SEQUENCING OF FLAX LIS-1 INSERTION SITE IN THE ALBIDUM GENOTYPE

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Jana Žiarovská

2012-12-01

Full Text Available The paper presents a methodology of identifying the insertion site of LIS-1-1 (Linum Insertion Sequence 1 element in flax Albidum variety when growing under the in vitro combined with environmental stress conditions. Abiotic stress was induced by a reduced nutrient content in a growth medium. The LIS-1 insertion site amplification was reaLIS-1ed using the forward LIS-L: 5'-GGG CAG TTT AAC TGT AAC GAA - 3 'and revers LIS-R: 5'-GCT TGG ATT TAG ACT TGG CAA C - 3' primers by PCR. PCR product was sequenced by direct sequencing method to proove the nucleotide sequence for matching with database LIS-1 sequence. A comparison has been matched with the sequence of the amplified segment in the database for all nucleotides except the 11-position in the 5'-3 ' direction, where instead of the three adenine pair is a couple in the Albidum variety. Changes caused by mobile elements or insertion sequences result in common flax in variability that can be used for the purposes of development of effective marker identification or environment based markers development.

Poly(A)-tag deep sequencing data processing to extract poly(A) sites.

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Wu, Xiaohui; Ji, Guoli; Li, Qingshun Quinn

2015-01-01

Polyadenylation [poly(A)] is an essential posttranscriptional processing step in the maturation of eukaryotic mRNA. The advent of next-generation sequencing (NGS) technology has offered feasible means to generate large-scale data and new opportunities for intensive study of polyadenylation, particularly deep sequencing of the transcriptome targeting the junction of 3'-UTR and the poly(A) tail of the transcript. To take advantage of this unprecedented amount of data, we present an automated workflow to identify polyadenylation sites by integrating NGS data cleaning, processing, mapping, normalizing, and clustering. In this pipeline, a series of Perl scripts are seamlessly integrated to iteratively map the single- or paired-end sequences to the reference genome. After mapping, the poly(A) tags (PATs) at the same genome coordinate are grouped into one cleavage site, and the internal priming artifacts removed. Then the ambiguous region is introduced to parse the genome annotation for cleavage site clustering. Finally, cleavage sites within a close range of 24 nucleotides and from different samples can be clustered into poly(A) clusters. This procedure could be used to identify thousands of reliable poly(A) clusters from millions of NGS sequences in different tissues or treatments.

High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

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Kretz, Colin A; Tomberg, Kärt; Van Esbroeck, Alexander; Yee, Andrew; Ginsburg, David

2018-02-12

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10 7 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites.

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Richard M Sharpe

Full Text Available High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3'UTR sequencing, and cleaved amplified polymorphic sequence (CAPS molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

Sequence Capture versus Restriction Site Associated DNA Sequencing for Shallow Systematics.

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Harvey, Michael G; Smith, Brian Tilston; Glenn, Travis C; Faircloth, Brant C; Brumfield, Robb T

2016-09-01

Sequence capture and restriction site associated DNA sequencing (RAD-Seq) are two genomic enrichment strategies for applying next-generation sequencing technologies to systematics studies. At shallow timescales, such as within species, RAD-Seq has been widely adopted among researchers, although there has been little discussion of the potential limitations and benefits of RAD-Seq and sequence capture. We discuss a series of issues that may impact the utility of sequence capture and RAD-Seq data for shallow systematics in non-model species. We review prior studies that used both methods, and investigate differences between the methods by re-analyzing existing RAD-Seq and sequence capture data sets from a Neotropical bird (Xenops minutus). We suggest that the strengths of RAD-Seq data sets for shallow systematics are the wide dispersion of markers across the genome, the relative ease and cost of laboratory work, the deep coverage and read overlap at recovered loci, and the high overall information that results. Sequence capture's benefits include flexibility and repeatability in the genomic regions targeted, success using low-quality samples, more straightforward read orthology assessment, and higher per-locus information content. The utility of a method in systematics, however, rests not only on its performance within a study, but on the comparability of data sets and inferences with those of prior work. In RAD-Seq data sets, comparability is compromised by low overlap of orthologous markers across species and the sensitivity of genetic diversity in a data set to an interaction between the level of natural heterozygosity in the samples examined and the parameters used for orthology assessment. In contrast, sequence capture of conserved genomic regions permits interrogation of the same loci across divergent species, which is preferable for maintaining comparability among data sets and studies for the purpose of drawing general conclusions about the impact of

Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

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Artyom A Alekseyenko

Full Text Available The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE. However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex and female Kc cells (which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

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

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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.

Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

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Amy L Bauer

2010-11-01

Full Text Available An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF. Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

Noninvasive imaging of three-dimensional cardiac activation sequence during pacing and ventricular tachycardia.

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Han, Chengzong; Pogwizd, Steven M; Killingsworth, Cheryl R; He, Bin

2011-08-01

Imaging cardiac excitation within ventricular myocardium is important in the treatment of cardiac arrhythmias and might help improve our understanding of arrhythmia mechanisms. This study sought to rigorously assess the imaging performance of a 3-dimensional (3D) cardiac electrical imaging (3DCEI) technique with the aid of 3D intracardiac mapping from up to 216 intramural sites during paced rhythm and norepinephrine (NE)-induced ventricular tachycardia (VT) in the rabbit heart. Body surface potentials and intramural bipolar electrical recordings were simultaneously measured in a closed-chest condition in 13 healthy rabbits. Single-site pacing and dual-site pacing were performed from ventricular walls and septum. VTs and premature ventricular complexes (PVCs) were induced by intravenous NE. Computed tomography images were obtained to construct geometry models. The noninvasively imaged activation sequence correlated well with invasively measured counterpart, with a correlation coefficient of 0.72 ± 0.04, and a relative error of 0.30 ± 0.02 averaged over 520 paced beats as well as 73 NE-induced PVCs and VT beats. All PVCs and VT beats initiated in the subendocardium by a nonreentrant mechanism. The averaged distance from the imaged site of initial activation to the pacing site or site of arrhythmias determined from intracardiac mapping was ∼5 mm. For dual-site pacing, the double origins were identified when they were located at contralateral sides of ventricles or at the lateral wall and the apex. 3DCEI can noninvasively delineate important features of focal or multifocal ventricular excitation. It offers the potential to aid in localizing the origins and imaging activation sequences of ventricular arrhythmias, and to provide noninvasive assessment of the underlying arrhythmia mechanisms. Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Prediction of protein-protein interaction sites in sequences and 3D structures by random forests.

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Mile Sikić

2009-01-01

Full Text Available Identifying interaction sites in proteins provides important clues to the function of a protein and is becoming increasingly relevant in topics such as systems biology and drug discovery. Although there are numerous papers on the prediction of interaction sites using information derived from structure, there are only a few case reports on the prediction of interaction residues based solely on protein sequence. Here, a sliding window approach is combined with the Random Forests method to predict protein interaction sites using (i a combination of sequence- and structure-derived parameters and (ii sequence information alone. For sequence-based prediction we achieved a precision of 84% with a 26% recall and an F-measure of 40%. When combined with structural information, the prediction performance increases to a precision of 76% and a recall of 38% with an F-measure of 51%. We also present an attempt to rationalize the sliding window size and demonstrate that a nine-residue window is the most suitable for predictor construction. Finally, we demonstrate the applicability of our prediction methods by modeling the Ras-Raf complex using predicted interaction sites as target binding interfaces. Our results suggest that it is possible to predict protein interaction sites with quite a high accuracy using only sequence information.

Activation and clustering of a Plasmodium falciparum var gene are affected by subtelomeric sequences.

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Duffy, Michael F; Tang, Jingyi; Sumardy, Fransisca; Nguyen, Hanh H T; Selvarajah, Shamista A; Josling, Gabrielle A; Day, Karen P; Petter, Michaela; Brown, Graham V

2017-01-01

The Plasmodium falciparum var multigene family encodes the cytoadhesive, variant antigen PfEMP1. P. falciparum antigenic variation and cytoadhesion specificity are controlled by epigenetic switching between the single, or few, simultaneously expressed var genes. Most var genes are maintained in perinuclear clusters of heterochromatic telomeres. The active var gene(s) occupy a single, perinuclear var expression site. It is unresolved whether the var expression site forms in situ at a telomeric cluster or whether it is an extant compartment to which single chromosomes travel, thus controlling var switching. Here we show that transcription of a var gene did not require decreased colocalisation with clusters of telomeres, supporting var expression site formation in situ. However following recombination within adjacent subtelomeric sequences, the same var gene was persistently activated and did colocalise less with telomeric clusters. Thus, participation in stable, heterochromatic, telomere clusters and var switching are independent but are both affected by subtelomeric sequences. The var expression site colocalised with the euchromatic mark H3K27ac to a greater extent than it did with heterochromatic H3K9me3. H3K27ac was enriched within the active var gene promoter even when the var gene was transiently repressed in mature parasites and thus H3K27ac may contribute to var gene epigenetic memory. © 2016 Federation of European Biochemical Societies.

Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

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Oscar Harari

2010-07-01

Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments

DEFF Research Database (Denmark)

Jessen, Leon Ivar; Hoof, Ilka; Lund, Ole

2013-01-01

Site does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set......) using a set of human immunodeficiency virus protease-inhibitor genotype–phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found...

Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids.

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Wu, Xiuyun; Tian, Zhennan; Jiang, Xukai; Zhang, Qun; Wang, Lushan

2018-01-01

XynB from Aspergillus niger ATCC1015 (AnXynB) is a mesophilic glycoside hydrolase (GH) family 11 xylanase which holds great potentials in a wide variety of industrial applications. In the present study, the catalytic activity and stability of AnXynB were improved by a combination of computational and experimental approaches. Virtual mutation and molecular dynamics simulations indicated that the introduction of Glu and Asn altered the interaction network at the - 3 subsite. Interestingly, the double mutant S41N/T43E displayed 72% increase in catalytic activity when compared to the wild type (WT). In addition, it also showed a better thermostability than the WT enzyme. Kinetic determination of the T43E and S41N/T43E mutants suggested that the higher xylanase activity is probably due to the increasing binding affinity of enzyme and substrate. Consequently, the enzyme activity and thermostability of AnXynB was both increased by selective site-directed mutagenesis at the - 3 subsite of its active site architecture which provides a good example for a successfully engineered enzyme for potential industrial application. Moreover, the molecular evolution approach adopted in this study led to the design of a library of sequences that captures a meaningful functional diversity in a limited number of protein variants.

The Use of Non-Variant Sites to Improve the Clinical Assessment of Whole-Genome Sequence Data.

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Alberto Ferrarini

Full Text Available Genetic testing, which is now a routine part of clinical practice and disease management protocols, is often based on the assessment of small panels of variants or genes. On the other hand, continuous improvements in the speed and per-base costs of sequencing have now made whole exome sequencing (WES and whole genome sequencing (WGS viable strategies for targeted or complete genetic analysis, respectively. Standard WGS/WES data analytical workflows generally rely on calling of sequence variants respect to the reference genome sequence. However, the reference genome sequence contains a large number of sites represented by rare alleles, by known pathogenic alleles and by alleles strongly associated to disease by GWAS. It's thus critical, for clinical applications of WGS and WES, to interpret whether non-variant sites are homozygous for the reference allele or if the corresponding genotype cannot be reliably called. Here we show that an alternative analytical approach based on the analysis of both variant and non-variant sites from WGS data allows to genotype more than 92% of sites corresponding to known SNPs compared to 6% genotyped by standard variant analysis. These include homozygous reference sites of clinical interest, thus leading to a broad and comprehensive characterization of variation necessary to an accurate evaluation of disease risk. Altogether, our findings indicate that characterization of both variant and non-variant clinically informative sites in the genome is necessary to allow an accurate clinical assessment of a personal genome. Finally, we propose a highly efficient extended VCF (eVCF file format which allows to store genotype calls for sites of clinical interest while remaining compatible with current variant interpretation software.

Prediction of glutathionylation sites in proteins using minimal sequence information and their experimental validation.

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Pal, Debojyoti; Sharma, Deepak; Kumar, Mukesh; Sandur, Santosh K

2016-09-01

S-glutathionylation of proteins plays an important role in various biological processes and is known to be protective modification during oxidative stress. Since, experimental detection of S-glutathionylation is labor intensive and time consuming, bioinformatics based approach is a viable alternative. Available methods require relatively longer sequence information, which may prevent prediction if sequence information is incomplete. Here, we present a model to predict glutathionylation sites from pentapeptide sequences. It is based upon differential association of amino acids with glutathionylated and non-glutathionylated cysteines from a database of experimentally verified sequences. This data was used to calculate position dependent F-scores, which measure how a particular amino acid at a particular position may affect the likelihood of glutathionylation event. Glutathionylation-score (G-score), indicating propensity of a sequence to undergo glutathionylation, was calculated using position-dependent F-scores for each amino-acid. Cut-off values were used for prediction. Our model returned an accuracy of 58% with Matthew's correlation-coefficient (MCC) value of 0.165. On an independent dataset, our model outperformed the currently available model, in spite of needing much less sequence information. Pentapeptide motifs having high abundance among glutathionylated proteins were identified. A list of potential glutathionylation hotspot sequences were obtained by assigning G-scores and subsequent Protein-BLAST analysis revealed a total of 254 putative glutathionable proteins, a number of which were already known to be glutathionylated. Our model predicted glutathionylation sites in 93.93% of experimentally verified glutathionylated proteins. Outcome of this study may assist in discovering novel glutathionylation sites and finding candidate proteins for glutathionylation.

On-site and off-site activities

International Nuclear Information System (INIS)

Martin, H.D.

1986-01-01

Design principles for NPP training programs. Effects of NPP contracts. Effects of domestic industrial activities. The role of international bodies. Requirements for on-site training. Training abroad, technical, financial and social aspects. Training center on-site, an evaluation. (orig.)

Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing

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Leaché, Adam D.; Chavez, Andreas S.; Jones, Leonard N.; Grummer, Jared A.; Gottscho, Andrew D.; Linkem, Charles W.

2015-01-01

Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both “recent” and “deep” timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

Imaging cardiac activation sequence during ventricular tachycardia in a canine model of nonischemic heart failure.

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Han, Chengzong; Pogwizd, Steven M; Yu, Long; Zhou, Zhaoye; Killingsworth, Cheryl R; He, Bin

2015-01-15

Noninvasive cardiac activation imaging of ventricular tachycardia (VT) is important in the clinical diagnosis and treatment of arrhythmias in heart failure (HF) patients. This study investigated the ability of the three-dimensional cardiac electrical imaging (3DCEI) technique for characterizing the activation patterns of spontaneously occurring and norepinephrine (NE)-induced VTs in a newly developed arrhythmogenic canine model of nonischemic HF. HF was induced by aortic insufficiency followed by aortic constriction in three canines. Up to 128 body-surface ECGs were measured simultaneously with bipolar recordings from up to 232 intramural sites in a closed-chest condition. Data analysis was performed on the spontaneously occurring VTs (n=4) and the NE-induced nonsustained VTs (n=8) in HF canines. Both spontaneously occurring and NE-induced nonsustained VTs initiated by a focal mechanism primarily from the subendocardium, but occasionally from the subepicardium of left ventricle. Most focal initiation sites were located at apex, right ventricular outflow tract, and left lateral wall. The NE-induced VTs were longer, more rapid, and had more focal sites than the spontaneously occurring VTs. Good correlation was obtained between imaged activation sequence and direct measurements (averaged correlation coefficient of ∼0.70 over 135 VT beats). The reconstructed initiation sites were ∼10 mm from measured initiation sites, suggesting good localization in such a large animal model with cardiac size similar to a human. Both spontaneously occurring and NE-induced nonsustained VTs had focal initiation in this canine model of nonischemic HF. 3DCEI is feasible to image the activation sequence and help define arrhythmia mechanism of nonischemic HF-associated VTs. Copyright © 2015 the American Physiological Society.

Noninvasive reconstruction of the three-dimensional ventricular activation sequence during pacing and ventricular tachycardia in the canine heart.

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Han, Chengzong; Pogwizd, Steven M; Killingsworth, Cheryl R; He, Bin

2012-01-01

Single-beat imaging of myocardial activation promises to aid in both cardiovascular research and clinical medicine. In the present study we validate a three-dimensional (3D) cardiac electrical imaging (3DCEI) technique with the aid of simultaneous 3D intracardiac mapping to assess its capability to localize endocardial and epicardial initiation sites and image global activation sequences during pacing and ventricular tachycardia (VT) in the canine heart. Body surface potentials were measured simultaneously with bipolar electrical recordings in a closed-chest condition in healthy canines. Computed tomography images were obtained after the mapping study to construct realistic geometry models. Data analysis was performed on paced rhythms and VTs induced by norepinephrine (NE). The noninvasively reconstructed activation sequence was in good agreement with the simultaneous measurements from 3D cardiac mapping with a correlation coefficient of 0.74 ± 0.06, a relative error of 0.29 ± 0.05, and a root mean square error of 9 ± 3 ms averaged over 460 paced beats and 96 ectopic beats including premature ventricular complexes, couplets, and nonsustained monomorphic VTs and polymorphic VTs. Endocardial and epicardial origins of paced beats were successfully predicted in 72% and 86% of cases, respectively, during left ventricular pacing. The NE-induced ectopic beats initiated in the subendocardium by a focal mechanism. Sites of initial activation were estimated to be ∼7 mm from the measured initiation sites for both the paced beats and ectopic beats. For the polymorphic VTs, beat-to-beat dynamic shifts of initiation site and activation pattern were characterized by the reconstruction. The present results suggest that 3DCEI can noninvasively image the 3D activation sequence and localize the origin of activation of paced beats and NE-induced VTs in the canine heart with good accuracy. This 3DCEI technique offers the potential to aid interventional therapeutic procedures for

ICRPfinder: a fast pattern design algorithm for coding sequences and its application in finding potential restriction enzyme recognition sites

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Stafford Phillip

2009-09-01

Full Text Available Abstract Background Restriction enzymes can produce easily definable segments from DNA sequences by using a variety of cut patterns. There are, however, no software tools that can aid in gene building -- that is, modifying wild-type DNA sequences to express the same wild-type amino acid sequences but with enhanced codons, specific cut sites, unique post-translational modifications, and other engineered-in components for recombinant applications. A fast DNA pattern design algorithm, ICRPfinder, is provided in this paper and applied to find or create potential recognition sites in target coding sequences. Results ICRPfinder is applied to find or create restriction enzyme recognition sites by introducing silent mutations. The algorithm is shown capable of mapping existing cut-sites but importantly it also can generate specified new unique cut-sites within a specified region that are guaranteed not to be present elsewhere in the DNA sequence. Conclusion ICRPfinder is a powerful tool for finding or creating specific DNA patterns in a given target coding sequence. ICRPfinder finds or creates patterns, which can include restriction enzyme recognition sites, without changing the translated protein sequence. ICRPfinder is a browser-based JavaScript application and it can run on any platform, in on-line or off-line mode.

Phyloscan: locating transcription-regulating binding sites in mixed aligned and unaligned sequence data.

Science.gov (United States)

Palumbo, Michael J; Newberg, Lee A

2010-07-01

The transcription of a gene from its DNA template into an mRNA molecule is the first, and most heavily regulated, step in gene expression. Especially in bacteria, regulation is typically achieved via the binding of a transcription factor (protein) or small RNA molecule to the chromosomal region upstream of a regulated gene. The protein or RNA molecule recognizes a short, approximately conserved sequence within a gene's promoter region and, by binding to it, either enhances or represses expression of the nearby gene. Since the sought-for motif (pattern) is short and accommodating to variation, computational approaches that scan for binding sites have trouble distinguishing functional sites from look-alikes. Many computational approaches are unable to find the majority of experimentally verified binding sites without also finding many false positives. Phyloscan overcomes this difficulty by exploiting two key features of functional binding sites: (i) these sites are typically more conserved evolutionarily than are non-functional DNA sequences; and (ii) these sites often occur two or more times in the promoter region of a regulated gene. The website is free and open to all users, and there is no login requirement. Address: (http://bayesweb.wadsworth.org/phyloscan/).

Noninvasive reconstruction of the three-dimensional ventricular activation sequence during pacing and ventricular tachycardia in the rabbit heart.

Science.gov (United States)

Han, Chengzong; Pogwizd, Steven M; Killingsworth, Cheryl R; He, Bin

2011-01-01

Ventricular arrhythmias represent one of leading causes for sudden cardiac death, a significant problem in public health. Noninvasive imaging of cardiac electric activities associated with ventricular arrhythmias plays an important role in better our understanding of the mechanisms and optimizing the treatment options. The present study aims to rigorously validate a novel three-dimensional (3-D) cardiac electrical imaging (3-DCEI) technique with the aid of 3-D intra-cardiac mapping during paced rhythm and ventricular tachycardia (VT) in the rabbit heart. Body surface potentials and intramural bipolar electrical recordings were simultaneously measured in a closed-chest condition in thirteen healthy rabbits. Single-site pacing and dual-site pacing were performed from ventricular walls and septum. VTs and premature ventricular complexes (PVCs) were induced by intravenous norepinephrine (NE). The non-invasively imaged activation sequence correlated well with invasively measured counterparts, with a correlation coefficient of 0.72 and a relative error of 0.30 averaged over all paced beats and NE-induced PVCs and VT beats. The averaged distance from imaged site of initial activation to measured site determined from intra-cardiac mapping was ∼5mm. These promising results suggest that 3-DCEI is feasible to non-invasively localize the origins and image activation sequence of focal ventricular arrhythmias.

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

International Nuclear Information System (INIS)

Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

1980-01-01

N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[ 14 C 2 ]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene

Maximum-likelihood model averaging to profile clustering of site types across discrete linear sequences.

Directory of Open Access Journals (Sweden)

Zhang Zhang

2009-06-01

Full Text Available A major analytical challenge in computational biology is the detection and description of clusters of specified site types, such as polymorphic or substituted sites within DNA or protein sequences. Progress has been stymied by a lack of suitable methods to detect clusters and to estimate the extent of clustering in discrete linear sequences, particularly when there is no a priori specification of cluster size or cluster count. Here we derive and demonstrate a maximum likelihood method of hierarchical clustering. Our method incorporates a tripartite divide-and-conquer strategy that models sequence heterogeneity, delineates clusters, and yields a profile of the level of clustering associated with each site. The clustering model may be evaluated via model selection using the Akaike Information Criterion, the corrected Akaike Information Criterion, and the Bayesian Information Criterion. Furthermore, model averaging using weighted model likelihoods may be applied to incorporate model uncertainty into the profile of heterogeneity across sites. We evaluated our method by examining its performance on a number of simulated datasets as well as on empirical polymorphism data from diverse natural alleles of the Drosophila alcohol dehydrogenase gene. Our method yielded greater power for the detection of clustered sites across a breadth of parameter ranges, and achieved better accuracy and precision of estimation of clusters, than did the existing empirical cumulative distribution function statistics.

Sequence Matters but How Exactly? A Method for Evaluating Activity Sequences from Data

Science.gov (United States)

Doroudi, Shayan; Holstein, Kenneth; Aleven, Vincent; Brunskill, Emma

2016-01-01

How should a wide variety of educational activities be sequenced to maximize student learning? Although some experimental studies have addressed this question, educational data mining methods may be able to evaluate a wider range of possibilities and better handle many simultaneous sequencing constraints. We introduce Sequencing Constraint…

Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

International Nuclear Information System (INIS)

Porter, M.A.; Hartman, F.C.

1986-01-01

The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

Science.gov (United States)

Lee, Ciaran M; Davis, Timothy H; Bao, Gang

2018-04-01

What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

Explorability and predictability of the paleozoic sedimentary sequence beneath the Bruce nuclear site

International Nuclear Information System (INIS)

Parmenter, A.; Jensen, M.; Crowe, R.; Raven, K.

2011-01-01

Ontario Power Generation (OPG) is proposing to develop a Deep Geologic Repository (DGR) for the long-term management of its Low and Intermediate Level Waste (L&ILW) at the Bruce nuclear site located in the Municipality of Kincardine, Ontario. A 4-year program of geoscientific studies to assess the suitability of the 850 m thick Palaeozoic age sedimentary sequence beneath the site to host the DGR was completed in 2010. The studies provide evidence of a geologic setting in which the DGR concept would be safely implemented at a nominal depth of 680 m within the argillaceous limestone of the Cobourg Formation. This paper describes the geologic framework of the Bruce nuclear site with a focus on illustrating the high degree of stratigraphic continuity and traceability at site-specific and regional scales within the Ordovician sediments proposed to host and enclose the DGR. As part of the site-specific studies, a program of deep drilling/coring (6 boreholes) and in-situ testing through the sedimentary sequence was completed from 4 drill sites situated beyond the DGR footprint, approximately 1 km apart. Core logging reveals that the stratigraphic sequence comprises 34 distinct bedrock formations/members/units consistent with the known regional stratigraphic framework. These layered sedimentary formations dip 0.6 o (~10 m/km) to the southwest with highly uniform thicknesses both at the site- and regional-scale, particularly, the Ordovician sediments, which vary on the order of metres. The occurrence of steeply-dipping faults within the sedimentary sequence is not revealed through surface outcrop fracture mapping, micro-seismic (M ≥ 1) monitoring, inclined borehole coring or intersection of hydrothermal type dolomitized reservoir systems. Potential fault structures, interpreted from a 2-D seismic survey, were targeted by angled boreholes which found no evidence for their existence. Formation specific continuity is also evidence by the lateral traceability of physical rock

DOE site performance assessment activities

International Nuclear Information System (INIS)

1990-07-01

Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions

Anoxic degradation of nitrogenous heterocyclic compounds by activated sludge and their active sites.

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Xu, Peng; Han, Hongjun; Zhuang, Haifeng; Hou, Baolin; Jia, Shengyong; Wang, Dexin; Li, Kun; Zhao, Qian

2015-05-01

The potential for degradation of five nitrogenous heterocyclic compounds (NHCs), i.e., imidazole, pyridine, indole, quinoline, and carbazole, was investigated under anoxic conditions with acclimated activated sludge. Results showed that NHCs with initial concentration of 50 mg/L could be completely degraded within 60 hr. The degradation of five NHCs was dependent upon the chemical structures with the following sequence: imidazole>pyridine>indole>quinoline>carbazole in terms of their degradation rates. Quantitative structure-biodegradability relationship studies of the five NHCs showed that the anoxic degradation rates were correlated well with highest occupied molecular orbital. Additionally, the active sites of NHCs identified by calculation were confirmed by analysis of intermediates using gas chromatography and mass spectrometry. Copyright © 2015. Published by Elsevier B.V.

Prediction of protein hydration sites from sequence by modular neural networks

DEFF Research Database (Denmark)

Ehrlich, L.; Reczko, M.; Bohr, Henrik

1998-01-01

The hydration properties of a protein are important determinants of its structure and function. Here, modular neural networks are employed to predict ordered hydration sites using protein sequence information. First, secondary structure and solvent accessibility are predicted from sequence with two...... separate neural networks. These predictions are used as input together with protein sequences for networks predicting hydration of residues, backbone atoms and sidechains. These networks are teined with protein crystal structures. The prediction of hydration is improved by adding information on secondary...... structure and solvent accessibility and, using actual values of these properties, redidue hydration can be predicted to 77% accuracy with a Metthews coefficient of 0.43. However, predicted property data with an accuracy of 60-70% result in less than half the improvement in predictive performance observed...

Molecular dynamics explorations of active site structure in designed and evolved enzymes.

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Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

2015-04-21

This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

DEFF Research Database (Denmark)

Uren, Anthony G; Mikkers, Harald; Kool, Jaap

2009-01-01

sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

International Nuclear Information System (INIS)

Vlahos, C.J.

1987-01-01

The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with [ 14 C]pyruvate in the presence of NaCNBH 3 results in the incorporation of one mol of 14 C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with [3- 14 C]bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved

Importance of Viral Sequence Length and Number of Variable and Informative Sites in Analysis of HIV Clustering.

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Novitsky, Vlad; Moyo, Sikhulile; Lei, Quanhong; DeGruttola, Victor; Essex, M

2015-05-01

To improve the methodology of HIV cluster analysis, we addressed how analysis of HIV clustering is associated with parameters that can affect the outcome of viral clustering. The extent of HIV clustering and tree certainty was compared between 401 HIV-1C near full-length genome sequences and subgenomic regions retrieved from the LANL HIV Database. Sliding window analysis was based on 99 windows of 1,000 bp and 45 windows of 2,000 bp. Potential associations between the extent of HIV clustering and sequence length and the number of variable and informative sites were evaluated. The near full-length genome HIV sequences showed the highest extent of HIV clustering and the highest tree certainty. At the bootstrap threshold of 0.80 in maximum likelihood (ML) analysis, 58.9% of near full-length HIV-1C sequences but only 15.5% of partial pol sequences (ViroSeq) were found in clusters. Among HIV-1 structural genes, pol showed the highest extent of clustering (38.9% at a bootstrap threshold of 0.80), although it was significantly lower than in the near full-length genome sequences. The extent of HIV clustering was significantly higher for sliding windows of 2,000 bp than 1,000 bp. We found a strong association between the sequence length and proportion of HIV sequences in clusters, and a moderate association between the number of variable and informative sites and the proportion of HIV sequences in clusters. In HIV cluster analysis, the extent of detectable HIV clustering is directly associated with the length of viral sequences used, as well as the number of variable and informative sites. Near full-length genome sequences could provide the most informative HIV cluster analysis. Selected subgenomic regions with a high extent of HIV clustering and high tree certainty could also be considered as a second choice.

NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility

DEFF Research Database (Denmark)

Hansen, Jan Erik; Lund, Ole; Tolstrup, Niels

1998-01-01

-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predicition of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O...... structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. charged residues were disfavoured at postition -1 and +3......-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based...

Genome-wide identification and characterization of Notch transcription complex-binding sequence paired sites in leukemia cells

Science.gov (United States)

Severson, Eric; Arnett, Kelly L.; Wang, Hongfang; Zang, Chongzhi; Taing, Len; Liu, Hudan; Pear, Warren S.; Liu, X. Shirley; Blacklow, Stephen C.; Aster, Jon C.

2018-01-01

Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and are linked to the Notch-responsiveness of a few genes, but their overall contribution to Notch-dependent gene regulation is unknown. To address this issue, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay, and applied insights from these in vitro studies to Notch-“addicted” leukemia cells. We find that SPSs contribute to the regulation of approximately a third of direct Notch target genes. While originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates HES5. Our work provides a general method for identifying sequence-paired sites in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells. PMID:28465412

Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

Science.gov (United States)

Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

2011-02-01

RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Predicting protein amidation sites by orchestrating amino acid sequence features

Science.gov (United States)

Zhao, Shuqiu; Yu, Hua; Gong, Xiujun

2017-08-01

Amidation is the fourth major category of post-translational modifications, which plays an important role in physiological and pathological processes. Identifying amidation sites can help us understanding the amidation and recognizing the original reason of many kinds of diseases. But the traditional experimental methods for predicting amidation sites are often time-consuming and expensive. In this study, we propose a computational method for predicting amidation sites by orchestrating amino acid sequence features. Three kinds of feature extraction methods are used to build a feature vector enabling to capture not only the physicochemical properties but also position related information of the amino acids. An extremely randomized trees algorithm is applied to choose the optimal features to remove redundancy and dependence among components of the feature vector by a supervised fashion. Finally the support vector machine classifier is used to label the amidation sites. When tested on an independent data set, it shows that the proposed method performs better than all the previous ones with the prediction accuracy of 0.962 at the Matthew's correlation coefficient of 0.89 and area under curve of 0.964.

Amino acid sequence surrounding the chondroitin sulfate attachment site of thrombomodulin regulates chondroitin polymerization.

Science.gov (United States)

Izumikawa, Tomomi; Kitagawa, Hiroshi

2015-05-01

Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of cis-regulatory sequences that activate transcription in the suspensor of plant embryos.

Science.gov (United States)

Kawashima, Tomokazu; Wang, Xingjun; Henry, Kelli F; Bi, Yuping; Weterings, Koen; Goldberg, Robert B

2009-03-03

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the scarlet runner bean (Phaseolus coccineus) G564 gene to understand how genes are activated specifically within the suspensor during early embryo development. Previously, we showed that the G564 upstream region has a block of tandem repeats, which contain a conserved 10-bp motif (GAAAAG(C)/(T)GAA), and that deletion of these repeats results in a loss of suspensor transcription. Here, we use gain-of-function (GOF) experiments with transgenic globular-stage tobacco embryos to show that only 1 of the 5 tandem repeats is required to drive suspensor-specific transcription. Fine-scale deletion and scanning mutagenesis experiments with 1 tandem repeat uncovered a 54-bp region that contains all of the sequences required to activate transcription in the suspensor, including the 10-bp motif (GAAAAGCGAA) and a similar 10-bp-like motif (GAAAAACGAA). Site-directed mutagenesis and GOF experiments indicated that both the 10-bp and 10-bp-like motifs are necessary, but not sufficient to activate transcription in the suspensor, and that a sequence (TTGGT) between the 10-bp and the 10-bp-like motifs is also necessary for suspensor transcription. Together, these data identify sequences that are required to activate transcription in the suspensor of a plant embryo after fertilization.

Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

Science.gov (United States)

Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

2015-03-01

Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

Mason: a JavaScript web site widget for visualizing and comparing annotated features in nucleotide or protein sequences.

Science.gov (United States)

Jaschob, Daniel; Davis, Trisha N; Riffle, Michael

2015-03-07

Sequence feature annotations (e.g., protein domain boundaries, binding sites, and secondary structure predictions) are an essential part of biological research. Annotations are widely used by scientists during research and experimental design, and are frequently the result of biological studies. A generalized and simple means of disseminating and visualizing these data via the web would be of value to the research community. Mason is a web site widget designed to visualize and compare annotated features of one or more nucleotide or protein sequence. Annotated features may be of virtually any type, ranging from annotating transcription binding sites or exons and introns in DNA to secondary structure or domain boundaries in proteins. Mason is simple to use and easy to integrate into web sites. Mason has a highly dynamic and configurable interface supporting multiple sets of annotations per sequence, overlapping regions, customization of interface and user-driven events (e.g., clicks and text to appear for tooltips). It is written purely in JavaScript and SVG, requiring no 3(rd) party plugins or browser customization. Mason is a solution for dissemination of sequence annotation data on the web. It is highly flexible, customizable, simple to use, and is designed to be easily integrated into web sites. Mason is open source and freely available at https://github.com/yeastrc/mason.

Metagenome sequencing of the microbial community of two Brazilian anthropogenic Amazon dark earth sites, Brazil.

Science.gov (United States)

Lemos, Leandro Nascimento; de Souza, Rosineide Cardoso; de Souza Cannavan, Fabiana; Patricio, André; Pylro, Victor Satler; Hanada, Rogério Eiji; Mui, Tsai Siu

2016-12-01

The Anthropogenic Amazon Dark Earth soil is considered one of the world's most fertile soils. These soils differs from conventional Amazon soils because its higher organic content concentration. Here we describe the metagenome sequencing of microbial communities of two sites of Anthropogenic Amazon Dark Earth soils from Amazon Rainforest, Brazil. The raw sequence data are stored under Short Read Accession number: PRJNA344917.

Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

Directory of Open Access Journals (Sweden)

Jian Sun

Full Text Available We used a next-generation, Illumina-based sequencing approach to characterize the bacterial community development of apple rhizosphere soil in a replant site (RePlant and a new planting site (NewPlant in Beijing. Dwarfing apple nurseries of 'Fuji'/SH6/Pingyitiancha trees were planted in the spring of 2013. Before planting, soil from the apple rhizosphere of the replant site (ReSoil and from the new planting site (NewSoil was sampled for analysis on the Illumina MiSeq platform. In late September, the rhizosphere soil from both sites was resampled (RePlant and NewPlant. More than 16,000 valid reads were obtained for each replicate, and the community was composed of five dominant groups (Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria. The bacterial diversity decreased after apple planting. Principal component analyses revealed that the rhizosphere samples were significantly different among treatments. Apple nursery planting showed a large impact on the soil bacterial community, and the community development was significantly different between the replanted and newly planted soils. Verrucomicrobia were less abundant in RePlant soil, while Pseudomonas and Lysobacter were increased in RePlant compared with ReSoil and NewPlant. Both RePlant and ReSoil showed relatively higher invertase and cellulase activities than NewPlant and NewSoil, but only NewPlant soil showed higher urease activity, and this soil also had the higher plant growth. Our experimental results suggest that planting apple nurseries has a significant impact on soil bacterial community development at both replant and new planting sites, and planting on new site resulted in significantly higher soil urease activity and a different bacterial community composition.

GenProBiS: web server for mapping of sequence variants to protein binding sites.

Science.gov (United States)

Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

2017-07-03

Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Random Tagging Genotyping by Sequencing (rtGBS, an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome.

Directory of Open Access Journals (Sweden)

Elena Hilario

Full Text Available Genotyping by sequencing (GBS is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al.some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS. By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145 of BamH I sites shared with the reference genome, compared to only 14% (11,513 by stdGBS.

eMatchSite: sequence order-independent structure alignments of ligand binding pockets in protein models.

Directory of Open Access Journals (Sweden)

Michal Brylinski

2014-09-01

Full Text Available Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4-9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite.

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

Science.gov (United States)

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

Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor

International Nuclear Information System (INIS)

Dewji, N.N.; Wenger, D.A.; O'Brien, J.S.

1987-01-01

Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a λ gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of ≅ 2 kilobases (λ-S-1.2 and λ-S-1.3) and both were both homologous with a previously isolated clone (λ-S-1.1) for mature SAP-1. The authors report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is ≅ 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide mature SAP-1 is generated by removing an additional 7 amino acids from the amino terminus and ≅ 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which they designate as P-2

Conserved antigenic sites between MERS-CoV and Bat-coronavirus are revealed through sequence analysis.

Science.gov (United States)

Sharmin, Refat; Islam, Abul B M M K

2016-01-01

MERS-CoV is a newly emerged human coronavirus reported closely related with HKU4 and HKU5 Bat coronaviruses. Bat and MERS corona-viruses are structurally related. Therefore, it is of interest to estimate the degree of conserved antigenic sites among them. It is of importance to elucidate the shared antigenic-sites and extent of conservation between them to understand the evolutionary dynamics of MERS-CoV. Multiple sequence alignment of the spike (S), membrane (M), enveloped (E) and nucleocapsid (N) proteins was employed to identify the sequence conservation among MERS and Bat (HKU4, HKU5) coronaviruses. We used various in silico tools to predict the conserved antigenic sites. We found that MERS-CoV shared 30 % of its S protein antigenic sites with HKU4 and 70 % with HKU5 bat-CoV. Whereas 100 % of its E, M and N protein's antigenic sites are found to be conserved with those in HKU4 and HKU5. This sharing suggests that in case of pathogenicity MERS-CoV is more closely related to HKU5 bat-CoV than HKU4 bat-CoV. The conserved epitopes indicates their evolutionary relationship and ancestry of pathogenicity.

A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data.

Science.gov (United States)

Park, Doori; Park, Su-Hyun; Ban, Yong Wook; Kim, Youn Shic; Park, Kyoung-Cheul; Kim, Nam-Soo; Kim, Ju-Kon; Choi, Ik-Young

2017-08-15

Genetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms. To detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment. Herein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9-5, SNU-Bt9-30, and SNU-Bt9-109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses. NGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.

Prediction of transcriptional regulatory sites in the complete genome sequence of Escherichia coli K-12.

Science.gov (United States)

Thieffry, D; Salgado, H; Huerta, A M; Collado-Vides, J

1998-06-01

As one of the best-characterized free-living organisms, Escherichia coli and its recently completed genomic sequence offer a special opportunity to exploit systematically the variety of regulatory data available in the literature in order to make a comprehensive set of regulatory predictions in the whole genome. The complete genome sequence of E.coli was analyzed for the binding of transcriptional regulators upstream of coding sequences. The biological information contained in RegulonDB (Huerta, A.M. et al., Nucleic Acids Res.,26,55-60, 1998) for 56 different transcriptional proteins was the support to implement a stringent strategy combining string search and weight matrices. We estimate that our search included representatives of 15-25% of the total number of regulatory binding proteins in E.coli. This search was performed on the set of 4288 putative regulatory regions, each 450 bp long. Within the regions with predicted sites, 89% are regulated by one protein and 81% involve only one site. These numbers are reasonably consistent with the distribution of experimental regulatory sites. Regulatory sites are found in 603 regions corresponding to 16% of operon regions and 10% of intra-operonic regions. Additional evidence gives stronger support to some of these predictions, including the position of the site, biological consistency with the function of the downstream gene, as well as genetic evidence for the regulatory interaction. The predictions described here were incorporated into the map presented in the paper describing the complete E.coli genome (Blattner,F.R. et al., Science, 277, 1453-1461, 1997). The complete set of predictions in GenBank format is available at the url: http://www. cifn.unam.mx/Computational_Biology/E.coli-predictions ecoli-reg@cifn.unam.mx, collado@cifn.unam.mx

Solar-Type Activity in Main-Sequence Stars

CERN Document Server

Gershberg, Roald E

2005-01-01

Solar-type activity over the whole range of the electromagnetic spectrum is a phenomenon inherent in the majority of low- and moderate-mass main sequence stars. In this monograph observational results are summarized in a systematic and comprehensive fashion. The analysis of the various manifestations of such stellar activity leads to the identification of these phenomena with macroscopic non-linear processes in a magnetized plasma. Comparative study of flare stars and the Sun has become increasingly fruitful and is presently an active field of research involving stellar and solar physicists, experts in plasma physics and high-energy astrophysicists. This book will provide them with both an introduction and overview of observational results from the first optical photometry and spectroscopy, from the satellite telescopes International Ultraviolet Explorer to Hubble Space Telescope, XMM-Newton and Chandra, as well as with the present physical interpretation of solar-type activity in main sequence stars. Gershbe...

Folding and activity of hybrid sequence, disulfide-stabilized peptides

Energy Technology Data Exchange (ETDEWEB)

Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E. (Univ. of California, Berkeley (USA))

1990-08-01

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a {beta}-turn and an {alpha}-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the {alpha}-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences.

Folding and activity of hybrid sequence, disulfide-stabilized peptides

International Nuclear Information System (INIS)

Pease, J.H.B.; Storrs, R.W.; Wemmer, D.E.

1990-01-01

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a β-turn and an α-helix. These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. The authors show that in S peptide the α-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure - function relationships for many biologically active sequences

Site directed recombination

Science.gov (United States)

Jurka, Jerzy W.

1997-01-01

Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

SiteFind: A software tool for introducing a restriction site as a marker for successful site-directed mutagenesis

Directory of Open Access Journals (Sweden)

Evans Paul M

2005-12-01

Full Text Available Abstract Background Site-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed. However, this method provides no means for verifying its success other than sequencing the putative mutant construct: This can quickly become an expensive method for screening for successful mutations. An alternative to sequencing is to simultaneously introduce a restriction site near the point mutation in manner such that the restriction site has no effect on the translated amino acid sequence. Thus, the novel restriction site can be used as a marker for successful mutation which can be quickly and easily assessed. However, finding a restriction site that does not disturb the corresponding amino acid sequence is a time-consuming task even for experienced researchers. A fast and easy to use computer program is needed for this task. Results We wrote a computer program, called SiteFind, to help us design a restriction site within the mutation primers without changing the peptide sequence. Because of the redundancy of genetic code, a given peptide can be encoded by many different DNA sequences. Since the list of possible restriction sites for a given DNA sequence is not always obvious, SiteFind automates this task. The number of possible sequences a computer program must search through increases exponentially as the sequence length increases. SiteFind uses a novel "moving window" algorithm to reduce the number of possible sequences to be searched to a manageable level. The user enters a nucleotide sequence, specifies what amino acid residues should be changed in the mutation, and SiteFind generates a list of possible restriction sites and what nucleotides must be changed to introduce that site. As a demonstration of its use, we successfully generated a single point mutation

Activities on the site during construction phase

International Nuclear Information System (INIS)

Fickel, O.F.

1977-01-01

A survey is given of the work done on the site from site-opening till turn over of the plant to the client. After a short introduction to time schedules, manpower on site, site facilities and civil work and constructions, the commissioning and trial operation phase is discussed in detail. This phase begins with finishing the assembly of individual systems and components and ends with the trial operation and the acceptance measurement. During this period the subsystems are started-up in a useful sequence, first from cold, then from hot conditions and are finally operated as a total with nuclear energy. The single steps are: a) commissioning of indivudal systems; b) hot functional test 1 (without fuels) c) baseline inspection at the reactor pressure vessel; d) hot functional test 2 (with fuels); e) preparation for first criticality; f) postcriticality test program; g) trial operation: h) acceptance measurement. (HP) [de

Utility of two types of MR cisternography for patency evaluation of aqueduct and third ventriculostomy site: Three dimentsional sagittal fast spin echo sequence and steady-state coherent fast gradient echo sequence

Energy Technology Data Exchange (ETDEWEB)

Park, Jung Hyun; Kim, Eun Hee; Park, Jong Bin; Kim, Jae Hyoung; Choi, Byung Se; Jung, Cheol Kyu; Bae, Yun Jung; Lee, Kyung Mi [Dept. of Radiology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of)

2015-07-15

We aimed to evaluate the utility of two types of MR cisternography [fast spin echo sequence and steady-state coherent gradient echo (GRE) sequence] in addition to phase contrast-cine imaging (PC-cine), for assessing patency at the aqueduct and third ventriculostomy site. 43 patients (35 patients with suspected aqueductal stenosis and 8 patients with third ventriculostomy) were retrospectively analyzed. PC-cine, 3 dimensional sagittal fast spin echo sequence [driven-equilibrium imaging (DRIVE) or volumetric isotrophic T2-weighted acquisition (T2 VISTA)] and steady-state coherent fast GRE sequence (balanced turbo field echo; bTFE) imaging were performed in all patients. The patency of the aqueduct or third ventriculostomy site was scored. Some pitfalls of each sequence were also analyzed in individual cases. 93% of all cases showed consistent scores in PC-cine, DRIVE/T2 VISTA, and bTFE imaging. DRIVE/T2 VISTA imaging provided functional information of cerebrospinal fluid flow with flow-related artifacts, while bTFE imaging allowed direct visualization of the aqueduct or ventriculostomy site. However, evaluation of anatomical structures was difficult in three cases with strong flow-related artifacts on DRIVE/T2 VISTA and in 2 cases with susceptibility artifacts on bTFE. Both DRIVE/T2 VISTA and bTFE imaging have complementary roles in evaluating the patency of the aqueduct and 3rd ventriculostomy site.

Utility of two types of MR cisternography for patency evaluation of aqueduct and third ventriculostomy site: Three dimentsional sagittal fast spin echo sequence and steady-state coherent fast gradient echo sequence

International Nuclear Information System (INIS)

Park, Jung Hyun; Kim, Eun Hee; Park, Jong Bin; Kim, Jae Hyoung; Choi, Byung Se; Jung, Cheol Kyu; Bae, Yun Jung; Lee, Kyung Mi

2015-01-01

We aimed to evaluate the utility of two types of MR cisternography [fast spin echo sequence and steady-state coherent gradient echo (GRE) sequence] in addition to phase contrast-cine imaging (PC-cine), for assessing patency at the aqueduct and third ventriculostomy site. 43 patients (35 patients with suspected aqueductal stenosis and 8 patients with third ventriculostomy) were retrospectively analyzed. PC-cine, 3 dimensional sagittal fast spin echo sequence [driven-equilibrium imaging (DRIVE) or volumetric isotrophic T2-weighted acquisition (T2 VISTA)] and steady-state coherent fast GRE sequence (balanced turbo field echo; bTFE) imaging were performed in all patients. The patency of the aqueduct or third ventriculostomy site was scored. Some pitfalls of each sequence were also analyzed in individual cases. 93% of all cases showed consistent scores in PC-cine, DRIVE/T2 VISTA, and bTFE imaging. DRIVE/T2 VISTA imaging provided functional information of cerebrospinal fluid flow with flow-related artifacts, while bTFE imaging allowed direct visualization of the aqueduct or ventriculostomy site. However, evaluation of anatomical structures was difficult in three cases with strong flow-related artifacts on DRIVE/T2 VISTA and in 2 cases with susceptibility artifacts on bTFE. Both DRIVE/T2 VISTA and bTFE imaging have complementary roles in evaluating the patency of the aqueduct and 3rd ventriculostomy site

Sequence requirement of the ade6-4095 meiotic recombination hotspot in Schizosaccharomyces pombe.

Science.gov (United States)

Foulis, Steven J; Fowler, Kyle R; Steiner, Walter W

2018-02-01

Homologous recombination occurs at a greatly elevated frequency in meiosis compared to mitosis and is initiated by programmed double-strand DNA breaks (DSBs). DSBs do not occur at uniform frequency throughout the genome in most organisms, but occur preferentially at a limited number of sites referred to as hotspots. The location of hotspots have been determined at nucleotide-level resolution in both the budding and fission yeasts, and while several patterns have emerged regarding preferred locations for DSB hotspots, it remains unclear why particular sites experience DSBs at much higher frequency than other sites with seemingly similar properties. Short sequence motifs, which are often sites for binding of transcription factors, are known to be responsible for a number of hotspots. In this study we identified the minimum sequence required for activity of one of such motif identified in a screen of random sequences capable of producing recombination hotspots. The experimentally determined sequence, GGTCTRGACC, closely matches the previously inferred sequence. Full hotspot activity requires an effective sequence length of 9.5 bp, whereas moderate activity requires an effective sequence length of approximately 8.2 bp and shows significant association with DSB hotspots. In combination with our previous work, this result is consistent with a large number of different sequence motifs capable of producing recombination hotspots, and supports a model in which hotspots can be rapidly regenerated by mutation as they are lost through recombination.

Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

Science.gov (United States)

Clifford, Jacob; Adami, Christoph

2015-09-02

Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

Directory of Open Access Journals (Sweden)

Kareem Mohideen-Abdul

2017-06-01

Full Text Available Most nuclear receptors (NRs bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR belongs to the steroid hormone nuclear receptor (SHR family and shares strong similarity in its DNA-binding domain (DBD with that of the estrogen receptor (ER. In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3, but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS, non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3, such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3, where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site

Predicting sumoylation sites using support vector machines based on various sequence features, conformational flexibility and disorder.

Science.gov (United States)

Yavuz, Ahmet Sinan; Sezerman, Osman Ugur

2014-01-01

Sumoylation, which is a reversible and dynamic post-translational modification, is one of the vital processes in a cell. Before a protein matures to perform its function, sumoylation may alter its localization, interactions, and possibly structural conformation. Abberations in protein sumoylation has been linked with a variety of disorders and developmental anomalies. Experimental approaches to identification of sumoylation sites may not be effective due to the dynamic nature of sumoylation, laborsome experiments and their cost. Therefore, computational approaches may guide experimental identification of sumoylation sites and provide insights for further understanding sumoylation mechanism. In this paper, the effectiveness of using various sequence properties in predicting sumoylation sites was investigated with statistical analyses and machine learning approach employing support vector machines. These sequence properties were derived from windows of size 7 including position-specific amino acid composition, hydrophobicity, estimated sub-window volumes, predicted disorder, and conformational flexibility. 5-fold cross-validation results on experimentally identified sumoylation sites revealed that our method successfully predicts sumoylation sites with a Matthew's correlation coefficient, sensitivity, specificity, and accuracy equal to 0.66, 73%, 98%, and 97%, respectively. Additionally, we have showed that our method compares favorably to the existing prediction methods and basic regular expressions scanner. By using support vector machines, a new, robust method for sumoylation site prediction was introduced. Besides, the possible effects of predicted conformational flexibility and disorder on sumoylation site recognition were explored computationally for the first time to our knowledge as an additional parameter that could aid in sumoylation site prediction.

Identification of sequence motifs significantly associated with antisense activity

Directory of Open Access Journals (Sweden)

Peek Andrew S

2007-06-01

Full Text Available Abstract Background Predicting the suppression activity of antisense oligonucleotide sequences is the main goal of the rational design of nucleic acids. To create an effective predictive model, it is important to know what properties of an oligonucleotide sequence associate significantly with antisense activity. Also, for the model to be efficient we must know what properties do not associate significantly and can be omitted from the model. This paper will discuss the results of a randomization procedure to find motifs that associate significantly with either high or low antisense suppression activity, analysis of their properties, as well as the results of support vector machine modelling using these significant motifs as features. Results We discovered 155 motifs that associate significantly with high antisense suppression activity and 202 motifs that associate significantly with low suppression activity. The motifs range in length from 2 to 5 bases, contain several motifs that have been previously discovered as associating highly with antisense activity, and have thermodynamic properties consistent with previous work associating thermodynamic properties of sequences with their antisense activity. Statistical analysis revealed no correlation between a motif's position within an antisense sequence and that sequences antisense activity. Also, many significant motifs existed as subwords of other significant motifs. Support vector regression experiments indicated that the feature set of significant motifs increased correlation compared to all possible motifs as well as several subsets of the significant motifs. Conclusion The thermodynamic properties of the significantly associated motifs support existing data correlating the thermodynamic properties of the antisense oligonucleotide with antisense efficiency, reinforcing our hypothesis that antisense suppression is strongly associated with probe/target thermodynamics, as there are no enzymatic

The predictable nature of the Paleozoic sedimentary sequence beneath the Bruce nuclear site in Southern Ontario, Canada

International Nuclear Information System (INIS)

Parmenter, Andrew; Jensen, Mark; Crowe, Richard

2012-01-01

Document available in extended abstract form only. A key aspect of a Deep Geologic Repository (DGR) safety case is the ability to develop and communicate an understanding of the geologic stability and resilience to change at time frames relevant to demonstrating repository performance. As part of an on-going Environmental Assessment, Ontario Power Generation (OPG) recently completed site-specific investigations within an 850 m thick Paleozoic sedimentary sequence beneath the Bruce nuclear site for the proposed development of a DGR for Low and Intermediate Level Waste (L and ILW). As envisioned, the shaft-accessed DGR would be excavated at a nominal depth of 680 m within the low permeability Ordovician argillaceous limestone of the Cobourg Formation, which is overlain by more than 200 m of low permeability Ordovician shale. The geo-scientific investigations revealed a relatively undeformed and laterally continuous architecture within the sedimentary sequence at the repository scale (1.5 km 2 ) and beyond. This paper explores the predictable nature of the sedimentary sequence that has contributed to increasing confidence in an understanding of the spatial distribution of groundwater system properties, deep groundwater system evolution and natural barrier performance. Multi-disciplinary geo-scientific investigations of the Bruce nuclear site were completed in 3 phases between 2006 and 2010. The sub-surface investigations included a deep drilling, coring and in-situ testing program and, the completion of a 19.7 km (9 lines) 2-D seismic reflection survey. The drilling program involved 6 (150 mm dia.) deep boreholes (4-vertical; 2 inclined) that were extended through the sedimentary sequence from 4 drill sites, arranged around the 0.3 km 2 footprint of the proposed repository. The more than 3.8 km of rock core (77 mm dia.) retrieved have provided, in part, a strong basis to understand bedrock lithology and mineralogy, facies assemblages, structure, and oil and gas

Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure.

Directory of Open Access Journals (Sweden)

Masfique Mehedi

Full Text Available Ebolavirus (EBOV, the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure.

Science.gov (United States)

Mehedi, Masfique; Hoenen, Thomas; Robertson, Shelly; Ricklefs, Stacy; Dolan, Michael A; Taylor, Travis; Falzarano, Darryl; Ebihara, Hideki; Porcella, Stephen F; Feldmann, Heinz

2013-01-01

Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

DEFF Research Database (Denmark)

Kielpinski, Lukasz J; Boyd, Mette; Sandelin, Albin

2013-01-01

Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput...... of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR...

Efficient oxygen electrocatalysis on special active sites

DEFF Research Database (Denmark)

Halck, Niels Bendtsen

throughout this thesis to understand these local structure effects and their influence on surface reactions. The concept of these special active sites is used to explain how oxygen evolution reaction (OER) catalysts can have activities beyond the limits of what was previously thought possible. The concept...... stored in these bonds in an eco-friendly fashion in fuel cells. This thesis explores catalysts for oxygen electrocatalysis and how carefully designed local structures on catalysts surfaces termed special active sites can influence the activity. Density functional theory has been used as a method...... is used to explain the increase in activity observed for the OER catalyst ruthenium dioxide when it is mixed with nickel or cobalt. Manganese and cobalt oxides when in the vicinity of gold also display an increase in OER activity which can be explained by locally created special active sites. Density...

Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo

DEFF Research Database (Denmark)

Mikkelsen, J G; Lund, Anders Henrik; Duch, M

2000-01-01

synthesis in newly infected cells. We have previously shown that template shifts within the 5' leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single......-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site...... specificity of recombination within the highly structured 5' leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA...

Brain activation during anticipation of sound sequences.

Science.gov (United States)

Leaver, Amber M; Van Lare, Jennifer; Zielinski, Brandon; Halpern, Andrea R; Rauschecker, Josef P

2009-02-25

Music consists of sound sequences that require integration over time. As we become familiar with music, associations between notes, melodies, and entire symphonic movements become stronger and more complex. These associations can become so tight that, for example, hearing the end of one album track can elicit a robust image of the upcoming track while anticipating it in total silence. Here, we study this predictive "anticipatory imagery" at various stages throughout learning and investigate activity changes in corresponding neural structures using functional magnetic resonance imaging. Anticipatory imagery (in silence) for highly familiar naturalistic music was accompanied by pronounced activity in rostral prefrontal cortex (PFC) and premotor areas. Examining changes in the neural bases of anticipatory imagery during two stages of learning conditional associations between simple melodies, however, demonstrates the importance of fronto-striatal connections, consistent with a role of the basal ganglia in "training" frontal cortex (Pasupathy and Miller, 2005). Another striking change in neural resources during learning was a shift between caudal PFC earlier to rostral PFC later in learning. Our findings regarding musical anticipation and sound sequence learning are highly compatible with studies of motor sequence learning, suggesting common predictive mechanisms in both domains.

Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

Directory of Open Access Journals (Sweden)

Wang Nian

2012-08-01

Full Text Available Abstract Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison.

Integrin activation dynamics between the RGD-binding site and the headpiece hinge.

Science.gov (United States)

Puklin-Faucher, Eileen; Vogel, Viola

2009-12-25

Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII(10)-bound alpha(V)beta(3) integrin headpiece how the binding pocket and interdomain betaA/hybrid domain hinge on the distal end of the betaA domain are allosterically linked via a hydrophobic T-junction between the middle of the alpha1 helix and top of the alpha7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca(2+) in place of Mg(2+) at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca(2+) at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.

Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

International Nuclear Information System (INIS)

Zull, Lawrence M.; Yeniscavich, William

2008-01-01

The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites

Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity

DEFF Research Database (Denmark)

Nørgaard, P; Winther, Jakob R.

2001-01-01

to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight...... into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild...

A ChIP-Seq benchmark shows that sequence conservation mainly improves detection of strong transcription factor binding sites.

Directory of Open Access Journals (Sweden)

Tony Håndstad

Full Text Available BACKGROUND: Transcription factors are important controllers of gene expression and mapping transcription factor binding sites (TFBS is key to inferring transcription factor regulatory networks. Several methods for predicting TFBS exist, but there are no standard genome-wide datasets on which to assess the performance of these prediction methods. Also, it is believed that information about sequence conservation across different genomes can generally improve accuracy of motif-based predictors, but it is not clear under what circumstances use of conservation is most beneficial. RESULTS: Here we use published ChIP-seq data and an improved peak detection method to create comprehensive benchmark datasets for prediction methods which use known descriptors or binding motifs to detect TFBS in genomic sequences. We use this benchmark to assess the performance of five different prediction methods and find that the methods that use information about sequence conservation generally perform better than simpler motif-scanning methods. The difference is greater on high-affinity peaks and when using short and information-poor motifs. However, if the motifs are specific and information-rich, we find that simple motif-scanning methods can perform better than conservation-based methods. CONCLUSIONS: Our benchmark provides a comprehensive test that can be used to rank the relative performance of transcription factor binding site prediction methods. Moreover, our results show that, contrary to previous reports, sequence conservation is better suited for predicting strong than weak transcription factor binding sites.

Meaningful spatial and temporal sequences of activities in dwelling

NARCIS (Netherlands)

Hematalikeikha, M.A.; Coolen, H.C.C.H.; Pourdeihimi, S.

2014-01-01

Human activities based on human needs are affected by affordances and meanings that occur in the dwelling. Activities over time and space have meaningful sequences. The meaningfulness of activities in the cultural framework is conditioned by its special temporality and spatiality. Also, temporal or

Deduction of upstream sequences of Xanthomonas campestris flagellar genes responding to transcription activation by FleQ

International Nuclear Information System (INIS)

Hu, R.-M.; Yang, T.-C.; Yang, S.-H.; Tseng, Y.-H.

2005-01-01

Xanthomonas campestris pv. campestris (Xcc), a close relative to Pseudomonas aeruginosa, is the pathogen causing black rot in cruciferous plants. In P. aeruginosa, FleQ serves as a cognate activator of σ 54 in transcription from several σ 54 -dependent promoters of flagellar genes. These P. aeruginosa promoters have been analyzed for FleQ-binding sequences; however, no consensus was deduced. Xcc, although lacks fleSR, has a fleQ homologue residing among over 40 contiguously clustered flagellar genes. A fleQ mutant, Xc17fleQ, constructed by insertional mutation is deficient in FleQ protein, non-flagellated, and immobile. Transcriptional fusion assays on six putative σ 54 -dependent promoters of the flagellar genes, fliE, fliQ, fliL, flgG, flgB, and flhF, indicated that each of them is also FleQ dependent. Each of these promoters has a sequence with weak consensus to 5'-gaaacCCgccgCcgctTt-3', immediately upstream of the predicted σ 54 -binding site, with an imperfect inverted repeat containing a GC-rich center flanked by several A and T at 5'- and 3'-ends, respectively. Replacing this region in fliE promoter with a HindIII recognition sequence abolished the transcription, indicating that this region responds to transcription activation by FleQ

Identification of metal ion binding sites based on amino acid sequences.

Science.gov (United States)

Cao, Xiaoyong; Hu, Xiuzhen; Zhang, Xiaojin; Gao, Sujuan; Ding, Changjiang; Feng, Yonge; Bao, Weihua

2017-01-01

The identification of metal ion binding sites is important for protein function annotation and the design of new drug molecules. This study presents an effective method of analyzing and identifying the binding residues of metal ions based solely on sequence information. Ten metal ions were extracted from the BioLip database: Zn2+, Cu2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Na+, K+ and Co2+. The analysis showed that Zn2+, Cu2+, Fe2+, Fe3+, and Co2+ were sensitive to the conservation of amino acids at binding sites, and promising results can be achieved using the Position Weight Scoring Matrix algorithm, with an accuracy of over 79.9% and a Matthews correlation coefficient of over 0.6. The binding sites of other metals can also be accurately identified using the Support Vector Machine algorithm with multifeature parameters as input. In addition, we found that Ca2+ was insensitive to hydrophobicity and hydrophilicity information and Mn2+ was insensitive to polarization charge information. An online server was constructed based on the framework of the proposed method and is freely available at http://60.31.198.140:8081/metal/HomePage/HomePage.html.

Magnetic nanoparticle imaging by random and maximum length sequences of inhomogeneous activation fields.

Science.gov (United States)

Baumgarten, Daniel; Eichardt, Roland; Crevecoeur, Guillaume; Supriyanto, Eko; Haueisen, Jens

2013-01-01

Biomedical applications of magnetic nanoparticles require a precise knowledge of their biodistribution. From multi-channel magnetorelaxometry measurements, this distribution can be determined by means of inverse methods. It was recently shown that the combination of sequential inhomogeneous excitation fields in these measurements is favorable regarding the reconstruction accuracy when compared to homogeneous activation . In this paper, approaches for the determination of activation sequences for these measurements are investigated. Therefor, consecutive activation of single coils, random activation patterns and families of m-sequences are examined in computer simulations involving a sample measurement setup and compared with respect to the relative condition number of the system matrix. We obtain that the values of this condition number decrease with larger number of measurement samples for all approaches. Random sequences and m-sequences reveal similar results with a significant reduction of the required number of samples. We conclude that the application of pseudo-random sequences for sequential activation in the magnetorelaxometry imaging of magnetic nanoparticles considerably reduces the number of required sequences while preserving the relevant measurement information.

Dashboard applications to monitor experiment activities at sites

Energy Technology Data Exchange (ETDEWEB)

Andreeva, Julia; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciaba, Andrea [CERN, European Organization for Nuclear Research (Switzerland); Belforte, Stefano [INFN Trieste (Italy); Boehm, Max [EDS, an HP Company, Plano, TX (United States); Casajus, Adrian [Universitat de Barcelona (Spain); Flix, Josep [PIC, Port d' Informacio CientIfica, Bellaterra (Spain); Tsaregorodtsev, Andrei, E-mail: Elisa.Lanciotti@cern.c, E-mail: Pablo.Saiz@cern.c [CPPM Marseille (France)

2010-04-01

In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

Dashboard applications to monitor experiment activities at sites

International Nuclear Information System (INIS)

Andreeva, Julia; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciaba, Andrea; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Tsaregorodtsev, Andrei

2010-01-01

In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments

International Nuclear Information System (INIS)

Sage, E.; Moustacchi, E.

1987-01-01

The photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. The authors took advantage of the blockage by bulky adducts of the 3'-5'-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T < TT << TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT << TA ∼ TAT << ATA < ATAT < ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5'-TpA sites. The results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA

Sequence analysis reveals how G protein-coupled receptors transduce the signal to the G protein.

NARCIS (Netherlands)

Oliveira, L.; Paiva, P.B.; Paiva, A.C.; Vriend, G.

2003-01-01

Sequence entropy-variability plots based on alignments of very large numbers of sequences-can indicate the location in proteins of the main active site and modulator sites. In the previous article in this issue, we applied this observation to a series of well-studied proteins and concluded that it

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

Directory of Open Access Journals (Sweden)

Elela Sherif

2006-01-01

Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

Mapping membrane activity in undiscovered peptide sequence space using machine learning.

Science.gov (United States)

Lee, Ernest Y; Fulan, Benjamin M; Wong, Gerard C L; Ferguson, Andrew L

2016-11-29

There are some ∼1,100 known antimicrobial peptides (AMPs), which permeabilize microbial membranes but have diverse sequences. Here, we develop a support vector machine (SVM)-based classifier to investigate ⍺-helical AMPs and the interrelated nature of their functional commonality and sequence homology. SVM is used to search the undiscovered peptide sequence space and identify Pareto-optimal candidates that simultaneously maximize the distance σ from the SVM hyperplane (thus maximize its "antimicrobialness") and its ⍺-helicity, but minimize mutational distance to known AMPs. By calibrating SVM machine learning results with killing assays and small-angle X-ray scattering (SAXS), we find that the SVM metric σ correlates not with a peptide's minimum inhibitory concentration (MIC), but rather its ability to generate negative Gaussian membrane curvature. This surprising result provides a topological basis for membrane activity common to AMPs. Moreover, we highlight an important distinction between the maximal recognizability of a sequence to a trained AMP classifier (its ability to generate membrane curvature) and its maximal antimicrobial efficacy. As mutational distances are increased from known AMPs, we find AMP-like sequences that are increasingly difficult for nature to discover via simple mutation. Using the sequence map as a discovery tool, we find a unexpectedly diverse taxonomy of sequences that are just as membrane-active as known AMPs, but with a broad range of primary functions distinct from AMP functions, including endogenous neuropeptides, viral fusion proteins, topogenic peptides, and amyloids. The SVM classifier is useful as a general detector of membrane activity in peptide sequences.

Application of SCALE 6.1 MAVRIC Sequence for Activation Calculation in Reactor Primary Shield Concrete

International Nuclear Information System (INIS)

Kim, Yong IL

2014-01-01

Activation calculation requires flux information at desired location and reaction cross sections for the constituent elements to obtain production rate of activation products. Generally it is not an easy task to obtain fluxes or reaction rates with low uncertainties in a reasonable time for deep penetration problems by using standard Monte Carlo methods. The MAVRIC (Monaco with Automated Variance Reduction using Importance Calculations) sequence in SCALE 6.1 code package is intended to perform radiation transport on problems that are too challenging for standard, unbiased Monte Carlo methods. And the SCALE code system provides plenty of ENDF reaction types enough to consider almost all activation reactions in the nuclear reactor materials. To evaluate the activation of the important isotopes in primary shield, SCALE 6.1 MAVRIC sequence has been utilized for the KSNP reactor model and the calculated results are compared to the isotopic activity concentration of related standard. Related to the planning for decommission, the activation products in concrete primary shield such as Fe-55, Co-60, Ba-133, Eu-152, and Eu-154 are identified as important elements according to the comparisons with related standard for exemption. In this study, reference data are used for the concrete compositions in the activation calculation to see the applicability of MAVRIC code to the evaluation of activation inventory in the concrete primary shield. The composition data of trace elements as shown in Table 1 are obtained from various US power plant sites and accordingly they have large variations in quantity due to the characteristics of concrete composition. In practical estimation of activation radioactivity for a specific plant related to decommissioning, rigorous chemical analysis of concrete samples of the plant would first have to be performed to get exact information for compositions of concrete. Considering the capability of solving deep penetration transport problems and richness

Simultaneous activation of parallel sensory pathways promotes a grooming sequence in Drosophila

Science.gov (United States)

Hampel, Stefanie; McKellar, Claire E

2017-01-01

A central model that describes how behavioral sequences are produced features a neural architecture that readies different movements simultaneously, and a mechanism where prioritized suppression between the movements determines their sequential performance. We previously described a model whereby suppression drives a Drosophila grooming sequence that is induced by simultaneous activation of different sensory pathways that each elicit a distinct movement (Seeds et al., 2014). Here, we confirm this model using transgenic expression to identify and optogenetically activate sensory neurons that elicit specific grooming movements. Simultaneous activation of different sensory pathways elicits a grooming sequence that resembles the naturally induced sequence. Moreover, the sequence proceeds after the sensory excitation is terminated, indicating that a persistent trace of this excitation induces the next grooming movement once the previous one is performed. This reveals a mechanism whereby parallel sensory inputs can be integrated and stored to elicit a delayed and sequential grooming response. PMID:28887878

Exploring functionally related enzymes using radially distributed properties of active sites around the reacting points of bound ligands

Directory of Open Access Journals (Sweden)

Ueno Keisuke

2012-04-01

Full Text Available Abstract Background Structural genomics approaches, particularly those solving the 3D structures of many proteins with unknown functions, have increased the desire for structure-based function predictions. However, prediction of enzyme function is difficult because one member of a superfamily may catalyze a different reaction than other members, whereas members of different superfamilies can catalyze the same reaction. In addition, conformational changes, mutations or the absence of a particular catalytic residue can prevent inference of the mechanism by which catalytic residues stabilize and promote the elementary reaction. A major hurdle for alignment-based methods for prediction of function is the absence (despite its importance of a measure of similarity of the physicochemical properties of catalytic sites. To solve this problem, the physicochemical features radially distributed around catalytic sites should be considered in addition to structural and sequence similarities. Results We showed that radial distribution functions (RDFs, which are associated with the local structural and physicochemical properties of catalytic active sites, are capable of clustering oxidoreductases and transferases by function. The catalytic sites of these enzymes were also characterized using the RDFs. The RDFs provided a measure of the similarity among the catalytic sites, detecting conformational changes caused by mutation of catalytic residues. Furthermore, the RDFs reinforced the classification of enzyme functions based on conventional sequence and structural alignments. Conclusions Our results demonstrate that the application of RDFs provides advantages in the functional classification of enzymes by providing information about catalytic sites.

Integrin Activation Dynamics between the RGD-binding Site and the Headpiece Hinge*

Science.gov (United States)

Puklin-Faucher, Eileen; Vogel, Viola

2009-01-01

Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII10-bound αVβ3 integrin headpiece how the binding pocket and interdomain βA/hybrid domain hinge on the distal end of the βA domain are allosterically linked via a hydrophobic T-junction between the middle of the α1 helix and top of the α7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca2+ in place of Mg2+ at the site adjacent to the metal ion-dependent adhesion site (“ADMIDAS”). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca2+ at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated. PMID:19762919

Metal active site elasticity linked to activation of homocysteine in methionine synthases

Energy Technology Data Exchange (ETDEWEB)

Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L. (Michigan)

2008-04-02

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

Neural correlates of skill acquisition: decreased cortical activity during a serial interception sequence learning task.

Science.gov (United States)

Gobel, Eric W; Parrish, Todd B; Reber, Paul J

2011-10-15

Learning of complex motor skills requires learning of component movements as well as the sequential structure of their order and timing. Using a Serial Interception Sequence Learning (SISL) task, participants learned a sequence of precisely timed interception responses through training with a repeating sequence. Following initial implicit learning of the repeating sequence, functional MRI data were collected during performance of that known sequence and compared with activity evoked during novel sequences of actions, novel timing patterns, or both. Reduced activity was observed during the practiced sequence in a distributed bilateral network including extrastriate occipital, parietal, and premotor cortical regions. These reductions in evoked activity likely reflect improved efficiency in visuospatial processing, spatio-motor integration, motor planning, and motor execution for the trained sequence, which is likely supported by nondeclarative skill learning. In addition, the practiced sequence evoked increased activity in the left ventral striatum and medial prefrontal cortex, while the posterior cingulate was more active during periods of better performance. Many prior studies of perceptual-motor skill learning have found increased activity in motor areas of the frontal cortex (e.g., motor and premotor cortex, SMA) and striatal areas (e.g., the putamen). The change in activity observed here (i.e., decreased activity across a cortical network) may reflect skill learning that is predominantly expressed through more accurate performance rather than decreased reaction time. Copyright © 2011 Elsevier Inc. All rights reserved.

Quantum mechanical design of enzyme active sites.

Science.gov (United States)

Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

2008-02-01

The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

The genealogy of sequences containing multiple sites subject to strong selection in a subdivided population.

Science.gov (United States)

Nordborg, Magnus; Innan, Hideki

2003-03-01

A stochastic model for the genealogy of a sample of recombining sequences containing one or more sites subject to selection in a subdivided population is described. Selection is incorporated by dividing the population into allelic classes and then conditioning on the past sizes of these classes. The past allele frequencies at the selected sites are thus treated as parameters rather than as random variables. The purpose of the model is not to investigate the dynamics of selection, but to investigate effects of linkage to the selected sites on the genealogy of the surrounding chromosomal region. This approach is useful for modeling strong selection, when it is natural to parameterize the past allele frequencies at the selected sites. Several models of strong balancing selection are used as examples, and the effects on the pattern of neutral polymorphism in the chromosomal region are discussed. We focus in particular on the statistical power to detect balancing selection when it is present.

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

Science.gov (United States)

Kwong, M. Y.; Harris, R. J.

1994-01-01

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox.

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Julien Hiblot

Full Text Available Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263 that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability.

Position-specific prediction of methylation sites from sequence conservation based on information theory.

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Shi, Yinan; Guo, Yanzhi; Hu, Yayun; Li, Menglong

2015-07-23

Protein methylation plays vital roles in many biological processes and has been implicated in various human diseases. To fully understand the mechanisms underlying methylation for use in drug design and work in methylation-related diseases, an initial but crucial step is to identify methylation sites. The use of high-throughput bioinformatics methods has become imperative to predict methylation sites. In this study, we developed a novel method that is based only on sequence conservation to predict protein methylation sites. Conservation difference profiles between methylated and non-methylated peptides were constructed by the information entropy (IE) in a wider neighbor interval around the methylation sites that fully incorporated all of the environmental information. Then, the distinctive neighbor residues were identified by the importance scores of information gain (IG). The most representative model was constructed by support vector machine (SVM) for Arginine and Lysine methylation, respectively. This model yielded a promising result on both the benchmark dataset and independent test set. The model was used to screen the entire human proteome, and many unknown substrates were identified. These results indicate that our method can serve as a useful supplement to elucidate the mechanism of protein methylation and facilitate hypothesis-driven experimental design and validation.

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus.

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May, Jared; Johnson, Philip; Saleem, Huma; Simon, Anne E

2017-04-15

To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA in vivo Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation. IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary

POLYAR, a new computer program for prediction of poly(A sites in human sequences

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Qamar Raheel

2010-11-01

Full Text Available Abstract Background mRNA polyadenylation is an essential step of pre-mRNA processing in eukaryotes. Accurate prediction of the pre-mRNA 3'-end cleavage/polyadenylation sites is important for defining the gene boundaries and understanding gene expression mechanisms. Results 28761 human mapped poly(A sites have been classified into three classes containing different known forms of polyadenylation signal (PAS or none of them (PAS-strong, PAS-weak and PAS-less, respectively and a new computer program POLYAR for the prediction of poly(A sites of each class was developed. In comparison with polya_svm (till date the most accurate computer program for prediction of poly(A sites while searching for PAS-strong poly(A sites in human sequences, POLYAR had a significantly higher prediction sensitivity (80.8% versus 65.7% and specificity (66.4% versus 51.7% However, when a similar sort of search was conducted for PAS-weak and PAS-less poly(A sites, both programs had a very low prediction accuracy, which indicates that our knowledge about factors involved in the determination of the poly(A sites is not sufficient to identify such polyadenylation regions. Conclusions We present a new classification of polyadenylation sites into three classes and a novel computer program POLYAR for prediction of poly(A sites/regions of each of the class. In tests, POLYAR shows high accuracy of prediction of the PAS-strong poly(A sites, though this program's efficiency in searching for PAS-weak and PAS-less poly(A sites is not very high but is comparable to other available programs. These findings suggest that additional characteristics of such poly(A sites remain to be elucidated. POLYAR program with a stand-alone version for downloading is available at http://cub.comsats.edu.pk/polyapredict.htm.

Mesozoic tectonomagmatic activity and uranium metallogenetic sequence in mid-Nanling tectonic belt

International Nuclear Information System (INIS)

Deng Ping; Shu Liangshu

2002-01-01

Based on the synthesis and analysis of the relationship of various Mesozoic intrusive massifs, the tectonic activity, and the hydrothermal veins, as well as data of isotopic geochronology, the author makes a time sequence of the tectonomagmatic activities, the hydrothermal activities and uranium mineralization, and summarizes characteristics of tectonomagmatic and hydrothermal activities of different stages, and discusses the time sequence of various ore-controlling factors for granite-type uranium metallogeny. Finally, authors conclude that uranium metallogeny shows a very close spatial and temporal relationship to Mesozoic tectonomagmatic and hydrothermal activities

The sequence specificity of UV-induced DNA damage in a systematically altered DNA sequence.

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Khoe, Clairine V; Chung, Long H; Murray, Vincent

2018-06-01

The sequence specificity of UV-induced DNA damage was investigated in a specifically designed DNA plasmid using two procedures: end-labelling and linear amplification. Absorption of UV photons by DNA leads to dimerisation of pyrimidine bases and produces two major photoproducts, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). A previous study had determined that two hexanucleotide sequences, 5'-GCTC*AC and 5'-TATT*AA, were high intensity UV-induced DNA damage sites. The UV clone plasmid was constructed by systematically altering each nucleotide of these two hexanucleotide sequences. One of the main goals of this study was to determine the influence of single nucleotide alterations on the intensity of UV-induced DNA damage. The sequence 5'-GCTC*AC was designed to examine the sequence specificity of 6-4PPs and the highest intensity 6-4PP damage sites were found at 5'-GTTC*CC nucleotides. The sequence 5'-TATT*AA was devised to investigate the sequence specificity of CPDs and the highest intensity CPD damage sites were found at 5'-TTTT*CG nucleotides. It was proposed that the tetranucleotide DNA sequence, 5'-YTC*Y (where Y is T or C), was the consensus sequence for the highest intensity UV-induced 6-4PP adduct sites; while it was 5'-YTT*C for the highest intensity UV-induced CPD damage sites. These consensus tetranucleotides are composed entirely of consecutive pyrimidines and must have a DNA conformation that is highly productive for the absorption of UV photons. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Multi-site risk-based project planning, optimization, sequencing and budgeting process and tool for the integrated facility disposition project - 59394

International Nuclear Information System (INIS)

Nelson, Jerel; Castillo, Carlos; Huntsman, Julie; Lucek, Heather; Marks, Tim

2012-01-01

Document available in abstract form only. Full text of publication follows: Faced with the DOE Complex Transformation, NNSA was tasked with developing an integrated plan for the decommissioning of over 400 facilities and 300 environmental remediation units, as well as the many reconfiguration and modernization projects at the Oak Ridge National Laboratory (ORNL) and Y-12 Complex. Manual scheduling of remediation activities is time-consuming, labor intensive, and inherently introduces bias and unaccounted for aspects of the scheduler or organization in the process. Clearly a tool was needed to develop an objective, unbiased baseline optimized project sequence and schedule with a sound technical foundation for the Integrated Facility Disposition Project (IFDP). In generating an integrated disposition schedule, each project (including facilities, environmental sites, and remedial action units) was identified, characterized, then ranked relative to other projects. Risk matrices allowed for core project data to be extrapolated into probable contamination levels, relative risks to the public, and other technical and risk parameters to be used in the development of an overall ranking. These matrices ultimately generated a complete data set that were used in the Ranking and Sequencing Model (RSM), commonly referred to as the SUPER model, for its numerous abilities to support D and D planning, prioritization, and sequencing

THE 'MAIN SEQUENCE' OF EXPLOSIVE SOLAR ACTIVE REGIONS: DISCOVERY AND INTERPRETATION

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Falconer, David A; Moore, Ronald L; Adams, Mitzi [Space Science Office, VP62, Marshall Space Flight Center, Huntsville, AL 35812 (United States); Gary, G. Allen [Center for Space Plasma and Aeronomic Research, University of Alabama in Huntsville, Huntsville, AL 35899 (United States)], E-mail: David.falconer@msfc.nasa.gov

2009-08-01

We examine the location and distribution of the production of coronal mass ejections (CMEs) and major flares by sunspot active regions in the phase space of two whole-active-region magnetic quantities measured from 1897 SOHO/MDI magnetograms. These magnetograms track the evolution of 44 active regions across the central disk of radius 0.5 R {sub Sun}. The two quantities are {sup L}WL{sub SG}, a gauge of the total free energy in an active region's magnetic field, and {sup L}{phi}, a measure of the active region's total magnetic flux. From these data and each active region's history of production of CMEs, X flares, and M flares, we find (1) that CME/flare-productive active regions are concentrated in a straight-line 'main sequence' in (log {sup L}WL{sub SG}, log {sup L}{phi}) space, (2) that main-sequence active regions have nearly their maximum attainable free magnetic energy, and (3) evidence that this arrangement plausibly results from equilibrium between input of free energy to an explosive active region's magnetic field in the chromosphere and corona by contortion of the field via convection in and below the photosphere and loss of free energy via CMEs, flares, and coronal heating, an equilibrium between energy gain and loss that is analogous to that of the main sequence of hydrogen-burning stars in (mass, luminosity) space.

THE 'MAIN SEQUENCE' OF EXPLOSIVE SOLAR ACTIVE REGIONS: DISCOVERY AND INTERPRETATION

International Nuclear Information System (INIS)

Falconer, David A.; Moore, Ronald L.; Adams, Mitzi; Gary, G. Allen

2009-01-01

We examine the location and distribution of the production of coronal mass ejections (CMEs) and major flares by sunspot active regions in the phase space of two whole-active-region magnetic quantities measured from 1897 SOHO/MDI magnetograms. These magnetograms track the evolution of 44 active regions across the central disk of radius 0.5 R Sun . The two quantities are L WL SG , a gauge of the total free energy in an active region's magnetic field, and L Φ, a measure of the active region's total magnetic flux. From these data and each active region's history of production of CMEs, X flares, and M flares, we find (1) that CME/flare-productive active regions are concentrated in a straight-line 'main sequence' in (log L WL SG , log L Φ) space, (2) that main-sequence active regions have nearly their maximum attainable free magnetic energy, and (3) evidence that this arrangement plausibly results from equilibrium between input of free energy to an explosive active region's magnetic field in the chromosphere and corona by contortion of the field via convection in and below the photosphere and loss of free energy via CMEs, flares, and coronal heating, an equilibrium between energy gain and loss that is analogous to that of the main sequence of hydrogen-burning stars in (mass, luminosity) space.

SitesIdentify: a protein functional site prediction tool

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Doig Andrew J

2009-11-01

Full Text Available Abstract Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify, based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/

Prediction of Human Activity by Discovering Temporal Sequence Patterns.

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Li, Kang; Fu, Yun

2014-08-01

Early prediction of ongoing human activity has become more valuable in a large variety of time-critical applications. To build an effective representation for prediction, human activities can be characterized by a complex temporal composition of constituent simple actions and interacting objects. Different from early detection on short-duration simple actions, we propose a novel framework for long -duration complex activity prediction by discovering three key aspects of activity: Causality, Context-cue, and Predictability. The major contributions of our work include: (1) a general framework is proposed to systematically address the problem of complex activity prediction by mining temporal sequence patterns; (2) probabilistic suffix tree (PST) is introduced to model causal relationships between constituent actions, where both large and small order Markov dependencies between action units are captured; (3) the context-cue, especially interactive objects information, is modeled through sequential pattern mining (SPM), where a series of action and object co-occurrence are encoded as a complex symbolic sequence; (4) we also present a predictive accumulative function (PAF) to depict the predictability of each kind of activity. The effectiveness of our approach is evaluated on two experimental scenarios with two data sets for each: action-only prediction and context-aware prediction. Our method achieves superior performance for predicting global activity classes and local action units.

Full-Length Sequence of Mouse Acupuncture-Induced 1-L (Aig1l Gene Including Its Transcriptional Start Site

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Mika Ohta

2011-01-01

Full Text Available We have been investigating the molecular efficacy of electroacupuncture (EA, which is one type of acupuncture therapy. In our previous molecular biological study of acupuncture, we found an EA-induced gene, named acupuncture-induced 1-L (Aig1l, in mouse skeletal muscle. The aims of this study consisted of identification of the full-length cDNA sequence of Aig1l including the transcriptional start site, determination of the tissue distribution of Aig1l and analysis of the effect of EA on Aig1l gene expression. We determined the complete cDNA sequence including the transcriptional start site via cDNA cloning with the cap site hunting method. We then analyzed the tissue distribution of Aig1l by means of northern blot analysis and real-time quantitative polymerase chain reaction. We used the semiquantitative reverse transcriptase-polymerase chain reaction to examine the effect of EA on Aig1l gene expression. Our results showed that the complete cDNA sequence of Aig1l was 6073 bp long, and the putative protein consisted of 962 amino acids. All seven tissues that we analyzed expressed the Aig1l gene. In skeletal muscle, EA induced expression of the Aig1l gene, with high expression observed after 3 hours of EA. Our findings thus suggest that the Aig1l gene may play a key role in the molecular mechanisms of EA efficacy.

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

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Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao; Perez, Zhanita N.; Ghirlando, Rodolfo; Chandler, Michael; Dyda, Fred (Centre Nat); (NIH)

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalent intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.

Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

Science.gov (United States)

Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

1999-03-01

The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

Optimal rotation sequences for active perception

Science.gov (United States)

Nakath, David; Rachuy, Carsten; Clemens, Joachim; Schill, Kerstin

2016-05-01

One major objective of autonomous systems navigating in dynamic environments is gathering information needed for self localization, decision making, and path planning. To account for this, such systems are usually equipped with multiple types of sensors. As these sensors often have a limited field of view and a fixed orientation, the task of active perception breaks down to the problem of calculating alignment sequences which maximize the information gain regarding expected measurements. Action sequences that rotate the system according to the calculated optimal patterns then have to be generated. In this paper we present an approach for calculating these sequences for an autonomous system equipped with multiple sensors. We use a particle filter for multi- sensor fusion and state estimation. The planning task is modeled as a Markov decision process (MDP), where the system decides in each step, what actions to perform next. The optimal control policy, which provides the best action depending on the current estimated state, maximizes the expected cumulative reward. The latter is computed from the expected information gain of all sensors over time using value iteration. The algorithm is applied to a manifold representation of the joint space of rotation and time. We show the performance of the approach in a spacecraft navigation scenario where the information gain is changing over time, caused by the dynamic environment and the continuous movement of the spacecraft

Salt site performance assessment activities

International Nuclear Information System (INIS)

Kircher, J.F.; Gupta, S.K.

1983-01-01

During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables

Detection of nucleic acid sequences by invader-directed cleavage

Science.gov (United States)

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.

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

Science.gov (United States)

Wang, Meng; Rada, Cristina; Neuberger, Michael S

2010-01-18

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

Active site of Zn2+-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

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Jin-Suk Han

2005-01-01

Full Text Available The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261 is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

An overview of the prediction of protein DNA-binding sites.

Science.gov (United States)

Si, Jingna; Zhao, Rui; Wu, Rongling

2015-03-06

Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

Evolutionary rates at codon sites may be used to align sequences and infer protein domain function

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Hazelhurst Scott

2010-03-01

Full Text Available Abstract Background Sequence alignments form part of many investigations in molecular biology, including the determination of phylogenetic relationships, the prediction of protein structure and function, and the measurement of evolutionary rates. However, to obtain meaningful results, a significant degree of sequence similarity is required to ensure that the alignments are accurate and the inferences correct. Limitations arise when sequence similarity is low, which is particularly problematic when working with fast-evolving genes, evolutionary distant taxa, genomes with nucleotide biases, and cases of convergent evolution. Results A novel approach was conceptualized to address the "low sequence similarity" alignment problem. We developed an alignment algorithm termed FIRE (Functional Inference using the Rates of Evolution, which aligns sequences using the evolutionary rate at codon sites, as measured by the dN/dS ratio, rather than nucleotide or amino acid residues. FIRE was used to test the hypotheses that evolutionary rates can be used to align sequences and that the alignments may be used to infer protein domain function. Using a range of test data, we found that aligning domains based on evolutionary rates was possible even when sequence similarity was very low (for example, antibody variable regions. Furthermore, the alignment has the potential to infer protein domain function, indicating that domains with similar functions are subject to similar evolutionary constraints. These data suggest that an evolutionary rate-based approach to sequence analysis (particularly when combined with structural data may be used to study cases of convergent evolution or when sequences have very low similarity. However, when aligning homologous gene sets with sequence similarity, FIRE did not perform as well as the best traditional alignment algorithms indicating that the conventional approach of aligning residues as opposed to evolutionary rates remains the

Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

International Nuclear Information System (INIS)

Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

1986-01-01

The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme

Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motive

Science.gov (United States)

Parish, David; Benach, Jordi; Liu, Goahua; Singarapu, Kiran Kumar; Xiao, Rong; Acton, Thomas; Su, Min; Bansal, Sonal; Prestegard, James H.; Hunt, John; Montelione, Gaetano T.

2010-01-01

The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Eschericia coli was determined by NMR. The two proteins belong to Pfam [1] PF07449, which currently comprises 50 members, and belongs itself to the ‘thioredoxin-like clan’. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides. PMID:19039680

Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motif.

Science.gov (United States)

Parish, David; Benach, Jordi; Liu, Goahua; Singarapu, Kiran Kumar; Xiao, Rong; Acton, Thomas; Su, Min; Bansal, Sonal; Prestegard, James H; Hunt, John; Montelione, Gaetano T; Szyperski, Thomas

2008-12-01

The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.

Protein Chaperones Q8ZP25_SALTY from Salmonella Typhimurium and HYAE_ECOLI from Escherichia coli Exhibit Thioredoxin-like Structures Despite Lack of Canonical Thioredoxin Active Site Sequence Motif

Energy Technology Data Exchange (ETDEWEB)

Parish, D.; Benach, J; Liu, G; Singarapu, K; Xiao, R; Acton, T; Hunt, J; Montelione, G; Szyperski, T; et. al.

2008-01-01

The structure of the 142-residue protein Q8ZP25 SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE ECOLI was previously classified as a (NiFe) hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.

Managing Siting Activities for Nuclear Power Plants

International Nuclear Information System (INIS)

2012-01-01

One of the IAEA's statutory objectives is to ''seek to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world''. One way this objective is achieved is through the publication of a range of technical series. Two of these are the IAEA Nuclear Energy Series and the IAEA Safety Standards Series. According to Article III.A.6 of the IAEA Statute, the safety standards establish 'standards of safety for protection of health and minimization of danger to life and property.' The safety standards include the Safety Fundamentals, Safety Requirements and Safety Guides. These standards are written primarily in a regulatory style, and are binding on the IAEA for its own programmes. The principal users are the regulatory bodies in Member States and other national authorities. The IAEA Nuclear Energy Series comprises reports designed to encourage and assist R and D on, and application of, nuclear energy for peaceful uses. This includes practical examples to be used by owners and operators of utilities in Member States, implementing organizations, academia, and government officials, among others. This information is presented in guides, reports on technology status and advances, and best practices for peaceful uses of nuclear energy based on inputs from international experts. The IAEA Nuclear Energy Series complements the IAEA Safety Standards Series. The introduction of nuclear power brings new challenges to States - one of them being the selection of appropriates sites. It is a project that needs to begin early, be well managed, and deploy good communications with all stakeholders; including regulators. This is important, not just for those States introducing nuclear power for the first time, but for any State looking to build a new nuclear power plant. The purpose of the siting activities goes beyond choosing a suitable site and acquiring a licence. A large part of the project is about producing and maintaining a validated

Managing Siting Activities for Nuclear Power Plants

Energy Technology Data Exchange (ETDEWEB)

NONE

2012-06-15

One of the IAEA's statutory objectives is to ''seek to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world''. One way this objective is achieved is through the publication of a range of technical series. Two of these are the IAEA Nuclear Energy Series and the IAEA Safety Standards Series. According to Article III.A.6 of the IAEA Statute, the safety standards establish 'standards of safety for protection of health and minimization of danger to life and property.' The safety standards include the Safety Fundamentals, Safety Requirements and Safety Guides. These standards are written primarily in a regulatory style, and are binding on the IAEA for its own programmes. The principal users are the regulatory bodies in Member States and other national authorities. The IAEA Nuclear Energy Series comprises reports designed to encourage and assist R and D on, and application of, nuclear energy for peaceful uses. This includes practical examples to be used by owners and operators of utilities in Member States, implementing organizations, academia, and government officials, among others. This information is presented in guides, reports on technology status and advances, and best practices for peaceful uses of nuclear energy based on inputs from international experts. The IAEA Nuclear Energy Series complements the IAEA Safety Standards Series. The introduction of nuclear power brings new challenges to States - one of them being the selection of appropriates sites. It is a project that needs to begin early, be well managed, and deploy good communications with all stakeholders; including regulators. This is important, not just for those States introducing nuclear power for the first time, but for any State looking to build a new nuclear power plant. The purpose of the siting activities goes beyond choosing a suitable site and acquiring a licence. A large part of the project is about producing and maintaining a validated

Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site.

Science.gov (United States)

Bause, E; Wesemann, M; Bartoschek, A; Breuer, W

1997-02-15

Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asu-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own 'suicide' inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation.

Spatiotemporal dynamics and epistatic interaction sites in dengue virus type 1: a comprehensive sequence-based analysis.

Directory of Open Access Journals (Sweden)

Pei-Yu Chu

Full Text Available The continuing threat of dengue fever necessitates a comprehensive characterisation of its epidemiological trends. Phylogenetic and recombination events were reconstructed based on 100 worldwide dengue virus (DENV type 1 genome sequences with an outgroup (prototypes of DENV2-4. The phylodynamic characteristics and site-specific variation were then analysed using data without the outgroup. Five genotypes (GI-GV and a ladder-like structure with short terminal branch topology were observed in this study. Apparently, the transmission of DENV1 was geographically random before gradual localising with human activity as GI-GIII in South Asia, GIV in the South Pacific, and GV in the Americas. Genotypes IV and V have recently shown higher population densities compared to older genotypes. All codon regions and all tree branches were skewed toward a negative selection, which indicated that their variation was restricted by protein function. Notably, multi-epistatic interaction sites were found in both PrM 221 and NS3 1730. Recombination events accumulated in regions E, NS3-NS4A, and particularly in region NS5. The estimated coevolution pattern also highlights the need for further study of the biological role of protein PrM 221 and NS3 1730. The recent transmission of emergent GV sublineages into Central America and Europe mandates closely monitoring of genotype interaction and succession.

Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase

International Nuclear Information System (INIS)

Hjalmarsson, K.; Marklund, S.L.; Engstroem, A.; Edlund, T.

1987-01-01

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase has been isolated and the nucleotide sequence determined. The cDNA has a very high G + C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (∼ 50%) with the final two-thirds of the sequences of all know eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergencies are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved form the CuZn SODs before the evolution of fungi and plants

Accurate identification of RNA editing sites from primitive sequence with deep neural networks.

Science.gov (United States)

Ouyang, Zhangyi; Liu, Feng; Zhao, Chenghui; Ren, Chao; An, Gaole; Mei, Chuan; Bo, Xiaochen; Shu, Wenjie

2018-04-16

RNA editing is a post-transcriptional RNA sequence alteration. Current methods have identified editing sites and facilitated research but require sufficient genomic annotations and prior-knowledge-based filtering steps, resulting in a cumbersome, time-consuming identification process. Moreover, these methods have limited generalizability and applicability in species with insufficient genomic annotations or in conditions of limited prior knowledge. We developed DeepRed, a deep learning-based method that identifies RNA editing from primitive RNA sequences without prior-knowledge-based filtering steps or genomic annotations. DeepRed achieved 98.1% and 97.9% area under the curve (AUC) in training and test sets, respectively. We further validated DeepRed using experimentally verified U87 cell RNA-seq data, achieving 97.9% positive predictive value (PPV). We demonstrated that DeepRed offers better prediction accuracy and computational efficiency than current methods with large-scale, mass RNA-seq data. We used DeepRed to assess the impact of multiple factors on editing identification with RNA-seq data from the Association of Biomolecular Resource Facilities and Sequencing Quality Control projects. We explored developmental RNA editing pattern changes during human early embryogenesis and evolutionary patterns in Drosophila species and the primate lineage using DeepRed. Our work illustrates DeepRed's state-of-the-art performance; it may decipher the hidden principles behind RNA editing, making editing detection convenient and effective.

Sequence of ligand binding and structure change in the diphtheria toxin repressor upon activation by divalent transition metals.

Science.gov (United States)

Rangachari, Vijayaraghavan; Marin, Vedrana; Bienkiewicz, Ewa A; Semavina, Maria; Guerrero, Luis; Love, John F; Murphy, John R; Logan, Timothy M

2005-04-19

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2). Equilibrium binding energetics for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 microM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism spectroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants.

Identification of S-glutathionylation sites in species-specific proteins by incorporating five sequence-derived features into the general pseudo-amino acid composition.

Science.gov (United States)

Zhao, Xiaowei; Ning, Qiao; Ai, Meiyue; Chai, Haiting; Yang, Guifu

2016-06-07

As a selective and reversible protein post-translational modification, S-glutathionylation generates mixed disulfides between glutathione (GSH) and cysteine residues, and plays an important role in regulating protein activity, stability, and redox regulation. To fully understand S-glutathionylation mechanisms, identification of substrates and specific S-Glutathionylated sites is crucial. Experimental identification of S-glutathionylated sites is labor-intensive and time consuming, so establishing an effective computational method is much desirable due to their convenient and fast speed. Therefore, in this study, a new bioinformatics tool named SSGlu (Species-Specific identification of Protein S-glutathionylation Sites) was developed to identify species-specific protein S-glutathionylated sites, utilizing support vector machines that combine multiple sequence-derived features with a two-step feature selection. By 5-fold cross validation, the performance of SSGlu was measured with an AUC of 0.8105 and 0.8041 for Homo sapiens and Mus musculus, respectively. Additionally, SSGlu was compared with the existing methods, and the higher MCC and AUC of SSGlu demonstrated that SSGlu was very promising to predict S-glutathionylated sites. Furthermore, a site-specific analysis showed that S-glutathionylation intimately correlated with the features derived from its surrounding sites. The conclusions derived from this study might help to understand more of the S-glutathionylation mechanism and guide the related experimental validation. For public access, SSGlu is freely accessible at http://59.73.198.144:8080/SSGlu/. Copyright © 2016 Elsevier Ltd. All rights reserved.

Direct instrumental identification of catalytically active surface sites

Science.gov (United States)

Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

2017-09-01

The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences

International Nuclear Information System (INIS)

Klimov, Eugene; Vinokourova, Svetlana; Moisjak, Elena; Rakhmanaliev, Elian; Kobseva, Vera; Laimins, Laimonis; Kisseljov, Fjodor; Sulimova, Galina

2002-01-01

In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

Directory of Open Access Journals (Sweden)

Md Zahid Kamal

Full Text Available Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Active sites environmental monitoring Program - Program Plan: Revision 2

International Nuclear Information System (INIS)

Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

1994-05-01

The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results

Sequence learning in differentially activated dendrites

DEFF Research Database (Denmark)

Nielsen, Bjørn Gilbert

2003-01-01

. It is proposed that the neural machinery required in such a learning/retrieval mechanism could involve the NMDA receptor, in conjunction with the ability of dendrites to maintain differentially activated regions. In particular, it is suggested that such a parcellation of the dendrite allows the neuron......Differentially activated areas of a dendrite permit the existence of zones with distinct rates of synaptic modification, and such areas can be individually accessed using a reference signal which localizes synaptic plasticity and memory trace retrieval to certain subregions of the dendrite...... to participate in multiple sequences, which can be learned without suffering from the 'wash-out' of synaptic efficacy associated with superimposition of training patterns. This is a biologically plausible solution to the stability-plasticity dilemma of learning in neural networks....

Functional brain activation differences in stuttering identified with a rapid fMRI sequence

Science.gov (United States)

Kraft, Shelly Jo; Choo, Ai Leen; Sharma, Harish; Ambrose, Nicoline G.

2011-01-01

The purpose of this study was to investigate whether brain activity related to the presence of stuttering can be identified with rapid functional MRI (fMRI) sequences that involved overt and covert speech processing tasks. The long-term goal is to develop sensitive fMRI approaches with developmentally appropriate tasks to identify deviant speech motor and auditory brain activity in children who stutter closer to the age at which recovery from stuttering is documented. Rapid sequences may be preferred for individuals or populations who do not tolerate long scanning sessions. In this report, we document the application of a picture naming and phoneme monitoring task in three minute fMRI sequences with adults who stutter (AWS). If relevant brain differences are found in AWS with these approaches that conform to previous reports, then these approaches can be extended to younger populations. Pairwise contrasts of brain BOLD activity between AWS and normally fluent adults indicated the AWS showed higher BOLD activity in the right inferior frontal gyrus (IFG), right temporal lobe and sensorimotor cortices during picture naming and and higher activity in the right IFG during phoneme monitoring. The right lateralized pattern of BOLD activity together with higher activity in sensorimotor cortices is consistent with previous reports, which indicates rapid fMRI sequences can be considered for investigating stuttering in younger participants. PMID:22133409

Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity.

Science.gov (United States)

Moghadam, Morteza Shojaei; Albersmeier, Andreas; Winkler, Anika; Cimmino, Lorenzo; Rise, Kjersti; Hohmann-Marriott, Martin Frank; Kalinowski, Jörn; Rückert, Christian; Wentzel, Alexander; Lale, Rahmi

2016-02-16

Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42%, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Site Effects Study In Athens (greece) Using The 7th September 1999 Earthquake Aftershock Sequence

Science.gov (United States)

Serpetsidaki, A.; Sokos, E.

On 7 September 1999 at 11:56:50 GMT, an earthquake of Mw=5.9 occurred at Athens capital of Greece. The epicenter was located in the Northwest area of Parnitha Moun- tain at 18km distance from the city centre. This earthquake was one of the most de- structive in Greece during the modern times. The intensity of the earthquake reached IX in the Northwest territories of the city and caused the death of 143 people and seri- ous structural damage in many buildings. On the 13th of September the Seismological Laboratory of Patras University, installed a seismic network of 30 stations in order to observe the evolution of the aftershock sequence. This temporary seismic network remained in the area of Attika for 50 days and recorded a significant part of the af- tershock sequence. In this paper we use the high quality recordings of this network to investigate the influence of the surface geology to the seismic motion, on sites within the epicentral area, which suffered the most during this earthquake. We applied the horizontal-to-vertical (H/V) spectral ratio method on noise and on earthquake records and the obtained results exhibit very good agreement. Finally we compare the results with the geological conditions of the study area and the damage distribution. Most of the obtained amplification levels were low with an exemption in the site of Ano Liosia were a significant amount of damage was observed and the results indicate that the earthquake motion was amplified four times. Based on the above we conclude that the damages in the city of Athens were due to source effects rather than site effects.

Molecular Cloning and Sequence Analysis of a Phenylalanine Ammonia-Lyase Gene from Dendrobium

Science.gov (United States)

Cai, Yongping; Lin, Yi

2013-01-01

In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum. PMID:23638048

Fidelity of target site duplication and sequence preference during integration of xenotropic murine leukemia virus-related virus.

Directory of Open Access Journals (Sweden)

Sanggu Kim

Full Text Available Xenotropic murine leukemia virus (MLV-related virus (XMRV is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity.

Bifunctional cis-Abienol Synthase from Abies balsamea Discovered by Transcriptome Sequencing and Its Implications for Diterpenoid Fragrance Production*

Science.gov (United States)

Zerbe, Philipp; Chiang, Angela; Yuen, Macaire; Hamberger, Björn; Hamberger, Britta; Draper, Jason A.; Britton, Robert; Bohlmann, Jörg

2012-01-01

The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production. PMID:22337889

An Overview of the Prediction of Protein DNA-Binding Sites

Directory of Open Access Journals (Sweden)

Jingna Si

2015-03-01

Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

Science.gov (United States)

Currin, Andrew; Swainston, Neil; Day, Philip J.

2015-01-01

The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (K d) and catalytic (k cat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving k cat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole

Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

Directory of Open Access Journals (Sweden)

Selbig Joachim

2009-04-01

Full Text Available Abstract Background Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites. Results We characterized the spatial context of phosphorylation sites and assessed its usability for improved phosphorylation site predictions. We identified 750 non-redundant, experimentally verified sites with three-dimensional (3D structural information available in the protein data bank (PDB and grouped them according to their respective kinase family. We studied the spatial distribution of amino acids around phosphorserines, phosphothreonines, and phosphotyrosines to extract signature 3D-profiles. Characteristic spatial distributions of amino acid residue types around phosphorylation sites were indeed discernable, especially when kinase-family-specific target sites were analyzed. To test the added value of using spatial information for the computational prediction of phosphorylation sites, Support Vector Machines were applied using both sequence as well as structural information. When compared to sequence-only based prediction methods, a small but consistent performance improvement was obtained when the prediction was informed by 3D-context information. Conclusion While local one-dimensional amino acid sequence information was observed to harbor most of the discriminatory power, spatial context information was identified as

Promoter proximal polyadenylation sites reduce transcription activity

DEFF Research Database (Denmark)

Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

2012-01-01

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site......, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...... on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

Energy transfer at the active sites of heme proteins

International Nuclear Information System (INIS)

Dlott, D.D.; Hill, J.R.

1995-01-01

Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes

Multi-site risk-based project planning, optimization, sequencing, & budgeting process and tool for the integrated facility disposition project

International Nuclear Information System (INIS)

Nelson, J.G.; Castillo, C.; Huntsman, J.; Killoy, S.; Lucek, H.; Marks, T.C.

2011-01-01

Faced with the Department of Energy (DOE) Complex Transformation, National Nuclear Security Administration (NNSA) was tasked with developing an integrated plan for the decommissioning of over 400 facilities and 300 environmental remediation units, as well as the many reconfiguration and modernization projects at the Oak Ridge National Laboratory (ORNL) and Y-12 Complex. Manual scheduling of remediation activities is time-consuming and inherently introduces bias of the scheduler or organization into the process. Clearly a well-defined process, quantitative risk-based tool was needed to develop an objective, unbiased baseline sequence and schedule with a sound technical foundation for the Integrated Facility Disposition Project (IFDP). Faced with limited available data, innovation was needed to extrapolate intelligent relative data for key risk parameters based on known data elements. The IFDP Supermodel was customized and expanded to provide this capability for conceptual planning of diverse project portfolios and multiple sites. (author)

In silico Analysis of 3′-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

Science.gov (United States)

Tanaka, Mizuki; Sakai, Yoshifumi; Yamada, Osamu; Shintani, Takahiro; Gomi, Katsuya

2011-01-01

To investigate 3′-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3′-untranslated region (3′ UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3′ UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3′ UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15–30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3′-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3′-end-processing signals are similar to those in yeast and plants, some notable differences exist between them. PMID:21586533

Identification and positional distribution analysis of transcription factor binding sites for genes from the wheat fl-cDNA sequences.

Science.gov (United States)

Chen, Zhen-Yong; Guo, Xiao-Jiang; Chen, Zhong-Xu; Chen, Wei-Ying; Wang, Ji-Rui

2017-06-01

The binding sites of transcription factors (TFs) in upstream DNA regions are called transcription factor binding sites (TFBSs). TFBSs are important elements for regulating gene expression. To date, there have been few studies on the profiles of TFBSs in plants. In total, 4,873 sequences with 5' upstream regions from 8530 wheat fl-cDNA sequences were used to predict TFBSs. We found 4572 TFBSs for the MADS TF family, which was twice as many as for bHLH (1951), B3 (1951), HB superfamily (1914), ERF (1820), and AP2/ERF (1725) TFs, and was approximately four times higher than the remaining TFBS types. The percentage of TFBSs and TF members showed a distinct distribution in different tissues. Overall, the distribution of TFBSs in the upstream regions of wheat fl-cDNA sequences had significant difference. Meanwhile, high frequencies of some types of TFBSs were found in specific regions in the upstream sequences. Both TFs and fl-cDNA with TFBSs predicted in the same tissues exhibited specific distribution preferences for regulating gene expression. The tissue-specific analysis of TFs and fl-cDNA with TFBSs provides useful information for functional research, and can be used to identify relationships between tissue-specific TFs and fl-cDNA with TFBSs. Moreover, the positional distribution of TFBSs indicates that some types of wheat TFBS have different positional distribution preferences in the upstream regions of genes.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

Science.gov (United States)

Morellet, Nelly; Li, Xianghong; Wieninger, Silke A; Taylor, Jennifer L; Bischerour, Julien; Moriau, Séverine; Lescop, Ewen; Bardiaux, Benjamin; Mathy, Nathalie; Assrir, Nadine; Bétermier, Mireille; Nilges, Michael; Hickman, Alison B; Dyda, Fred; Craig, Nancy L; Guittet, Eric

2018-01-01

Abstract The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure–function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5′-TGCGT-3′/3′-ACGCA-5′ motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity. PMID:29385532

Active-site modification of mammalian DNA polymerase β with pyridoxal 5'-phosphate: Mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

International Nuclear Information System (INIS)

Basu, A.; Kedar, P.; Wilson, S.H.; Modak, M.J.

1989-01-01

Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase β. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [ 3 H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate

Bromopyruvate, an active site-directed inactivator of E. coli 2-keto-4-hydroxyglutarate(KHG) aldolase, modifies glutamic acid residue-45

International Nuclear Information System (INIS)

Vlahos, C.J.; Dekker, E.E.

1987-01-01

E. coli KHG-aldolase (2-keto-4-hydroxyglutarate ↔ pyruvate + glyoxylate), a novel trimeric Class I aldolase, requires one active-site lysine residue (Lys 133)/subunit for Schiff-base formation as well as one arginine residue (Arg 49)/subunit for catalytic activity. The substrate analog, 3-bromopyruvate (BRPY), causes a time- and concentration-dependent loss of KHG-aldolase activity. This inactivation is regarded as active site-directed since: (a) BRPY modification results in complete loss of enzymatic activity; (b) saturation kinetics are exhibited, suggesting that a reversible complex is formed between the aldolase and BRPY prior to the rate-limiting inactivation step; (c) over 90% of the initial aldolase activity is protected by either substrate, pyruvate or KHG; (d) 1.1 mol of 14 C-BRPY is bound/enzyme subunit. Peptide isolation and sequencing show that the incorporated radioactivity is associated with residue Glu-45. Denaturation of the enzyme with guanidine x HCl following treatment with excess 14 C-BRPY allows for the incorporation of carbon-14 at Cys-159 and Cys-180 as well. The presence of pyruvate protects Glu-45 from being esterified but does not prevent the alkylation of the two cysteine residues. These results suggest that Glu-45 is essential for the catalytic activity of E. coli KHG-aldolase, most likely functioning as the active-site amphoteric proton donor/acceptor moiety that is involved in the overall mechanism of the reaction catalyzed by this enzyme

Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.

Science.gov (United States)

Yonemoto, Isaac T; Weyman, Philip D

2017-01-01

Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.

Active Sites Environmental Monitoring Program: Mid-FY 1991 report

Energy Technology Data Exchange (ETDEWEB)

Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

1991-10-01

This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

Klipperaas study site. Scope of activities and main results

International Nuclear Information System (INIS)

Ahlbom, K.; Andersson, J.E.; Andersson, Peter; Ittner, T.; Tiren, S.; Ljunggren, C.

1992-09-01

During the period from 1977 - 1986 SKB (Swedish Nuclear Fuel and Waste Management Co.) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection rpogramme will be to perform detailed characterisation, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if any of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favorable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concern the Klipperaas study site. The main topics are the scope of activities, geologic model, geohydrological model, groundwater chemistry, assessment of solute transport, and rock mechanics

Active Sites Environmental Monitoring Program: FY 1990 annual report

International Nuclear Information System (INIS)

Wickliff, D.S.; Morrissey, C.M.; Ashwood, T.L.

1991-10-01

Chapter 3 of US Department of Energy (DOE) Order 5820.2A (DOE 1988) sets forth requirements for environmental monitoring of active low-level waste (LLW) disposal sites. Active sites are defined as those LLW facilities that were in use on or after the date of the order (September 1988). The transuranic (TRU) waste storage areas in Solid Waste Storage Area (SWSA) 5 North are covered by Chap. 2 of the order. In both chapters, monitoring is required to provide for early warning of leaks before those leaks pose a threat to human health or the environment. Chapter 3 also requires that monitoring be conducted to evaluate the short- and long-term performance of LLW disposal facilities. In accordance with this order, the Solid Waste Operations Department at Oak Ridge National Laboratory (ORNL) has established an Active Sites Environmental Monitoring Program (ASEMP) that is implemented by staff of the Environmental Sciences Division (ESD) at ORNL. This report summarizes data from ASEMP monitoring activities for the final 6 months of FY 1990. A brief summary of the monitoring methodology for each site is presented also

Cernavoda Unit 2: - BOP 3D model proposal for a possible organization of site activities

International Nuclear Information System (INIS)

Ghiesura, G.; Scarsin, Sioli; Orlandi, S.

1999-01-01

The scope of this activity is to define characteristics and advantages of the 3D model of Cernavoda BOP to this set up at site for engineering and construction activities. This model will provide a modern and proven tool able to strongly support the site activities with particular regard to the following: 1. engineering activities, - plant arrangement 'double check' for resolution of clashing; - easy management of future design changes; - real time plant configuration updating as soon as any design modification is approved and integrated in the model; - preparation of high quality documentation for procurement, construction and commissioning; - prompt availability of the as built configuration of the plant as soon as the last modification is frozen; 2. material procurement activities, - definition of the priorities in the construction material procurement according to the construction planning by area; - inventory list of equipment, pipes, fittings, valves, cable trays and ventilation ducts to be installed in each construction area; 3. construction activities, - definition of construction sequences, with particular reference in the congested areas, for piping cable trays (electrical and C-and-I) and ventilations ducts; - definition of piping spools by construction contractors; - follow-up of the activities in each area (i.e. construction, painting, insulation, flushing, pressure testing, etc); 4. turn-over and commissioning, - check of the progress. The success of this approach is based on the following: i) proper management of the remote workstations providing easy and reliable access to the model; ii) subdivision of the Integrated Building in construction areas, whose detail design may be allotted to Romanian organizations with multidisciplinary tasks; iii) integration in the model of the remote developed engineering in order to validate the details of the design. (authors)

The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

Directory of Open Access Journals (Sweden)

Suneeth F Mathew

Full Text Available HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon' contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

Tritium activities in selected wells on the Nevada Test Site

International Nuclear Information System (INIS)

Lyles, B.F.

1993-05-01

Literature and data were reviewed related to radionuclides in groundwater on and near the Nevada Test Site. No elevated tritium activities have been reported outside of the major testing regions of the Nevada Test Site. Three wells were identified as having water with above-background (>50 pCi/l) tritium activities: UE-15d Water Well; USGS Water Well A; and USGS Test Well B Ex. Although none of these wells have tritium activities greater than the Nevada State Drinking Water standard (20,000 pCi/l), their time-series tritium trends may be indicative to potential on-site radionuclide migration

Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands.

Science.gov (United States)

Limmer, Katja; Aschenbrenner, Daniela; Gaub, Hermann E

2013-04-01

Malfunction of protein translation causes many severe diseases, and suitable correction strategies may become the basis of effective therapies. One major regulatory element of protein translation is the nuclease Dicer that cuts double-stranded RNA independently of the sequence into pieces of 19-22 base pairs starting the RNA interference pathway and activating miRNAs. Inhibiting Dicer is not desirable owing to its multifunctional influence on the cell's gene regulation. Blocking specific RNA sequences by small-molecule binding, however, is a promising approach to affect the cell's condition in a controlled manner. A label-free assay for the screening of site-specific interference of small molecules with Dicer activity is thus needed. We used the Molecular Force Assay (MFA), recently developed in our lab, to measure the activity of Dicer. As a model system, we used an RNA sequence that forms an aptamer-binding site for paromomycin, a 615-dalton aminoglycoside. We show that Dicer activity is modulated as a function of concentration and incubation time: the addition of paromomycin leads to a decrease of Dicer activity according to the amount of ligand. The measured dissociation constant of paromomycin to its aptamer was found to agree well with literature values. The parallel format of the MFA allows a large-scale search and analysis for ligands for any RNA sequence.

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

Science.gov (United States)

Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

2018-01-16

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

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

International Nuclear Information System (INIS)

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

1989-01-01

Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the β-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

Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

Directory of Open Access Journals (Sweden)

Qing Jin

Full Text Available In this study, a phenylalanine ammonia-lyase (PAL gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748 has 2,458 bps and contains a complete open reading frame (ORF of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

Science.gov (United States)

da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

2014-01-01

The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland

Science.gov (United States)

Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Čapek, Petr; Kaiser, Christina; Torsvik, Vigdis L.; Richter, Andreas; Schleper, Christa; Urich, Tim

2014-01-01

Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation (“buried topsoils”), resulting from a decrease in fungal abundance compared to recent (“unburied”) topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation. PMID

HIVBrainSeqDB: a database of annotated HIV envelope sequences from brain and other anatomical sites

Directory of Open Access Journals (Sweden)

O'Connor Niall

2010-12-01

Full Text Available Abstract Background The population of HIV replicating within a host consists of independently evolving and interacting sub-populations that can be genetically distinct within anatomical compartments. HIV replicating within the brain causes neurocognitive disorders in up to 20-30% of infected individuals and is a viral sanctuary site for the development of drug resistance. The primary determinant of HIV neurotropism is macrophage tropism, which is primarily determined by the viral envelope (env gene. However, studies of genetic aspects of HIV replicating in the brain are hindered because existing repositories of HIV sequences are not focused on neurotropic virus nor annotated with neurocognitive and neuropathological status. To address this need, we constructed the HIV Brain Sequence Database. Results The HIV Brain Sequence Database is a public database of HIV envelope sequences, directly sequenced from brain and other tissues from the same patients. Sequences are annotated with clinical data including viral load, CD4 count, antiretroviral status, neurocognitive impairment, and neuropathological diagnosis, all curated from the original publication. Tissue source is coded using an anatomical ontology, the Foundational Model of Anatomy, to capture the maximum level of detail available, while maintaining ontological relationships between tissues and their subparts. 44 tissue types are represented within the database, grouped into 4 categories: (i brain, brainstem, and spinal cord; (ii meninges, choroid plexus, and CSF; (iii blood and lymphoid; and (iv other (bone marrow, colon, lung, liver, etc. Patient coding is correlated across studies, allowing sequences from the same patient to be grouped to increase statistical power. Using Cytoscape, we visualized relationships between studies, patients and sequences, illustrating interconnections between studies and the varying depth of sequencing, patient number, and tissue representation across studies

Active sites environmental monitoring program FY 1997 annual report

International Nuclear Information System (INIS)

Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

1998-03-01

This report summarizes the activities conducted by the Active Sites Environmental Monitoring Program (ASEMP) from October 1996 through September 1997. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North. This report continues a series of annual and semiannual reports that present the results of ASEMP monitoring activities. This report details monitoring results for fiscal year (FY) 1997 from SWSA 6, including the Interim Waste Management Facility (IWMF) and the Hillcut Disposal Test Facility (HDTF), and (2) TRU-waste storage areas in SWSA 5 N. This report presents a summary of the methodology used to gather data for each major area along with the FY 1997 results. Figures referenced in the text are found in Appendix A and data tables are presented in Appendix B

Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

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Abraham P. Fong

2015-03-01

Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

Protein model discrimination using mutational sensitivity derived from deep sequencing.

Science.gov (United States)

Adkar, Bharat V; Tripathi, Arti; Sahoo, Anusmita; Bajaj, Kanika; Goswami, Devrishi; Chakrabarti, Purbani; Swarnkar, Mohit K; Gokhale, Rajesh S; Varadarajan, Raghavan

2012-02-08

A major bottleneck in protein structure prediction is the selection of correct models from a pool of decoys. Relative activities of ∼1,200 individual single-site mutants in a saturation library of the bacterial toxin CcdB were estimated by determining their relative populations using deep sequencing. This phenotypic information was used to define an empirical score for each residue (RankScore), which correlated with the residue depth, and identify active-site residues. Using these correlations, ∼98% of correct models of CcdB (RMSD ≤ 4Å) were identified from a large set of decoys. The model-discrimination methodology was further validated on eleven different monomeric proteins using simulated RankScore values. The methodology is also a rapid, accurate way to obtain relative activities of each mutant in a large pool and derive sequence-structure-function relationships without protein isolation or characterization. It can be applied to any system in which mutational effects can be monitored by a phenotypic readout. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sequence Analysis and Characterization of Active Human Alu Subfamilies Based on the 1000 Genomes Pilot Project.

Science.gov (United States)

Konkel, Miriam K; Walker, Jerilyn A; Hotard, Ashley B; Ranck, Megan C; Fontenot, Catherine C; Storer, Jessica; Stewart, Chip; Marth, Gabor T; Batzer, Mark A

2015-08-29

The goal of the 1000 Genomes Consortium is to characterize human genome structural variation (SV), including forms of copy number variations such as deletions, duplications, and insertions. Mobile element insertions, particularly Alu elements, are major contributors to genomic SV among humans. During the pilot phase of the project we experimentally validated 645 (611 intergenic and 34 exon targeted) polymorphic "young" Alu insertion events, absent from the human reference genome. Here, we report high resolution sequencing of 343 (322 unique) recent Alu insertion events, along with their respective target site duplications, precise genomic breakpoint coordinates, subfamily assignment, percent divergence, and estimated A-rich tail lengths. All the sequenced Alu loci were derived from the AluY lineage with no evidence of retrotransposition activity involving older Alu families (e.g., AluJ and AluS). AluYa5 is currently the most active Alu subfamily in the human lineage, followed by AluYb8, and many others including three newly identified subfamilies we have termed AluYb7a3, AluYb8b1, and AluYa4a1. This report provides the structural details of 322 unique Alu variants from individual human genomes collectively adding about 100 kb of genomic variation. Many Alu subfamilies are currently active in human populations, including a surprising level of AluY retrotransposition. Human Alu subfamilies exhibit continuous evolution with potential drivers sprouting new Alu lineages. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties.

Science.gov (United States)

Pan, Gaofeng; Jiang, Limin; Tang, Jijun; Guo, Fei

2018-02-08

DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods-especially machine learning methods-have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k -gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria-area under the receiver operating characteristic curve (AUC), Matthew's correlation coefficient (MCC), accuracy (ACC), sensitivity (SN), and specificity-are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

A Novel Computational Method for Detecting DNA Methylation Sites with DNA Sequence Information and Physicochemical Properties

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Gaofeng Pan

2018-02-01

Full Text Available DNA methylation is an important biochemical process, and it has a close connection with many types of cancer. Research about DNA methylation can help us to understand the regulation mechanism and epigenetic reprogramming. Therefore, it becomes very important to recognize the methylation sites in the DNA sequence. In the past several decades, many computational methods—especially machine learning methods—have been developed since the high-throughout sequencing technology became widely used in research and industry. In order to accurately identify whether or not a nucleotide residue is methylated under the specific DNA sequence context, we propose a novel method that overcomes the shortcomings of previous methods for predicting methylation sites. We use k-gram, multivariate mutual information, discrete wavelet transform, and pseudo amino acid composition to extract features, and train a sparse Bayesian learning model to do DNA methylation prediction. Five criteria—area under the receiver operating characteristic curve (AUC, Matthew’s correlation coefficient (MCC, accuracy (ACC, sensitivity (SN, and specificity—are used to evaluate the prediction results of our method. On the benchmark dataset, we could reach 0.8632 on AUC, 0.8017 on ACC, 0.5558 on MCC, and 0.7268 on SN. Additionally, the best results on two scBS-seq profiled mouse embryonic stem cells datasets were 0.8896 and 0.9511 by AUC, respectively. When compared with other outstanding methods, our method surpassed them on the accuracy of prediction. The improvement of AUC by our method compared to other methods was at least 0.0399 . For the convenience of other researchers, our code has been uploaded to a file hosting service, and can be downloaded from: https://figshare.com/s/0697b692d802861282d3.

Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites

International Nuclear Information System (INIS)

Serrano, Pedro; Pedrini, Bill; Geralt, Michael; Jaudzems, Kristaps; Mohanty, Biswaranjan; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

2010-01-01

Tools for systematic comparisons of NMR and crystal structures developed by the JCSG were applied to two proteins with known functions: the T. maritima anti-σ factor antagonist TM1081 and the mouse γ-glutamylamine cyclotransferase A2LD1 (gi:13879369). In an attempt to exploit the complementarity of crystal and NMR data, the combined use of the two structure-determination techniques was explored for the initial steps in the challenge of searching proteins of unknown functions for putative active sites. The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Å resolution and at 100 K and 1.9 Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the

Plans for characterization of salt sites

International Nuclear Information System (INIS)

Heim, G.E.; Matthews, S.C.; Kircher, J.F.; Kennedy, R.K.

1983-01-01

This basic salt site characterization program has been designed to provide the data required to support the design, performance assessment, and licensing of each of the principal project elements: the repository, the shafts, and the surface facilities. The work has been sequenced to meet the design and licensing schedule. It is anticipated that additional characterization activities will be performed to address site-specific considerations and to provide additional information to address questions which arise during the evaluation of characterization data. 3 figures, 3 tables

Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems.

Science.gov (United States)

Yao, Yining; Yang, Qinrui; Shao, Chengchen; Liu, Baonian; Zhou, Yuxiang; Xu, Hongmei; Zhou, Yueqin; Tang, Qiqun; Xie, Jianhui

2018-01-01

Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex ® 21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR ® . Identifiler ® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex ® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets. Copyright © 2017 Elsevier B.V. All rights reserved.

Computational identification of developmental enhancers:conservation and function of transcription factor binding-site clustersin drosophila melanogaster and drosophila psedoobscura

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Berman, Benjamin P.; Pfeiffer, Barret D.; Laverty, Todd R.; Salzberg, Steven L.; Rubin, Gerald M.; Eisen, Michael B.; Celniker, SusanE.

2004-08-06

The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. Measuring conservation of sequence features closely linked to function--such as binding-site clustering--makes better use of comparative sequence data than commonly used methods that examine only sequence identity.

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

Science.gov (United States)

Freimuth, P; Anderson, C W

1993-03-01

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants

OpenAIRE

Lanciano, Sophie; Carpentier, M. C.; Llauro, C.; Jobet, E.; Robakowska-Hyzorek, D.; Lasserre, E.; Ghesquière, Alain; Panaud, O.; Mirouze, Marie

2017-01-01

Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposon...

Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

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Yu Zhang

Full Text Available Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL from Geobacillus kaustophilus HTA426 (GkaP exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

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Agarwala Usha

2011-06-01

Full Text Available Abstract Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6 are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. Results A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP. An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. Conclusion These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with

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

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Javier Villacreses

2015-04-01

Full Text Available 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.

Better estimation of protein-DNA interaction parameters improve prediction of functional sites

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O'Flanagan Ruadhan A

2008-12-01

Full Text Available Abstract Background Characterizing transcription factor binding motifs is a common bioinformatics task. For transcription factors with variable binding sites, we need to get many suboptimal binding sites in our training dataset to get accurate estimates of free energy penalties for deviating from the consensus DNA sequence. One procedure to do that involves a modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment method designed to produce many such sequences. Results We analyzed low stringency SELEX data for E. coli Catabolic Activator Protein (CAP, and we show here that appropriate quantitative analysis improves our ability to predict in vitro affinity. To obtain large number of sequences required for this analysis we used a SELEX SAGE protocol developed by Roulet et al. The sequences obtained from here were subjected to bioinformatic analysis. The resulting bioinformatic model characterizes the sequence specificity of the protein more accurately than those sequence specificities predicted from previous analysis just by using a few known binding sites available in the literature. The consequences of this increase in accuracy for prediction of in vivo binding sites (and especially functional ones in the E. coli genome are also discussed. We measured the dissociation constants of several putative CAP binding sites by EMSA (Electrophoretic Mobility Shift Assay and compared the affinities to the bioinformatics scores provided by methods like the weight matrix method and QPMEME (Quadratic Programming Method of Energy Matrix Estimation trained on known binding sites as well as on the new sites from SELEX SAGE data. We also checked predicted genome sites for conservation in the related species S. typhimurium. We found that bioinformatics scores based on SELEX SAGE data does better in terms of prediction of physical binding energies as well as in detecting functional sites. Conclusion We think that training binding site detection

Interactions of Chromatin Context, Binding Site Sequence Content, and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

OpenAIRE

Su, Dan; Wang, Xuting; Campbell, Michelle R.; Song, Lingyun; Safi, Alexias; Crawford, Gregory E.; Bell, Douglas A.

2015-01-01

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, ...

Disulfide bond within mu-calpain active site inhibits activity and autolysis.

Science.gov (United States)

Lametsch, René; Lonergan, Steven; Huff-Lonergan, Elisabeth

2008-09-01

Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.

Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

Science.gov (United States)

2015-01-01

Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

The dapE-encoded N-succinyl-L,L-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae Contains two Active Site Histidine Residues

Science.gov (United States)

Gillner, Danuta M.; Bienvenue, David L.; Nocek, Boguslaw P.; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C.

2009-01-01

The catalytic and structural properties of the H67A and H349A altered dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from H. influenzae were investigated. Based on sequence alignment with CPG2 both H67 and H349 were predicted to be Zn(II) ligands. Catalytic activity was observed for the H67A altered DapE enzyme which exhibited kcat = 1.5 ± 0.5 sec−1 and Km = 1.4 ± 0.3 mM. No catalytic activity was observed for H349A under the experimental conditions used. The EPR and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to WT DapE after the addition of a single Co(II) ion. The addition of one equivalent of Co(II) to H67A altered DapE provides spectra that are very different from the first Co(II) binding site of the WT enzyme, but similar to the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from N. meningitidis as a template and superimposed on the structure of AAP. This homology structure confirms the assignment of H67 and H349 as active site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP, all of the amino acid residues of DapE are nearly identical. PMID:18712420

UET: a database of evolutionarily-predicted functional determinants of protein sequences that cluster as functional sites in protein structures.

Science.gov (United States)

Lua, Rhonald C; Wilson, Stephen J; Konecki, Daniel M; Wilkins, Angela D; Venner, Eric; Morgan, Daniel H; Lichtarge, Olivier

2016-01-04

The structure and function of proteins underlie most aspects of biology and their mutational perturbations often cause disease. To identify the molecular determinants of function as well as targets for drugs, it is central to characterize the important residues and how they cluster to form functional sites. The Evolutionary Trace (ET) achieves this by ranking the functional and structural importance of the protein sequence positions. ET uses evolutionary distances to estimate functional distances and correlates genotype variations with those in the fitness phenotype. Thus, ET ranks are worse for sequence positions that vary among evolutionarily closer homologs but better for positions that vary mostly among distant homologs. This approach identifies functional determinants, predicts function, guides the mutational redesign of functional and allosteric specificity, and interprets the action of coding sequence variations in proteins, people and populations. Now, the UET database offers pre-computed ET analyses for the protein structure databank, and on-the-fly analysis of any protein sequence. A web interface retrieves ET rankings of sequence positions and maps results to a structure to identify functionally important regions. This UET database integrates several ways of viewing the results on the protein sequence or structure and can be found at http://mammoth.bcm.tmc.edu/uet/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Site characterization techniques used in environmental remediation activities

International Nuclear Information System (INIS)

Kostelnik, K.M.

2000-01-01

As a result of decades of nuclear energy research, weapons production, as well as ongoing operations, a significant amount of radioactive contamination has occurred throughout the United States Department of Energy (DOE) complex. DOE facility are in the process of assessing and potentially remediating various sites according to the regulations imposed by a Federal Facility Agreement and Consent order (FFA/CO) between DOE, the state in which the facility is located, and the U.S. Environmental Protection Agency (EPA). In support of these active site remediation efforts, the DOE has devoted considerable resources towards the development of innovative site characterization techniques that support environmental restoration activities. These resources and efforts have focused on various aspects of this complex problem. Research and technology development conducted at the Idaho National Engineering and Environmental Laboratory (INEEL) has resulted in the ability and state-of-the-art equipment required to obtain real-time, densely spaced, in situ characterization data (i.e. detection, speciation, and location) of various radionuclides and contaminants. The Remedial Action Monitoring System (RAMS), developed by the INEEL, consists of enhanced sensor technology, measurement modeling and interpretation techniques, and a suite of deployment platforms which can be interchanged to directly support remedial cleanup and site verification operations. In situ characterization techniques have advanced to the point where they are being actively deployed in support of remedial operations. The INEEL has deployed its system at various DOE and international sites. The deployment of in situ characterization systems during environmental restoration operations has shown that this approach results in several significant benefits versus conventional sampling techniques. A flexible characterization system permits rapid modification to satisfy physical site conditions, available site resources

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

OpenAIRE

Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

2015-01-01

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modificati...

Actinobacterial diversity in limestone deposit sites in Hundung, Manipur (India and their antimicrobial activities

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Salam eNimaichand

2015-05-01

Full Text Available Studies on actinobacterial diversity in limestone habitats are scarce. This paper reports profiling of actinobacteria isolated from Hundung limestone samples in Manipur, India using ARDRA as the molecular tool for preliminary classification. A total of 137 actinobacteria were clustered into 31 phylotypic groups based on the ARDRA pattern generated and representative of each group was subjected to 16S rRNA gene sequencing. Generic diversity of the limestone isolates consisted of Streptomyces (15 phylotypic groups, Micromonospora (4, Amycolatopsis (3, Arthrobacter (3, Kitasatospora (2, Janibacter (1, Nocardia (1, Pseudonocardia (1 and Rhodococcus (1. Considering the antimicrobial potential of these actinobacteria, 19 showed antimicrobial activities against at least one of the bacterial and candidal test pathogens, while 45 exhibit biocontrol activities against at least one of the rice fungal pathogens. Out of the 137 actinobacterial isolates, 118 were found to have at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, NRPS. The results indicate that 86% of the strains isolated from Hundung limestone deposit sites possessed biosynthetic gene clusters of which 40% exhibited antimicrobial activities. It can, therefore, be concluded that limestone habitat is a promising source for search of novel secondary metabolites.

Active sites in char gasification: Final technical report

Energy Technology Data Exchange (ETDEWEB)

Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

1987-09-01

Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

Human β-globin locus control region: Analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice

International Nuclear Information System (INIS)

Caterina, J.J.; Ryan, T.M.; Pawlik, K.M.; Townes, T.M.; Brinster, R.L.; Behringer, R.R.; Palmiter, R.D.

1991-01-01

The human β-globin locus control region (LCR) is essential for high-level expression of human var-epsilon-, γ-, and β-globin genes. Developmentally stable DNase I hypersensitive sites (designated HS) mark sequences within this region that are important for LCR activity. A 1.9-kilobase (kb) fragment containing the 5' HS 2 site enhances human β-globin gene expression 100-fold in transgenic mice and also confers position-independent expression. To further define important sequences within this region, deletion mutations of the 1.9-kb fragment were introduced upstream of the human β-globin gene, and the constructs were tested for activity in transgenic mice. Although enhancer activity was gradually lost with deletion of both 5' and 3' sequences, a 373-base-pair (BP) fragment retained the ability to confer relative position-independent expression. Three prominent DNase I footprints were observed in this region with extracts from the human erythroleukemia cell line K-562, one of which contained duplicated binding sites for transcription factor AP-1 (activator protein 1). When the 1.9-kb fragment containing an 19-bp deletion of the AP-1 binding sites was tested in transgenic mice, enhancer activity decreased 20-fold but position-independent expression was retained

Widespread alternative and aberrant splicing revealed by lariat sequencing

Science.gov (United States)

Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

2015-01-01

Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

Relation of chromospheric activity to convection, rotation, and pre-main-sequence evolution

International Nuclear Information System (INIS)

Gilliland, R.L.

1986-01-01

Pre-main-sequence, or T Tauri, stars are characterized by much larger fluxes of nonradiative origin than their main-sequence counterparts. As a class, the T Tauri stars have only moderate rotation rates, making an explanation of their chromospheric properties based on rapid rotation problematic. The recent success of correlating nonradiative fluxes to the Rossby number, Ro = P/sub rot//tau/sub conv/, a central parameter of simple dynamo theories of magnetic field generation, has led to the suggestion that the same relation might be of use in explaining the pre-main-sequence (PMS) stars if tau/sub conv/ is very large. We show that tau/sub conv/ does depend strongly on evolutionary effects above the main sequence (MS), but that this dependence alone cannot account for the high observed nonradiative fluxes. The acoustic flux is also strongly dependent on PMS evolutionary state, and when coupled to the parameterization of magnetic activity based on Ro, these two mechanisms seem capable of explaining the high observed level of chromospheric activity in T Tauri stars. The moment of inertia decreases by two to three order of magnitude during PMS evolution. Since young MS stars do not rotate two to three orders of magnitude faster than PMS stars, rapid loss or redistribution of angular momentum must occur

Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus.

Science.gov (United States)

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 Pyrococcus furiosus (Pf), a hyperthermophillic archeon. Sequence analysis of the Pf gene indicated an open reading frame specifying a protein of 485 amino acids (aa) with a calculated M(r) of 52900. Canonical Archaea promoter elements, Box A and Box B, are located -49 and -17 nucleotides (nt), respectively, upstream of the putative start codon. The sequence of the putative active-site region conforms to the IMPDH signature motif and contains a putative active-site cysteine. Phylogenetic relationships derived by using all available IMPDH sequences are consistent with trees developed for other molecules; they do not precisely resolve the history of Pf IMPDH but indicate a close similarity to bacterial IMPDH proteins. The phylogenetic analysis indicates that a gene duplication occurred prior to the division between rodents and humans, accounting for the Type I and II isoforms identified in mice and humans.

Mapping the active site of vaccinia virus RNA triphosphatase

International Nuclear Information System (INIS)

Gong Chunling; Shuman, Stewart

2003-01-01

The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called ''triphosphate tunnel''). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery

SABER: a computational method for identifying active sites for new reactions.

Science.gov (United States)

Nosrati, Geoffrey R; Houk, K N

2012-05-01

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. Copyright © 2012 The Protein Society.

GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

Science.gov (United States)

Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

2017-07-03

Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

Science.gov (United States)

Jia, Xianbo; Lin, Xinjian; Chen, Jichen

2017-11-02

Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

Total sequence decomposition distinguishes functional modules, "molegos" in apurinic/apyrimidinic endonucleases

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Braun Werner

2002-11-01

Full Text Available Abstract Background Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos", that can eventually be related to function. Here, the approach is applied to the apurinic/apyrimidinic endonuclease (APE DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. The APEs, DNase-1 and inositol 5'-polyphosphate phosphatases (IPP form a superfamily that catalyze metal ion based phosphorolysis, but recognize different substrates. Results MASIA decomposition of APE yielded 12 sequence motifs, 10 of which are also structurally conserved within the family and are designated as molegos. The 12 motifs include all the residues known to be essential for DNA cleavage by APE. Five of these molegos are sequentially and structurally conserved in DNase-1 and the IPP family. Correcting the sequence alignment to match the residues at the ends of two of the molegos that are absolutely conserved in each of the three families greatly improved the local structural alignment of APEs, DNase-1 and synaptojanin. Comparing substrate/product binding of molegos common to DNase-1 showed that those distinctive for APEs are not directly involved in cleavage, but establish protein-DNA interactions 3' to the abasic site. These additional bonds enhance both specific binding to damaged DNA and the processivity of APE1. Conclusion A modular approach can improve structurally predictive alignments of homologous proteins with low sequence identity and reveal residues peripheral to the traditional "active site" that control the specificity of enzymatic activity.

Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Tachibana, C; Winther, Jakob R.

1997-01-01

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC......-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth....

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance.

Science.gov (United States)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-07-05

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance

International Nuclear Information System (INIS)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-01-01

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable

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

DEFF Research Database (Denmark)

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...... 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...... 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...

Facebook, Twitter Activities Sites, Location and Students' Interest in Learning

Science.gov (United States)

Igbo, J. N.; Ezenwaji, Ifeyinwa; Ajuziogu, Christiana U.

2018-01-01

This study was carried out to ascertain the influence of social networking sites activities (twitter and Facebook) on secondary school students' interest in learning It also considered the impact of these social networking sites activities on location of the students. Two research questions and two null hypotheses guided the study. Mean and…

BIOPEP database and other programs for processing bioactive peptide sequences.

Science.gov (United States)

Minkiewicz, Piotr; Dziuba, Jerzy; Iwaniak, Anna; Dziuba, Marta; Darewicz, Małgorzata

2008-01-01

This review presents the potential for application of computational tools in peptide science based on a sample BIOPEP database and program as well as other programs and databases available via the World Wide Web. The BIOPEP application contains a database of biologically active peptide sequences and a program enabling construction of profiles of the potential biological activity of protein fragments, calculation of quantitative descriptors as measures of the value of proteins as potential precursors of bioactive peptides, and prediction of bonds susceptible to hydrolysis by endopeptidases in a protein chain. Other bioactive and allergenic peptide sequence databases are also presented. Programs enabling the construction of binary and multiple alignments between peptide sequences, the construction of sequence motifs attributed to a given type of bioactivity, searching for potential precursors of bioactive peptides, and the prediction of sites susceptible to proteolytic cleavage in protein chains are available via the Internet as are other approaches concerning secondary structure prediction and calculation of physicochemical features based on amino acid sequence. Programs for prediction of allergenic and toxic properties have also been developed. This review explores the possibilities of cooperation between various programs.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

DEFF Research Database (Denmark)

Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

2016-01-01

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

Diversity and characterization of sulfate-reducing bacteria in groundwater at a uranium mill tailings site

International Nuclear Information System (INIS)

Chang, Yun-Juan; Peacock, A D.; Long, Philip E.; Stephen, John R.; McKinley, James P.; Mcnaughton, Sarah J.; Hussain, A K M A.; Saxton, A M.; White, D C.

2000-01-01

Microbially mediated reduction and immobilization of U(VI) to U(TV) plays a role in both natural attenuation and accelerated bioremediation of uranium contaminated sites. To realize bioremediation potential and accurately predict natural attenuation, it is important to first understand the microbial diversity of such sites. In this paper, the distribution of sulfate-reducing bacteria (SRB) in contaminated groundwater associated with a uranium mill tailings disposal site at Shiprock, N.Mex,, was investigated. Two culture-independent analyses were employed: sequencing of clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) gene fragments and phospholipid fatty acid (PLFA) biomarker analysis. A remarkable diversity among the DSR sequences was revealed, including sequences from F-Proteobacteria, gram-positive organisms, and the Nitrospira division. PLFA analysis detected at least,52 different mid-chain-branched saturate PLFA and included a high proportion of 10me16:0, Desulfotomaculum and Desulfotomaculum-like sequences were the most dominant DSR genes detected. Those belonging to SRB within F-Proteobacteria were mainly recovered from low-uranium (less than or equal to 302 ppb) samples. One Desulfotomaculum like sequence cluster overwhelmingly dominated high-U (> 1,500 ppb) sites. Logistic regression showed a significant influence of uranium concentration over the dominance of this cluster of sequences (P= 0.0001), This strong association indicates that Desulfotomaculum has remarkable tolerance and adaptation to high levels of uranium and suggests the organism's possible involvement in natural attenuation of uranium. The in situ activity level of Desulfotomaculum in uranium-contaminated environments and its comparison to the activities of other SRB and other functional groups should be an important area for future research

Poisoning Experiments Aimed at Discriminating Active and Less-Active Sites of Silica-Supported Tantalum Hydride for Alkane Metathesis

KAUST Repository

Saggio, Guillaume

2010-10-04

Only 50% of the silica-supported tantalum hydride sites are active in the metathesis of propane. Indeed, more than 45% of the tantalum hydride can be eliminated by a selective oxygen poisoning of inactive sites with no significant decrease in the global turnover. Conversely, cyclopentane induces no such selective poisoning. Hence, the active tantalum hydride sites that show greater resistance to oxygen poisoning correspond to the νTa-H bands of higher wavenumbers, particularly that at 1860cm-1. These active tantalum hydride sites should correspond to tris- or monohydride species relatively far from silica surface oxygen atoms. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro site selection of a consensus binding site for the Drosophila melanogaster Tbx20 homolog midline.

Directory of Open Access Journals (Sweden)

Nima Najand

Full Text Available We employed in vitro site selection to identify a consensus binding sequence for the Drosophila melanogaster Tbx20 T-box transcription factor homolog Midline. We purified a bacterially expressed T-box DNA binding domain of Midline, and used it in four rounds of precipitation and polymerase-chain-reaction based amplification. We cloned and sequenced 54 random oligonucleotides selected by Midline. Electromobility shift-assays confirmed that 27 of these could bind the Midline T-box. Sequence alignment of these 27 clones suggests that Midline binds as a monomer to a consensus sequence that contains an AGGTGT core. Thus, the Midline consensus binding site we define in this study is similar to that defined for vertebrate Tbx20, but differs from a previously reported Midline binding sequence derived through site selection.

Antiprotozoal activities of benzimidazoles and correlations with beta-tubulin sequence.

Science.gov (United States)

Katiyar, S K; Gordon, V R; McLaughlin, G L; Edlind, T D

1994-01-01

Benzimidazoles have been widely used since the 1960s as anthelmintic agents in veterinary and human medicine and as antifungal agents in agriculture. More recently, selected benzimidazole derivatives were shown to be active in vitro against two protozoan parasites, Trichomonas vaginalis and Giardia lamblia, and clinical studies with AIDS patients have suggested that microsporidia are susceptible as well. Here, we first present in vitro susceptibility data for T. vaginalis and G. lamblia using an expanded set of benzimidazole derivatives. Both parasites were highly susceptible to four derivatives, including mebendazole, flubendazole, and fenbendazole (50% inhibitory concentrations of 0.005 to 0.16 microgram/ml). These derivatives also had lethal activity that was time dependent: 90% of T. vaginalis cells failed to recover following a 20-h exposure to mebendazole at 0.17 microgram/ml. G. lamblia, but not T. vaginalis, was highly susceptible to five additional derivatives. Next, we examined in vitro activity of benzimidazoles against additional protozoan parasites: little or no activity was observed against Entamoeba histolytica, Leishmania major, and Acanthamoeba polyphaga. Since the microtubule protein beta-tubulin has been identified as the benzimidazole target in helminths and fungi, potential correlations between benzimidazole activity and beta-tubulin sequence were examined. This analysis included partial sequences (residues 108 to 259) from the organisms mentioned above, as well as the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi and the sporozoan Cryptosporidium parvum. beta-tubulin residues Glu-198 and, in particular, Phe-200 are strong predictors of benzimidazole susceptibility; both are present in Encephalitozoon spp. but absent in C. parvum. PMID:7811023

Concept for calculating dose rates from activated groundwater at accelerator sites

CERN Document Server

Prolingheuer, N; Vanderborght, J; Schlögl, B; Nabbi, R; Moormann, R

Licensing of particle accelerators requires the proof that the groundwater outside of the site will not be significantly contaminated by activation products formed below accelerator and target. In order to reduce the effort for this proof, a site independent simplified but conservative method is under development. The conventional approach for calculation of activation of soil and groundwater is shortly described on example of a site close to Forschungszentrum Juelich, Germany. Additionally an updated overview of a data library for partition coefficients for relevant nuclides transported in the aquifer at the site is presented. The approximate model for transport of nuclides with ground water including exemplary results on nuclide concentrations outside of the site boundary and of resulting effective doses is described. Further applications and developments are finally outlined.

Sequence alignment reveals possible MAPK docking motifs on HIV proteins.

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Perry Evans

Full Text Available Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs. MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme.

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Patrick E F O'Rourke

Full Text Available 4-Amino-4-deoxychorismate lyase (PabC catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5'-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp(2 to sp(3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of

Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method

Directory of Open Access Journals (Sweden)

Bingfu Guo

2016-07-01

Full Text Available Molecular characterization of sequences flanking exogenous fragment insertions is essential for safety assessment and labeling of genetically modified organisms (GMO. In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS method. About 21 Gb sequence data (~21× coverage for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundary of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767-50543792 and Chr17: 7980527-7980541 in these two transgenic lines. Identification of the genomic insertion site of the G2-EPSPS and GAT transgenes will facilitate the use of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS is a cost-effective and rapid method of identifying sites of T-DNA insertions and flanking sequences in soybean.

Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.

Directory of Open Access Journals (Sweden)

Rick S Mitchell

2004-08-01

Full Text Available The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV, avian sarcoma-leukosis virus (ASLV, and murine leukemia virus (MLV. Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.

Remediation of uranium contaminated sites: clean-up activities in Serbia

International Nuclear Information System (INIS)

Raicevic, S.; Raicevic, J. . E-mail address of corresponding author: raich@beotel.yu; Raicevic, S.)

2005-01-01

One of the serious environmental problems in Serbia represent sites contaminated with depleted uranium (DU) during past war activities. According to UNEP reports and our findings there are two types of contamination: (i) localized points of high, concentrated contamination where DU penetrators enter the soil, and (ii) low level of widespread DU contamination, which indicates that during the conflict DU dust was dispersed into the environment. Remediation of these sites is an urgent need because they represent a permanent threat to the population living in this area. Here we give a brief description of approaches commonly used in remediation of DU contaminated sites, and an overview of current clean-up activities performed in Serbia. (author)

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

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Thorsten Pauly

Full Text Available Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs. Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

Science.gov (United States)

Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

2008-07-16

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

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Melissa K Boles

2009-12-01

Full Text Available An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn, inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

International Nuclear Information System (INIS)

Flores-Riveros, J.R.; Lane, M.D.

1987-01-01

Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with [γ- 32 P]ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the 32 P-labeled β-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total 32 P radioactivity is found in site I and the rate of 32 P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the β-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress

The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues.

Science.gov (United States)

Gillner, Danuta M; Bienvenue, David L; Nocek, Boguslaw P; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C

2009-01-01

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 A of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.

Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing

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Haoran Wang

2018-03-01

Full Text Available Detection of complex splice sites (SSs and polyadenylation sites (PASs of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS have revealed prevalent alternative splicing (AS and alternative polyadenylation (APA in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3′-cDNA ends (3′-RACE and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2 transcription factor genes of Populus trichocarpa were analyzed by 3′-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3′-UTR PAS-rich markers were revealed by 3′-RACE-seq. Using 3′-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3′-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage.

Scanning mutagenesis of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

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Nagib eAhsan

2012-07-01

Full Text Available The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform scanning mutagenesis of the residues flanking the site of phosphorylation. Consistent with the results from phylogenetic analysis of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

Active Sites Environmental Monitoring Program. FY 1993: Annual report

International Nuclear Information System (INIS)

Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

1994-08-01

This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993

Essential histidyl residues at the active site(s) of sucrose-phosphate synthase from Prosopis juliflora.

Science.gov (United States)

Sinha, A K; Pathre, U V; Sane, P V

1998-11-10

Chemical modification of sucrose-phosphate synthase (EC 2.4.1.14) from Prosopis juliflora by diethyl pyrocarbonate (DEP) and photo-oxidation in the presence of rose bengal (RB) which modify the histidyl residues of the protein resulted in the inactivation of the enzyme activity. This inactivation was dependent on the concentration of the modifying reagent and the time of incubation and followed pseudo-first order kinetics. For both the reagents, the inactivation was maximum at pH 7.5, which is consistent with the involvement and presence of histidine residues at the active site of the enzyme. Substrates, UDPG and F6P protected the enzyme against the inactivation by the modifying reagents suggesting that the histidine residues may be involved in the binding of these substrates and are essential for the catalytic activity. Specificity of DEP was indicated by an increase in absorbance at 240 nm along with concomitant inactivation of the enzyme and reactivation of the modified enzyme by hydroxylamine. These results strongly suggest the presence of histidine residue(s) at or near the active site of the enzyme.

Disturbance opens recruitment sites for bacterial colonization in activated sludge.

Science.gov (United States)

Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

2016-01-01

Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Gideaa study site. Scope of activities and main results

International Nuclear Information System (INIS)

Ahlbom, K.; Andersson, J.E.; Nordqvist, R.; Ljunggren, C.; Tiren, S.; Voss, C.

1991-10-01

During the period from 1977-1986 SKB (Swedish Nuclear Fuel and Waste Management Co) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other site with geological and/or socio-economical characteristics judged more favourable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concerns the Gideaa study site. (au)

Kamlunge study site. Scope of activities and main results

International Nuclear Information System (INIS)

Ahlbom, K.; Andersson, J.E.; Andersson, P.; Ittner, T.; Tiren, S.; Ljunggren, C.

1992-05-01

During the period from 1977-1986 SKB (Swedish nuclear Fuel and Waste Management Co.) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favourable may very well be selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concerns the Kamlunge study site. (79 refs.) (au)

Fjaellveden study site. Scope of activities and main results

International Nuclear Information System (INIS)

Ahlbom, K.; Andersson, J.E.; Nordqvist, R.; Ljunggren, C.; Tiren, S.; Voss, C.

1991-10-01

During the period from 1977-1986 SKB (Swedish Nuclear Fuel and Waste Management CO) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favourable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need for complementary investigations. This report concerns the Fjaellveden study site. (au)

Context based computational analysis and characterization of ARS consensus sequences (ACS of Saccharomyces cerevisiae genome

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Vinod Kumar Singh

2016-09-01

Full Text Available Genome-wide experimental studies in Saccharomyces cerevisiae reveal that autonomous replicating sequence (ARS requires an essential consensus sequence (ACS for replication activity. Computational studies identified thousands of ACS like patterns in the genome. However, only a few hundreds of these sites act as replicating sites and the rest are considered as dormant or evolving sites. In a bid to understand the sequence makeup of replication sites, a content and context-based analysis was performed on a set of replicating ACS sequences that binds to origin-recognition complex (ORC denoted as ORC-ACS and non-replicating ACS sequences (nrACS, that are not bound by ORC. In this study, DNA properties such as base composition, correlation, sequence dependent thermodynamic and DNA structural profiles, and their positions have been considered for characterizing ORC-ACS and nrACS. Analysis reveals that ORC-ACS depict marked differences in nucleotide composition and context features in its vicinity compared to nrACS. Interestingly, an A-rich motif was also discovered in ORC-ACS sequences within its nucleosome-free region. Profound changes in the conformational features, such as DNA helical twist, inclination angle and stacking energy between ORC-ACS and nrACS were observed. Distribution of ACS motifs in the non-coding segments points to the locations of ORC-ACS which are found far away from the adjacent gene start position compared to nrACS thereby enabling an accessible environment for ORC-proteins. Our attempt is novel in considering the contextual view of ACS and its flanking region along with nucleosome positioning in the S. cerevisiae genome and may be useful for any computational prediction scheme.

Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

Science.gov (United States)

Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

2017-01-01

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

Nuclear waste: Status of DOE's nuclear waste site characterization activities

International Nuclear Information System (INIS)

1987-01-01

Three potential nuclear waste repository sites have been selected to carry out characterization activities-the detailed geological testing to determine the suitability of each site as a repository. The sites are Hanford in south-central Washington State, Yucca Mountain in southern Nevada, and Deaf Smith in the Texas Panhandle. Two key issues affecting the total program are the estimations of the site characterization completion data and costs and DOE's relationship with the Nuclear Regulatory Commission which has been limited and its relations with affected states and Indian tribes which continue to be difficult

cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

[Neuronal activity of monkey dorso-lateral premotor cortex during tasks of figure recognition guided motor sequence vs memorized spatial motor sequence].

Science.gov (United States)

Chen, Y C; Huang, F D; Chen, N H; Shou, J Y; Wu, L

1998-04-01

In the last 2-3 decades the role of the premotor cortex (PM) of monkey in memorized spatial sequential (MSS) movements has been amply investigated. However, it is as yet not known whether PM participates in the movement sequence behaviour guided by recognition of visual figures (i.e. the figure-recognition sequence, FRS). In the present work three monkeys were trained to perform both FRS and MSS tasks. Postmortem examination showed that 202 cells were in the dorso-lateral premotor cortex. Among 111 cells recorded during the two tasks, more than 50% changed their activity during the cue periods in either task. During the response period, the ratios of cells with changes of firing rate in both FRS and MSS were high and roughly equal to each other, while during the image period, the proportion in the FRS (83.7%) was significantly higher than that in the MSS (66.7%). Comparison of neuronal activities during same motor sequence of two different tasks showed that during the image periods PM neuronal activities were more closely related to the FRS task, while during the cue periods no difference could be found. Analysis of cell responses showed that the neurons with longer latency were much more in MSS than in FRS in either cue or image period. The present results indicate that the premotor cortex participates in FRS motor sequence as well as in MSS and suggest that the dorso-lateral PM represents another subarea in function shared by both FRS and MSS tasks. However, in view of the differences of PM neuronal responses in cue or image periods of FRS and MSS tasks, it seems likely that neural networks involved in FRS and MSS tasks are different.

Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry

DEFF Research Database (Denmark)

Gallina, Serafina; Cunsolo, Vincenzo; Saletti, Rosaria

2016-01-01

Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography...... characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis...... of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476....

Viewing multiple sequence alignments with the JavaScript Sequence Alignment Viewer (JSAV).

Science.gov (United States)

Martin, Andrew C R

2014-01-01

The JavaScript Sequence Alignment Viewer (JSAV) is designed as a simple-to-use JavaScript component for displaying sequence alignments on web pages. The display of sequences is highly configurable with options to allow alternative coloring schemes, sorting of sequences and 'dotifying' repeated amino acids. An option is also available to submit selected sequences to another web site, or to other JavaScript code. JSAV is implemented purely in JavaScript making use of the JQuery and JQuery-UI libraries. It does not use any HTML5-specific options to help with browser compatibility. The code is documented using JSDOC and is available from http://www.bioinf.org.uk/software/jsav/.

What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

Science.gov (United States)

Zhang, Ni; Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

2017-06-22

Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. ©Ni Zhang, Shelly Campo, Jingzhen Yang, Petya Eckler, Linda Snetselaar, Kathleen Janz, Emily Leary. Originally published in the Journal of Medical

Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato.

Science.gov (United States)

Shirasawa, Kenta; Hirakawa, Hideki; Isobe, Sachiko

2016-04-01

Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction sites from the genome sequence is recognized as an effective approach for choosing the restriction enzymes to be used, few reports have evaluated the in silico predictions in actual experimental data. In this study, we designed and demonstrated a workflow for in silico and empirical ddRAD-Seq analysis in tomato, as follows: (i)in silico prediction of optimum restriction enzymes from the reference genome, (ii) verification of the prediction by actual ddRAD-Seq data of four restriction enzyme combinations, (iii) establishment of a computational data processing pipeline for high-confidence single nucleotide polymorphism (SNP) calling, and (iv) validation of SNP accuracy by construction of genetic linkage maps. The quality of SNPs based on de novo assembly reference of the ddRAD-Seq reads was comparable with that of SNPs obtained using the published reference genome of tomato. Comparisons of SNP calls in diverse tomato lines revealed that SNP density in the genome influenced the detectability of SNPs by ddRAD-Seq. In silico prediction prior to actual analysis contributed to optimization of the experimental conditions for ddRAD-Seq, e.g. choices of enzymes and plant materials. Following optimization, this ddRAD-Seq pipeline could help accelerate genetics, genomics, and molecular breeding in both model and non-model plants, including crops. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Topoisomerase I tyrosine phosphorylation site and the DNA-interactive site

International Nuclear Information System (INIS)

Roll, D.; Durban, E.

1986-01-01

Phosphorylation of topoisomerase I (topo I) at serine by NII kinase is accompanied by stimulation of enzymatic activity. In contrast, phosphorylation at tyrosine by tyrosine kinase seems to inhibit enzymatic activity. This inhibition may be caused by interference of the phosphorylated tyrosine residue with the interaction of topo I with DNA. To test this, topo I was labeled with crude membrane fraction enriched for EGF-receptor kinase in presence of γ-P32-ATP and electrophoresed on SDS-polyacrylamide gels. Stained topo I bands were excised, dried, digested with trypsin and analyzed on a C18 reverse-phase HPLC column. One major peak of radioactivity eluted at fraction 23 with 20% acetonitrile. To obtain the DNA-interactive site, topo I was incubated with pBR322 DNA labeled by nick-translation followed by DNase I treatment, and electrophoresis on SDS-polyacrylamide gels. Tryptic peptides were generated and analyzed by reverse-phase HPLC. A major peak of radioactivity eluted at fraction 16-18 with 15.5-17% acetonitrile. Studies are in progress to resolve whether (a) the two peptides are different, i.e. the tyrosine-P site and DNA-tyrosine interactive site are localized at different regions of the topo I or (b) the peptide sequences are identical but the covalent attachment of deoxynucleotides altered the peptide's elution from the HPLC column

MOLECULAR CLONING, SEQUENCING, EXPRESSION AND BIOLOGICAL ACTIVITY OF GIANT PANDA (AILUROPODA MELANOLEUCA) INTERFERON-GAMMA.

Science.gov (United States)

Zhu, Hui; Wang, Wen-Xiu; Wang, Bao-Qin; Zhu, Xiao-Fu; Wu, Xu-Jin; Ma, Qing-Yi; Chen, De-Kun

2012-06-29

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. Interferon-gamma (IFN-γ) is the only member of type □ IFN and is vital for the regulation of host adapted immunity and inflammatory response. Little is known aboutthe FN-γ gene and its roles in giant panda.In this study, IFN-γ gene of Qinling giant panda was amplified from total blood RNA by RT-CPR, cloned, sequenced and analysed. The open reading frame (ORF) of Qinling giant panda IFN-γ encodes 152 amino acidsand is highly similar to Sichuan giant panda with an identity of 99.3% in cDNA sequence. The IFN-γ cDNA sequence was ligated to the pET32a vector and transformed into E. coli BL21 competent cells. Expression of recombinant IFN-γ protein of Qinling giant panda in E. coli was confirmed by SDS-PAGE and Western blot analysis. Biological activity assay indicated that the recombinant IFN-γ protein at the concentration of 4-10 µg/ml activated the giant panda peripheral blood lymphocytes,while at 12 µg/mlinhibited. the activation of the lymphocytes.These findings provide insights into the evolution of giant panda IFN-γ and information regarding amino acid residues essential for their biological activity.

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site.

Science.gov (United States)

Vyhlidal, C; Samudio, I; Kladde, M P; Safe, S

2000-06-01

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.

Increase in posterior alpha activity during rehearsal predicts successful long-term memory formation of word sequences.

Science.gov (United States)

Meeuwissen, Esther B; Takashima, Atsuko; Fernández, Guillén; Jensen, Ole

2011-12-01

It is becoming increasingly clear that demanding cognitive tasks rely on an extended network engaging task-relevant areas and, importantly, disengaging task-irrelevant areas. Given that alpha activity (8-12 Hz) has been shown to reflect the disengagement of task-irrelevant regions in attention and working memory tasks, we here ask if alpha activity plays a related role for long-term memory formation. Subjects were instructed to encode and maintain the order of word sequences while the ongoing brain activity was recorded using magnetoencephalography (MEG). In each trial, three words were presented followed by a 3.4 s rehearsal interval. Considering the good temporal resolution of MEG this allowed us to investigate the word presentation and rehearsal interval separately. The sequences were grouped in trials where word order either could be tested immediately (working memory trials; WM) or later (LTM trials) according to instructions. Subjects were tested on their ability to retrieve the order of the three words. The data revealed that alpha power in parieto-occipital regions was lower during word presentation compared to rehearsal. Our key finding was that parieto-occipital alpha power during the rehearsal period was markedly stronger for successfully than unsuccessfully encoded LTM sequences. This subsequent memory effect demonstrates that high posterior alpha activity creates an optimal brain state for successful LTM formation possibly by actively reducing parieto-occipital activity that might interfere with sequence encoding. Copyright © 2010 Wiley Periodicals, Inc.

Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.

Science.gov (United States)

Imamura, Haruna; Wagih, Omar; Niinae, Tomoya; Sugiyama, Naoyuki; Beltrao, Pedro; Ishihama, Yasushi

2017-04-07

Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

Energy Technology Data Exchange (ETDEWEB)

Anderson, Courtney M. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Hu, Jianxin [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Thomas, Reuben [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Gainous, T. Blair [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Celona, Barbara [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Sinha, Tanvi [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Dickel, Diane E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Heidt, Analeah B. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Xu, Shan-Mei [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Bruneau, Benoit G. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pollard, Katherine S. [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pennacchio, Len A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Black, Brian L. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Dept. of

2017-03-28

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.

Complete cDNA sequence and amino acid analysis of a bovine ribonuclease K6 gene.

Science.gov (United States)

Pietrowski, D; Förster, M

2000-01-01

The complete cDNA sequence of a ribonuclease k6 gene of Bos Taurus has been determined. It codes for a protein with 154 amino acids and contains the invariant cysteine, histidine and lysine residues as well as the characteristic motifs specific to ribonuclease active sites. The deduced protein sequence is 27 residues longer than other known ribonucleases k6 and shows amino acids exchanges which could reflect a strain specificity or polymorphism within the bovine genome. Based on sequence similarity we have termed the identified gene bovine ribonuclease k6 b (brk6b).

Possible interaction between B1 retrotransposon-containing sequences and β(major) globin gene transcriptional activation during MEL cell erythroid differentiation.

Science.gov (United States)

Vizirianakis, Ioannis S; Tezias, Sotirios S; Amanatiadou, Elsa P; Tsiftsoglou, Asterios S

2012-01-01

Repetitive sequences consist of >50% of mammalian genomic DNAs and among these SINEs (short interspersed nuclear elements), e.g. B1 elements, account for 8% of the mouse genome. In an effort to delineate the molecular mechanism(s) involved in the blockade of the in vitro differentiation program of MEL (murine erythroleukaemia) cells by treatment with methylation inhibitors, we detected a DNA region of 559 bp in chromosome 7 located downstream of the 3'-end of the β(major) globin gene (designated B1-559) with unique characteristics. We have fully characterized this B1-559 region that includes a B1 element, several repeats of ATG initiation codons and consensus DNA-binding sites for erythroid-specific transcription factors NF-E2 (nuclear factor-erythroid-derived 2), GATA-1 and EKLF (erythroid Krüppel-like factor). Fragments derived from B1-559 incubated with nuclear extracts form protein complexes in both undifferentiated and differentiated MEL cells. Transient reporter-gene experiments in MEL and human erythroleukaemia K-562 cells with recombinant constructs containing B1-559 fragments linked to HS-2 (hypersensitive site-2) sequences of human β-globin gene LCR (locus control region) indicated potential cooperation upon erythropoiesis and globin gene expression. The possible interaction between the B1-559 region and β(major) globin gene transcriptional activation upon execution of erythroid MEL cell differentiation programme is discussed. © The Author(s) Journal compilation © 2012 Portland Press Limited

Predicting protein-ATP binding sites from primary sequence through fusing bi-profile sampling of multi-view features

Directory of Open Access Journals (Sweden)

Zhang Ya-Nan

2012-05-01

Full Text Available Abstract Background Adenosine-5′-triphosphate (ATP is one of multifunctional nucleotides and plays an important role in cell biology as a coenzyme interacting with proteins. Revealing the binding sites between protein and ATP is significantly important to understand the functionality of the proteins and the mechanisms of protein-ATP complex. Results In this paper, we propose a novel framework for predicting the proteins’ functional residues, through which they can bind with ATP molecules. The new prediction protocol is achieved by combination of sequence evolutional information and bi-profile sampling of multi-view sequential features and the sequence derived structural features. The hypothesis for this strategy is single-view feature can only represent partial target’s knowledge and multiple sources of descriptors can be complementary. Conclusions Prediction performances evaluated by both 5-fold and leave-one-out jackknife cross-validation tests on two benchmark datasets consisting of 168 and 227 non-homologous ATP binding proteins respectively demonstrate the efficacy of the proposed protocol. Our experimental results also reveal that the residue structural characteristics of real protein-ATP binding sites are significant different from those normal ones, for example the binding residues do not show high solvent accessibility propensities, and the bindings prefer to occur at the conjoint points between different secondary structure segments. Furthermore, results also show that performance is affected by the imbalanced training datasets by testing multiple ratios between positive and negative samples in the experiments. Increasing the dataset scale is also demonstrated useful for improving the prediction performances.

Immobilized low-activity waste site borehole 299-E17-21

International Nuclear Information System (INIS)

Reidel, S.P.; Reynolds, K.D.; Horton, D.G.

1998-08-01

The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area

Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

Science.gov (United States)

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

Science.gov (United States)

Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

2012-05-01

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

Sternoe study site. Scope of activities and main results

International Nuclear Information System (INIS)

Ahlbom, K.; Andersson, J.E.; Nordqvist, R.; Tiren, S.; Ljunggren, C.; Voss, C.

1992-01-01

During the period from 1977-1986 SKB (Swedish Nuclear Fuel and Waste Management Co) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the previously investigated study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favourable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concerns the Sternoe study site. This site was one of the first sites to be investigated by SKB . The studies at Sternoe were made under severe time-constraints and with prototype borehole instrumentations. These limitations should be kept in mind when reading the report. (41 refs., 16 figs., 12 tabs.) (au)

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

Science.gov (United States)

Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

2011-05-31

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

Energy Technology Data Exchange (ETDEWEB)

Miao, Yinglong [ORNL; Baudry, Jerome Y [ORNL

2011-01-01

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

Science.gov (United States)

Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

2014-07-01

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

Active site mutations change the cleavage specificity of neprilysin.

Directory of Open Access Journals (Sweden)

Travis Sexton

Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

Transcriptional activation signals found in the Epstein-Barr virus (EBV) latency C promoter are conserved in the latency C promoter sequences from baboon and Rhesus monkey EBV-like lymphocryptoviruses (cercopithicine herpesviruses 12 and 15).

Science.gov (United States)

Fuentes-Pananá, E M; Swaminathan, S; Ling, P D

1999-01-01

The Epstein-Barr virus (EBV) EBNA2 protein is a transcriptional activator that controls viral latent gene expression and is essential for EBV-driven B-cell immortalization. EBNA2 is expressed from the viral C promoter (Cp) and regulates its own expression by activating Cp through interaction with the cellular DNA binding protein CBF1. Through regulation of Cp and EBNA2 expression, EBV controls the pattern of latent protein expression and the type of latency established. To gain further insight into the important regulatory elements that modulate Cp usage, we isolated and sequenced the Cp regions corresponding to nucleotides 10251 to 11479 of the EBV genome (-1079 to +144 relative to the transcription initiation site) from the EBV-like lymphocryptoviruses found in baboons (herpesvirus papio; HVP) and Rhesus macaques (RhEBV). Sequence comparison of the approximately 1,230-bp Cp regions from these primate viruses revealed that EBV and HVP Cp sequences are 64% conserved, EBV and RhEBV Cp sequences are 66% conserved, and HVP and RhEBV Cp sequences are 65% conserved relative to each other. Approximately 50% of the residues are conserved among all three sequences, yet all three viruses have retained response elements for glucocorticoids, two positionally conserved CCAAT boxes, and positionally conserved TATA boxes. The putative EBNA2 100-bp enhancers within these promoters contain 54 conserved residues, and the binding sites for CBF1 and CBF2 are well conserved. Cp usage in the HVP- and RhEBV-transformed cell lines was detected by S1 nuclease protection analysis. Transient-transfection analysis showed that promoters of both HVP and RhEBV are responsive to EBNA2 and that they bind CBF1 and CBF2 in gel mobility shift assays. These results suggest that similar mechanisms for regulation of latent gene expression are conserved among the EBV-related lymphocryptoviruses found in nonhuman primates.

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

Science.gov (United States)

Waszak, Sebastian M; Kilpinen, Helena; Gschwind, Andreas R; Orioli, Andrea; Raghav, Sunil K; Witwicki, Robert M; Migliavacca, Eugenia; Yurovsky, Alisa; Lappalainen, Tuuli; Hernandez, Nouria; Reymond, Alexandre; Dermitzakis, Emmanouil T; Deplancke, Bart

2014-01-15

High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter

Communication activities for NUMO's site selection process

International Nuclear Information System (INIS)

Takeuchi, Mitsuo; Okuyama, Shigeru; Kitayama, Kazumi; Kuba, Michiyoshi

2004-01-01

A siting program for geological disposal of high-level radioactive waste (HLW) in Japan has just started and is moving into a new stage of communication with the public. A final repository site will be selected via a stepwise process, as stipulated in the Specified Radioactive Waste Final Disposal Act promulgated in June 2000. Based on the Act, the site selection process of the Nuclear Waste Management Organization of Japan (NUMO, established in October 2000) will be carried out in the three steps: selection of Preliminary Investigation Areas (PIAs), selection of Detailed Investigation Areas (DIAs) and selection of the Repository Site. The Act also defines NUMO's responsibilities in terms of implementing the HLW disposal program in an open and transparent manner. NUMO fully understands the importance of public participation in its activities and is aiming to promote public involvement in the process of site selection based on a fundamental policy, which consists of 'adopting a stepwise approach', 'respecting the initiative of municipalities' and 'ensuring transparency in information disclosure'. This policy is clearly reflected in the adoption of an open solicitation approach for volunteer municipalities for Preliminary Investigation Areas (PIAs). NUMO made the official announcement of the start of its open solicitation program on 19 December 2002. This paper outlines how NUMO's activities are currently carried out with a view to encouraging municipalities to volunteer as PIAs and how public awareness of the safety of the HLW disposal is evaluated at this stage

Seismic activity parameters of the Finnish potential repository sites

International Nuclear Information System (INIS)

Saari, J.

2000-10-01

Posiva Oy has started a project for estimating the possible earthquake induced rock movements on the deposition holes containing canisters of spent nuclear fuel. These estimates will be made for the four investigation sites, Romuvaara, Kivetty, Olkiluoto and Haestholmen. This study deals with the current and future seismicity associated with the above mentioned sites. Seismic belts that participate the seismic behaviour of the studied sites have been identified and the magnitude-frequency distributions of these belts have been estimated. The seismic activity parameters of the sites have been deduced from the characteristics of the seismic belts in order to forecast the seismicity during the next 100,000 years. The report discusses the possible earthquakes induced by future glaciation. The seismic interpretation seems to indicate that the previous postglacial faults in Finnish Lapland have been generated in compressional environment. The orientation of the rather uniform compression has been NW-SE, which coincide with the current stress field. It seems that, although the impact of postglacial crustal rebound must have been significant, the impact of plate tectonics has been dominant. A major assumption of this study has been that future seismicity will generally resemble the current seismicity. However, when the postglacial seismicity is concerned, the magnitude-frequency distribution is likely different and the expected maximum magnitude will be higher. Maximum magnitudes of future postglacial earthquakes have been approximated by strain release examinations. Seismicity has been examined within the framework of the lineament maps, in order to associate the future significant earthquakes with active fault zones in the vicinity of the potential repository sites. (orig.)

TargetM6A: Identifying N6-Methyladenosine Sites From RNA Sequences via Position-Specific Nucleotide Propensities and a Support Vector Machine.

Science.gov (United States)

Li, Guang-Qing; Liu, Zi; Shen, Hong-Bin; Yu, Dong-Jun

2016-10-01

As one of the most ubiquitous post-transcriptional modifications of RNA, N 6 -methyladenosine ( [Formula: see text]) plays an essential role in many vital biological processes. The identification of [Formula: see text] sites in RNAs is significantly important for both basic biomedical research and practical drug development. In this study, we designed a computational-based method, called TargetM6A, to rapidly and accurately target [Formula: see text] sites solely from the primary RNA sequences. Two new features, i.e., position-specific nucleotide/dinucleotide propensities (PSNP/PSDP), are introduced and combined with the traditional nucleotide composition (NC) feature to formulate RNA sequences. The extracted features are further optimized to obtain a much more compact and discriminative feature subset by applying an incremental feature selection (IFS) procedure. Based on the optimized feature subset, we trained TargetM6A on the training dataset with a support vector machine (SVM) as the prediction engine. We compared the proposed TargetM6A method with existing methods for predicting [Formula: see text] sites by performing stringent jackknife tests and independent validation tests on benchmark datasets. The experimental results show that the proposed TargetM6A method outperformed the existing methods for predicting [Formula: see text] sites and remarkably improved the prediction performances, with MCC = 0.526 and AUC = 0.818. We also provided a user-friendly web server for TargetM6A, which is publicly accessible for academic use at http://csbio.njust.edu.cn/bioinf/TargetM6A.

Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants.

Directory of Open Access Journals (Sweden)

Sophie Lanciano

2017-02-01

Full Text Available Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes.

Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants.

Science.gov (United States)

Lanciano, Sophie; Carpentier, Marie-Christine; Llauro, Christel; Jobet, Edouard; Robakowska-Hyzorek, Dagmara; Lasserre, Eric; Ghesquière, Alain; Panaud, Olivier; Mirouze, Marie

2017-02-01

Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes.

Amplification of a transcriptionally active DNA sequence in the human brain

International Nuclear Information System (INIS)

Yakovlev, A.G.; Sazonov, A.E.; Spunde, A.Ya.; Gindilis, V.M.

1986-01-01

The authors present their findings of tissue-specific amplification of a DNA fragment actively transcribed in the human brain. This genome fragment was found in the library complement of cDNA of the human brain and evidently belongs to a new class of moderate repetitions of DNA with an unstable copying capacity in the human genome. The authors isolated total cell RNA from various human tissues (brain, placenta), and rat tissues (brain, liver), by the method of hot phenol extraction with guanidine thiocynate. The poly(A + ) RNA fraction was isolated by chromatography. Synthesis of cDNA was done on a matrix of poly(A + ) RNA of human brain. The cDNA obtained was cloned in plasmid pBR322 for the PstI site using (dC/dG) sequences synthesized on the 3' ends of the vector molecule and cDNA respectively. In cloning 75 ng cDNA, the authors obtained approximately 10 5 recombinant. This library was analyzed by the hybridization method on columns with two radioactive ( 32 P) probes: the total cDNA preparation and the total nuclear DNA from the human brain. The number of copies of the cloned DNA fragment in the genome was determined by dot hybridization. Restricting fragments of human and rat DNA genomes homologous to the cloned cDNA were identified on radio-autographs. In each case, 10 micrograms of EcoRI DNA hydrolyzate was fractionated in 1% agarose gel. The probe was also readied with RNA samples fractionated in agarose gel with formaldehyde and transferred to a nitrocellulose filter under weak vacuum. The filter was hybridized with 0.1 micrograms DNA pAG 02, labeled with ( 32 P) to a specific activity of 0.5-1 x 10 9 counts/min x microgram. The autograph was exposed with amplifying screens at -70 0 C for 2 days

On multi-site damage identification using single-site training data

Science.gov (United States)

Barthorpe, R. J.; Manson, G.; Worden, K.

2017-11-01

This paper proposes a methodology for developing multi-site damage location systems for engineering structures that can be trained using single-site damaged state data only. The methodology involves training a sequence of binary classifiers based upon single-site damage data and combining the developed classifiers into a robust multi-class damage locator. In this way, the multi-site damage identification problem may be decomposed into a sequence of binary decisions. In this paper Support Vector Classifiers are adopted as the means of making these binary decisions. The proposed methodology represents an advancement on the state of the art in the field of multi-site damage identification which require either: (1) full damaged state data from single- and multi-site damage cases or (2) the development of a physics-based model to make multi-site model predictions. The potential benefit of the proposed methodology is that a significantly reduced number of recorded damage states may be required in order to train a multi-site damage locator without recourse to physics-based model predictions. In this paper it is first demonstrated that Support Vector Classification represents an appropriate approach to the multi-site damage location problem, with methods for combining binary classifiers discussed. Next, the proposed methodology is demonstrated and evaluated through application to a real engineering structure - a Piper Tomahawk trainer aircraft wing - with its performance compared to classifiers trained using the full damaged-state dataset.

An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.

Science.gov (United States)

Bland, Michael J; Ducos-Galand, Magaly; Val, Marie-Eve; Mazel, Didier

2017-07-14

Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla). The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

[Study on method of tracking the active cells in image sequences based on EKF-PF].

Science.gov (United States)

Tang, Chunming; Liu, Ying

2013-02-01

In cell image sequences, due to the nonlinear and nonGaussian motion characteristics of active cells, the accurate prediction and tracking is still an unsolved problem. We applied extended Kalman particle filter (EKF-PF) here in our study, attempting to solve the problem. Firstly we confirmed the existence and positions of the active cells. Then we established a motion model and improved it via adding motion angle estimation. Next we predicted motion parameters, such as displacement, velocity, accelerated velocity and motion angle, in region centers of the cells being tracked. Finally we obtained the motion traces of active cells. There were fourteen active cells in three image sequences which have been tracked. The errors were less than 2.5 pixels when the prediction values were compared with actual values. It showed that the presented algorithm may basically reach the solution of accurate predition and tracking of the active cells.

Site identification: environmental and radiological considerations

International Nuclear Information System (INIS)

Waite, D.A.

1980-01-01

Radiological and environmental considerations are recognized as being of utmost importance in planning, siting, licensing, operating, and decommissioning a high-level nuclear waste repository. In such a complex undertaking, it is important to identify the major concerns anticipated to arise in all of these phases in order to address them as early as possible in the program. Three representative activities/studies are summarized which will identify some of the important radiological and environmental considerations which must be addressed through this prolonged sequence of events and will indicate how these considerations are being addressed. It should be emphasized that these are only three of many which could have been chosen. The three key activities/studies are: (1) the NWTS Program criteria for identifying repository sites, (2) the generic guide for preparing environmental evaluations for deep drilling and (3) a preliminary environmental assessment for disposal of mined rock during excavation of a repository

Copper-zinc-superoxide dismutase (CuZnSOD), an antioxidant gene from seahorse (Hippocampus abdominalis); molecular cloning, sequence characterization, antioxidant activity and potential peroxidation function of its recombinant protein.

Science.gov (United States)

Perera, N C N; Godahewa, G I; Lee, Jehee

2016-10-01

Copper-zinc-superoxide dismutase (CuZnSOD) from Hippocampus abdominalis (HaCuZnSOD) is a metalloenzyme which belongs to the ubiquitous family of SODs. Here, we determined the characteristic structural features of HaCuZnSOD, analyzed its evolutionary relationships, and identified its potential immune responses and biological functions in relation to antioxidant defense mechanisms in the seahorse. The gene had a 5' untranslated region (UTR) of 67 bp, a coding sequence of 465 bp and a 3' UTR of 313 bp. The putative peptide consists of 154 amino acids. HaCuZnSOD had a predicted molecular mass of 15.94 kDa and a theoretical pI value of 5.73, which is favorable for copper binding activity. In silico analysis revealed that HaCuZnSOD had a prominent Cu-Zn_superoxide_dismutase domain, two Cu/Zn signature sequences, a putative N-glycosylation site, and several active sites including Cu(2+) and Zn(2+) binding sites. The three dimensional structure indicated a β-sheet barrel with 8 β-sheets and two short α-helical regions. Multiple alignment analyses revealed many conserved regions and active sites among its orthologs. The highest amino acid identity to HaCuZnSOD was found in Siniperca chuatsi (87.4%), while Maylandia zebra shared a close relationship in the phylogenetic analysis. Functional assays were performed to assess the antioxidant, biophysical and biochemical properties of overexpressed recombinant (r) HaCuZnSOD. A xanthine/XOD assay gave optimum results at pH 9 and 25 °C indicating these may be the best conditions for its antioxidant action in the seahorse. An MTT assay and flow cytometry confirmed that rHaCuZnSOD showed peroxidase activity in the presence of HCO3(-). In all the functional assays, the level of antioxidant activity of rHaCuZnSOD was concentration dependent; metal ion supplementation also increased its activity. The highest mRNA expressional level of HaCuZnSOD was found in blood. Temporal assessment under pathological stress showed a delay

Bottom-up driven involuntary auditory evoked field change: constant sound sequencing amplifies but does not sharpen neural activity.

Science.gov (United States)

Okamoto, Hidehiko; Stracke, Henning; Lagemann, Lothar; Pantev, Christo

2010-01-01

The capability of involuntarily tracking certain sound signals during the simultaneous presence of noise is essential in human daily life. Previous studies have demonstrated that top-down auditory focused attention can enhance excitatory and inhibitory neural activity, resulting in sharpening of frequency tuning of auditory neurons. In the present study, we investigated bottom-up driven involuntary neural processing of sound signals in noisy environments by means of magnetoencephalography. We contrasted two sound signal sequencing conditions: "constant sequencing" versus "random sequencing." Based on a pool of 16 different frequencies, either identical (constant sequencing) or pseudorandomly chosen (random sequencing) test frequencies were presented blockwise together with band-eliminated noises to nonattending subjects. The results demonstrated that the auditory evoked fields elicited in the constant sequencing condition were significantly enhanced compared with the random sequencing condition. However, the enhancement was not significantly different between different band-eliminated noise conditions. Thus the present study confirms that by constant sound signal sequencing under nonattentive listening the neural activity in human auditory cortex can be enhanced, but not sharpened. Our results indicate that bottom-up driven involuntary neural processing may mainly amplify excitatory neural networks, but may not effectively enhance inhibitory neural circuits.

Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

Energy Technology Data Exchange (ETDEWEB)

Storrs, Richard Wood [Univ. of California, Berkeley, CA (United States)

1992-08-01

Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by ³¹P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 Å of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an α-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

Energy Technology Data Exchange (ETDEWEB)

Storrs, R.W.

1992-08-01

Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors

Energy Technology Data Exchange (ETDEWEB)

Su, Hua-Poo; Yan, Youwei; Prasad, G. Sridhar; Smith, Robert F.; Daniels, Christopher L.; Abeywickrema, Pravien D.; Reid, John C.; Loughran, H. Marie; Kornienko, Maria; Sharma, Sujata; Grobler, Jay A.; Xu, Bei; Sardana, Vinod; Allison, Timothy J.; Williams, Peter D.; Darke, Paul L.; Hazuda, Daria J.; Munshi, Sanjeev (Merck)

2010-09-02

HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

New findings on the d(TGGGAG) sequence: Surprising anti-HIV-1 activity.

Science.gov (United States)

Romanucci, Valeria; Zarrelli, Armando; Liekens, Sandra; Noppen, Sam; Pannecouque, Christophe; Di Fabio, Giovanni

2018-02-10

The biological relevance of tetramolecular G-quadruplexes especially as anti-HIV agents has been extensively reported in the literature over the last years. In the light of our recent results regarding the slow G-quadruplex folding kinetics of ODNs based on d(TGGGAG) sequence, here we report a systematic anti-HIV screening to investigate the impact of the G-quadruplex folding on their anti-HIV activity. In particular, varying the single stranded concentrations of ODNs, it has been tested a pool of ODN sample solutions with different G-quadruplex concentrations. The anti-HIV assays have been designed favouring the limited kinetics involved in the tetramolecular G4-association based on the d(TGGGAG) sequence. Aiming to determine the stoichiometry of G-quadruplex structures in the same experimental conditions of the anti-HIV assays, a native gel electrophoresis was performed. The gel confirmed the G-quadruplex formation for almost all sample solutions while showing the formation of high order G4 structures for the more concentrated ODNs solutions. The most significant result is the discovery of a potent anti-HIV activity of the G-quadruplex formed by the natural d(TGGGAG) sequence (IC 50  = 14 nM) that, until now, has been reported to be completely inactive against HIV infection. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences.

Science.gov (United States)

Yadav, Shailendra; Kundu, Sharbadeb; Ghosh, Sankar K; Maitra, S S

2015-01-01

Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about "methanogenic archaea composition" and "abundance" in the contrasting ecosystems like "landfill" and "marshland" may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences

Directory of Open Access Journals (Sweden)

Shailendra Yadav

2015-01-01

Full Text Available Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic and Thaumarchaeota (mesophilic, were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

A near-infrared survey for pre-main sequence stars in Taurus

Science.gov (United States)

Gomez, Mercedes; Kenyon, Scott J.; Hartmann, Lee

1994-01-01

We present a near-infrared survey of approximately 2 sq deg covering parts of L1537, L1538, and Heiles cloud 2 in the Taurus-Auriga molecular cloud. Although this study is more sensitive than previous attempts to identify pre-main sequence stars in Taurus-Auriga, our survey regions contain only one new optically visible, young star. We did find several candidate embedded protostars; additional 10 micrometer photometry is necessary to verify the pre-main sequence nature of these sources. Our results--combined with those of previous surveys--show that the L1537/L1538 clouds contain no pre-main sequence stars. These two clouds are less dense than the active star formation sites in Taurus-Auriga, which suggests a cloud must achieve a threshold density to form stars.

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

Science.gov (United States)

Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

2004-01-01

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

Settings for Physical Activity – Developing a Site-specific Physical Activity Behavior Model based on Multi-level Intervention Studies

DEFF Research Database (Denmark)

Troelsen, Jens; Klinker, Charlotte Demant; Breum, Lars

Settings for Physical Activity – Developing a Site-specific Physical Activity Behavior Model based on Multi-level Intervention Studies Introduction: Ecological models of health behavior have potential as theoretical framework to comprehend the multiple levels of factors influencing physical...... to be taken into consideration. A theoretical implication of this finding is to develop a site-specific physical activity behavior model adding a layered structure to the ecological model representing the determinants related to the specific site. Support: This study was supported by TrygFonden, Realdania...... activity (PA). The potential is shown by the fact that there has been a dramatic increase in application of ecological models in research and practice. One proposed core principle is that an ecological model is most powerful if the model is behavior-specific. However, based on multi-level interventions...

Synthesis of a molecularly defined single-active site heterogeneous catalyst for selective oxidation of N-heterocycles.

Science.gov (United States)

Zhang, Yujing; Pang, Shaofeng; Wei, Zhihong; Jiao, Haijun; Dai, Xingchao; Wang, Hongli; Shi, Feng

2018-04-13

Generally, a homogeneous catalyst exhibits good activity and defined active sites but it is difficult to recycle. Meanwhile, a heterogeneous catalyst can easily be reused but its active site is difficult to reveal. It is interesting to bridge the gap between homogeneous and heterogeneous catalysis via controllable construction of a heterogeneous catalyst containing defined active sites. Here, we report that a molecularly defined, single-active site heterogeneous catalyst has been designed and prepared via the oxidative polymerization of maleimide derivatives. These polymaleimide derivatives can be active catalysts for the selective oxidation of heterocyclic compounds to quinoline and indole via the recycling of -C=O and -C-OH groups, which was confirmed by tracing the reaction with GC-MS using maleimide as the catalyst and by FT-IR analysis with polymaleimide as the catalyst. These results might promote the development of heterogeneous catalysts with molecularly defined single active sites exhibiting a comparable activity to homogeneous catalysts.

The status of siting activities for a low level waste repository in the Philippines

International Nuclear Information System (INIS)

Valdezco, E.M.; Visitacion, M.; Palattao, B.; Marcelo, E.A.; Venida, L.L.

2001-01-01

The process of site selection for a low level waste repository was initiated in 1976 when the Philippine Government decided to go nuclear and constructed the first Philippine Nuclear Power Plant in the Bataan Peninsula. However, all siting activities were suspended when the nuclear power plant was mothballed and the final decision was made to convert the plant into a combined cycle power plant. In 1995, an inter-agency committee was created under the Nuclear Power Steering Committee and mandated to conduct studies on siting of radioactive waste disposal facilities, and at the same time, perform R and D activities in support of the project. This paper describes the various siting activities carried out to date. (author)

Atypical Thioredoxins in Poplar: The Glutathione-Dependent Thioredoxin-Like 2.1 Supports the Activity of Target Enzymes Possessing a Single Redox Active Cysteine1[W

Science.gov (United States)

Chibani, Kamel; Tarrago, Lionel; Gualberto, José Manuel; Wingsle, Gunnar; Rey, Pascal; Jacquot, Jean-Pierre; Rouhier, Nicolas

2012-01-01

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways. PMID:22523226

Correlated mutations in protein sequences: Phylogenetic and structural effects

Energy Technology Data Exchange (ETDEWEB)

Lapedes, A.S. [Los Alamos National Lab., NM (United States). Theoretical Div.]|[Santa Fe Inst., NM (United States); Giraud, B.G. [C.E.N. Saclay, Gif/Yvette (France). Service Physique Theorique; Liu, L.C. [Los Alamos National Lab., NM (United States). Theoretical Div.; Stormo, G.D. [Univ. of Colorado, Boulder, CO (United States). Dept. of Molecular, Cellular and Developmental Biology

1998-12-01

Covariation analysis of sets of aligned sequences for RNA molecules is relatively successful in elucidating RNA secondary structure, as well as some aspects of tertiary structure. Covariation analysis of sets of aligned sequences for protein molecules is successful in certain instances in elucidating certain structural and functional links, but in general, pairs of sites displaying highly covarying mutations in protein sequences do not necessarily correspond to sites that are spatially close in the protein structure. In this paper the authors identify two reasons why naive use of covariation analysis for protein sequences fails to reliably indicate sequence positions that are spatially proximate. The first reason involves the bias introduced in calculation of covariation measures due to the fact that biological sequences are generally related by a non-trivial phylogenetic tree. The authors present a null-model approach to solve this problem. The second reason involves linked chains of covariation which can result in pairs of sites displaying significant covariation even though they are not spatially proximate. They present a maximum entropy solution to this classic problem of causation versus correlation. The methodologies are validated in simulation.

Human Splice-Site Prediction with Deep Neural Networks.

Science.gov (United States)

Naito, Tatsuhiko

2018-04-18

Accurate splice-site prediction is essential to delineate gene structures from sequence data. Several computational techniques have been applied to create a system to predict canonical splice sites. For classification tasks, deep neural networks (DNNs) have achieved record-breaking results and often outperformed other supervised learning techniques. In this study, a new method of splice-site prediction using DNNs was proposed. The proposed system receives an input sequence data and returns an answer as to whether it is splice site. The length of input is 140 nucleotides, with the consensus sequence (i.e., "GT" and "AG" for the donor and acceptor sites, respectively) in the middle. Each input sequence model is applied to the pretrained DNN model that determines the probability that an input is a splice site. The model consists of convolutional layers and bidirectional long short-term memory network layers. The pretraining and validation were conducted using the data set tested in previously reported methods. The performance evaluation results showed that the proposed method can outperform the previous methods. In addition, the pattern learned by the DNNs was visualized as position frequency matrices (PFMs). Some of PFMs were very similar to the consensus sequence. The trained DNN model and the brief source code for the prediction system are uploaded. Further improvement will be achieved following the further development of DNNs.

Decrease in gamma-band activity tracks sequence learning

Science.gov (United States)

Madhavan, Radhika; Millman, Daniel; Tang, Hanlin; Crone, Nathan E.; Lenz, Fredrick A.; Tierney, Travis S.; Madsen, Joseph R.; Kreiman, Gabriel; Anderson, William S.

2015-01-01

Learning novel sequences constitutes an example of declarative memory formation, involving conscious recall of temporal events. Performance in sequence learning tasks improves with repetition and involves forming temporal associations over scales of seconds to minutes. To further understand the neural circuits underlying declarative sequence learning over trials, we tracked changes in intracranial field potentials (IFPs) recorded from 1142 electrodes implanted throughout temporal and frontal cortical areas in 14 human subjects, while they learned the temporal-order of multiple sequences of images over trials through repeated recall. We observed an increase in power in the gamma frequency band (30–100 Hz) in the recall phase, particularly in areas within the temporal lobe including the parahippocampal gyrus. The degree of this gamma power enhancement decreased over trials with improved sequence recall. Modulation of gamma power was directly correlated with the improvement in recall performance. When presenting new sequences, gamma power was reset to high values and decreased again after learning. These observations suggest that signals in the gamma frequency band may play a more prominent role during the early steps of the learning process rather than during the maintenance of memory traces. PMID:25653598

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

Science.gov (United States)

Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Identification of a highly reactive threonine residue at the active site of γ-glutamyl transpeptidase

International Nuclear Information System (INIS)

Stole, E.; Seddon, A.P.; Wellner, D.; Meister, A.

1990-01-01

γ-Glutamyl transpeptidase an enzyme of major importance in glutathione metabolism, was inactivated by treating it with L-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-[3- 14 C]isoxazoleacetic acid. This selective reagent binds stoichiometrically to the enzyme; more than 90% of the label was bound to its light subunit. Enzymatic digestion of the light subunit gave a 14 C-labeled peptide that corresponds to amino acid residues 517-527 of the enzyme and two incomplete digestion products that contain this labeled peptide moiety. The radioactivity associated with this peptide was released with threonine-523 during sequencing by the automated gas-phase Edman method. The light subunit contains 14 other threonine residues and a total of 19 serine residues; these were not labeled. Threonine-523 is situated in the enzyme in an environment that greatly increases its reactivity, indicating that other amino acid residues of the enzyme must also participate in the active-site chemistry of the enzyme

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

Science.gov (United States)

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

Directed PCR-free engineering of highly repetitive DNA sequences

Directory of Open Access Journals (Sweden)

Preissler Steffen

2011-09-01

Full Text Available Abstract Background Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. Results For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. Conclusion Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

Hydrogenation active sites of unsupported molybdenum sulfide catalysts for hydroprocessing heavy oils

Energy Technology Data Exchange (ETDEWEB)

Iwata, Y.; Araki, Y.; Honna, K. [Tsukuba-branch, Advanced Catalyst Research Laboratory, Petroleum Energy Center, 1-1 Higashi, Tsukuba, 305-8565 Ibaraki (Japan); Miki, Y.; Sato, K.; Shimada, H. [National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, 305-8565 Ibaraki (Japan)

2001-02-20

The purpose of the present study was to elucidate the nature of the hydrogenation active sites on unsupported molybdenum sulfide catalysts, aimed at the improvement of the catalysts for the slurry processes. The number of hydrogenation active sites was found to relate to the 'inflection' on the basal plane of the catalyst particles. The comparison of the catalytic activity to that of an oil-soluble catalyst in the hydroprocessing of heavy oils suggests that the performance of the oil-soluble catalyst was near the maximum, unless another component such as Ni or Co was incorporated.

Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

Science.gov (United States)

De Rocquigny, H; Ficheux, D; Gabus, C; Allain, B; Fournie-Zaluski, M C; Darlix, J L; Roques, B P

1993-02-25

The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger.

Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

Directory of Open Access Journals (Sweden)

Annie Nguyen

Full Text Available Cystatin C (Cys C is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer's disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP, were all secreted as efficiently from immortalized human cells as their wild-type (WT counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively. Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as 'site 2' cleavage. However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K mutation, A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 ('site 1' or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease

Active site characterization and molecular cloning of Tenebrio molitor midgut trehalase and comments on their insect homologs.

Science.gov (United States)

Gomez, Ana; Cardoso, Christiane; Genta, Fernando A; Terra, Walter R; Ferreira, Clélia

2013-08-01

The soluble midgut trehalase from Tenebrio molitor (TmTre1) was purified after several chromatographic steps, resulting in an enzyme with 58 kDa and pH optimum 5.3 (ionizing active groups in the free enzyme: pK(e1) = 3.8 ± 0.2 pK(e2) = 7.4 ± 0.2). The purified enzyme corresponds to the deduced amino acid sequence of a cloned cDNA (TmTre1-cDNA), because a single cDNA coding a soluble trehalase was found in the T. molitor midgut transcriptome. Furthermore, the mass of the protein predicted to be coded by TmTre1-cDNA agrees with that of the purified enzyme. TmTre1 has the essential catalytic groups Asp 315 and Glu 513 and the essential Arg residues R164, R217, R282. Carbodiimide inactivation of the purified enzyme at different pH values reveals an essential carboxyl group with pKa = 3.5 ± 0.3. Phenylglyoxal modified a single Arg residue with pKa = 7.5 ± 0.2, as observed in the soluble trehalase from Spodoptera frugiperda (SfTre1). Diethylpyrocarbonate modified a His residue that resulted in a less active enzyme with pK(e1) changed to 4.8 ± 0.2. In TmTre1 the modified His residue (putatively His 336) is more exposed than the His modified in SfTre1 (putatively His 210) and that affects the ionization of an Arg residue. The architecture of the active site of TmTre1 and SfTre1 is different, as shown by multiple inhibition analysis, the meaning of which demands further research. Trehalase sequences obtained from midgut transcriptomes (pyrosequencing and Illumina data) from 8 insects pertaining to 5 different orders were used in a cladogram, together with other representative sequences. The data suggest that the trehalase gene went duplication and divergence prior to the separation of the paraneopteran and holometabolan orders and that the soluble trehalase derived from the membrane-bound one by losing the C-terminal transmembrane loop. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Survey of Agreement Rate between Simple MTC and Post Contrast T1 Sequence MRI for Diagnosing Active Multiple Sclerosis Plaques

Directory of Open Access Journals (Sweden)

N. Farshchian

2016-07-01

Full Text Available Introduction & Objective: MS is the most common disabling neurological disorder. Identifying new active MS plaques at the onset and clinical status and faster onset of treatment as well as evaluating the response to treatment is important and MRI with contrast is the best indicator for these measures. Materials & Methods: This study was cross-sectional including 62 patients with diagnosed MS. Whose clinical symptoms suggested the recurrence of MS. They were referred to the radiol-ogy department to undergo brain MRI with injection for the diagnosis of active plaques by a neurologist,The Data were analyzed using statistical tests and SPSS 21 software. Results: Based on the sequences of post contrast T1, pre contrast MTC and post contrast MTC 74, 272 and 271 plaques were respectively discovered. Detection of active MS plaques on T1 sequences after injection were in poor accordance and had significant difference with MTC before and after injection. Moreover, detection of active MS plaques on MTC sequences be-fore injection were in good accordance and did not show significant difference with MTC se-quences after injection. Conclusion: Based on these results, it seems that the purpose of MRI in MS patients is deter-mining the amount of active plaques. Sequences of pre contrast and post contrast MTC are significantly more than sequences of post contrast T1. Therefore, using sequences of MTC can be helpful in MRI. (Sci J Hamadan Univ Med Sci 2016; 23 (2:97-102

Modeling ChIP sequencing in silico with applications.

Directory of Open Access Journals (Sweden)

Zhengdong D Zhang

2008-08-01

Full Text Available ChIP sequencing (ChIP-seq is a new method for genomewide mapping of protein binding sites on DNA. It has generated much excitement in functional genomics. To score data and determine adequate sequencing depth, both the genomic background and the binding sites must be properly modeled. To develop a computational foundation to tackle these issues, we first performed a study to characterize the observed statistical nature of this new type of high-throughput data. By linking sequence tags into clusters, we show that there are two components to the distribution of tag counts observed in a number of recent experiments: an initial power-law distribution and a subsequent long right tail. Then we develop in silico ChIP-seq, a computational method to simulate the experimental outcome by placing tags onto the genome according to particular assumed distributions for the actual binding sites and for the background genomic sequence. In contrast to current assumptions, our results show that both the background and the binding sites need to have a markedly nonuniform distribution in order to correctly model the observed ChIP-seq data, with, for instance, the background tag counts modeled by a gamma distribution. On the basis of these results, we extend an existing scoring approach by using a more realistic genomic-background model. This enables us to identify transcription-factor binding sites in ChIP-seq data in a statistically rigorous fashion.

Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

Science.gov (United States)

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

2011-01-01

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

Full-length sequencing and identification of novel polymorphisms in ...

Indian Academy of Sciences (India)

The aim of this work was to sequence the entirecoding region of ACACA gene in Valle del Belice sheep breed to identify polymorphic sites. A total of 51 coding exons of ACACA gene were sequenced in 32 individuals of Valle del Belice sheep breed. Sequencing analysis and alignment of obtained sequences showed the ...

Air pathway analysis for cleanup at the chemical plant area of the Weldon Spring site

International Nuclear Information System (INIS)

Chang, Y.S.

1994-01-01

The Weldon Spring site is a mixed waste site located in St. Charles County, Missouri. Cleanup of the site is in the planning and design stage, and various engineering activities were considered for remedial action, including excavating soils, dredging sludge, treating various contaminated media in temporary facilities, transporting and staging supplies and contaminated material, and placing waste in an engineered disposal cell. Both contaminated and uncontaminated emissions from these activities were evaluated to assess air quality impacts and potential health effects for workers and the general public during the cleanup period. A site-specific air quality modeling approach was developed to address several complex issues, such as a variety of emission sources, an array of source/receptor configurations, and complicated sequencing/scheduling. This approach can be readily adapted to reflect changes in the expected activities as engineering plans are finalized

Poisoning Experiments Aimed at Discriminating Active and Less-Active Sites of Silica-Supported Tantalum Hydride for Alkane Metathesis

KAUST Repository

Saggio, Guillaume; Taoufik, Mostafa; Basset, Jean-Marie; Thivolle-Cazat, Jean

2010-01-01

Only 50% of the silica-supported tantalum hydride sites are active in the metathesis of propane. Indeed, more than 45% of the tantalum hydride can be eliminated by a selective oxygen poisoning of inactive sites with no significant decrease

Genome Sequencing and Analysis Conference IV

Energy Technology Data Exchange (ETDEWEB)

1993-12-31

J. Craig Venter and C. Thomas Caskey co-chaired Genome Sequencing and Analysis Conference IV held at Hilton Head, South Carolina from September 26--30, 1992. Venter opened the conference by noting that approximately 400 researchers from 16 nations were present four times as many participants as at Genome Sequencing Conference I in 1989. Venter also introduced the Data Fair, a new component of the conference allowing exchange and on-site computer analysis of unpublished sequence data.

Primary structure of human pancreatic elastase 2 determined by sequence analysis of the cloned mRNA

International Nuclear Information System (INIS)

Fletcher, T.S.; Shen, W.F.; Largman, C.

1987-01-01

A cDNA encoding elastase 2 has been cloned from a human pancreatic cDNA library. The cDNA contains a translation initiation site and a poly(A) recognition site and encodes a protein of 269 amino acids, including a proposed 16-residue signal peptide. The amino acid sequence of the deduced mature protein contains a 12-residue activation peptide containing a cysteine at residue 1 similar to that of chymotryspin. The proposed active enzyme contains all of the characteristic active-site amino acids, including His-57, Asp-102, and Ser-195. The S1 binding pocket is bounded by Gly-216 and Ser-226, making this pocket intermediate in size between chymotrypsins and elastase 1 or protease E, consistent with the substrate specificity of elastase 2 for long-chain aliphatic or aromatic amino acids. Computer modeling studies using the amino acid sequence of elastase 2 superimposed on the X-ray structure of porcine elastase 1 suggest that a change of Gln-192 in elastase 1 to Asn-192 in elastase 2 may account for the lower catalytic efficiency of the latter enzyme. Several basic residues appear to be near the ends of the extended binding pocket of elastases which might serve to anchor the enzyme to the elastin substrate. These studies indicate that elastases 2 and elastase 1 both contain an Arg-65A as well as a basic dipeptide at 223/224 which is not present in chymotrypsins. In addition, Arg-217A is present in humaan elastase 2 but absent in rat pancreatic protein which has been proposed to be an elastase 2 on the basis of sequence homology, but which was not isolated during screening of rat pancreatic tissue extracts for elastolytic activity

Isolation and complete amino acid sequence of human thymopoietin and splenin

International Nuclear Information System (INIS)

Audhya, T.; Schlesinger, D.H.; Goldstein, G.

1987-01-01

Human thymopoietin and splenin were isolated from human thymus and spleen, respectively, by monitoring tissue fractionation with a bovine thymopoietin RIA cross-reactive with human thymopoietin and splenin. Bovine thymopoietin and splenin are 49-amino acid polypeptides that differ by only 2 amino acids at positions 34 and 43; the change at position 34 in the active-site region changes the receptor specificities and biological activities. The complete amino acid sequences of purified human thymopoietin and splenin were determined and shown to be 48-amino acid polypeptides differing at four positions. Ten amino acids, constant within each species for thymopoietin and splenin, differ between the human and bovine polypeptides. The pentapeptide active side of thymopoietin (residues 32-36) is constant between the human and bovine thymopoietins, but position 34 in the active site of splenin has changed from glutamic acid in bovine splenin to alanine in human splenin, accounting for the biological activity of the human but not the bovine splenin on the human T-cell line MOLT-4

Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

Science.gov (United States)

Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.

2005-09-01

The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

Science.gov (United States)

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

Science.gov (United States)

Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

2013-05-10

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Threatened and endangered wildlife species of the Hanford Site related to CERCLA characterization activities

Energy Technology Data Exchange (ETDEWEB)

Fitzner, R.E. [Pacific Northwest Lab., Richland, WA (United States); Weiss, S.G.; Stegen, J.A. [Westinghouse Hanford Co., Richland, WA (United States)

1994-06-01

The US Department of Energy`s (DOE) Hanford Site has been placed on the National Priorities List, which requires that it be remediated under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) or Superfund. Potentially contaminated areas of the Hanford Site were grouped into operable units, and detailed characterization and investigation plans were formulated. The DOE Richland Operations Office requested Westinghouse Hanford Company (WHC) to conduct a biological assessment of the potential impact of these characterization activities on the threatened, endangered, and sensitive wildlife species of the Hanford Site. Additional direction for WHC compliances with wildlife protection can be found in the Environmental Compliance Manual. This document is intended to meet these requirements, in part, for the CERCLA characterization activities, as well as for other work comparable in scope. This report documents the biological assessment and describes the pertinent components of the Hanford Site as well as the planned characterization activities. Also provided are accounts of endangered, threatened, and federal candidate wildlife species on the Hanford Site and information as to how human disturbances can affect these species. Potential effects of the characterization activities are described with recommendations for mitigation measures.

Antitumor activity of sequence-specific alkylating agents: pyrolle-imidazole CBI conjugates with indole linker.

Science.gov (United States)

Shinohara, Ken-ichi; Bando, Toshikazu; Sasaki, Shunta; Sakakibara, Yogo; Minoshima, Masafumi; Sugiyama, Hiroshi

2006-03-01

DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (Ralkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.

A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

Science.gov (United States)

Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

2018-06-01

The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

Predicting human activities in sequences of actions in RGB-D videos

Science.gov (United States)

Jardim, David; Nunes, Luís.; Dias, Miguel

2017-03-01

In our daily activities we perform prediction or anticipation when interacting with other humans or with objects. Prediction of human activity made by computers has several potential applications: surveillance systems, human computer interfaces, sports video analysis, human-robot-collaboration, games and health-care. We propose a system capable of recognizing and predicting human actions using supervised classifiers trained with automatically labeled data evaluated in our human activity RGB-D dataset (recorded with a Kinect sensor) and using only the position of the main skeleton joints to extract features. Using conditional random fields (CRFs) to model the sequential nature of actions in a sequence has been used before, but where other approaches try to predict an outcome or anticipate ahead in time (seconds), we try to predict what will be the next action of a subject. Our results show an activity prediction accuracy of 89.9% using an automatically labeled dataset.

Decommissioning and decontamination activity, Gnome Site, Eddy County, New Mexico

International Nuclear Information System (INIS)

1978-10-01

The purpose of this assessment is to present a brief description of the proposed activity and its potential impacts on the environment. This assessment will constitute an evaluation as to whether or not a formal Environmental Statement need be prepared. As background to the proposed activity, Project Gnome was an underground nuclear test conducted in December 1961 as part of the PLOWSHARE Program. The project site is located about 25 miles southeast of Carlsbad, New Mexico. By means of an excavated shaft and tunnel, a 3-kiloton nuclear explosive was emplaced and detonated in a salt bed about 1200 feet below the surface. The uncontaminated rock and salt muck from the original excavation and subsequent contaminated muck and minor construction debris from reentry activities into the nuclear cavity is commingled and stored in a pile near the Gnome/Coach Shaft. Other areas on the site are known to have been contaminated. In 1969, a program was conducted to cleanup and dispose of all surface contamination to whatever depth it occurred in excess of 0.1 mR/hr. Contaminated materials and soil were collected and disposed into the Gnome shaft, which was filled and sealed. Since then, NV has proposed to DOE/HQ much lower criteria for residual radioactive contamination for the Gnome Site. These proposed criteria were to collect and dispose of surficial materials which contain more than 2 x 10 -5 microcuries per gram of soil for beta/gamma emitters and 3 x 10 -2 microcuries per milliliter of tritium in soil moisture. According to the latest reconnaissance in 1972, low concentrations of Cs-137, Sr-90 and tritium were present at various locations on the site in excess of these proposed guidelines. Other operational areas within the site are suspected of containing radioactive contamination in much lesser volume, which are to be determined by careful probing and monitoring, as described in the next section

UMTRA [Uranium Mill Tailings Remedial Action] Project site management manual

International Nuclear Information System (INIS)

1990-10-01

The purpose of this manual is to summarize the organizational interfaces and the technical approach used to manage the planning, design development, National Environmental Policy Act (NEPA) compliance, engineering, and remedial action required to stabilize and control the designated Uranium Mill Tailings Remedial Action (UMTRA) Project sites. This manual describes the Project's objective, participants' roles and responsibilities, technical approach for accomplishing the objective, and planning and managerial controls to be used in performing the site work. The narrative follows the flow of activities depicted in Figure 1.1, which provides the typical sequence of key Project activities. A list of acronyms used is presented at the end of the manual. The comparable manual for UMTRA Project vicinity properties is the ''Vicinity Properties Management and Implementation Manual'' (VPMIM) (UMTRA-DOE/AL-050601). Together, the two manuals cover the remedial action activities associated with UMTRA Project sites. The UMTRA Project's objective is to stabilize and control the uranium mill tailings, vicinity property materials, and other residual radioactive materials at the designated sites (Figure 1.2) in a safe and environmentally sound manner in order to minimize radiation health hazards to the public. 26 figs., 6 tabs

The fitness landscapes of cis-acting binding sites in different promoter and environmental contexts.

Directory of Open Access Journals (Sweden)

Ryan K Shultzaberger

2010-07-01

Full Text Available The biophysical nature of the interaction between a transcription factor and its target sequences in vitro is sufficiently well understood to allow for the effects of DNA sequence alterations on affinity to be predicted. But even in relatively simple in vivo systems, the complexities of promoter organization and activity have made it difficult to predict how altering specific interactions between a transcription factor and DNA will affect promoter output. To better understand this, we measured the relative fitness of nearly all Escherichia coli sigma(70 -35 binding sites in different promoter and environmental contexts by competing four randomized -35 promoter libraries controlling the expression of the tetracycline resistance gene (tetagainst each other in increasing concentrations of drug. We sequenced populations after competition to determine the relative enrichment of each -35 sequence. We observed a consistent relationship between the frequency of recovery of each -35 binding site and its predicted affinity for sigma(70 that varied depending on the sequence context of the promoter and drug concentration. Overall the relative fitness of each promoter could be predicted by a simple thermodynamic model of transcriptional regulation, in which the rate of transcriptional initiation (and hence fitness is dependent upon the overall stability of the initiation complex, which in turn is dependent upon the energetic contributions of all sites within the complex. As implied by this model, a decrease in the free energy of association at one site could be compensated for by an increase in the binding energy at another to produce a similar output. Furthermore, these data show that a large and continuous range of transcriptional outputs can be accessed by merely changing the -35, suggesting that evolved or engineered mutations at this site could allow for subtle and precise control over gene expression.

Delta Learning Rule for the Active Sites Model

OpenAIRE

Lingashetty, Krishna Chaithanya

2010-01-01

This paper reports the results on methods of comparing the memory retrieval capacity of the Hebbian neural network which implements the B-Matrix approach, by using the Widrow-Hoff rule of learning. We then, extend the recently proposed Active Sites model by developing a delta rule to increase memory capacity. Also, this paper extends the binary neural network to a multi-level (non-binary) neural network.

Methodology for contaminated sites of military activity territories restoration

International Nuclear Information System (INIS)

Khrushchov, D. P.; Yushchenko, Yu. V.; Shekhunova, S. B.

2002-01-01

Major part of Eastern Europe countries meet environmental problems related to sites of military activity. Major part of these sites is characterised with degradation of natural landscapes and contamination of geological environment with toxic and hazardous waste representing actual and potential danger for population and environment. Actual danger is caused with localisation of toxic waste, hazardous materials and waste which are preventing normal land use. Potential danger is related to successive dispersion of contamination in biosphere as well as origin of new derivatives and products having toxic and hazardous properties. The list of such sites and objects comprises bases of land, air and naval forces. These objects include a network of infrastructures: storages of fuels and lubricants (surface, underground), filling stations, pipe lines, reparation stations, garages, decontamination stations, underground storages of different purposes, depots (for ammunition, chemical products), hospitals, constructions, firing grounds (tank, artillery, aircraft bombing etc.) and waste disposal sites. Special programs aimed at military industries and bases contaminated sites remediation have been carrying out in developed countries (USA, United Kingdom, Germany etc.). This experience was used in the frames of joint programs having been founded in several countries of Central and Eastern Europe (Chesh Republic, Slovakia, Lithuania etc.). (author)

Active chemisorption sites in functionalized ionic liquids for carbon capture.

Science.gov (United States)

Cui, Guokai; Wang, Jianji; Zhang, Suojiang

2016-07-25

Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

Structural and functional analysis of an enhancer GPEI having a phorbol 12-O-tetradecanoate 13-acetate responsive element-like sequence found in the rat glutathione transferase P gene.

Science.gov (United States)

Okuda, A; Imagawa, M; Maeda, Y; Sakai, M; Muramatsu, M

1989-10-05

We have recently identified a typical enhancer, termed GPEI, located about 2.5 kilobases upstream from the transcription initiation site of the rat glutathione transferase P gene. Analyses of 5' and 3' deletion mutants revealed that the cis-acting sequence of GPEI contained the phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE)-like sequence in it. For the maximal activity, however, GPEI required an adjacent upstream sequence of about 19 base pairs in addition to the TRE-like sequence. With the DNA binding gel-shift assay, we could detect protein(s) that specifically binds to the TRE-like sequence of GPEI fragment, which was possibly c-jun.c-fos complex or a similar protein complex. The sequence immediately upstream of the TRE-like sequence did not have any activity by itself, but augmented the latter activity by about 5-fold.

Intraspecific variations in Cyt b and D-loop sequences of Testudine species, Lissemys punctata from south Karnataka

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R. Lalitha

2018-01-01

Full Text Available The freshwater Testudine species have gained importance in recent years, as most of their population is threatened due to exploitation for delicacy and pet trade. In this regard, Lissemys punctata, a freshwater terrapin, predominantly distributed in Asian countries has gained its significance for the study. A pilot study report on mitochondrial markers (Cyt b and D-loop conducted on L. punctata species from southern Karnataka, India was presented in this investigation. A complete region spanning 1.14 kb and ∼1 kb was amplified by HotStart PCR and sequenced by Sanger sequencing. The Cyt b sequence revealed 85 substitution sites, no indels and 17 parsimony informative sites, whereas D-loop showed 189 variable sites, 51 parsimony informative sites with 5′ functional domains TAS, CSB-F, CSBs (1, 2, 3 preceding tandem repeat at 3′ end. Current data highlights the intraspecific variations in these target regions and variations validated using suitable evolutionary models points out that the overall point mutations observed in the region are transitions leading to no structural and functional alterations. The mitochondrial data generated uncover the genetic diversity within species and conservationist can utilize the data to estimate the effective population size or for forensic identification of animal or its seizures during unlawful trade activities.

Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

DEFF Research Database (Denmark)

Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer

2015-01-01

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example......, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an 18F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[18F]fluorobenzoate, and the [18F]ASIS was purified on a PD-10 desalting...... column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [18F]ASIS to TF and to a specific anti-factor VII...

Antimicrobial Activity of Truncated and Polyvalent Peptides Derived from the FKCRRQWQWRMKKGLA Sequence against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923

Directory of Open Access Journals (Sweden)

Nataly de Jesús Huertas

2017-06-01

Full Text Available Peptides derived from LfcinB were designed and synthesized, and their antibacterial activity was tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Specifically, a peptide library was constructed by systemically removing the flanking residues (N or C-terminal of Lfcin 17–31 (17FKCRRWQWRMKKLGA31, maintaining in all peptides the 20RRWQWR25 sequence that corresponds to the minimal antimicrobial motif. For this research, also included were (i a peptide containing an Ala instead of Cys ([Ala19]-LfcinB 17–31 and (ii polyvalent peptides containing the RRWQWR sequence and a non-natural amino acid (aminocaproic acid. We established that the lineal peptides LfcinB 17–25 and LfcinB 17–26 exhibited the greatest activity against E. coli ATCC 25922 and S. aureus ATCC 25923, respectively. On the other hand, polyvalent peptides, a dimer and a tetramer, exhibited the greatest antibacterial activity, indicating that multiple copies of the sequence increase the activity. Our results suggest that the dimeric and tetrameric sequence forms potentiate the antibacterial activity of lineal sequences that have exhibited moderate antibacterial activity.

Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis.

Science.gov (United States)

Gebhardt, Yvonne Helen; Witte, Simone; Steuber, Holger; Matern, Ulrich; Martens, Stefan

2007-07-01

Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues.

Science.gov (United States)

Desideri, A; Falconi, M; Polticelli, F; Bolognesi, M; Djinovic, K; Rotilio, G

1992-01-05

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.

Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.

Directory of Open Access Journals (Sweden)

Silvia Schumann

Full Text Available Mouse aldehyde oxidase (mAOX1 forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

Sequence walkers: a graphical method to display how binding proteins interact with DNA or RNA sequences | Center for Cancer Research

Science.gov (United States)

A graphical method is presented for displaying how binding proteins and other macromolecules interact with individual bases of nucleotide sequences. Characters representing the sequence are either oriented normally and placed above a line indicating favorable contact, or upside-down and placed below the line indicating unfavorable contact. The positive or negative height of each letter shows the contribution of that base to the average sequence conservation of the binding site, as represented by a sequence logo.

Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

Science.gov (United States)

deCamp, Allan C; Rolland, Morgane; Edlefsen, Paul T; Sanders-Buell, Eric; Hall, Breana; Magaret, Craig A; Fiore-Gartland, Andrew J; Juraska, Michal; Carpp, Lindsay N; Karuna, Shelly T; Bose, Meera; LePore, Steven; Miller, Shana; O'Sullivan, Annemarie; Poltavee, Kultida; Bai, Hongjun; Dommaraju, Kalpana; Zhao, Hong; Wong, Kim; Chen, Lennie; Ahmed, Hasan; Goodman, Derrick; Tay, Matthew Z; Gottardo, Raphael; Koup, Richard A; Bailer, Robert; Mascola, John R; Graham, Barney S; Roederer, Mario; O'Connell, Robert J; Michael, Nelson L; Robb, Merlin L; Adams, Elizabeth; D'Souza, Patricia; Kublin, James; Corey, Lawrence; Geraghty, Daniel E; Frahm, Nicole; Tomaras, Georgia D; McElrath, M Juliana; Frenkel, Lisa; Styrchak, Sheila; Tovanabutra, Sodsai; Sobieszczyk, Magdalena E; Hammer, Scott M; Kim, Jerome H; Mullins, James I; Gilbert, Peter B

2017-01-01

Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing

Directory of Open Access Journals (Sweden)

Doori Park

2015-09-01

Full Text Available Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF in genetically modified rice cells. A total of 29.3 Gb (~72× coverage was sequenced with a 2 × 150 bp paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

Anti-nuclear activities and critics concerning nuclear power plant sites

International Nuclear Information System (INIS)

Rhee, We-Beg

2000-01-01

Korea has dynamic nuclear power expansion programs, operating 16 nuclear units producing 13710 MW in total located on 4 different sites. Last year, nuclear power supplied over 40 % of national total electricity demands. In 1998, Korean government initiated re-designation work investigating circumstance changes to rule out the unnecessary sites in consideration of a long-term power supply. Korean government has determined to expand the Ulchin site and to designate one point of Woolju county as a new candidate site, and ruled out the rest candidate sites at the end of 1998. About such a governmental measure, the two areas show different reactions. Ulchin where nuclear power plant has been operated safely for about 10 years was likely to accept the governmental determination in spite of some opposition and called for several financial supports for local development. WooIju county, however, showed a strong opposition among local environmental groups and autonomous politicians, and they presented a variety of anti-nuclear activities including demonstrations mainly at the neighbouring metropolis, Ulsan city

Effects of resource activities upon repository siting and waste containment with reference to bedded salt

International Nuclear Information System (INIS)

Ashby, J.; Rowe, J.

1980-02-01

The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases

Disturbance opens recruitment sites for bacterial colonization in activated sludge

OpenAIRE

Marr, Junko; Spear, John; Drewes, Jörg; Vuono, David

2015-01-01

Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we sh...

Site-SpecificCu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N), Using Copper Free Click Chemistry

DEFF Research Database (Denmark)

Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H

2018-01-01

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migrati...

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

Directory of Open Access Journals (Sweden)

Soares Alexei S

2007-11-01

Full Text Available Abstract Background Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. Results We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol. The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. Conclusion We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

Science.gov (United States)

Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

2018-05-30

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

An evaluation of sequence tagged microsatellite site markers for genetic analysis within Citrus and related species.

Science.gov (United States)

Kijas, J M; Fowler, J C; Thomas, M R

1995-04-01

Microsatellites, also called sequence tagged microsatellite sites (STMSs), have become important markers for genome analysis but are currently little studied in plants. To assess the value of STMSs for analysis within the Citrus plant species, two example STMSs were isolated from an intergeneric cross between rangpur lime (Citrus x limonia Osbeck) and trifoliate orange (Poncirus trifoliata (L.) Raf.). Unique flanking primers were constructed for polymerase chain reaction amplification both within the test cross and across a broad range of citrus and related species. Both loci showed length variation between test cross parents with alleles segregating in a Mendelian fashion to progeny. Amplification across species showed the STMS flanking primers to be conserved in every genome tested. The traits of polymorphism, inheritance, and conservation across species mean that STMS markers are ideal for genome mapping within Citrus, which contains high levels of genetic variability.

Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

Directory of Open Access Journals (Sweden)

Ting Wei

Full Text Available The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa-long hydrophobic region (termed TM2. However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G. Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s oriented parallel to the membrane inner surface.

Carcinogen-DNA interaction study by base sequence footprinting. Progress report, July 1, 1985-January 21, 1986

International Nuclear Information System (INIS)

Bases, R.

1986-01-01

Acetyl-aminofluorene (AAF) modified plasmid pSV 2 CAT is being studied to learn how the adducts influence expression of chloramphenicol acetyltransferase (CAT) genes. phi X-174 RF DNA exhibits specific base sequence abnormalities induced by the formation of AAF adducts. The DNAase I sensitive state of AAF modified DNA sequences could presumably lead to enhanced expression of genes since it is a well-known characteristic of active or potentially active derepressed genes. DNAase I hypersensitive sites are necessary but not sufficient for transcription. We observed enhanced expression of CAT genes in CV-1 cells after transfection with modified plasmids, using electroporation to introduce the plasmids into the cells. 34 refs., 2 figs

Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

OpenAIRE

Martin, M E; Piette, J; Yaniv, M; Tang, W J; Folk, W R

1988-01-01

The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription fac...

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site.

Science.gov (United States)

Wu, Fei; Shao, Yong; Ma, Kun; Cui, Qinghua; Liu, Guiying; Xu, Shujuan

2012-04-28

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe. This journal is © The Royal Society of Chemistry 2012

Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

Science.gov (United States)

Asakura, H; Makino, S; Kobori, H; Watarai, M; Shirahata, T; Ikeda, T; Takeshi, K

2001-08-01

Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans.

The nature of the active site in heterogeneous metal catalysis

DEFF Research Database (Denmark)

Nørskov, Jens Kehlet; Bligaard, Thomas; Larsen, Britt Hvolbæk

2008-01-01

This tutorial review, of relevance for the surface science and heterogeneous catalysis communities, provides a molecular-level discussion of the nature of the active sites in metal catalysis. Fundamental concepts such as "Bronsted-Evans-Polanyi relations'' and "volcano curves'' are introduced...

Spreadsheet-based program for alignment of overlapping DNA sequences.

Science.gov (United States)

Anbazhagan, R; Gabrielson, E

1999-06-01

Molecular biology laboratories frequently face the challenge of aligning small overlapping DNA sequences derived from a long DNA segment. Here, we present a short program that can be used to adapt Excel spreadsheets as a tool for aligning DNA sequences, regardless of their orientation. The program runs on any Windows or Macintosh operating system computer with Excel 97 or Excel 98. The program is available for use as an Excel file, which can be downloaded from the BioTechniques Web site. Upon execution, the program opens a specially designed customized workbook and is capable of identifying overlapping regions between two sequence fragments and displaying the sequence alignment. It also performs a number of specialized functions such as recognition of restriction enzyme cutting sites and CpG island mapping without costly specialized software.

Improved ethanol electrooxidation performance by shortening Pd-Ni active site distance in Pd-Ni-P nanocatalysts

Science.gov (United States)

Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

2017-01-01

Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (~5 nm) Pd-Ni-P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni-P heterodimers into Pd-Ni-P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites.

Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

Science.gov (United States)

Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

2015-04-01

On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

Biophysical and structural considerations for protein sequence evolution

Directory of Open Access Journals (Sweden)

Grahnen Johan A

2011-12-01

Full Text Available Abstract Background Protein sequence evolution is constrained by the biophysics of folding and function, causing interdependence between interacting sites in the sequence. However, current site-independent models of sequence evolutions do not take this into account. Recent attempts to integrate the influence of structure and biophysics into phylogenetic models via statistical/informational approaches have not resulted in expected improvements in model performance. This suggests that further innovations are needed for progress in this field. Results Here we develop a coarse-grained physics-based model of protein folding and binding function, and compare it to a popular informational model. We find that both models violate the assumption of the native sequence being close to a thermodynamic optimum, causing directional selection away from the native state. Sampling and simulation show that the physics-based model is more specific for fold-defining interactions that vary less among residue type. The informational model diffuses further in sequence space with fewer barriers and tends to provide less support for an invariant sites model, although amino acid substitutions are generally conservative. Both approaches produce sequences with natural features like dN/dS Conclusions Simple coarse-grained models of protein folding can describe some natural features of evolving proteins but are currently not accurate enough to use in evolutionary inference. This is partly due to improper packing of the hydrophobic core. We suggest possible improvements on the representation of structure, folding energy, and binding function, as regards both native and non-native conformations, and describe a large number of possible applications for such a model.

A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

Directory of Open Access Journals (Sweden)

Agata Jacewicz

Full Text Available Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A, that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

Engineering of a DNA Polymerase for Direct m6 A Sequencing.

Science.gov (United States)

Aschenbrenner, Joos; Werner, Stephan; Marchand, Virginie; Adam, Martina; Motorin, Yuri; Helm, Mark; Marx, Andreas

2018-01-08

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N 6 -methyladenosine (m 6 A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m 6 A-containing RNA prior to sequencing, since m 6 A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m 6 A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N 6 -methylation. We identified a mutant that exhibits increased misincorporation opposite m 6 A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m 6 A sites directly from the sequencing data of untreated RNA samples. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

Energy Technology Data Exchange (ETDEWEB)

Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

1994-09-01

The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

International Nuclear Information System (INIS)

Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

1994-09-01

The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria

Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

International Nuclear Information System (INIS)

Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

2013-01-01

The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

Energy Technology Data Exchange (ETDEWEB)

Beres, Christopher M.; Fort, E. Joseph [Cabrera Services, Inc., 473 Silver Lane, East Hartford, CT 06118 (United States); Boyle, James D. [United States Army Corps of Engineers - Buffalo, 1776 Niagara Street, Buffalo, NY 14207 (United States)

2013-07-01

The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

Pattern recognition in complex activity travel patterns : comparison of Euclidean distance, signal-processing theoretical, and multidimensional sequence alignment methods

NARCIS (Netherlands)

Joh, C.H.; Arentze, T.A.; Timmermans, H.J.P.

2001-01-01

The application of a multidimensional sequence alignment method for classifying activity travel patterns is reported. The method was developed as an alternative to the existing classification methods suggested in the transportation literature. The relevance of the multidimensional sequence alignment