, function, and influence on nutrient metabolism in hosts, it was crucial to assess the genetic potential of gut microbial community in yaks of different ages. Methods The denaturing gradient gel electrophoresis (DGGE) profiles and Illumina-based metagenomic sequencing on colon contents of 15 semi-domestic yaks were investigated. Unweighted pairwise grouping method with mathematical averages (UPGMA) clustering and principal component analysis (PCA) were used to analyze the DGGE fingerprint. The Illumina sequences were assembled, predicted to genes and functionally annotated, and then classified by querying protein sequences of the genes against the Kyoto encyclopedia of genes and genomes (KEGG) database. Results Metagenomic sequencing showed that more than 85% of ribosomal RNA (rRNA) gene sequences belonged to the phylum Firmicutes and Bacteroidetes, indicating that the family Ruminococcaceae (46.5%), Rikenellaceae (11.3%), Lachnospiraceae (10.0%), and Bacteroidaceae (6.3%) were dominant gut microbes. Over 50% of non-rRNA gene sequences represented the metabolic pathways of amino acids (14.4%), proteins (12.3%), sugars (11.9%), nucleotides (6.8%), lipids (1.7%), xenobiotics (1.4%), coenzymes, and vitamins (3.6%). Gene functional classification showed that most of enzyme-coding genes were related to cellulose digestion and amino acids metabolic pathways. Conclusion Yaks’ age had a substantial effect on gut microbial composition. Comparative metagenomics of gut microbiota in 0.5-, 1.5-, and 2.5-year-old yaks revealed that the abundance of the class Clostridia, Bacteroidia, and Lentisphaeria, as well as the phylum Firmicutes, Bacteroidetes, Lentisphaerae, Tenericutes, and Cyanobacteria, varied more greatly during yaks’ growth, especially in young animals (0.5 and 1.5 years old). Gut microbes, including Bacteroides, Clostridium, and Lentisphaeria, make a contribution to the energy metabolism and synthesis of amino acid, which are essential to the normal growth of yaks. PMID:28183172

Isolation and Selection of Microalgal Strains from Natural Water Sources in Viet Nam with Potential for Edible Oil Production.

PubMed

Thao, Tran Yen; Linh, Dinh Thi Nhat; Si, Vo Chi; Carter, Taylor W; Hill, Russell T

2017-06-23

Industrial vegetable oil production in Viet Nam depends on oil seeds and crude plant oils that are currently more than 90% imported. As the first step in investigating the feasibility of using microalgae to provide Viet Nam with a domestic source of oil for food and edible oil industries, fifty lipid-producing microalgae were isolated and characterized. The microalgae were isolated from water sources ranging from freshwater to brackish and marine waters from a wide geographic distribution in Viet Nam. Initial analyses showed that 20 of the 50 strains had good growth rates, produced high biomass and had high lipid content, ranging up to 50% of dry weight biomass. 18S rRNA gene sequence analyses of the 50 strains showed a great diversity in this assemblage of microalgae, comprising at least 38 species and representatives of 25 genera : Chlamydomonas , Poterioochromonas , Scenedesmus , Desmodesmus , Chlorella , Bracteacoccus , Monoraphidium , Selenastrum , Acutodesmus , Mychonastes , Ankistrodesmus , Kirchneriella , Raphidocelis , Dictyosphaerium , Coelastrella , Schizochlamydella , Oocystidium , Nannochloris , Auxenochlorella , Chlorosarcinopsis , Stichococcus , Picochlorum , Prasinoderma , Chlorococcum , and Marvania. Some of the species are closely related to well-known lipid producers such as Chlorella sorokiniana , but some other strains are not closely related to the strains found in public sequence databases and likely represent new species. Analysis of oil quality showed that fatty acid profiles of the microalgal strains were very diverse and strain-dependent. Fatty acids in the microalgal oils comprised saturated fatty acids (SFAs), poly-unsaturated fatty acids (PUFAs), and mono-unsaturated fatty acids (MUFAs). The main SFA was palmitic acid. MUFAs and PUFAs were dominated by oleic acid, and linoleic and linolenic acids, respectively. Some strains were especially rich in the essential fatty acid α-linolenic acid (ALA), which comprised more than 20% of the fatty acids in these strains. Other strains had fatty acid compositions similar to that of palm oil. Several strains have been selected on the basis of their suitable fatty acid profiles and high lipid content for further chemical and physical characterization, toxicity and organoleptic tests of their oils, and for scale-up.

Isolation and Selection of Microalgal Strains from Natural Water Sources in Viet Nam with Potential for Edible Oil Production

PubMed Central

Thao, Tran Yen; Linh, Dinh Thi Nhat; Si, Vo Chi; Carter, Taylor W.; Hill, Russell T.

2017-01-01

Industrial vegetable oil production in Viet Nam depends on oil seeds and crude plant oils that are currently more than 90% imported. As the first step in investigating the feasibility of using microalgae to provide Viet Nam with a domestic source of oil for food and edible oil industries, fifty lipid-producing microalgae were isolated and characterized. The microalgae were isolated from water sources ranging from freshwater to brackish and marine waters from a wide geographic distribution in Viet Nam. Initial analyses showed that 20 of the 50 strains had good growth rates, produced high biomass and had high lipid content, ranging up to 50% of dry weight biomass. 18S rRNA gene sequence analyses of the 50 strains showed a great diversity in this assemblage of microalgae, comprising at least 38 species and representatives of 25 genera: Chlamydomonas, Poterioochromonas, Scenedesmus, Desmodesmus, Chlorella, Bracteacoccus, Monoraphidium, Selenastrum, Acutodesmus, Mychonastes, Ankistrodesmus, Kirchneriella, Raphidocelis, Dictyosphaerium, Coelastrella, Schizochlamydella, Oocystidium, Nannochloris, Auxenochlorella, Chlorosarcinopsis, Stichococcus, Picochlorum, Prasinoderma, Chlorococcum, and Marvania. Some of the species are closely related to well-known lipid producers such as Chlorella sorokiniana, but some other strains are not closely related to the strains found in public sequence databases and likely represent new species. Analysis of oil quality showed that fatty acid profiles of the microalgal strains were very diverse and strain-dependent. Fatty acids in the microalgal oils comprised saturated fatty acids (SFAs), poly-unsaturated fatty acids (PUFAs), and mono-unsaturated fatty acids (MUFAs). The main SFA was palmitic acid. MUFAs and PUFAs were dominated by oleic acid, and linoleic and linolenic acids, respectively. Some strains were especially rich in the essential fatty acid α-linolenic acid (ALA), which comprised more than 20% of the fatty acids in these strains. Other strains had fatty acid compositions similar to that of palm oil. Several strains have been selected on the basis of their suitable fatty acid profiles and high lipid content for further chemical and physical characterization, toxicity and organoleptic tests of their oils, and for scale-up. PMID:28644408

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

PubMed

Chen, Guiqian; Qiu, Yuan; Zhuang, Qingye; Wang, Suchun; Wang, Tong; Chen, Jiming; Wang, Kaicheng

2018-05-09

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli.

PubMed

Le Chevanton, L; Leblon, G

1989-04-15

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.

Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus.

PubMed

Sakata, Junko; Kawatsu, Kentaro; Iwasaki, Tadashi; Kumeda, Yuko

2015-09-01

To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epitopes recognized by mAb-VP34 and mAb-VP109 were mapped to sequences of eight ((47)LLTSSFSA(54)) and six amino acid residues ((16)FDFAVD(21)), respectively. An amino acid sequence similarity search of the NCBI database using BLASTP showed that both epitopic amino acid sequences were present together only in V. parahaemolyticus. When 124 V. parahaemolyticus strains and 94 strains of 27 other Vibrio species or 35 non-Vibrio species were tested using the VP-ICA, the VP-ICA identified V. parahaemolyticus with 100% accuracy. The VP-ICA rapidly and simply identified the pathogen directly from a single agar colony within 30 min, indicating that VP-ICA will greatly reduce labor and time required to identify V. parahaemolyticus compared with conventional biochemical tests. Copyright © 2015. Published by Elsevier B.V.

Overdispersion of the Molecular Clock: Temporal Variation of Gene-Specific Substitution Rates in Drosophila

PubMed Central

Hartl, Daniel L.

2008-01-01

Simple models of molecular evolution assume that sequences evolve by a Poisson process in which nucleotide or amino acid substitutions occur as rare independent events. In these models, the expected ratio of the variance to the mean of substitution counts equals 1, and substitution processes with a ratio greater than 1 are called overdispersed. Comparing the genomes of 10 closely related species of Drosophila, we extend earlier evidence for overdispersion in amino acid replacements as well as in four-fold synonymous substitutions. The observed deviation from the Poisson expectation can be described as a linear function of the rate at which substitutions occur on a phylogeny, which implies that deviations from the Poisson expectation arise from gene-specific temporal variation in substitution rates. Amino acid sequences show greater temporal variation in substitution rates than do four-fold synonymous sequences. Our findings provide a general phenomenological framework for understanding overdispersion in the molecular clock. Also, the presence of substantial variation in gene-specific substitution rates has broad implications for work in phylogeny reconstruction and evolutionary rate estimation. PMID:18480070

Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio).

PubMed

Ken, C F; Lin, C T; Wu, J L; Shaw, J F

2000-06-01

A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.

Xuhuai goat H-FABP gene clone, subcellular localization of expression products and the preparation of transgenic mice.

PubMed

Yin, Yan-hui; Li, Bi-chun; Wei, Guang-hui; Zhu, Cai-ye; Li, Wei; Zhang, Ya-ni; Du, Li-xin; Cao, Wen-guang

2012-05-01

The aim of this study was to clone the heart-type fatty acid binding protein (H-FABP) gene of Xuhuai goat, to explore it bioinformatically, and analyze the subcellular localization using enhanced green fluorescent protein (EGFP). The results showed that the coding sequence (CDS) length of Xuhuai goat H-FABP gene was 402 bp, encoding 133 amino acids (GenBank accession number AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding region of human, chicken, brown rat, cow, wild boar, donkey, and zebrafish. The similarity were 89%, 76%, 85%, 84%, 93%, 91%, 70%, respectively. For the corresponding amino acid sequences, the similarity were 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. This study did not find the signal peptide region in the H-FABP protein; it revealed that H-FABP protein might be a nonsecreted protein. H-FABP expression was detected in vitro by reverse transcription-polymerase chain reaction (RT-PCR), and the EGFP-H-FABP fusion protein was localized to the cytoplasm. The gene could also be transiently and permanently expressed in mice.

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

G-quadruplex prediction in E. coli genome reveals a conserved putative G-quadruplex-Hairpin-Duplex switch.

PubMed

Kaplan, Oktay I; Berber, Burak; Hekim, Nezih; Doluca, Osman

2016-11-02

Many studies show that short non-coding sequences are widely conserved among regulatory elements. More and more conserved sequences are being discovered since the development of next generation sequencing technology. A common approach to identify conserved sequences with regulatory roles relies on topological changes such as hairpin formation at the DNA or RNA level. G-quadruplexes, non-canonical nucleic acid topologies with little established biological roles, are increasingly considered for conserved regulatory element discovery. Since the tertiary structure of G-quadruplexes is strongly dependent on the loop sequence which is disregarded by the generally accepted algorithm, we hypothesized that G-quadruplexes with similar topology and, indirectly, similar interaction patterns, can be determined using phylogenetic clustering based on differences in the loop sequences. Phylogenetic analysis of 52 G-quadruplex forming sequences in the Escherichia coli genome revealed two conserved G-quadruplex motifs with a potential regulatory role. Further analysis revealed that both motifs tend to form hairpins and G quadruplexes, as supported by circular dichroism studies. The phylogenetic analysis as described in this work can greatly improve the discovery of functional G-quadruplex structures and may explain unknown regulatory patterns. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase.

PubMed

Tabor, C W; Tabor, H

1987-11-25

We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822).

Identification, Classification, and Phylogeny of the Pathogenic Species Exophiala jeanselmei and Related Species by Mitochondrial Cytochrome b Gene Analysis

PubMed Central

Wang, Li; Yokoyama, Koji; Miyaji, Makoto; Nishimura, Kazuko

2001-01-01

We analyzed a 402-bp sequence of the mitochondrial cytochrome b gene of 34 strains of Exophiala jeanselmei and 16 strains representing 12 related species. The strains of E. jeanselmei were classified into 20 DNA types and 17 amino acid types. The differences between these strains were found in 1 to 60 nucleotides and 1 to 17 amino acids. On the basis of the identities and similarities of nucleotide and amino acid sequences, some strains were reidentified: i.e., two strains of E. jeanselmei var. hetermorpha and one strain of E. castellanii as E. dermatitidis (including the type strain), three strains of E. jeanselmei as E. jeanselmei var. lecanii-corni (including the type strain), three strains of E. jeanselmei as E. bergeri (including the type strain), seven strains of E. jeanselmei as E. pisciphila (including the type strain), seven strains of E. jeanselmei as E. jeanselmei var. jeanselmei (including the type strain), one strain of E. jeanselmei as Fonsecaea pedrosoi (including the type strain), and one strain of E. jeanselmei as E. spinifera (including the type strain). Some E. jeanselmei strains showed distinct nucleotide and amino acid sequences. The amino-acid-based UPGMA (unweighted pair group method with the arithmetic mean) tree exhibited nearly the same topology as those of the DNA-based trees obtained by neighbor joining, maximum parsimony, and maximum likelihood methods. PMID:11724862

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.

Blends of cysteine-containing proteins

NASA Astrophysics Data System (ADS)

Barone, Justin

2005-03-01

Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

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).

Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

PubMed Central

Wu, S; Kriz, A L; Widholm, J M

1994-01-01

The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions. PMID:7972490

PIPI: PTM-Invariant Peptide Identification Using Coding Method.

PubMed

Yu, Fengchao; Li, Ning; Yu, Weichuan

2016-12-02

In computational proteomics, the identification of peptides with an unlimited number of post-translational modification (PTM) types is a challenging task. The computational cost associated with database search increases exponentially with respect to the number of modified amino acids and linearly with respect to the number of potential PTM types at each amino acid. The problem becomes intractable very quickly if we want to enumerate all possible PTM patterns. To address this issue, one group of methods named restricted tools (including Mascot, Comet, and MS-GF+) only allow a small number of PTM types in database search process. Alternatively, the other group of methods named unrestricted tools (including MS-Alignment, ProteinProspector, and MODa) avoids enumerating PTM patterns with an alignment-based approach to localizing and characterizing modified amino acids. However, because of the large search space and PTM localization issue, the sensitivity of these unrestricted tools is low. This paper proposes a novel method named PIPI to achieve PTM-invariant peptide identification. PIPI belongs to the category of unrestricted tools. It first codes peptide sequences into Boolean vectors and codes experimental spectra into real-valued vectors. For each coded spectrum, it then searches the coded sequence database to find the top scored peptide sequences as candidates. After that, PIPI uses dynamic programming to localize and characterize modified amino acids in each candidate. We used simulation experiments and real data experiments to evaluate the performance in comparison with restricted tools (i.e., Mascot, Comet, and MS-GF+) and unrestricted tools (i.e., Mascot with error tolerant search, MS-Alignment, ProteinProspector, and MODa). Comparison with restricted tools shows that PIPI has a close sensitivity and running speed. Comparison with unrestricted tools shows that PIPI has the highest sensitivity except for Mascot with error tolerant search and ProteinProspector. These two tools simplify the task by only considering up to one modified amino acid in each peptide, which results in a higher sensitivity but has difficulty in dealing with multiple modified amino acids. The simulation experiments also show that PIPI has the lowest false discovery proportion, the highest PTM characterization accuracy, and the shortest running time among the unrestricted tools.

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

PubMed Central

Brown, D P; Idler, K B; Katz, L

1990-01-01

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Images FIG. 2 FIG. 3 PMID:2180909

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.

Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases.

PubMed

Westin, Maria A K; Alexson, Stefan E H; Hunt, Mary C

2004-05-21

Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mainly chain-shortened and then transported to mitochondria for further metabolism. We have now identified and characterized two peroxisomal acyl-CoA thioesterases, named PTE-Ia and PTE-Ic, that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A. PTE-Ia and PTE-Ic show 82% sequence identity at the amino acid level, and a putative peroxisomal type 1 targeting signal of -AKL was identified at the carboxyl-terminal end of both proteins. Localization experiments using green fluorescent fusion protein showed PTE-Ia and PTE-Ic to be localized in peroxisomes. Despite their high level of sequence identity, we show that PTE-Ia is mainly active on long-chain acyl-CoAs, whereas PTE-Ic is mainly active on medium-chain acyl-CoAs. Lack of regulation of enzyme activity by free CoASH suggests that PTE-Ia and PTE-Ic regulate intraperoxisomal levels of acyl-CoA, and they may have a function in termination of beta-oxidation of fatty acids of different chain lengths. Tissue expression studies revealed that PTE-Ia is highly expressed in kidney, whereas PTE-Ic is most highly expressed in spleen, brain, testis, and proximal and distal intestine. Both PTE-Ia and PTE-Ic were highly up-regulated in mouse liver by treatment with the peroxisome proliferator WY-14,643 and by fasting in a peroxisome proliferator-activated receptor alpha-dependent manner. These data show that PTE-Ia and PTE-Ic have different functions based on different substrate specificities and tissue expression.

Lion (Panthera leo) and cheetah (Acinonyx jubatus) IFN-gamma sequences.

PubMed

Maas, Miriam; Van Rhijn, Ildiko; Allsopp, Maria T E P; Rutten, Victor P M G

2010-04-15

Cloning and sequencing of the full length lion and cheetah interferon-gamma (IFN-gamma) transcript will enable the expression of the recombinant cytokine, to be used for production of monoclonal antibodies and to set up lion and cheetah-specific IFN-gamma ELISAs. These are relevant in blood-based diagnosis of bovine tuberculosis, an important threat to lions in the Kruger National Park. Alignment of nucleotide and amino acid sequences of lion and cheetah and that of domestic cats showed homologies of 97-100%. Copyright 2009 Elsevier B.V. All rights reserved.

Nucleotide Sequence of the blaRTG-2 (CARB-5) Gene and Phylogeny of a New Group of Carbenicillinases

PubMed Central

Choury, Daniele; Szajnert, Marie-France; Joly-Guillou, Marie-Laure; Azibi, Kemal; Delpech, Marc; Paul, Gérard

2000-01-01

We determined the nucleotide sequence of the bla gene for the Acinetobacter calcoaceticus β-lactamase previously described as CARB-5. Alignment of the deduced amino acid sequence with those of known β-lactamases revealed that CARB-5 possesses an RTG triad in box VII, as described for the Proteus mirabilis GN79 enzyme, instead of the RSG consensus characteristic of the other carbenicillinases. Phylogenetic studies showed that these RTG enzymes constitute a new, separate group, possibly ancestors of the carbenicillinase family. PMID:10722515

Quantum-Sequencing: Fast electronic single DNA molecule sequencing

NASA Astrophysics Data System (ADS)

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

2014-03-01

A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

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

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

The Diversity Present in 5140 Human Mitochondrial Genomes

PubMed Central

Pereira, Luísa; Freitas, Fernando; Fernandes, Verónica; Pereira, Joana B.; Costa, Marta D.; Costa, Stephanie; Máximo, Valdemar; Macaulay, Vincent; Rocha, Ricardo; Samuels, David C.

2009-01-01

We analyzed the current status (as of the end of August 2008) of human mitochondrial genomes deposited in GenBank, amounting to 5140 complete or coding-region sequences, in order to present an overall picture of the diversity present in the mitochondrial DNA of the global human population. To perform this task, we developed mtDNA-GeneSyn, a computer tool that identifies and exhaustedly classifies the diversity present in large genetic data sets. The diversity observed in the 5140 human mitochondrial genomes was compared with all possible transitions and transversions from the standard human mitochondrial reference genome. This comparison showed that tRNA and rRNA secondary structures have a large effect in limiting the diversity of the human mitochondrial sequences, whereas for the protein-coding genes there is a bias toward less variation at the second codon positions. The analysis of the observed amino acid variations showed a tolerance of variations that convert between the amino acids V, I, A, M, and T. This defines a group of amino acids with similar chemical properties that can interconvert by a single transition. PMID:19426953

Expression of arginine kinase enzymatic activity and mRNA in gills of the euryhaline crabs Carcinus maenas and Callinectes sapidus.

PubMed

Kotlyar, S; Weihrauch, D; Paulsen, R S; Towle, D W

2000-08-01

Phosphagen kinases catalyze the reversible dephosphorylation of guanidino phosphagens such as phosphocreatine and phosphoarginine, contributing to the restoration of adenosine triphosphate concentrations in cells experiencing high and variable demands on their reserves of high-energy phosphates. The major invertebrate phosphagen kinase, arginine kinase, is expressed in the gills of two species of euryhaline crabs, the blue crab Callinectes sapidus and the shore crab Carcinus maenas, in which energy-requiring functions include monovalent ion transport, acid-base balance, nitrogen excretion and gas exchange. The enzymatic activity of arginine kinase approximately doubles in the ion-transporting gills of C. sapidus, a strong osmoregulator, when the crabs are transferred from high to low salinity, but does not change in C. maenas, a more modest osmoregulator. Amplification and sequencing of arginine kinase cDNA from both species, accomplished by reverse transcription of gill mRNA and the polymerase chain reaction, revealed an open reading frame coding for a 357-amino-acid protein. The predicted amino acid sequences showed a minimum of 75 % identity with arginine kinase sequences of other arthropods. Ten of the 11 amino acid residues believed to participate in arginine binding are completely conserved among the arthropod sequences analyzed. An estimation of arginine kinase mRNA abundance indicated that acclimation salinity has no effect on arginine kinase gene transcription. Thus, the observed enhancement of enzyme activity in C. sapidus probably results from altered translation rates or direct activation of pre-existing enzyme protein.

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.

A novel cry2Ab gene from the indigenous isolate Bacillus thuringiensis subsp. kurstaki.

PubMed

Sevim, Ali; Eryüzlü, Emine; Demirbağ, Zihni; Demir, Ismail

2012-01-01

A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.

Molecular epidemiology demonstrated three emerging clusters of human immunodeficiency virus type 1 subtype B infection in Hong Kong.

PubMed

Leung, Tommy W C; Mak, Darwin; Wong, K H; Wang, Y; Song, Y H; Tsang, D N C; Wong, C; Shao, Y M; Lim, W L

2008-07-01

We conducted a molecular epidemiological study on newly diagnosed human immunodeficiency virus type 1 (HIV-1)-infected patients in Hong Kong to identify the epidemiological linkage of HIV-1 infection in the locality. Reverse transcription polymerase chain reaction (RT-PCR) for HIV-1 was performed on newly diagnosed HIV-1-positive sera collected from January 2002 to December 2006. PCR products correspond to the env C2V3V4 region and gag p17/p24 junction of the HIV-1 genome were nucleotide sequenced. Phylogenetic analyses performed on the acquired nucleotide sequences revealed that CRF01_AE and subtype B were the two dominant HIV-1 subtypes. Analyses also demonstrated the presence of three emerging HIV-1 clusters among the subtype B sequences in Hong Kong. Individual cluster possesses a unique cluster-specific amino acid signature for identification. Data show that one of the clusters (Cluster I) is rapidly expanding. In addition to the unique cluster-specific amino acid signature, the majority of sequences in Cluster I harbor a 6-amino acid insertion at the gag p17/p24 junction in a region that is thought to be closely associated with HIV-1 infectivity.

Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

PubMed Central

Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

1995-01-01

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method.

PubMed

Liu, Wei; Dong, Derong; Yang, Zhan; Zou, Dayang; Chen, Zeliang; Yuan, Jing; Huang, Liuyu

2015-07-29

In this study, we report a novel isothermal nucleic acid amplification method only requires one pair of primers and one enzyme, termed Polymerase Spiral Reaction (PSR) with high specificity, efficiency, and rapidity under isothermal condition. The recombinant plasmid of blaNDM-1 was imported to Escherichia coli BL21, and selected as the microbial target. PSR method employs a Bst DNA polymerase and a pair of primers designed targeting the blaNDM-1 gene sequence. The forward and reverse Tab primer sequences are reverse to each other at their 5' end (Nr and N), whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The PSR method was performed at a constant temperature 61 °C-65 °C, yielding a complicated spiral structure. PSR assay was monitored continuously in a real-time turbidimeter instrument or visually detected with the aid of a fluorescent dye (SYBR Greenı), and could be finished within 1 h with a high accumulation of 10(9) copies of the target and a fine sensitivity of 6 CFU per reaction. Clinical evaluation was also conducted using PSR, showing high specificity of this method. The PSR technique provides a convenient and cost-effective alternative for clinical screening, on-site diagnosis and primary quarantine purposes.

Complete Genome Sequences of Two Plant-Associated Pseudomonas putida Isolates with Increased Heavy-Metal Tolerance.

PubMed

Nesme, Joseph; Cania, Barbara; Zadel, Urška; Schöler, Anne; Płaza, Grażyna A; Schloter, Michael

2017-11-22

We report here the complete genome sequences of two Pseudomonas putida isolates recovered from surface-sterilized roots of Sida hermaphrodita The two isolates were characterized by an increased tolerance to zinc, cadmium, and lead. Furthermore, the strains showed typical plant growth-promoting properties, such as the production of indole acetic acid, cellulolytic enzymes, and siderophores. Copyright © 2017 Nesme et al.

Gene cloning and overexpression of a geranylgeranyl diphosphate synthase of an extremely thermophilic bacterium, Thermus thermophilus.

PubMed

Ohto, C; Ishida, C; Koike-Takeshita, A; Yokoyama, K; Muramatsu, M; Nishino, T; Obata, S

1999-02-01

A geranylgeranyl diphosphate (GGPP) synthase gene of an extremely thermophilic bacterium, Thermus thermophilus, was cloned and sequenced. T. thermophilus GGPP synthase, overexpressed in Escherichia coli cells as a glutathione S-transferase fusion protein, was purified and characterized. The fusion protein, retaining thermostability, formed a homodimer, and showed higher specific activity than did a partially purified thermostable enzyme previously reported. Optimal reaction conditions and kinetic parameters were also examined. The deduced amino acid sequence indicated that T. thermophilus GGPP synthase was excluded from the group of bacterial type GGPP synthases and lacked the insertion amino acid residues in the first aspartate-rich motif as do archaeal and eukaryotic short-chain prenyltransferases.

Molecular identification of catalases from Nicotiana plumbaginifolia (L.).

PubMed

Willekens, H; Villarroel, R; Van Montagu, M; Inzé, D; Van Camp, W

1994-09-19

We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3-aminotriazole, and salicylic acid. 3-Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

[Characterization and comparison of interferon reference standards using UPLC-MS].

PubMed

Tao, Lei; Pei, De-ning; Han, Chun-mei; Chen, Wei; Rao, Chun-ming; Wang, Jun-zhi

2015-01-01

The study aims to characterize and compare interferon reference standards from 5 manufacturers. By testing molecular mass and trypsin-digested peptide mass mapping, the amino acid sequence was verified and post-translational modifications such as disulfide bond were identified. Results show that the molecular mass and amino acid sequence were consistent with theory; the disulfide bonds of 4 lots of interferon were Cys1-Cys98/Cys29-Cys138, 1 lot was Cys29-Cys139/Cys86-Cys99; N-terminal "+Met", acetyl N-terminal and Met oxidation were identified in part of the sample. UPLC-MS can be used to characterize and compare interferon reference standards from different manufacturers.

In vitro Inhibition of Pancreatic Lipase by Polyphenols:
A Kinetic, Fluorescence Spectroscopy and Molecular Docking Study

PubMed Central

2017-01-01

Summary The inhibitory activity and binding characteristics of caffeic acid, p-coumaric acid, quercetin and capsaicin, four phenolic compounds found in hot pepper, against porcine pancreatic lipase activity were studied and compared to hot pepper extract. Quercetin was the strongest inhibitor (IC50=(6.1±2.4) µM), followed by p-coumaric acid ((170.2±20.6) µM) and caffeic acid ((401.5±32.1) µM), while capsaicin and a hot pepper extract had very low inhibitory activity. All polyphenolic compounds showed a mixed-type inhibition. Fluorescence spectroscopy studies showed that polyphenolic compounds had the ability to quench the intrinsic fluorescence of pancreatic lipase by a static mechanism. The sequence of Stern-Volmer constant was quercetin, followed by caffeic and p-coumaric acids. Molecular docking studies showed that caffeic acid, quercetin and p-coumaric acid bound near the active site, while capsaicin bound far away from the active site. Hydrogen bonds and π-stacking hydrophobic interactions are the main pancreatic lipase-polyphenolic compound interactions observed. PMID:29540986

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

Fatty Acid Profile and Unigene-Derived Simple Sequence Repeat Markers in Tung Tree (Vernicia fordii)

PubMed Central

Zhang, Lin; Jia, Baoguang; Tan, Xiaofeng; Thammina, Chandra S.; Long, Hongxu; Liu, Min; Wen, Shanna; Song, Xianliang; Cao, Heping

2014-01-01

Tung tree (Vernicia fordii) provides the sole source of tung oil widely used in industry. Lack of fatty acid composition and molecular markers hinders biochemical, genetic and breeding research. The objectives of this study were to determine fatty acid profiles and develop unigene-derived simple sequence repeat (SSR) markers in tung tree. Fatty acid profiles of 41 accessions showed that the ratio of α-eleostearic acid was increasing continuously with a parallel trend to the amount of tung oil accumulation while the ratios of other fatty acids were decreasing in different stages of the seeds and that α-eleostearic acid (18∶3) consisted of 77% of the total fatty acids in tung oil. Transcriptome sequencing identified 81,805 unigenes from tung cDNA library constructed using seed mRNA and discovered 6,366 SSRs in 5,404 unigenes. The di- and tri-nucleotide microsatellites accounted for 92% of the SSRs with AG/CT and AAG/CTT being the most abundant SSR motifs. Fifteen polymorphic genic-SSR markers were developed from 98 unigene loci tested in 41 cultivated tung accessions by agarose gel and capillary electrophoresis. Genbank database search identified 10 of them putatively coding for functional proteins. Quantitative PCR demonstrated that all 15 polymorphic SSR-associated unigenes were expressed in tung seeds and some of them were highly correlated with oil composition in the seeds. Dendrogram revealed that most of the 41 accessions were clustered according to the geographic region. These new polymorphic genic-SSR markers will facilitate future studies on genetic diversity, molecular fingerprinting, comparative genomics and genetic mapping in tung tree. The lipid profiles in the seeds of 41 tung accessions will be valuable for biochemical and breeding studies. PMID:25167054

Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

NASA Astrophysics Data System (ADS)

Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

2013-11-01

Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

A new esterase for the cleavage of pivalic acid-containing prodrug esters of cephalosporins.

PubMed

Sauber, K; Aretz, W; Meiwes, J; Wollmann, T

1996-07-01

An extracellular esterase from the actinomycetes Amycolatopsis orientalis was found by screening. It is capable of splitting the isomeric mixture (K/J) of (I, Scheme 1) into 7-amino-3-methoxymethyl-3-cephem-4-carboxylic acid, pivalic acid, and acetaldehyde with a high yield. The purified enzyme of 55.4 Kd by SDS-PAGE shows an N-terminal sequence of VRTCADLVRTYDLPGAVTH. The isoelectric point is 8.9 +/- 0.1. It can be immobilized with good yield to VA-Epoxy Biosynth. Besides the above-mentioned reaction, the esterase cleaves many other esters such as methyl-2-chloropropionic acid.

Characterisation of a large family of polymorphic collagen-like proteins in the endospore-forming bacterium Pasteuria ramosa.

PubMed

McElroy, Kerensa; Mouton, Laurence; Du Pasquier, Louis; Qi, Weihong; Ebert, Dieter

2011-09-01

Collagen-like proteins containing glycine-X-Y repeats have been identified in several pathogenic bacteria potentially involved in virulence. Recently, a collagen-like surface protein, Pcl1a, was identified in Pasteuria ramosa, a spore-forming parasite of Daphnia. Here we characterise 37 novel putative P. ramosa collagen-like protein genes (PCLs). PCR amplification and sequencing across 10 P. ramosa strains showed they were polymorphic, distinguishing genotypes matching known differences in Daphnia/P. ramosa interaction specificity. Thirty PCLs could be divided into four groups based on sequence similarity, conserved N- and C-terminal regions and G-X-Y repeat structure. Group 1, Group 2 and Group 3 PCLs formed triplets within the genome, with one member from each group represented in each triplet. Maximum-likelihood trees suggested that these groups arose through multiple instances of triplet duplication. For Group 1, 2, 3 and 4 PCLs, X was typically proline and Y typically threonine, consistent with other bacterial collagen-like proteins. The amino acid composition of Pcl2 closely resembled Pcl1a, with X typically being glutamic acid or aspartic acid and Y typically being lysine or glutamine. Pcl2 also showed sequence similarity to Pcl1a and contained a predicted signal peptide, cleavage site and transmembrane domain, suggesting that it is a surface protein. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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.

Identification of the allergen Psi c 2 from the basidiomycete Psilocybe cubensis as a fungal cyclophilin.

PubMed

Horner, W E; Reese, G; Lehrer, S B

1995-01-01

Basidiospores are a prevalent and frequent cause of respiratory allergies, yet their allergens remain poorly defined; thus, we have attempted a molecular characterization of representative basidiomycete allergens. A Psilocybe cubensis mycelial cDNA library was immunoscreened with patient serum. A clone was isolated that expressed a 23-kD recombinant allergen as a fusion protein and inhibited a 16-kD band (Psi c 2) in immunoprints of P. cubenis extract, indicating antigenic identity. Sequence (cDNA) analysis of the clone indicates homology with cyclophilin and the deduced amino acid sequence of Psi c 2 showed 78% identity and 4% similarity with the amino acid sequence of Schizosaccharomyces pombe cyclophilin. This recombinant allergen is a useful model for epitope analysis of basidiospore allergens and fungal allergen cross-reactivity, and may provide an improved reagent for basidiospore allergy diagnosis and treatment.

Evidence for a vast peptide overlap between West Nile virus and human proteomes.

PubMed

Capone, Giovanni; Pagoni, Maria; Delfino, Antonella Pesce; Kanduc, Darja

2013-10-01

The primary amino acid sequence of West Nile virus (WNV) polyprotein, GenBank accession number M12294, was analyzed by computional biology. WNV is a mosquito-borne neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans. Using pentapeptides as scanning units and the perfect peptide match program from PIR International Protein Sequence Database, we compared the WNV polyprotein and the human proteome. WNV polyprotein showed significant sequence similarities to a number of human proteins. Several of these proteins are involved in embryogenesis, neurite outgrowth, cortical neuron branching, formation of mature synapses, semaphorin interactions, and voltage dependent L-type calcium channel subunits. The biocomputional study suggest that common amino acid segments might represent a potential platform for further studies on the neurological pathophysiology of WNV infections. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids.

PubMed

Niedźwiecka, Agnieszka; Cisnetti, Federico; Lebrun, Colette; Delangle, Pascale

2012-05-07

The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P(22), which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb(3+). To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P(HD2) (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH(2)) and P(HD5) (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH(2)) led to octadentate Tb(3+) complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P(HD5) promotes the formation of polymetallic species in excess of Ln(3+), P(HD2) forms exclusively the mononuclear complex. The octadentate coordination of Tb(3+) by both P(HD) leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P(HD2) forms a unique octadentate Tb(3+) complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP(22) complex by a factor of 4.

In silico identification and analysis of phytoene synthase genes in plants.

PubMed

Han, Y; Zheng, Q S; Wei, Y P; Chen, J; Liu, R; Wan, H J

2015-08-14

In this study, we examined phytoene synthetase (PSY), the first key limiting enzyme in the synthesis of carotenoids and catalyzing the formation of geranylgeranyl pyrophosphate in terpenoid biosynthesis. We used known amino acid sequences of the PSY gene in tomato plants to conduct a genome-wide search and identify putative candidates in 34 sequenced plants. A total of 101 homologous genes were identified. Phylogenetic analysis revealed that PSY evolved independently in algae as well as monocotyledonous and dicotyledonous plants. Our results showed that the amino acid structures exhibited 5 motifs (motifs 1 to 5) in algae and those in higher plants were highly conserved. The PSY gene structures showed that the number of intron in algae varied widely, while the number of introns in higher plants was 4 to 5. Identification of PSY genes in plants and the analysis of the gene structure may provide a theoretical basis for studying evolutionary relationships in future analyses.

Geodermatophilus sabuli sp. nov., a γ-radiation-resistant actinobacterium isolated from desert limestone.

PubMed

Hezbri, Karima; Ghodhbane-Gtari, Faten; Montero-Calasanz, Maria del Carmen; Sghaier, Haïtham; Rohde, Manfred; Schumann, Peter; Klenk, Hans-Peter; Gtari, Maher

2015-10-01

A novel γ-radiation-resistant and Gram-staining-positive actinobacterium designated BMG 8133T was isolated from a limestone collected in the Sahara desert of Tunisia. The strain produced dry, pale-pink colonies with an optimum growth at 35–40 °C and pH 6.5–8.0. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The main polar lipids were phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine and one unspecified glycolipid. MK-9(H4) was the dominant menaquinone. Galactose and glucose were detected as diagnostic sugars. The major cellular fatty acids were branched-chain saturated acids iso-C16 : 0 and iso-C15 : 0. The DNA G+C content of the novel strain was 74.5 %. The 16S rRNA gene sequence showed highest sequence identity with Geodermatophilus ruber (98.3 %). Based on phenotypic results and 16S rRNA gene sequence analysis, strain BMG 8133T is proposed to represent a novel species, Geodermatophilus sabuli sp. nov. The type strain is BMG 8133T ( = DSM 46844T = CECT 8820T).

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

1992-01-01

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

A new earthworm cellulase and its possible role in the innate immunity.

PubMed

Park, In Yong; Cha, Ju Roung; Ok, Suk-Mi; Shin, Chuog; Kim, Jin-Se; Kwak, Hee-Jin; Yu, Yun-Sang; Kim, Yu-Kyung; Medina, Brenda; Cho, Sung-Jin; Park, Soon Cheol

2017-02-01

A new endogenous cellulase (Ean-EG) from the earthworm, Eisenia andrei and its expression pattern are demonstrated. Based on a deduced amino acid sequence, the open reading frame (ORF) of Ean-EG consisted of 1368 bps corresponding to a polypeptide of 456 amino acid residues in which is contained the conserved region specific to GHF9 that has the essential amino acid residues for enzyme activity. In multiple alignments and phylogenetic analysis, the deduced amino acid sequence of Ean- EG showed the highest sequence similarity (about 79%) to that of an annelid (Pheretima hilgendorfi) and could be clustered together with other GHF9 cellulases, indicating that Ean-EG could be categorized as a member of the GHF9 to which most animal cellulases belong. The histological expression pattern of Ean-EG mRNA using in situ hybridization revealed that the most distinct expression was observed in epithelial cells with positive hybridization signal in epidermis, chloragogen tissue cells, coelomic cell-aggregate, and even blood vessel, which could strongly support the fact that at least in the earthworm, Eisenia andrei, cellulase function must not be limited to digestive process but be possibly extended to the innate immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Endo-β-1,3-Glucanase GLU1, from the Fruiting Body of Lentinula edodes, Belongs to a New Glycoside Hydrolase Family ▿ †

PubMed Central

Sakamoto, Yuichi; Nakade, Keiko; Konno, Naotake

2011-01-01

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128). PMID:21965406

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

Immunoglobulin from Antarctic fish species of Rajidae family.

PubMed

Coscia, Maria Rosaria; Cocca, Ennio; Giacomelli, Stefano; Cuccaro, Fausta; Oreste, Umberto

2012-03-01

Immunoglobulins (Ig) of Chondroichthyes have been extensively studied in sharks; in contrast, in skates investigations on Ig remain scarce and fragmentary despite the high occurrence of skates in all of the major oceans of the world. To focus on Rajidae Igμ, the most abundant heavy chain isotype, we have chosen the Antarctic species Bathyraja eatonii, Bathyraja albomaculata, Bathyraja brachyurops, and Amblyraja georgiana which live at high latitudes in the Southern Ocean, and at very low temperatures. We prepared mRNA from the spleen of individuals of each species and performed RT-PCR experiments using two oligonucleotides designed on the alignment of various elasmobranch Igμ heavy chain sequences available in GenBank. The PCR products, about 1400-nt long, were cloned and sequenced. Nucleotide sequence identities calculated for the constant region domains ranged from 88.5% to 97.5% between species, and from 91.1% to 99.7% within species. In a distance tree, including also Raja erinacea sequences, two major branches were obtained, one containing Arhynchobatinae sequences, the other one Rajinae sequences. Four presumptive D gene segments were identified in the region of the VH/D/JH recombination; two different D segments were often found in the same sequence. Moreover, 5-15 genomic fragments of different lengths, carrying the gene locus encoding Igμ chain were revealed by Southern blotting analysis. B. eatonii amino acid sequences were analyzed for the positional diversity by Shannon entropy analysis, showing CH4 as the most conserved domain, and CH3 as the most variable one. B. eatonii CDR3 region length varied between 11 and 15 amino acid residues; the mean length (13.4 aa) was greater than that of Leucoraja eglanteria sequences (7.7 aa). An alignment of representative sequences of Antarctic species and R. erinacea showed that more cysteine residues not involved in the intradomain disulfide bridges were present in Antarctic species. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

A charge-dependent mechanism is responsible for the dynamic accumulation of proteins inside nucleoli.

PubMed

Musinova, Yana R; Kananykhina, Eugenia Y; Potashnikova, Daria M; Lisitsyna, Olga M; Sheval, Eugene V

2015-01-01

The majority of known nucleolar proteins are freely exchanged between the nucleolus and the surrounding nucleoplasm. One way proteins are retained in the nucleoli is by the presence of specific amino acid sequences, namely nucleolar localization signals (NoLSs). The mechanism by which NoLSs retain proteins inside the nucleoli is still unclear. Here, we present data showing that the charge-dependent (electrostatic) interactions of NoLSs with nucleolar components lead to nucleolar accumulation as follows: (i) known NoLSs are enriched in positively charged amino acids, but the NoLS structure is highly heterogeneous, and it is not possible to identify a consensus sequence for this type of signal; (ii) in two analyzed proteins (NF-κB-inducing kinase and HIV-1 Tat), the NoLS corresponds to a region that is enriched for positively charged amino acid residues; substituting charged amino acids with non-charged ones reduced the nucleolar accumulation in proportion to the charge reduction, and nucleolar accumulation efficiency was strongly correlated with the predicted charge of the tested sequences; and (iii) sequences containing only lysine or arginine residues (which were referred to as imitative NoLSs, or iNoLSs) are accumulated in the nucleoli in a charge-dependent manner. The results of experiments with iNoLSs suggested that charge-dependent accumulation inside the nucleoli was dependent on interactions with nucleolar RNAs. The results of this work are consistent with the hypothesis that nucleolar protein accumulation by NoLSs can be determined by the electrostatic interaction of positively charged regions with nucleolar RNAs rather than by any sequence-specific mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

PubMed

Kutz, Russell; Okwumabua, Ogi

2008-10-01

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

Chemical property based sequence characterization of PpcA and its homolog proteins PpcB-E: A mathematical approach

PubMed Central

Pal Choudhury, Pabitra

2017-01-01

Periplasmic c7 type cytochrome A (PpcA) protein is determined in Geobacter sulfurreducens along with its other four homologs (PpcB-E). From the crystal structure viewpoint the observation emerges that PpcA protein can bind with Deoxycholate (DXCA), while its other homologs do not. But it is yet to be established with certainty the reason behind this from primary protein sequence information. This study is primarily based on primary protein sequence analysis through the chemical basis of embedded amino acids. Firstly, we look for the chemical group specific score of amino acids. Along with this, we have developed a new methodology for the phylogenetic analysis based on chemical group dissimilarities of amino acids. This new methodology is applied to the cytochrome c7 family members and pinpoint how a particular sequence is differing with others. Secondly, we build a graph theoretic model on using amino acid sequences which is also applied to the cytochrome c7 family members and some unique characteristics and their domains are highlighted. Thirdly, we search for unique patterns as subsequences which are common among the group or specific individual member. In all the cases, we are able to show some distinct features of PpcA that emerges PpcA as an outstanding protein compared to its other homologs, resulting towards its binding with deoxycholate. Similarly, some notable features for the structurally dissimilar protein PpcD compared to the other homologs are also brought out. Further, the five members of cytochrome family being homolog proteins, they must have some common significant features which are also enumerated in this study. PMID:28362850

Sequentially distant but structurally similar proteins exhibit fold specific patterns based on their biophysical properties.

PubMed

Rajendran, Senthilnathan; Jothi, Arunachalam

2018-05-16

The Three-dimensional structure of a protein depends on the interaction between their amino acid residues. These interactions are in turn influenced by various biophysical properties of the amino acids. There are several examples of proteins that share the same fold but are very dissimilar at the sequence level. For proteins to share a common fold some crucial interactions should be maintained despite insignificant sequence similarity. Since the interactions are because of the biophysical properties of the amino acids, we should be able to detect descriptive patterns for folds at such a property level. In this line, the main focus of our research is to analyze such proteins and to characterize them in terms of their biophysical properties. Protein structures with sequence similarity lesser than 40% were selected for ten different subfolds from three different mainfolds (according to CATH classification) and were used for this analysis. We used the normalized values of the 49 physio-chemical, energetic and conformational properties of amino acids. We characterize the folds based on the average biophysical property values. We also observed a fold specific correlational behavior of biophysical properties despite a very low sequence similarity in our data. We further trained three different binary classification models (Naive Bayes-NB, Support Vector Machines-SVM and Bayesian Generalized Linear Model-BGLM) which could discriminate mainfold based on the biophysical properties. We also show that among the three generated models, the BGLM classifier model was able to discriminate protein sequences coming under all beta category with 81.43% accuracy and all alpha, alpha-beta proteins with 83.37% accuracy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

PubMed Central

Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

2015-01-01

This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

An atypical topoisomerase II sequence from the slime mold Physarum polycephalum.

PubMed

Hugodot, Yannick; Dutertre, Murielle; Duguet, Michel

2004-01-21

We have determined the complete nucleotide sequence of the cDNA encoding DNA topoisomerase II from Physarum polycephalum. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic enzymes, a 250-bp fragment was polymerase chain reaction (PCR) amplified. This fragment was used as a probe to screen a Physarum cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. Rapid amplification of cDNA ends (RACE)-PCR was employed to isolate the remaining portion of the gene. The complete sequence of 4613 bp contains an open reading frame of 4494 bp that codes for 1498 amino acid residues with a theoretical molecular weight of 167 kDa. The predicted amino acid sequence shares similarity with those of other eukaryotes and shows the highest degree of identity with the enzyme of Dictyostelium discoideum. However, the enzyme of P. polycephalum contains an atypical amino-terminal domain very rich in serine and proline, whose function is unknown. Remarkably, both a mitochondrial targeting sequence and a nuclear localization signal were predicted respectively in the amino and carboxy-terminus of the protein, as in the case of human topoisomerase III alpha. At the Physarum genomic level, the topoisomerase II gene encompasses a region of about 16 kbp suggesting a large proportion of intronic sequences, an unusual situation for a gene of a lower eukaryote, often free of introns. Finally, expression of topoisomerase II mRNA does not appear significantly dependent on the plasmodium cycle stage, possibly due to the lack of G1 phase or (and) to a mitochondrial localization of the enzyme.

Analysis of the vhoGAC and vhtGAC operons from Methanosarcina mazei strain Gö1, both encoding a membrane-bound hydrogenase and a cytochrome b.

PubMed

Deppenmeier, U; Blaut, M; Lentes, S; Herzberg, C; Gottschalk, G

1995-01-15

DNA encompassing the structural genes of two membrane-bound hydrogenases from Methanosarcina mazei Gö1 was cloned and sequenced. The genes, arranged in the order vhoG and vhoA as well as vhtG and vhtA, were identified as those encoding the small and the large subunits of the NiFe hydrogenases [Deppenmeier, U., Blaut, M., Schmidt, B. & Gottschalk, G. (1992) Arch. Microbiol. 157, 505-511]. Northern-blot analysis revealed that the structural genes formed part of two operons, both containing one additional open reading frame (vhoC and vhtC) which codes for a cytochrome b. This conclusion was drawn from the homology of the deduced N-terminal amino acid sequences of vhoC and vhtC and the N-terminus of a 27-kDa cytochrome isolated from Ms. mazei C16. VhoC and VhtC contain four tentative hydrophobic segments which might span the cytoplasmic membrane. Hydropathy plots suggest that His23 and His50 are involved in heme coordination. The comparison of the sequencing data of vhoG and vhtG with the experimentally determined N-terminus of the small subunit indicate the presence of a 48-amino-acid leader peptide in front of the polypeptides. VhoA and VhtA contained the conserved sequence DPCXXC in the C-terminal region, which excludes the presence of a selenocysteine residue in these hydrogenases. Promoter sequences were found upstream of vhoG and vhtG, respectively. Downstream of vhoC, a putative terminator sequence was identified. Alignments of the deduced amino acid sequences of the gene clusters vhoGAC and vhtGAC showed 92-97% identity. Only the C-termini of VhoC and VhtC were not similar.

[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.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene.

PubMed

Huang, Rong; Sun, Xiao-Feng; Hu, Wei; Wang, Ya-Ping; Guo, Qiong-Lin

2008-05-01

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.